REVIEW Design and production of recombinant subunit vaccines

Biotechnol. Appl. Biochem. (2000) 32, 95–107 (Printed in Great Britain) 95
REVIEW
Design and production of recombinant subunit vaccines
Marianne Hansson, Per-A � ke Nygren and Stefan Sta� hl 1
Department of Biotechnology, Kungliga Tekniska Ho� gskolan, SE-100 44 Stockholm, Sweden
The development of subunit vaccines is presently the
main strategy being evaluated for prevention of infectious
diseases. The use of recombinant-DNA techniques
has facilitated the development of new principles
for design and production of subunit vaccines. First of
all, the properties of a target protein immunogen can
be improved by the use of gene-fusion technology or by
the creation of specific changes, to generate ‘secondgeneration
protein vaccines’. Properties that can be
modified include protein solubility, protein stability, in
vivo half-lives, etc. In addition, for subunit protein
vaccine candidates,the immunogenic properties can be
significantly augmented by the addition of immunopotentiating
tags or by means of targeting to immunoreactive
sites. The recombinant subunit vaccine
can furthermore be adaptedby gene-fusion technology,
to be efficiently incorporated into immunopotentiating
adjuvant systems. Also in passive vaccination strategies,
i.e. the use of antibodies or antibody fragments for
prevention of infectious diseases, the recombinant
strategies have become increasingly important.
Humanized antibodies and antibody fusion proteins
represent common present anti-infectious-disease
agents. The selected examples will indicate that recombinant
strategies will indeed have an impact on the
design, selection and production of recombinant proteins
to be used in the prevention of infectious diseases.
Introduction to vaccinology
The development of vaccines has had great impact on the
public health of the world [1]. The first vaccine for human
use, the smallpox vaccine developed by Edward Jenner, was
introduced in 1798. Since the introduction of this first
vaccine a little more than 200 years ago, vaccination has
controlled nine major diseases, at least in parts of the world:
smallpox, diphtheria, pertussis, tetanus, yellow fever, poliomyelitis,
measles, mumps and rubella; and, in the case of
smallpox, the dream of eradication has been fulfilled.
Vaccines can be divided into two broad categories: active
and passive. An active vaccine is intended to stimulate the
body’s immune system to produce specific antibodies
(humoral response), cellular immune responses (e.g. cytotoxic
T-lymphocytes), or both, with the aim of protecting
against or eliminating a pathogen. A passive vaccine is a
preparation of antibodies that is protective against a
pathogen or disease and is administered before, at or around
the time of known or potential exposure.
Active vaccines can in turn be divided in three main
categories, live attenuated vaccines, killed vaccines and
subunit vaccines (for a comparison see Table 1). As will be
discussed, it is today possible also to produce subunit
vaccines by recombinant means.
Live vaccines can potentially replicate in the host, but
are typically attenuated in their pathogenicity in order not to
cause disease. Live vaccines may elicit both humoral and
cellular immunity, and only a single or a few doses may give
lifelong protection. One main drawback of attenuated live
vaccines is, of course, the risk of reversion into their original
pathogenic forms, especially in immunocompromised individuals
and infants. Moreover, it is possible that some live
vaccine strains can be transmitted from the vaccinee to an
unvaccinated individual.
Killed vaccines should not be capable of replicating in
the host and can therefore not multiply or revert to
pathogenicity, or transmit the disease to another person.
The immunogenicity of a killed vaccine usually has to be
enhanced by its presentation by an adjuvant system, and
multiple doses are normally necessary for obtaining longterm
protective immunity. Killed vaccines usually function by
stimulating the humoral immune response, as well as by
priming for immunological memory. The production of such
vaccines requires large-scale culturing in vitro of the diseasecausing
micro-organism, which can be associated with both
safety risks and problems to achieve cost-efficient production.
In the preparation of killed vaccines, one problem is
to achieve effective killing without complete destruction of
the protective antigens. With the present requirements
from regulatory agencies, e.g. the U. S. Food and Drug
Key words: fusion protein, live delivery system, nucleic acid vaccine, protein
immunogen, targeting.
Abbreviations used: LTB, binding subunit of heat-labile toxin; HBsAg,
hepatitis B surface antigen; SpA, Staphylococcus aureus Protein A; SpG,
streptococcal protein G; RSV, respiratory syncytial virus; CTB, cholera
toxin B; iscom; immunostimulating complex.
1 To whom correspondence should be addressed
(e-mail stefans�biochem.kth.se).
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96 M. Hansson, P.-A� . Nygren and