Experimental infection and protection against ... - TI Pharma

Experimental infection and protection 

against Plasmodium falciparum malaria in 

humans 

Meta Roestenberg

ISBN 978-90-9027235-1 

Cover photo by Iris Spaink, www.irisspaink.nl 

Printed by Ridderprint 

Copyright 2013© Meta Roestenberg 

All rights reserved. No part of this book may be reproduced or transmitted in 

any form or by any means, electronic of mechanical, including photocopying, 

recording, or by any information storage and retrieval system without express 

written permission from the autor, or where appropriate, the publisher of the 

articles

Experimental infection and protection 

against Plasmodium falciparum malaria in 

humans 

Proefschrift 

ter verkrijging van de graad van doctor 

aan de Radboud Universiteit Nijmegen 

op gezag van de rector magnificus prof. mr. S.C.J.J.Kortman, 

volgens besluit van het college van Decanen 

In het openbaar te verdedigen op maandag 14 januari 2013 

om 15.30 uur precies 

Door 

Meta Roestenberg 

Geboren op 24 januari 1981 

te De Bilt

Promotoren: 

Prof. dr. R.W. Sauerwein 

Prof. dr. A.J.A.M. van der Ven 

Manuscriptcommissie: 

Prof. dr. P.A.B.M. Smits 

Prof. dr. C.G. Figdor 

Prof. dr. J.T. van Dissel, Leiden University

Contents 

Contents 5 

Chapter 1 - Introduction 7 

Section 1 - Apical Membrane Antigen 1 29 

Chapter 2 - Safety and Immunogenicity of a Recombinant Plasmodium 

falciparum AMA1 Malaria vaccine Adjuvanted with Alhydrogel TM, Montanide 

ISA 720 or AS02. 31 

Chapter 3 - Humoral immune responses to a single allele PfAMA1 vaccine in 

healthy malaria-naïve adults. 57 

Chapter 4 - A saturation of antibody avidity and concentration induced by 

malaria vaccine candidate Apical Membrane Antigen 1 81 

Section 2 - Controlled human malaria infection model 95 

Chapter 5 - Comparison of clinical and parasitological data from experimental 

human malaria challenge trials 97 

Chapter 6 - Efficacy of pre-erythrocytic and blood-stage malaria vaccines can be 

assessed in small sporozoite challenge trials in human volunteers 117 

Chapter 7 - NF135.C10: a new Plasmodium falciparum clone for controlled 

human malaria infections 129 

Chapter 8 - Induction of malaria in volunteers by intradermal injection of 

cryopreserved Plasmodium falciparum sporozoites 151 

Section 3 - Whole parasite inoculation 171 

Chapter 9 - Protection against a Malaria Challenge by Sporozoite Inoculation 173 

Chapter 10 - Long-term protection against malaria after experimental sporozoite 

inoculation 193 

Chapter 11 - General Discussion 215 

Chapter 12 - Summary, Samenvatting, List of publications, Dankwoord, C.V. 243

Chapter 1 

Introduction

Introduction 9 

Malaria 

Malaria is one of the world's most common infectious diseases with an annual 

225 million cases leading to an estimated 655 000 deaths in 2010, mainly among 

young children. Malaria morbidity and mortality is particularly severe in Sub- 

Saharan Africa, where 91% of deaths occur [1](Figure 1). 

Figure 1. Malaria, countries or areas at risk for transmission, 2010. 

www.who.int/malaria 

Malaria is responsible for an enormous economic burden due to medical 

expenses, missed education and lost productivity. It has been estimated that the 

GDP of countries endemic for malaria grows at 1.3% per year slower than 

countries which are malaria-free, even after other factors such as initial income 

level, overall health and tropical location are taken into account [2]. Moreover, 

in highly endemic settings up to 50% of low birth weight (LBW) deliveries can be 

attributed to malaria in pregnancy, leading to approximately 100 000 infant 

deaths annually [3]. 

Plasmodium falciparum (Pf) is the malaria parasite species responsible for 

almost all global malaria deaths. It is one of five human malaria parasite species 

transmitted by the bites of infected Anopheles mosquitoes. A clinical Pf infection 

in humans is characterised by non-specific symptoms such as fever, headache, 

myalgia, fatigue and abdominal discomfort. Neurological deficits such as coma 

or convulsions and severe anemia are signs of severe disease and have high 

mortality rates [4]. Plasmodium vivax, ovale, malariae and knowlesi cause a

10 Chapter 1 

generally milder disease that is rarely fatal, but, in the case of vivax and ovale 

malaria, that may remain in a dormant form for years. 

Malaria control receives high priority from the international community, which 

has been established in the United Nation's Millennium Development Goal 6 

aiming at the reduction of the incidence of malaria and other major diseases by 

2015. Malaria control is also central to Millennium Development Goal 4, 

targeting a two-third reduction in the mortality rate among children under the 

age of five [5]. 

Current control strategies are based on early diagnosis and treatment of malaria 

infections combined with preventive measures aimed at mosquito control. The 

development of widespread resistance to the anti-malarial drugs chloroquine 

and sulfadoxine-pyrimethamine has re-directed treatment strategies to 

artemisinine-based combination therapies, such as artemether plus 

lumefantrine, artesunate plus amodiaquine, artesunate plus mefloquine or 

artesunate plus sulfadoxine-pyrimethamine, which are now the WHOrecommended 

treatment regimens. Unfortunately, Pf resistance has been 

observed to many of the currently used antimalarial drugs (amodiaquine, 

chloroquine, mefloquine, quinine and sulfadoxine-pyrimethamine) and, more 

recently, also to artemisinine derivatives [6]. To date, there are no new 

medicines in advanced stages of development to replace the artimisinins [7]. 

Vector control is the primary intervention for reducing malaria transmission at 

the community level. When universal vector control coverage is achieved by 

impregnating bednets and spraying indoor surfaces of houses with insecticides, 

transmission can be reduced to close to zero. However, the increasing resistance 

of mosquitoes to insecticides including dichlorodiphenyltrichloroethane (DDT) 

and pyrethroids, particularly in Africa, poses challenges to current prevention 

policies [8]. 

The current control strategies permitted the interruption of malaria 

transmission in low transmission countries, particularly in those with a robust 

institutional infrastructure and well-functioning health systems, and in those 

neighbouring malaria-free areas [8]. However, the lack of new effective antimalarial 

drugs and insecticides places malaria control and elimination efforts at 

considerable risk. In order to reach the ultimate goal of malaria eradication, 

much greater gains could be achieved with currently available tools, including 

elimination from a number of countries and regions, but even with maximal 

effort we will fall short of elimination in many areas and of global eradication


Preclinical Clinical 

Phase 1A Phase 1B Phase 2A Phase 2B Phase 3 

Aim Safety, dose, efficacy, long-term Safety & Safety & Efficacy & Efficacy & Adverse 

effects 

dose dose side effects side effects reactions 

and longterm 

Population Rodent and/or simian Non- Endemic Non- Endemic Endemic 

endemicendemic 

Duration 3-6 years 3-12 3-12 3-24 3-24 3 years 

months months months months 

Typical size of 

~10 ~10 ~10 100-300 10,000test 

group 

RTS,S/AS01E 

AdCh63/MVA ME-TRAP 

PfSPZ: metabolically active, nonreplicating 

malaria sporozoite vaccine 

Adenovirus (Ad35) vectored CS 

polyepitope DNA EP1300 

CSVAC 

Pf CelTOS FMP012 

Ad35.CS.01/Ad26.CS.01 

(Heterologous adenovirus primeboost 

strategy) 

Two-component paediatric malaria 

vaccine (CS) 

Pf GAP p52-/p32- (Genetically 

Attenuated Sporozoites) 

RTS,S/AS02A 

DNA/MVA prime-boost Multi-Epitope 

(ME) string + TRAP 

FP9 MVA prime-boost ME-TRAP 

FP9 CSP + LSA-1 epitope/ MVA-CSP + 

LSA-1 epitope 

MuStDO5 (Multi-Stage DNA vaccine 

Operation, 5 antigens) 

NMRC-MV-Ad-PfC 

CSP long synthetic peptide 

LSA-3 (inactive) 

FMP011/AS01B (LSA-1 E. coliexpressed 

in AS01B adjuvant) 

FMP011/AS02A (LSA-1 E. coliexpressed 

in AS02A adjuvant) 

DNA/MVA CSP 

HepB Core-Ag CSP VLP 

RTS,S/AS02 and SSP2/TRAP 

RTS,S/AS02 and MVA CSP 

RTS,S/AS02 and DNA CSP 

CSP DNA immunization 

50,000 

Pre-erythrocytic vaccine candidates 

Figure 2: Malaria vaccines in (pre-)clinical development. Vaccines in lighter shades have 

stopped or halted in development. [16] 

[7]. Current efforts should thus be supplemented with a structured research 

agenda taking a multidisciplinary global approach [9]. An effective malaria 

vaccine is a key tool of critical research that is needed to support malaria control 

and eradication [9]. Ambitious goals in this regard have been set by the Malaria 

Vaccine Technology Roadmap, which aims to develop and license a malaria 

vaccine with more than 80% protective efficacy against clinical disease that lasts 

longer than four years, by the year 2025 [10]. However, despite extensive

12 Chapter 1 

Preclinical Clinical 

Phase 1A Phase 1B Phase 2A Phase 2B Phase 3 

Aim Safety, dose, efficacy, long-term effects Safety & dose Safety & dose Efficacy & side Efficacy & side Adverse 

effects effects reactions and 

long-term 

Population Rodent and/or simian Non-endemic Endemic Non-endemic Endemic Endemic 

Duration 3-6 years 3-12 months 3-12 months 3-24 months 3-24 months 3 years 

Typical size of 

test group 

~10 ~10 ~10 100-300 10,000-50,000 

FMP2.1/AS02A (AMA-1 3D7 E. coli-expressed in 

Erythrocytic vaccine candidates 

Combined vaccine 

candidates 

Transmissio 

n blocking 

vaccine 

candidates 

GMZ2 

MSP3 [181-276] 

AMA1-C1/Alhydrogel ® + CPG 7909 

AdCh63/MVA MSP1 

FMP2.1/AS01B (AMA-1 3D7 E. coli expressed 

in ASO1B adjuvant) 

AdCh63 AMA1/MVA AMA1 

EBA175 RII 

FMP010/AS01B (MSP-1 42 FVO E. coliexpressed 

in AS01B adjuvant) 

SE36 

BSAM-2/Alhydrogel®+CPG 7909 

JAIVAC (MSP1 19/EBA175) 

P27A 

pfAMA1 DiCo 

PAM VAR2CSA 

Var2-CSA DBL2/3-X 

MSP1 full length 

FMP1/AS02A (MSP-1 42 3D7 E. coli-expressed 

in AS02A adjuvant) 

MSP2-C1/ISA720 

AMA1-FVO [25-545] 

PfCP2.9 (MSP-1 19/AMA-1 chimera) 

GLURP [85-213] 

MSP1-C1/AlOH/AlOH + CpG 

AMA1-C1/ ISA720 

AMA-1 (Australia) 

NMRC-M3V-Ad-PfCA 

CSP, AMA1 virosomes (PEV 301,302) 

SR11.1 

NMRC-M3V-Ad-Pf5 

RESA, MSP1, MSP2 (combination B) 

FP9/MVA Polyprotein 

RTS,S/AS02 and FMP1/AS02 

NMRC-M3V-Ad-PfCA in Adjuvant (7DW8-5) 

Pf10C-MBP 

Pfs25-rEPA/Alhydrogel 

Pfs25-Pfs25 conjugate vaccine 

Pfs25 

Figure 2 (continued): Malaria vaccines in (pre-)clinical development. Vaccines in lighter 

shades have stopped or halted in development. [16] 

research efforts and a significant increase in the number of vaccine candidates 

being developed, there is no licensed malaria vaccine yet. 

Malaria vaccine development 

Developments in molecular techniques have advanced the development of 

malaria vaccine candidates to a current number of approximately 36 Pf 

candidate (sub-unit) malaria vaccines in clinical development [11], an overview is 

provided in Figure 2. Only a fraction of lead vaccine candidates entering the


pipeline of clinical development will ultimately be licensed for clinical use. 

Clinical development is time-consuming and costly and starts with safety and 

immunogenicity trials. If satisfactory, clinical trials aimed at obtaining efficacy 

profiles will be undertaken. Whereas the first two criteria can generally be 

assessed in a small initial Phase I trial, vaccine efficacy in the field can only be 

assessed in larger study cohorts with at least a few hundred volunteers in 

malaria-endemic areas (Phase IIb trials). With only a limited number of 

competent field trial sites and a downward trend in malaria incidence in several 

endemic areas, the size and costs of Phase IIb trials are rising [12], underpinning 

the need to develop tools for downstream selection of vaccine candidates. 

Conventional vaccine technologies rely on the production of a single 

recombinant or synthetic subunit protein or peptide vaccine in a yeast or 

bacterial vector or administer (attenuated) whole organisms [13]. Proteins may 

also be combined together to enhance protection or increase the breath of the 

immune response. Alternatively, DNA vaccines have been tried, but they proved 

to be less immunogenic [14]. In order to increase or modify the vaccine-elicited 

immune response, vaccines are often administered in conjunction with an 

adjuvant. Adjuvants can consist of many different materials that are either 

mixed with or linked to the vaccine. Moreover, delivery platforms can deliver a 

vaccine to a certain site, such as (Adeno-)viral vectors or liposomes that can take 

care of intracellular delivery of the vaccine. In such cases, the DNA-sequence 

vaccine is expressed by the host cell. Particularly combinations of a viralvectored 

DNA prime and protein booster injections have proven to be successful 

[15]. 

Malaria vaccine candidates 

The vaccine candidates currently under pre-clinical or clinical development are 

categorized according to the life cycle stage of antigen expression (Figure 3). The 

pre-erythrocytic stage begins when a female Anopheles mosquito inoculates Pf 

sporozoites into the human skin. These sporozoites travel to the human liver 

and invade hepatocytes, where they develop into thousands of asexual parasites 

called merozoites. This process takes approximately one week and is clinically 

silent. Pre-erythrocytic stage vaccines aim to prevent the passage of parasites 

through the human liver and subsequent blood-stage infection. Ideally, such 

vaccines should be 100% effective to prevent disease, since any merozoite 

released from the liver is potentially capable of exponential growth by invasion

14 Chapter 1 

Pre-erythrocytic stage 

Asexual 

erythrocytic stage 

Merozoites 

invade 

erythrocytes 

Asexual 

blood stages 

Sexual and 

mosquito stage 

Blood vessel 

Exo-erythrocytic 

schizont 

Sporozoites released 

into bloodstream 

Sporozoite 

Sporozoites 

enter liver cells 

Some merozoites 

develop into 

gametocytes 

Lymph 

node 

Merozome 

Liver cells and 

merosomes rupture 

and merozoites 

released into 

bloodstream 

Merozoite 

Erythrocytic 

schizont 

Mosquito 

Salivary 

gland 

Gut 

Zygote 

Ookinete 

Gametes 

Gametocytes taken 

up by mosquito in 

bloodmeal 

Mosquito 

Sporozoites 

released and 

travel to 

salivary glands 

Sporozoite 

Oocyst 

Male and female 

gametes form 

zygotes and 

oocysts develop 

Figure 3. Plasmodium falciparum life cycle showing the three developmental stages of 

the parasite that are targeted by malaria vaccine candidates [12]. 

of red blood cells (erythrocytic or blood stage). This developmental stage is 

responsible for the clinical signs and symptoms of malaria infection. Erythrocytic 

stage vaccines focus on inhibiting parasite multiplication, thereby decreasing the 

probability of complications and (severe) disease. In contrast to pre-erythrocytic 

vaccines, an erythrocytic vaccine may therefore be only partially efficacious and 

can still protect against severe disease. Following asexual multiplication in 

erythrocytes, a small percentage of merozoites convert to sexual forms, the 

gametocytes. Gametocytes are infectious to Anopheles mosquitoes that bite the 

infected host. Transmission blocking vaccines contain sexual blood stage or 

mosquito stage antigens that prevent infection of mosquitoes and thus further 

transmission of the parasite, reducing the spread of malaria in the population.


The division of antigen expression according to parasite life-cycle stages, 

although useful, may be a simplification of reality since many antigens are 

shared between stages [17-19]. 

Pre-erythrocytic vaccine candidates 

The development of pre-erythrocytic vaccines started with the observation by 

Ruth Nussenzweig that vaccination of mice with irradiated sporozoites results in 

protection from sporozoite challenge. [20]. Following this observation, the 

exploration of sporozoite antigens lead to the identification of the 

circumsporozoite protein (CS), which was capable of inducing immunity in mice 

[21]. Further development of this antigen, particularly by the Walter Reed Army 

Institute of Research in the United States, has resulted in the most advanced 

vaccine candidate so far: GlaxoSmithKline's RTS,S vaccine. This candidate 

consists of a Hepatitis B surface antigen fused to the Pf circumsporozoite 

protein, adjuvanted by GlaxoSmithKline's proprietary adjuvant AS01 containing 

the immunostimulants MPL and QS21 in liposomes. It is currently in phase III 

clinical development and has consistently shown 30-60% clinical protection [22- 

27]. 

Other promising pre-erythrocytic antigens include thrombospondin-related 

adhesion protein (TRAP) and liver stage antigen 1 and 3 (LSA). Cytokine 

responses to LSA-1 were associated with parasitemia and clinical malaria in 

endemic human populations and the antigen is safe and immunogenic in 

primates and humans [28-30]. Unfortunately, a phase IIa efficacy trial with the 

AS01/AS02 adjuvanted recombinant LSA1 product did not show any protection 

or delay in prepatent period [31]. The vaccine candidate LSA-3 was safe and 

immunogenic in humans, but did not protect malaria naïve volunteers in a 

controlled human malaria infection trial (Nieman et al. manuscript in 

preparation). A vaccine based on multiple T- and B-cell epitopes, amongst which 

LSA-1 and LSA-3, fused to the TRAP antigen protected malaria-naïve subjects 

against subsequent challenge with infectious mosquitoes, when administered as 

a heterologous prime-boost schedule whereby the plasmid DNA vaccine is 

boosted by a viral-vectored vaccine [32], but did not show any efficacy in field 

trials in both adult and children [33, 34]. 

The most successful immunization strategy so far, however, was directly derived 

from the irradiated sporozoites used in the murine models of Ruth Nussenzweig. 

Infected, laboratory-reared Anopheles stephensi mosquitoes were irradiated and

16 Chapter 1 

allowed to bite on human volunteers. Irradiation attenuated the sporozoites 

located in the mosquito salivary gland. It was shown that more than a 1000 of 

such bites could protect humans from subsequent challenge [35]. The technical 

challenge of whole-sporozoite immunization is the manufacturing process, since 

the sporozoite parasite forms cannot be cultured outside their mosquito host. 

The production of whole-sporozoite vaccines that comply with current vaccine 

regulations for registration thus requires the standardized isolation, purification 

and preservation of parasites from mosquito salivary glands. This procedure 

meets numerous technical hurdles [36]. 

Blood stage vaccine candidates 

Despite the fact that blood stage parasite multiplication is the only parasite 

developmental stage that causes disease, relatively few blood-stage antigens are 

in clinical development as vaccines thus far. Nevertheless, erythrocytic stage 

vaccines are considered valuable, because they may not only be used as single 

antigen vaccines but also protect against break-through infections in a multistage 

vaccine with pre-erythrocytic vaccine components [37]. Erythrocytic stage 

malaria vaccine candidates include Apical Membrane Antigen 1, (AMA1), 

Erythrocyte-Binding Antigen-175 (EBA-175), Glutamate-Rich Protein (GLURP), 

Merozoite Surface Protein 1 (MSP1), MSP2, MSP3 and Serine-Repeat Antigen 5 

(SERA5), all of which are highly expressed on the merozoite surface [11]. To 

date, none of these candidates have demonstrated protection against clinical 

outcomes [37]. Efficacy of these candidates may be hampered by the genetic 

diversity of the parasite surface proteins, which is likely due to the selective 

pressure exerted by the human immune response [37]. 

AMA1 is one of the most advanced blood stage malaria vaccine candidates. 

Animal data consistently show that AMA1 antibodies alone can protect mice and 

monkeys against subsequent challenge, raising high hopes for the vaccine [38- 

40]. The protein is maximally expressed in late schizony of erythrocytic 

development and relocates from the parasite microneme to the merozoite 

surface, where it plays a role in erythrocyte invasion [41]. Possibly, AMA1 is also 

involved in liver cell invasion [42], making the antigen an attractive target 

particularly for antibodies. Unfortunately, AMA1 is also highly polymorphic with 

hundreds of haplotypes identified [43]. Another potential target for blood-stage 

vaccines is the Pf erythrocyte membrane protein 1 (PfEMP1) family, a highly 

polymorphic protein encoded by a great number of genes. The PfEMP1s are


Figure 4. Population indices of immunity to malaria in Kilifi, Kenya. [53] 

expressed on the surface of infected erythrocytes and are essential for 

adherence of the parasite to vascular endothelium to avoid destruction in the 

spleen [11, 44]. As one parasite clone expressing one PfEMP1 is detected by the 

immune system, another parasite clone expressing another PfEMP1 takes over. 

Despite its diversity, efforts are undertaken to identify conserved domains or 

induce antibodies against PfEMP1s, since these antigens are responsible for 

specific disease manifestations such as placental or cerebral malaria [45, 46]. 

Placental malaria is mediated by parasites expressing a specific variant surface 

antigen, var2CSA [47]. Acquired immunity to placental malaria in multigravid 

women corresponds with acquisition of var2CSA-specific antibodies [48]. 

Transmission blocking vaccine candidates 

Transmission Blocking Vaccines consist of molecules eliciting the production of 

antibodies that will interfere with the parasite development in the mosquito. 

Four Pf antigens have attracted most attention as potential components of 

mosquito stage vaccines: gametocyte/gamete antigens Pfs230 and Pfs48/45 and 

zygote/ookinete antigens Pfs25 and Pfs28. Naturally occurring antibodies 

directed against these proteins can be found in people living in endemic areas 

[49, 50]. Nonetheless, these antigen targets show very limited sequence 

diversity compared to blood stage antigens. A major practical difficulty has 

been in expressing the proteins in the correct conformation to achieve 

immunogenicity [51]. A phase I trial of Pfs25 in healthy human volunteers was 

halted prematurely because of systemic reactogenicity [52].

18 Chapter 1 

Malaria immunity 

Central to the hampering vaccine development is our limited understanding of 

precisely what constitutes immunity to Pf malaria. In areas of natural exposure, 

clinical immunity to malaria slowly develops in repeatedly exposed humans. In 

such populations, immunity to severe disease develops in early childhood, 

whereas immunity to mild disease is not typically acquired until late adolescence 

[53, 54] (Figure 4). Immunity is never sterile and the cumulative incidence of 

parasitemia may approach 100% in adults [55]. Instead, natural acquired 

immunity appears to be directed at the control of parasite replication and 

limited parasite clearance. 

Both humoral and cellular mechanisms contribute to the immunological 

responses to Pf parasites, depending on the stage of the life cycle. Although 

antibodies can inhibit the invasion of parasites into hepatocytes [56], rodent 

studies suggest that pre-erythrocytic immunity is dependent mainly upon 

cascades of cellular and cytokine interactions [57, 58]. CD8+ T cells have been 

implicated as the principal effector cells, and IFN-γ as a critical effector molecule 

[59, 60]. 

Given the absence of antigen processing in erythrocytes, immunity to blood 

stage malaria parasites is primarily conferred by humoral immune responses 

[61], although T-cells may be able to also limit blood stage parasite growth by 

enhancing phagocytosis by macrophages [62]. The importance of antibodies in 

blood stage parasite inhibition was originally demonstrated by passive transfer 

experiments [63, 64], whereby the transfer of antibodies from exposed 

volunteers suppressed parasitemia in Pf patients. Antibodies may act 

independently in inhibiting parasite growth, as shown by in vitro parasite growth 

inhibition assays (GIA) [65], or they may assist the cellular killing of parasites, an 

in vitro phenomenon called antibody-dependent cell-mediated cytotoxicity [66]. 

Ideally, immunological assays should be able to predict the efficacy of malaria 

vaccines. Unfortunately, immune correlates of protection are lacking and thus 

malaria vaccine development depends on a trial-and-error approach [12]. 

Controlled malaria infections 

A controlled malaria infection provides the opportunity of obtaining preliminary 

efficacy data for a vaccine candidate in a selected small group of malaria naïve 

volunteers and act as the key tool for downstream selection of vaccine


candidates. Initially developed as a therapy for neurosyphilis in the 1920’s [67], 

controlled malaria infections were established in their present form in the 

1980s, when techniques to culture parasites and obtain laboratory-reared 

infectious mosquitoes were available [68-70]. In controlled infection trials, 

human (malaria-naïve) volunteers are deliberately exposed to bites of a predefined 

number of infectious mosquitoes. Subjects are subsequently followed, 

mostly on an out-patient basis, for clinical and parasitological endpoints. As soon 

as parasites are detected in the circulation by microscopy, volunteers are 

treated with anti-malarials for obvious safety reasons. As such, parasitemia is 

generally kept below 0.0004% (~10-20 Pf/µl) and severe malaria does not occur 

[71-73]. By comparing the proportion of infected subjects between vaccine and 

control groups, preliminary efficacy data can be obtained. If clinical protection is 

not achieved, the pre-patent period may be used as a surrogate marker 

preceding clinical protection. The recent development of molecular techniques 

for the detection and quantification of parasites [74] has further refined the 

parasitological endpoints to include a detailed analysis of kinetics of blood stage 

parasite growth. In addition, statistical models have become available to analyse 

these data [75, 76]. Such analysis may be helpful to identify the mechanism of 

immunity induced by the vaccine candidate [77, 78]. 

Controlled malaria infection studies have halted the development of candidate 

vaccines LSA1 and PfCS102 [31, 79], whereas a ~30-50% protection induced by 

the vaccine candidate RTS,S in an initial controlled infection study was translated 

into a similar protectivity against clinical malaria in the field [27, 80-83]. 

Controlled malaria infection trials are thought particularly suitable for evaluation 

of pre-erythrocytic malaria vaccine candidates, since they employ the natural 

route of infection (mosquito bite) and allow full pre-erythrocytic stage 

development. For asexual erythrocytic stage vaccines, the use of controlled 

malaria infections is controversial, because only low levels of blood stage 

parasitemia are induced until the trial is interrupted by treatment [12]. Vaccines 

would thus have to exert significant inhibition in a very small time window. 

Because controlled human malaria infection trials require the combined 

technical ability to rear Pf infected mosquitoes and the clinical facility to closely 

monitor healthy human volunteers, such trials are currently routinely carried out 

in only five institutions worldwide: the US Military Malaria Vaccine Program; the 

University of Maryland, USA; the Radboud University Nijmegen Medical Centre,

20 Chapter 1 

The Netherlands; The University of Oxford, UK; and, more recently, Seattle 

Biomed, USA [84]. 

Thesis outline 

The current thesis aims at exploring the induction of protection against 

Plasmodium falciparum malaria in humans with specific attention for three 

research questions: 

What is the safety and immunogenicity of malaria vaccine candidate Apical 

Membrane Antigen 1 (AMA1) in humans? 

Apical Membrane Antigen 1 is a promising vaccine candidate. It has been 

produced as a recombinant protein, covering amino acids 25-545, in Pichia 

pastoris and has shown to be immunogenic with satisfactory 

pharmacotoxicology in animal models. As with most asexual erythrocytic stage 

antigens, AMA1 is highly polymorphic and relies on the induction of antibodies 

for conveying protection. We will address the following objectives in its first 

phase of clinical development: 

- to determine the safety profile of PfAMA1 FVO (25-545) when 

administered to humans in two different doses with three different 

adjuvants (Chapter 2) 

- to determine the quantity and quality of antibodies induced by PfAMA1 

FVO (25-545) in humans (Chapter 2,3,4) 

How can we optimize controlled human malaria infections in order to 

maximize safety and scientific information? 

Controlled human malaria infections (CHMI) are currently used to assess 

preliminary efficacy of pre-erythrocytic vaccine candidates in a limited number 

of institutions worldwide. Because controlled infections seem an efficient tool 

for investigating malaria immunology and candidate vaccine efficacy, we will 

investigate how to optimize its protocol. We will explore the comparability and 

power of the current model with the following specific objectives: 

- to identify parameters influencing the comparability of CHMI safety and 

parasitological outcome in different institutions (Chapter 5)


- to identify parameters influencing the power of CHMI to detect vaccine 

efficacy of pre-erythrocytic and erythrocytic malaria vaccines (Chapter 

6) 

We next will address two important aspects of improvement to the CHMI model: 

- to increase the portfolio of Pf strains that can be used for infection 

(Chapter 7) 

- to investigate the administration of parasites by needle (Chapter 8) 

Can protection to malaria in humans be efficiently induced by inoculation of 

live whole sporozoites? 

The inoculation of irradiated sporozoites by mosquito bite has shown to induce 

full protection in humans, albeit inefficiently and with limited longevity. We will 

explore whether viable intact sporozoites induce protection more efficiently, 

eventually aiming to create a model for malaria immunity. The administration of 

chloroquine prophylaxis will prevent clinical disease whilst the exposure to 

viable sporozoites allows exposure of parasite antigen. We address the following 

specific objectives: 

- to induce protection to Pf malaria in malaria naïve volunteers by 

exposure to parasites whilst taking chloroquine prophylaxis (Chapter 9) 

- to investigate the longevity of induced protection (Chapter 10)

22 Chapter 1 

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Section 1 

Apical Membrane Antigen 1

Chapter 2 

Safety and Immunogenicity of a 

Recombinant Plasmodium falciparum 

AMA1 Malaria vaccine Adjuvanted with 

Alhydrogel TM , Montanide ISA 720 or AS02. 

Meta Roestenberg* 1 , Ed Remarque 2 , Erik de Jonge 1 , Rob Hermsen 1 , Hildur 

Blythman 3 , Odile Leroy 3 , Egeruan Imoukhuede 3 , Soren Jepsen 3 , Opokua Ofori- 

Anyinam 4 , Bart Faber 2 , Clemens H. M. Kocken 2 , Miranda Arnold 2 , Vanessa 

Walraven 2 , Karina Teelen 1 , Will Roeffen 1 , Quirijn de Mast 1 , W. Ripley Ballou 4,5 , 

Joe Cohen 4 , Marie Claude Dubois 4 , Stéphane Ascarateil 6 , Andre van der Ven 1 , 

Alan Thomas 2 , Robert Sauerwein 1 

1 Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 

2 Biomedical Primate Research Centre, Rijswijk, The Netherlands 

3 European Malaria Vaccine Initiative, Copenhagen, Denmark 

4 GlaxoSmithKline Biologicals, Rixensart, Belgium 

5 Present address: Bill and Melinda Gates Foundation, Seattle, USA 

6 SEPPIC, Paris, France 

PLoS ONE 2008; 3:e3960

Chapter 2 32 

Abstract 

Plasmodium falciparum Apical Membrane Antigen 1 (PfAMA1) is a candidate 

vaccine antigen expressed by merozoites and sporozoites. It plays a key role in 

red blood cell and hepatocyte invasion that can be blocked by antibodies. 

We assessed the safety and immunogenicity of recombinant PfAMA1 in a doseescalating, 

phase Ia trial. PfAMA1 FVO strain, produced in Pichia pastoris, was 

reconstituted at 10 µg and 50 µg doses with three different adjuvants, 

Alhydrogel, Montanide ISA720 and AS02 Adjuvant System. Six randomised 

groups of healthy male volunteers, 8-10 volunteers each, were scheduled to 

receive three immunisations at 4-week intervals. Safety and immunogenicity 

data were collected over one year. 

Transient pain was the predominant injection site reaction (80-100%). 

Induration occurred in the Montanide 50 µg group, resulting in a sterile abscess 

in two volunteers. Systemic adverse events occurred mainly in the AS02 groups 

lasting for 1-2 days. Erythema was observed in 22% of Montanide and 59% of 

AS02 group volunteers. After the second dose, six volunteers in the AS02 group 

and one in the Montanide group who reported grade 3 erythema (>50mm) were 

withdrawn as they met the stopping criteria. All adverse events resolved. There 

were no vaccine-related serious adverse events. 

Humoral responses were highest in the AS02 groups. Antibodies showed activity 

in an in vitro growth inhibition assay up to 80%. Upon stimulation with the 

vaccine, peripheral mononuclear cells from all groups proliferated and secreted 

IFNγ and IL-5 cytokines. 

In conclusion, all formulations showed distinct reactogenicity profiles. All 

formulations with PfAMA1 were immunogenic and induced functional 

antibodies.

Safety and Immunogenicity of a Recombinant Plasmodium falciparum AMA1 

Malaria vaccine Adjuvanted with Alhydrogel TM, Montanide ISA 720 or AS02 

Introduction 

In sub-Saharan Africa the burden of death and disease from Plasmodium 

falciparum malaria is particularly severe. To date, there are no approved 

vaccines to help reduce this burden, although a number of candidate vaccines 

have been put forward. The majority of the candidates target the preerythrocytic 

circumsporozoite protein (CSP) and the merozoite proteins 

Merozoite Surface Protein 1 (MSP1) and Apical Membrane Antigen 1 (AMA1)[1]. 

The RTS,S candidate vaccine has shown efficacy in infants and children [2,3] and 

a phase III clinical trial is planned. The MSP1 and AMA1 candidate vaccines are in 

early stage clinical development and efficacy trials will provide information to 

determine whether these antigens are suitable targets, and whether they can be 

deployed singly or as components of a multivalent malaria vaccine. 

Following an infected mosquito bite, P. falciparum sporozoites migrate to 

hepatocytes, each developing over a period of a week to release several 

thousand merozoites. These initiate cyclical asexual blood stage development, 

producing merozoites that invade erythrocytes. AMA1 is an integral membrane 

protein of merozoites and sporozoites and has a central role in parasite invasion 

of erythrocytes and potentially hepatocytes that can be inhibited by anti-AMA1 

antibody [4–6]. In merozoites, AMA1 is synthesised as an 83kDa molecule 

originally localised to the microneme. Around the time of merozoite release and 

the subsequent rapid erythrocyte invasion, the protein is N-terminally cleaved to 

a 66 kDa form. This translocates to the merozoite surface and undergoes 

secondary proteolytic processing, shedding soluble fragments (44 or 48kDa) [7]. 

Immunisation with AMA1 can provide protection against infection in 

experimental animal models, and can induce antibodies that show functionality 

in in vitro growth inhibition assays (GIA). However, AMA1 is polymorphic and 

immune responses have varying degrees of strain specificity and growth 

inhibition [8]. 

Previous Phase I trials have shown that growth inhibitory antibodies can be 

induced by immunisation with PfAMA1 [9,10], but immunogenicity varied 

depending on the vaccine formulation. In particular, the choice of adjuvant has a 

major effect on the safety, stability, immunogenicity and, presumably, eventual 

efficacy of a vaccine [11]. Adjuvants can be tools that channel the immune 

response to generate high levels of the desired type of long-lived immunity. 

33

34 Chapter 2 

Alhydrogel, an aluminium salt, is the most widely used adjuvant in licensed 

human vaccines and is therefore used as a standard to compare other adjuvants. 

Unfortunately, in combination with malaria antigens, it has generally induced 

poor responses [10,12–14]. Montanide ISA 720, a squalene based water-in-oil 

adjuvant formulation has shown promising results in previous malaria vaccine 

trials [15–18], possibly due to the slow-release capacity of the inert water-in-oil 

emulsion and immune stimulating effects of its components [19]. AS02, a 

proprietary Adjuvant System from GlaxoSmithKline Biologicals based on an oilin-water 

formulation, contains 50 µg each of the immunostimulants 

monophosphoryl lipid A (MPL) and Quillaja saponaria 21 (QS21) [20]. It has been 

used to adjuvant the RTS,S malaria candidate vaccine that targets CSP. To date, 

this candidate is the only malaria vaccine that has induced protection in adults, 

children and infants in natural field trials [20–23]. When combined with 

Alhydrogel, RTS,S did not convey protection in a combined phase I/IIa trial 

[24]. AS02 is capable of eliciting high antibody titres along with strong cellmediated 

immunity [25], both of which are believed to contribute to the efficacy 

of the RTS,S candidate vaccine [26]. 

Because of their central role in vaccine formulation, the development of 

adjuvants and delivery systems have become increasingly important. This study 

aims at comparing the safety and immunogenicity of PfAMA1 in two dosages 

formulated with three different adjuvants in a phase Ia trial. 

Materials and Methods 

Vaccine preparation 

Clinical grade PfAMA1-FVO[25-545] was developed [27] and produced [28] as 

previously reported. In brief, FVO strain PfAMA1 was codon adapted to 

expression in the methylotrophic yeast Pichia pastoris. Glycosylation sites were 

conservatively mutated, and the ectodomain comprising amino acids 25-545 was 

expressed. PfAMA1-FVO[25-545] preparation was manufactured and lyophilised 

according to current good manufacturing practice in multidose vials containing 

either 120 µg (44 µg EDTA, 180 µg sucrose and 120 µg NaHCO3, lot B) or 62.5 µg 

(23 µg EDTA, 25 mg saccharose, 226 µg K2HPO4 and 187 µg NaH2PO4, lot C) of 

AMA1 that were stored between -18°C and -30 °C and between +2 and +8°C 

respectively. Quality control and stability data are described by Faber et al. [28]. 

Reconstitution and mixing of vaccine with adjuvant was performed under sterile 

conditions under responsibility of the hospital pharmacist.



PfAMA1 vaccine at 50 µg (high dose) and 10 µg (low dose) PfAMA1 per 

injection (0.5 ml) was formulated with three different adjuvants and, after 

preparation, was kept at a constant temperature of +4°C for a maximum of six 

hours until injection. For the Alhydrogel formulation, 1.2 ml aluminium 

hydroxide suspension at 2 mg/ml (Statens Serum Institut (SSI), Copenhagen, 

Denmark) was added to the 120 µg PfAMA1 vial (lot B) to obtain a high dose (50 

µg in 0.5 ml) and 6 ml was added to obtain a low dose (10 µg in 0.5 ml) 

formulation. The resulting amount of aluminium in each vaccine was 0.5 mg. 

Stability studies confirmed adsorption of 99.9% of the antigen to the aluminium. 

Montanide formulations were prepared by dissolving the contents of the 120 µg 

PfAMA1 vial (lot B) in sterile phosphate buffered saline (145mM NaCl, 5mM 

Phosphate, pH7.4), 0.32 ml for the high dose and 1.6 ml for the low dose 

formulation. Montanide ISA 720 (SEPPIC, Paris, France) was subsequently added, 

0.88 ml for the high dose to obtain 1.2 ml of formulation (50 µg PfAMA1 in 0.5 

ml) and 4.4 ml for the low dose to obtain 6 ml of formulation of which five 10 µg 

PfAMA1 in 0.5 ml doses could be prepared. The suspension was prepared by 

manually pushing through a 22 gauge syringe coupling piece (3038068 Omnilabo 

International, Breda, The Netherlands) at +20°C for twenty up and down strokes. 

The suspension was confirmed to be homogeneous and reached a median 

droplet size of approximately 1.5 µm (SD 0.17 µm) by particle size 

measurements with the Malvern Mastersizer S by SEPPIC. 

For the AS02 formulation, the contents of one vial of lyophilized PfAMA1 

containing 62.5 µg of antigen (lot C) was mixed by gentle shaking with AS02 

(approximately 0.6 ml) [29]. A 0.5 ml dose contained approximately 50 µg AMA- 

1 in 500 µl AS02 (high dose). For low dose preparations (10 µg) five times more 

AS02 adjuvant was added to the 62.5 µg vial of AMA1, from which five 0.5 ml 

low vaccine doses could be obtained. 

Study design 

This study was designed as a dose-escalating phase Ia trial to assess the safety 

and immunogenicity of two dosages of PfAMA1 with three different adjuvants. 

Volunteers were thus randomised into six different groups, each of which was 

aimed to constitute of a limited number of 10 volunteers for safety reasons. 

Randomisation was performed by an external statistician in six blocks through a 

computer program. Block randomization were used to ensure equal distribution 

of adjuvants among the immunisation groups. There was no stratification for sex 

and/or age. The randomization list was provided to the pharmacy departments. 

35

36 Chapter 2 

The clinical investigators allocated the next available number on entry into the 

trial. The code was revealed to the researchers once recruitment, data 

collection, and laboratory analyses were complete. The immunisations were 

thus performed blind, so neither volunteers, nor investigator or laboratory 

personnel were aware of the adjuvant allocation. Because of the dose-escalating 

design, the trial could not be blinded for dose. 

For logistical reasons, the AS02 adjuvanted groups were immunised nine months 

after the Alhydrogel and Montanide groups, breaking the blind for this trial 

arm. A subsequent bias cannot formally be excluded but seems unlikely, since all 

trial procedures were identical. All immunisations were performed 

intramuscularly in the deltoid region of alternate arms at 0, 4 and 8 weeks. 

Participants 

We aimed to recruit 60 healthy, malaria naïve male volunteers, aged between 

18 to 45 years through advertisements at the Radboud University Nijmegen 

Medical Centre. Potential volunteers provided a medical history and a physical 

examination was conducted with routine laboratory tests consisting of full blood 

count, serum biochemistries and serologic assays for human immunodeficiency 

virus, hepatitis C and B virus. Volunteers were excluded from participation if 

they had any symptoms, signs or laboratory values suggestive of systemic illness, 

including renal, hepatic, cardiovascular, pulmonary, skin, immunodeficiency, 

psychiatric and other conditions, which could interfere with the interpretation of 

the study results or compromise the health of the volunteers, or received 

chronic medication, had a history of drug or alcohol abuse interfering with social 

function one year prior to enrolment, or a known hypersensitivity to any of the 

vaccine components. Additional reasons for exclusion were a history of malaria 

or residence in malaria endemic areas within the past six months, previous 

participation in a malaria vaccine trial or receiving vaccines other than the study 

vaccines. Furthermore, volunteers were not enrolled in any other clinical trial, 

and agreed to remain available to be closely monitored. All volunteers provided 

written informed consent. The study was approved by the Institutional Review 

Board (CMO Regio Arnhem-Nijmegen, 2005/015). The study was conducted in 

accordance with the Declaration of Helsinki principles for the conduct of clinical 

trials and the International Committee of Harmonization Good Clinical Practice 

Guidelines [30] and registered at www.clinicaltrials.gov (NCT00730782).



Assessment of safety 

Volunteers were observed for 30 minutes and evaluated on days 1, 3, 7 and 14 

after every immunisation. At each visit, local and systemic reactogenicity was 

assessed by a physician and findings recorded and scored as follows: grade 1, 

mild reaction (easily tolerated), grade 2, moderate reaction (interferes with 

normal activity), or grade 3, severe reaction (prevents normal activity). Redness, 

swelling and induration (according to Brighton collaboration definitions, 

www.brightoncollaboration.org) were measured with a ruler, and categorised 

according to the longest diameter as grade 1: 20 and 50mm. Temperature was measured with an oral thermometer; fever 

intensity was defined as grade 1 (37.5°C to 38°C), grade 2 (>38°C to 39°C) or 

grade 3 (>39°C). The following adverse events were solicited and recorded 

routinely during the 14 days after immunisation: injection site pain, redness and 

swelling, systemic fatigue, fever, headache, malaise, myalgia, joint pain, 

gastrointestinal symptoms and contralateral local reactions. 

Blood samples 

Safety was also determined by serial laboratory evaluations of clinical chemistry 

and haematology on blood samples collected 7 and 28 days after immunisation. 

For evaluation of immunogenicity, blood was collected in Vacutainer CPT tubes 

(Becton and Dickinson) and processed within two hours after collection on 

immunisation days, one month after each immunisation and on Days 140 and 

365. Plasma was collected after centrifugation (2000 g 15’) aliquoted and stored 

at –20°C for antibody analysis (ELISA, Immuno Fluorescence Assay (IFA) and 

Growth Inhibition Assay (GIA)). Peripheral blood mononuclear cells (PBMC) were 

collected, washed in PBS (800 g, 10 min) and immediately used for assays 

(lymphocyte stimulation assay and ELISPOT). 

Measurement of anti-AMA1 antibodies by ELISA and IFA 

Antibody to PfAMA1 was measured using a standardized ELISA protocol. All 

procedures used Phosphate buffered saline (PBS) and for washing steps 0.05% 

Tween 20 (Sigma-Aldrich). Briefly, wells in 96-well polystyrene plates (NUNC 

Maxisorp, Sanbio), were coated overnight (100 µl, 0.5 µg/ml PfAMA1, 4°C), 

washed (3x), blocked (60 min, 3% BSA (Sigma-Aldrich)) and washed (3x) before 

addition of 100 µL from duplicate dilution series (diluted in PBS-Tween BSA, one 

hour, +37°C). After washing (3x) goat anti-human IgG alkaline phosphate (Perbio 

Science) diluted 1:1250 in 0.5% BSA, 0.05% Tween was added, (one hour, +37°C). 

Plates were washed and 100 μl of 1 mg/ml para-nitro-phenyl-phosphatase 

37

38 Chapter 2 

(Fluka, Sigma-Aldrich) substrate was added (30 minutes, room temperature). A 

human plasma pool from a malaria endemic area was used as reference positive 

control, whereas a plasma pool from eight healthy malaria-naive Dutch 

volunteers was used as a negative control. Optical density was measured at 

405nm. Variation between duplicates was set to a maximum of 15%. 

Measurements with a greater variation were repeated. The standard curve of 

human plasma pool from a malaria endemic area, defined to contain 400 

Arbitrary Units, was fitted to a four-parameter hyperbolic function, using the 

ADAMSEL program (E. Remarque, unpublished work). Using this standard curve, 

optical density from samples were converted to Arbitrary Units (AU). Test 

samples that did not fall within the linear part of the optical density range of the 

standard were tested at alternate dilutions. 

IFA was performed on cultured P. falciparum parasitized red blood cells. Ten 

well black slides (30-966-A black, Nutacon, The Netherlands) were coated with a 

washed parasite suspension of 3 x 10 6 parasites/ml, air dried and kept at –80°C 

until used. FCR3 parasites, expressing an AMA1 protein with one amino-acid 

difference from the FVO parasites, and NF54 strain parasites, with 26 amino-acid 

difference in AMA1 protein were used to prepare slides. 

Based on antibody titres by ELISA on day 84, a representative sample of fifteen 

sera was selected for IFA, containing at least two samples from each adjuvant 

group and at least three samples with low, intermediate or high ELISA titres. 

Before use slides were brought to room temperature in an evacuated exicator. 

Plasma was diluted in PBS (1:40, 1:80, 1:160, 1:320, 1:640) and a final volume of 

20 µL was added to the wells and incubated for 0.5 hour, at room temperature. 

As for the ELISA protocol, the malaria-naïve blood bank donor plasma pool was 

used as a negative control and human malaria endemic plasma was used as a 

positive control. After washing (2x in PBS) and air drying, slide samples were 

incubated with rabbit anti-human Immunoglobin FITC (F0200, DAKO, Denmark) 

in 0.05% w.v Evans Blue (3169, Merck), PBS for 30 minutes at room 

temperature. Slides were washed twice and incubated for 15 minutes with DAPI 

(4’-6-Diamindino-2-phenylindole, 24653, Merck, Darmstadt, Germany), 5 µg/ml 

in PBS. After washing (2x in PBS) slides were mounted with Vectashield 

Mounting Medium (H-1000, Brunschwig, Amsterdam), covered with a deck-slide 

and read immediately by two independent blinded examiners. Examiners 

identified the highest dilution still showing a staining pattern above the 

background of pre-immunisation samples. Differences between examiners were 

never greater than one dilution and the mean of both dilutions was taken.



ELISPOT for IFNy and IL-5 

ELISPOT was performed according to manufacturer’s instructions (Becton and 

Dickinson Elispot Set Human IFNγ or IL-5). In summary, plates provided in the set 

were coated with either IFNγ or IL-5 capture antibody (5 µg/ml, overnight, 4°C). 

After blocking with complete medium solution (RPMI 1640 (Invitrogen) 

containing 10% Fetal Bovine Serum (FBS Invitrogen, Breda, The Netherlands), 1% 

Glutamax (Invitrogen), 1% Penicillin-Streptomycin (GIBCO-BRL, Invitrogen), 1% 

MEM) 100 µl of 105 PBMC suspension and 100 µl of PfAMA1 containing either 

60 µg, 12 µg or 2.4 µg was added per well. Positive controls were stimulated 

with Tetanus Toxoid 10 µg/ml (RIVM, Bilthoven, The Netherlands) and 

phytohaemagglutinin 5 µg/ml (PHA-L Sigma-Aldrich) end concentration. 

Negative controls were incubated with complete medium solution (mean 

SFC/10 5 cells 31±17 for IFNγ and 9±7 for IL-5). After incubation (40 hours, 37°C in 

humidified 5% CO2), biotinylated anti human IFNγ and IL-5 (0.25 µg/ml and 2 

µg/ml, respectively), containing 10% FBS was added (two hours at room 

temperature). Streptavidin-HRP was used as an enzyme conjugate. Detection 

was performed with the Becton and Dickinson AEC Substrate Reagent Set, 

according to manufacturer’s instruction. Spot-forming cell numbers were 

counted by ELISPOT reader (4 Microtitre Plate Reader, AELVIS, Sanquin, 

Amsterdam) and analysed by the ELISPOT Analysis Software Version 4.0 

(Sanquin, Amsterdam). All measurements were performed in triplo. Variation 

between triplicates was set to a maximum of 20%. 

Lymphocyte Stimulation Assay 

Lymphocyte stimulation assays were performed as described previously [31]. 

Peripheral blood mononuclear cell suspension (PBMC) was diluted to 1 x 10 6 

PBMC per ml in Dulbecco’s MEM (DMEM) with Glutamax-I, 2 mM pyruvate and 

high Glucose (GIBCO BRL, Invitrogen) supplemented with 10 mM HEPES buffer 

(GIBCO BRL, Invitrogen), 100 IU/mL Penicillin-Streptomycin (GIBCO BRL, 

Invitrogen), 100 µM non-essential amino acids (GIBCO BRL, Invitrogen) and 2.5% 

human AB serum (AB) (Bodinco BV, Alkmaar, The Netherlands). 100 µl of PBMC 

was added to 100 µl PfAMA1 (30, 6 or 1.2 µg/ml in PBS) in 96 well Nunclon 

surface flat plates (Life Technology). Plates were incubated (six days, +37°C, 

humidified 0.5%CO2) before labelling (10 µl 3H thymidine, 0.25 uCi per well, 24 

hours) and harvested onto Wallac filter mats using the Wallac Beta plate 

harvester. Incorporated 3H-thimidine was determined using a Wallac Beta Plate 

counter. Stimulation indices (SI) were calculated relative to control wells to 

which no PfAMA1 had been added. PBMC were tested in parallel for their 

39

40 Chapter 2 

Figure 1. Study flow chart showing number of volunteers randomised, withdrawn and 

completing follow-up. Coding for adjuvant as follows: Alum = Alhydrogel, Mon = 

Montanide. Reasons for withdrawal are given: “rash” = allergic rash unrelated to study 

procedure, “erythema” = grade 3 injection site erythema leading to withdrawal, “other 

vac.” = concomitant Hepatitis B vaccination leading to exclusion. 

ability to be stimulated with Tetanus Toxoid (Purified Tetanus Toxoid 150 Lf/ml, 

RIVM, Bilthoven, The Netherlands) and phytohaemagglutinin 5 µg/ml (PHA-L, 

Sigma-Aldrich). 

In vitro parasite growth inhibition 

Antibodies to be used for parasite inhibition assays were purified on protein A 

columns (Immunopure Plus Pierce, St Louis, MO, USA) using standard protocols, 

exchanged into RPMI 1640 using Amicon Ultra-15 concentrators (30 kDa cut-off, 

Millipore, Ireland), filter-sterilised and stored at -20°C until use. IgG 

concentrations were determined using a Nanodrop ND-1000 spectrophotometer 

(Nanodrop Technologies, Wilmington, DE, USA). 

P. falciparum strain FCR3 was cultured in vitro using standard P. falciparum 

culture techniques in an atmosphere of 5% CO2, 5% O2 and 90% N2. FCR3 AMA1 

(accession no. M34553) differs by one amino acid in the pro-sequence from FVO 

AMA1 (accession no. AJ277646).



The effect of purified IgG antibodies on parasite invasion was evaluated with 

two IgG concentrations (5 and 10 mg/mL, respectively) in triplicate using 96 well 

flat-bottomed plates (Greiner) with synchronized cultures of P. falciparum 

schizonts at a starting parasitemia of 0.2-0.4%, a haematocrit of 2.0% and a final 

volume of 100 µL containing 10% control non-immune human serum, 20 µg /ml 

gentamicin in RPMI 1640. After 40 to 42 hours, cultures were resuspended, and 

50 µL was transferred into 200 µL ice-cold PBS. The cultures were then 

centrifuged, the supernatant removed and the plates were frozen. Inhibition of 

parasite growth was estimated using the pLDH assay as previously described 

[14]. Parasite growth inhibition, reported as a percentage, was calculated as 

follows: 100 - ((Odexperimental - Od background)/ (Odcontrol - Odbackground) 

x 100). IgG purified from plasma before immunisation was used as a control, and 

culture medium was used to measure the background Od. 

Statistical methods 

Safety analyses were based on intention to treat data selection (n=56). For 

immunology assays, per protocol analyses were used (n=47). Between group 

differences were calculated by one-way ANOVA, using post-hoc Bonferroni 

when p

42 Chapter 2 

Adjuvant Alhydrogel Montanide AS02 Total 

PfAMA1 

dose 

10µg 50µg 10µg 50µg 10µg 50µg 

N 10 10 10 9 9 8 56 

Total 8 

10 

9 9 

9 8 53 

LOCAL 

(80.0 %) (100 %) (90.0 %) (100 %) (100 %) (100 %) (94.6 %) 

Pain 

8 

(80.0 %) 

10 

(100 %) 

8 

(80.0 %) 

9 

(100 %) 

9 

(100 %) 

8 

(100 %) 

52 

(92.9 %) 

Erythema 

- - 

2 

(20.0 %) 

2 

(22.2%) 

4 

(44.4%) 

6 

(75.0%) 

14 

(25%) 

Swelling 

- - 

1 

(10.0 %) 

- 

3 

(33.3 %) 

1 

(12.5 %) 

5 

(8.9 %) 

Induration 

- - 

1 

(10.0 %) 

2 

(22.2 %) 

- - 

3 

(5.4 %) 

Sterile 

abscess 

SYSTEMIC 

- - - 

2 

(22.2 %) 

- - 

2 

(3.6 %) 

Headache 

1 

(10.0 %) 

- 

2 

(20.0 %) 

- 

6 

(66.7 %) 

7 

(87.5 %) 

16 

(28.6 %) 

Malaise 

- - - 

1 

(11.1 %) 

6 

(66.7 %) 

7 

(87.5 %) 

14 

(25.0 %) 

Fever 

- - - - 

5 

(55.6 %) 

5 

(62.5 %) 

10 

(17.9 %) 

Myalgia 

- - - - 

4 

(44.4 %) 

2 

(25.0 %) 

6 

(10.7 %) 

Nausea 

1 

(10.0 %) 

- - - 

1 

(11.1 %) 

2 

(25.0 %) 

4 

(7.1 %) 

Fatigue 

- - - - - 

2 

(25.0 %) 

2 

(3.6 %) 

Arthralgia 

- - - - 

1 

(11.1 %) 

- 

1 

(1.8 %) 

Abdominal 

pain 

- - - - 

1 

(11.1 %) 

- 

1 

(1.8 %) 

Table 2. Number of volunteers reporting vaccine related adverse events per dose and 

adjuvant group. 

be closely monitored for social, geographic or psychological reasons. Table 1 

shows the demographics of volunteers per randomised group. The mean age 

was 23 years old (range 18 – 42 years) and all but one were Caucasian. 

Safety and reactogenicity 

No serious adverse events occurred that were definitely, probably, or possibly 

related to immunisation. No clinically relevant changes in vital signs or 

laboratory values were reported throughout the study. Forty-seven volunteers 

(84%) received all three immunisations; nine were excluded for one or more 

immunisations (Figure 1). Two of these were excluded for reasons unrelated to 

the trial procedures. One (Alhydrogel 10 µg group) developed a generalised 

rash assessed as unrelated to the vaccine between the first and second 

immunisations and one (Montanide 10 µg group) received a concomitant 

hepatitis B immunisation. Seven volunteers were excluded because they 

developed grade 3 erythema (diameter >50mm) after the second immunisation;



one in the Montanide 10 µg group, the other six in the AS02 groups (two in the 

10 µg group, four in the 50 µg group). 

Volunteers in all groups presented with local injection site reactions, the most 

predominant being transient mild to moderate pain (80-100%, table 2). 

Erythema was commonly observed (10 of 17 volunteers) in the AS02 adjuvanted 

groups, occurring after the second and third immunisation. In the Montanide 

group 4 of 18 volunteers developed erythema. Seven volunteers reported 

grade 3 erythema and were withdrawn from further immunisation after dose 2. 

The skin in grade 3 (diameter >50 mm) erythema was not painful and did not 

limit daily activities. Episodes of erythema generally lasted 2-3 days. 

Induration at the site of injection occurred in three volunteers in the course of 

the study (table 2). One volunteer, in the Montanide 10 µg group, developed 

moderate induration 15 days after the first immunisation, lasting for five days. 

The second and third immunisations in this volunteer were well tolerated; 

induration did not re-appear. Another volunteer developed induration starting 

nine days after the first immunisation in the left arm with 50 µg PfAMA1 in 

Montanide, lasting 25 days. The second immunisation was well tolerated, but 

the left arm induration re-appeared one day after the third immunisation, 

accompanied by pain and induration at the previous immunisation site in the 

contralateral (right) arm. Four weeks later, the induration became soft and 

fluctuated, indicating abscess formation. A total of 63 ml of opaque, brown fluid 

was aspirated by two subsequent punctures, after which the abscess and 

induration resolved spontaneously and disappeared completely at 81 days post 

third immunisation. The third volunteer, also in the 50 µg PfAMA1 adjuvanted 

with Montanide group, developed moderate induration nine days after the 

second immunisation which lasted approximately one week. Six days after the 

third immunisation he developed induration at his left arm (the site of the first 

and last immunisation) which eventually started fluctuating. A total of 130 ml 

brown, opaque fluid was collected by means of two punctures. Thereafter, 

spontaneous percutaneous drainage occurred and the lesion resolved 57 days 

after the third immunisation. Both volunteers did not have any systemic 

symptoms such as fever during this time period. Abscesses were only mildly 

painful, but limited volunteers daily activities because of their size. 

The aspirated fluids from both volunteers were abundant in red blood cells and 

lymphocytes with low Creatinine Kinase (CK) levels. Repeated cultures did not 

reveal any bacterial contamination. Serum CK levels were normal. Circulating 

43

44 Chapter 2 

Figure 2. Mean log anti-AMA-1 titres with standard deviation for low and 

high dose per adjuvant group. Anti-AMA-1 titres were determined by ELISA 

for the six different groups, immunized with Alhydrogel, Montanide and 

AS02 adjuvanted PfAMA1 vaccine. Dashed lines represent high dose of 

PfAMA1 (50 µg), continuous lines represent low dose groups (10 µg). 

Measurements were performed at baseline, 28 days after the first, second 

and third immunisation (day 28, 56 and 84 respectively) and day 140 and 

365. 

levels of C-reactive protein remained below detection levels (indicating that the 

reaction was a local response). Ultrasound examination suggested an 

intramuscular and subcutaneous localisation of the fluid-filled cavity. 

Systemic reactions were infrequent in the Alhydrogel and Montanide groups 

and occurred mainly in the AS02 groups. The systemic adverse events occurred 

within 24 hours of immunisation and usually resolved within two days. The most 

prevalent systemic adverse events were headache (77.8-87.5%) and malaise 

(66.7-87.5%) in the AS02 groups. Four of those volunteers reported grade 3 

headache or malaise. Most of the systemic adverse events occurred after dose 

2. There was no effect of antigen dose on reactogenicity. No changes in blood 

pressure were noted in any of these volunteers.



Figure 3. Representative immunofluorescent microscopy picture, showing 

recognition of native antigen on merozoites by induced anti-AMA1 antibodies. 

Immunofluorescence picture of merozoites incubated with 40x diluted anti-AMA1 

plasma from an immunized volunteer one month after final immunisation stained 

with Rabbit anti-human immunoglobulin FITC (A) and DAPI (B). Photo was taken at 

magnification 400x. Incubation with monoclonal antibody 4G2, a pan-specific anti- 

AMA1 antibody, confirmed the surface staining pattern (not shown). 

Humoral immune response 

Peak antibody titres were observed one month after the final immunisation. 

100% of volunteers in the 10 and 50 µg AS02 and 50 µg Montanide groups 

showed a greater than four-fold increase in antibody titre over preimmunisation 

compared to 60% in the 10 µg Alhydrogel, 80% in the 50 µg 

Alhydrogel and 90% in the 10 µg Montanide groups (Figure 2). All vaccinees 

had reached IgG titres comparable to or higher than semi immune sera. Two and 

four months post final immunisation both AS02 groups and the Montanide 50 µg 

group showed the highest IgG titres but given the small sample sizes there was 

no power to detect statistical differences between groups. Antibody titres 

decreased further one year post immunisation, with the steepest decline being 

in the Montanide groups, to a level comparable with the reference Alhydrogel 

groups. 

One year post vaccination, titres in the 10 µg AS02 group were significantly 

higher as compared to the reference group (post hoc Bonferroni when 

compared with low dose Alhydrogel reference group p

46 Chapter 2 

Figure 4. ELISPOT assay for IFNγ (A) and IL-5 (B) after stimulation with 6 µg PfAMA1. 

Peripheral Blood Mononuclear Cells from immunised volunteers 28 days after the 

second immunisation and 28 days after the third immunisation (day 56 and 84 

respectively) were stimulated with 6 µg of PfAMA1 vaccine. Production of IFNγ and IL- 

5 was measured by counting spots in ELISPOT plates. Box plots and whiskers show the 

range and the 25th, 50th and 75th percentile of spots per 2x10 5 cells. Circles 

represent outliers.



Figure 5. Stimulation indices in response to 6 µg/ml PfAMA1 presented as box 

plots and whiskers. Peripheral Blood Mononuclear Cells from immunised 

volunteers 28 days after the second immunisation and 28 days after the third 

immunisation (Day 56 and 84 respectively) were stimulated with 6 µg of 

PfAMA1 vaccine. Cell proliferation was measured by adding 3H thymidine and 

calculated relative to control wells. Box plots and whiskers show the range and 

the 25th, 50th and 75th percentile. Circles represent outliers. Measurements 

were performed at baseline, 28 days after the second immunisation and 28 

days after the third immunisation (day 56 and 84 respectively). 

Sera from vaccinees could be shown to recognise the native PfAMA1 by 

immunofluorescence in a dose dependent manner. Eight of fifteen samples were 

positive in IFA, amongst which were four samples with the highest antibody 

titres. The staining pattern found in positive samples localised to the same 

structures as 4G2 rat monoclonal antibody (Figure 3). 

Cellular immune response 

In all groups, induction of IFNγ and IL-5 cytokines could be demonstrated (Figure 

4). The magnitude of cytokine production was not dose dependent or depen- 

47

48 Chapter 2 

Figure 6. Percentage of growth inhibition of FVO-strain P. falciparum 

parasites after addition of 5 or 10 mg/ml IgG. Serum samples from volunteers 

immunised with PfAMA1 were obtained four weeks after the final 

immunisation by per protocol analysis and included in a merozoite growth 

inhibition assay. Growth inhibition is expressed as a percentage to control. 

Boxes show 25th, 50th and 75th percentile growth inhibition, whiskers show 

the range, circles are outliers. 

dent on the number of immunisations. Rather, IFNγ production in many samples 

decreased after the third immunisation. For both cytokines, PfAMA1 induction 

was comparable or higher than that following stimulation with 5 µg Tetanus 

Toxoid (data not shown). Cytokine production in the different groups did not 

differ significantly from each other (for IFNγ p = 0.18, for IL-5 p = 0.14). Ratio’s of 

IFNγ / IL-5 production were also not significantly different between adjuvant 

groups (data not shown), but showed a trend towards higher ratio in the 

Montanide and AS02 groups (Day 84 mean ratio Alhydrogel: 1.16 (95% CI: 0.08 

to 2.23), Montanide: 2.82 (95% CI: 1.44 to 4.21), AS02: 2.66 (95% CI: 0.57 to 

4.76)). 

All groups showed Peripheral Blood Mononuclear Cell proliferation upon 

stimulation with PfAMA1 (Figure 5). Stimulation indices between PBMC’s 

stimulated with 30, 6 or 1.2 µg/ml PfAMA1 were similar. All groups of volunteers



showed significant increase in proliferation upon stimulation with PfAMA1 after 

the second immunisation. After the third immunisation none of the groups 

showed a further increase in stimulation index, rather the 10 µg Montanide 

group showed a significant decrease after the third immunisation (p=0.03). 

There were no significant differences in stimulation index between the different 

adjuvant or dose groups. 

In vitro parasite growth inhibition 

To estimate functionality of the induced antibodies, an in vitro GIA was 

performed. Results are shown as percentage inhibition compared to prevaccination 

sera from the same individual (Figure 6). At a concentration of 10 

mg/ml, the median growth inhibition in the Montanide 50 µg and AS02 groups 

was about 30% and 50% respectively. In the Alhydrogel and Montanide 10 µg 

groups median inhibition was lower, ranging from 4 to 17%. Only differences 

between Alhydrogel and AS02 groups were significant (p=0.002). 

Discussion 

This trial demonstrates that reactogenicity of PfAMA1-FVO[25-545] varies, 

depending on the adjuvant. Immunogenicity at both high and low doses and in 

all adjuvant formulations is good, although the type and magnitude of immune 

response varied among different adjuvant groups. 

PfAMA1-FVO[25-545] mixed with the adjuvants Alhydrogel, Montanide and 

AS02 tended to be locally reactogenic, mainly causing short lasting injection site 

pain when administered to healthy adult volunteers. Most post immunisations 

adverse events were mild-to-moderate in intensity and have been seen 

previously with other vaccines [32–37]. Because this was the first time Pichia 

pastoris produced FVO PfAMA1 antigen was being given to humans, the 

occurrence of a grade 3 adverse event was a stopping criterion, which led to 

withdrawal of seven subjects post dose 2 for grade 3 (>50mm) erythema. 

However, the erythema observed resolved spontaneously within three days of 

onset without any sequelae. The erythema is not considered a hindrance for 

further vaccination with the AS02 adjuvant. In terms of systemic adverse events, 

most were related to the AS02 adjuvant and transient resolving within two days 

with no sequelae. The pattern of transient, primarily mild to moderate systemic 

adverse events has been reported with another AMA-1 antigen adjuvanted with 

AS02 [36]. 

49

50 Chapter 2 

Three immunisations with 50 µg PfAMA1 adjuvanted by Montanide induced a 

sterile abscess in two of ten volunteers. Progression of induration to a sterile 

abscess has been previously reported before after immunisation with 

Montanide [38–40]. In all reports the development of an abscess followed 

intramuscular immunisation and was accompanied by enhanced 

immunogenicity. The increased reactogenicity of Montanide-adjuvanted 

vaccines has been attributed to a combination of antigen dose and the 

formation of a vaccine depot that may persist locally and that is inherent to 

water-in-oil emulsions [41]. Similarly, induration at the previous immunisation 

site has been attributed to persistent antigen in previous trials [31]. A less 

condensed vaccination regimen and avoidance of the same injection sites may 

be measures to avoid induration. 

To date, there are four other reports on clinical phase Ia trials of a PfAMA1 

vaccine. These trials employed different PfAMA1 constructs and utilized 

different adjuvants. The constructs were of P. pastoris or E. coli origin or used a 

virally vectored delivery system [9,10,36,42]. 

The P. pastoris-produced PfAMA1 comprised recombinant proteins based on 

sequences from the ectodomains of FVO and 3D7 strain AMA1 adjuvanted with 

Alhydrogel have been tested both in a phase Ia and Ib trial. As with previous 

studies in which malarial antigens have been adjuvanted with Alhydrogel, this 

candidate vaccine showed an acceptable reactogenicity profile but a limited 

immune response. The Malkin et al. phase Ia trial shows a GIA response in only 4 

of 22 subjects despite high seroconversion rates [10], similar to the data 

obtained here with Alhydrogel. Interestingly, in our study, the P. pastoris 

PfAMA1 combined with Alhydrogel was much less reactogenic and did not 

produce any erythema or induration, even though the doses were comparable. 

The lower Alhydrogel dose (500 µg per immunisation) used in this trial, as 

compared to Malkin et al. (800 µg) may also play a role in its decreased 

reactogenicity. 

There are two trials utilizing the E. coli-produced 3D7 strain AMA1, one 

reconstituted in Montanide and a second formulated in AS02. The AMA1- 

Montanide combination was considered safe but the trial was compromised by 

apparent loss of potency [42]. The AMA1-AS02 combination [36] showed 

comparable local and systemic reactogenicity. Although Polhemus et al. were 

able to show recognition of the native antigen by IFA, growth inhibition results 

were approximately two fold lower than those found in this study.



Lastly, PfAMA1 has also been evaluated in a multi-antigen malaria vaccine 

delivered in an attenuated vaccinia virus. Although weak protective effects were 

found, immunogenicity in that trial was poor [9]. 

After one year follow-up, we found antibody levels still to be significantly higher 

than baseline for all groups. This is in sharp contrast to the results of Malkin et 

al. [10] who reported detectable antibodies in only 50-90% of volunteers by day 

364, even though they had been boosted much later (on Day 180). This suggests 

that a more condensed immunisation regime may affect the persistence of 

antibodies. 

In this trial we have shown that the combination of clinical grade PfAMA1 FVO 

[25-545] P. pastoris expressed material with either Montanide or AS02 is 

significantly more immunogenic than previous PfAMA1 formulations, being 

capable of inducing high levels of antibodies for both dosages in both adjuvant 

groups. A positive trend between antigen dose, antibody response and in vitro 

parasite growth inhibition could be detected, although the effect of antigen 

dose on immunogenicity was negligible compared to the effect of varying the 

adjuvant. The wide variety of immune responses found in different adjuvant 

formulations stresses the importance of adjuvants as a critical component in 

malaria vaccine development. 

The functionality of vaccine induced antibodies was assessed by growth 

inhibition assay. Although this assay has not been validated as a correlate of 

protection, this trial demonstrates that the standardised assay is able to 

demonstrate recognition of the native protein and thus functionality in vitro. 

Different adjuvants are known to prompt immune responses towards Th1 or 

Th2. It has been previously reported that AS02 induces an immune response 

skewed towards Th1 [37], with production of primarily IFNγ. In contrast 

Alhydrogel is known to be Th2 inducer [43]. In this study, ratio’s of cytokine 

production at day 84 showed relatively more IFNγ over IL-5 production in the 

Montanide and AS02 groups suggesting a pro-Th1 response, although 

statistically non-significant. Interestingly, the additional third immunisation 

generally did not lead to a further increase in IFNγ or IL-5 production or in 

lymphocyte proliferation. Rather, many volunteers showed a reduction in the 

response after the third immunisation. This difference could not be explained by 

inter-test variability. It remains to be investigated if it indicates a shift in the 

relative balance between immediate effector cells and long-lived memory cells. 

51

52 Chapter 2 

Although this phase I trial is limited with respect to the size and generalizibility 

to the target population, it met its objectives to outline a generalizable safety 

profile. Specifically the direct comparison of the safety profile of different 

adjuvants is valuable for future development of AMA1 and other malaria 

vaccines. Furthermore, the malaria vaccine candidate AMA1 provides the 

possibility of assessing functionality of the immune response by a parasite 

growth inhibition assay. However, it must be noted that the growth inhibition 

assay is not validated as a correlate of protection, and is as such a limited 

predictor for efficacy. 

With this study we have shown that the PfAMA1 vaccine combined with 

different adjuvants, Alhydrogel, Montanide and AS02 provided distinct 

reactogenicity profiles. All vaccine formulation were immunogenic at both 

dosages. Growth inhibition results indicate that induction of functional immune 

responses is probably dependent on adjuvant, underscoring the need for strong 

immunopotentiators for malaria vaccines. Altogether, these results are 

promising for a future development of a PfAMA1 malaria vaccine. 

Acknowledgements 

We thank Dominique Lemoine and Natalie Imbault for their comments and 

contribution to the manuscript 

Funding 

This work was supported by a grant from the European Malaria Vaccine 

Initiative.



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efficacy of a recombinant DNA Plasmodium falciparum sporozoite vaccine. Lancet 

1987; 1:1277-1281. 

14. Amador R, Moreno A, Valero V, Murillo L, Mora AL et al. The first field trials of the 

chemically synthesized malaria vaccine SPf66: safety, immunogenicity and 

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15. Lawrence G, Cheng QQ, Reed C, Taylor D, Stowers A et al. Effect of vaccination 

with 3 recombinant asexual-stage malaria antigens on initial growth rates of 

Plasmodium falciparum in non-immune volunteers. Vaccine 2000; 18:1925-1931. 

16. Lawrence GW, Saul A, Giddy AJ, Kemp R, Pye D. Phase I trial in humans of an oilbased 

adjuvant SEPPIC Montanide ISA 720. Vaccine 1997; 15:176-178. 

17. Saul A, Lawrence G, Smillie A, Rzepczyk CM, Reed C et al. Human phase I vaccine 

trials of 3 recombinant asexual stage malaria antigens with Montanide ISA720 

adjuvant. Vaccine 1999; 17:3145-3159. 

18. Genton B, Betuela I, Felger I, Al-Yaman F, Anders RF et al. A recombinant bloodstage 

malaria vaccine reduces Plasmodium falciparum density and exerts selective 

pressure on parasite populations in a phase 1-2b trial in Papua New Guinea. J 

Infect Dis 2002; 185:820-827. 

19. Aucouturier J, Dupuis L, Deville S, Ascarateil S, Ganne V. Montanide ISA 720 and 

51: a new generation of water in oil emulsions as adjuvants for human vaccines. 

Expert Rev Vaccines 2002; 1:111-118. 

20. Heppner DG, Jr., Kester KE, Ockenhouse CF, Tornieporth N, Ofori O et al. Towards 

an RTS,S-based, multi-stage, multi-antigen vaccine against falciparum malaria: 

progress at the Walter Reed Army Institute of Research. Vaccine 2005; 23:2243- 

2250. 

21. Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E et al. Duration of protection 

with RTS,S/AS02A malaria vaccine in prevention of Plasmodium falciparum 

disease in Mozambican children: single-blind extended follow-up of a randomised 

controlled trial. Lancet 2005; 366:2012-2018. 

22. Aponte JJ, Aide P, Renom M, Mandomando I, Bassat Q et al. Safety of the 

RTS,S/AS02D candidate malaria vaccine in infants living in a highly endemic area of 

Mozambique: a double blind randomised controlled phase I/IIb trial. Lancet 2007; 

370:1543-1551. 

23. Bojang KA, Milligan PJ, Pinder M, Vigneron L, Alloueche A et al. Efficacy of 

RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semiimmune 

adult men in The Gambia: a randomised trial. Lancet 2001; 358:1927- 

1934. 

24. Gordon DM, McGovern TW, Krzych U, Cohen JC, Schneider I et al. Safety, 

immunogenicity, and efficacy of a recombinantly produced Plasmodium



falciparum circumsporozoite protein-hepatitis B surface antigen subunit vaccine. J 

Infect Dis 1995; 171:1576-1585. 

25. Sun P, Schwenk R, White K, Stoute JA, Cohen J et al. Protective immunity induced 

with malaria vaccine, RTS,S, is linked to Plasmodium falciparum circumsporozoite 

protein-specific CD4+ and CD8+ T cells producing IFN-gamma. J Immunol 2003; 

171:6961-6967. 

26. Stoute JA, Slaoui M, Heppner DG, Momin P, Kester KE et al. A preliminary 

evaluation of a recombinant circumsporozoite protein vaccine against 

Plasmodium falciparum malaria. RTS,S Malaria Vaccine Evaluation Group. N Engl J 

Med 1997; 336:86-91. 

27. Kocken CH, Withers-Martinez C, Dubbeld MA, Van Der WA, Hackett F et al. Highlevel 

expression of the malaria blood-stage vaccine candidate Plasmodium 

falciparum apical membrane antigen 1 and induction of antibodies that inhibit 

erythrocyte invasion. Infect Immun 2002; 70:4471-4476. 

28. Faber BW, Remarque EJ, Kocken CH, Cheront P, Cingolani D et al. Production, 

quality control, stability and pharmacotoxicity of cGMP-produced Plasmodium 

falciparum AMA1 FVO strain ectodomain expressed in Pichia pastoris. Vaccine 

2008; 26:6143-6150. 

29. Kester KE, McKinney DA, Tornieporth N, Ockenhouse CF, Heppner DG, Jr. et al. A 

phase I/IIa safety, immunogenicity, and efficacy bridging randomized study of a 

two-dose regimen of liquid and lyophilized formulations of the candidate malaria 

vaccine RTS,S/AS02A in malaria-naive adults. Vaccine 2007; 25:5359-5366. 

30. Human D, Crawley F, IJesselmuiden C. Revised declaration of Helsinki. WMA will 

continue to revise policy as medicine and research changes. BMJ 2001; 323:283- 

284. 

31. Hermsen CC, Verhage DF, Telgt DS, Teelen K, Bousema JT et al. Glutamate-rich 

protein (GLURP) induces antibodies that inhibit in vitro growth of Plasmodium 

falciparum in a phase 1 malaria vaccine trial. Vaccine 2007; 25:2930-2940. 

32. Zepp F, Knuf M, Habermehl P, Mannhardt-Laakmann W, Howe B et al. Safety of 

reduced-antigen-content tetanus-diphtheria-acellular pertussis vaccine in 

adolescents as a sixth consecutive dose of acellular pertussis-containing vaccine. J 

Pediatr 2006; 149:603-610. 

33. Kshirsagar N, Mur N, Thatte U, Gogtay N, Viviani S et al. Safety, immunogenicity, 

and antibody persistence of a new meningococcal group A conjugate vaccine in 

healthy Indian adults. Vaccine 2007; 25 suppl1:A101-A107 

34. Malkin E, Long CA, Stowers AW, Zou L, Singh S et al. Phase 1 Study of Two 

Merozoite Surface Protein 1 (MSP1(42)) Vaccines for Plasmodium falciparum 

Malaria. PLoS Clin Trials 2007; 2:e12. 

55

56 Chapter 2 

35. Kester KE, McKinney DA, Tornieporth N, Ockenhouse CF, Heppner DG, Jr. et al. A 

phase I/IIa safety, immunogenicity, and efficacy bridging randomized study of a 

two-dose regimen of liquid and lyophilized formulations of the candidate malaria 

vaccine RTS,S/AS02A in malaria-naive adults. Vaccine 2007; 25:5359-5366. 

36. Polhemus ME, Magill AJ, Cummings JF, Kester KE, Ockenhouse CF et al. Phase I 

dose escalation safety and immunogenicity trial of Plasmodium falciparum apical 

membrane protein (AMA-1) FMP2.1, adjuvanted with AS02A, in malaria-naive 

adults at the Walter Reed Army Institute of Research. Vaccine 2007; 25:4203- 

4212. 

37. Vandepapeliere P, Rehermann B, Koutsoukos M, Moris P, Garcon N et al. Potent 

enhancement of cellular and humoral immune responses against recombinant 

hepatitis B antigens using AS02A adjuvant in healthy adults. Vaccine 2005; 

23:2591-2601. 

38. Langermans JA, Schmidt A, Vervenne RA, Birkett AJ, Calvo-Calle JM et al. Effect of 

adjuvant on reactogenicity and long-term immunogenicity of the malaria Vaccine 

ICC-1132 in macaques. Vaccine 2005; 23:4935-4943. 

39. Langermans JA, Hensmann M, van GM, Zhang D, Pan W et al. Preclinical 

evaluation of a chimeric malaria vaccine candidate in Montanide ISA 720: 

immunogenicity and safety in rhesus macaques. Hum Vaccin 2006; 2:222-226. 

40. Toledo H, Baly A, Castro O, Resik S, Laferte J et al. A phase I clinical trial of a multiepitope 

polypeptide TAB9 combined with Montanide ISA 720 adjuvant in non-HIV- 

1 infected human volunteers. Vaccine 2001; 19:4328-4336. 

41. Miles AP, McClellan HA, Rausch KM, Zhu D, Whitmore MD et al. Montanide ISA 

720 vaccines: quality control of emulsions, stability of formulated antigens, and 

comparative immunogenicity of vaccine formulations. Vaccine 2005; 23:2530- 

2539. 

42. Saul A, Lawrence G, Allworth A, Elliott S, Anderson K et al. A human phase 1 

vaccine clinical trial of the Plasmodium falciparum malaria vaccine candidate 

apical membrane antigen 1 in Montanide ISA720 adjuvant. Vaccine 2005; 

23:3076-3083. 

43. Ulanova M, Tarkowski A, Hahn-Zoric M, Hanson LA The Common vaccine adjuvant 

aluminum hydroxide up-regulates accessory properties of human monocytes via 

an interleukin-4-dependent mechanism. Infect Immun 2001; 69:1151-1159.

Chapter 3 

Humoral immune responses to a single 

allele PfAMA1 vaccine in healthy malarianaïve 

adults. 

Edmond J. Remarque* 1 , Meta Roestenberg 2 , Sumera Younis 1 , Vanessa 

Walraven 1 , Nicole van der Werff 1 , Bart W. Faber 1 , Odile Leroy 3 , Robert 

Sauerwein 2 , Clemens H.M. Kocken 1 Alan W. Thomas 1 

Biomedical Primate Research Centre, Rijswijk, The Netherlands 

Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 

European Vaccine Initiative, Heidelberg, Germany 

PLoS ONE, 2012;7(6):e38898.

Chapter 3 58 

Abstract 

Plasmodium falciparum apical membrane antigen 1 (AMA1) is a candidate 

malaria vaccine antigen expressed on merozoites and sporozoites. The 

polymorphic nature of AMA1 may compromise vaccine induced protection. 

The humoral response induced by two dosages (10 and 50 µg) of a single allele 

AMA1 antigen formulated with Alhydrogel, Montanide ISA 720 or AS02 was 

investigated in 47 malaria-naïve adult volunteers. Volunteers were vaccinated 3 

times at 4 weekly intervals and serum samples obtained four weeks after the 

third immunization were analysed for (i) antibody responses to various allelic 

variants, (ii) domain specificity, (iii) avidity, (iv) IgG subclass levels, by ELISA and 

(v) functionality of antibody responses by Growth Inhibition Assay. 

About half of the antibodies induced by vaccination cross reacted with 

heterologous AMA1 alleles. The choice of adjuvant determined the magnitude of 

the antibody response, but had only a marginal influence on specificity, avidity, 

domain recognition or subclass responses. The highest antibody responses were 

observed for AMA1 formulated with AS02. The Growth Inhibition Assay (GIA) 

activity of the antibodies was proportional to the amount of antigen specific IgG 

and the functional capacity of the antibodies was similar for heterologous 

AMA1-expressing laboratory strains.

Humoral immune responses to a single allele PfAMA1 vaccine in healthy malarianaïve 

adults. 

Introduction 

Malaria is a serious public health problem causing high levels of morbidity and 

mortality in malaria-endemic regions [1]. An effective malaria vaccine will 

contribute to reducing the burden of malaria in addition to existing measures 

like insecticide-treated bed nets, antimalarials and vector control. 

Apical Membrane Antigen 1 (AMA1) is a promising malaria vaccine candidate 

(reviewed in [2]), expressed on merozoites and sporozoites as a type I integral 

membrane protein [3,4] and is initially located in the micronemes [3,5]. In 

Plasmodium falciparum AMA1 is expressed as an 83 kDa protein and relocates 

as a 66 kDa protein to the parasite surface following cleavage of the prosequence 

at the time of invasion [6], where it is subsequently shed as 44 and 48 

kDa forms [6]. The AMA1 ectodomain consists of an N-terminal pro-sequence 

and three tightly interacting domains [7,8]. AMA1 is believed to play an essential 

role in red blood cell invasion [9] and may also be implicated in liver cell invasion 

by sporozoites [4]. The protective efficacy of AMA1-based vaccines has been 

demonstrated in numerous mouse and simian models (reviewed in [2]). 

AMA1 is a polymorphic antigen [10], and this polymorphism is entirely due to 

single amino acid substitutions [7]. These polymorphisms have been found to be 

restricted to the surface of AMA1, predominantly mapping to one molecular 

face [7,8,11]. Studies with the rodent malaria P. chabaudi have shown that 

polymorphism in AMA1 can ablate vaccine efficacy [12]. Rabbit immunisation 

studies have demonstrated that, although antibodies raised against one PfAMA1 

allele show excellent inhibition of strains expressing a homologous allele, strains 

expressing a heterologous PfAMA1 allele are inhibited to a variable lesser 

degree depending on the antigenic differences and their locations [13–17], 

suggesting that PfAMA1 polymorphism reduces the efficacy of PfAMA1 based 

vaccines. Results from a Phase IIb vaccine study in Malian children suggests that 

the specificity of the AMA1 immune response is crucial in protection [18]. 

Most of the data addressing the impact of polymorphism have been generated 

in laboratory animals and relatively little is known from human vaccination 

studies. A phase Ia study with 10 or 50 µg of a single allele vaccine (PfAMA1 FVO 

25-545) formulated with three adjuvants [19] in malaria-naïve subjects offers a 

unique opportunity to perform an in depth analysis of antibody responses in 

humans. Total IgG and GIA responses to the homologous antigen have been 

59

60 Chapter 3 

reported previously [19]. The analysis reported here comprises ELISA and 

Growth Inhibition Assay (GIA) titres to the homologous and heterologous AMA1 

antigens, domain specificity, subclass distribution, avidity and the relation 

between GIA and Elisa titres after 3 immunisations with PfAMA1 FVO 25-545. In 

addition, it also offers the opportunity to investigate the effect of various 

adjuvants on the quality and quantity of the humoral immune response. 

Adjuvant Dose N Age Minimum Maximum 

Alhydrogel 10 9 23.3 ± 2.9 21.1 29.5 

Alhydrogel 50 10 23.2 ± 2.1 19.4 26.9 

ISA720 10 8 23.1 ± 5.2 18.5 33.7 

ISA720 50 9 23.2 ± 3.6 19.7 31.1 

AS02 10 7 24.5 ± 8.3 20.0 42.8 

AS02 50 4 24.3 ± 4.8 20.5 31.3 

Table 1: Characteristics of the subjects at the time of first vaccination (per 

protocol) 


Participants 

Fifty-six malaria-naïve healthy male study participants were recruited at the 

Radboud University Nijmegen Medical Centre as previously reported [19]. A 

total of 47 subjects completing all 3 immunisations were included for the per 

protocol analysis in the current paper [19]. The characteristics of the subjects at 

the time of first vaccination are presented in Table 1. All volunteers provided 

written informed consent. The study was approved by the Institutional Review 

Board (CMO Regio Arnhem-Nijmegen, 2005/015). The study was conducted in 

accordance with the Declaration of Helsinki principles for the conduct of clinical 

trials and the International Committee of Harmonization Good Clinical Practice 

Guidelines and registered at www.clinicaltrials.gov (NCT00730782). 

Vaccines, vaccination and blood samples 

Clinical grade PfAMA1 FVO[25-545] consisting of P. falciparum FVO-strain AMA1 

ectodomain (amino acids 25 to 545) was produced as previously described [20]. 

The cGMP produced PfAMA1 FVO[25-545] was available in multidose vials 

containing 120 µg lyophilised AMA1 (44 µg EDTA, 180 µg saccharose and 120 µg 

NaHCO3, Lot B) or 62.5 µg lyophilised AMA1 (23 µg EDTA, 25 mg saccharose, 226 

µg K2HPO4 and 187 µg NaH2PO4, Lot C). PfAMA1 vaccines of 0.5 mL were 

prepared at two dosages (10 and 50 µg) with three different adjuvants


adults. 

*----30---*----40---* ---50 --*----60---*----70---*----80---*----90---*----100--*----110--*---- 

120-- 

FVO ∆ Glyc 

QNYWEHPYQKSDVYHPINEHREHPKEYEYPLHQEHTYQQEDSGEDENTLQHAYPIDHEGAEPAPQEQNLFSSIEIVERSNYMGNPWTEYMAKYDIEEVHG 

3D7 ∆.Glyc ---------N----R---------------------------------------------------------------------------------- 

--- 

HB3 ∆ Glyc ---------N----R--------------------------------------------------------------------------------- 

K--- 

CAMP ∆ Glyc ---------N—N---------------Q--------------------------------------------------------------------- 

--- 

*----130--*----140--*----150--*----160--*----170--*----180--*----190--*----200--*----210--*---- 

220-- 

FVO ∆ Glyc 

SGIRVDLGEDAEVAGTQYRLPSGKCPVFGKGIIIENSKTTFLKPVATGNQDLKDGGFAFPPTNPLISPMTLNGMRDFYKNNEYVKNLDELTLCSRHAGNM 

3D7 ∆ Glyc ------------------------------------------T-------Y-----------E—-M-----DE—-H---D-K--------------- 

--- 

HB3 ∆ Glyc ------------------------------------------T----E--------------E--------DQ—-HL—-D----------------- 

--- 

CAMP ∆ Glyc --------------------------------------------------------------E---------------------------------- 

--- 

*----230--*----240--*----250--*----260--*----270--*----280--*----290--*----300--*----310--*---- 

320-- 

FVO ∆ Glyc 

NPDNDKNSNYKYPAVYDYNDKKCHILTIAAQENNGPRYCNKDQSKRNSMFCFRPAKDKLFENYVYLSKNVVDNWEEVCPRKNLENAKFGLWVDGNCEDIP 

3D7 ∆ Glyc I----------------DK-----------------------E--------------IS-Q--------------K-------Q------------- 

--- 

HB3 ∆ Glyc ------------------E-----------------------E------------------------------------------------------ 

--- 

CAMP ∆ Glyc ---K-E-----------DK-----------------------E---------------S-Q--------------K--------------------- 

--- 

*----330--*----340--*----350--*----360--*----370--*----380--*----390--*----400--*----410--*---- 

420-- 

FVO ∆ Glyc 

HVNEFSANDLFECNKLVFELSASDQPKQYEQHLTDYEKIKEGFKNKNADMIKSAFLPTGAFKADRYKSHGKGYNWGNYNRETQKCEIFNVKPTCLINDKS 

3D7 ∆ Glyc -----P-I-----------------------------------------------------------------------T----------------- 

--- 

HB3 ∆ Glyc --------------------------------------------------------------------R----------T----------------- 

-- 

CAMP ∆ Glyc --------------------------------------------------------------------------------K-H-------------- 

--- 

*----430--*----440--*----450--*----460--*----470--*----480--*----490--*----500--*----510--*---- 

520-- 

FVO ∆ Glyc 

YIATTALSHPIEVEHNFPCSLYKDEIKKEIERESKRIKLNDNDDEGNKKIIAPRIFISDDKDSLKCPCDPEMVSQSTCRFFVCKCVERRAEVTSNNEVVV 

3D7 ∆ Glyc --------------N-----------M---------------------------------------------------------------------- 

--- 

HB3 ∆ Glyc ----------N---N--------------------------------------------------------I------N--------K--------- 

--- 

CAMP ∆ GlyC --------------N--------N—-M---------------------------------------------------------------------- 

---- 

*----530--*----540--* 

FVO ∆ Glyc KEEYKEDYADIPEHKPTYDNM 

3D7 ∆ Glyc -------------------K- 

HB3 ∆ Glyc --------------------- 

CAMP ∆ Glyc --------------------- 

Figure 1: Alignment of AMA1 protein sequences amino acids 25 through 545 used for Elisa 

measurements. Potential N-glycosylation sited were changed (six for FVO, 3D7 and CAMP 

and five for HB3) to avoid unwanted N-glycosylation: N 162 K (For FVO, 3D7 and CAMP. 

HB3 AMA1 has K at position 162). T 288 V, S 273 D, N 422 D, S 423 K and N 499 Q. 

(Alhydrogel, Montanide ISA 720 or AS02) as previously described [19]. 

Alhydrogel and Montanide ISA 720 vaccines were prepared with lot B PfAMA1 

and AS02 vaccines were prepared with lot C PfAMA1. Formulated vaccines were 

kept at 4°C for a maximum of six hours until administration. Vaccine 

formulations were tested for stability as previously described [20] and all 

fulfilled the pre-set specifications. Immunisations were given intramuscularly in 

the deltoid region of alternate arms at days 0, 28 and 56. Blood was collected in 

VacutainerTM CPT tubes (Becton and Dickinson), plasma collected after 

centrifugation and stored at -20°C. 

61

62 Chapter 3 

Quantification ELISA for total IgG and IgG subclasses 

Enzyme-linked immunosorbent assay (ELISA) was performed in duplicate on 

plasma samples in 96 well flat-bottomed microtitre plates (Greiner, Alphen a/d 

Rijn, The Netherlands), coated with 1 µg/mL purified AMA1 antigens according 

to published methods [10]. The sequences of the four Pichia pastoris-expressed 

AMA1 proteins used for ELISA are shown in Figure 1, potential N-glycosylation 

sites were removed as previously described [10,15] and proteins were produced 

as previously described [20]. The P. pastoris-expressed AMA1 from 3D7, HB3 and 

CAMP used in the ELISA’s differ by 26 (2, 17, 5 and 2 for prodomain and domains 

I, II and III, respectively), 20 (2, 11, 4, 3 for prodomain and domains I, II and III, 

respectively) and 17 (3, 9, 3, 2 for prodomain and domains I, II and III, 

respectively) amino acid positions in the ectodomains (aa 25-545) (Figure 1). The 

secondary antibody was goat anti-human IgG conjugated to alkaline 

phosphatase (Pierce, Rockford, IL), or mouse anti-human IgG subclass 

conjugated to horseradish peroxidase (Sanquin, Amsterdam, The Netherlands). 

A standard curve was included on each plate and antibody levels in the 

unknowns were calculated using a four-parameter fit. Titres are expressed as 

arbitrary units, where 1 AU yields an OD of 1 over background. Thus the amount 

of AU of a sample is the reciprocal dilution at which an OD of 1 over background 

will be achieved. 

Competition Elisa 

Competition ELISA was performed as previously described [16]. Dilutions that 

resulted in 2 AU were calculated for each plasma sample and used in the 

subsequent antigen competition assay. The assay involved co-incubation of 

different allelic forms of PfAMA1, or PfAMA1-FVO domains with test plasma in 

plates coated with PfAMA1 FVO allele, such that competition occurs between 

the added (competitor) antigens and the coated antigen for binding to test IgG’s. 

For the AMA1 allelic forms the competitor/soluble antigens were titrated 3-fold 

over 7 wells from 100 to 0.137 µg /ml with PfAMA1 from the FVO, 3D7, HB3 or 

CAMP alleles (Figure 1). For the PfAMA1-FVO domains competition ELISA, plates 

were coated with FVO AMA1 amino acids 97 through 545 (Domains I, II and III) a 

fixed concentration of 30 µg/ml of various FVO AMA1 domain constructs were 

used as competitors (pro-DI-II-III aa 25-545, DI-II aa 97 - 442, DII-III aa 303 - 545 

and DII aa 303 - 442). The appropriately diluted plasma was then added and 

after incubation for 2 h, plates were developed as described above. Values are 

presented as the fraction remaining relative to the initial amount.


adults. 

Avidity ELISA 

The avidities of the antibodies were determined by sodium isothiocyanate 

(NaSCN) elution ELISA as previously described [16]. Briefly, microtitre plates 

were coated with AMA1 variant proteins (Figure 1) as described above, and after 

blocking, incubated with a pre-determined titre (1 AU) of sera for 1 h. Plates 

were then washed and incubated with an increasing concentration of NaSCN 

(ranging from 0 to 3.0 M in 0.25 M steps) in duplicate wells for 15 min. Plates 

were washed and developed with goat anti-human IgG alkaline phosphatase 

conjugate and substrate as previously described [16]. The avidity index is 

expressed as the concentration of NaSCN required for 50% dissociation of bound 

antibodies (relative to duplicate wells without NaSCN). 

Growth Inhibition Assay (GIA) 

Antibodies used for growth inhibition assays (GIA) were purified from CPT 

plasma on protein A columns (Immunopure Plus Pierce, St Louis, MO, USA), 


Millipore, Ireland), filter-sterilised and stored at -20°C until use. IgG 


(Nanodrop Technologies, Wilmington, DE, USA). P. falciparum strains FCR3, 3D7 

and HB3 were cultured in vitro using standard culture techniques in an 

atmosphere of 5% CO2, 5% O2 and 90% N2. FCR3 AMA1 (accession no. M34553) 

differs by one amino acid in the pro-sequence (D36G) from FVO AMA1 

(accession no. AJ277646). The ectodomain (amino acids 25-545) of 3D7 

(accession no. U65407) differs by 26 amino acids (2, 17, 5 and 2 for prodomain 

and domains I, II and III, respectively) from FVO and the ectodomain of HB3 

(accession no. U33277) differs by 21 amino acids (2, 12, 4 and 3 for prodomain 

and domains I, II and III, respectively) from FVO. 

The GIA was performed as previously described [14]. Briefly, the effect of 

purified IgG antibodies on in vitro parasite growth was evaluated at two IgG 

concentrations (5 and 10 mg/mL, respectively) and each participants preimmune 

IgG was used as negative control. A IgG concentration of 10 mg /ml 

approximates the amount of IgG (9.5 to 11.5 mg /ml) found in undiluted human 

plasma [21]. Samples were run in triplicate using 96 well flat-bottomed plates 

with alanine-synchronized cultures of P. falciparum schizonts at an initial 

parasitemia of 0.2–0.4%, a haematocrit of 2.0% and a final volume of 100 µL. 

After 40 to 42 hours, cultures were resuspended, and 50 µL was transferred into 

200 µL ice-cold PBS. The cultures were then centrifuged, the supernatant 

63

64 Chapter 3 

AMA1 IgG (AU/mL) 

65536 

32768 

16384 

8192 

4096 

2048 

1024 

512 

FVO 

Alhydrogel 10 

Alhydrogel 50 

ISA 720 10 

ISA 720 50 

AS02 10 

AS02 50 

3D7 

Alhydrogel 10 

Alhydrogel 50 

ISA 720 10 

ISA 720 50 

AS02 10 

AS02 50 

Alhydrogel 10 

Alhydrogel 50 

ISA 720 10 

ISA 720 50 

AS02 10 

AS02 50 

Treatment 

removed and the plates were frozen. Parasite growth was assessed by 

measuring parasite lactate dehydrogenase levels with the lactate diaphorase 

APAD substrate system, and plates were read at 655 nm after 30 min incubation 

in the dark. Parasite growth inhibition was expressed as: 100 x (1 – (OD655 

Day84 – OD655 RBC)/(OD655 Day0 – OD655 RBC)), Where: OD655Day84 is the 

OD655 for IgG purified from day 84 plasma, OD655Day0 is the OD655 for IgG 

purified from day 0 plasma and OD655RBC is the average OD655 of RBC control 

wells. The data are presented as the arithmetic mean % inhibition from each 

sample triplicate. 

Statistics 

Antibody titres (IgG and IgG subclasses) were log-transformed to obtain 

normality and are presented as geometric means with 95% confidence intervals. 

GIA titres, IgG avidity and levels of depletion were approximately normally 

distributed and are therefore presented as arithmetic means with 95% confi- 

HB3 

CAMP 

Alhydrogel 10 

Alhydrogel 50 

ISA 720 10 

ISA 720 50 

AS02 10 

AS02 50 

Figure 2: IgG levels to the vaccine allele and three variant AMA1 alleles in serum 

obtained on day 84. IgG antibody levels to the vaccine antigen (FVO) and 3 

heterologous AMA1 alleles (3D7, HB3 and CAMP). Values for each subject within a 

treatment group are indicated by a specific symbol (same symbol within each 

treatment group is same subject throughout graphs 2, 4, 5, 6 and 7), boxes indicate 

median and quartile ranges. Treatment groups are indicated by Adjuvant name and 

AMA1 dose, respectively.


adults. 

Fraction of IgG remaining (%) 

100 

80 

60 

40 

20 

0 

100 

80 

60 

40 

20 

0 

100 

80 

60 

40 

20 

0 

0 

0.14 

0.41 

Alhydrogel 10 Alhydrogel 50 

ISA 720 10 ISA 720 50 

AS02 10 AS02 50 

1.23 

3.7 

11.11 

33.33 

100 

0 

Competitor concentration (µg/mL) 

Figure 3. Competition ELISA using four different competitor antigens on FVO coated 

plates in serum obtained on day 84. 

Concentration of competitor antigen is depicted on the x-axis (µg /ml) and the fraction 

remaining bound to the ELISA plate is depicted on the y-axis (%). Light Blue = FVO AMA1 

(homologous), Orange = HB3 AMA1, Green = CAMP AMA1 and Dark Blue = 3D7 AMA1. 

Treatment groups are indicated by Adjuvant name and AMA1 dose, respectively. 

0.14 

0.41 

1.23 

3.7 

11.11 

33.33 

100 

65

66 Chapter 3 

dence intervals. The statistical significance of between group differences was 

initially evaluated using one way (comparing the 6 adjuvant-dose groups 

directly) Analysis of Variance (ANOVA). 

Significant between group differences were further evaluated by Tukey’s Honest 

Significant Difference post-hoc test which applies a correction on the p-value for 

multiple comparisons and provides estimates with 95% confidence intervals 

(95% CI) for the between group comparisons. 

The relation between GIA and IgG titres was investigated by non linear least 

squares regression using the following formula: GIA = 100 * 1 / [1 + Exp(( Ln IC50 

– Ln IgG ) * Slope) ], where IgG is the antibody titre, IC50 represents the IgG 

concentration yielding 50% inhibition and slope is a parameter indicating the 

steepness of the curve, with steeper curves at higher (absolute) values. As Slope 

is rather difficult to interpret, it is expressed as the fold-increase in IgG required 

to raise GIA levels from 10 to 50% and, as the sigmoid is symmetrical around 

50%, also from 50 to 90%. It can be shown algebraically that this is 

approximately e(2.197 / Slope). 

Results 

IgG responses to AMA1 alleles 

All plasma samples obtained 4 weeks after the third vaccination (Day 84) were 

titrated in a single laboratory run on 4 different AMA1 variants; IgG antibody 

titres are depicted in Figure 2. Results obtained for the FVO AMA1 are in 

agreement with those previously reported (r = 0.916, p < 0.0001)[19]. IgG 

antibody levels were lowest in the Alhydrogel groups (10 and 50 µg) and in the 

Montanide ISA 720 10 µg group as compared to the Montanide ISA 720 50 µg 

and both AS02 groups, this trend was consistently observed for all AMA1 

variants under investigation (Figure 2). The IgG antibody levels to the 

heterologous AMA1 (3D7, HB3 and CAMP) variants, were approximately 50% 

lower as compared to the homologous FVO AMA1 (Figure 2). IgG antibody levels 

to all AMA1 variants under investigation were 4 to 5 fold higher in the 

Montanide ISA 720 50 µg and AS02 10 µg groups as compared to the Alhydrogel 

10 µg group (Figure 2, all p < 0.02). 

The specificity of the IgG responses in day 84 samples was further characterised 

using competition ELISA, where increasing amounts of competitor antigen were 

co-incubated with a fixed amount of antibodies. Figure 3 shows that the addition


adults. 

Figure 4. Competition ELISA using AMA1 domain constructs in serum obtained on 

day 84.The fraction of antibodies remaining bound after competition with 30 µg 

of the indicated competitor are depicted on the y-axis, boxes indicate median and 

quartile ranges. P-DI-II-III = FVO AMA1 ectodomain amino acids 25 – 545, DI-II = 

FVO-AMA1 amino acids 97 – 442, DII-III = FVO AMA1 amino acids 303 – 545 and 

DII = FVO AMA1 amino acids 303 – 442. Treatment groups are indicated by 

Adjuvant name and AMA1 dose, respectively. 

of increasing amounts of antigen reduces the amount of bound antibody for all 

competitor antigens, with the most pronounced reduction observed for the 

homologous antigen; the heterologous antigens also reduced the amount of IgG 

remaining bound, but less efficiently as compared to the homologous antigen. 

The depletion curves per competitor antigen were similar, as judged by IC50 and 

slope parameters, for the adjuvant and dose groups, suggesting neither adjuvant 

nor dose markedly influenced antibody specificities (Figure 3). The amount of 

competitor antigen required to remove 50% of antibodies (IC50) was lowest for 

FVO at 0.23 µg /mL (95% CI 0.21 to 0.26), 1.47 µg/ml (95% CI 1.08 to 2.02) for 

HB3, 4.39 µg /mL-(95% CI 2.93 to 6.57) for CAMP and 20.15 µg/mL (95% CI 14.17 

to 28.65) for 3D7 (Figure 3). The increase in competitor antigen concentration 

required to reduce the remaining amount of bound antibodies from 50 to 10% 

was 38-fold (95% CI 34 to 43) for FVO AMA1. The slopes for the heterologous 

AMA1 proteins indicated that a much larger increase in competitor concentra- 

67

68 Chapter 3 

AMA1 IgG avidity (M NaSCN) 

2.5 

2.0 

1.5 

1.0 

0.5 

FVO 

Alhydrogel 10 

Alhydrogel 50 

ISA 720 10 

ISA 720 50 

AS02 10 

AS02 50 

3D7 

Alhydrogel 10 

Alhydrogel 50 

ISA 720 10 

ISA 720 50 

AS02 10 

AS02 50 

Alhydrogel 10 

Treatment 

HB3 

Alhydrogel 50 

ISA 720 10 

ISA 720 50 

AS02 10 

AS02 50 

CAMP 

Alhydrogel 10 

Alhydrogel 50 

ISA 720 10 

ISA 720 50 

AS02 10 

AS02 50 

Figure 5. Antibody avidity to the vaccine allele and three variant alleles in serum 

obtained on day 84, boxes indicate median and quartile ranges. Antibody avidity is 

expressed as the amount of NaSCN required to dissociate 50% of bound antibodies. 

Treatment groups are indicated by Adjuvant name and AMA1 dose, respectively. 

tion was required to reduce the amount of bound antibodies from 50 to 

10%: 1015-fold (95% CI 702 to 1535), 1328-fold (95% CI 920 to 2001) and 910fold 

(95% CI 471 to 2058) for HB3, CAMP and 3D7 AMA1, respectively (Figure 3). 

IgG responses to AMA1 domains 

The domain specificity of the antibody response at day 84 was investigated using 

competition ELISA’s, where fixed amounts (30 µg) of PfAMA1-FVO domains were 

added. Figure 4 shows that 7.2% (95% CI 3.3 to 11.2) of the IgG remained bound 

after the addition of 30 µg of the full length antigen, indicating over 90% 

depletion. The fraction of IgG remaining after the addition of the full length 

antigen (p-DI-II-III) did not differ for the groups (p=0.36). After addition of 

domains I-II approximately 19.0% (95% CI 17.6 to 20.5) of the IgG remained 

bound, indicating more than 80% depletion. The addition of domains I-II resulted 

in significant between group differences in the fraction remaining bound (p = 

0.0009, ANOVA). More IgG remained bound In the Montanide ISA 720 10 µg 

group when compared to the Alhydrogel 50 µg and AS02 50 µg groups, (7.8 % 

(95% CI 2.0 to 13.6, p = 0.0030, Tukey HSD) and 10.9 (95% CI 3.4 to 18.4. p =


adults. 

AMA1 IgGx (AU/mL) 

2048 

512 

128 

32 

8 

2 

0.5 

0.125 

0.03125 

IgG1 

Alhydrogel 10 

Alhydrogel 50 

ISA 720 10 

ISA 720 50 

AS02 10 

AS02 50 

IgG2 

Alhydrogel 10 

Alhydrogel 50 

ISA 720 10 

ISA 720 50 

AS02 10 

AS02 50 

Alhydrogel 10 

Alhydrogel 50 

ISA 720 10 

ISA 720 50 

AS02 10 

AS02 50 

Treatment 

IgG3 

Alhydrogel 10 

Alhydrogel 50 

ISA 720 10 

ISA 720 50 

AS02 10 

AS02 50 

Figure 6. IgG subclasses to the vaccine antigen in serum obtained on day 84, boxes 

indicate median and quartile ranges. Treatment groups are indicated by Adjuvant 

name and AMA1 dose, respectively. 

0.0011, Tukey HSD). A similar trend, albeit not significant, was observed when 

comparing the Montanide ISA 720 50 µg group with the AS02 50 µg group 

(7.2 % difference, 95% CI -0.17 to 14.5, p = 0.059, Tukey HSD). This suggests 

that Montanide ISA 720 induces more antibodies directed against domain III. No 

significant differences were observed between the treatment groups following 

depletion with domains II-III or domain II only (p values 0.16 and 0.051, 

respectively, ANOVA). The average amount of IgG remaining bound was 21.1% 

(95% CI 19.2 to 23.0) and 61.5% (95% CI 57.1 to 66.0) for domains II-III and 

domain II, respectively. 

Antibody Avidity 

Antibody avidity in plasma samples collected on day 84 was quantified using a 

sodium thiocyanate (NaSCN) elution ELISA and expressed as the concentration 

of NaSCN required to reduce the amount of bound antibody by 50%, shown in 

Figure 5. Adjuvant or antigen dose did not significantly influence the average 

avidity to the AMA1 variants under investigation (p=0.169, p=0.725, p=0.864 and 

p=0.609 for FVO, 3D7, HB3 and CAMP, respectively). The average avidity was 

0.694 M NaSCN (95% CI 0.660 to 0.725) for the homologous FVO AMA1. Average 

avidities for the heterologous antigens were: 0.609 M (95% CI 0.542 to 0.676), 

IgG4 

69

70 Chapter 3 

0.711 M (95% CI 0.602 to 0.820) and 0.929 M (95% CI 0.775 to 1.083) for 3D7, 

HB3 and CAMP AMA1, respectively. Antibody avidity to the 3D7 AMA1 was 

significantly lower than to the homologous FVO AMA1 (0.085 M NaSCN, 95% CI 

0.025 to 0.1435, p = 0.006, paired t-test). By contrast, antibody avidity to the 

CAMP AMA1 was significantly higher than to the homologous FVO AMA1 (0.235 

M NaSCN, 95% CI 0.0885 to 0.3821, p = 0.002, paired t-test). Antibody avidities 

to the HB3 allele were similar to those observed for the FVO allele (p = 0.742, 

paired t-test). 

Subclass distribution 

IgG subclass levels to the homologous FVO AMA1 allele determined in plasma 

samples taken on day 84 are shown in Figure 6. The ranking of IgG subclasses in 

descending titre order was IgG1 > IgG3 ≈ IgG4 > IgG2. IgG1 and IgG3 antibody 

levels showed a similar pattern as to what was observed for total IgG, with 

lowest titres in the Alhydrogel groups (10 and 50 µg) and in the Montanide ISA 

720 10 µg group as compared to the Montanide ISA 720 50 µg and both AS02 

groups (Figure 6). Interestingly, titre differences between the treatment arms 

were slightly higher for IgG3 as compared to IgG1 (Figure 6). 

GIA 

The results of the Growth Inhibition Assay (GIA) for three laboratory strains 

obtained with IgG fractions purified from plasma samples collected at day 84 are 

shown in Figure 7. GIA titres determined on the FCR3 laboratory strain 

expressing an AMA1 similar to the vaccine antigen were below 20% in most 

(22/27) subjects in the Alhydrogel (10 and 50 µg) and Montanide ISA 720 10 µg 

groups; only 5 out of 27 subjects had GIA titres higher than 20% at 10 mg/ ml 

total IgG. By contrast, 14 out of 20 subjects in the Montanide ISA 720 (50 µg) 

and AS02 (10 and 50 µg) groups had GIA titres exceeding 20% at 10 mg /ml total 

IgG (Figure 7). Average GIA responses were significantly higher (38.7 %-points, 

95% CI 4.7 to 72.6%, p = 0.018, Tukey HSD) in the AS02 10 µg group as compared 

to the Alhydrogel 10 µg group. The GIA values at 10 mg /ml total IgG in 

Montanide ISA 720 and AS02 50 µg groups were also higher than the Alhydrogel 

10 µg group, but this failed to achieve statistical significance (p=0.17 and 0.20, 

respectively, Tukey HSD, Figure 7). The GIA titres determined on the FCR3 strain 

at 5 mg/ ml total IgG were lower and showed a ranking similar to what was 

observed at 10 mg/ ml, but none of the between group comparisons reached 

statistical significance (p=0.070, ANOVA, Figure 7).


adults. 

Growth Inhibition (%) 

80 

60 

40 

20 

0 

−20 

FCR3 

400 

800 

1600 

3200 

6400 

12800 

25600 

51200 

102400 

3D7 

400 

800 

1600 

3200 

6400 

12800 

25600 

51200 

AMA1 IgG (AU/mL) 

102400 

400 

800 

1600 

3200 

6400 

12800 

25600 

51200 

102400 

Figure 8. Relation between AMA1-specific IgG titre and GIA titre for the 

homologous (FCR3) and two heterologous strains (3D7 and HB3). The GIA value 

for the designated strain measured at 10 mg /mL total IgG is depicted on the yaxis 

and the corresponding Elisa titre to the corresponding AMA1 allele is 

depicted on the x-axis. Estimated IC50 values are indicated by dashed lines. 

Symbols represent treatment groups: white circles = Alhydrogel 10 µg, black 

circles = Alhydrogel 50 µg, white squares = Montanide ISA 720 10 µg, black 

squares = Montanide ISA 720 10 µg, white diamonds = AS02 10 µg and black 

diamonds = AS02 50 µg, where the 10 or 50 µg refers to the AMA1 dose. 

GIA titres determined on the 3D7 laboratory strain at 10 mg/ml IgG were lower 

than those to the FCR3 strain at the same total IgG concentration and no 

significant differences were observed between the treatment groups (p=0.095, 

ANOVA). Interestingly, GIA titres on the 3D7 strain at 10 mg/ml were similar to 

GIA titres to the homologous FCR3 strain at 5 mg/ml total IgG (Figure 7). GIA 

titres on the 3D7 laboratory strain at 5 mg/ml IgG were low; only 2 out of 47 

subjects had GIA titres higher than 20% (Figure 7). 

GIA titres determined on the HB3 laboratory strain at 10 mg /ml IgG were lower 

than to the FCR3 strain and similar to the titres observed for the 3D7 strain. 

Significant treatment group differences were observed (p=0.004, ANOVA). GIA 

titres in the AS02 10 µg group were significantly higher than in the Alhydrogel 10 

µg group (24.6 %-points, 95% CI 3.4 to 45.8%, p = 0.014, Tukey HSD) and GIA 

titres in the AS02 10 µg group tended to be higher as compared to the 

Alhydrogel 50 µg group (20.5%-points, 95% CI -0.2 to 41.3%, p = 0.053, Tukey 

HB3 

71

72 Chapter 3 

GIA (%) 

80 

60 

40 

20 

0 

−20 

80 

60 

40 

20 

0 

−20 

Alhydrogel 10 

Alhydrogel 50 

10 

FCR3 

5 

FCR3 

ISA 720 10 

ISA 720 50 

AS02 10 

AS02 50 

Alhydrogel 10 

Alhydrogel 50 

10 

3D7 

5 

3D7 

ISA 720 10 

ISA 720 50 

Treatment 

AS02 10 

AS02 50 

Alhydrogel 10 

Alhydrogel 50 

10 

HB3 

5 

HB3 

. Figure 7. GIA to 3 laboratory strains (FCR3, homologous and 3D7 and HB3, 

heterologous) at 10 and 5 mg /ml total IgG obtained at day 84. Top row: GIA at 

10 mg /ml and bottom row GIA at 5 mg /ml IgG. Boxes indicate median and 

quartile ranges. Treatment groups are indicated by Adjuvant name and AMA1 

dose, respectively 

ISA 720 10 

ISA 720 50 

AS02 10 

AS02 50


adults. 

HSD) and the Montanide ISA 720 10 µg group (21.2%-points, 95% CI -0.6 to 

42.9%, p = 0.061, Tukey HSD). GIA titres on the HB3 laboratory strain at 5 mg /ml 

total IgG were low, with only 4 out of 47 subjects having GIA titres of over 20%. 

GIA titres on the HB3 laboratory strain at 5 mg /ml IgG in the AS02 10 µg group 

tended to be higher, similar to what was observed at 10 mg ml total IgG, but this 

failed to reach statistical significance (p=0.091, ANOVA). 

Relation between IgG and GIA titres 

The relation between AMA1 variant-specific IgG and GIA was investigated by 

modelling sigmoid curves using non-linear least squares regression. The data are 

graphically presented in Figure 8. The correlation coefficients were 0.911 (95% 

CI 0.845 to 0.950, p < 0.0001) for FCR3, 0.795 (95% CI 0.658 to 0.8810, p < 

0.0001) for 3D7 and 0.794 (95% CI 0.657 to 0.881, p < 0.0001) for HB3. The IC50 

values, i.e. the amount of AMA1 specific IgG required for 50% inhibition were 

estimated at 31.3 kAU/ ml (95% CI 27.7 to 35.4) for FCR3, 39.8 kAU/ ml (95% CI 

30.8 to 51.3) for 3D7 and 38.9 kAU/ ml (95% CI 28.9 to 52.5) for HB3 (Figure 8), 

which was not significantly different from the value for the FCR3 strain (p = 0.08 

and 0.20, respectively). The fold-increase in AMA1 variant specific IgG required 

to raise the GIA titre from 10 to 50% or from 50 to 90% was 3.5-fold (95% CI 2.7 

to 5.0) for FCR3, 2.9-fold (95% CI 2.2 to 5.7) for 3D7 and 4.5-fold (95% CI 3.1 to 

9.5) for HB3, the values for the 3D7 and HB3 strains were not significantly 

different from the value for the FCR3 strain (p = 0.50 and p = 0.33, respectively). 

Discussion 

The main finding of this study is that AMA1 antibody responses induced by 

vaccination of malaria-naïve human volunteers with a single AMA1 variant are 

biased towards the vaccine allele. This is in agreement with previously published 

results from a similar human Phase I vaccine trial [22] and observations in 

rabbits [14,16]. Approximately half of the IgG induced by vaccination of human 

volunteers reacts in ELISA with heterologous AMA1 variants, similar to what has 

been observed in rabbits [14,16]. The functionality of the antibody response 

(GIA titre) to heterologous AMA1 expressing strains reflects differences in the 

quantity of IgG; suggesting that, although absolute antibody levels to 

heterologous AMA1 variants are lower, these antibodies are, on per mass basis, 

equally capable of inhibiting parasite growth. 

73

74 Chapter 3 

The data presented here also demonstrate that both adjuvant and dose can 

have a profound impact on antibody levels, while other aspects of the antibody 

response like avidity, domain recognition, breadth and subclass distribution, are 

much less influenced by adjuvant or dose. This is, with exception of the subclass 

distribution, in agreement with what has been observed in rabbit studies [23]. 

The data presented here show that the widely used adjuvant Alhydrogel yields 

relatively low antibody levels, and increasing the antigen dose from 10 to 50 µg 

only marginally improved antibody responses. The data confirm that Alhydrogel 

is not potent enough to induce high levels of functional antibodies in malaria 

naïve subjects, as was previously reported [24,25]. The low potency of 

Alhydrogel at inducing a functional response is even more pronounced when the 

functionality of the antibody response is evaluated on strains expressing 

heterologous AMA1 alleles. Therefore adjuvants more potent than Alhydrogel 

are definitively required for the induction of functional antibody responses to 

AMA1 variants not included in a vaccine. Compared to Alhydrogel, the water in 

oil emulsion Montanide ISA 720, appeared to yield improved antibody responses 

when combined with a 50 µg AMA1 dose, but also gave increased numbers of 

adverse events [19]. Moreover, Montanide ISA 720 can chemically modify the 

vaccine antigen [26], potentially resulting in loss of potency and thus vaccine 

failure [27]. The proprietary adjuvant AS02 yielded relatively high (functional) 

antibody responses at both antigen doses tested, in agreement with previously 

published results [22, 28]. 

Recently, vaccination induced efficacy against the AMA1 vaccine allele 

formulated with AS02 in a Phase IIb study was estimated at 64%, whereas 

overall vaccine efficacy was estimated at 17%, indicating the importance of 

covering AMA1 variants [18]. Thus, the challenge for an AMA1-based vaccine 

appears to be with covering AMA1 polymorphism. Theoretically, two options 

would be available: i) The induction of a broad antibody response and ii) The 

maximisation of heterologous antibody responses by using potent adjuvants or 

prime boost strategies combined with a potent adjuvant, such that the response 

induced with a single variant would be high enough to also be sufficiently 

functional against heterologous strains. As the latter may not be possible with 

adjuvants currently available, a more practical approach would be the 

combination of the induction of the broadest response possible with the highest 

response possible.


adults. 

The breadth of the antibody response can be improved by vaccination with a 

mixture of several AMA1 variants, either naturally occurring [14, 16, 23, 29], or 

artificial ones [10, 29]. Three artificial diversity covering (DiCo) AMA1 sequences 

[10] have recently been produced under cGMP and will enter clinical testing in 

the near future. 

The magnitude of the antibody response can be improved by the use of potent 

adjuvants, as shown here. A novel proprietary adjuvant, CoVaccine HT, has 

yielded promising results in rhesus macaques and rabbits [23, 30]. Moreover, P. 

knowlesi AMA1 formulated with CoVaccine HT has induced protection against 

blood-stage challenge with P. knowlesi in the rhesus macaque model and the 

degree of protection was correlated with GIA titre [31]. It would therefore be 

interesting to combine DiCo AMA1 with CoVaccine HT in a Phase I trial. 

Antibody avidity was determined with a sodium thiocyanate elution ELISA and 

average antibody avidities ranged between 0.6 and 0.9 M for the various 

antigens. These values are lower than what was previously observed in rabbits 

(0.9 to 1.4 M) [16]. Of note is that the average avidity to the heterologous CAMP 

AMA1 was higher than the homologous antigen. This could possibly be explained 

by the fact that half of FVO AMA1-specific antibodies bound to CAMP AMA1 and 

that this fraction may bind with higher avidity. Conversely, avidities to the 

heterologous 3D7 AMA1 were lower than the homologous avidities. This may 

represent antigenic relatedness of the respective AMA1 molecules, with CAMP 

being more close to FVO AMA1 and 3D7 more distant. 

The antibody response to AMA1 appears to be mainly directed against domains I 

and II, as competition with a construct comprising these domains removes about 

80% of antibodies bound. Competition with a domain II-III construct removes a 

similar amount of antibodies as the I-II construct, suggesting that the majority of 

the response would be directed against domain II. This is, however, not 

supported by the depletion obtained by a domain II construct. Of note here is 

that only constructs including domains I-II induce functionally active antibodies 

in rabbits [32, 33], suggesting conformational authenticity. The Domain II-III and 

the domain II constructs both failed to elicit functionally active antibodies in 

rabbits [32, 33]. The results obtained here warrant the statement that the 

majority of the antibody response to AMA1 is directed against domains I and II 

and a further subdivision for these two domains is not possible with the data 

hitherto obtained. The importance of domains I and II in the antibody response 

is in agreement with what has been found in rabbits [32,33]. 

75

76 Chapter 3 

The subclass distribution found in vaccinated malaria naive volunteers was 

similar to what was observed in exposed children [34, 35] and reflects the 

expected subclass distribution for a protein antigen [36]. Antigen dose or 

adjuvant only marginally influenced the subclass distribution. 

In conclusion, vaccination with a single allele AMA1 vaccine induces a humoral 

immune response that is biased towards the vaccine allele. The magnitude of 

the response can be enhanced by a potent adjuvant, in contrast other 

parameters of the humoral response like breadth, avidity and subclass 

distribution appear much less influenced by the adjuvant. Future vaccine 

development should focus on improving both breadth and magnitude of 

antibody responses to AMA1. 


The authors wish to thank Glaxo Smith Kline, Rixensart, Belgium for supplying 

AS02 and SEPPIC, Paris, France for supplying Montanide ISA 720. 

Funding 


Initiative.


adults. 

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responses to Plasmodium falciparum blood-stage antigens and association with 

incidence of clinical malaria in children living in an area of seasonal malaria 

transmission in Burkina Faso, West Africa. Infect Immun 2008; 76:759-766. 

36. Hammarstrom L, Smith CIE. IgG subclass changes in response to vaccination. In: 

Skakib F, editors. Monographs in allergy (vol 19): Basic and clinical aspects of 

IgG subclasses. Basel: Karcher. 1986; pp. 241-251. 

79

Chapter 4 

A saturation of antibody avidity and 

concentration induced by malaria vaccine 

candidate Apical Membrane Antigen 1 

Meta Roestenberg 1 , Ed Remarque 2 , Bart Faber 2 , Clemens Kocken 2 , Alan 

Thomas 2 , Cornelus C. Hermsen 1 , Will Roeffen 1 , Robert W. Sauerwein 1 

1 Radboud University Nijmegen Medical Centre, department of Medical 

Microbiology 268, P.O Box 9101, 6500 HB, Nijmegen, The Netherlands 

2 Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ, Rijswijk, The 

Netherlands 

Manuscript in preparation

Chapter 4 82 

Abstract 

The functional activity of antibodies is determined by their quantity and quality. 

Antibody avidity describes the quality of antigen-antibody binding, but this 

parameter has received only limited attention in vaccine design. The asexual 

erythrocytic stage antigen Apical Membrane Antigen 1 (AMA1) is a potential 

Plasmodium falciparum vaccine candidate because of its critical role in red blood 

cell invasion. AMA1 induces antibody-mediated protection against infection in 

animals by blocking invasion of the parasite. Assuming a simple 1:1 interaction 

model of immunoglobulin with antigen, we determined anti-AMA1 antibody 

concentration and avidity in rabbit and human sera post-vaccination by Surface 

Plasmon Resonance (SPR) technology. We show that anti-AMA1 antibody 

concentration and avidity are inversely correlated and become saturated at a 

maximum fraction of bound antigen. The matured AMA1 antibody response, 

whether induced through vaccination or natural exposure, thus always has a 

maximum saturated, constant capacity to bind AMA1 antigen, although there is 

some interindividual variation in the balance between concentration and avidity. 

The functionality of these antibodies in vitro was tested by the parasite growth 

inhibition assay. Growth inhibition in vitro correlated with antibody 

concentration, but not with avidity. The relevance of these findings in vivo 

warrants further research, as the appreciation of a saturated antibody response 

may guide AMA1 vaccine development.

A saturation of antibody avidity and concentration induced by malaria vaccine 


Introduction 

The functional activity of antibodies depends on epitope specificity, titre and 

affinity. The avidity of antibodies defines the sum of the individual affinities 

between the antibody binding sites and single epitopes [1, 2] and is an 

important parameter defining antibody quality. Both concentration and avidity 

of antibodies should be addressed in vaccines that depend on humoral 

responses to convey protection. One candidate immunogen is Plasmodium 

falciparum Apical Membrane Antigen 1 (PfAMA1). Located at the surface of 

blood-stage malaria parasites, AMA1 plays an essential role in the process of red 

blood cell invasion [3-5]. Functional antibody binding has been shown to prevent 

parasite invasion [6], protecting mice and monkeys against parasite 

multiplication and disease [7]. The full-length ectodomain recombinant PfAMA1 

(amino acids 25-545) of the Plasmodium falciparum FVO strain has been 

expressed under cGCP in Pichia pastoris [8]. The product was formulated with 

Alhydrogel, Montanide ISA 720 and AS02A and tested for safety and 

immunogenicity in humans [9]. We investigated the concentration and avidity of 

antibodies induced by recombinant AMA1 in rabbits and human immunizations 

studies by Surface Plasmon Resonance. We correlated these data with the 

capacity of sera to functionally inhibit parasite growth in vitro. 


Antigens 

Six different recombinant PfAMA1 constructs were prepared as described 

previously [10]. Pf3mH comprises the prosequence, subdomain I and subdomain 

II, Pf8mH comprises subdomain II only, Pf9mH comprises subdomain II and 

subdomain III and Pf10mH comprises subdomain III only, covering amino-acid 

residues 25-442, 303-442, 303-544 and 419-544 of the FVO AMA1 antigen 

respectively (Figure 1). The Pf4mH construct comprises the prosequence, 

subdomain I, subdomain II and subdomain III, representing residues 25 to 544 

from the Pf FVO strain as described by Kocken et al. [11]. Construct Pf11.0 

comprises amino-acid residues 25-545, but lacks the additional Myc-His tag 

(Figure 1). Based on rabbit immunization studies, the latter construct was used 

for a human phase I trial [9]. 

83

84 Chapter 4 

Figure 1. Schematic representation of the different AMA1 antigen 

constructs used for the Rabbit immunizations. Construct Pf11.0 was 

used for the Phase I clinical trial. 

Sera 

Immunizations of rabbits were performed as described previously [11]. Briefly, 

injection of 100 μg of purified FVO strain AMA1 antigen with Freund's complete 

adjuvant was followed by three booster injections of 100 μg AMA1 at days 14, 

28, and 56 with Freund's incomplete adjuvant. Rabbits were immunized with the 

different AMA1 constructs shown in Figure 1. A total number of 18 rabbits were 

immunized, with minimum sets of 2 rabbits receiving the same recombinant 

antigen. Antisera obtained 4 weeks after the last injection were used for 

antibody evaluation. Sera from rabbits immunized with the same antigen were 

pooled to obtain enough material for kinetic analysis. Pre-immunization sera 

were used as a negative control. 

Human sera were obtained from a previously published phase I trial [9]. In short, 

healthy malaria-naïve Dutch volunteers were immunized with either 10 or 50µg 

of PfAMA1-FVO [25-545] adjuvanted with either Alhydrogel TM , Montanide ISA 

720 (SEPPIC, Paris, France) or AS02A (a proprietary Adjuvant System from 

GlaxoSmithKline Biologicals) at three occasions with monthly intervals. Sera 

were obtained one month after the third immunisation. For SPR analysis 

immunoglobulins (Ig) from a random selection of six human sera from every trial 

arm (18 total) were purified according to manufacturer’s instructions using 

HiTrap TM MabSelect SuRe TM (GE Healthcare, Diegem, Belgium) and concentrated 

to their original volume by Vivaspin® 20 (GE Healthcare, Diegem, Belgium). Sera 

from ten volunteers living in malaria endemic areas in Burkina Faso, Came-



Figure 2. Example of a BIAcore sensorgram. The sensorgram shows 

immobilization of Pf4mH starting at 1, injection of a serum sample at 2 

and initiation of regeneration with glycine pH2.0 at 3. 

roon, Gambia, Madagascar, Senegal and Tanzania, previously obtained for other 

purposes, were pooled for analysis. 

Surface Plasmon Resonance (SPR) analysis 

A Biacore TM 2000 (Biacore International SA, Uppsala, Sweden) was used for SPR 

measurements at 25°C using CM5 chips (Carboxylmethylated dextrane, Biacore 

International SA, Uppsala, Sweden). Rabbit anti-myc antibodies (PA1-981 Affinity 

BioReagents, Golden USA) were immobilized until approximately 13,000 

resonance units (RU) using standard amine coupling in 10 mM acetate pH 5.0. 

according to manufacturer’s instructions, after which the immobilized surface 

was washed with injections of 50 mM HCl. All subsequent measurements on the 

biosensor were performed in duplicate. Pre-immunization sera were used as a 

negative control. Correction for non-specific binding was performed by 

subtraction of the control channel. 

Chips were coated with approximately 350 RU Pf4mH construct. Samples were 

serially diluted ranging from 1:25 to 1:800, and 25 µl of diluted sample were 

injected at 5 µl/min. Measurement of antibody concentrations was performed 

by evaluating the association rate 25 seconds after initiation injection, 

measuring the concentration of binding antibodies only. Regeneration of the 

anti-myc antibody chip surface was performed with 50 µl glycine pH 2.0 at a flow 

rate of 30µl/min. Calibration curves were prepared with the rat monoclonal 

antibody 4G2 [12]. 

For analysis of antibody avidity, Pf4mH was injected at 20 µl/min for 1.5 minute 

in the experimental flow cells to obtain a total immobilization level of approx 

85

86 Chapter 4 

Concentration 

(mg/ml, range) 

Rabbit 1.31 (0.58 – 2.98) 

Human 

0.066 (0.007 - 0.50) 

Mean ka 

(1/Ms, range) 

1.26x10 5 

(9.1x10 3 – 3.7x10 5 ) 

1.50 x 10 5 

(1.19x10 4 – 6.22 x 10 5 ) 

Mean kd 

(1/s, range) 

2.80x10 -3 

(1.10 x10 -3 – 5.53 x10 -3 ) 

2.65 x 10 -3 

(2.41 – 9.45 x 10 -3 ) 

Table 1. Mean antibody concentration, association constant (ka) and 

dissociation constant (kd) for rabbit (n=38) and human (n=72) sera. 

1/Ms = per Molar per second, 1/s = per second. 

150 RU. The abovementioned dilution series of samples was injected at 30 

µl/min. An example of a Biacore sensorgram is provided in figure 2. The 

association rate constant (ka) and the dissociation rate constant (kd) were 

obtained by fitting the sensorgrams with the use of BIAevaluation software 3.2 

(Biacore International SA, Uppsala, Sweden). Curves were fitted according to a 

simple 1:1 Langmuir binding model, with correction for a linear drifting baseline 

to adjust for slow dissociation of the Pf4mH coat from anti-myc antibodies. 

Rabbit sera and human sera were each measured using one chip. 

Growth inhibition assay 

Antibodies used for parasite inhibition assays were purified on protein A 

columns (Immunopure Plus Pierce, St Louis, MO, USA) using standard protocols, 


Millipore, Ireland), filter-sterilised and stored at -20 o C until use. IgG 


(Nanodrop Technologies, Wilmington, DE, USA). 

Pf strain FCR3 was cultured in vitro using standard Pf culture techniques in an 

atmosphere of 5% CO2, 5% O2 and 90% N2. FCR3 AMA1 (accession no. M34553) 

differs by one amino acid in the pro-sequence from FVO AMA1 (accession no. 

AJ277646). 

The effect of purified IgG antibodies on parasite invasion was evaluated in 

triplicate using 96-well flat-bottom plates (Greiner Bio-One, Alphen a/d Rijn, The 

Netherlands) with synchronized cultures of Pf schizonts at a starting parasitemia 

of 0.2-0.4% and a haematocrit of 2.0% in a final volume of 100 µL containing 

10% control non-immune human serum, 20 µg /ml gentamicin in RPMI 1640 and 

5 mg/mL purified IgG. After 40 to 42 hours, cultures were resuspended, and 50 

µL was transferred into 200 µL ice-cold PBS. The cultures were then centrifuged, 

the supernatant removed and the plates were frozen. Inhibition of parasite 

growth was estimated using the pLDH assay as previously described [13].



Figure 3: AMA1 antibody concentration and association constant (ka) of pooled rabbit 

sera (A) or human sera (B). Dashed lines show inverse curve fit of data in panel A. 

A: Inverse correlation between concentration and ka of AMA1 antibodies (clear circles) 

and concentration and ka of antibodies measured against heterologous AMA1 sequences 

from strain 3D7 (grey) or HB3 (black) 

B: Inverse correlation between concentration and ka of AMA1 antibodies in naturally 

exposed semi-immunes (stars) and concentration and ka of AMA1 antibodies in human 

volunteers post-vaccination with FVO PfAMA1 [25-545] vaccine (construct Pf11.0) 

adjuvanted by Alhydrogel® (white), Montanide ISA 720 (grey) or AS02A (black). 

Parasite growth inhibition was calculated based on optical density (OD) 

measurements as follows: 100 - ((ODexperimental - ODbackground)/ (ODcontrol - 

ODbackground) x 100). IgG purified from pre- immunization plasma was used as a 

control and culture medium was used to measure the background OD. 

Statistical Analysis 

Statistical fitting of interaction kinetics was performed using BIAevaluation 

software 3.2, whereby the closeness of fit is represented by the statistical value 

chi2. Chi2 reduces to the average squared residual per data point. All other 

statistical analyses were performed in SPSS 16.0 (SPSS Inc.). For comparison 

between means of independent data sets the Kruskall-Wallis was used for three 

or more groups, the Mann-Whitney U for two independent groups and Wilcoxon 

singed ranks for paired samples. Non-parametric Spearman analyses were used 

to describe correlations. Curve fits were assessed by ANOVA. All statistical 

analysis were two-tailed and p values less than 0.05 were considered significant. 

Results 

Sera obtained from rabbit immunization studies and purified immunoglobulins 

(Ig) obtained from volunteers in a phase I clinical trial were tested for their 

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88 Chapter 4 

Figure 4: Correlation between residue number of the vaccine construct and AMA1 

antibody concentration (A) or association constant ka (B) post-vaccination in rabbit sera. 

binding kinetics to the full length homologous ectodomain of recombinant 

PfAMA1 by Surface Plasmon Resonance (construct Pf4mH, Figure 1). Association 

constants (ka) and dissociation constants (kd) of immunoglobulin-antigen (IgAg) 

interactions were determined separately by fitting kinetic curves to a simple 1:1 

Langmuir binding model. Fits to the model showed an average chi2 of 2.98 for 

rabbit samples (n=38 measurements) and 0.85 for human samples (n=72 

measurements). Two rabbit samples could not be fitted with the model and 

were excluded from analyses. Mean ka and kd values of all other measurements 

are shown in Table 1. Kinetic constants of rabbit and human samples covered a 

similar range. Values for kd were considerably smaller than for ka (~10 8 fold 

difference), reflecting a stable antigen-antibody complex. 

Antibody concentration (of binding antibodies) and ka values of pooled rabbit 

sera were inversely correlation (Figure 3A), approximated by inverse curve fit 

(p=0.002, R 2 = 0.871). The implication of these results are best illustrated by 

analysis of a simple non-covalent interaction system, whereby the increase in 

IgAg over time (δ[IgAg]/ δt) is described by concentration of immunoglobulin 

[Ig], antigen [Ag] and ka and kd as follows: δ[IgAg]/ δt = ka*[Ig]*[Ag] – 

kd*[IgAg]. 

In a state of equilibrium, when the increase in immunoglobulin-antigen complex 

over time is zero, concentration and binding constants are balanced 

(ka*[Ig]*[Ag] = kd*[IgAg]). Algebraic rearrangement will result in ka/kd*[Ig] = 

[IgAg]/[Ag], where [IgAg]/[Ag] represents the fraction of antigen bound by 

antibody. Assuming kd is negligible, the observed inverse relationship in rabbit 

sera (Figure 3A) thus suggests that the fraction of bound antigen is constant and 

identical for all investigated sera.



Figure 5. Correlation between in vitro growth inhibition and AMA1 antibody 

concentration (A) or association constant ka (B) in human sera post-vaccination with 

FVO PfAMA1 [25-545] vaccine (construct Pf11.0) adjuvanted by Alhydrogel® (white), 

Montanide ISA 720 (grey) or AS02A (black). 

The balance between concentration and ka was influenced by the vaccine 

construct, whereby the number of amino-acid residues of the antigen used for 

immunization was positively correlated with the induced concentration of 

antibodies and inversely related to ka (p=0.007 R 2 =0.89 and p=0.003, R 2 =-0.92 

respectively using Pf4mH for all measurements, Figure 4). PfAMA1-FVO 

subdomain I, being the largest subdomain, was the major contributor to this 

effect and immunizations using antigen constructs spanning the entire domain 

typically induced high concentrations and lower avidity antibodies (p=0.03 for 

both parameters). When the same sera were tested against PfAMA1 antigen 

from heterologous strains 3D7 and HB3, the total fraction of bound antigen was 

generally lower (Figure 3A). Antibodies binding the 3D7 strain PfAMA1 showed a 

(non-significant) trend towards lower ka, corresponding with an increased 

number of discordant residues (FVO vs 3D7 25 residues, FVO vs HB3 18 residues 

[14]). 

PfAMA1 antibodies from subjects participating in a phase I vaccine trial also 

adhered to the inverse relation similar to rabbit sera. Of note, a number of 

samples, particularly from the Alhydrogel TM adjuvanted group, showed a lower 

total fraction of bound antigen and did not follow this inverse correlation (Figure 

3B). We additionally studied human AMA1 antibodies from highly-exposed 

individuals living in malaria endemic areas and again found a similar inverse 

relationship between concentration and antibody avidity (Figure 3B). 

Finally, human post-immunization sera were tested for their capacity to inhibit 

asexual parasite growth in vitro. Growth inhibiting capacity of antibodies 

89

90 Chapter 4 

positively correlated with anti-AMA1 antibody concentration but not with the 

mean ka (p



thus establish an equilibrium depending on their competition for common 

resources, of which antigen is only one [17]. The availability of other resources, 

such as growth factors, interleukins and the presence of adhesion molecules 

may provide critical advantage preserving diversity and preventing dominance of 

one B-cell pool [18]. This is an interesting hypothesis, because it suggests that 

possible addition of resources to the antigen (such as cytokine adjuvants) would 

offer an opportunity to increase the specific B-cell population beyond the 

current maximum. 

Few sera did not reach the saturated level, particularly in the Alhydrogel® 

adjuvanted group. We hypothesize that the antibody response in these sera may 

not have undergone full maturation and have not (yet) reached a maximum 

fraction of bound antigen. Similarly, allergen-specific IgE from atopic children 

also lacked a negative relationship between concentration and avidity [19], 

whereas this was apparent for the more matured antibody responses in adults 

[16]. 

These data are particularly relevant for vaccine development, since the avidity of 

antibodies may determine susceptibility to diseases such as the Respiratory 

Syncytial virus or meningococcal disease [20-22]. For AMA1 antibodies, we 

found a relation between in vitro parasite growth inhibition and antibody 

concentration, but not avidity. Caution should be taken, however, when 

concluding that the functionality of AMA1 antibodies in vitro seems to be 

governed primarily by concentration of antibodies, as epitope functionality has 

not been taken into account in this study. Particularly PfAMA1 domain I and 

domain II antibodies are thought to harbour the major targets for inhibitory 

responses [7]. Moreover, the results of in vitro growth inhibition so far do not 

correlate with in vivo susceptibility [23]. 

In conclusion, our data illustrate an inverse relation between concentration and 

avidity, that becomes saturated at a maximum, constant capacity to bind 

antigen. Any increase in antibody concentration at this saturated level will be 

balanced by a decrease in antibody avidity. The appreciation of a saturated 

antibody response may be crucial for the future development of PfAMA1 

vaccine, since it provides guidance to the search for more effective antigens, 

adjuvants or delivery platforms. 

91

92 Chapter 4 


The authors wish to thank GlaxoSmithKline Biologicals, Rixensart, Belgium for 

supplying AS02A and SEPPIC, Paris, France for supplying Montanide ISA 720. 

Funding 


Initiative.



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thiocyanate elution: a simple dose-response approach. J Immunol Methods 

1995; 187:297-305. 

2. Kaattari SL, Zhang HL, Khor IW, Kaattari IM, Shapiro DA. Affinity maturation in 

trout: clonal dominance of high affinity antibodies late in the immune response. 

Dev Comp Immunol 2002; 26:191-200. 




72:154-8. 

4. Dutta S, Haynes JD, Barbosa A, et al. Mode of Action of Invasion-Inhibitory 

Antibodies Directed against Apical Membrane Antigen 1 of Plasmodium 

falciparum. Infect Immun 2005; 73:2116-22. 

5. Thomas AW, Deans JA, Mitchell GH, Alderson T, Cohen S. The Fab fragments of 

monoclonal IgG to a merozoite surface antigen inhibit Plasmodium knowlesi 

invasion of erythrocytes. Mol Biochem Parasitol 1984; 13:187-99. 

6. Treeck M, Tamborrini M, Daubenberger CA, Gilberger TW, Voss TS. Caught in 

action: mechanistic insights into antibody-mediated inhibition of Plasmodium 

merozoite invasion. Trends Parasitol 2009; 25:494-7. 



8. Faber BW, Remarque EJ, Kocken CH, et al. Production, quality control, stability 

and pharmacotoxicity of cGMP-produced Plasmodium falciparum AMA1 FVO 

strain ectodomain expressed in Pichia pastoris. Vaccine 2008; 26:6143-50. 

9. Roestenberg M, Remarque E, de JE, et al. Safety and immunogenicity of a 

recombinant Plasmodium falciparum AMA1 malaria vaccine adjuvanted with 

Alhydrogel, Montanide ISA 720 or AS02. PLoS One 2008; 3:e3960. 

10. Polley SD, Mwangi T, Kocken CH, et al. Human antibodies to recombinant 

protein constructs of Plasmodium falciparum Apical Membrane Antigen 1 

(AMA1) and their associations with protection from malaria. Vaccine 2004; 

23:718-28. 

11. Kocken CH, Withers-Martinez C, Dubbeld MA, et al. High-level expression of the 

malaria blood-stage vaccine candidate Plasmodium falciparum apical 

membrane antigen 1 and induction of antibodies that inhibit erythrocyte 

invasion. Infect Immun 2002; 70:4471-6. 

12. Kocken CH, van der Wel AM, Dubbeld MA, et al. Precise timing of expression of 

a Plasmodium falciparum-derived transgene in Plasmodium berghei is a critical 

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determinant of subsequent subcellular localization. J Biol Chem 1998; 

273:15119-24. 

13. Amador R, Moreno A, Valero V, et al. The first field trials of the chemically 

synthesized malaria vaccine SPf66: safety, immunogenicity and protectivity. 

Vaccine 1992; 10:179-84. 




Infect Immun 2008; 76:2660-70. 

15. de Voer RM, van der Klis FR, Schepp RM, Rijkers GT, Sanders EA, Berbers GA. 

Age-Related Immunity to Meningococcal Serogroup C Vaccination: An Increase 

in the Persistence of IgG2 Correlates with a Decrease in the Avidity of IgG. PLoS 

One 2011; 6:e23497. 

16. Pierson-Mullany LK, Jackola DR, Blumenthal MN, Rosenberg A. Evidence of an 

affinity threshold for IgE-allergen binding in the percutaneous skin test reaction. 

Clin Exp Allergy 2002; 32:107-16. 

17. McLean AR, Rosado MM, Agenes F, Vasconcellos R, Freitas AA. Resource 

competition as a mechanism for B cell homeostasis. Proc Natl Acad Sci U S A 

1997; 94:5792-7. 

18. Freitas AA, Rosado MM, Viale AC, Grandien A. The role of cellular competition 

in B cell survival and selection of B cell repertoires. Eur J Immunol 1995; 

25:1729-38. 

19. Jackola DR, Liebeler CL, Lin CY, Chiu YK, Blumenthal MN, Rosenberg A. Evidence 

that negative feedback between antibody concentration and affinity regulates 

humoral response consolidation to a non-infectious antigen in infants. Mol 

Immunol 2005; 42:19-30. 

20. Freitas GR, Silva DA, Yokosawa J, et al. Antibody response and avidity of 

respiratory syncytial virus-specific total IgG, IgG1, and IgG3 in young children. J 

Med Virol 2011; 83:1826-33. 

21. Granoff DM, Maslanka SE, Carlone GM, et al. A modified enzyme-linked 

immunosorbent assay for measurement of antibody responses to 

meningococcal C polysaccharide that correlate with bactericidal responses. Clin 

Diagn Lab Immunol 1998; 5:479-85. 

22. Delgado MF, Coviello S, Monsalvo AC, et al. Lack of antibody affinity maturation 

due to poor Toll-like receptor stimulation leads to enhanced respiratory 

syncytial virus disease. Nat Med 2009; 15:34-41. 

23. Spring MD, Cummings JF, Ockenhouse CF, et al. Phase 1/2a study of the malaria 


adjuvant system AS01B or AS02A. PLoS One 2009; 4:e5254.

Section 2 

Controlled human malaria infection model

Chapter 5 

Comparison of clinical and parasitological 

data from experimental human malaria 

challenge trials 

Meta Roestenberg 1 , Geraldine A. O’Hara 2 , Christopher J.A. Duncan 2 , Judith E. 

Epstein 4 , Nick J. Edwards 2 , Anja Scholzen 1 , André J.A.M. van der Ven 3 , Cornelus 

C. Hermsen 1 , Adrian V.S. Hill 2 , Robert W. Sauerwein 1 

1 Radboud University Nijmegen Medical Centre, Department of Medical 

Microbiology, Nijmegen, The Netherlands 

2 Centre for Clinical Vaccinology and Tropical Medicine and Jenner Institute, 

Churchill Hospital, University of Oxford. 

3Radboud University Nijmegen Medical Centre, Department of General 

Internal Medicine, Nijmegen, The Netherlands 

4Naval Medical Research Centre, US Military Malaria Vaccine Program, 

Maryland, United States 

PLoS ONE, 2012;7(6):e38434

Chapter 5 98 

Abstract 

Exposing healthy human volunteers to Plasmodium falciparum-infected 

mosquitoes is an accepted tool to evaluate preliminary efficacy of malaria 

vaccines. To accommodate the demand of the malaria vaccine pipeline, 

controlled infections are carried out in an increasing number of centres 

worldwide. We assessed their safety and reproducibility. 

We reviewed safety and parasitological data from 128 malaria-naïve subjects 

participating in controlled malaria infection trials conducted at the University of 

Oxford, UK, and the Radboud University Nijmegen Medical Centre, The 

Netherlands. Results were compared to a report from the US Military Malaria 

Vaccine Program. 

We show that controlled human malaria infection trials are safe and 

demonstrate a consistent safety profile with minor differences in the 

frequencies of arthralgia, fatigue, chills and fever between institutions. But 

prepatent periods show significant variation. Detailed analysis of Q-PCR data 

reveals highly synchronous blood stage parasite growth and multiplication rates. 

Procedural differences can lead to some variation in safety profile and parasite 

kinetics between institutions. Further harmonization and standardization of 

protocols will be useful for wider adoption of these cost-effective small-scale 

efficacy trials. Nevertheless, parasite growth rates are highly reproducible, 

illustrating the robustness of controlled infections as a valid tool for malaria 

vaccine development.

Comparison of clinical and parasitological data from experimental human malaria 


Introduction 

Deliberate exposure of healthy human volunteers to the bites of laboratoryreared 

Plasmodium falciparum (Pf)-infected mosquitoes in a controlled 

experimental setting is an accepted tool in malaria vaccine development. Such 

controlled human malaria infection (CHMI) trials can be used to investigate Pf 

immunology [1] or to provide data on the efficacy of malaria vaccine candidates 

[2] as a precursor to more costly and logistically challenging Phase IIb field 

efficacy trials. In CHMI, development of blood stage parasites in test subjects is 

assessed by blood smears at regular time points and anti-malarial treatment is 

given as soon as blood stage parasites are detected microscopically, keeping 

blood stage parasitemia low (treatment threshold: four parasites/µl) and 

confined to a short (two to eight day) period [3]. A comparison of the interval 

between exposure and parasite detection (prepatent period) among vaccinated 

and control subjects, together with sterile efficacy rates in vaccinees, provides 

an important efficacy estimate for the candidate vaccine. Because prepatent 

periods without information on parasite growth rates provide only an estimate 

of vaccine efficacy, molecular techniques have been developed to more 

accurately quantify blood parasites and provide parasite kinetic data [3-5]. 

Decades of extensive efforts to find an efficacious malaria vaccine have lead to 

the development of about 38 Pf candidate (sub-unit) malaria vaccines or vaccine 

components (www.who.int/vaccine_research/links/Rainbow/en/index.html). To 

meet the demands of the growing malaria vaccine development pipeline, CHMI 

will likely be conducted in an increasing number of sites worldwide. We have 

performed a comparative analysis of safety and parasitological data from trials 

performed at the Radboud University Nijmegen Medical Centre (RUNMC), The 

Netherlands, and the University of Oxford in the United Kingdom, two of a total 

of five different institutions and the only non-US institutions currently routinely 

performing CHMI. Where possible, data were compared with a previously 

published report from the US Military Malaria Vaccine Program (USMMVP), 

Naval Medical Research Centre Component, Silver Spring, Maryland [6]. Based 

on this data, we provide a perspective on future strengthening of and 

improvements to the CHMI model. 

99

100 Chapter 5 

Cohort RUNMC I RUNMC II Oxford USMMVP 

Number of volunteers 20 43 65 47 

Demographics 

Mean age (stdev) 29 (8.3) 22 (2.5) 27 (6.2) 27 (UNK) 

Sex (males) 10 14 32 27 

Immunized, non-protected volunteers 0 12 0 31 

Methodology 

Mosquito strain NF54 NF54 3D7 NF54 

Number of infected mosquitoes 4-7 5 5 5 

Exposure time to mosq. (min) 10 10 5 5 

Threshold microscopy (parasites/ul) 2 4 2 3 

Clinical follow-up frequency (times daily) 3 3 2 1 

Anti-malarial treatment 

Chloroquine 20 30 47 

Artemether/lumefantrine 33 35 

Atovaquone/proguanil 10 

Parasitological data 

Median prepatent period (days) 9,0 10,0 11,2 11,0 

Range prepatent period (days) 7.3-10.3 7.0-12.3 8.0-14.5 9.0-14.0 

Geometric mean peak parasitemia (Pf/ml) 7076 15901 9055 

Geometric mean parasitemia first cycle (Pf/ml) 567 456 48 

Geometric multiplication factor 11,8 11,1 11,6 

Laboratory safety parameters 

Mean platelet count day 7-10 (x10e9/l) 242 261 239 

References [7] [8-10] [11-17] [6] 

Table 1: Cohort characteristics. 

Methods 

Volunteers participating in CHMI studies performed at the RUNMC in Nijmegen, 

The Netherlands and the Centre for Clinical Vaccinology and Tropical Medicine 

at the University of Oxford, United Kingdom were included from 1999 until 2010 

and from 2000 to 2010 respectively. Data were compared with a previously 

published report of trials performed between 1998 and 2002 at the USMMVP, 

Silver Spring, Maryland [6]. 

Patient population 

Data from three different cohorts were assessed. A summary of the cohort 

characteristics is provided in Table 1. Data from eight studies at RUNMC were 

analyzed in two cohorts [7-10]. The first cohort (RUNMC I) has been previously 

described by Verhage et al [7]. Five volunteers from this cohort received anti-



malarial treatment with 48 hours delay after parasites were detected by 

microscopy. The RUNMC II cohort includes volunteers from 2004 onwards, when 

more stringent cardiovascular inclusion criteria (based on SCORE cardiovascular 

risk [11]) were adapted following a case of myocardial infarction in a malarianegative 

volunteer [7] and an increased threshold for microscopic parasite 

detection was implemented. RUNMC II includes 12 volunteers who participated 

in a candidate malaria vaccine trial, but were not protected (Nieman et al. 

manuscript in preparation). All other volunteers were unimmunized In Oxford, 

65 infectivity control volunteers participated in 14 studies [12-18]. 

All included subjects were healthy, male and female, malaria-naïve volunteers 

between the ages of 18 and 50 years (Table 1). Malaria naiveté was confirmed 

by medical and travel history. Volunteers from the RUNMC cohorts were also 

confirmed negative for antibodies against blood-stage Pf by ELISA [19]. 

Volunteers were excluded in case of known allergies to anti-malarials, 

pregnancy, systemic disease or chronic use of medication. Volunteers were 

screened by a physician based on medical history, physical examination, 

complete blood count, liver and renal function tests, pregnancy test and 

serological testing for HIV, hepatitis B and C. Volunteers provided written 

informed consent and all studies were approved by either the RUNMC 

Committee on Research involving Human Subjects or Central Committee on 

Research involving Human Subjects (CMO 0004-0090, 0011-0262, 2001/203, 

2002/170, 2004/129, 2006/207, NL14715.000.06, NL24193.091.09) or the 

Oxfordshire Research Ethics Committee or the UK Gene Therapy Advisory 

Committee (C01.111, C02.069, C02.152, C02.153, C02.266, C02.268, C02.293, 

C02.305, CL03.100, C03.088, 04/Q1604/93, 06/Q1604/55, 05/Q1604/69, GTAC 

160-02). 

Infection procedures. 

Anopheles stephensi mosquitoes were infected with the NF54 strain of Pf 

(RUNMC) or 3D7, a clone originally derived from NF54, (Oxford) following 

previously described procedures [20]. Both strains are chloroquine sensitive 

(data not shown). 

Fixed numbers of mosquitoes were allowed to bite volunteers during five 

(Oxford) or ten (RUNMC) minutes. Fully blood-engorged mosquitoes were 

confirmed positive for salivary gland sporozoites by dissection (a threshold of 

>10 sporozoites/gland was used in all centres). If necessary, feeding sessions 

were repeated until exactly the predefined number of infected mosquitoes were 

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102 Chapter 5 

fully engorged, i.e. five mosquitoes, except for the RUNMC I cohort, where 

volunteers were exposed to the bites of four to seven mosquitoes (Table 1). 

Monitoring took place twice (Oxford) or thrice daily (RUNMC) using microscopy 

of Giemsa-stained blood smears starting on day five at RUNMC or on the 

afternoon of day six at Oxford. Volunteers were treated with a standard 

therapeutic regimen of chloroquine, arthemether/lumefantrine or 

atovaquone/proguanil as soon as microscopy confirmed the presence of 

parasites or by the discretion of the physician. 

Trial volunteers were followed on an outpatient basis and lived in the vicinity of 

the hospital. An active tracking policy using mobile phones and/or home visits 

was operational at both institutions during the monitoring period. Adverse 

events were recorded at every visit. Investigators evaluated the potential 

relation of adverse events with trial procedures. All probable or possible related 

events were included in the analysis, with exception of the five volunteers for 

whom anti-malarial treatment was delayed by 48 hours. The USMMVP report 

included adverse events from day seven after challenge [6]. Severity of 

symptoms in RUNMC II and the USMMVP report were assessed according to 

standard guidelines (http://www.fda.gov/BiologicsBloodVaccines/GuidanceCom 

plianceRegulatoryInformation/Guidances/Vaccines/ucm074775.htm). 

Mild symptoms (grade 1) did not interfere with daily activities, moderate 

symptoms (grade 2) interfered with daily activities, severe symptoms (grade 3) 

prevented daily activity. Symptom severity was not consistently assessed in the 

other cohorts, with exception of fever, which was graded mild when 37.5 to 

37.9°C, moderate when 38 to 38.9°C and severe when ≥39°C in all cohorts. All 

centres recorded oral temperatures, which were measured at least once daily, 

either by volunteers themselves or by the attending physician at the clinical site. 

RUNMC also recorded auricular temperatures at the clinical site up to three 

times daily. Serious adverse events (grade 4) were defined according to 

International Conference of Harmonization Good Clinical Practice Guidelines. 

Clinical haematological laboratory data were available on a daily basis from day 

five post-challenge until three days after anti-malaria treatment for RUNMC 

cohorts. Biochemical parameters in RUNMC cohorts were assessed once, at 

three days after anti-malarial treatment. Clinical laboratory parameters in 

Oxford were not routinely recorded during challenge in any trials. For the 

USMMVP cohort, clinical laboratory parameters were reported at days 10-12 

after challenge.



Parasitological data 

Prepatent period was defined as time from exposure to positive thick smear. The 

threshold for microscopic detection of parasites varied between cohorts 

depending on the local standard operating procedure (Table 1). Centres use 

different microscopes, blood volume and slide surface area. RUNMC and Oxford 

readers complete 200 fields, yielding a threshold of approximately two parasites 

per μl blood in RUNMC I and Oxford (slide was deemed positive if one parasite 

was found), which was confirmed by Q-PCR in Oxford. A threshold of four 

parasites per μl blood was achieved in RUNMC II (slide was deemed positive if 

two parasites were found). USMMVP readers completed 5 passes (72 

fields/pass), yielding a threshold of approximately 3 parasites per μl blood (slide 

positive if two parasites were found). In all centres, slides were read by two 

independent readers at 1000x magnification. 

Simultaneously, a quantitative PCR (Q-PCR) for Pf was used in Oxford to support 

microscopy. Parasite densities were measured by Q-PCR for RUNMC and Oxford 

cohorts as previously described [3, 5]. Although methodology of the Q-PCR 

differed, there was no inter-institutional difference in measured densities, 

confirmed by an exchange of samples between both institutions (data not 

shown). Peak parasitemia was defined as the highest parasite density during 

infection measured by Q-PCR. Any cycle threshold above 45 was plotted as zero 

parasitemia. For calculations, these samples were given a value of half the 

detection threshold (ten parasites/ml). 

Data analysis 

Data were assessed in SPSS 16.0 with correction for multiple analyses. 

Differences between frequencies and prepatent period were compared by 

Kruskal-Wallis tests when comparing multiple groups or Mann-Whitney U tests 

when comparing two groups. Dunn’s multiple comparisons test was performed 

as post-hoc analysis when appropriate. Analysis of parasitological PCR data was 

performed on log-transformed data using independent-samples t-test when 

comparing two groups and one-way ANOVA when comparing three groups. The 

multiplication rate of blood stage parasites was calculated by the ratio of the 

geometric mean parasitemia in the second cycle with the first cycle (day 8.6-10.5 

vs 6.6-8.5) or the third cycle with the second cycle (day 10.6-12.5 vs 8.6-10.5), or 

if possible, the mean of both ratios. 

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104 Chapter 5 

p-value* 

0.44 



Correlations were assessed by Pearson’s correlation when parametric and 

Spearman’s when non-parametric. Two-sided p-values below 0.05 were 

considered significant unless stated otherwise. 

Results 

Demographics 

A total of 128 volunteers were grouped into three different cohorts: RUNMC I 

and II and Oxford and compared to published data from 47 infected individuals 

at USMMVP [6], for a total of 175 volunteers. Individuals were generally 20-40 

years old, with equal distribution between male and female participants (Table 

1). There were no significant differences in sex distribution between the 

different cohorts (Kruskal-Wallis, p=0.22), but RUNMC II volunteers were 

significantly younger (Kruskal-Wallis, p37.5°C) occurred with significant higher frequency in 

RUNMC and USMMVP. 

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106 Chapter 5 

Figure 1. Time to microscopically detected parasitemia by cohort 

Survival curve for four cohorts: RUNMC I (orange), RUNMC II (blue), USMMVP 

(grey) and Oxford (green). 

Unsolicited adverse events were infrequent and never severe, and all resolved 

spontaneously. Events included vasovagal collapse, epistaxis, flatulence, 

insomnia, tinnitus, hyperesthesia, psychiatric complaints associated with 

chloroquine [7], pleuritic chest pain, sore throat, migraine, gingivitis, 

palpitations, numbness in fingers, dizziness, drowsiness, photosensitivity and 

stiff neck. 

Laboratory safety parameters 

Clinical laboratory parameters in RUNMC and USMMVP did not show significant 

abnormalities in haemoglobin content; parameters were not routinely available 

in Oxford. The majority of volunteers experienced a mild to moderate decrease 

in leucocyte count, none of which was severe (



Figure 2: Geometric mean parasite density by Q-PCR per cohort 

Three cohorts are depicted RUNMC I (orange), RUNMC II (blue) and Oxford 

(green). 

No abnormalities of urea and creatinine were found, but USMMVP reported six 

cases of haemoglobinuria and one case of proteinuria [6]. Urinary parameters 

were not checked in Oxford or RUNMC. Incidental increases particularly in 

alanine aminotransferase and aspartate aminotransferase were reported in both 

RUNMC and USMMVP. Severe increases (>2.5x ULN) were found in five cases, 

three from USMMVP (twice at day 10, once at day 14-16) and two from RUNMC 

II (day 16 and 17). All tests normalized at the end of the trial. 

Parasitemia by microscopy 

Prepatent period between the four cohorts was significantly different (Kruskall- 

Wallis p

108 Chapter 5 

Figure 3. Statistics of Q-PCR parasitemia per cohort. 

Peak parasitemia (A, one-way ANOVA p=0.13), geometric mean parasitemia during 

the first blood stage parasite multiplication cycle, day 6.5 to 8.5 (B, one-way 

ANOVA p45 was assigned a parasitemia of 10 parasites/ml. 

3A), although there seemed to be a trend towards higher parasitemia at RUNMC 

after the threshold for microscopic parasite detection changed. The first blood 

stage parasite growth cycle was significantly lower in Oxford as compared to 

RUNMC (p



Figure 4. Correlation of Q-PCR parasitemia with prepatent period 

Correlation between peak parasitemia (A) or geometric mean 

parasitemia during the first multiplication cycle from day 6.5 to day 8.5 

(B) with prepatent period (R 2 = 0.27 and -0.73, p=0.006 and p=

110 Chapter 5 

in time to microscopically detected parasitemia whereas clinical symptoms are 

broadly similar. Although there are several methodological differences between 

the CHMI protocols that limit direct comparability, this difference in prepatency 

likely results from variation in the mean parasite burden in the first blood stage, 

possibly as a result of different inoculum size, whilst blood stage multiplication 

factors are equal. 

The analysis of adverse events from 175 non-immune participants of sporozoite 

challenge trials shows that serious adverse events (grade 4) are rare. One 

serious cardiac adverse event was reported; the true nature and pathophysiological 

explanation of that event remains unclear [21]. Severe adverse 

events (grade 3) related to clinical malaria occur in up to half of the volunteers 

and persist for several days. We conclude that CHMI are generally safe, but may 

lead to severe (grade 3) symptoms, though not serious adverse events, in a 

significant proportion of subjects. Several precautions are taken in both 

institutions to ensure safety of volunteers, such as 24-hour phone access, medicalert 

cards and emergency contact procedures. Nevertheless, the exposure of 

volunteers to the likelihood of some severe adverse events should be carefully 

weighed against the benefits of the information to be gained [22]. 

We find significant differences in frequencies of fever, fatigue, arthralgia and 

chills between institutions. Cohorts with a longer prepatent period or a higher 

peak parasitemia do not consistently show a higher frequency of adverse events. 

These data, combined with methodological differences in the recording of 

adverse events (e.g. home-monitoring of oral temperature in RUNMC), lead us 

to conclude that biologically relevant variation in patho-physiology between 

institutions seems unlikely. Nevertheless, standardized assessment of adverse 

events and harmonization of solicited events would advance the interpretability 

and comparability of CHMI in different settings worldwide. 

Laboratory safety parameters show a severe decrease in platelet count 

(



An increase in threshold for microscopic detection of parasites leads to a 

prolonged prepatent period but not an increase in the number of adverse 

events, as illustrated by comparing the RUNMC I and II cohorts. Standardized 

reading of blood smears is thus essential to harmonize trial endpoints 

worldwide; recent efforts by the WHO have resulted in a proposed 

harmonization document (Laurens MB, Roestenberg M, and Moorthy VS 

manuscript in preparation). Variability in prepatent period among institutions, 

however, cannot be explained solely by microscopy methodology. A detailed 

analysis of parasitemia by Q-PCR revealed a ~10 fold difference in the parasite 

burden during the first blood stage growth cycle. Taking into account a 

replication factor of approximately 10 every 48 hours, this difference accounts 

for a 48 hour shorter prepatent period at RUNMC. The variation in parasite load 

may reflect variation in liver stage development of the parasites or, 

alternatively, the number of inoculated parasites. The number of inoculated 

parasites is estimated to vary widely (ranging from 5-10 [26] to 100-300 

sporozoites per bite [27, 28]). Whether the intensity of mosquito infection (e.g. 

sporozoite salivary gland load) or exposure time influences the number of 

sporozoites inoculated is controversial [29, 30]. However, also a formal relation 

between the number of parasites inoculated and prepatent period has never 

been established [7, 28, 30-33]. Similarly, the role of mosquito infectivity (i.e. 

number of sporozoites per mosquito), parasite strain (3D7 vs NF54), exposure 

time (5 vs 10 minutes) or viability of inoculated sporozoites in determining the 

inoculated dose is unclear [30, 31]. Efforts to standardize the sporozoite dose 

should ideally be tested for their impact on comparability and reproducibility of 

CHMI. Standardization may be achieved by harmonization of mosquito breeding 

and feeding protocols or by needle injection of cryopreserved sporozoites. CHMI 

trials are underway to test the infectiousness of cryopreserved sporozoites by 

needle injection (NCT01086917) [34]. 

We show that blood stage parasite growth is cyclical and highly synchronous 

within and between institutions. Importantly, the duration of parasite liver stage 

development as well as the blood stage multiplication rate are highly 

reproducible. Thus vaccine efficacy can be robustly evaluated in any of the CHMI 

centres if a non-protected, malaria-naïve control group is included. 

CHMI trials do not fully mimic conditions in endemic regions where pre-existing 

immunity may augment or impair vaccine efficacy. A limited number of 

comparisons between Phase IIa preliminary efficacy trials and Phase IIb field 

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112 Chapter 5 

efficacy trials shows that results are generally in line, but more comparisons are 

required before definite conclusions can be drawn [2]. Another potential 

difference is reflected by the almost instant delivery of parasites by five infected 

mosquitoes, which has been considered unnatural and a stringent test for 

vaccine-induced immune responses [35]. However, although the frequency of 

infectious mosquito bites is generally lower in malaria-endemic areas, intense 

transmission can occur. A person may be subjected to 35–96 mosquito bites per 

night, and in certain areas approximately 10% of mosquitoes are infected with Pf 

[36]. 

The present data show that CHMI can be safely conducted, but will lead to grade 

3 adverse events in a proportion of volunteers. The primary parasitological 

outcome of such experiments is highly reproducible within institutions but may 

vary between trial centres. With an increasing number of CHMI centres being 

installed, priority should be given to initiatives to standardize challenge 

procedures [37]. The implementation of guidelines will enhance the 

comparability of CHMI; a critical and indispensable component of malaria 

vaccine development worldwide. 


We would like to acknowledge Geert-Jan van Gemert, Marga van de Vegte- 

Bolmer, Matthew McCall, An-Emmie Nieman, Theo Arens, Pieter Beckers, Karina 

Teelen, Jorien Wiersma and the staff of the Clinical Research Center Nijmegen 

for their continuing support of controlled malaria infection trials at the RUNMC. 

We thank the clinical staff of the CCVTM and scientists of Jenner Institute Labs 

who contributed to these trials in Oxford. We thank all participating volunteers. 

Judith Epstein is a military service member. This work was prepared as part of 

her official duties. The views expressed in this article are those of the authors 

and do not necessarily reflect the official policy or position of the Department of 

the Navy, Department of Defense, nor the U.S. Government. Title 17 U.S.C. §105 

provides that ‘Copyright protection under this title is not available for any work 

of the United States Government.’ Title 17 U.S.C. §101 defines a U.S. 

Government work as a work prepared by a military service member or employee 

of the U.S. Government as part of that person’s official duties.



Funding 

This work was supported by Top Institute Pharma [grant number T4-102] and 

Dioraphte Foundation. The UK trials were supported by the UK Medical Research 

Council, the Wellcome Trust, the European Commission framework 

programmes, the European Vaccine Initiative, the PATH Malaria Vaccine 

Initiative and the UK National Institute for Health Research. 

113

114 Chapter 5 

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3. Andrews L, Andersen RF, Webster D et al. Quantitative real-time polymerase 

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Med 2005; 63:52-58. 

8. McCall MB, Beynon AJ, Mylanus EA, van d, V, Sauerwein RW. No hearing loss 

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9. Roestenberg M, McCall M, Hopman J et al. Protection against a malaria 

challenge by sporozoite inoculation. N Engl J Med 2009; 361:468-477. 

10. Roestenberg M, Teirlinck AC, McCall MB et al. Long-term protection against 

malaria after experimental sporozoite inoculation: an open-label follow-up 

study. Lancet 2011. 

11. Conroy RM, Pyorala K, Fitzgerald AP et al. Estimation of ten-year risk of fatal 

cardiovascular disease in Europe: the SCORE project. Eur Heart J 2003; 24:987- 

1003. 

12. Thompson FM, Porter DW, Okitsu SL et al. Evidence of blood stage efficacy with 


13. Dunachie SJ, Walther M, Epstein JE et al. A DNA prime-modified vaccinia virus 

ankara boost vaccine encoding thrombospondin-related adhesion protein but 

not circumsporozoite protein partially protects healthy malaria-naive adults 

against Plasmodium falciparum sporozoite challenge. Infect Immun 2006; 

74:5933-5942. 

14. Walther M, Thompson FM, Dunachie S et al. Safety, immunogenicity, and 

efficacy of prime-boost immunization with recombinant poxvirus FP9 and 

modified vaccinia virus Ankara encoding the full-length Plasmodium falciparum 

circumsporozoite protein. Infect Immun 2006; 74:2706-2716.



15. Webster DP, Dunachie S, Vuola JM et al. Enhanced T cell-mediated protection 

against malaria in human challenges by using the recombinant poxviruses FP9 

and modified vaccinia virus Ankara. Proc Natl Acad Sci U S A 2005; 102:4836- 

4841. 

16. Walther M, Dunachie S, Keating S et al. Safety, immunogenicity and efficacy of a 

pre-erythrocytic malaria candidate vaccine, ICC-1132 formulated in Seppic ISA 

720. Vaccine 2005; 23:857-864. 

17. McConkey SJ, Reece WH, Moorthy VS et al. Enhanced T-cell immunogenicity of 

plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara 

in humans. Nat Med 2003; 9:729-735. 

18. Porter DW, Thompson FM, Berthoud TK et al. A human Phase I/IIa malaria 

challenge trial of a polyprotein malaria vaccine. Vaccine 2011; 29:7514-7522. 

19. Bousema JT, Roeffen W, van der KM et al. Rapid onset of transmission-reducing 

antibodies in javanese migrants exposed to malaria in Papua,Iindonesia. Am J 

Trop Med Hyg 2006; 74:425-431. 




21. Nieman AE, de MQ, Roestenberg M et al. Cardiac complication after 

experimental human malaria infection: a case report. Malar J 2009; 8:277. 

22. Visser HK. Experimental malaria in human volunteers: ethical aspects. Neth J 

Med 2005; 63:41-42. 

23. de Mast Q, Groot E, Lenting PJ et al. Thrombocytopenia and release of activated 

von Willebrand Factor during early Plasmodium falciparum malaria. J Infect Dis 

2007; 196:622-628. 

24. de Mast Q, de Groot PG, van Heerde WL et al. Thrombocytopenia in early 

malaria is associated with GP1b shedding in absence of systemic platelet 

activation and consumptive coagulopathy. Br J Haematol 2010; 151:495-503. 

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and the spectrum of clinical illness in falciparum malaria. J Coll Physicians Surg 

Pak 2008; 18:362-368. 

26. Beier JC, Davis JR, Vaughan JA, Noden BH, Beier MS. Quantitation of 

Plasmodium falciparum sporozoites transmitted in vitro by experimentally 

infected Anopheles gambiae and Anopheles stephensi. Am J Trop Med Hyg 

1991; 44:564-570. 

27. Jin Y, Kebaier C, Vanderberg J. Direct microscopic quantification of dynamics of 

Plasmodium berghei sporozoite transmission from mosquitoes to mice. Infect 

Immun 2007; 75:5532-5539. 

28. Frischknecht F, Baldacci P, Martin B et al. Imaging movement of malaria 

parasites during transmission by Anopheles mosquitoes. Cell Microbiol 2004; 

6:687-694. 

29. Pumpuni CB, Mendis C, Beier JC. Plasmodium yoelii sporozoite infectivity varies 

as a function of sporozoite loads in Anopheles stephensi mosquitoes. J Parasitol 

1997; 83:652-655. 

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30. Ponnudurai T, Lensen AH, van Gemert GJ, Bolmer MG, Meuwissen JH. Feeding 

behaviour and sporozoite ejection by infected Anopheles stephensi. Trans R Soc 

Trop Med Hyg 1991; 85:175-180. 

31. Rickman LS, Jones TR, Long GW et al. Plasmodium falciparum-infected 

Anopheles stephensi inconsistently transmit malaria to humans. Am J Trop Med 

Hyg 1990; 43:441-445. 

32. Jeffery GM, Young MD, Burgess RW, Eyles DE. Early activity in sporozoiteinduced 

Plasmodium falciparum infections. Ann Trop Med Parasitol 1959; 

53:51-58. 

33. Rosenberg R, Wirtz RA, Schneider I, Burge R. An estimation of the number of 

malaria sporozoites ejected by a feeding mosquito. Trans R Soc Trop Med Hyg 

1990; 84:209-212. 



Hum Vaccin 2010; 6:97-106. 




36. Trape JF, Zoulani A. Malaria and urbanization in central Africa: the example of 

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37. Moorthy VS, Diggs C, Ferro S et al. Report of a consultation on the optimization 



Chapter 6 

Efficacy of pre-erythrocytic and blood-stage 

malaria vaccines can be assessed in small 

sporozoite challenge trials in human 

volunteers 

Meta Roestenberg 1 , Sake J. de Vlas 3 , An-Emmie Nieman 2 , Robert W. Sauerwein 1 , 

Cornelus C. Hermsen 1 

1 

Radboud University Nijmegen Medical Centre, dept. of Medical Microbiology, 

Nijmegen, The Netherlands 

2 

VU Medical Centre, dept. of Medical Microbiology, Amsterdam, The 


3 

Erasmus Medical Centre, dept. of Public Health, Rotterdam, The Netherlands 

J. Infect. Dis. 2012 Aug; 206(3):319-23

Chapter 6 118 

Abstract 

The development of a vaccine against malaria has public health priority. In a 

controlled setting, preliminary data on the efficacy of Plasmodium falciparum 

(Pf) vaccine candidates can be obtained by exposing immunized human 

volunteers to the bites of laboratory-reared Pf-infected mosquitoes. Using 

empirical data we show that these trials, with small numbers of volunteers, are 

sufficiently powered to detect protective biological effects induced by preerythrocytic 

and/or blood-stage candidate vaccines if parasitemia is measured 

daily by quantitative PCR. Sporozoite challenge trials are thus a powerful tool for 

early selection of candidates that warrant efficacy of trials in the field.

Efficacy of pre-erythrocytic and blood-stage malaria vaccines can be assessed in small 

sporozoite challenge trials in human volunteers 

Introduction 

The development of an effective vaccine against malaria has public health 

priority. With an increasing number of candidate Plasmodium falciparum (Pf) 

vaccines and only a limited number of field trial sites available, human 

sporozoite challenges are used to assess preliminary vaccine efficacy before 

proceeding to phase IIb trials [1]. In such phase IIa challenge trials malaria-naïve 

volunteers are immunized with a candidate vaccine and subsequently exposed 

to the bites of laboratory-reared Pf-infected mosquitoes. Traditionally, a 

comparison of the prepatent period (time from challenge until positive bloodslide) 

between controls and vaccinees provides an efficacy estimate. The 

development of molecular techniques (Q-PCR) allow for a detailed analysis of 

blood-stage parasite growth [2]. 

Sporozoite challenge trials are thought suitable for testing pre-erythrocytic (liver 

stage) vaccines, because of the natural route of exposure (mosquito bite) and 

the full pre-erythrocytic development of Pf parasites in volunteers. To date, 30 

reports of combined phaseI/IIa sporozoite challenge trials are available, of which 

three pre-erythrocytic vaccine candidates induced full protection [1]. One of the 

three candidates is currently in phase III clinical development [3], whereas 

disappointing preliminary efficacy data have halted the clinical development of 

others [4, 5]. This illustrates the importance of challenge trials in the clinical 

development path of pre-erythrocytic vaccines. 

Preliminary efficacy testing of asexual erythrocytic (blood-stage) stage vaccines 

is more complex, since vaccine efficacy can only be obtained by evaluating 

blood-stage parasite growth over a sufficient lengthy period of time. However, 

blood-stage parasitemia is terminated by curative anti-malarial treatment at 

0.0001% infected erythrocytes, limiting parasitemia to an interval of one to six 

days only (mean 1.7 multiplication cycles) [6]. Immunological effects have to 

exert significant parasite inhibition in this short period in order to ensure 

detectable vaccine efficacy, making the use of challenge trials for erythrocytic 

vaccine candidates controversial. To date, only two erythrocytic malaria vaccine 

candidates have been subjected to sporozoite challenge (Apical Membrane 

Antigen 1 and Merozoite Surface Protein 1) [7, 8]. Analysis of parasitological 

data from two of these trials revealed an apparent pre-erythrocytic inhibiting 

effect, but could not show blood-stage inhibition (S.H. Sheehy pers. comm.) [7]. 

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120 Chapter 6 

It is thus unclear whether blood-stage parasite growth inhibition can be 

determined with sufficient precision after sporozoite challenge. 

The advent of molecular techniques allows for quantification of submicroscopic 

parasitemia [2]. In addition to the number of protected volunteers, Q-PCR allows 

for estimations of liver stage parasite load (for pre-erythrocytic vaccines) or 

blood-stage multiplication rate (for erythrocytic vaccines), which may be 

particularly useful in vaccines that do not induce full protection, e.g. “leaky 

vaccines” [9]. Here we use Q-PCR data from 11 years of experience of sporozoite 

challenge trials at the Radboud University Nijmegen Medical Centre (RUNMC) to 

determine inter-individual variation in parasite kinetics and calculate the power 

of sporozoite challenge trials for both pre-erythrocytic and asexual erythrocytic 

vaccines. 

Methods 

Data and study population 

Data were available from 48 volunteers participating in seven different 

sporozoite challenge trials at the RUNMC from 1999 to 2010. The data set 

included subjects from immunological studies (N=20), infectivity controls from 

immunisation trials (N=16) and subjects from a malaria vaccine trial not showing 

any protection (N= 12). All volunteers were challenged by the bites of four to 

seven infected mosquitoes for ten minutes. Mosquitoes were laboratory-reared 

and infected with the NF54 strain of Pf [10]. Presence of sporozoites in 

mosquitoes was confirmed by salivary gland dissection. Trial subjects were 

followed two to three times daily from day five after challenge until three days 

after start of antimalarial treatment. At every visit, blood samples were collected 

and assessed for presence of parasites by microscopy (threshold 4 Pf/µl) and 

quantified by Q-PCR (threshold 20-100 Pf/ml) [2]. Antimalarial treatment was 

provided as soon parasites were detected by microscopy. Ethical approval was 

obtained for each trial separately (Review board numbers: 0011-0262, 

2001/203, 2002/170, 2004/129, 2006/207, NL14715.000.06, NL24193.091.09). 

Statistical analysis 

Analyses were performed using log-transformed data. We calculated geometric 

mean parasitemia for every cycle in individual volunteers as well as group 

geometric mean and standard deviation (SD). We determined individual parasite 

multiplication rate by division of parasite densities of two subsequent cycles and



calculated the geometric mean multiplication rate and SD. Samples with a Q-PCR 

CT >45 were estimated at half the detection limit (10 or 50 Pf/ml). 

Geometric mean parasitemia in the first cycle and parasite multiplication rates 

showed a normal distribution, so power calculations were performed using a 

two-sample t test power analysis with α=0.05 and β=0.80. We separately 

determined the power using all three daily time points if available (8:00am, 

4:00pm and 10:00pm, n=33), two daily time points (8:00am and 4:00pm, n=48) 

or one daily time point (8:00am, n=48). 

Power calculations estimated the group size required to detect significant 

differences between vaccinees and controls. Vaccine and control group were 

assumed of equal size. Pre-erythrocytic vaccines were assumed to reduce the 

geometric mean parasitemia of the first multiplication cycle, but not alter the 

parasite multiplication rate. Erythrocytic stage vaccines were assumed to reduce 

the parasite blood-stage multiplication rate, reducing parasite load from the first 

cycle onwards. Vaccine effects were simulated by beta distribution with an 

assumed mean inhibition of 70, 80, 90 and 95% and SD of 5 or 10%. Vaccine 

inhibition was simulated 100 times for every individual subject and repeated ten 

times based on random sampling from the distribution. We assumed a 

continuous infectious dose of merozoites, as opposed to the actual release of 

discrete batches of merozoites from infected hepatocytes. Interindividual 

variation masked the discrete nature of infection in empirical situations with 

high numbers of infected hepatocytes. In simulations with low numbers of 

infected hepatocytes, where a discrete variable would give a binary outcome 

(i.e. protected or non-protected), the resulting blood-stage parasitemia was 

always below the Q-PCR detection limit. In such cases, values were corrected to 

a standard value of half the Q-PCR detection limit. The number of subjects with 

simulated parasitemia ≤ 1Pf/ml or multiplication rate ≤1 was counted. For some 

subjects second cycle data were missing because they had already received 

antimalarial treatment for safety reasons: eight volunteers in the group with 

samples thrice daily (24%) and nine volunteers in the group with samples twice 

daily (19%). The group size for erythrocytic vaccines was corrected for these 

missing data by addition of the respective proportion of volunteers. Similarly, 

the power for pre-erythrocytic vaccines was corrected for the number of 

volunteers with the first cycle data points below the detection limit: two 

volunteers in the group with samples once daily (4%). 

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122 Chapter 6 

Figure 1. (A) Parasite density in blood measured by quantitative PCR in 48 volunteers 

participating in seven sporozoite challenge trials at the Radboud University Medical 

Centre Nijmegen. Grey lines are measurements for individual volunteers, the black line 

shows geometric mean parasitemia. Q-PCR cycle threshold above 45 was plotted as zero. 

(B) Geometric mean parasite density per multiplication cycle for individual volunteers. 

(C) Multiplication rate for every individual by taking the ratio of geometric mean 

parasitemia from two cycles. Shown are the ratio of multiplication cycle 2 with cycle 1, 

cycle 3 with cycle 2 and square root of the ratio of cycle 3 with cycle 1. Lines indicate 

geometric mean of ratios. 

Results 

Parasite densities showed a consistent cyclical pattern of blood-stage parasite 

growth in all volunteers (Figure 1A). The following multiplication cycle intervals 

were identified: first cycle from day 6.5 until day 8.5, second cycle from day 8.5 

until day 10.5 and third cycle from day 10.5 until day 12.5. All individuals deve-



Assumed 

mean 

Assumed 

inhibition 

Corresponding Corresponding Group size (N) 

subjects with 

1st cycle mean 

subjects with 

multiplication 

Pre-erythrocytic 

vaccine 

Asexual erythrocytic 

vaccine 

inhibition (%) s.d. (%) ≤ 1 Pf/ml (%) rate ≤ 1 (%) Once Twice Thrice Once Twice Thrice 

70 5 2 9 24 21 20 12 12 11 

80 5 5 20 13 12 12 7 7 6 

90 5 16 47 6 6 6 4 4 4 

95 5 40 77 4 3 3 3 3 3 

70 10 3 11 22 20 19 12 11 11 

80 10 7 24 12 11 11 7 7 7 

90 10 29 59 6 5 5 4 4 4 

95 10 65 82 3 3 3 3 3 3 

Table 1. Sporozoite challenge group size required for determining parasite inhibition 

induced by pre-erythrocytic and asexual erythrocytic malaria candidate vaccines when 

measuring parasitemia by PCR once, twice or three times a day. For pre-eythrocytic 

vaccines a threshold of 1Pf/ml was set somewhat arbitrarily to estimate the percentage 

of subjects considered protected. For erythrocytic vaccines, the percentage of subjects 

with multiplication rate ≤ 1 is provided. Power calculations were based on data from 48 

volunteers participating in seven sporozoite challenge trials at the Radboud University 

Medical Centre Nijmegen. 

loped microscopically detectable parasitemia (range day 7-12.3). No individuals 

were lost to follow-up. 

Parasite densities in the first cycle varied from 23 to 5273 Pf/ml (1.37 to 3.17 

log), with a geometric mean of 500 Pf/ml (2.70 log, SD 0.60, n=48, Figure 1B). 

Group geometric mean parasite density in the first cycle was not significantly 

different between the seven trials (ANOVA p=0.5), so data were pooled for 

analysis. In the second and third cycle, geometric mean parasite density 

increased to 4485 Pf/ml (3.65 log, SD 0.62, n=40) and 11369 Pf/ml (4.06 log, SD 

0.48, n=9) respectively. Only limited data were available for the third 

multiplication cycle, because the majority had crossed the blood-slide threshold 

for antimalarial treatment. Volunteers for whom data were available in the third 

cycle started with a lower parasite load in the first cycle (geometric mean 

density first cycle: 126 Pf/ml, t-test p=0.001). Generally, multiplication rates 

between volunteers were similar, as is clear from the parallel growth in Figure 

1B. 

Blood-stage multiplication rate was calculated by dividing the mean parasitemia 

in the second cycle by the first cycle (subtraction of log-transformed values) for 

every individual volunteer. The group geometric mean multiplication rate was 

10.9 (1.04 log, SD 0.33, n=40). Similarly, the ratio of the third with the second 

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124 Chapter 6 

cycle gave a multiplication rate of 10.8 (1.03 log, SD 0.21, n=9) and the square 

root of third with the first cycle ratio gave an estimate of 8.9 (0.94 log, SD 0.25, 

n=9), Figure 1C. Multiplication rates were not significantly different between 

trials (ANOVA p=0.1) nor between multiplication cycles (ANOVA p=0.6). 

Results for power calculations are shown in Table 1. The vaccine effect SD hardly 

affected group size. The power of small sporozoite challenge trials to detect 

vaccine inhibition was slightly higher for erythrocytic vaccines as compared to 

pre-erythrocytic vaccines, due to smaller inter-individual variation in blood-stage 

multiplication rate. Sporozoite challenge trials with a group size of 6-7 subjects 

could be powered to detect 80% inhibiting erythrocytic and pre-erythrocytic 

vaccines. Drawing additional blood samples up to thrice daily enhanced the 

sensitivity of sporozoite challenge trials, allowing a reduced group size 

particularly in less efficacious vaccines. 

In order to translate biological effects into clinical efficacy, we estimated the 

threshold of inhibition necessary to give clinical protection. Clinical protection 

was defined as volunteers that do not develop microscopic blood-stage 

parasitemia. For erythrocytic vaccines, we estimate that 90% inhibition results in 

a multiplication rate of ≤1 [6]. Depending on variation in vaccine effect, 20-25% 

of volunteers will cross this threshold in a trial with seven volunteers per group. 

Similarly, we hypothesize that parasitemia will have to be reduced to a 

somewhat arbitrary value 1 Pf/ml in pre-erythrocytic vaccines for clinical 

protection; the remaining low blood-stage parasite loads may possibly induce 

erythrocytic immunity [11]. Depending on the variation of vaccine effect, 16-30% 

of volunteers will cross this threshold in trials with seven volunteers per group 

(Table 1). 

Discussion 

The current data show that small sporozoite challenge trials, typically including 

seven subjects per group, are powered to detect biological effects induced by 

both pre-erythrocytic and erythrocytic candidate vaccines that may herald 

clinical protection. We thus show that sporozoite challenge trials are a very 

sensitive tool to evaluate preliminary efficacy of any pre-erythrocytic or 

erythrocytic candidate malaria vaccine.



The decreasing malaria incidence in several endemic areas [12] has increased 

the size and costs of Phase IIb malaria vaccine field trials. Sporozoite challenges 

can be used for preliminary efficacy selection of candidates. Efficacy goals set by 

the Malaria Vaccine Roadmap and the target product profile of RTS,S, the only 

candidate currently in phase III clinical development, dictate a protective 

efficacy of more than 80% against clinical disease [13] and a 50% reduction of 

clinical attacks due to Pf respectively [14]. So called all-or-nothing vaccines or 

very effective “leaky” vaccines will induce full protection and can thus easily be 

tested by controlled infection. However, in addition to these promising vaccines, 

vaccine developers may be interested in less efficacious vaccines when 

combining antigens. Such approach is currently followed, for example, for the 

malaria vaccine ME-TRAP [15]. We show that in these vaccines, Q-PCR may be 

useful to improve the power of sporozoite challenge trials to detect 80-90% 

parasite inhibition. These biological effects correspond with clinical protection in 

a smaller proportion of subjects. 

We show that the sensitivity of sporozoite challenge trials is limited by variation 

in vaccine effect, inter-individual variation in parasite development and the 

limited time window during which blood-stage parasitemia can be assessed. 

Inter-individual variation is the most important determinant. Three factors may 

account for this variation: the parasite inoculum size, parasite fitness and human 

(innate) immune factors. Standardizations of these points would likely decrease 

variance. For this purpose, clinical trials testing the needle administration of a 

predefined number of viable sporozoites are currently planned (NCT01086917). 

As an alternative, challenge trials using low numbers of erythrocytic stage 

parasites are conducted, allowing for a longer time window of follow-up [16]. 

Careful analyses of Q-PCR data from several of such trials are needed to quantify 

their benefit. 

We performed calculations using simplified methodology based on crude 

empirical data, taking into account a limited number of variables. The power of 

sporozoite challenge trials may be higher if inhibiting effects transcend parasite 

developmental stage when antigens are shared or combined between stages, or 

when inter-individual variation is limited. More complicated statistical models, 

such as previously described by Hermsen et al. [6] and Bejon et al. [17], take into 

account more variables and may subsequently generate data with higher 

precision and power, but also require more assumptions and may be less 

reliable. 

125

126 Chapter 6 

The community is in urgent need of criteria for selection of vaccine candidates 

that warrant clinical efficacy trials in the field. Knowing that sporozoite challenge 

trials can not only evaluate pre-erythrocytic vaccines, but can also confidently 

assess asexual erythrocytic malaria vaccine candidates is an assuring 

consummation to the malaria vaccine development community. 


We acknowledge the support of the team from the Clinical Centre for Malaria 

Studies and the volunteers participating in the challenge trials. We would like to 

thank Teun Bousema for his critical review of the manuscript.



References 



Immunol 2011; 11:57-64. 

2. Hermsen CC, Telgt DS, Linders EH, et al. Detection of Plasmodium falciparum 


2001; 118:247-251. 

3. Olotu A, Lusingu J, Leach A, et al. Efficacy of RTS,S/AS01E malaria vaccine and 

exploratory analysis on anti-circumsporozoite antibody titres and protection in 

children aged 5-17 months in Kenya and Tanzania: a randomised controlled 

trial. Lancet Infect Dis 2011; 11:102-109. 

4. Cummings JF, Spring MD, Schwenk RJ, et al. Recombinant Liver Stage Antigen-1 

(LSA-1) formulated with AS01 or AS02 is safe, elicits high titer antibody and 

induces IFN-gamma/IL-2 CD4+ T cells but does not protect against experimental 

Plasmodium falciparum infection. Vaccine 2010; 28:5135-5144. 

5. Genton B, D'Acremont V, Lurati-Ruiz F, et al. Randomized double-blind 







7. Spring MD, Cummings JF, Ockenhouse CF, et al. Phase 1/2a study of the malaria 



8. Heppner DG, Jr., Kester KE, Ockenhouse CF, et al. Towards an RTS,S-based, 

multi-stage, multi-antigen vaccine against falciparum malaria: progress at the 

Walter Reed Army Institute of Research. Vaccine 2005; 23:2243-2250. 

9. White MT, Griffin JT, Drakeley CJ, Ghani AC. Heterogeneity in malaria exposure 

and vaccine response: implications for the interpretation of vaccine efficacy 

trials. Malar J 2010; 9:82. 




11. Pombo DJ, Lawrence G, Hirunpetcharat C, et al. Immunity to malaria after 

administration of ultra-low doses of red cells infected with Plasmodium 

falciparum. Lancet 2002; 360:610-617. 

12. World Malaria Report. Available at: http://www.who.int/malaria. Accessed 28 

July 2011. 

13. Malaria Vaccine Roadmap. Available at: 

http://www.malariavaccine.org/malvac-roadmap.php. Accessed 28 July 2011. 

14. Target Product Profile RTS`S. Available at: http://www.who.int/vaccinesdocuments/DocsPDF04/www773.pdf. 

Accessed 28 July 2011. 

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128 Chapter 6 

15. Dunachie SJ, Walther M, Epstein JE, et al. A DNA prime-modified vaccinia virus 




74:5933-5942. 

16. Sanderson F, Andrews L, Douglas AD, Hunt-Cooke A, Bejon P, Hill AV. Bloodstage 

challenge for malaria vaccine efficacy trials: a pilot study with discussion 

of safety and potential value. Am J Trop Med Hyg 2008; 78:878-883. 

17. Bejon P, Andrews L, Andersen RF, et al. Calculation of liver-to-blood inocula, 



malaria sporozoites. J Infect Dis 2005; 191:619-626.

Chapter 7 

NF135.C10: a new Plasmodium falciparum 

clone for controlled human malaria 

infections 

Anne C. Teirlinck 1 *, Meta Roestenberg 1 * ± , Marga van de Vegte-Bolmer 1 , Anja 

Scholzen 1 , Moniek J.L. Heinrichs 1 , Rianne Siebelink-Stoter 1 , Wouter Graumans 1 , 

Geert-Jan van Gemert 1 , Karina Teelen 1 , Martijn W. Vos 1 , Krystelle Nganou- 

Makamdop 1 , Steffen Borrmann 2± , Yolanda P.A. Rozier 3 , Marianne A.A. Erkens 3 , 

Adrian J.F. Luty 1± , Cornelus C. Hermsen 1 , B. Kim Lee Sim 6 , Lisette van Lieshout 3,4 , 

Stephen L. Hoffman 6 , Leo G. Visser 5 , Robert W. Sauerwein 1 

*The authors contributed equally to this work 

1 

Radboud University Nijmegen Medical Centre, Department of Medical 


2 

Department of Infectious Diseases, Heidelberg University School of Medicine, 

Heidelberg, Germany 

3 

Department of Medical Microbiology (Clinical Microbiology Laboratory), Leiden 

University Medical Centre, Leiden, The Netherlands 

4 

Department of Parasitology, Leiden University Medical Centre, Leiden, The 


5 

Department of Infectious Diseases, Leiden University Medical Centre, Leiden, The 


6 Sanaria Inc., Rockville, Maryland, United States of AmericaJ. 

J.Infect. Dis 2012

130 Chapter 7 

Abstract 

The portfolio of Plasmodium falciparum (Pf) strains for controlled human 

malaria infections (CHMI) primarily consists of NF54 and its clone 3D7. 

Availability of additional strains will allow better evaluation of efficacy of 

experimental vaccines and drugs, and understanding of immunological 

protection mechanisms. 

NF135 was isolated from a Dutch patients who acquired malaria whilst travelling 

in Cambodia. Parasites were adapted to continuous culture and cloned. Clone 

NF135.C10 consistently produced gametocytes and generated substantial 

numbers of sporozoites in Anopheles mosquitoes. NF135.C10 was tested in a 

CHMI trial in comparison with NF54 for parasitological, immunological and 

clinical parameters. Two groups of five malaria-naive volunteers were exposed 

to bites of five NF54 or NF135.C10 infected mosquitoes. 

NF135.C10 parasites diverged from NF54 parasites by drug sensitivity and 

genetic marker profiles. Both NF135.C10 and NF54 were infectious to humans. 

Three out of five volunteers challenged with NF135.C10 and four out of five 

challenged with NF54 developed parasitemia after seven to eleven days as 

detected by microscopy. The two strains showed great similarity in prepatent 

period, kinetics of parasitemia, clinical features and cellular immunological 

responses. 

In conclusion, we successfully established a new field strain of Pf that can be 

used in CHMI and vaccine studies.

NF135.C10: a new Plasmodium falciparum clone for controlled human malaria 


Introduction 

Malaria caused an estimated 216 million cases and around one million deaths in 

2010 [1, 2], mainly in sub-Saharan Africa where most cases are caused by 

Plasmodium falciparum (Pf). Development of vaccines and new drugs, and 

better understanding of immunological processes are essential in order to tackle 

this immense problem. Controlled Human Malaria Infection (CHMI), in which 

healthy volunteers are exposed to bites of Pf-infected mosquitoes, is a powerful 

tool to address questions regarding Pf drug and vaccine efficacy, clinical 

properties, parasite kinetics and human immunology. Since the first CHMI by 

mosquitoes fed on cultures of Pf [3], more than 1300 healthy volunteers have 

been exposed to CHMI [4] with mainly either the Nijmegen Falciparum strain 

NF54 [5] or its clone 3D7 [6]. Strain NF54 stably produces sexual stages required 

for production of infectious mosquitoes. Parasites have been adapted to 

laboratory conditions by continuous in vitro culture for more than three 

decades. In the field, Pf isolates display a wide genetic diversity, which is 

currently not represented by the available laboratory strains for CHMI. Other 

strains, such as the South American 7G8 Pf clone [7, 8] of the Brazilian isolate 

IMTM22 [9] have been sporadically used in limited number of volunteers [10- 

12]. We therefore aimed to identify and test an additional Pf strain that can be 

used in CHMIs, and developed several qualification criteria: i) The strain must 

consistently produce gametocytes and sporozoites. ii) The strain should be 

cloned to create a single genetically homogenous parasite population and 

should be genetically characterized. iii) The clone should be sensitive to 

commonly administered antimalarials. iv) It should be of non-African origin in 

order to be geographically and genetically distinct from the NF54 strain, an 

airport strain that probably originates from Africa [13, 14]. Here we report the 

generation, characterization and first CHMI with NF135.C10, a new Cambodian 

clone, including drug sensitivity, microsatellite profile, kinetics of parasitemia, 

clinical features and immunological properties in a direct comparison with NF54. 

Methods 

Culturing of NF135.C10 and NF54 parasites 

Parasites were cultured as described previously [15]. Briefly, NF135.C10 and 

NF54 Pf asexual blood-stage parasites, regularly screened for mycoplasma 

contamination, were grown in RPMI-1640 medium containing 10% human A + 

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132 Chapter 7 

serum at 5% haematocrit in a semi-automated suspension culture system, in the 

absence of antibiotics and in an atmosphere containing 4% CO2 and 3% O2. 

Cloning of NF135 was performed in 96-wells plates by limiting dilution [16]. 

Characterization of NF135.C10 and NF54 

Genetic identity of NF135.C10 and NF54 was defined by PCR and microsatellite 

mapping. The polymorphic regions of three Pf antigen genes were assessed in a 

method adapted from Snounou et al. [17]. Briefly, parasite DNA was isolated 

using QIAamp DNA Blood Mini Kit (Qiagen) and amplified with thermoperfect 

taq polymerase using specific primers for GLURP, MSP1 K1, MSP1 MAD20 and 

MSP2 IC (all primers from Invitrogen). For microsatellite mapping, the repetitive 

element rif MS (pfRRM), a polymorphic microsatellite marker [18] was used to 

compare NF135.C10 with NF54. The NF135.C10 and NF54 genomic DNA used 

was derived from respective master cell banks produced in compliance with 

cGMPs at Sanaria. PCR amplification was performed using fluorescently labelled 

forward primer 5’-TACGTTACATTATGTTTTA-3’ and reverse primer 5’- 

ATATGTATTGCGCTTTTA-3’. PCR products generated from each sample were 

separated by capillary electrophoresis on an Applied Biosystems 3100 Genetic 

Analyzer. A spectral image was generated with the Genemapper software V4.0. 

with each individual peak in the spectral image representing a PCR product. The 

base pair (bp) size of the amplified sequence products gave a pattern 

(fingerprint) that was unique to each malaria strain [18, 19]. Sensitivity of 

NF135.C10 and NF54 to dihydroartemisinin (DHA; SigmaTau), chloroquine 

diphosphate salt (Sigma-Aldrich), proguanil (British Pharmacopia), atovaquone 

(GSK) and lumefantrine (Novartis) was tested by the Malaria SYBR Green I- 

Based Fluorescence Assay in triplicate experiments [20]. 

Production of NF135.C10 and NF54 infected mosquitoes 

Gametocyte suspensions of NF135.C10 or NF54 were fed to Anopheles stephensi 

mosquitoes reared according to standard operating procedures as described 

previously [21]. To assess the rate of infection, salivary glands of ten mosquitoes 

were dissected for each strain to confirm the presence of sporozoites. 

Inclusion, infection and clinical follow-up of volunteers 

Volunteers, aged 18-35, were recruited public by advertisement and screened at 

the Leiden University Medical Centre (LUMC) for eligibility based on medical and 

family history, physical examination and general haematological and 

biochemical tests including HIV, hepatitis B and hepatitis C serology, urine



NF135.C10 

Session 

# mosquitoes 

offered 

# mosquitoes 

with infected 

bites 

1060-09 s1 5 5 

total 5 5 

1065-10 s1 5 3 

s2 2 1 

s3 1 1 

total 8 5 

1083-15 s1 5 4 

s2 1 0 

s3 1 1 

total 7 5 

1109-19 s1 5 4 

s2 1 1 

total 6 5 

1114-21 s1 5 4 

s2 1 1 

total 6 5 

NF54 

1004-03 s1 5 5 

total 5 5 

1089-18 s1 5 5 

total 5 5 

1094-16 s1 5 4 

s2 1 1 

total 6 5 

1095-25 s1 5 5 

total 5 5 

1100-26 s1 5 5 

total 5 5 

Supplementary Table 1. Feeding sessions on volunteers by mosquitoes infected 

with either NF135.C10 or NF54 parasites. Sessions for every individual volunteer 

are displayed for either NF135.C10 (upper panel) or NF54 (lower panel) groups. 

Sessions were repeated with a smaller number of mosquitoes until precisely five 

infected mosquitoes per volunteer had bitten. 

toxicology and pregnancy. Main exclusion criteria were residence in a malaria 

endemic area within the previous six months, positive Pf serology [22] or an 

estimated ten year risk of >5% of developing a cardiac event as estimated by the 

Systematic Coronary Evaluation system. All volunteers gave written informed 

consent prior to inclusion. 

Ten Dutch malaria-naïve volunteers were included and after randomization two 

groups of five volunteers each were exposed to bites of Anopheles stephensi 

mosquitoes infected with either NF54 or NF135.C10 for ten minutes at the 

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134 Chapter 7 

insectary of the Radboud University Nijmegen Medical Centre. Feeding sessions 

were repeated when necessary, with a smaller number of mosquitoes until each 

volunteer had been exposed to exactly five mosquitoes that took a blood meal 

and had Pf sporozoites in their salivary glands. One feeding session was 

sufficient for one and four volunteers in the NF135.C10 and NF54 groups 

respectively, two and one volunteers required two sessions and three feeding 

sessions were needed in three volunteers of the NF135.C10 group 

(Supplementary Table 1). Starting from day five post-infection, volunteers were 

subjected to intensive follow-up with up to thrice daily visits to the LUMC outpatient 

clinical research department. All signs and symptoms (solicited and 

unsolicited) were recorded and graded by the attending physician as follows: 

mild (easily tolerated), moderate (interferes with normal activity), or severe 

(prevents normal activity), or in case of fever grade 1 (>37.5°C – 38.0°C), grade 2 

(>38.0°C – 39.0°C) or grade 3 (>39.0°C). Haematological and biochemical 

parameters were monitored daily. Because of a previously reported serious 

cardiac adverse event after a malaria challenge infection in a separate study 

[23], particular attention was paid to markers of coagulation or cardiac damage 

with daily follow-up of highly sensitive troponin, platelets, d-dimer and lactate 

dehydrogenase during the period of expected blood stage parasitemia. 

Whenever abnormal, blood samples were checked for the presence of 

fragmentocytes and von Willebrand cleaving protease activity. Promptly after 

identification of a positive blood smear, volunteers were treated with a curative 

regimen of four tablets of 250/100mg atovaquone/proguanil once daily for three 

days. Volunteers whose blood smears remained free of parasites until day 21 

after challenge presumptively received the same curative treatment with followup 

to the end of the study at day 28. Complete cure was always confirmed by 

two consecutive parasite-negative blood smears. The trial was performed in 

accordance with Good Clinical Practice and approved by the Central Committee 

for Research Involving Human Subjects of The Netherlands (CCMO 

NL30350.058.09). Clinicaltrials.gov identifier: NCT01002833. 

Parasitological Outcomes 

Thick blood smears were examined by microscopy twice daily on days five and 

six post-challenge, thrice daily on days seven to eleven, twice daily on days 12- 

15 and once daily on days 16-21 post-challenge. 15µl of EDTA-anti-coagulated 

blood was spread over the standardised surface of one well of a 3-well glass 

slide (CEL-LINE Diagnostic Microscope Slides, 30-12A-black-CE24). After drying, 

wells were stained with Giemsa for 30 minutes. Slides were read at 1000x



magnification by assessing 123 high-power fields, equal to approximately 0.5µl 

of blood. A smear was considered positive if two unambiguously identifiable 

parasites were found. Pre-patent period was defined as the period between 

exposure to infected mosquitoes and first positive blood smear. Additionally, 

parasitemia was measured by real-time quantitative PCR (qPCR), performed 

retrospectively on all samples collected after challenge, as described previously 

[24] with minor changes: using the MGB probe AAC AAT TGG AGG GCA AG 

instead of the turbo TaqMan probe sequence. 

In vitro immunological assays 

For antigen preparation, asynchronous asexual-stage cultures of NF135.C10 and 

NF54 parasites were harvested at parasitemias of approximately 5-10% and 

mature asexual stages were purified by centrifugation on a 27% and 63% Percoll 

density gradient [25] resulting in preparations of 80-90% parasitemia with >95% 

schizonts/mature trophozoites. Preparations of parasitized red blood cells 

(PfRBC) were washed twice in PBS and cryopreserved at 150x10 6 /ml in 15% 

glycerol/PBS in aliquots for use in individual stimulation assays. Mock-cultured 

uninfected erythrocytes (uRBC) were obtained similarly and served as controls. 

For cellular immunology, venous whole blood was collected into citrated 

vacutainer CPT cell preparation tubes (Becton and Dickinson) on the day prior to 

challenge (C-1), on days 5, 35 and 140 after challenge and on the first day of 

treatment (DT). Peripheral blood mononuclear cells (PBMCs) were isolated by 

density gradient centrifugation, washed twice in PBS, enumerated, frozen in 

foetal-calf serum containing 10% dimethylsulfoxide and stored in liquid nitrogen. 

After thawing, 500.000 PBMCs were cultured in the presence of NF135.C10 or 

NF54 PfRBC at a 1:2 (PBMC:PfRBC) ratio for 24 hours with addition of Brefeldin A 

(Sigma-Aldrich) for the last four hours. Cells were harvested and subsequently 

stained for viability (Live/Dead fixable dead cell stain kit Aqua, Invitrogen) and 

surface markers; either 1) CD4 PE (SK3, BioLegend), CD45RO ECD (UCHL1, 

Beckman-Coulter), CD3 PerCP (UCHT1, BioLegend), CD62L PeCy7 (DREG-56, 

eBioscience) and CD8a Alexa Fluor 700 (HIT8A, BioLegend) or 2) anti-TCR Pan 

γ/δ-PE (IMMU510, Beckman-Coulter), CD45RO ECD, CD3 PerCP, CD4 PECy7 

(RPA-T4, BioLegend), Biotin CD56 (HCD56, BioLegend) and CD8a Alexa Fluor 700. 

Cells were washed and the second staining panel was incubated with 

Streptavidin eFluor450 (eBioscience). After washing, all cells were incubated 

with fixation Medium A (Caltag) and subsequently stained with either 1) IFNγ 

FITC (4S.B3, eBioscience), TNF Pacific Blue (MAb11, BioLegend), IL-2 APC (MQ1- 

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136 Chapter 7 

NF135.C10 NF54 

Re-started cultures until CHMI 7 306 

Period 2009-2010 

Infection 74% (62-87) 86% (78-94) 

Oocysts 12 (7.3-16) 27 (22-33) 

Sporozoites/mosquito x10 3 39 (18-60) 99 (74-124) 

CHMI (April 2010) 

Infection 100% 100% 

Oocysts 5.6 17 

Sporozoites/mosquito x10 3 12.5 69 

Gametocyte male: female ratio 1:5 1:3 

Drug sensitivity (IC50) 

dihydroartemisinin 3.4 nM 9.9 nM 

lumefantrine 89 nM 78 nM 

proguanil 21 µM 27 µM 

atovaquone 0.3 nM 0.6 nM 

chloroquine 201 nM 24 nM 

Table 1. Mosquito infection and drug sensitivity profile of NF135.C10 and NF54 in 

the period 2009-2010 and for the specific batches used in this CHMI. Data are 

displayed for the period 2009-2010 (mean (95% CI)) after 26 and 39 standard 

dissections respectively, from 10 mosquitoes per dissection. Half-inhibitory 

concentrations (IC50) are means from three independent experiments. 

17H12, eBioscience )) or 2) IFNγ FITC, in permeabilization Medium B (Caltag). 

Cells were read on a CyAn ADP 9-color flow cytometer (Beckman-Coulter) and 

analysed using FlowJo software (Tree Star, Inc.) version 9.2. Gating of cytokinepositive 

cells was performed based on the Median Fluorescent Intensity (MFI) of 

cytokine negative PBMCs for each volunteer, time point and stimulus. 


Data analysis was performed using GraphPad Prism5 software. Differences in 

parasite kinetics between subjects in the NF135.C10 and NF54 group were 

analysed using the non-parametric Mann-Whitney test. A two-sided P value of 

less than 0.05 was considered statistically significant. 

Results 

Generation and characterization of NF135.C10 

Clinical isolates of asexual Pf parasites were obtained by culturing blood from Pfmalaria 

patients from hospitals in The Netherlands. We adapted 74 different



Figure 1. Genetic characterization of Plasmodium falciparum parasite isolates. 

PCR was performed to assess allelic variation on (A) MSP1 K1 and MSP1 MAD20, (B) 

MSP2 IC and (C) GLURP for the Pf strains NF135.C10 and NF54. Rif repetitive element 

(PfRR) were characterized by the microsatellite identity test on the genomic DNA of the Pf 

parasite strains (D) NF135.C10 and (E) NF54. The spectral image generated in the Gene 

Mapper showing a strain-specific microsatellite fingerprint comprising a unique peak 

pattern. 

field strains to culture, of which 21 produced gametocytes and 16 were able to 

successfully infect mosquitoes. Based on their ability to stably produce mature 

gametocytes, exflagellate and to be transmitted to mosquitoes, seven strains 

were cloned and two strains produced at least five oocysts and 30 000 

sporozoites in more than 70% of mosquitoes after feeding. Clone NF135.C10 was 

isolated by limiting dilution from isolate NF135, which was obtained from a 

Dutch traveller to Cambodia diagnosed with Pf malaria in February 1993. Culture 

characteristics of NF135.C10 and NF54 are displayed in Table 1. 

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138 Chapter 7 

Figure 2. Parasite kinetics of strains NF135.C10 and NF54 assessed by 

thick smear and quantitative real-time PCR. Volunteers were infected by 

bites of mosquitoes infected with either NF135.C10 or NF54. (A) Percentage 

of volunteers infected with NF135.C10 (red, n=5) or NF54 (black, n=5) 

becoming thick smear positive, followed up up to 21 days after infection. 

(B) Parasitemia of volunteers until thick smear positivity, measured by 

qPCR, is shown as geometric mean and 95% confidence interval for 

volunteers infected with NF135.C10 (red) and NF54 (black), and historical 

controls infected with NF54 (grey area, n=48). 

The drug sensitivity profile of NF135.C10 is similar to NF54 for atovaquone, 

proguanil, DHA and lumefantrine, but NF135.C10 is more than 8-fold less sensitive 

to chloroquine than NF54. Comparison of NF135.C10 and NF54 genotypes



ANY ADVERSE 

EVENT 

NF135.C10(n=3) NF54(n=4) 

frequency 

Mean duration 

days (stdev) 

frequency 

Mean duration 

days (stdev) 

Smear negative(n=3) 

frequency 

Mean duration 

days (stdev) 

Abdominal pain 

Arthralgia 

Chills 

2 0 (0.0) 

Fatigue 3 2.4 (1.9) 1 3.0 -- 2 13.5 (9.3) 

Fever 1 0.2 -- 2 0.7 (0.8) 

Headache 3 1.5 (2.4) 4 2.3 (2.0) 3 4.6 (3.8) 

Itching 

4 3.1 (1.5) 2 5.2 (0.3) 

Malaise 

4 2.5 (3.3) 

Myalgia 1 2.7 -- 3 1.3 (1.4) 2 2.7 (2.4) 

Nausea 1 0.1 -- 3 2 (2.0) 1 0.0 -- 

Vomiting 

1 0.4 -- 

Any 3 1.3 (1.7) 4 2.2 (1.9) 3 5.7 (5.8) 

GRADE 3 

ADVERSE EVENT 

Headache 

1 4.6 -- 

Malaise 

1 0.4 -- 

Vomiting 

1 0.4 -- 

Any 

1 1.8 (2.4) 

Table 2. Reported solicited adverse events that were considered possibly, probably, or 

definitely related to the trial procedures. Data is displayed as frequency per event (n) 

with mean duration in days (n (stdev)), separately for volunteers infected with NF135.C10 

(first column) or NF54 (second column) or volunteers that did not became positive by 

thick smear and PCR (third column). 

using PCR and rifin microsatellite mapping showed distinct genetic differences 

between the two strains (Figure 1). 

Controlled human malaria infection 

Ten Dutch malaria-naive volunteers were exposed to bites of mosquitoes 

infected with either NF135.C10 (n=5) or NF54 (n=5). Daily follow-up until day 21 

revealed positive thick smears in three out of five volunteers infected with 

NF135.C10 and four out of five volunteers infected with NF54 parasites. The 

remaining three smear-negative volunteers were presumptively treated 21 days 

post-infection (Figure 2A). 

All blood samples from these three smear negative volunteers were also 

negative for Pf as retrospectively assessed by qPCR. In Pf positive volunteers, 

kinetics of parasitemia of both strains were comparable to historical controls 

(n=48) infected with NF54 (grey area represents 95% CI, Figure 2B [26]). Patent 

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140 Chapter 7 

parasitemia for NF135.C10 seemed to occur slightly earlier than for NF54 as 

measured by thick smear (median of 7.0 days after infection; range 7.0-9.0 

versus median of 10.6; range 10.6-11; p=0.05; Mann-Whitney test) and qPCR 

(median day 7.0; range 6.3-7.0 versus median day 7.3; range 7.0-7.3; p=0.1 

Mann-Whitney test, Figure 2B). Particularly the peak of the first cycle seemed 

higher for NF135.C10 (Geometric Mean (GM) 1.2; 95% CI 0.61-2.4 parasites/µL) 

than NF54 (GM 0.16; 95% CI 0.055-0.46; p=0.06 Mann-Whitney test). The GM of 

peak parasitemia was 11 parasites/µL (95% CI 1.8-73) and 30 parasites/µL (95% 

CI 7.7-120) (p=0.4) for the positive volunteers of the NF135.C10 and NF54 

infected groups, respectively. PCR identity of both strains was confirmed by 

culture of smear-positive samples from several randomly selected infected 

volunteers. 

Safety 

All volunteers, including the smear-negative volunteers, reported solicited 

adverse events (AEs) that were considered possibly or probably related to the 

trial procedures (Table 2). All volunteers reported headache with a mean 

duration of 1.5 (NF135.C10) versus 2.3 (NF54) days and most volunteers 

experienced fatigue, myalgia and nausea. One volunteer infected with NF54 

reported grade three adverse events (malaise, headache and vomiting). 

Duration and frequency of AEs were not different between NF135.C10 and NF54 

infected volunteers in this limited number of volunteers. One infected volunteer 

of the NF135.C10 group showed a decreased platelet count of 146x10 9 /L at day 

3 after treatment (cut-off 150x10 9 /L), which returned to normal values at 

routine check-up on day 28. D-dimers did not increase in any of the volunteers 

before thick smear positivity. As can be expected, four volunteers had increased 

d-dimers (range 632-1100 ng/mL) during treatment, that decreased to normal 

levels after treatment. Highly sensitive troponin T values were always below 

0.05 µg/L. 

Cellular immune responses 

We next compared the induction of cellular recall immune responses between 

volunteers infected with NF135.C10 or NF54. In 24-hour in vitro stimulation 

assays, T-lymphocytes of volunteers successfully infected with either NF135.C10 

or NF54 showed similarly increased IFNγ, TNF and IL-2 responses 35 days after 

infection (Figure 3A-C). 

Furthermore, cellular responses showed the same kinetics for both homologous 

(NF135.C10 infected/NF135.C10 stimulated, NF54 infected/NF54 stimulated)



Figure 3. Production of IFNγ, TNF and IL-2 by CD3+ T-lymphocytes after in vitro restimulation 

with NF135.C10 or NF54 determined before and after challenge of 

volunteers. Time points displayed are: before challenge (C-1), five days after challenge 

(C+5), on day of treatment (DT) and 35 and 140 days after challenge. PBMCs were 

stimulated for 24-hour with asexual stage parasites (PfRBC) of NF135.C10 or NF54. 

Numbers of (A,D,G) IFNγ (B,E,H) TNF and (C,F,I) IL-2 producing cells are depicted as 

percentages of total T-lymphocytes. (A-C) Production of cytokines after homologous 

stimulation of cells taken before and after challenge. Cells of volunteers infected with 

NF135.C10 (open red circles) were stimulated with NF135.C10 PfRBC. Cells of volunteers 

infected with NF54 (closed black circles) were stimulated with NF54 PfRBC. (D-F) 

Production of cytokines after homologous (NF135.C10, closed red circles) or 

heterologous (NF54, open grey circles) stimulation of PBMCs of volunteers before and 

after challenge with NF135.C10. (G-I) Production of cytokines after homologous (NF54, 

closed black circles) or heterologous (NF135.C10, open grey circles) stimulation of PBMCs 

of volunteers before and after challenge with NF54. Symbols indicate individual values 

from volunteers (A-C) or represent group medians with interquartile range (D-I). 

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142 Chapter 7 

Figure 4. Contribution of different cell types to the total number of IFNγ-producing 

cells upon stimulation with either homologous or heterologous PfRBC. Mean 

percentage contribution to the total response are displayed for PBMCs of volunteers 

successfully infected with NF135.C10 (n=3) or NF54 (n=4) when re-stimulated in 

vitro with either NF135.C10 or NF54 PfRBC. Columns show mean percentage 

contribution on day 35 post-challenge of (A) NK cells (CD3 - CD56 + ), NK-T γδ-T + cells 

(CD3 + γδ-T + CD56 + ), NK-T cells (CD3 + γδ-T - CD56 + ), γδ-T cells (CD3 + γδ-T + CD56 - ), αβ-T 

CD4 cells (CD3 + γδ-T - CD4 + ), and αβ-T CD8 cells (CD3 + γδ-T - CD8 + ) and (B) effector 

memory (EM) cells (CD3 + , CD45RO + , CD62L - ), central memory (CM) cells (CD3 + , 

CD45RO + , CD62L + ) and naive T-lymphocytes (CD3 + , CD45RO - ). Responses to 

uninfected red blood cells are subtracted from responses to PfRBC per contributing 

cell type.



and heterologous (NF54 infected/NF135.C10 stimulated, NF135.C10 

infected/NF54 stimulated) in vitro re-stimulation (Figure 3D-I). IL-2 

responses seemed slightly higher when stimulated with NF135.C10 but this was 

similar for NF135.C10 and NF54 infected volunteers. IFNγ-producing cells were 

found in both the innate compartment (γδ-T, NK, NK-T) and the adaptive 

compartment (CD4 and CD8) and mainly displayed an effector memory 

phenotype (Figure 4). Despite inter-individual differences in relative contribution 

of various subsets, the composition of responding cells was highly consistent 

over time for every individual (data not shown). Upon homologous restimulation 

of cells on C+35, the vast majority of responding lymphocytes were 

single positive for IFNγ (mean 71%), TNF (9.4%) or double positive for IFNγ and 

TNF (18%). Additional responding lymphocytes were single positive for IL-2 

(0.40%), double positive IFNγ + IL-2 + (0.21%) or IL-2 + TNF + (0.61%) or triple 

positive (0.50%) (data not shown). 

Discussion 

We identified and characterized NF135.C10 as the first Pf clone of Asian origin 

for successful infection of malaria-naive human volunteers by CHMI. Clone 

NF135.C10 consistently produced gametocytes in culture, and was able to 

generate infections in laboratory-reared mosquitoes with high yields of 

sporozoites. NF135.C10 parasites were clearly distinct from NF54 parasites by 

drug sensitivity and established genetic marker profiles. Clinical presentation 

after CHMI and characteristics of PfRBC-specific recall (T-)lymphocyte responses 

in vitro were similar to NF54. 

Selection and identification of field strain parasites for CHMI poses technical 

difficulties, because of insufficient and unstable production of infectious sexual 

and sporogonic stages. For manufacturing purposes, cultures should ideally 

produce gametocytes that consistently infect at least 75% of the mosquitoes 

with at least ten oocysts resulting in 10-30 thousand sporozoites/mosquito. Only 

after extensive effort on more than seventy isolates were we able to successfully 

identify a parasite clone, NF135.C10, that met these criteria. Until now, the 

culture of NF135.C10 has only been re-started (using previous cryopreserved 

batches) seven times, as compared to 306 times for the NF54, and is therefore 

more closely related to the original clone. Also the geographical and molecular 

divergence from the NF54 strain makes this new clone an attractive candidate 

for use in CHMI trials. The vast majority of CHMI trials have been carried out 

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144 Chapter 7 

with either the NF54 strain or its clone 3D7 (~1300 volunteers [4, 27]) as 

opposed to only 42 volunteers challenged with strain 7G8 of South American 

origin [7, 8, 27]. 3D7 is derived from NF54 and cannot be distinguished from 

NF54 by drug sensitivity testing, simple genetic markers, or microsatellite 

mapping (Sim et al., unpublished). Thus, clinical trials with more genetically 

distinct parasite strains including 7G8 or NF135.C10 will be important to 

complement current knowledge on heterologous Pf strains. Parasitological and 

clinical findings after CHMI with NF54 or 3D7 were recently compared in two 

meta-analyses (Roestenberg et al. PLoS One, in press)[28]. Here we show that 

clinical signs and symptoms induced by NF135.C10 or NF54 do not show any 

differences in successfully infected volunteers. We found slight differences in 

infectivity of the parasites; in these small groups of volunteers, NF135.C10 

showed a marginally shorter prepatent period, but parasite kinetics of both 

NF135.C10 and NF54 were both within the limits of historical NF54 controls. 

Notably, not all volunteers exposed to NF54 infected mosquitoes became 

parasitemic, as assessed by both microscopy and qPCR in contrast to 22 previous 

CHMI trials infecting 128 naive volunteers with NF54 parasites (Roestenberg et 

al. PLoS One, in press). Unsuccessful infection after bites of five mosquitoes has 

been described previously for 3D7 [29, 30]. Although the exact reason is unclear, 

this might due to the unusual low NF135.C10 and NF54 oocyst and sporozoite 

counts per mosquito obtained for this trial compared to our routinely obtained 

counts (table 1). A technical disturbance in our cultures, leading to suboptimal 

culture circumstances prior to this study was most likely the reason for this 

relatively low mosquito infection with both strains. However, a formal 

relationship between sporozoite counts and mosquito infectivity has never been 

established [31] and only small percentages of sporozoites in salivary glands are 

injected during a blood meal of a mosquito [32-34]. Surprisingly, all three 

unsuccessfully infected volunteers reported AEs that were considered possibly, 

probably, or definitely related to the trial procedures. Symptoms reported by 

these volunteers might have been the result of over-reporting in an intense 

follow-up schedule, concurring with our findings from a previous study, in which 

volunteers reported malaria-related symptoms after exposure to bites of only 

uninfected mosquitoes [35]. 

Previously, we reported sustained IFNγ re-call responses in vitro in both αβ-T 

cells and γδ-T cells upon homologous PfRBC re-stimulation with NF54 asexual 

stage parasites after a single CHMI, suggestive of cross talk between innate and 

adaptive compartments with induction of memory [36, 37]. Here we show 

similar kinetics and composition of IFNγ responses upon homologous Pf54 and



Pf135.C10 re-stimulation. Notably, responses to heterologous re-stimulation 

were very similar to homologous responses, both in kinetics and magnitude. 

Considerable genetic diversity has been found in a number of important malaria 

antigens and vaccine candidates, especially in blood stages [38], but also in preerythrocytic 

stages [39]. Specific (conserved) antigens may be responsible for 

induction and maintenance of heterologous memory responses against Pf [40, 

41]; conserved epitopes have indeed been found in many immunogenic proteins 

[42-44], although some being cryptic and therefore not available for the immune 

system [45]. Albeit tested in only a small number of volunteers, (partial) 

protection has previously been reported after heterologous challenge infection 

following immunisation with radiation-attenuated sporozoites [10, 46, 47] or a 

previous experimental infection [48]. Whether the heterologous T-lymphocyte 

responses observed in our volunteers also translate into or represent crossstrain 

protective immunity in vivo remains to be investigated. 

The availability of NF135.C10 increases the portfolio of Pf parasites that can be 

used in CHMI. Obtaining additional multiple genetically distinct, fully 

characterized and sequenced Pf clones will be important in the evaluation 

process of diversity covering sub-unit vaccines or whole-parasite based vaccine 

approaches [4], intended to protect against all Pf parasite strains in nature [49]. 

Additionally, if immunization with whole sporozoite vaccines based on one 

single Pf strain does not fully protect against CHMI with heterologous Pf 

parasites, it will be necessary to determine whether immunization with 

sporozoites from a combination of clones are required to achieve such 

protection. Finally, to take advantage of our recent demonstration that CHMIs 

can also be successfully conducted by needle and syringe inoculation of aseptic, 

purified, cryopreserved Pf sporozoites called PfSPZ Challenge (Roestenberg et 

al., submitted), we have established the master and working cell banks, and 

manufacturing process required to produce PfSPZ Challenge using NF135.C10 

parasites (Sim et al., unpublished). 

In conclusion, increasing the portfolio of new Pf parasite strains, as achieved 

here for NF135.C10, will accelerate the evaluation of malaria vaccines 

candidates by facilitating the downstream selection process for further clinical 

vaccine development. Although more trials will be necessary to fine-tune the 

heterologous CHMI model with strain NF135.C10, the current results will boost 

the continued application of CHMIs as a crucial tool for malaria vaccine 

development. 

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146 Chapter 7 


We thank the volunteers for their enthusiastic participation in this trial and Kitty 

Suijk for her nursing support. We thank Laura Pelser, Jolanda Klaassen, Astrid 

Pouwelsen and Jacqueline Kuhnen for their work in the culturing and dissection 

of mosquitoes. We are indebted to all the slide readers in Leiden: Jan Kromhout, 

Jaco Verweij, Meriam Beljon, Jolanda van Schie, Jaqueline Schelfaut, Jeanette 

van der Slot, Heleen Gerritsma, Fons van der Sande, Eric Brienen, and Els van 

Oorschot. We thank Adriana Ahumada at Protein Potential, Jianbing Mu and Xinzhuan 

Su at Laboratory of Malaria and Vector Research, NIAID, NIH for the 

microsatellite mapping studies. Moreover, we thank Chris Janse, Shahid Khan 

and the malaria team for their hospitality in their laboratory in Leiden. We thank 

safety monitors Sandra Arend and Mark de Boer, and independent physician 

Frank Kroon for their continuing support. 

Funding 

This work was supported by of Top Institute Pharma [grant number T4-102]. ACT 

was funded by the European Malaria Vaccine Development Association, AS by a 

long term EMBO fellowship and KN by NWO Mozaiek [grant number 

017.005.011].



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Infect Immun 2008; 76:2660-70.

Chapter 8 

Induction of malaria in volunteers by 

intradermal injection of cryopreserved 

Plasmodium falciparum sporozoites 

Meta Roestenberg a,1 , Else M. Bijker a,1 , B. Kim Lee Sim b,c , Peter F. Billingsley b , Eric 

R. James b , Guido J.H. Bastiaens a , Anne C. Teirlinck a , Dunja Zimmerman a , Karina 

Teelen a , Theo Arens a , Pieter Beckers a , Annemieke Jansens a , Anja Scholzen a , 

Adrian J.F. Luty a,* , Krystelle Nganou-Makamdop a , Jorien Wiersma a , André J.A.M. 

van der Ven d , Anusha Gunasekera b , Adam Richman b , Sumana Chakravarty b , 

Soundarapandian Velmurugan b , Tao Li b , Anita Manoj b , Abraham G. Eappen b , 

Minglin Li c , Richard E. Stafford b,c , Cornelus C. Hermsen a , Robert W. Sauerwein a,2 , 

Stephen L. Hoffman b,2 1 contributed equally to this study 

2 contributed equally to this study 

a 

Radboud University Nijmegen Medical Center, Department of Medical 


b 

Sanaria Inc., Rockville, USA 

c 

Protein Potential LLC, Rockville, USA 

d 

Radboud University Nijmegen Medical Center, Department of General Internal 

Medicine, Nijmegen, The Netherlands 

* 

Current address: Institut de Recherche pour le Développement, UMR 216 Mère 

et enfant face aux infections tropicales, Paris, France/Faculté de Pharmacie, 

Université Paris Descartes, Sorbonne Paris Cité, France. 

Am. J. Trop. Med. Hyg. 2012; nov.13

152 Chapter 8 

Abstract 

Vaccines and new drugs are needed to prevent the approximately one million 

deaths and hundreds of millions of cases caused by Plasmodium falciparum (Pf) 

malaria annually. To test these vaccines and drugs, volunteers are infected by 

the bites of Pf sporozoite (SPZ)-infected mosquitoes under controlled conditions. 

Such infections are limited to the few centres with production of PfSPZ-infected 

mosquitoes. We assessed the capacity to infect volunteers by intradermal (ID) 

injection of aseptic, purified, vialed, cryopreserved PfSPZ (PfSPZ Challenge). 

Dutch volunteers received ID injections of PfSPZ that had been cryopreserved 

for 27-30 months. In a dose escalation study volunteers (N=6/group) received 

2,500, 10,000, or 25,000 PfSPZ. Detection of blood-stage parasites by 

microscopy was the primary outcome. Kinetics of parasitemia were 

retrospectively assessed by quantitative PCR. Fifteen of eighteen volunteers 

(84%) developed Pf parasitemia, 5/6 volunteers at each dose. Infection was safe. 

There were no differences between groups in time until parasitemia by 

microscopy and PCR, parasite kinetics, clinical symptoms and signs or laboratory 

values. 

Aseptic, purified, vialed, cryopreserved PfSPZ manufactured in compliance with 

regulatory standards and administered ID successfully infected volunteers. 

Following optimization of administration, such PfSPZ could be used to assess 

efficacy of vaccines and drugs in clinical trial centres worldwide. In addition 

PfSPZ Challenge can also be used for immunization strategies against malaria. 

For example, 100% protection against Pf infection in volunteers immunized by 

exposure to PfSPZ-infected mosquitoes while taking chloroquine was recently 

demonstrated. PfSPZ Challenge could be used to translate this artificial 

immunization protocol into an implementable vaccine.

Induction of malaria in volunteers by intradermal injection of cryopreserved 


Introduction 

Malaria caused by Plasmodium falciparum [Pf] causes approximately one million 

deaths and 250 million clinical cases annually [1-2]. Implementation of 

insecticide impregnated bednets, residual insecticide spraying, and 

combinations of antimalarial drugs, has reduced malaria associated morbidity 

and mortality in many areas [1]. Questions related to sustainability of this effort, 

however, have led to a recent delineation of requirements for new tools [3]. A 

safe, long-acting anti-malarial drug and a highly effective malaria vaccine would 

be powerful tools for control and elimination of Pf malaria. 

Progress has been facilitated by the capacity to infect volunteers under 

controlled conditions in order to test new vaccines and drugs. Volunteers are 

infected by exposure to laboratory-reared Anopheles spp. mosquitoes 

transmitting Pf sporozoites (SPZ) [4], which was first introduced for treatment of 

neurosyphilis in the 1920s [5]. The development of drugs such as chloroquine 

[6], primaquine [7], and atovaquone [8] were facilitated by these controlled 

human malaria infections. The ability to culture Pf gametocytes [9-10] enhanced 

the capacity to produce infected mosquitoes for controlled human malaria 

infection (CHMI) studies. Although potentially serious or even lethal, Pf malaria 

can be radically cured at the earliest stages of blood infection when risks of 

complications are virtually absent. CHMIs are restricted to a few specialized 

centres that can produce PfSPZ-infected mosquitoes, where more than 1,300 

volunteers have been safely infected by the bite of PfSPZ-infected mosquitoes 

since 1986, primarily for clinical trials of malaria vaccines [4, 11-15], but also for 

trials of drugs [8] and diagnostic tests [16], and studying human immune 

responses to Pf [17]. 

In addition to the use of CHMIs for testing vaccines and drugs, controlled 

infections can also be used to immunize against malaria. For example, 

immunization with radiation-attenuated PfSPZ by bites of mosquitoes protects > 

90% of volunteers [18-20]. And recently 100% protection against CHMI was 

achieved by immunization of volunteers with PfSPZ by mosquito bites while 

taking chloroquine [21-22]. 

These highly protective immunization strategies have not been translated into 

an implementable vaccine, because they require inoculation of SPZ by mosquito 

bites. Inoculation of SPZ by injection would be a more feasible method and was 

performed through the early 1950s. The SPZ preparations used, however, were 

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contaminated with bacteria and rates of infection with frozen and thawed SPZ 

were highly variable [23-26]. A contemporary approach to production of SPZ for 

vaccination requires generating aseptic SPZ-infected mosquitoes, purifying SPZ 

from mosquito tissues, vialing, preserving, and administering the SPZ by needle 

and syringe. Sanaria has met these requirements to produce an attenuated 

(irradiated) PfSPZ vaccine [27-28] and infectious (non-irradiated) aseptic, 

purified, vialed, cryopreserved PfSPZ (PfSPZ Challenge). Here we report infection 

of volunteers with PfSPZ Challenge administered intradermally (ID) by needle 

and syringe. 

Methods 

Study population and study design 

This open label, Phase 1 clinical trial was performed at Radboud University 

Nijmegen Medical Centre, the Netherlands, from October 2010 to July 2011. 

Volunteers aged 18-35 years were screened for eligibility by assessing medical 

history, physical examination, haematological, biochemical, urine toxicology, and 

pregnancy tests, and malaria, HIV, hepatitis B and hepatitis C serology. The main 

exclusion criteria were pregnancy, residence in a malaria endemic area within 

the previous six months, positive Pf serology, symptoms, physical signs or 

laboratory test results suggestive of systemic disorders, and history of drug or 

alcohol abuse interfering with normal social function. All volunteers gave written 

informed consent. 

Eighteen healthy malaria-naïve volunteers were recruited for this trial. Groups of 

six volunteers were injected ID with 2,500, 10,000, or 25,000 PfSPZ Challenge. 

The sample size of six per group had a power of 75% to show a difference 

between 2 of 6 volunteers infected in the 2,500 PfSPZ group and 6/6 volunteers 

infected in the 25,000 PfSPZ group. Dose escalation was done at a minimum 

interval of 3.5 weeks. 

The trial was performed in accordance with Good Clinical Practice and an 

Investigational New Drug application filed with the U.S. Food and Drug 

Administration, and approved by the Central Committee for Research Involving 

Human Subjects of The Netherlands (CCMO NL31858.091.10). Clinicaltrials.gov 

identifier: NCT 01086917.



Figure 1. Liver-stage Pf parasite expressing PfMSP-1 after six days in culture. PfSPZ 

from the lot of PfSPZ Challenge used in this clinical trial were used to infect a human 

hepatocyte line in vitro (see Table S1 for methods). Six days later the parasites were 

stained with an antibody to PfMSP-1. PfMSP-1 is the major protein on the surface of 

merozoites, and is required for merozoite invasion of erythrocytes. It is only 

expressed in mature liver stage parasites and blood stage parasites, and is required 

for the survival of blood stage parasites. Potency of lots of PfSPZ Challenge is 

assessed by expression of PfMSP-1 in vitro (see Table S1). Image captured with a 

Zeiss LSM510 META laser scanning confocal microscope. 

Study treatment (PfSPZ Challenge) 

PfSPZ Challenge contains aseptic, purified, cryopreserved PfSPZ isolated from 

salivary glands of aseptically reared mosquitoes [28]. A. stephensi mosquitoes 

were raised under aseptic conditions, then fed on cultured Stage V gametocytes 

of the NF54 strain of Pf [38]. Approximately two weeks later, mosquito salivary 

glands containing PfSPZ were dissected, and PfSPZ were purified, formulated, 

vialed (15,000 PfSPZ per vial) and cryopreserved in liquid nitrogen vapour phase 

at -140°C to -196°C [28]. PfSPZ Challenge released for clinical use met quality 

control specifications including sterility (USP 71 compendial assay), purity (Fig. 

S1), and potency (Table S1). The latter was assessed by quantification of late 

liver stage parasites expressing Pf merozoite surface protein 1 (PfMSP-1) [39] in 

cultured human hepatocytes (HC-04 cells) six days after addition of PfSPZ (Figure 

1, Table S1). Membrane integrity of PfSPZ was used to assess cell viability (Table 

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156 Chapter 8 

S1). The lot of PfSPZ Challenge used in this study had been cryopreserved for 27 

(dose of 2,500 PfSP)-30 months (dose of 25,000 PfSPZ) before administration. 

Immediately prior to use, a vial of PfSPZ Challenge was thawed and diluted with 

phosphate buffered saline (PBS) containing human serum albumin (HSA). 

Volunteers were injected within 30 minutes of thawing. 

Controlled human malaria infection (CHMI) 

Three groups of six volunteers each were injected ID with PfSPZ Challenge over 

the deltoid muscle, one injection in each upper arm. Each injection of 50 μl 

contained half the total dose. After injection, volunteers were observed for at 

least 60 minutes. Inoculations of volunteers were spaced 60 minutes apart. In 

each dose group, two volunteers were inoculated three days prior to the 

remaining four volunteers. 

Volunteers made at least one daily outpatient clinical visit beginning five days 

after inoculation of PfSPZ Challenge. All symptoms and signs (solicited and 

unsolicited) were recorded and graded by the attending physician as follows: 

mild (easily tolerated), moderate (interferes with normal activity), or severe 

(prevents normal activity); fever was recorded as grade 1 (>37.5°C – 38.0°C), 

grade 2 (>38.0°C – 39.0°C) or grade 3 (>39.0°C). Haematological and biochemical 

parameters were monitored daily. Because of a previous cardiac related serious 

adverse event following CHMI with Pf infection [40], markers of cardiac damage 

and coagulation were assessed. Troponin, lactate dehydrogenase, platelets, and 

d-dimer were assessed daily during the period when blood stage parasitemia 

was expected, and for three days after initiating curative treatment with 

atovaquone/proguanil. If d-dimer or LDH were abnormal, blood samples were 

tested for fragmentocytes and Von Willebrand cleaving protease activity, as 

markers for vascular endothelial cell activation [41]. Final follow-up visits were 

on days 35 and 140 after infection. 

As soon as parasites were detected by microscopic examination of blood smears, 

volunteers were treated with atovaquone/proguanil (1000/400 mg) 

administered orally once daily for three days. Complete cure was confirmed in 

all volunteers by two consecutive parasite-negative blood-slides after treatment. 

Volunteers who did not develop parasitemia by day 21 after challenge were 

presumptively treated with the same regimen.



Thick Smear qPCR 

Volunteer Pre patent Parasite qPCR positive Parasite Parasite 

code period (day) density at (day) density at first density by 

diagnosis 

day positive qPCR at time of 

(Pf/μl) 

(Pf/μl) diagnosis by 

thick smear 

(Pf/μl) 

Group 1 – 2,500PfSPZ 

696-18 12.3 4 9.6 0.08 5 

711-08 14.0 16 12 0.16 71 

795-06 N/A N/A N/A N/A N/A 

935-01 14.0 124 10.6 0.03 89 

937-20 12.3 6 10.6 0.12 43 

940-14 12.3 6 10.3 0.06 35 

Geom. mean 13.0 12 10.59 0.1 35 

# of positives 5/6 

Group 2 – 10,000 PfSPZ 

119-03 12.6 24 9.6 0.68 6 

603-11 13.0 8 11 0.17 2 

736-04 11.0 6 9.6 0.04 3 

783-25 13.3 6 10.6 0.03 15 

788-21 14.0 26 11 1.12 6 

925-26 N/A N/A N/A N/A N/A 

Geom. mean 12.7 11 10.34 0.2 5 

# of positives 5/6 

Group 3 – 25,000 PfSPZ 

647-30 14.0 512 9.3 0.32 759 

720-13 12.3 6 10.3 0.32 162 

789-15 N/A N/A N/A N/A N/A 

806-09 12.3 8 9 0.25 48 

909-29 14.3 48 11.3 0.13 102 

926-24 12.3 6 10 0.19 68 

Geom. mean 13.0 23 9.95 0.2 132 

Table 1. Parasitemia data by thick blood smear and quantitative polymerase chain 

reaction (Q-PCR) 

N/A: not applicable; thick-smear negative volunteers were presumptively treated on day 

21 after infection. 

Outcomes 

The primary outcome was occurrence of Pf parasitemia detected by microscopic 

examination of blood smears. Sampling was done twice daily on days 5 and 6 

post-inoculation, thrice daily on days 7-11, twice daily on days 12-15, once daily 

on days 16-21, and for two days after initiation of treatment for positive smears. 

To make thick blood smears, 15 μL of EDTA-anti-coagulated blood was spread on 

one well of a 3-well glass slide (CEL-LINE Diagnostic Microscope Slides, 30-12A- 

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158 Chapter 8 

Figure 2. Parasite density as measured by Q-PCR in the 2,500 (A), 10,000 (B) and 25,000 

(C) PfSPZ Challenge dose groups. Panels A, B and C show geometric mean parasite 

density of positive volunteers per group with confidence intervals (N=5 for all groups) 

from day of inoculation through last day of positivity after initiation of treatment. Panel 

D shows an overlay of geometric mean parasite densities of positive volunteers in each 

group. 

black-CE24). After drying, wells were stained with Giemsa for 45 minutes, and 

examined at 1000× magnification to assess 0.5 μL of blood. The smear was 

scored as positive if two unambiguous parasites were found. Thus, volunteers 

could be diagnosed with as few as 4 parasites/μL of blood. Pre-patent period 

was defined as the period between inoculation of PfSPZ Challenge and 

appearance of first positive blood smear. 

Retrospectively, parasitemias were determined by real-time quantitative PCR 

(qPCR), performed on all samples collected after challenge, as previously 

described [42]. The sensitivity of qPCR was 20 parasites/mL of blood. 


Data analysis was performed using SPSS software version 16.0. Q-PCR results 

were assessed by ANOVA on log-transformed data.



Figure 3. Number of possibly, probably, or definitely related solicited and unsolicited 

adverse events reported over time in the 2,500 (black ), 10,000 (red ) and 25,000 PfSPZ 

Challenge dose (green ) groups. 

Results 

Parasitemia after injection of PfSPZ Challenge 

Thirty-six healthy, malaria-naïve volunteers were screened and eighteen were 

included. All volunteers completed follow-up (Figure S2). After ID injection of 

PfSPZ Challenge, 15 of the 18 volunteers developed a positive blood smear for 

Pf, five of six volunteers from each group (Table 1). The slide-negative volunteers 

in each group were presumptively treated with atovaquone/proguanil at 21 days 

post-infection. 

Blood slides were first positive 11 to 14.3 days after administration of PfSPZ 

Challenge. The geometric mean (GM) pre-patent period was similar for all 

groups i.e. 13.0, 12.7, and 13.0 days for the groups receiving 2,500, 10,000, and 

25,000 PfSPZ Challenge, respectively (ANOVA p=0.92). The GM parasite densities 

by microscopy at the time of diagnosis were 12.4, 11.2, and 23.4 parasites/μL 

blood (ANOVA p=0.69 on log-transformed data) (Table 1). 

Quantitative PCRs (qPCRs) were first positive 9⋅0 to 12 days after challenge 

(Table 1). Volunteers in the 2,500, 10,000, and 25,000 PfSPZ Challenge groups 

had similar GM times to first detection of parasites by qPCR of 10.6, 10.3, and 

9.9 days (ANOVA p=0.486) at a GM parasite density of 0.07, 0.2 and 0.2 

parasites/μL blood (ANOVA p=0.24), respectively. The GM parasite densities by 

PCR at the time of thick smear diagnosis were 35, 5, and 132 parasites/μL 

(ANOVA p=0⋅23). Thick smear and qPCR were negative throughout the 21-day 

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160 Chapter 8 

Adverse-event 

Number of 

volunteers 

2,500 (n=6) 10,000 (n=6) 25,000 (n=6) 

Mean 

Duration 

±SD (days) 

Number of 

volunteers 

Mean 

Duration 

±SD (days) 

Number of 

volunteers 

Mean 

Duration 

±SD (days) 

Abdominal pain 1 2.9 1 0.04 2 0.3±0.1 

Arthralgia 0 N/A 0 N/A 0 N/A 

Chest pain 1 0.04 0 N/A 0 N/A 

Chills 1 2.0 2 0.3±0.2 2 0.9±0.6 

Diarrhea 0 N/A 0 N/A 1 0.8 

Fatigue 5 2.9±3.3 3 2.5±1.7 5 3.0±3.9 

Fever 3 1.6±1.5 2 1.8±0.6 4 0.8±0.4 

Headache 6 1.1±1.1 6 1.5±1.6 6 1.4±2.6 

Malaise 2 2.2±2.4 5 1.8±1.4 1 0.7 

Myalgia 2 3.7±3.2 2 1.3±0.5 2 0.8±0.1 

Nausea 3 1.7±1.3 5 0.9±0.9 3 1.0±0.9 

Vomiting 0 N/A 2 0.01±0.0 0 N/A 

Any 6 2.0±1.4 6 1.1±0.8 6 1.1±1.0 

Grade 3 adverse event 

Fatigue 0 N/A 0 N/A 1 2.2 

Fever 0 N/A 1 1.2 0 N/A 

Headache 2 3.0±0.4 0 N/A 0 N/A 

Malaise 1 4.8 0 N/A 1 0.1 

Vomiting 0 N/A 2 0.01±0.0 0 N/A 

Any 2 3.9±0.2 3 0.6±0.0 2 1.2±0.0 

Table 2. Numbers of volunteers reporting solicited adverse events possibly, probably, or 

definitely related to administration of PfSPZ Challenge, with mean duration of events 

N/A: not applicable 

follow-up for the three slide-negative volunteers. Parasite growth was cyclical, 

and was similar in all dose groups (Figure 2). 

Safety. 

All volunteers, including the three volunteers who did not develop parasitemia, 

reported solicited adverse events (AEs) considered possibly, probably, or 

definitely related to the trial procedures (clinical malaria) (Table 2). Headache 

was the most frequently reported AE, and occurred in all volunteers including 

the three who did not develop parasitemia. There were no significant 

differences among the groups in solicited AEs, which were most frequently 

reported between days 12 and 18 post-injection. The total number of solicited 

and unsolicited AEs reported over time is shown in Figure 3. 

Routine daily laboratory tests showed no clinically significant abnormalities 

before initiation of anti-malarial treatment. Three or four days after receiving 

the first dose of atovaquone/proguanil, four volunteers had thrombocyte levels 

in the range 78-95 × 10 9 /L, which was below the lower limit of normal (120 ×



10 9 /L). In thirteen volunteers, D-dimers were above 500 ng/mL, the upper limit 

of normal (ULN), at one or two days after initiation of anti-malarial treatment 

(range of peaks: 540 to 10,200 ng/mL). In all volunteers, D-dimer levels 

normalized without complications. One volunteer had abnormal liver function 

tests at day 2 post atovaquone/proguanil initiation. Maximum values were 526 

units/L ASAT (ULN 40 units/L), 745 units/L ALAT (ULN 45 units/L), 777 units/L 

LDH (ULN 450 units/L), and 74 units/L γGT (ULN 50 units/L). Bilirubin and alkaline 

phosphatase were normal. Abnormal values had returned to baseline levels at 

day 100 after infection. 

One serious adverse event (SAE) occurred in a volunteer who reported chest 

pain one day after the first dose of atovaquone/proguanil. Based on medical 

history, the chest pain was initially considered possibly consistent with angina 

pectoris. Pain resolved within one hour without treatment. The volunteer was 

admitted to the cardiac care unit for monitoring for 6.5 hours. The first 

electrocardiogram (ECG) had a negative T-wave in V2, which was absent at time 

of study initiation. All subsequent ECGs, beginning 2.5 hours after the first ECG, 

were comparable to baseline, with a negative T in V1 only. Troponin T levels 

were normal at the time of chest pain, 6 and 17 hours later, daily for the next 

three days and at trial days 28 and 35. As per protocol, the trial was put on hold, 

and the event was reported to the Safety Monitoring Committee (SMC) and 

regulatory authorities. The SMC concurred with the PI’s attribution of the chest 

pain as “possibly related” to participation in the trial. The SMC concluded that 

while the cause of chest pain was not clear, the clinical data suggested that the 

SAE was not a serious cardiac event, and recommended resumption of the trial 

within three days of the event. The regulatory authorities concurred. 

Discussion 

We report for the first time that healthy, malaria-naïve volunteers can be 

infected with P. falciparum malaria by injection of aseptic, purified, 

cryopreserved PfSPZ manufactured in compliance with regulatory standards. 

Five of six volunteers became infected when 2500, 10,000 or 25,000 PfSPZ were 

inoculated ID. AEs were comparable with those in mosquito bite challenge trials 

[29-31]. 

In the 1950s Jeffery and colleagues were able to infected volunteers with 

cryopreserved PfSPZ [20]. They cryopreserved PfSPZ-infected salivary glands and 

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162 Chapter 8 

inoculated the thawed salivary glands from 2.5 to 15 mosquitoes intravenously 

and achieved Pf parasitemia in 13 of 14 volunteers. The only volunteer who did 

not develop Pf parasitemia received salivary glands from 2.5 PfSPZ-infected 

mosquitoes, the lowest dose. Because the preparations were likely highly 

contaminated with bacteria they administered penicillin concomitantly with 

PfSPZ-infected salivary gland preparations. 

Since 1986 CHMIs have been performed by exposing volunteers to bites of 

laboratory-reared mosquitoes infected by feeding on Pf gametocyte-infected 

erythrocytes grown in culture [11]. When volunteers are challenged by bites of 

five PfSPZ-infected mosquitoes, essentially all develop Pf parasitemia [4, 11, 29, 

31]. When numbers are reduced to one or two mosquitoes success rates drop to 

50% [30, 32]. Recently, 5 of 6 volunteers became infected when bitten by one 

aseptic, PfSPZ-infected mosquito produced in compliance with regulatory 

standards [33]. ID inoculation of 2,500 cryopreserved PfSPZ Challenge in the 

current study was at least as effective as the bites of 1-2 infected mosquitoes in 

infecting volunteers. 

The capacity to infect volunteers with PfSPZ Challenge is a function of the 

infectiousness of the cryopreserved PfSPZ and the efficiency of administration. 

We use in vitro assays of potency and viability to estimate infectiousness of fresh 

vs. cryopreserved SPZ. These in vitro assays predicted a maximum difference of 

25-30% (Table S1). Rodent model in vivo data, however, suggested that the in 

vitro assays underestimate the reduction in infectivity associated with SPZ 

cryopreservation (Table S2). 

Increasing the dose of PfSPZ Challenge from 2,500 PfSPZ to 25,000 PfSPZ 

administered ID neither increased the percentage of infected volunteers nor 

reduced time until blood stage parasites were detectable. Parasite density at 

diagnosis was lower in the 10,000 PfSPZ group compared to both the lower and 

the higher dose groups. This is probably a reflection of natural variability 

between the volunteers. Apparently, increasing the dose did not result in higher 

numbers of PfSPZ developing in hepatocytes, and invading erythrocytes. A 

possible explanation for this lack of dose response may be trapping of PfSPZ at 

the inoculation site. Mosquitoes deposit SPZ in the dermis and subcutaneous 

tissues in much smaller volumes (< 0.5 μL) than the inocula used in this study (50 

μL). This interpretation is consistent with recent studies that showed that 

approximately 90% of P. yoelii sporozoites did not reach the liver when 

administered ID [34]. Additionally, mosquitoes also inoculate SPZ directly into



the circulation [35]. The fact that Jeffery et al. achieved infection in 13 of 14 

volunteers by intravenous injection of PfSPZ-infected salivary glands [25], and 

the only negative case was at the lowest does of salivary glands, supports this 

interpretation. To closer mimic the SPZ delivery of mosquitoes, administration of 

PfSPZ Challenge will be optimized by modifying route of administration (e.g. ID, 

subcutaneous, intramuscular, intravenous), inoculation volume, numbers of 

inoculations, and sites of injection. Our sample sizes of 6 per group, in this firstin-humans 

study, were not powered to be able show a modest difference 

between groups. Thus, another explanation for the lack of difference in 

infectivity or prepatent period between as the dose was increased 10-fold from 

2,500 to 25,000 PfSPZ is that that sample sizes were too small to detect small 

differences between groups. However, they should have been adequate to 

detect a 10-fold difference, if the PfSPZ actually reached the bloodstream. Since 

there was no difference as we increased the dose, it is possible that 2,500 PfSPZ 

was above the threshold necessary to achieve infection, and even lower doses 

might be equally successful. 

Once administration of PfSPZ Challenge is optimized, the global capacity to 

conduct CHMIs can be further expanded including sites in malaria endemic 

areas. This will be critical for meeting the demand to assess the increasing 

numbers of malaria vaccine candidates and anti-malarial drugs in development 

[36-37]. Comparative analysis of CHMI by mosquito bite has shown that 

variability of parasites may lead to significant variation in the primary outcome 

variables between sites. By using needle administration of defined quantities of 

PfSPZ Challenge from the same lot, variation in infectivity in CHMI will be 

reduced, allowing comparisons of parallel and sequential clinical trials at 

multiple sites, including in malaria endemic areas. 

In addition to its use in CHMI, needle and syringe administration of 

cryopreserved SPZ is critical for development of whole SPZ vaccines. PfSPZ 

protective efficacy in humans was originally established by controlled exposure 

of volunteers to bites of irradiated PfSPZ-infected mosquitoes [18-20]. The 

potential impact of a radiation-attenuated PfSPZ vaccine was reappraised and 

significant progress has been made in manufacturing and clinically testing such a 

vaccine [27-28]. The aseptic, purified, cryopreserved PfSPZ Vaccine is 

manufactured identically to PfSPZ Challenge, except that the parasites in the 

vaccine are attenuated by irradiation and cannot fully develop in the liver. In the 

first clinical trial the PfSPZ Vaccine was administered ID or subcutaneously. It 

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was safe and well tolerated, but immunogenicity and protective efficacy were 

not nearly to the levels found after mosquito bite immunization [28]. The data 

reported herein on PfSPZ Challenge administered ID demonstrated that the 

manufacturing process produces viable and potent SPZ. These data, along with 

non-human primate immunization studies with PfSPZ Vaccine, informed the 

design of the next clinical trial of the PfSPZ Vaccine, which is being administered 

by intravenous injection [28]. 

Another approach to whole SPZ vaccination was recently established [21-22]. 

Healthy malaria-naïve volunteers were protected for 28 months against CHMI 

after being immunized by exposure to bites of PfSPZ-infected mosquitoes while 

taking the antimalarial drug chloroquine. This protection was induced using > 

20-fold fewer PfSPZ-infected mosquitoes [36 to 45] than needed for protection 

with radiation attenuated PfSPZ (> 1000) [21-22]. PfSPZ Challenge can replace 

mosquito bites, allowing for rapid translation of this experimental research 

finding into a potential vaccine. We are planning the first clinical trials of PfSPZ 

Challenge administered to volunteers taking chloroquine for 2012. 

In summary, we have established that aseptic, purified, vialed, cryopreserved 

PfSPZ (PfSPZ Challenge) are infectious to humans for at least 2.5 years after 

cryopreservation. These data provide the rationale and foundation for a clinical 

trials program aimed at significantly increasing the scale of CHMI in clinical trials 

of malaria vaccines and new drugs, and producing, testing, and licensing a highly 

effective, long-acting PfSPZ malaria vaccine. 


We thank the trial volunteers, the staff from the Clinical Research Centre 

Nijmegen and the staff from the RUNMC Pharmacy who made this study 

possible; Wendy Arts, Nanny Huiberts, Chantal Siebes, Marlou Kooreman, Paul 

Daemen and Ella Driessen for reading many thick smears; and Dr. Gheorghe Pop 

for his cardiac monitoring of the trial volunteers. We thank Bas van Haren for his 

support with the ADAMTS13 measurements and Jody van den Ouweland for the 

performance of the Troponin T measurements. We thank the members of the 

Safety Monitoring Committee, Dr. Barney Graham, Dr. David Diemert and Dr. 

Alexander Rennings for their participation and for their guidance and safety 

recommendations throughout the trial. We thank the entire Sanaria 

Manufacturing Team including Gametocyte Production: Yonas Abebe, Asha Patil, 

Yeab Getachew, Mark Loyvesky, Bingbing Deng; Mosquito Production: Steve



Matheny, Yingda Wen, Keith Nelson, James Overby, Virak Pich, Thomas Mitchell; 

Dissection: Richard Fan; Purification: Lixin Gao, Ray Xu; Formulation and 

Cryopreservation: Adam Ruben, Aderonke Awe, Quality Assurance and Quality 

Control: Irina Belyakova, Mary King; Assays: Chinnamma Chakiath; Rana 

Chattopadhyay, Charles Anderson, Solomon Conteh, Gwynne Roth; Regulatory 

and Clinical Affairs: Tooba Murshedkar. We are grateful for Sanaria’s Operations 

and Legal teams, especially Robert Thompson, Alexander Hoffman, and David 

Dolberg. We thank the entire Protein Potential Team including, Adriana 

Ahumada, Maria Socorro Orozco, Bing Jiang, Emily Chen. We also thank MSource 

(Belgium, now CromSource, Italy) for clinical monitoring. 

Funding 

Manufacturing of PfSPZ Challenge and this clinical trial were funded by 

TIPharma, grant number T4-102. ACT is funded by the European Malaria Vaccine 

Development Association, KNM was funded by a Mozaiek grant from the 

Netherlands Organisation for Scientific Research. The development and 

manufacturing of PfSPZ Challenge was also supported by Small Business 

Innovation Research (SBIR) grants R44AI058375-03,04,05,05S1 from NIAID/NIH 

and through Agreement 07984 from the PATH Malaria Vaccine Initiative. 

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34. Inoue M & Culleton RL The intradermal route for inoculation of sporozoites of 

rodent malaria parasites for immunological studies. Parasite Immunol. 2011; 

33:137-142. 

35. Fairley NH. Sidelights on malaria in man obtained by subinoculation 

experiments. Trans R Soc Trop Med Hyg 1947; 40:621-76. 

36. Alonso PL, Djimde A, Kremsner P, et al. A research agenda for malaria 

eradication: drugs. PLoS Med 2011;8:e1000402. 

37. WHO (2011) Malaria Vaccine Rainbow Tables. 

http://www.who.int/vaccine_research /links/Rainbow/en/index.html. 

38. Ponnudurai T, Lensen AHW, Van Gemert GJ, Bensink MPE, Bolmer M, 

Meuwissen JH. Infectivity of cultured Plasmodium falciparum gametocytes to 

mosquitoes. Parasitol 1989; 98:165-73. 

39. Holder AA. The carboxy-terminus of merozoite surface protein 1: structure, 

specific antibodies and immunity to malaria. Parasitology 2009; 136:1445-56. 

40. Nieman AE, de Mast Q, Roestenberg M, et al. Cardiac complication after 


41. de Mast Q, Groot E, Lenting PJ, et al. Thrombocytopenia and release of 

activated von Willebrand Factor during early Plasmodium falciparum malaria. J 

Infect Dis 2007; 196:622-8. 


malaria parasites in vivo by real-time quantitative PCR. Mol. Biochem. 

Parasitol.2001;118:247-251.



Supplementary Data 

Time point Potency 

(# of parasites expressing 

PfMSP-1/well) 

% Viability 

(sporozoite membrane 

integrity assay) 

Fresh 27 ± 4.6 ND* 

Release 20 ± 1.7 83.3% ± 6.5% 

6 Month 18 ± 2.1 86.6% ± 1.9% 

9 Month 20 ± 2.1 83.7% ± 8.4% 

12 Month 21 ± 1.5 84.8% ± 3.0% 

18 Month 20 ± 0.6 83.7% ± 4.2% 

24 Month 18 ± 1.0 86.0% ± 1.5% 

Pre-1st clinical dose (26 

Month) 

17 ± 0.6 79.4% ± 6.5% 

Post-last clinical dose (30 

Month) 

16 ± 2.6 87.4% ± 1.9% 

Table S1. Results of potency and sporozoite membrane integrity assays on 

the lot of PfSPZ 

* Not done 

Date Status of PySPZ Viability 

(SMIA) 

Number of PySPZ 

Inoculated (IV) 

ID50 (Number of 

PySPZ) 

Oct 2009 fresh 96.3% 24-12-6-3 8.9 

Dec 2009 cryopreserved 72.7% 200-100-50-25 33.1 

Dec 2009 cryopreserved 68.3% 200-100-50-25 62.1 

Jan 2010 cryopreserved 67.7% 400-200-100-50-25 103.8 

Feb 2010 cryopreserved 67.1% 400-200-100-50-25 55.2 

Feb 2010 cryopreserved 71.6% 400-200-100-50-25 107 

Feb 2010 cryopreserved 73.9% 400-200-100-50-25 34.5 

Mean cryopreserved 70.2% 66.0 

Difference between fresh 

and cryopreserved PySPZ 

26.1% 7.4-fold 

Table S2. Effect of cryopreservation on sporozoite membrane integrity and infectivity 

in mice inoculated intravenously with the same lot of P. yoelii sporozoites. Infectivity 

was the number of sporozoites required to infect 50% of BALB/c mice. 

169

170 Chapter 8 

Before Purification (14 x 10 6 PfSPZ/ml) After Purification (1.6 x 10 6 PfSPZ/ml) 

Figure S1. PfSPZ before and after purification. Photomicrograph (200 x) of the PfSPZ in 

the lot of PfSPZ Challenge used in the clinical trial prior to purification and after 

purification. The purification process reduced the amount of salivary gland material in 

the PfSPZ samples by greater than 99.9%. 

Figure S2: Clinical trial profile

Section 3 

Whole parasite inoculation

Chapter 9 

Protection against a Malaria Challenge by 

Sporozoite Inoculation 

Meta Roestenberg 1 *, Matthew McCall 1 *, Joost Hopman 1 , Jorien Wiersma 1 , 

Adrian J.F. Luty 1 , Geert Jan van Gemert 1 , Marga van de Vegte-Bolmer 1 , Ben van 

Schaijk 1 , Karina Teelen 1 , Theo Arens 1 , Lopke Spaarman 1 , Quirijn de Mast 2 , Will 

Roeffen 1 , Georges Snounou 3 , Laurent Rénia 4 , Andre van der Ven 2 , Cornelus C. 

Hermsen 1 , Robert Sauerwein 1 

*These authors contributed equally 

1 

Department of Medical Microbiology Radboud University Nijmegen Medical 

Center, Nijmegen, the Netherlands 

2 

Department of General Internal Medicine, Radboud University Nijmegen 

Medical Center, Nijmegen, the Netherlands 

3 

Department of Parasitology, INSERM Unité 511, Hôpital Pitié– Salpêtrière, and 

the Université Pierre et Marie Curie, Paris, France 

4 

Laboratory of Malaria Immunobiology, Singapore Immunology Network, 

Agency for Technology and Research, Biopolis, Singapore [L.R.). 

N Engl J Med 2009; 361:468-77.

174 Chapter 9 

Abstract 

An effective vaccine for malaria is urgently needed. Naturally acquired immunity 

to malaria develops slowly, and induction of protection in humans can be 

achieved artificially by the inoculation of radiation-attenuated sporozoites by 

means of more than 1000 infective mosquito bites. 

We exposed 15 healthy volunteers — with 10 assigned to a vaccine group and 5 

assigned to a control group — to bites of mosquitoes once a month for 3 months 

while they were receiving a prophylactic regimen of chloroquine. The vaccine 

group was exposed to mosquitoes that were infected with Plasmodium 

falciparum, and the control group was exposed to mosquitoes that were not 

infected with the malaria parasite. One month after the discontinuation of 

chloroquine, protection was assessed by homologous challenge with five 

mosquitoes infected with P. falciparum. We assessed humoral and cellular 

responses before vaccination and before the challenge to investigate correlates 

of protection. 

All 10 subjects in the vaccine group were protected against a malaria challenge 

with the infected mosquitoes. In contrast, patent parasitemia (i.e., parasites 

found in the blood on microscopic examination) developed in all five control 

subjects. Adverse events were mainly reported by vaccinees after the first 

immunization and by control subjects after the challenge; no serious adverse 

events occurred. In this model, we identified the induction of parasite-specific 

pluripotent effector memory T cells producing interferon-γ tumour necrosis 

factor α, and interleukin-2 as a promising immunologic marker of protection. 

In conclusion, protection against a homologous malaria challenge can be 

induced by the inoculation of intact sporozoites.

Protection against a Malaria Challenge by Sporozoite Inoculation 175 

Introduction 

Malaria is responsible for a significant burden of morbidity and mortality in the 

developing world and an effective vaccine against this disease is urgently 

required [1]. Despite decades of research a licensed vaccine is still not available, 

largely because immunity to Plasmodium falciparum malaria is considered 

difficult to acquire, whether through natural exposure or artificially through 

vaccination. A further critical factor is our incomplete understanding of precisely 

what constitutes protective anti-malarial immunity in humans. 

The possibility of vaccinating humans against P. falciparum malaria was raised 

originally by the success of the radiation-attenuated sporozoite model 

developed several decades ago [2,3]. Irradiation of infectious mosquitoes 

disrupts the gene expression of sporozoites, which remain capable of 

hepatocyte invasion but no longer of complete liver-stage maturation or 

progression to the pathogenic blood-stage [4]. Infection of human volunteers 

with irradiated sporozoites thus exposes them to liver-stage antigens and 

generates pre-erythrocytic immunity. However, the requirement of a minimum 

of one thousand bites by irradiated mosquitoes, over five or more immunization 

sessions, in order to successfully induce sterile immunity in humans [5], 

precludes this method for routine immunization. 

Research turned thus to developing a subunit vaccine based on antigens 

expressed by pre-erythrocytic, intra-erythrocytic or sexual stages of the parasite. 

Unfortunately, results of many such subunit vaccines in humans have been 

disappointing and to date only one candidate, based on the circum-sporozoite 

protein (CSP) and known as RTS,S, has progressed to phase III field trials. The 

protection induced by this vaccine is encouraging but the ultimate success of 

this approach remains to be determined [6-9]. 

It has been shown that, in rodent models, sterile protection against malaria can 

also be achieved by inoculation of intact sporozoites whilst treating the animals 

concomitantly with chloroquine (CQ) [10], a drug that kills asexual blood-stage 

but not pre-erythrocytic parasites [11], with an efficacy significantly higher than 

that of the radiation-attenuated sporozoite model [12]. We therefore designed a 

proof-of-concept study in malaria-naive volunteers to investigate whether 

protection can be equally effectively induced by this approach in humans and to 

explore the immune responses elicited.

176 Chapter 9 

Figure 1. Study design and enrolment 

Immunological assessment was performed 1 day before the first immunization 

(day I-1) and 1 day before the challenge infection (day C-1).A final challenge 

with infectious mosquito bites was performed 28 days after the 

discontinuation of chloroquine prophylaxis. 

Methods 

Volunteers 

Fifteen healthy volunteers were recruited, aged 18-45 years old, without history 

of malaria or of residence in a malaria endemic area in the six months prior to 

the trial onset. Only one volunteer had ever been in an endemic area, several 

years earlier. All volunteers underwent routine physical examination, 

haematology and biochemistry screening at the Clinical Research Centre 

Nijmegen. Serology for HIV, hepatitis B and C and asexual P. falciparum parasites 

was negative in all volunteers. All volunteers gave written informed consent 

prior to inclusion. The trial was approved by the Institutional Review Board of


the Radboud University Nijmegen Medical Centre (CMO 2006/207). 

Clinicaltrials.gov identifier: NCT00442377. 

Protocol 

Volunteers were randomized double blind into two groups (Figure 1), 10 vera 

and 5 controls. Chloroquine(CQ) was provided to all volunteers in a standard 

prophylactic regimen of 300mg base weekly, starting with 600mg in two days, 

for a total duration of 13 weeks. Whilst under CQ prophylaxis, vera were 

exposed on three occasions, at four-weekly intervals, to bites of 12-15 P. 

falciparum-infected mosquitoes per session, for a total exposure of 36-45 

infected mosquitoes per volunteer. Controls received bites from an equal 

number of uninfected mosquitoes on the same occasions. Anopheles stephensi 

mosquitoes were reared according to standard procedures at our insectary. 

Infected mosquitoes were obtained by feeding on gametocytes of NF54, a 

chloroquine sensitive strain of P. falciparum, as described previously [13]. NF54 

is genetically homogeneous, but has not been formally cloned. Only the 

insectary technicians preparing the mosquitoes were aware of the infectivity 

status of the mosquitoes allocated to the volunteers. Blood-engorged 

mosquitoes were dissected to confirm the presence of sporozoites. If necessary, 

feeding sessions were repeated until precisely the predefined number of 

infected mosquitoes had fed. However, a single feeding session was sufficient in 

49/60 of all instances of immunization or challenge, whereas a second session 

was required in just 10 instances and a third session in only 1 instance. 

From day 6-10 after each mosquito exposure, all volunteers were followed on an 

outpatient basis and blood was drawn for standard whole blood counts and daily 

thick smears. Any signs and symptoms were recorded by the attending physician 

as follows: mild (easily tolerated), moderate (interferes with normal activity), or 

severe (prevents normal activity). 

Eight weeks after the last immunization dose and 4 weeks after discontinuation 

of CQ prophylaxis, all 15 volunteers were challenged by exposure to the bites of 

five homologous NF54 strain P. falciparum-infected mosquitoes. This period was 

considered to be sufficient for CQ levels to drop below levels which might be 

inhibitory to parasite multiplication [14]. Volunteers were checked daily on an 

outpatient basis from day 5 to day 21 for symptoms and signs of malaria, 

haematological parameters and thick smears. 

As soon as thick smear positive, volunteers were treated with a standard 

curative regimen of artemether/lumefantrine (starting dose of 80/480 mg,

178 Chapter 9 

followed by five identical doses at 8, 24, 36, 48 and 60 hours) and followed 

closely for three days. Complete cure was confirmed by thick blood smears. All 

volunteers still negative by day 21 post-challenge were presumptively treated 

with artemether/lumefantrine. 

Haematological and biochemical parameters were determined in routine fashion 

at the hospital’s central clinical laboratory. Nucleic Acid Sequence Base 

Amplification (NASBA) and real-time QT-PCR to determine the densities of P. 

falciparum parasites have been described in detail [15,16]. Chloroquine levels 

were measured by liquid chromatography as previously described [17,18]. 

Minimum therapeutic concentrations for plasma chloroquine levels maintained 

by the laboratory were 30 µg/l, in accordance with Rombo et al. [14]. 

Immunological analysis 

Venous whole blood was collected into CPT vacutainers (Becton and Dickinson, 

Basel) prior to first immunization and again prior to challenge. Plasma was 

collected and stored at -70°C. Peripheral Blood Mononuclear Cells [PBMCs) 

were isolated by density gradient centrifugation, frozen down in 10% DMSO/FCS 

and stored in liquid nitrogen. Antibody titres were assessed by ELISA and 

immunofluorescence assay according standard protocols as previously described 

[19-21]. Cellular responses to cryopreserved asexual parasites were assessed by 

24-hour in vitro PBMC cell stimulation assays as previously described [22], 

followed by intracellular cytokine staining (Fix & Perm kit, Caltag Laboratories) 

and flow-cytometry. A more detailed description of these immunological assays 

is provided in the web-only supplementary methods. 


Flowcytometric analysis was performed using CellQuest and data analyzed in 

SPSS; differences in responses within volunteers between time points and 

between vera and control volunteers were analyzed by non-parametric tests 

(Wilcoxon and Mann-Whitney-U, respectively), two-sided p-values


Abdominal pain mild 2 

moderate 

severe 

Vera [n=10) Controls [n=5) 

Post immunization 

Post 

Post immunization 

challenge 

Post 

challenge 

I II III I II III 

Fatigue mild 1 1 7 1 2 

moderate 2 1 1 2 

severe 1 

Fever mild 2 

moderate 1 

severe 2 5 

Headache mild 4 7 2 3 2 

moderate 2 1 1 1 1 3 

severe 1 1 

Loss of appetite mild 1 

moderate 

severe 

Malaise mild 1 1 1 

moderate 2 1 

severe 1 1 5 

Myalgia mild 1 2 3 

moderate 2 1 4 

severe 1 

Nausea mild 3 1 1 1 1 

moderate 1 1 1 1 

severe 1 

Vomiting mild 1 

moderate 1 

severe 

Overall† mild 4 

[40%) 

moderate 3 

[30%) 

severe 2 

[20%) 

1 

[10%) 

1 

[10%) 

8 

[80%) 

1 

[10%) 

1 3 

[20%) [60%) 

1 2 1 

[20%) [40%) [20%) 

1 

[20%) 

5 

[100%) 

Table 1. Adverse events after the first, second, and third exposures to immunizing 

mosquito bites and after challenge with infectious mosquito bites. 

* Subjects could have more than one adverse event and reports of events could have 

been either solicited or unsolicited. Only adverse events possibly or probably related to 

the study are listed.† The highest-grade event is listed per subject per infection.† Highest 

grade event listed per volunteer per infection.

180 Chapter 9 

Figure 2. Parasitemia in the Vaccine Group and the Control Group. 

Panel A shows the mean number of Plasmodium falciparum parasites per millilitre, as 

measured by nucleic acid sequence–based amplification (NASBA), after each of three 

immunizations on days before infection and during expected blood-stage parasitemia 

(days 6 to 10) in the vaccine group. The numbers of subjects who had positive results 

on peripheral blood smears for malarial parasites (MPS) and NASBA are shown below 

the graph. Panel B shows the mean number of P. falciparum parasites in 5 subjects in 

the control group and 10 subjects in the vaccine group, as determined by real-time 

polymerase-chain-reaction assay before treatment with artemether–lumefantrine. 

The I bars denote standard errors.


No parasites were seen in the thick blood-smears of any of the 10 vera following 

each of the three immunization sessions under CQ prophylaxis, but after the first 

immunization a brief sub-microscopic parasitemic episode was detected in all 

vera (Figure 2a). This was not unexpected, since CQ has no effect against either 

sporozoites, liver-stage parasites, or the early ring forms of the first generation 

of blood-stage parasites caused by merozoites released from mature hepatic 

schizonts [11]. Following each of the subsequent two immunizations, a 

progressively reduced incidence and burden of sub-microscopic parasitemia was 

seen. 

In line with these findings, all vera reported solicited or unsolicited adverse 

events at least once during the immunization phase. When excluding local 

itching after the mosquito bites, adverse events were most commonly reported 

after the first immunization (9/10 volunteers), with headache being the most 

frequent (7/10) (Table 1). Only few adverse events occurred subsequently (0/10 

and 2/10 volunteers after the second and third immunization respectively). 

Severe adverse events were reported by three vera: two experienced fever 

above 39°C after the first immunization, one reported severe malaise after the 

last immunization. 

Following challenge with homologous NF54 strain of P. falciparum, asexual 

blood-stage parasites were detected in thick blood smears of all 5 control 

volunteers between days 7 and 10.6 post-exposure (mean pre-patent period 9.2 

days). Real-time PCR analyses revealed the expected cyclical multiplication of 

blood-stage parasites (Figure 2b). Clinical course and kinetics of parasite 

multiplication were identical to those in previous studies involving naïve 

volunteers [23,24], with all controls reporting severe events, in particular fever 

above 39°C and malaise (Table 1). In marked contrast, there was no evidence of 

blood-stage parasites in any of the vera at any time during the post-challenge 

follow-up period till day 20, either by repeated microscopy of thick blood smears 

or by real-time PCR analyses (Figure 2b). Interestingly, 9/10 vera did report mild 

to moderate events in the week following challenge. No serious adverse events 

occurred during any part of the trial and all 15 volunteers completed follow-up 

according to protocol. 

Mean peak chloroquine and desbutyl-chloroquine levels measured 24 hours 

after administration were 76 µg/l (range 58-104 µg/l) and 13 µg/l (5-33 µg/l) 

respectively and did not differ between control and vera. The day before 

challenge plasma levels had dropped to 8 µg/l (range

182 Chapter 9 

Test Day I-1 Day C-1 

Vaccine group 

(N=10) 

Control group 

(N=5) 

Vaccine group 

(N=10) 

Control group 

(N=5) 

No. of Median 

No. of 

Antibody 

Median 

No. of 

Antibody 

Median 

Antibody 

No. of Median 

Antibody 

Subjects Titre Subjects Titre Subjects Titre Subjects Titre 

AU AU AU AU 

CSP 0


Figure 3 The proportion of lymphocytes that produced interferon-γ (IFN-γ) and interleukin-2 (Panel A), 

tumour necrosis factor α (TNF-α) and interleukin-2 (Panel B), or IFN-γ, TNF-α, and interleukin-2 (Panel 

C) after in vitro stimulation with erythrocytes infected with the homologous strain of P. falciparum 

(PfRBC), with uninfected erythrocytes (uRBC), or with phytohemagglutinin (PHA) as a positive control 

are shown before immunization (day I-1) and before malaria challenge (day C-1). Dashed lines 

represent the proportion of positive cells in unstimulated wells (culture medium only). The geometric 

mean fluorescence intensity of cells producing IFN-γ (Panel D), TNF-α (Panel E), and interleukin- 

2(Panel F) that were isolated from vaccinees on day C-1 is shown after stimulation in vitro with 

infected erythrocytes. Cells are grouped according to their positivity or negativity for each of the 

other two cytokines. In Panels G, H, and I, the proportion of lymphocytes that produced IFN-γ and 

interleukin-2 in response to infected erythrocytes are shown on day I-1 and day C-1 for lymphocyte 

phenotypes, including naive T cells (CD3+CD45RO−), memory T cells (CD3+CD45RO+), and non-T 

lymphocytes (CD3−CD45RO−) (Panel G); for T-cell phenotypes, including helper T cells (CD4+CD8−), 

cytotoxic T cells (CD4−CD8+), and other lymphocytes (CD4−CD8−) (Panel H); and for memory 

phenotypes, including naive T cells (CD62L+CD45RO−), central memory T cells (CD62L+CD45RO+), 

effector memory T cells (CD62L−CD45RO+), and other lymphocytes (CD62L−CD45RO−) (Panel I). The 

proportions of lymphocytes that produced IFN-γ and interleukin-2 after stimulation with uninfected 

erythrocytes were below 0.005% (not shown). All P values are for the comparison between the 

vaccine group and the control group and were calculated with the use of the Mann–Whitney test. The 

T bars represent standard errors..

184 Chapter 9 

strain P. falciparum-infected erythrocytes (PfRBC) or controls (Figure 3 and 

supplementary figures 2+3). Whereas cellular responses to uninfected 

erythrocytes (uRBC) never differed in any experiment from those to culture 

medium alone, stimulation with PfRBC elicited small percentages of lymphocytes 

producing IFNγ or TNFα, but not IL-2, in both groups of volunteers prior to 

immunization (day I-1, Supplementary figure 2). Although no statistically 

significant increment was seen in the overall percentage of cells producing 

individual cytokines (IFNγ+ or TNFα+) in either group following immunization 

(day C-1), a marked and significant increase was observed in the percentages of 

cells producing multiple cytokines in response to PfRBC in vera (IFNγ+IL-2+ 

p=0.026, Figure 3a; TNFα+IL-2+ p=0.046, Figure 3b; IFNγ+TNFα+IL-2+ p=0.032, 

Figure 3c). The importance of these pluripotent lymphocytes in acquired 

immune protection is suggested by their higher cytokine content and possibly as 

a consequence a better effector function (Figure 3d-f). The major contributors to 

this increase in PfRBC-responding pluripotent lymphocytes were 

[CD3+CD45RO+) memory-like T cells (p=0.025 vs. day I-1, Figure 3g and 

supplementary figure 3), in particular CD4+CD8- cells (p=0.005 vs. day I-1, Figure 

3h). Most noticeably, these new pluripotent lymphocytes were predominantly of 

the effector memory (CD62L-CD45RO+) phenotype (p=0.005 vs. day I-1), 

although there was also a small but significant (p=0.017) increase in the 

numbers of responding central memory (CD62L+CD45RO+) cells in vera (Figure 

3i). 

Discussion 

Our study shows that sterile protection to an homologous challenge with P. 

falciparum malaria can be much more efficiently induced in comparison to 

irradiation-attenuated sporozoite immunization. In the endemic situation, nonsterile 

semi-immunity is acquired only after years of repeated natural exposure. 

The improved efficiency of our approach we believe to be due to a critical 

balance of exposure to pre- and intra-erythrocytic antigens. In contrast to 

irradiated sporozoites that arrest early during liver-stage development [4], intact 

sporozoites under CQ cover mature fully and develop into a first generation of 

blood-stage parasites [11], thus presenting to the host’s immune system a 

markedly broader palette of pre-erythrocytic and additionally, albeit at relatively 

low-dose, erythrocytic-stage antigens. The contribution of intra-erythrocytic 

antigens to the development of protective immunity is suggested by Pombo et 

al., who reported that repeated intravenous injection of ultra-low densities of


blood-stage parasites followed by drug-cure with atovaquone/proguanil induced 

protection in human volunteers against a similarly low-dose blood-stage 

challenge [25]. However, caution needs to be exercised when interpreting the 

latter results, since residual anti-malarial drug concentrations may partially or 

even fully have accounted for the observed protection [26]. 

In the field, in contrast, patent parasitemia typically develops before patients 

seek treatment. In these individuals, acute blood-stage infection may suppress 

the induction of protective pre-erythrocytic immunity, as has been shown in 

rodent models [27]. Indeed, parasitemic episodes or febrile malaria attacks in 

Kenyan children are prospectively associated with a poorer induction and more 

rapid attrition of cellular ex vivo and memory responses to a pre-erythrocytic P. 

falciparum antigen [28]. Thus, patent parasitemia and probably also chronic subpatent 

parasitemia, as are experienced regularly by children in endemic areas, 

appear to induce inhibitory mechanisms that delay the generation of protective 

anti-parasite immunity. Meta-analysis of intermittent preventative therapy in 

infancy (IPTi) studies has decreased concerns of a rebound-effect and in some 

cases even indicates sustained protection following discontinuation of 

prophylaxis [29], thus further indicating that the acquisition and maintenance of 

protective immunity is not dependent on chronic blood-stage exposure. The 

salient feature of our approach we believe therefore to be the exposure of the 

immune system to the full palette of pre-and intra-erythrocytic antigens, whilst 

restricting the development of symptomatic and potentially immunosuppressive 

parasitemia [30]. Since NF54 is known to be a CQ-sensitive strain in vitro [31], 

we cannot formally exclude a synergistic effect of residual sub-therapeutic 

chloroquine levels on immunological parasite clearance. However, chloroquine 

levels prior to challenge approached or fell below the limit of detection and had 

no measureable parasitocidal effect in control volunteers. Of more importance, 

the longevity of immunological responses, both naturally-acquired and vaccineinduced, 

remains a critical issue in malaria and follow-up studies are planned to 

address this issue. 

We have identified pluripotent effector memory T cell responses as associated 

with protection. Undefined lymphocyte subsets with the same cytokine profile 

have been associated with the induction and maintenance of antigen-specific T 

cell memory in individuals immunized with pre-erythrocytic malaria vaccine 

candidates, but this study did not explore associations with protection [32]. The 

potent effector function of pluripotent cells, as suggested by their high cytokine 

content, has however been noted in other investigations demonstrating their

186 Chapter 9 

protective role in other infectious diseases [33,34]. Further detailed 

investigations will be necessary to ascertain the longevity of this immunological 

response, its association with central memory-type T cell activity and its ability 

to serve as a true correlate of protection. 

Since the magnitude of the first wave of parasitemia is thought to directly reflect 

the burden of erupting mature liver schizonts, the step-wise decrease of such 

following each subsequent immunizing infection, culminating in the total 

absence of PCR-detectable parasitemia following challenge, would suggest that 

the protection in our model is primarily due to pre-erythrocytic immunity. 

However, a component of blood-stage immunity, i.e. the inhibition of 

erythrocyte invasion and maturation of sub-PCR liver-derived merozoite inocula, 

is also possible. Indeed we found cellular responses to asexual blood-stage 

parasites [PfRBC) prior to challenge to be an excellent discriminative marker of 

exposure and protection in our volunteers and similar immune responses may 

have contributed to protection in the rodent model [12]. It must be borne in 

mind, however, that many of the best-studied P. falciparum antigens conferring 

protective immunity are shared between sporozoite, liver-stage and blood-stage 

parasites [35,36]. Thus it is plausible that our findings represent the response to 

a broad antigenic repertoire that transcends parasite developmental stages [37], 

making a dichotomy into pre-erythrocytic or intra-erythrocytic immunity 

inappropriate. At present the stage-specificity of the protective immune 

response must thus remain formally unresolved, although one way to further 

address this issue in future studies would be a blood-stage challenge. 

Whereas the methodology described here does not itself represent a widely 

implementable vaccine strategy, the induction of sterile protection against an 

homologous malaria challenge suggests that the concept of a whole parasite 

malaria vaccine warrants further consideration. In addition, this model allows 

the nature of protective immune responses against malaria, both stage- and 

antigen-specific, to be further investigated. 


Foremost, we are indebted to the study volunteers for their participation. We 

thank K. Nganou Makamdop for help with RT-PCR, J. Bakkers & W. Melchers for 

parasite genotyping, W. Arts, N. Huibers & P. Beckers for blood slide reading, P. 

Houze & D. Mazier for chloroquine measurements and J. Klaassen, L. Pelser- 

Posthumus, J. Kuhnen & A. Pouwelsen for technical assistance generating


infected mosquitoes. We acknowledge the safety monitors on this study, B. 

Schouwenberg & P. Smits and independent physician A. Brouwer. 

Funding 

Supported by the Dioraphte Foundation, by a fellowship from the European 

Union FP6 Network of Excellence (to Dr. McCall), and by grants from the 

NutsOhra Foundation (to Dr. van der Ven) and Agence Nationale de la Recherche 

in France (to Dr. Snounou).

188 Chapter 9 

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chloroquine and its three metabolites in human plasma, whole blood and urine 

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190 Chapter 9 

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Supplementary methods 

Serology 

To assess antibody titres, asexual ELISAs were performed as previously described 

[1]. CSP ELISAs were performed with (NANP)6 NA (kind gift from G. Corradin) 

according to published methods [2]. AMA-1 and GLURP ELISAs were performed 

according to a standard in-house protocol [2,3]. In all ELISAs a plasma pool of 8 

healthy malaria-naïve Dutch volunteers was used as a negative control to define 

sample positivity (above mean+2SD of negative control). A plasma pool of 

Tanzanian adults (n=100) living in a highly malaria endemic area was used as 

reference positive control, defined to contain 100 arbitrary units (AU). 

Immunofluorescence assays were performed by incubating plasma at 1/320 

dilution with air-dried whole sporozoites or at 1/40 dilution with asynchronous 

cultures of NF54 strain asexual P. falciparum parasites, as previously described 

[3]. 

Cellular immunology 

Cellular immune responses were assessed by in vitro stimulation assays. For use 

in these assays, Percoll-purified asynchronous asexual-stage cultures of NF54 

strain parasites of 80-90% parasitemia, consisting of more than 95% 

schizonts/mature trophozoites, were washed, aliquoted and cryopreserved in 

advance. Mock-cultured uninfected erythrocytes (uRBC) were obtained similarly 

and served as control. Cryopreserved PBMCs were thawed immediately prior to 

use in in vitro stimulation assays, washed and resuspended in RPMI 1640 culture 

medium containing 2mM glutamine, 1mM pyruvate, 50μg/mL gentamicine and 

10% pooled human AB+ serum (Sanquin, Nijmegen, NL), for a final concentration 

of 2.5x10 6 /mL. PBMCs were transferred into 96-well round-bottom plates 

(5x10 5 /well) and stimuli were added to duplicate wells. Stimuli included 

cryopreserved PfRBC (5x10 6 /mL), uRBC, PHA (10 μg/mL) or RPMI only. PBMCs 

were stimulated for 24 hours at 37°C/5%CO2. 4 hours prior to cell harvest, 

100μL/well supernatant was collected and replaced with 100 μL/well fresh 

culture medium containing 20 μg/mL brefeldin A (Sigma). PBMCs from both time 

points per volunteer were tested simultaneously and cells from two vaccinees 

and one control were always tested together. 

Following 24 hour in vitro stimulation, PBMCs were harvested, washed once in 

FACS buffer (0.5% BSA/PBS) and incubated for 15’ with fluorescent mAbs against 

cell surface markers. Cells were washed again and incubated for 15’ in fixation

192 Chapter 9 

medium A (Caltag Laboratories, Carlsbad, CA) according to the manufacturer’s 

instructions, washed again and incubated for 15’ with fluorescent mAbs against 

intracellular cytokines in permeabilization medium B. After a final wash step, 

cells were resuspended in FACS buffer and read on a FACScalibur flowcytometer. 

The following fluorescent mAbs were used: CD3-PerCP, CD4-PerCP, CD8-PE (BD, 

Breda, NL), IFNγ-FITC, TNFα-PE, IL-2-APC, CD45RO-PE, CD62L-PeCy7, mouse 

IgG1-FITC & IgG2a-PE and rat IgG2a-APC isotype controls (all Ebioscience, 

Uithoorn, NL). 

References 

1. Bousema JT, Roeffen W, van der KM et al. Rapid onset of transmissionreducing 

antibodies in javanese migrants exposed to malaria in Papua, 

Indonesia. Am J Trop Med Hyg 2006; 74:425-31. 

2. Remarque EJ, Faber BW, Kocken CH, Thomas AW. A diversity-covering 

approach to immunisation with Plasmodium falciparum AMA1 induces broader 

allelic recognition and growth inhibition responses in rabbits. Infect Immun 

2008. 

3. Hermsen CC, Verhage DF, Telgt DS et al. Glutamate-rich protein (GLURP) 

induces antibodies that inhibit in vitro growth of Plasmodium falciparum in a 

phase 1 malaria vaccine trial. Vaccine 2007; 25:2930-40.

Chapter 10 

Long-term protection against malaria after 

experimental sporozoite inoculation 

Meta Roestenberg 1 , Anne C. Teirlinck 1 , Matthew B.B. McCall 1 , Karina Teelen 1 , 

Krystelle Nganou Makamdop 1 , Jorien Wiersma 1 , Theo Arens 1 , Pieter Beckers 1 , 

GeertJan van Gemert 1 , Marga van de Vegte-Bolmer 1 , André J.A.M. van der Ven 2 , 

Adrian J.F. Luty 1 , Cornelus C. Hermsen 1 , Robert W. Sauerwein 1 

1 

Radboud University Nijmegen Medical Centre, Department of Medical 


2 

Radboud University Nijmegen Medical Centre, Department of Internal 

Medicine, Nijmegen, The Netherlands [A J A M van der Ven) 

Lancet 2011; 377:1770-1776

194 Chapter 10 

Abstract 

Induction of long-lived immunity to Plasmodium falciparum (Pf) is a major 

obstacle to malaria vaccine development. Recently we showed that immunity to 

Pf can be induced experimentally in 10/10 of malaria-naïve volunteers through 

immunisation by bites of Pf-infected mosquitoes whilst simultaneously 

preventing disease by chloroquine prophylaxis. This immunity was associated 

with parasite-specific production of IFNγ and IL-2 by pluripotent effector 

memory cells in vitro. Here we explore the persistence of protection and 

immune responses in the same volunteers. 

In an open-label study conducted 28 months after immunisation, six previously 

immune volunteers were re-challenged by the bites of five Pf-infected 

mosquitoes. Five naive volunteers served as infection controls. The primary 

outcome was detection of blood-stage parasitemia by microscopy. The kinetics 

of parasitemia were assessed by real-time quantitative PCR (Q-PCR) and clinical 

signs and symptoms were recorded. In vitro production of IFNγ and IL-2 by 

effector memory T cells was studied following stimulation with sporozoites and 

Pf-infected red blood cells. This study is registered with clinicaltrials.gov, number 

NCT00757887. 

Four of six immune volunteers were fully protected against re-challenge. Q-PCRbased 

detection of blood-stage parasites in these individuals was negative 

throughout follow-up. Patency in the remaining two immunised volunteers was 

markedly delayed. In vitro assays revealed the long-term persistence of parasitespecific 

pluripotent effector memory T cell responses in protected volunteers. 

We demonstrate that immunity to Pf in human volunteers can persist for more 

than two years using an experimental immunisation protocol. Immunity was 

paralleled by maintenance of Pf-specific pluripotent effector memory T cells. 

These findings are unprecedented and demonstrate that artificially induced 

immunity may be longer-lasting than generally observed after natural exposure. 

These results open a novel avenue for research into mechanisms of malaria 

immunity.

Long-term protection against malaria after experimental sporozoite inoculation 195 

Introduction 

In 2009, an estimated 225 million cases of Plasmodium falciparum malaria lead 

to 781,000 deaths worldwide, of which about 85% were children under the age 

of five (http://www.who.int/malaria/world_malaria_report_2010/en/index. 

html). Malaria is responsible for an enormous economic burden due to medical 

expenses, missed education and lost productivity. It has been estimated that the 

GDP of countries endemic for malaria grows at 1.3% per year slower than 

countries which are malaria-free [1]. Reducing the burden of malaria has been 

identified as a specific target within the United Nations Millennium 

Development Goal 4: reduce Child Mortality (www.un.org/millenniumgoals). 

Control programs have been successful in decreasing the incidence of malaria by 

more than 50% in 11 countries of the WHO African region over the past decade 

and are highly cost-effective [2]. The recent increase of malaria cases in some 

other African countries illustrates the fragility of current malaria control 

(http://www.who.int/malaria/world_malaria_report_2010/en/index.html). The 

urgent need for an effective malaria vaccine to complement these control 

programs is thus widely acknowledged. A major hurdle in vaccine development 

is the complex parasite-host interaction that seems to preclude the 

establishment of long-lasting, sterile anti-parasitic immunity [3]. This notion is 

supported by the lack of sustained protection acquired through natural malaria 

exposure. When lifelong residents move away from an endemic area, immunity 

is thought to wane quickly, rendering them susceptible to re-infection and 

disease when re-exposed. [4] Moreover, naturally acquired immunity controls 

parasitemia and prevents clinical symptoms but neither eliminates parasites 

completely from the blood nor prevents re-infection, and is therefore not 

sterilizing [4]. 

Immunity to Plasmodium falciparum (Pf) malaria in humans can be induced 

artificially by vaccination with subunit vaccine candidates or by repeated 

exposure to attenuated whole parasites [5,6]. The most successful subunit 

candidate vaccine to date, RTS,S, based on the circumsporozoite protein 

combined with hepatitis B surface antigen, is between 30-65% efficacious in 

delaying the first episode of clinical malaria in Africans [7]. Extended follow-up in 

Mozambican children demonstrated waning efficacy over a period of 20 months 

[8]. Immunity to Pf in humans can also be induced experimentally by exposure 

to the bites of more than 1000 irradiated sporozoite-infected mosquitoes. This 

results in protection of almost 100% volunteers, lasting up to 42 weeks in a few


Pre-erythrocytic stage 

Sporozoites 

Liver 

Blood stage 

Chloroquine 

mediated 

killing 

Merosome 

Merozoites 

individuals [5]. Recently we described a novel methodology for induction of 

immunity to Pf in humans, based on an immunisation procedure involving the 

repeated exposure to infectious mosquito bites under chloroquine prophylaxis 

[9]. Using this method, clinical disease was prevented by the drug that kills 

asexual blood-stage parasites, but allows complete liver-stage parasite 

development (Figure 1). Volunteers were protected from an experimental 

challenge with the homologous parasite strain two months later. Protected 

volunteers developed only low-level parasite-specific antibody activity, but 

strong parasite-specific effector memory T cell responses, characterized by the 

production of IFNγ and IL-2 after stimulation in vitro. 

The objective of the current follow-up study was to assess the persistence of 

protection against Pf infection after experimental immunisation. We rechallenged 

volunteers by the bites of sporozoite-infected mosquitoes after a 

period of 2.5 years. 

Skin 

Erythrocytes 

Lymph node 

Figure 1. Course of Plasmodium falciparum parasite development under 

chloroquine prophylaxis. Sporozoites are injected into the skin by an infected 

female Anopheles sp. mosquito. A proportion migrates through the bloodstream 

to the liver. Parasites develop and multiply in hepatocytes over a period of 6-7 

days. Subsequently, merosomes bud from infected hepatocytes into the sinusoid 

and the released merozoites invade red blood cells for further asexual 

multiplication. Chloroquine prophylaxis only targets and prevents replication of 

parasites in red blood cells, thereby allowing the induction of immune responses 

against preceding stages.


Methods 

Number of fully engorged mosquitoes [# of 

mosquitoes per cage) 

Volunteer number Session 1 Session 2 Session 3 

1 – Control 5 [5) 

2 – Control 5 [5) 

3 – Control 5 [5) 

4 – Control 3 [5) 2 [2) 

5 – Control 5 [5) 

6 – Immunised 4 [5) 1 [1) 

7 – Immunised 4 [5) 1 [1) 

8 – Immunised 5 [5) 

9 – Immunised 4 [5) 1 [1) 

10 – Immunised 3 [5) 2 [2) 

11 – Immunised 4 [5) 0 [1) 1 [1) 

Table 1. Number of blood feeding mosquitoes per volunteer. 

Participants and study design 

This open-label clinical trial follows a previous trial where immunity to Pf malaria 

was induced in ten healthy, malaria-naïve volunteers [9]. In short, volunteers 

took chloroquine prophylaxis weekly for a period of 13 weeks during which they 

were exposed to the bites of 12-15 infected mosquitoes on three occasions at 

monthly intervals. Figure 1 gives an overview of the course of development of 

parasites during these immunising infections. One month after stopping the 

prophylaxis, all ten volunteers were shown to be fully protected against a 

subsequent challenge infection via the bites of five Pf-infected mosquitoes, with 

complete absence of asexual parasitemia. 

The current trial was performed at the Radboud University Nijmegen Medical 

Centre, The Netherlands, from November to December 2009, twenty-eight 

months after the initiation of the previous challenge infection. All ten previously 

immune volunteers were invited to participate, six were eligible for 

participation. In addition, five malaria-naive volunteers were recruited as 

infectivity controls. Volunteers aged 18-35 years old were screened for eligibility 

based on medical and family history, physical examination and general 

haematological and biochemical screening including HIV, hepatitis B and 

hepatitis C serology, urine toxicology screening and a pregnancy test. The main 

exclusion criteria were residence in a malaria endemic area within the previous 

six months, positive Pf serology (all volunteers were tested according to


procedures described in [10]) or an estimated ten year risk of >5% of developing 

a cardiac event as estimated by the Systematic Coronary Evaluation (SCORE) 

system. All volunteers gave written informed consent prior to inclusion. The trial 

was performed in accordance with Good Clinical Practice and approved by the 

Central Committee for Research Involving Human Subjects of The Netherlands 

(CCMO NL24193.091.09). Clinicaltrials.gov identifier: NCT00757887. 

Procedures 

Both the six immunised, previously immune individuals, and the five malarianaive 

control volunteers were exposed to the bites of five Anopheles stephensi 

mosquitoes infected with the NF54 strain of Pf for ten minutes. Infected 

mosquitoes were obtained by feeding on gametocytes of NF54 as described 

previously [11]. The salivary glands of all blood-engorged mosquitoes were 

dissected, confirming the presence of sporozoites in 97%, with a mean of 88,000 

sporozoites per mosquito. When necessary, feeding sessions were repeated with 

a smaller number of mosquitoes until precisely five infected mosquitoes had 

bitten. (Five volunteers required one blood feeding session, five volunteers two 

sessions and one volunteer three sessions, raw data provided in Table 1). 

Starting from day five post-challenge infection, volunteers were subjected to an 

intense follow-up regime with multiple daily visits to our out-patient clinical 

research department. All signs and symptoms (solicited and unsolicited) were 

recorded and graded by the attending physician as follows: mild (easily 

tolerated), moderate (interferes with normal activity), or severe (prevents 

normal activity), or in case of fever grade 1 (>37.5°C – 38.0°C), grade 2 (>38.0°C – 

39.0°C) or grade 3 (>39.0°C). Haematological and biochemical parameters were 

monitored daily. Because of a previously reported serious cardiac adverse event 

after a malaria challenge infection in a separate study [12], particular attention 

was paid to markers of coagulation or cardiac damage with daily follow-up of 

highly-sensitive troponin, platelets, d-dimer and lactate dehydrogenase during 

the period when blood stage parasitemia could be expected. Whenever 

abnormal, blood samples were checked for the presence of fragmentocytes and 

von Willebrand cleaving protease activity, since previous reports have 

highlighted the occurrence of von Willebrand factor activation in experimental 

malaria [13]. 

As soon as a blood-slide was found to contain parasites, volunteers were treated 

with a curative regimen of atovaquone/proguanil 1000/400mg once daily for 

three days. Volunteers who remained free of parasites by blood-slide until day


21 after re-challenge presumptively received the same curative treatment. 

Complete cure was always confirmed by the occurrence of two consecutive 

parasite-negative blood-slides. 

Outcomes 

The primary outcome of the study was microscopic detection of parasites in a 

blood-slide, with sampling performed twice daily on days 5 and 6 post-challenge, 

thrice daily on days 7-11, twice daily on days 12-15 and once on days 16-21 postchallenge. 

Thick blood smears were made from 15µl of EDTA-anti-coagulated 

blood, spread over the standardised surface of one well of a 3-well glass slide 

(CEL-LINE Diagnostic Microscope Slides, 30-12A-black-CE24). After drying, wells 

were stained with Giemsa for 45 minutes. Slides were read at 1000x 

magnification by assessing 200 high-power fields, equal to approximately 0.5µl 

of blood. The smear was considered positive if two unambiguously identifiable 

parasites were found. The pre-patent period was defined as the period between 

challenge and first positive blood smear. Additionally, parasitemia was 

measured by real-time quantitative PCR (Q-PCR), performed retrospectively on 

all samples collected after challenge, as previously described [14]. 

Secondary outcomes comprised immunological parameters. After preparation of 

thick blood smears, remaining plasma was collected and stored at -70°C. 

Antibody titres on the day prior to challenge were assessed by ELISA as 

previously described [9]. Plasma concentrations of IL-6 and IFNγ were measured 

every other day using the Bio-Plex system (Bio-Rad) [15]. 

For cellular immunology, venous whole blood was collected into cell-preparation 

tubes (Becton and Dickinson, Basel) on the day before challenge. Peripheral 

blood mononuclear cells (PBMC) were isolated by density gradient 

centrifugation, frozen in fetal-calf serum containing 10% dimethylsulfoxide and 

stored in liquid nitrogen. Cellular responses against cryopreserved NF54 

sporozoite and NF54 asexual-stage parasites were assessed by 24-hour in vitro 

stimulation as previously described for the asexual-stage [9] followed by 

intracellular cytokine staining. 

Preparation of sporozoite- and asexual-stage parasites for immunological assays 

NF54 strain P. falciparum sporozoites (PfSpz) were obtained from Anopheles 

stephensi mosquitoes that were reared according to standard procedures in our 

insectary. Infected mosquitoes were obtained by feeding on gametocytecontaining 

cultures of NF54 strain P. falciparum, as described previously [15]. On 

day 21–28 after infection, the salivary glands of the mosquitoes were collected


by hand-dissection. Salivary glands were collected in RPMI-1640 medium (Gibco) 

and homogenized in a custom glass grinder. Free sporozoites were counted in a 

Bürker-Türk counting chamber using phase-contrast microscopy. PfSpz were 

cryopreserved at 16x10 6 /ml in 15% glycerol/PBS in aliquots for use in individual 

stimulation assays. Sporozoites that had undergone one freeze-thaw cycle were 

morphologically intact, but no longer able to glide (assay described in [16], using 

a FITC-3SP2 conjugated antibody). Salivary glands from an equal number of 

uninfected mosquitoes were used as a background control. 

NF54 strain P. falciparum asexual blood-stage parasites (PfRBC), regularly 

screened for mycoplasma contamination, were grown in RPMI-1640 medium 

containing 10% human A+ serum at 5% haematocrit in a semi-automated 

suspension culture system, in the absence of antibiotics and in an atmosphere 

containing 3% CO2 and 4% O2. For in vitro stimulation experiments, 

asynchronous asexual-stage cultures of NF54 strain parasites were harvested at 

a parasitemia of approximately 5-10% and mature asexual stages purified by 

centrifugation on a 27% and 63% Percoll density gradient [17]. This purification 

step results in preparations of 80-90% parasitemia, consisting of more than 95% 

schizonts/mature trophozoites. PfRBC were washed twice in PBS and 

cryopreserved at 150x10 6 /mL in 15% glycerol/PBS in aliquots for use in 

individual stimulation assays. Mock-cultured uninfected erythrocytes (uRBC) 

were obtained similarly and served as control. 

Stimulation assay and staining for flow cytometry 

After thawing, cells were stimulated with either cryopreserved NF54 PfRBC (as 

previously described [9]) at a final concentration of 5*10 6 /ml or cryopreserved 

PfSpz at a concentration of 4*10 5 /ml for 24 hours. Uninfected red blood cells 

(uRBC, 5*10 6 /ml) and uninfected mosquito salivary glands (MSG) preparations 

dissected from equal numbers of uninfected mosquitoes, respectively, were 

used as negative controls. For cell stimulations with sporozoites/MSG 0.8 µl/ml 

CD28/CD49d reagent (BD) was added to the culture as a co-stimulant. 

PMA/ionomycin stimulation (50ng/ml and 1µg/ml respectively, Sigma-Aldrich) 

was used as positive control and added four hours before harvest. Brefeldin A 

(final concentration 10μg/mL) was added to all wells four hours prior to harvest. 

For staining procedures, PBMC were transferred to a 96 V-wells micro-titre plate 

(500,000 cells/well), washed and incubated with LIVE/DEAD® cell stain kit Aqua 

(Invitrogen, Carlsbad, CA, USA) for 30 min at 4°C. Cells were washed in FACS 

buffer (PBS containing 0.5% Bovine Serum Albumin, Sigma-Aldrich Co.), and


Figure 2. Trial profile. Timelines of previous immunisation study in grey. 

stained with IQTest CD45RO-phycoerythrin Texas Red (Beckman-Coulter), PerCP 

anti-human CD3 (BioLegend, San Diego, CA, USA) and PE-Cy7 anti-human CD62L 

(eBioscience) for 20 min at 4°C. After washing, cells were incubated for 15 min at 

4°C with fixation Medium A (Caltag, S. San Francisco, CA, USA), washed and 

stained with FITC anti-human IFNγ (eBioscience) and APC anti-human IL-2 

(eBioscience) in permeabilization Medium B (Caltag) for 20 min at 4°C. 100,000 

events in the lymphocyte gate, based on forward- and side-scatter, were 

acquired on a CyAn ADP 9-color flow cytometer (Beckman-Coulter). Flow 

cytometry analysis was performed using FlowJo software version 9.1. Analysis 

was performed on CD3+ T cells and effector memory (EM) T cells. The IFNγ and 

IL-2 gates were set based on negative control samples. 


Data analysis was performed using SPSS software version 16.0. Differences in 

cellular immune responses between subjects in the vaccine group and control 

group were analyzed by the Mann–Whitney-U test. The correlation between 

peak temperature and peak d-dimer values was assessed by Pearson correlation 

analysis. A two-sided P value of less than 0.05 was considered to indicate 

statistical significance.


Figure 3. Mean number of adverse events per volunteer. Data from controls in black 

(n=5), volunteers that showed delayed patency in red (n=2) and protected volunteers in 

green (n=4). Only adverse events possibly or probably related to malaria are included. 

Results 

All ten volunteers previously immunised and protected against an experimental 

malaria infection, were contacted 32 months after the first immunisation [9]. Six 

of these volunteers passed screening for eligibility and were included in the 

current follow-up study. Three out of four excluded volunteers were unavailable 

and one had to be excluded because of a family history of myocardial infarction 

(second degree relative under 55 years of age). Five healthy volunteers were 

newly recruited as a control group. Figure 2 shows the trial profile. Eleven 

volunteers were challenged by the bites of five Pf-infected mosquitoes and all 

completed follow-up. The average age of participating volunteers was 23.6 years 

(range 21-28 years old), of whom four were male. Four controls and one 

immunised volunteer had previously (range 6 months to 9 years) travelled in 

malaria endemic areas during which time they all used malaria prophylaxis, none 

of the volunteers was a previous resident of a malaria endemic area. None of 

the volunteers reported having used antimalarials within the past 6 months and 

none showed positive serology for Pf. 

When re-challenged according to the standard protocol by bites of five infected 

mosquitoes, four out of six of the immunised volunteers never developed bloodstage 

parasitemia by microscopy during daily follow-up. One of these volunteers 

requested presumptive treatment with anti-malarials on day 14 post-infection 

for reasons unrelated to the trial. The remaining two volunteers showed a 

markedly delayed patent parasitemia on days 15 and 18, respectively.


Any Adverse 

event frequency 

Protected (n=4) Delayed patency (n=2) Controls (n=5) 

Mean 

duration ± 

SD (days) frequency 

Mean 

duration ± 

SD (days) frequency 

Mean 

duration ± 

SD (days) 

Abdominal pain 1 1.0±0 

Arthralgia 2 1.8±0 

Chills 2 0.8±0.6 

Fatigue 3 5.0±5.3 2 5.6±4.1 5 5.3±3.1 

Fever 1 0.1±0 2 2.0±0.5 2 1.3±1.0 

Headache 3 0.7±0.3 2 1.6±1.5 3 1.7±2.0 

Itching 2 1.5±2.0 2 3.6±3.6 3 3.4±2.4 

Malaise 2 3.2±2.1 

Myalgia 1 1.0±0 1 3.0±0 3 1.9±1.9 

Nausea 2 0.1±0.09 1 3.0±0 2 0.5±0.5 

Vomiting 1 0.04±0 

Any 4 1.8±3.1 2 2.7±2.8 5 5.7±2.2 

Grade 3 adverse event 

Fatigue 2 9.0±1.8 1 7.9±0 

Fever 2 1.8±0.6 

Itching 1 0.1±0 

Malaise 2 3.2±2.1 

Any 1 0.1±0 2 9.0±1.8 2 8.4±2.7 

Table 2. Number of individuals reporting possibly or probably related solicited 

adverse events in immune volunteers, volunteers with delayed patency and control 

volunteers. Unrelated adverse events were myalgia after sports, headache related to 

exams, common cold, a possible vasovagal reaction without collapse and a fracture of 

the scaphoid bone. 

The four protected volunteers reported several mild to moderate adverse 

events, of which short episodes of headache was the most common symptom 

(1-3 episodes per volunteer). Interestingly, one of these volunteers reported 

fever up to 38.0°C measured sub-lingual 19 days after the challenge infection. 

The two volunteers with delayed patency reported adverse events similar to 

those in the control group. The number of adverse events reported per 

volunteer over time is shown in Figure 3. 

Four of five control volunteers developed parasitemia detected by thick smear 

within the expected time frame of 7-12 days post-infection and were treated per 

protocol [9]. One control volunteer presumptively received anti-malarial 

treatment on day 9.3 in the absence of a parasite-positive blood-smear but 

based on clinical malaria symptoms and elevated d-dimer levels [1180 ng/ml), a 

pre-defined safety criterion. All control volunteers, including the latter, 

developed asexual blood-stage parasitemia detectable by Q-PCR retrospective-


Figure 4. Number of parasites as measured by Q-PCR in controls (A) and immunised 

volunteers (B). Parasitemia of control group (n=5) is shown as geometric mean and 

95% confidence interval, immunised volunteers are plotted individually (n=6). 

Parasitemia of volunteers with delayed patency is indicated by dashed lines. 

ly, and reported adverse events compatible with clinical malaria. Fatigue and 

headache were most commonly reported. An overview of all solicited possibly or 

probably related adverse events is provided in Table 2. 

For safety reasons related to a previously reported cardiac event [12], we 

measured biochemical cardiovascular indicators assiduously throughout the 

trial. Shortly after parasite detection by microscopy and initiation of antimalarial 

treatment, d-dimer levels became elevated in all volunteers. The maximum 

increase in d-dimer varied between 570 to 14,600 ng/ml, with a median peak of 

1,600 ng/ml (n=7). Von Willebrand cleaving protease activity was decreased at


Figure 5. In vitro production of IFNγ (A,B) or both IFNγ and IL-2 (C-F) by T-cells (A-D) or 

CD45RO+ and CD62L- effector memory (EM) T cells (E,F) on the day before (re-)challenge, 

upon stimulation with sporozoites (PfSpz, panel A,C,E) or asexual stage parasites (PfRBC, 

panel B,D,F). Numbers of cytokine producing cells are depicted as percentage of total T 

cells (A-D) or EM T cells (E-F). Symbols indicate individual values from immunised (n=6) 

and control (n=5) volunteers; horizontal lines represent group medians. Fully protected 

immunised volunteers and control volunteers are indicated as circles, immunised 

volunteers with delayed patency are indicated as triangles. 

this time in two of five volunteers with d-dimer above 1,000 ng/ml (58% and 

38% compared with 80% and 67% respectively in a resting state). 

Fragmentocytes were never detected. Peak d-dimer values correlated with peak


temperature (Pearson R=0.83, p=0.02). Platelets decreased slightly below 

120x10 9 /l in one volunteer (nadir 108x10 9 /l). Lactate dehydrogenase levels were 

never elevated above 1000 U/l. Highly sensitive troponin T was never abnormal 

(max 0.011 µg/l). None of the volunteers showed any bleeding or thrombotic 

complications. None of the protected volunteers showed an elevation of ddimers 

above detection level (250 ng/ml). 

Real-time quantitative PCR (Q-PCR) was performed on all collected samples 

retrospectively. Control volunteers showed cyclical parasite growth, identical to 

control volunteers in previous malaria challenge infection trials [9,14]. Asexual 

blood-stage parasites were never detected by Q-PCR (detection limit 20 

parasites/ml) in any of the four protected volunteers during the entire 21 day 

follow-up period. The remaining two immunised volunteers with delayed 

patency by microscopy also showed delayed appearance of blood-stage 

parasitemia by Q-PCR (Figure 4). 

Specific anti-parasite antibody responses were detectable at 28 months postimmunisation 

in only one of six immunised volunteers, i.e. against the major 

circumsporozoite antigen (NANP6 repeats) and crude blood-stage antigens (data 

not shown). Antibodies with specificity for individual asexual blood stage 

antigens (AMA-1 and GLURP) were not detectable. Blood stage parasitemia was 

accompanied by increased plasma concentrations of systemic inflammatory 

markers, including IFNγ (median peak 69 pg/ml, IQR 51-135), IL-6 (median peak 

9.2 pg/ml, IQR 6.4-14) and C-reactive protein (median peak 52 mg/l, IQR 26.5- 

57.5) with no difference between controls and volunteers with delayed patency. 

None of the protected volunteers showed any increase in plasma concentrations 

of IFNγ, IL-6 or CRP (max 5 pg/ml, 7 pg/ml and 2.5 mg/l respectively). 

In vitro stimulation assays showed a sustained Pf-specific T cell IFNγ re-call 

response to both pre-erythrocytic (sporozoite) and blood-stage parasites that 

persisted over years in all six immunised individuals (Figure 5). These responses 

remained significantly higher than those of control volunteers (p


Discussion 

Here we report for the first time the persistence of immunity in human 

volunteers to re-infection with Plasmodium falciparum more than two years 

after previous exposure. Using a recently developed artificial immunisation 

protocol [9] we observed long lasting protection in four of six re-challenged 

volunteers and markedly delayed patency in the two remaining volunteers. In 

addition, we describe the maintenance of Pf-specific T cell IFNγ responses. 

The notion that immunity to malaria is short-lived derives primarily from 

anecdotal reports of returning semi-immune immigrants. A critical review of the 

literature, however, reveals that such migrants appear to retain some 

protection, albeit only from severe disease and death [19]. The induction and 

persistence of clinical and parasitological immunity is more controversial, due to 

the great diversity in malaria transmission intensity, age, genetic background 

and study endpoints, complicating interpretation [4]. Protective immunity to 

clinical malaria can be acquired relatively rapidly [4], and in settings of epidemic 

malaria, some measure of clinical protection may be sustained over several 

years [20]. However, meta-analyses of intermittent preventive treatment studies 

in naturally exposed infants and children show renewed susceptibility following 

discontinuation of the intervention [21] and a historical review of patients 

treated for neurosyphilis by (repeated) artificial malaria infection revealed no 

sterile protective immunity against subsequent re-challenge [22]. Moreover, 

protection induced in volunteers by irradiated sporozoite inoculation lasted 42 

weeks in only a few subjects [5] and the protection induced by the most 

successful sub-unit vaccine to date, RTS,S, seems to wane [6]. In summary, 

naturally acquired immunity is far from optimal, but protective immunity to 

severe disease can be maintained [4,19,23]. 

The long-term protection against malaria in this study is thus surprising when 

compared with naturally acquired immunity. Several factors may account for 

this discrepancy. Firstly, the same Pf strain was used for both the immunisation 

protocol and the experimental challenges. Well-described target antigens for 

protective immunity exhibit high rates of genetic variation, circumventing crossprotective 

immunity in the field [24]. Secondly, our immunisation protocol 

prevents high blood-stage parasitemia, whilst repeated parasitemia in endemic 

areas is thought to suppress the development of immunity [25]. Thirdly, we 

immunised adult individuals, whereas natural exposure is first encountered by 

the immature immune system of infants4 or even in utero [26]. Finally, the


immune modulating effects of chloroquine might have enhanced the 

development of protective immune responses during the immunisation process 

[27]. 

While Pf-specific antibody responses were negligible and declining (data not 

shown), we found a sustained Pf-specific T cell IFNγ re-call response that 

persisted over years and paralleled the sustained protection in all six immunised 

volunteers. The two volunteers with delayed patency did not show a distinctly 

different T cell response as compared to the fully protected individuals. 

However, a three to six day delay of exponentially growing parasites equals one 

to three logs (e.g. >95%) reduction in parasite burden. The variation in the level 

of protection between the volunteers might thus be relatively small. A large 

body of evidence supports the view that IFNγ responses are protective against Pf 

infection, although naturally acquired T-cell responses to individual antigens 

wane after several years of non-exposure [28]. The functional importance of 

IFNγ–producing cells, however, still needs to be established in larger study 

groups. 

We detected cellular responses to both sporozoites and blood-stage parasites 

with remarkably similar dynamics, although responses to blood-stage parasites 

were consistently higher than to sporozoites. Whether such responses are 

directed against both pre-erythrocytic and blood-stage antigens or represent 

cross-reactivity, will require further detailed immunological analysis. In either 

case, the lack of blood-stage parasites in the fully protected volunteers seems to 

indicate a primarily pre-erythrocytic immunological effector mechanism. 

Experimental malaria infections provide a unique opportunity to study 

protection against Pf malaria in a controlled setting [29]. This trial reconfirms a 

solid correlation between the appearance of clinical symptoms and the presence 

of microscopically detectable blood stage parasites, including in volunteers with 

delayed patency. Clinical symptoms and the pattern of parasitemia by Q-PCR are 

very consistent between and within study groups illustrating the power of this 

experimental infection model (Chapter 6, [9]). Protected volunteers also 

reported some adverse events, without objective signs of inflammation. Possibly 

these events reflect natural fluctuations of subjective health perception. 

The occurrence of a cardiac event following experimental challenge after a 

phase I malaria vaccine trial in the past has necessitated an increased vigilance 

and surveillance of cardiac and coagulation markers [12]. We found increased 

concentrations of d-dimer that paralleled the increases in inflammatory markers,


but without overt clinical thrombotic microangiopathy or bleeding 

complications. In addition we report incidental decreases of von Willebrand 

cleaving protease, possibly as a result of increased consumption of the protease 

[30]. This is consistent with previous data concerning von Willebrand factor 

activation in experimental malaria [13]. The clinical relevance of these findings 

and their relation with the cardiac event is as yet unclear. 

Our relatively simple immunisation protocol represents a blueprint for induction 

of sustained anti-malarial immunity, providing a novel tool for exploring 

mechanisms of immunity and highlighting new research priorities. Precedence 

must be given to basic understanding of mechanisms of protection in this study 

and those factors which inhibit protection in naturally-exposed populations. 

Furthermore, strain specificity can be investigated with the use of heterologous 

challenge infections and in field studies. Given the efficacy of our immunisation 

protocol, however, equal emphasis on a pragmatic parallel approach to develop 

an implementable whole-organism-based vaccine is justified. 

In conclusion, the irrefutable evidence that long-term immunity against malaria 

is possible holds a promise for an urgently needed malaria vaccine. 


This trial was financed by a grant from the Dioraphte foundation. ACT is funded 

by the European Malaria Vaccine Development Association, MBBM was 

supported by an European Union FP6 Network of Excellence (BioMalPar) 

fellowship, KNM was funded by a Mozaiek grant from the Netherlands 

Organisation for Scientific Research. We thank the trial volunteers and the staff 

from the Clinical Research Centre Nijmegen who made this study possible. We 

thank the following individuals for their assistance during the trial: Laura Pelser, 

Jolanda Klaassen, Astrid Pouwelsen, Jacqueline Kuhnen for the mosquito 

infection and dissection work, Wendy Arts, Nanny Huiberts, Chantal Siebes, 

Marlou Kooreman, Paul Daemen and Ella Driessen for reading many thick 

smears. We would also like to acknowledge the contributions of Alexander 

Rennings for his help with monitoring volunteer safety and the cardiologists 

Gheorghe Pop and Marc Brouwer for their dedication to the cardiac monitoring 

of the trial volunteers. We acknowledge the work of Richard Huijbens for the


cytokine measurements by Luminex. We would like to thank Rob Woestenenk 

for use and support of the flowcytometer. 

Funding 

This trial was funded by the Dioraphte foundation.


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48.

Chapter 11 

General Discussion


The development of a highly efficacious malaria vaccine faces many hurdles, key 

to which is our limited knowledge of what exactly constitutes protection against 

Plasmodium falciparum malaria in humans. Malaria vaccine development relies 

on a trial-and-error approach, with many candidates in (pre-)clinical 

development awaiting efficacy testing in the field. This thesis focuses on the 

development of research tools that facilitate immunological and vaccine 

research into human protection against Plasmodium falciparum malaria in early 

stages of clinical development. We used the following approaches: 

Firstly, we tested an asexual erythrocytic stage subunit vaccine candidate (Apical 

Membrane Antigen 1) for its safety and immunogenicity in humans and 

performed in depth analysis of the humoral immune response following 

immunization. 

Secondly, we assessed and optimized an important tool for malaria vaccine and 

immunological research: controlled human malaria infections. In these trials, 

healthy malaria-naïve volunteers are exposed to infectious mosquito bites in 

order to test the preliminary efficacy of vaccine candidates or in order to 

perform immunological research. 

Thirdly, we developed a human model for protection against Plasmodium 

falciparum malaria to facilitate immunological research and facilitate the 

development of a whole-sporozoite vaccine for malaria. 

Apical Membrane Antigen 1 (AMA1) 

AMA1 is considered a promising vaccine candidate, primarily due to a large body 

of data from animal studies indicating that AMA1 can induce immune responses 

capable of inhibiting the blood stage parasite multiplication in vitro and in vivo 

(reviewed in [1]). Specifically, protection can be conveyed by transferring anti- 

AMA1 antibodies [2-5] and epidemiological studies in malaria endemic areas 

show that the presence of AMA1 antibodies correlate with malaria protection [6, 

7]. Subsequently, AMA1 has been produced as recombinant protein both in 

Pichia pastoris and E. Coli. Animal studies showed satisfactory pharmacotoxicity, 

so the whole-length (amino acid 25 to 545) Pichia pastoris-expressed AMA1 

from the FVO strain has been taken forward into the first phase of clinical 

testing. We showed that PfAMA1[25-545] FVO can be safely administered to 

humans and is immunogenic (Chapter 2). We also showed that both 

reactogenicity and immunogenicity are dependent on the adjuvant, with the

General Discussion 217 

occurrence of unwanted side-effects such as an abscess at the injection site in 

the higher dose Montanide ISA 720 group (Chapter 2). Moreover, we confirmed 

that the antibodies induced by the AMA1 vaccine have the capacity to inhibit the 

invasion of parasites into erythrocytes in vitro (Chapter 2, 3). Similar results were 

obtained with other AMA1 vaccine formulations [8-15]. 

Following these Phase I safety trials, several attempts were undertaken to test 

the efficacy of AMA1 formulations in the field (Phase IIb). Unfortunately, two 

separate field studies could not establish a significant effect of AMA1 

immunisation on primary parasitological endpoints [9, 16], although there was 

some evidence for homologous protection. There are two possible explanations 

for the apparent discrepancy between in vitro data, rodent and simian studies 

and in vivo human efficacy data: 1) allelic diversity or 2) insufficient immune 

responses. 

Ad 1). Allelic diversity may account for the failing efficacy of AMA1 in the field, 

as AMA1 is known to be highly polymorphic [17]. We showed that these allelic 

polymorphisms lead to reduced growth inhibition of antibodies measured 

against parasite strains expressing heterologous AMA1 in vitro (Chapter 3), but 

the translation of these in vitro effects to in vivo reduced efficacy was not found 

in controlled infection studies [18]. Detailed analysis of a field trial revealed 

specific efficacy against clinical episodes caused by parasites that expressed a 

form of AMA1 with the same residues as the vaccine strain (3D7) in the 

dominant cluster of polymorphisms in domain I [16]. However, the other field 

trial found no evidence of vaccine selection or strain-specific efficacy of a 

bivalent AMA1 vaccine [9], suggesting that the genetic diversity of AMA1 may 

not fully account for failure of the vaccine to provide protection [19]. Also blood 

stage infection of vaccines in a Phase I/IIa trial with homologous strain Pf did not 

show reduced parasite multiplication rates, indicating that failing efficacy could 

not be attributed to strain diversity [18]. In conclusion, the extent to which 

allelic diversity accounts for reduced efficacy of candidate AMA1 vaccines is 

debated, but the importance of inducing strain transcending immunity has been 

well-recognised and efforts into the development of multivalent AMA1 vaccines 

are increasing [20, 21]. 

Ad 2). A second possible explanation for failing efficacy of AMA1 vaccines is the 

quality of the immune response. Many AMA1 vaccines induce significant 

concentrations of antibodies but no protection, therefore antibody 

concentration alone does not suffice to protect. We explored the avidity of


antibodies induced by AMA1 in humans and rabbits and found that the avidity 

and concentration of AMA1-induced antibodies are linked by a negative 

feedback mechanism reaching saturation in most vaccinees (Chapter 4). Higher 

titre AMA1 antibodies may thus not necessarily implicate better binding 

efficiency and function (Chapter 4). The relevance of these findings for in vivo 

protection is unclear. A correlation between antibody avidity, as measured by 

thiocyanate ELISA, and protection was not found in a P. chabaudi rodent model, 

but the combination of titres to refolded and reduced/alkylated recombinant 

AMA1 proved an important predictor of protection [22]. These results illustrate 

the importance of understanding the nature of AMA1 immunogenic epitopes, 

their functionality and the antibody characteristics. Moreover, other Pf-specific 

antibodies may interfere with the biological activity of anti-AMA1 antibodies 

[23], underlining the fact that protection in the field is a composite of more than 

just anti-AMA1 antibodies. An immunological marker for protection is essential 

to assess immunogenicity of AMA1 vaccine candidates in an early stage of 

clinical development. 

The in vitro growth inhibition assay (GIA) has been developed as a functional 

marker that include all antibody properties. The GIA is a biological assay 

investigating the capacity of AMA-1 induced antibodies to inhibit parasite 

growth [24]. Although promising at first, the inhibitory capacity measured in 

vitro did not correlate with protection in rhesus monkeys [25] or in human trials 

[18, 26]. The fact that we found substantial inhibition of AMA1-induced 

antibodies in vitro, is thus no guarantee of protection in vivo. 

Nonetheless, protection from Plasmodium falciparum malaria can be transferred 

from one human to the other by the passive transfer of antibodies from malariaimmune 

to non-immune subjects. Since 1917, such experiments were 

performed in humans, when it was discovered that patients improved clinically 

when inoculated with serum obtained from “chronic” cases. In the 60’s 

experiments were repeated and antibodies taken from children in West Africa 

proved sufficient for the treatment of children in East Africa [27], indicating that 

antibodies can even overcome strain diversity. An antibody dependent cellular 

inhibiting effect (ADCI) has been proposed as an effector mechanism to this 

protection [28], relying on the help of cytokines such as IFNγ, IL-4 [28] and TNFα 

[29] to upregulate phagocytic function of polymorphonuclear leukocytes or 

monocytes. However, T-cells alone have also shown to adoptively transfer 

protection against malaria in rodent models [30] and the central role of IFNγ in


mediating this protection is increasingly appreciated [31]. Indeed, also in 

humans, repeated low grade blood stage infections can induce protection 

against Pf malaria in the absence of detectable antibodies [32]. In addition, 

multifunctional T-cell responses were identified following human AMA1 

vaccination [33] and T-cell memory responses to T-cell epitopes from the AMA1 

protein were detected in clinically immune subjects, although no clear-cut 

association with parasitemia was found [34, 35]. To conclude, AMA1-specific Tcells 

contribute to building an effective humoral response in mice [36-38], but 

antibodies are generally still thought to be the most critical effector for blood 

stage parasite inhibition [38, 39]. Apparently, not just the quantity of antibodies 

but possibly also the quality and the interplay between cellular and humoral 

responses play a role in building a protective immune response to AMA1. 

In order to understand the mechanism by which AMA1 directed immune 

responses effectuate parasite growth inhibition, more basic understanding of 

the function of AMA1 is essential. AMA1 is expressed in merozoites and is 

thought to help reorienting the merozoite when aligning with the erythrocyte 

membrane and subsequently attaching with erythrocyte membrane proteins [1]. 

More recent mechanistic in vitro studies, however, also highlight a possible role 

of AMA1 in parasite invasion into hepatocytes, showing expression of AMA1 in 

sporozoites and inhibition of AMA1 antibodies with invasion of hepatocytes [40]. 

The possible clinical relevance of this function was confirmed by two controlled 

malaria infection (CHMI) studies with human volunteers [18, 26], in which no 

clinically relevant effect on prepatency or blood stage parasite inhibition could 

be found in AMA1 immunized volunteers challenged by mosquito bite [26] or 

blood stage parasites[18], but possible reduction of liver load parasites could be 

detected by Q-PCR analysis of parasitemia in the former trial [26]. 

The versatility of AMA1 expression complicates the dissection of AMA1-induced 

inhibiting and enhancing immune responses. The induction of exactly the right 

quality and quantity immune responses, however, may be the key to AMA1 

success in humans. Adjuvants are a very heterogenic group of pharmacological 

or immunological agents that are co-administered with vaccines in order to 

potentiate and/or direct immune responses in humans. Particularly in malaria 

research adjuvants have proven to play a critical role: GlaxoSmithKline’s 

proprietary adjuvant AS01/2 was found crucial for enhancing responses to RTS,S 

to protective levels [41]. We found that adjuvants are also important 

determinants of cellular and humoral immune responses when combined with


the PfAMA1[25-545] FVO vaccine (Chapters 2, 3). Our work on AMA1 antibody 

avidity illustrates that the AS02A adjuvant directs the balance of antibody 

quantity and quality towards high titre antibody of lower avidity when compared 

to a conventional adjuvant such as Alhydrogel (Chapter 4). 

Recent advances in adjuvant research have led to the availability of many new 

adjuvants [42]. With the increased understanding of host/pathogen interactions 

new classes of adjuvants follow more rational design, such as Toll-like receptor 

agonists or cytokines. In addition, delivery platforms have been developed to 

ensure a specific presentation of the antigen to the immune system. For 

example, viral platforms are designed to direct the immune response from a 

classical humoral response following protein immunization to CD4+ or CD8+ 

responses induced by, for example, adenovirus, fowlpox or modified vaccinia 

viruses [43]. Also bacterium-like particles or nanoparticles have shown to 

provide benefit as carriers to potential malaria vaccines [44, 45]. Heterologous 

prime-boost strategies utilizing two different adjuvants with one antigen is 

another recent development that has shown to increase efficacy [46]. In 

addition to adjuvants and delivery platforms, which are co-administrated with 

the vaccine and designed to boost or direct the immune response, research has 

focussed on the development of especially designed delivery systems, aimed at 

the specific delivery of an antigen at a certain anatomical site. Nasal delivery, 

needle free jet devices and intrabuccal delivery systems are examples of the 

most recent developments (for example [47, 48]). Their specific advantages are 

yet to be investigated. 

Also AMA1 has been subjected to the addition of new adjuvants in an attempt to 

increase immunogenicity [49]. Thorough adjuvant research will be required in 

order to delineate the properties and optimal combination of antigen, adjuvant, 

delivery platform and delivery system, a complex task requiring huge effort and 

funds [42]. Direct comparisons of different adjuvants with one antigen, as we 

have described (Chapter 2), will prove to be instrumental to these 

developments. Unfortunately, the limited availability of newly developed 

adjuvants in the public domain has restricted the number of comparative trials 

so far. 

In conclusion, in vitro and animal studies have raised high hopes for AMA1 

vaccines, which could not be met in human efficacy trials. In addition, the phase 

III clinical development of RTS,S has set high standards for the development of 

any malaria vaccine, a challenge that also AMA1 has to face. The versatility of


AMA1 expression and immune responses complicate the development of an 

AMA1-induced correlate of protection, but may also hold the biggest promise 

for AMA1 vaccines, being able to induce both pre-erythrocytic and erythrocytic 

immunity. Possibly, the development of new adjuvants, delivery systems, strain 

covering approaches or the combination of AMA1 with other antigens will 

provide a means by which animal results can be translated into human efficacy. 

The availability of an AMA1 induced correlate of protection will most certainly 

reduce costs and accelerate the development of any AMA1 vaccine. A proof-ofprinciple 

human trial may thus be necessary to supply data for immunological 

research and provide a new basis for rejuvenated preclinical research for AMA1. 

Meanwhile, the characterisation of adjuvants and other malaria antigens 

facilitate the rational design of the most effective AMA1-based (combination-) 

vaccine. 

Controlled human malaria infections 

Adapted from Sauerwein et al [50]. 

Malaria vaccine candidate AMA1 illustrates the relevance of controlled malaria 

infection trials (CHMI) in acquiring novel insights on the mechanism by which a 

candidate vaccine can induce protection. A major strength of controlled malaria 

infections is the use of infectious mosquitoes, mimicking the natural route of 

infection. Moreover, these infections are carried out in a controlled 

environment, allowing detailed evaluation of parasite growth and immunological 

determinants, which make them suitable to investigate the efficacy of 

vaccine candidates and mechanisms of protection. The induction of immunity by 

exposure of malaria-naive volunteers to infectious mosquito bites while using 

chloroquine prophylaxis (Chapter 9) is one example. More basic research into 

immunological mechanisms following Pf infection in human is another [51]. The 

detailed analysis of parasitemia following infection may add to the 

understanding of immunological inhibiting effects in these trials. 

A real-time quantitative PCR (qPCR) assay based on 18S ribosomal RNA gene 

transcripts has been developed for tracking the kinetics of developing 

parasitemia before a positive diagnosis of infection can be made from a thick 

blood smear using microscopy [52]. This assay is becoming increasingly 

important for assessing very low parasite densities and incremental changes in 

density in small scale Phase IIa trials [53]. The detection of parasites below 

microscopy thresholds by qPCR allows for a detailed analysis of cyclical parasite


growth in the blood, albeit for a short time window of 2–3 days between liverstage 

infection and microscopic detection [52]. We have now formally shown 

that these molecular techniques enhance the power of controlled human 

infection trials to detect modest vaccine efficacy (which may not necessarily 

correspond with clinical protection) with only small numbers of volunteers 

(seven per group) (Chapter 6). Statistical models can be applied to further 

improve the discriminative power between control and test groups as well as to 

provide biological information about the parasite life cycle (including the 

duration of liver-stage maturation, number of infected hepatocytes, duration of 

blood-stage trophozoite maturation and multiplication rates [54-56]). 

Immediate treatment of volunteers at the earliest phase of microscopically 

detectable blood-stage infection ensures that the potential risks of 

complications associated with severe malaria are minimized to the greatest 

extent possible. Indeed, controlled human malaria infections have shown to be 

safe in the 1,343 volunteers challenged so far [57-59]. Recently, safety concerns 

were raised because of a cardiac event in a young volunteer shortly after 

treatment for diagnosed malaria following a controlled infection, although a 

definite relationship between the cardiac event and the controlled malaria 

infection was not established [60]. The chronology of the event with the malaria 

infection has raised discussion on a possible pathophysiological link between 

cardiac events and malaria. An ischemic cardiac event has previously been 

described after CHMI, however, this volunteer was never parasitemic [59]. There 

have been no other reports of ischemic cardiac events in the context of malaria, 

but events of myocarditis, although rare, have been found in several malaria 

patients [61-65]. Myocarditis has also been recognised as an immunological 

complication following all types of vaccinations, although particularly smallpox 

vaccines are renown [66-68]. Notwithstanding the aetiology, the identification of 

volunteers that may possibly develop cardiac complications before start of the 

CHMI and during the infection is important to safeguard volunteers. Therefore, 

it has been agreed that volunteers with an increased risk of cardiac disease 

should be excluded from such trials. Currently, the SCORE risk assessment [69] is 

used to identify those high risk volunteers, although risk factors for malaria 

induced ischemia may not resemble those for atherosclerotic processes. In 

addition, regular measurements of highly sensitive troponin are performed in 

order to detect cardiac damage in an early stage. However, the increased 

sensitivity of t-troponin detection parallels decreased clinical relevance with 

detection of minimal cardiac damage. For example, increased highly-sensitive


troponins can also be found in marathon runners [70], providing evidence for 

minimal cardiac damage that may not be clinically relevant. D-dimers and LDH 

have also been proposed as markers for ischemia, based on the hypothesis that 

endothelial cell activation and increased turnover of coagulation would lead to 

thrombotic events. Again, although highly sensitive, these markers lack 

specificity for clinically relevant cardiac damage. Highly elevated d-dimers, 

particularly after initiation of antimalarial-treatment, have been found in several 

CHMI volunteers since the initiation of monitoring without re-occurrence of 

cardiac damage. The correlation of d-dimers with peak temperature suggests 

that d-dimers are a bystander in inflammation rather than a predictor of cardiac 

damage (Chapter 10). In conclusion, the lack of knowledge on the aetiology of 

the cardiac event makes the identification of a “risk correlate” for cardiac 

damage, and the subsequent identification of volunteers at risk difficult, if not 

impossible. The continuous monitoring of volunteers for the occurrence of 

cardiac damage, by means of regular measurement of highly-sensitive troponins 

thus seems reasonable in order to detect any cardiac event in an early stage. 

Moreover, the occurrence of the cardiac event has, once again, stressed the 

importance of close and committed monitoring by the study physicians in order 

to detect any events in an early stage and prevent escalation. However, the 

apparently infrequent occurrence of serious adverse events in the large group of 

volunteers exposed to controlled infections so far and the wealth of data on 

clinical cases lacking evidence for cardiac damage, may be somewhat reassuring. 

In addition to the clinical manifestations, participation in a controlled malaria 

infection trial has a major impact on the daily life of volunteers. This is 

particularly due to the intense follow-up with blood sampling several times daily. 

Volunteers’ perception of their participation in such a trial depends mainly on 

whether they have realistic expectations of trial procedures and the severity of 

symptoms, indicating the importance of providing accurate and sufficient 

information to volunteers before the onset of the trial. 

As is true for any type of clinical research, risks must be minimized and scientific 

benefits maximized. We believe that the benefits of Phase IIa trials outweigh the 

potential risks in well-designed studies and will be essential to the development 

of an effective malaria vaccine, provided that all safeguards are in place for the 

safety of volunteers [71]. 

Differences between natural and controlled experimental infections signify the 

importance of validating the results of Phase IIa challenge trials with data from


Phase IIb field trials in malaria-endemic areas. Only four candidate vaccines have 

been assessed by both types of trial, allowing a comparison of the protective 

outcomes. The best studied candidate vaccine, RTS,S, which is currently in Phase 

III trials, has repeatedly demonstrated a protective efficacy of ~30–50% in Phase 

IIa trials with sterile protection as the study end point [41, 72, 73]. Interestingly, 

a similar ~30–50% efficacy of RTS,S was found in Phase IIb trials in the field using 

time to first clinical malaria episode as the primary study end point [74, 75]. A 

similar association between the results of Phase IIa and Phase IIb trials was 

found when testing long-term protection in adults [74, 75]. A second preerythrocytic 

stage candidate vaccine, ME-TRAP (a multi-epitope string fused to 

thrombospondin-related adhesion protein), delivered by a DNA prime and 

attenuated poxvirus boost, induced complete protection in only a few 

volunteers (three out of 74) in Phase IIa trials, and no protection was found in 

adult Phase IIb field studies in the Gambia [76, 77]. Artificial blood-stage 

challenge has been used in a Phase II trial after immunization with Combination 

B, a combination of merozoite surface protein 1 (MSP1), MSP2 and ring-infected 

erythrocyte surface antigen (RESA) in 17 volunteers, which resulted in no 

decrease in parasite growth rates [78]; this is in line with results from a Phase IIb 

trial of Combination B conducted in Papua New Guinea [79]. Finally, the vaccine 

candidate AMA1 (3D7 strain) did not show protection in a controlled infection 

trial [26] and parasitological outcome in an efficacy trial was also non-significant 

[16]. These limited data indicate that results obtained in controlled malaria 

infection trials are generally in line with results in the field, but more 

comparisons are required before definite conclusions can be drawn. 

The extrapolation of results from controlled infections to the field may also be 

limited by several potential differences between controlled human malaria 

infections and naturally acquired infections. One important difference is the 

delivery of parasites. In an experimental situation the parasite load is delivered 

almost instantly by five infected mosquitoes. Such a high parasite burden has 

been considered unnatural and might be an overly stringent test for the 

protective capacity of the vaccine-induced immune response [80]. However, 

although the frequency of infectious mosquito bites is generally less than this in 

malaria-endemic areas, intense transmission can occur. A person may be 

subjected to 35–96 mosquito bites per night, and in certain areas approximately 

10% of mosquitoes are infected with Pf [81]. 

Second, controlled malaria infections are carried out using one parasite strain 

only, whereas it is well known that Pf field strains are genetically diverse within


and between regions [82]. Genetic diversity of the parasite strains is a major 

challenge for vaccines that target strain-specific antigens, of which the vaccine 

candidate AMA1 is one example, and puts limitations on the direct translation of 

results from experimental infections into the field situation. The availability of a 

small portfolio of genetically well-characterized Pf strains for controlled malaria 

infections would be a major asset. We have taken the first step towards a field 

strain infection model by showing that NF135, a field strain from Cambodia is 

infectious to humans (Chapter 7). Future studies will need to establish whether 

the infectivity of NF135 is comparable to NF54 and the dose that should be 

administered to achieve 100% infection rates. Once several field strains are 

available, the controlled infection of different groups of volunteers with 

heterologous strain parasites would be highly instrumental to investigate the 

promiscuity of responses induced particularly with diversity covering vaccines or 

whole-parasite vaccine strategies. 

A final potential limitation of controlled malaria infection models relates to the 

uncontrolled number of sporozoites inoculated by biting mosquitoes. This 

number is generally thought to vary between 100-300 sporozoites per bite up to 

a maximum of several thousand sporozoites [83-86]. Use of a well-defined 

number of inoculated sporozoites will strengthen the power of the model 

(Chapter 6). In principle, the most accurate way of dosing sporozoites is to inject 

them directly by needle and syringe, as the number of sporozoites counted in 

mosquito salivary glands or the number of mosquito bites is a poor predictor of 

the number of sporozoites injected [84] 

Whole-sporozoite inoculation 

The concept of inoculation of whole sporozoites into humans is not novel. The 

first reports can be found in 1926, when five patients were injected with P. vivax 

in order to test antimalarial drugs [87]. More frequent records are found 

between 1937 and 1963 when malariatherapy was given to neurosyphilis 

patients, mostly by bites of infected mosquitoes but also by sporozoite injection 

[88, 89]. Infection of patients with P. falciparum or P. vivax sporozoites was 

primarily by intravenous route of freshly extracted sporozoites, since the 

viability was known to heavily depend on storage time, condition and handling 

[88]. Intradermal injection of sporozoites to investigate its relationship with 

prepatent period or clinical outcome were variably successful, mostly because of 

the unstable infection rates [90, 91]. Techniques to freeze and thaw sporozoites


were also tried at that time, sometimes accompanied by an intramuscular 

injection of penicillin to prevent bacterial co-infection [92-94]. The practice of 

malariatherapy stopped with the advent of antibiotics. Techniques to induce 

malaria were subsequently used in the vaccine research field, when the first 

available malaria vaccine candidates were tested [95, 96]. However, by that 

time, Pf gametocytes could be produced in culture and Anopheles mosquitoes 

were infected by feeding on culture material [97, 98]. Because these methods 

were safer and less costly, mosquitoes bites have been used for controlled 

infections ever since. 

The translation of mosquito-delivered inoculations into whole-sporozoite needle 

administration is a technical challenge because of difficulties to isolate, purify 

and cryopreserve sporozoites according to current regulatory standards. Recent 

progress has been made by Sanaria Inc., which has developed technology for the 

purification and cryopreservation of aseptic sporozoites for use in humans 

according to the current safety standards [99]. Initially developed as an 

attenuated sporozoite vaccine, these sporozoites were irradiated and tested for 

safety, immunogenicity and efficacy in a clinical vaccine trial. Unfortunately, 

although safe, the trial showed limited protectivity and immunogenicity [100]. 

We subsequently tested the unattenuated cryopreserved sporozoites for 

infectiousness by intradermal injection, proving their potency in five of six 

volunteers from each of three dose groups (Chapter 8). Future studies will focus 

on improving the administration of these sporozoites in order to achieve 100% 

infection rates. Results of these studies will not only attribute to the 

development of a standardized controlled human malaria infection model, but 

also advance the development of a whole-sporozoite vaccine. However, one 

must bear in mind that needle and syringe administration of a bolus of 

sporozoites is clearly different from mosquito bite delivery. Mosquitoes deliver a 

proportion of sporozoites intracapillary and a proportion intradermally [101]. 

Sporozoites are embedded in mosquito saliva when inoculated, components of 

which may possibly improve infectivity [102]. The volume of injection by 

mosquito is also considerably smaller than will ever be reached by needle and 

syringe. These factors may be important to consider particularly if sporozoites 

are injected for the testing sporozoite vaccines that aim to induce antibodies to 

immobilize sporozoites. 

Murine studies indicate that the intramuscular administration of sporozoites by 

needle may be more efficient than the intradermal delivery, possible due to 

better circulation of the muscular tissue (Ploemen pers comm.). The


immunological consequences of dermal versus intramuscular administration are 

currently being investigated (Nganou-Makamdop pers comm.). Whether or not 

the administration of local vasodilators or the use of smaller injection volumes 

improves the immunogenicity or infectivity of sporozoites will also be 

investigated. In any case, it will likely take several years before the needle 

administration of sporozoites has been optimized for human use. However 

elaborate, these studies are not only important to the development of a 

standardized controlled malaria infection model, but will also prove to be crucial 

to the translation of mosquito-delivered sporozoites into a vaccine strategy. 

Whole sporozoite vaccines 

The concept of whole-parasite immunisation originates from studies in which 

humans were immunized by irradiated infected mosquito bite and proved to be 

protected against subsequent Pf challenge (summarized in [103]). Irradiation of 

infectious mosquitoes disrupts the gene expression of sporozoites, which remain 

capable of hepatocyte invasion but are no longer capable of complete liver-stage 

maturation or progression to the pathogenic blood stage [104]. Exposure of 

humans to the antigen repertoire of the irradiated sporozoites proved to be 

sufficient to induce protection against subsequent challenge with viable 

sporozoites, which was shown to last 14 months in several instances [103]. The 

radiation dose was crucial: too much radiation reduced potency while too low 

radiation doses caused break-through infections [105]. 

Although these results were published already in the 1960’s they were not 

considered a vaccine strategy, because the production of a whole-sporozoite 

vaccine that would comply with regulatory standards seemed technically 

impossible. Moreover, the potency of these attenuated sporozoites was limited: 

high doses [> 1000 mosquito bites) were needed to achieve full protection [103]. 

This requirement for large immunisation doses might reflect the lack of normal 

intrahepatocytic differentiation of irradiated sporozoites into thousands of 

hepatic merozoites, an amplification process that is expected to result in an 

expanded antigen repertoire and thus drive immune responses qualitatively and 

quantitatively [106]. 

The protection of human volunteers from Pf challenge by mosquito-delivered 

whole sporozoite inoculation under chloroquine prophylaxis (CPS, Chapter 9) 

shed new light on the concept of whole-parasite immunisation, because this 

strategy proved to be much more efficient at inducing protection. In this case,


only ~45 infected mosquito bites were needed to induce full protection. 

Moreover, the follow-up study (Chapter 10) after 2.5 years proved that 

protection was long-lasting in at least a proportion of volunteers. Moreover, the 

CPS trial has led to the identification of pluripotent (IFNγ+IL-2+) effector memory 

responses as a potential immunological correlate of protection (Chapter 9). 

Serial in depth immunological analyses revealed that various lymphocyte subsets 

contribute to the IFNγ response, including αβT cells, γδT and NK cells. Both γδT 

cells and αβT cells were found to independently contribute to immunological 

memory [107]. Interestingly, individual antigens responsible for the induction of 

the IFNγ response (in vitro) could not be identified. Apparently, the induction of 

IFNγ by Pf is complex, not only involving several cell types with a possible central 

role for multifunctional T-cells, but also relying on a combination of multiple 

antigens. Moreover, interindividual variation in the quantity and quality of IFNγ 

responses reveals significant heterogeneity in Pf-induced immune responses. 

The true correlate of protection may thus not be universal, but also display 

diversity with several different immunological profiles correlating with 

protection. Whether or not multifunctional T-cells form part of this correlate, 

remains to be investigated. However, it seems likely that the true correlate of 

protection will be a composite of multiple responses that form an individual 

immunological fingerprint. 

Although the CPS model is not a formal vaccine strategy, the CPS studies raised 

the question of whether radiation alone produces the most efficient attenuated 

parasites. One possible explanation for the greater efficiency of the CPSapproach 

might be that the co-administration of chloroquine exhibits immune 

modulating effects [108]. Alternatively, the exposure of the immune system to a 

greater array of both pre-erythrocytic and intraerythrocytic antigens, while 

restricting the development of symptomatic and potentially immunosuppressive 

blood stage parasitemia may be critical to enhance the potency of wholesporozoite 

immunization. Indeed, there is some evidence to support the notion 

that late-liver stage arresting parasites might be more efficient in inducing 

protection than early-liver stage arresting parasites [109]. In addition, recent 

Plasmodium transcriptome and proteome analyses showed an increase in 

shared antigens between late-liver stage parasites and blood-stage parasites, 

supporting the use of late-liver stage arresting parasites as a tool to induce 

cross-stage protection [110]. To dissect the immunomodulatory capacity of 

chloroquine from its parasitocidal effect, it would be of interest to test the 

potency of alternative drugs, such as azithromycin or mefloquine when coadministered 

with live sporozoites in humans. Indeed, azithromycin has shown


to induce protection when co-administered with live sporozoites in a mouse 

model [111]. Interestingly, both drugs also exhibit immunoregulatory properties 

[112-114]. Alternatively, the passive transfer of antibodies directed against 

blood-stage antigens could also prevent clinical disease and modulate the 

immune response [115]. 

As an alternative to radiation, several other methods of attenuation, mainly in 

rodent models, have been developed. Treatment of sporozoites with the DNA 

alkylating agent centanamycin, a compound that particularly attenuates malaria 

parasites because it exploits the AT-richness of the parasite genome, impairs the 

infection of hepatocytes by sporozoites and arrests liver stage parasite 

development. Mice inoculated with centanamycin-treated sporozoites failed to 

produce blood stage infections and were protected against subsequent 

challenge with wild-type sporozoites [116]. Moreover, cross-species protection 

could be induced and parasite-specific antibodies and IFN-gamma-producing 

CD8(+) T cells were induced that were not significantly different from radiationattenuated 

sporozoites [116]. 

In fact, CPS immunisation also implies chemical attenuation, albeit in vivo. CPS as 

currently presented is not a widely implementable vaccine strategy, but one 

could envision the co-administration of a drug with a needle administered live 

vaccine product for a selected group of subjects. However, safety requirements 

for such in vivo attenuated product will likely be very stringent, e.g. full 

attenuation must be independent from compliance and pharmacokinetics of the 

drug. Moreover, in vivo attenuation precludes the ability to carry out viability 

checks before the vaccine is administered, necessitating extensive postvaccination 

follow-up. 

In addition to the random genetic attenuation induced by radiation or chemicals, 

targeted attenuation of sporozoites can be achieved by inactivation of specific 

genes by molecular techniques. The main advantage of Genetically Attenuated 

Parasites [GAP) over radiation and chemically attenuated parasites is the fact 

that a homogeneous parasite population with a defined phenotype is obtained. 

The recent availability of complete Plasmodium genome sequences permits the 

development of live-attenuated parasites by more precise and defined genetic 

manipulations [117]. Based on their expression profile and phenotype, 

approximately seven genes have been selected for attenuation and knock-out 

strains created primarily in rodent malaria parasites P. berghei and P. yoelii (p52, 

p36, uis3, uis4, fabb/f, fabz, sap1 [118-126]). Immunisation of mice with GAP


results in protective immune responses that are similar to those induced by 

irradiated sporozoites [127, 128]. To ensure safety of the GAP vaccine, the liver 

arrest of a genetically attenuated parasite vaccine needs to be complete and 

irreversible [129]. Occasional breakthrough infections in GAP lacking genes uis4 

and p52, emphasize that multiple genes will have to be removed in order to 

achieve complete arrest [119, 120]. Recently studies, however, show that also 

parasites lacking expression of both P52 and P36 are capable of developing 

through the liver stage into blood stage infections in vitro and in mouse models 

[130]. This raises concerns about the number of genes that need to be 

attenuated to ensure safety of a whole-sporozoite vaccine. The potency of a 

GAP, on the contrary, depends on development of the attenuated parasites into 

the late liver stage, engendering higher levels of T-cell responses and cross-stage 

and cross–species protection [124]. Whether or not safety and potency 

requirements can both be adequately met in one multiply-attenuated whole 

parasite product will have to be investigated. In addition, the GAP vaccines also 

require precautions with respect to the introduction of attenuated parasites into 

the environment, most likely necessitating the removal of foreign DNA 

sequences from the parasite clones [131]. 

Regardless of the method of attenuation, a live attenuated sporozoite vaccine 

will have to meet stringent safety regulations, as accidental parasite 

multiplication may induce a potentially fatal disease particularly if the vaccine is 

to be used in immunologically vulnerable populations: HIV-infected subjects or 

malnourished infants. An assay that could be reliably and repeatedly used to 

check the potency of sporozoites for vaccination would be an important asset. 

Such a potency assay could also potentially address the issue of translating the 

dose of mosquito-delivered inoculation into a needle-delivered vaccines. The 

only currently available potency assay is the in vitro hepatocyte invasion assay. 

This assay relies on the in vitro invasion of Pf sporozoites in the hepatoma cell 

line HepG2, the human transformed hepatocyte cell line HC04 or primary human 

hepatocytes, all of which are unfortunately limited by very low infection 

efficiencies (


Conclusions and perspectives 

Controlled human malaria infections (CHMI) provide a model to predict malaria 

vaccine efficacy in a well-controlled clinical setting. The CHMI model using Pfinfected 

mosquito bites is well established in several international sites and is 

increasingly used as a crucial check point for the clinical development of preerythrocytic 

stage malaria vaccines. The only candidate malaria vaccine showing 

protective efficacy in Phase IIb field trials so far is RTS,S. This candidate vaccine 

would almost certainly never have been developed without optimization after a 

series of Phase IIa trials. Moreover, efficacy data from Phase IIa trials can help to 

support the decision-making process by ethical boards and communities in 

malaria-endemic countries to justify further testing candidate vaccines in Phase 

IIb trials in susceptible populations. 

Several caveats in standardisation lead to variability in the primary endpoints of 

CHMI trials between institutions, which still need to be addressed. Nevertheless, 

small CHMI trials are sufficiently powered to evaluate >50% effective bloodstage 

vaccines (Chapter 6). CHMI can be strengthened by the administration of 

sporozoites by needle and the availability of several heterologous field strains. 

Particularly the needle administration of sporozoites can boost the setup of new 

clinical trial settings hosted by institutions in malaria-endemic countries, ideally 

mimicking local transmission and incorporating investigations on vaccine effects 

in semi-immune individuals [134]. However, the availability of live sporozoites 

for a larger number of trial centres also warrants increased efforts for 

standardisation and quality control of the infrastructure. After all, the stringent 

selection of volunteers and intense clinical follow-up schedule are important to 

ensure safety of volunteers. 

Clinical development of AMA1 vaccine development has also benefited from the 

CHMI model, confirming satisfactory immunogenicity but lack of significant 

effects on primary endpoints. Our incomplete understanding of immunological 

correlates to AMA1-induced protection in humans seems the main hurdle on the 

road to an effective AMA1 vaccine. CHMI can contribute to unravelling these 

immunological correlates, provided trials are well designed. For example, 

assuming that immunoglobulins are the main effectors to AMA1 induced 

immunity, these specific antibodies should be capable of passively transferring 

protection from one subject to the other, allowing for the identification of 

specific antibodies responsible for protection. Such antibodies could be the longsought 

for correlate of protection. Unfortunately, transfer of antigen specific


immunoglobulins has never been tried in malaria research. Such experiments 

are difficult to perform nowadays because of increased safety requirements 

associated with the transfer of blood products. Once proof of principle for AMA1 

is established, diversity covering [20, 21] or combination vaccines with the blood 

stage antigen MSP1 [135, 136], Circumsporozoite protein [137, 138] or a mix of 

more than two antigens [139], have better chances for success and the search 

for effective adjuvants, delivery platforms and delivery systems can be guided by 

a comparison of antibody responses. 

The inoculation of whole sporozoites is a very potent and efficient way of 

inducing protection against Pf malaria. However, the translation of this approach 

into a whole sporozoite vaccine faces several safety issues. In order to ensure 

safety the development of an in vitro sporozoite potency test will be of vital 

importance. Recent results with multiply-attenuated parasite lines leading to 

blood-infection, are a signal to scientists to take a prudent approach before 

administering live vaccine products to large populations. 

In conclusion, immunological research elucidating mechanisms of protection and 

enhancing efficacy through antigen-adjuvant combinations may be warranted 

for the subunit AMA1 vaccine candidate, whereas the promising efficacy results 

of the CPS immunisation strategy might stimulate a more pragmatic approach to 

develop an implementable whole-organism based vaccine. This thesis shows 

that clinical infection trials in small numbers of healthy human volunteers, 

although subject to rigorous ethical procedures, can provide valuable insight for 

the development of any type of malaria vaccine that would not have been 

acquired otherwise. Such tools may prove to be essential to meet the ambitious 

goals of the Malaria Vaccine Technology Roadmap by 2015–2025.


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Chapter 12 

Summary 

Samenvatting 

List of publications 

Dankwoord 

Curriculum vitae

Summary, Samenvatting, List of publications, Dankwoord, C.V. 245 

Summary 

Plasmodium falciparum (Pf) malaria is one of the most frequent infectious 

diseases and is responsible for severe malaria morbidity and mortality mainly 

among young children in Sub-Saharan Africa. Humans are infected through the 

bite of an infected Anopheles mosquito. An effective malaria vaccine is a key tool 

of critical research that is needed to support malaria control and eradication. 

Decades of research have led to the identification of approximately 36 antigens 

that have been put forward as vaccine candidates. These antigens are grouped 

according to their expression in the Pf lifecycle. Pre-erythrocytic antigens are 

expressed in the sporozoite forms of the parasite that invade the human liver 

causing subclinical infection. Blood stage antigens are expressed during the 

parasite development within the host red blood cells, where they are 

responsible for the clinical malaria disease, with fever, headache and myalgia. 

Transmission blocking antigens are derived from the parasite’s sexual stages, 

which will complete the lifecycle by reproduction within the mosquito host after 

biting the infectious human. 

This thesis focuses on the development of research tools that facilitate 

immunological and vaccine research into human protection against Plasmodium 

falciparum malaria in early stages of clinical development. 

In section 1, we describe the safety and immunogenicity of the Apical 

Membrane Antigen 1 (AMA1), which has been put forward as a promising blood 

stage vaccine candidate (Chapter 2). We combined AMA1 with different 

adjuvants, Alhydrogel, Montanide and AS02 and find distinct reactogenicity 

profiles, with all vaccine formulations being immunogenic. The magnitude of the 

humoral immune response can be enhanced by a more potent adjuvant, but 

breadth and subclass distribution appear much less influenced by the adjuvant 

(Chapter 3). We performed in depth analysis of the concentration and avidity of 

AMA1 antibodies induced and found an inverse relation between these 

parameters (Chapter 4). Since Phase II field efficacy trials with different AMA1 

formulations have been unsuccessful in inducing protection so far, the 

development of new adjuvants, delivery systems, strain covering approaches or 

the combination of AMA1 with other antigens may provide a means by which 

antibody responses can be boosted and animal results can be translated into 

human efficacy.


Controlled human malaria infections (CHMI) are trials in which healthy 

volunteers are exposed to malaria-infected mosquito bites, followed closely and 

treated as soon as blood-stage parasites are detected by microscopy. These 

studies are used to assess preliminary efficacy of primarily pre-erythrocytic 

vaccine candidates in a limited number of institutions worldwide. In section 2, 

we compared the safety and parasitological outcome of CHMI in different 

institutions and conclude that CHMI can be safely conducted, but will lead to 

grade 3 adverse events in a proportion of volunteers (Chapter 5). The primary 

parasitological outcome of such experiments is highly reproducible within 

institutions but may vary between trial centres. In addition, we evaluated the 

power of CHMI to detect vaccine efficacy of candidate malaria vaccines (Chapter 

6) and found that CHMI can confidently assess a reduction in parasitemia for 

both pre-erythrocytic and blood stage vaccines, possibly broadening its 

application in malaria vaccine research. 

We subsequently improved the protocol of CHMI trials by increasing the 

portfolio of Pf parasites for CHMI with the successful establishment of a new Pf 

strain from Cambodia, NF135.C10 (Chapter 7). The availability of this strain 

allows for future assessment of cross-strain immunity induced by candidate 

vaccines through heterologous CHMI trials. Furthermore, we tested 

unattenuated cryopreserved sporozoites for their infectivity by intradermal 

injection, proving potency in five of six volunteers from each of three dose 

groups (Chapter 8). The needle administration of sporozoites has advantages 

over mosquito delivery, in terms of dosing and expanding the global capacity to 

perform CHMI trials. However, future studies will need to focus on improving 

the administration of these sporozoites in order to achieve 100% infection rates. 

Moreover, with an increasing number of CHMI centres being installed, priority 

should be given to initiatives to standardize challenge procedures in order to 

ensure comparability of results worldwide. 

In section 3 we show that viable intact sporozoites are capable of inducing 

protection to Pf malaria in humans very efficiently, if high levels of blood stage 

parasitemia and clinical disease are prevented by co-administration of the drug 

chloroquine (CPS, Chapter 9). Chloroquine kills blood stage parasites but does 

not affect the hepatic development of malaria parasites. A protocol in which we 

exposed healthy, malaria-naïve volunteers to the bites of infected mosquitoes 

under chloroquine prophylaxis on three occasions with monthly intervals, 

rendered 10 of 10 volunteers protected against subsequent controlled infection. 

We found that protection was associated with an increased response of


pluripotent effector memory T-cells. We showed that protection lasts for more 

than two years in four of six re-challenged volunteers and found a markedly 

delayed patency in the two remaining volunteers (Chapter 10). Whereas the 

methodology described here does not itself represent a widely implementable 

vaccine strategy, the induction of protection against an homologous malaria 

challenge suggests that the concept of a whole parasite malaria vaccine 

warrants further consideration. However, the development of a whole 

sporozoite vaccine requires the development of an in vitro sporozoite potency 

test, in order to ensure the complete attenuation of the vaccine. Recent results 

with multiply genetically-attenuated parasite lines leading to blood-infection, 

are a signal to scientists to take a prudent approach before administering live 

vaccine products to large populations. 

In conclusion, a more careful approach incorporating immunological research 

elucidating mechanisms of protection and enhancing efficacy through antigenadjuvant 

combinations may be warranted for the subunit AMA1 vaccine 

candidate, whereas the promising efficacy results of the CPS immunisation 

strategy might stimulate a more pragmatic approach to develop an 

implementable whole-organism based vaccine. Controlled human infection trials 

performed in small numbers of subjects will provide a valuable tool accelerating 

the development of malaria vaccines.


Samenvatting 

Plasmodium falciparum (Pf) malaria is één van de meest voorkomende 

infectieziekten en verantwoordelijk voor veel ziekte en sterfte, in het bijzonder 

onder jonge kinderen in het Afrika ten zuiden van de Sahara. De mens wordt 

geïnfecteerd met malaria parasieten door de beet van een Anopheles mug. Een 

effectief malaria vaccin is essentieel onderzoek om de verspreiding van malaria 

te voorkomen en de ziekte uit te roeien. Tientallen jaren van onderzoek hebben 

geleid tot de identificatie van ongeveer 36 Pf antigenen, mogelijke vaccin 

kandidaten. Deze antigenen worden gegroepeerd naar het tijdstip van expressie 

in de parasitaire levenscyclus. Pre-erythrocytaire antigenen worden tot 

expressie gebracht in de zogenaamde sporozoiet. Deze sporozoiten worden door 

muggen overgedragen, dringen de menselijke lever binnen en veroorzaken een 

subklinische infectie. Bloed stadium antigenen komen tot expressie gedurende 

de ontwikkeling van de parasiet in de menselijke rode bloedcel, waar ze de 

klinische ziekte met koorts, hoofdpijn en spierpijn veroorzaken. Transmissie 

blokkerende antigenen worden verkregen uit de seksuele stadia van de parasiet, 

die de levenscyclus compleet maken door zich in de volgezogen muggenmaag 

voort te planten. 

Dit proefschrift richt zich op de ontwikkeling van onderzoeksinstrumenten 

waarmee immunologisch humaan vaccin onderzoek naar bescherming tegen 

Plasmodium falciparum malaria in een vroeg stadium verricht kan worden. 

In sectie 1 beschrijven wij de veiligheid en immunogeniciteit van Apical 

Membrane Antigen 1, een veelbelovende bloed stadium vaccin kandidaat 

(Hoofdstuk 2). Wij hebben AMA1 gecombineerd met verschillende adjuvantia 

(hulpstoffen), Alhydrogel, Montanide en AS02, en vonden een verschillend 

bijwerkingprofiel, terwijl alle formuleringen immunogeen waren. De omvang van 

de humorale immuun respons werd versterkt door toevoeging van een sterker 

adjuvans, maar de breedte en subklasse verdeling van de antilichamen leken 

veel minder beïnvloed te worden door de adjuvantia (Hoofdstuk 3). Wij 

onderzochten de concentratie en aviditeit van antilichamen geinduceerd tegen 

AMA1 en vonden een omgekeerd evenredige relatie tussen deze twee 

parameters (Hoofdstuk 4). Omdat Fase II veld studies met verschillende AMA1 

formuleringen tot nu toe geen bescherming lieten zien, zal het onderzoek zich


nu wellicht moeten richten op de ontwikkeling van nieuwe adjuvantia, 

toedieningsmethoden en manieren om parasitaire stamverschillen te 

overbruggen of een combinatie van AMA1 met andere antigenen. Dit alles in 

een poging om de antilichaamresponsen te versterken en veelbelovende 

dierexperimentele data te vertalen naar effectiviteit in mensen. 

Gecontroleerde humane malaria infecties (CHMI) zijn studies waarin gezonde 

vrijwilligers blootgesteld worden aan met malaria geïnfecteerde muggenbeten. 

Deze vrijwilligers worden op de voet gevolgd en behandeld met anti-malaria 

middelen zodra bloedstadium parasieten onder de microscoop zichtbaar zijn. 

Deze studies worden in een beperkt aantal centra wereldwijd toegepast om 

vroegtijdig effectiviteit van voornamelijk pre-erythrocytaire vaccin kandidaten 

aan te tonen. In sectie 2 vergelijken wij de veiligheid en parasitologische 

uitkomsten van CHMI in verschillende centra en concluderen wij dat CHMI veilig 

verricht kunnen worden, maar in een deel van de vrijwilligers wel tot graad 3 

symptomen leidt (Hoofdstuk 5). De primaire parasitologische uitkomst van zulke 

experimenten is zeer reproduceerbaar binnen een centrum, maar kan variëren 

tussen onderzoekscentra. Bovendien evalueerden wij de power van CHMI om 

vaccin effectiviteit te kunnen meten (Hoofdstuk 6) en vonden dat CHMI een 

reductie in parasieten-dichtheid voor zowel pre-erythrocytaire als erythrocytaire 

vaccin kandidaten kan aantonen, waarmee de toepassing van CHMI in het 

malaria onderzoek mogelijk uitgebreid kan worden. 

Vervolgens verbeterden wij het CHMI protocol door het portfolio aan Pf parasiet 

stammen aan te vullen met een nieuwe Cambodjaanse stam NF135.C10 

(Hoofdstuk 7). De beschikbaarheid van deze stam maakt het mogelijk om in de 

toekomst immuniteit tegen verschillende stammen te evalueren. Daarnaast 

hebben wij levende, onverzwakte, ingevroren sporozoieten getest op hun 

infectiviteit door middel van intradermale injectie, en hebben bewezen dat hier 

mee vijf van de zes vrijwilligers uit iedere dosis groep geïnfecteerd konden 

worden (Hoofdstuk 8). De toediening van sporozoieten met naald en spuit heeft 

voordelen ten opzichte van de toediening met muggen, omdat parasieten zo 

nauwkeurig gedoseerd kunnen worden en de wereldwijde capaciteit voor CHMI 

studies nu niet meer afhankelijk is van de plaatselijke productie van 

geïnfecteerde muggen. Echter toekomstig onderzoek zal nog nodig zijn om met 

methode 100% infectiviteit te bewerkstelligen. Bovendien zal met een 

toenemend aantal centra dat CHMI uitvoert prioriteit gegeven moeten worden 

aan het standaardiseren van studieprocedures zodat onderlinge resultaten 

vergelijkbaar zijn.


In sectie 3 laten wij zien dat levende, intacte sporozoiten heel efficiënt 

bescherming tegen Pf malaria in mensen kunnen induceren, indien grote 

aantallen bloed stadium parasieten en derhalve klinische ziekte voorkomen 

wordt door toediening van het geneesmiddel chloroquine (CPS, Hoofdstuk 9). 

Chloroquine doodt bloed stadium parasieten, maar tast de leverstadium 

ontwikkeling niet aan. Door middel van een protocol waarbij wij vrijwilligers 

maandelijks blootstelden aan de beten van infectieuze muggen terwijl zij 

chloroquine gebruikten, konden wij laten zien dat 10 van 10 vrijwilligers 

vervolgens beschermd waren tegen gecontroleerde infecties. Wij vonden een 

verband tussen bescherming en een toegenomen respons van pluripotente 

effector memory T-cellen. Wij lieten zien dat de bescherming in vier uit zes 

vrijwilligers meer dan twee jaar aanhield en vonden een vertraagde ontwikkeling 

van parasieten in de overige twee vrijwilligers (Hoofdstuk 10). De beschreven 

methode is geen implementeerbaar vaccin als zodanig, maar de efficiënte 

inductie van bescherming suggereert wel dat vaccin strategieën gebaseerd op de 

gehele parasiet als antigeen heroverwogen dienen te worden. Echter, de 

ontwikkeling van een zogenaamd “hele parasiet” vaccin vereist de ontwikkeling 

van een in vitro test voor de levendigheid van sporozoieten, zodat de 

verzwakking van dit soort vaccins in vitro getest kan worden. Recente resultaten 

met meervoudig genetisch verzwakte parasieten die vervolgens bloed stadium 

infectie veroorzaakten in diermodellen, zijn een signaal voor onderzoekers dat 

men voorzichtig moet zijn met het toedienen van vaccins gebaseerd op levende 

parasieten aan grote populaties. 

Concluderend lijkt een wat terughoudende aanpak voor kandidaat vaccin AMA1 

op zijn plaats, met de nadruk op immunologisch onderzoek naar mechanismen 

van bescherming en het versterken van immuniteit door antigeen-adjuvant 

combinaties, terwijl de veelbelovende bescherming van het CPS model wellicht 

een meer pragmatische aanpak rechtvaardigt om een implementeerbaar “hele 

parasiet” vaccin te ontwikkelen. Gecontroleerde humane infectie studies in 

kleine aantallen vrijwilligers vormen een belangrijk instrument om de 

ontwikkeling van malaria vaccins te versnellen.


List of publications 

Roestenberg M, Bijker EM, Sim BK, Billingsley PF, James ER, Bastiaens GJH, 

Teirlinck AC, Scholzen A, Teelen K, Arens T, van der Ven AJAM, Gunasekera A, 

Chakravarty S, Velmurugan S, Hermsen CC, Sauerwein RW, and Hoffman SL. 

Controlled human malaria infections by intradermal injection of cryopreserved 

Plasmodium falciparum sporozoites. Am J Trop Med Hyg. 2012 

Teirlinck AC, Roestenberg M, van de Vegte-Bolmer M, Scholzen A, Heinrichs 

MJL, Siebelink-Stoter R, Graumans W, van Gemert GJ, Teelen K, Vos MW, 

Nganou-Makamdop K, Borrmann S, Rozier YPA, Erkens MAA, Luty AJF, Hermsen 

CC, Sim BK, van Lieshout L, Hoffman SL, Visser LG, Sauerwein RW. NF135.C10: a 

new Plasmodium falciparum clone for controlled human malaria infections. J 

Infect Dis. 2012 

Nganou-Makamdop K, Ploemen I, Behet M, van Gemert GJ, Hermsen C, 

Roestenberg M, Sauerwein RW. Reduced Plasmodium berghei sporozoite liver 

load associates with low protective efficacy after intradermal immunization. 

Parasite Immunol. 2012 Aug 6. 

Remarque EJ, Roestenberg M, Younis S, Walraven V, van der Werff N, Faber BW, 

Leroy O, Sauerwein R, Kocken CH, Thomas AW. Humoral Immune Responses to a 

Single Allele PfAMA1 Vaccine in Healthy Malaria-Naïve Adults. PLoS One. 

2012;7(6):e38898. 

Roestenberg M, O'Hara GA, Duncan CJ, Epstein JE, Edwards NJ, Scholzen A, van 

der Ven AJ, Hermsen CC, Hill AV, Sauerwein RW. Comparison of clinical and 

parasitological data from controlled human malaria infection trials. PLoS One. 

2012;7(6):e38434. 

Laurens MB, Duncan CJ, Epstein JE, Hill AV, Komisar JL, Lyke KE, Ockenhouse CF, 

Richie TL, Roestenberg M, Sauerwein RW, Spring MD, Talley AK, Moorthy VS; 

The Consensus Group on Design of Clinical Trials of Controlled Human Malaria 

Infection. A consultation on the optimization of controlled human malaria 

infection by mosquito bite for evaluation of candidate malaria vaccines. Vaccine. 

2012 Aug 3;30(36):5302-5304. 

Roestenberg M, de Vlas SJ, Nieman AE, Sauerwein RW, Hermsen CC. Efficacy of 

preerythrocytic and blood-stage malaria vaccines can be assessed in small 

sporozoite challenge trials in human volunteers. J Infect Dis. 2012 

Aug;206(3):319-23. 

Teirlinck AC, McCall MB, Roestenberg M, Scholzen A, Woestenenk R, de Mast Q, 

van der Ven AJ, Hermsen CC, Luty AJ, Sauerwein RW. Longevity and composition 

of cellular immune responses following experimental Plasmodium falciparum 

malaria infection in humans. PLoS Pathog. 2011 Dec;7(12):e1002389.


Roestenberg M, Teirlinck AC, McCall MB, Teelen K, Makamdop KN, Wiersma J, 

Arens T, Beckers P, van Gemert G, van de Vegte-Bolmer M, van der Ven AJ, Luty 

AJ, Hermsen CC, Sauerwein RW. Long-term protection against malaria after 

experimental sporozoite inoculation: an open-label follow-up study. Lancet. 

2011 May 21;377(9779):1770-6. 

Sauerwein RW, Roestenberg M, Moorthy VS. Experimental human challenge 


Immunol. 2011 Jan;11(1):57-64. 

McCall MB, Roestenberg M, Ploemen I, Teirlinck A, Hopman J, de Mast Q, Dolo 

A, Doumbo OK, Luty A, van der Ven AJ, Hermsen CC, Sauerwein RW. Memorylike 

IFN-γ response by NK cells following malaria infection reveals the crucial role 

of T cells in NK cell activation by P. falciparum. Eur J Immunol. 2010 

Dec;40(12):3472-7. 

de Mast Q, de Groot PG, van Heerde WL, Roestenberg M, van Velzen JF, 

Verbruggen B, Roest M, McCall M, Nieman AE, Westendorp J, Syafruddin D, 

Fijnheer R, van Dongen-Lases EC, Sauerwein RW, van der Ven AJ. 

Thrombocytopenia in early malaria is associated with GP1b shedding in absence 

of systemic platelet activation and consumptive coagulopathy. Br J Haematol. 

2010 Dec;151(5):495-503. 

Nieman AE, de Mast Q, Roestenberg M, Wiersma J, Pop G, Stalenhoef A, Druilhe 

P, Sauerwein R, van der Ven A. Cardiac complication after experimental human 

malaria infection: a case report. Malar J. 2009 Dec 3;8:277. 

Roestenberg M, McCall M, Hopman J, Wiersma J, Luty AJ, van Gemert GJ, van de 

Vegte-Bolmer M, van Schaijk B, Teelen K, Arens T, Spaarman L, de Mast Q, 

Roeffen W, Snounou G, Rénia L, van der Ven A, Hermsen CC, Sauerwein R. 

Protection against a malaria challenge by sporozoite inoculation. N Engl J Med. 

2009 Jul 30;361(5):468-77. 

de Mast Q, van Dongen-Lases EC, Swinkels DW, Nieman AE, Roestenberg M, 

Druilhe P, Arens TA, Luty AJ, Hermsen CC, Sauerwein RW, van der Ven AJ. Mild 

increases in serum hepcidin and interleukin-6 concentrations impair iron 

incorporation in haemoglobin during an experimental human malaria infection. 

Br J Haematol. 2009 Jun;145(5):657-64. 

Roestenberg M, Remarque E, de Jonge E, Hermsen R, Blythman H, Leroy O, 

Imoukhuede E, Jepsen S, Ofori-Anyinam O, Faber B, Kocken CH, Arnold M, 

Walraven V, Teelen K, Roeffen W, de Mast Q, Ballou WR, Cohen J, Dubois MC, 

Ascarateil S, van der Ven A, Thomas A, Sauerwein R. Safety and immunogenicity 

of a recombinant Plasmodium falciparum AMA1 malaria vaccine adjuvanted 

with Alhydrogel, Montanide ISA 720 or AS02. PLoS One. 2008;3(12):e3960. 

Roestenberg M, McCall M, Mollnes TE, van Deuren M, Sprong T, Klasen I, 

Hermsen CC, Sauerwein RW, van der Ven A. Complement activation in


experimental human malaria infection. Trans R Soc Trop Med Hyg. 2007 

Jul;101(7):643-9. 

Hermsen CC, Verhage DF, Telgt DS, Teelen K, Bousema JT, Roestenberg M, Bolad 

A, Berzins K, Corradin G, Leroy O, Theisen M, Sauerwein RW. Glutamate-rich 

protein (GLURP) induces antibodies that inhibit in vitro growth of Plasmodium 

falciparum in a phase 1 malaria vaccine trial. Vaccine. 2007 Apr 12;25(15):2930- 

40. 

Damoiseaux J, van der Ven A, Hermsen R, Telgt D, Roestenberg M, Tervaert JW, 

Sauerwein R. Experimental infection with Plasmodium falciparum does not 

result in the induction of anticardiolipin antibodies in healthy volunteers. Ann 

Rheum Dis. 2005 Dec;64(12):1804-5.


Dankwoord 

Zoals dit proefschrift meer bloed, zweet en tranen heeft gekost dan het op het 

oog doet vermoeden, zo hebben ook veel meer mensen een bijdrage aan de 

diverse hoofdstukken geleverd dan alleen de auteurslijst aangeeft. 

Hoewel dit laatste hoofdstuk bedoeld is om dit recht te zetten, is dit bij voorbaat 

al een onmogelijke opgave. Acht jaar collegialiteit, vriendschap, lief en leed is 

niet te vatten in enkele pagina’s van woorden. Desalniettemin hierbij een 

poging. 

Allereerst ben ik natuurlijk enorme dank verschuldigd aan alle vrijwilligers. Het 

vertrouwen dat jullie mij gaven en jullie geloof in het belang van de studie was 

vaak overweldigend. Om maar niet te spreken over jullie talent om zelfs van de 

vroege ochtendbezoeken een feestje te maken en jullie doorzettingsvermogen 

ondanks het soms rigoureuze prikschema. Jullie zijn de echte helden van dit 

verhaal. 

Robert, het grote avontuur begon bij jou. André stelde mij aan je voor, je legde 

mij uit wat malaria was, en vanaf toen werden we samen meegenomen op een 

tumultueuze reis die promotie schijnt te heten. Ik ontwikkelde een talent om jou 

bij voorkeur ’s nachts of op vakantie te bellen, met immer urgente en 

onvoorziene ellende die á la minute opgelost dienden te worden. Op de meest 

cruciale momenten waren we afhankelijk van het telefoonnetwerk in Trinidad, 

Tanzania, Mali, of een willekeurige luchthaven ter wereld. Toch heb ik je niet 

één maal horen zuchten en heb ik me altijd, zelfs in die kleine uurtjes, door jou 

gesteund gevoeld. Het werk is nog lang niet af, en dit boekje slechts een 

spreekwoordelijke luchthaven, ons faciliterend in onze verdere reizen. Ik hoop 

dat we nog vaak samen zullen optrekken. 

Rob, na vele nachten of weken bikkelen kon ik altijd even bij jou binnenvallen 

voor goede raad. Jij, altijd geïnteresseerd in de mens achter de onderzoeker, 

stuurde me vaak naar huis, sprak me vaderlijk toe of nodigde zowel mij als 

Jeroen uit voor heerlijk eten, wat je samen met Elly bereidde. Ik waardeer je 

zeer, en hoop dat je ook trots bent op dit proefschrift. Het is immers een 

reflectie van niet alleen mijn, maar ook jouw “levenswerk”! 

Jorien, jij bent de personificatie van “malaria-moeder”. Je zorgde niet alleen 

voor alle vrijwilligers met een liefde en toewijding die grenzeloos leek, maar 

hebt ook mij onder je vleugels genomen. Ik mocht altijd bij jou in de keuken, 

achtertuin of op het CRCN even uitblazen onder het genot van een bak thee, 

terwijl jij me nieuwe moed gaf, alleen al door je grenzeloze geloof in het belang 

van het onderzoek. Zowel Jeroen als ik zijn je erg dankbaar en vinden het vooral


bijzonder dat je ook Simon in je hart gesloten hebt. We hopen dat je nog vaak bij 

ons in Wassenaar langs komt. 

Anne, wij deelden “gouden tulpen” in Leiden, leefden weken op enkele uurtjes 

slaap per nacht, en wisten de aspergesoep en Pavlov’s broodjes pas echt te 

waarderen. Nederland vierde Koninginnedag terwijl wij prikten, pipetteerde, 

centrifugeerden en eindeloze hoeveelheden droogijs verslonden en onze 

mannen met militaire precisie vele honderden epjes beplakten, intussen jullie 

huwelijk bekokstovend. Je bent een geweldige collega, vriendin en onderzoeker, 

je weet dat ik je openheid, integriteit en eerlijkheid waardeer. Kom je nog vaak 

bij ons “logeren”? 

Matthew, na negen maanden zwoegen, produceerden wij samen ons “gouden” 

kind, dat wij vervolgens, gelukkig succesvol, in een mandje op de rivier hebben 

losgelaten. Elke alinea kostte ons vele zondagen, op zoek naar perfectie. 

Gelukkig werd ons werken, koppigheid en eigenzinnigheid gewaardeerd en 

uiteindelijk beloond. Ik waardeer je als collega, vriend en briljant onderzoeker. 

Laten we hopen op nog vele “projectjes” samen! 

Karina, je bent haast een vanzelfsprekendheid op het lab, maar wij weten maar 

al te goed hoe onmisbaar je bent. Wat mij betreft ben jij de ultieme multitasker: 

je weet een druk gezin en gezellig sociaal leven te combineren met een baan die 

nauwelijks te plannen valt. Bij elke studie was je tot in de kleine uurtjes present. 

Bovendien ben jij de enige die foutloos drie batches extracties tegelijk kan 

runnen, gebruikmakend van diverse centrifuges die over verschillende labs 

verspreid zijn, zonder dat je humeur daar onder lijdt. Ik geloof niet dat ik dat ooit 

zal evenaren. 

Else, je kwam als geroepen toen het praktische werk van TIP2 op de drempel lag. 

Je ontpopte je tot een gezellige collega en stortte je vol enthousiasme in het 

malaria-avontuur waarbij de druk in zeer korte tijd opgevoerd werd en je 

soepeltjes verscheidene studies op verschillende lokaties tegelijk begeleidt. 

Gelukkig blijf je een ontnuchterend gevoel voor humor houden. Ik hoop nog 

altijd dat je je toekomstplannen wijzigt en we weer samen zullen werken ;) 

An-Emmie, vriendin en zeer gewaardeerde collega. Ik heb bewondering voor je 

flexibiliteit en durf om een andere richting te kiezen in je leven en ben je 

dankbaar voor vele uren aan steun in de flexplek. Fijn dat je het naar je zin hebt 

bij de microbiologie en vooral super dat je eigenlijk nog altijd een “intramurale” 

collega bent, al is het nu op iets meer "afstand". 

André, ik kwam als piepjonge dokter bij je binnen en je vroeg me wat ik leuk 

vond: “Malaria, HIV of TB?”. Je ontdekte malaria-Meta en stelde me aan Robert 

voor, waarbij je me verzekerde dat ook een fase I studie “uitdagend” kan zijn.


Dat bleek het zeker. Dank voor je bezielende klinische begeleiding, je 

betrokkenheid bij het welzijn van de vrijwilligers en je enthousiasme. Quirijn, als 

“opvolger” van André stond je aan de zijlijn om in te springen waar nodig. Je 

flexibiliteit is zeer gewaardeerd. 

Geert-Jan, Marga, Wouter en Rianne, zonder jullie was geen letter van dit 

proefschrift mogelijk geweest. Zonder overdrijven leveren jullie bij elke klinisch 

studie weer een topprestatie, die voor velen wellicht “gewoon” voelt, maar 

waarvan ik mij terdege bewust ben. Jullie zijn toppers van letterlijk 

wereldformaat! 

Ben, samen deelden wij vele jaren op het lab. Ik heb groot respect voor je 

doorzettingsvermogen en kundigheid. Maar de grootste uitdaging “is yet to 

come”. Count me in! Annemieke, ik kan me niet herinneren dat er ooit een 

moment was dat jij nog niet in Nijmegen werkte. Je bent een grote steun en 

toeverlaat voor mij, regelt altijd op het juiste moment de juiste zaken, niet in de 

laatste plaats een babybadje en luieremmer ;). Wat moet ik zonder je? Krystelle, 

je bent een fijne vriendin en collega, waarom moest je nou zo ver weg gaan 

wonen? Ivo, wat hebben we gelachen! Maar vergeet niet, als we “klaar” zijn met 

malaria, dan gaan we samen HIV “doen”, toch? Dunja, wat fijn om iemand te 

ontmoeten die blijkt te “houden” van het schuiven met papieren! 

Maarten, Martijn, Will, Joost, Adrian en alle andere lab-collegae. Ook al lijkt het 

werk eindeloos en soms herhaaldelijk te mislukken, vergeet niet dat elk 

experiment vooruitgang betekent! 

Leo, zelden smaakte een Starbucks’ “chai” zo goed als wanneer door jou op 

zondagochtend op sportschoenen gebracht. Je toewijding bleek grenzeloos en je 

voorzag niet alleen het onderzoek in Leiden van bezielende leiding, maar 

suppleerde ook al mijn broodnodige voedingsstoffen met heerlijk, “fatsoenlijk 

Vlaams” eten. Je bent een ongelooflijk goede arts, onderzoeker en vriendelijk 

mens. Ik ben er trots op dat je mijn opleider bent. 

Steve, Kim Lee, Pete, Eric, Anusha, Tooba and the Sanaria team. I absolutely 

loved working with you and am proud of what we have achieved so far. I cherish 

the memory of us sharing turkey with Thanksgiving or breaking scooter records 

in an attempt to reduce transport time. I have been very lucky to enjoy many 

aspects of your hospitality both in Maryland and in Holland where I felt part of 

the Sanaria-family. I hope we will get a chance to work again in the future to 

pursue our common dream and to show you some Dutch hospitality in Leiden. 

T_O, jij nam met veel aanstekelijk enthousiasme het “dikke druppel team 

Nijmegen” onder je hoede, zorgde voor de roosters en nam bovendien vaak het 

leeuwendeel van de “diensten” voor je rekening. Vele nachten en weekenden


hebben wij samen de juiste kleur M&M’s opgegeten, terwijl ik wachtte op jouw 

oordeel. Je bent een fijne en gezellige collega! 

Pieter, Nanny, Chantal, Wendy, Paul, Ella en Marlou, jullie keken vele, vele 

dikke druppels en toch waren jullie bij elke studie weer opnieuw enthousiast en 

allemaal aanwezig. Jullie betrokkenheid en deskundigheid waren onmisbaar bij 

al dit werk. Ik hoop dat jullie met evenveel trots en plezier terugkijken als ik. 

Lisette, Jaco, Eric, Marjan, Meriam, Els, Jolanda, Heleen, Fons en Jeanette jullie 

werden plots overvallen door een rollercoaster die het klinisch malaria 

onderzoek heet, maar stortte je hier samen met mij vol enthousiasme in. Ik heb 

jullie stuk voor stuk leren kennen als zeer kundige, zorgvuldige en gezellige 

collega’s, en ik ben vereerd dat ik nu vaker met jullie kom te werken. 

Chris, Shahid and the rest of the Leiden-malaria-team, the collaboration 

between Leiden and Nijmegen has become quite intense in the last few years 

and has proven to be fruitful. I admire your expertise and was astonished to find 

you at the lab at the most incredible hours of the night. I hope we will continue 

to be in touch over the next years and hopefully also share some “daylighthours” 

together. 

Ed, Bart, Alan, Clemens en andere BPRCers, ik heb de afgelopen jaren 

meermaals genoten van jullie gastvrijheid in Rijswijk, voel je welkom om eens in 

Leiden een biertje te komen drinken op het terras! 

Collega’s uit het CWZ, in het bijzonder Laura, Margreet, Pieter, Anja, Inge, jullie 

zijn allemaal hardwerkende, ondergewaardeerde schatten! Een steun en 

toeverlaat in “barre” tijden, ploeterend om de dienst/SEH/afdeling of poli te 

overleven. Wat zou het leuk zijn als we weer samen zouden kunnen werken! 

CWZ supervisoren, zonder jullie allen bij naam te noemen, draag ik jullie erfenis 

elke dag een klein beetje met me mee. Dank voor jullie kennis en enthousiasme 

om zelfs van een zeer eigenwijze arts-assistent een voorbeeldig arts te maken. 

Jacqueline de Graaf, jij bent een opleider pur sang. Altijd een luisterend oor 

paraat en altijd. Dank voor je steun en je geloof in mij, ik ben je veel dank 

verschuldigd. 

Yolanda, helaas verliet je de research om terug te gaan naar je stek bij de 

hematologie. Je was echter mijn grote steun en toeverlaat, een echte regelaar 

die nergens voor terug deinsde, aanpakte en zelfs om 6 uur ‘s ochtends een bus 

met “lachende” chauffeur wist te versieren om iedereen naar Nijmegen te 

vervoeren. Ik hoop dat ik je ooit nog eens kan verleiden om weer met me samen 

te werken… 

Kitty (en Jos), jullie flexibiliteit en hulp bij de TIP1 studie was onmisbaar. We zien 

elkaar op de vaccinatiepoli!


Harm, het kostte soms wat moeite voor je om te begrijpen hoe en wat ik wilde, 

maar het uiteindelijke resultaat mag er zijn! Ik ben trots op wat je gebouwd 

hebt, en hoop ook in de toekomst op je IT ondersteuning te kunnen vertrouwen. 

Sake, vele telefoontjes hebben toch zijn vruchten afgeworpen! We zullen vast in 

de toekomst nog wel vaker bellen, als ik weer een leuk “modelmatig” projectje 

bedenk. 

Audrey, Nicole en Sylvie van de Klinische Farmacie, ondanks de soms 

onmogelijke papieren rompslomp, hebben jullie mij altijd op zeer professionele 

wijze in de studies ondersteund. Hulde! 

Medewerkers van het Clinical Research Center Nijmegen, ondanks mijn soms 

onmogelijke korte-termijn planning en ingewikkelde prik-schema’s, lukte het 

jullie toch altijd weer om ons te faciliteren. Bovendien wisten wij ons samen 

verder te professionaliseren, opdat wij kwalitatief hoogwaardig onderzoek neer 

konden zetten. Ik kijk terug op een vruchtbare samenwerking! 

Safety monitors en independent physicians, zonder jullie bij naam te noemen, 

ben ik jullie absoluut veel dank verschuldigd. Dank voor het vertrouwen en jullie 

kundige support. 

Pa en ma Roestenberg, Timo en Liliane, Pa en ma Mulder, Thijs en Karin, dank 

voor jullie eindeloze steun en begrip. Vooral natuurlijk grote dank voor pa 

Roestenberg, die een formidabele prestatie leverde in de layout van dit boekje. 

Dankzij jouw inspanningen is het niet alleen af, maar ook leesbaar. 

Jeroen, je bent de eerste en de laatste, mijn rots in de branding en liefde van 

mijn leven. Jij stond aan de wieg van dit proefschrift, volgde het op de voet en 

steunde mij onvoorwaardelijk met een onwrikbaar je vertrouwen in mij en in het 

onderzoek. In de afgelopen jaren werden jij en ik “wij” en stond je opnieuw aan 

de wieg. Samen verleggen wij stenen.


Curriculum Vitae 

Meta Roestenberg was born on the 24 th of January 1981 in De Bilt, The 

Netherlands. She completed bilingual highschool at the Alberdingk Thijm College 

in Hilversum with the international baccalaureate diploma. She studied 

Medicine at the University of Maastricht, where she obtained her medical 

degree cum laude in 2004. During her medical training Meta had the opportunity 

to follow internships in Africa and South-East Asia, where she became fascinated 

by infectious diseases. She returned to India for additional training several 

months after her graduation and then decided to dedicate her professional 

carreer to poverty related infectious diseases. Upon returning to The 

Netherlands, she started malaria vaccine research at the department of Medical 

Microbiology of the Radboud University Medical Center. In 2006 she began with 

her training in internal medicine, first at the Radboud University Medical Center, 

later at the Canisius Wilhelmina Hospital, both in Nijmegen. In the meantime, 

she continued to work on her PhD, alternating between clinical work and 

research. In 2012 she started her subspecialisation for infectious diseases at the 

Leiden University Medical Center. In the future, Meta hopes to continue clinical 

research on vaccines to fight infectious diseases that cause significant morbidity 

and mortality among the poorest people in the world. 

Meta is married Jeroen Mulder and they have one son, Simon, born in 2011.

Experimental infection and protection against ... - TI Pharma

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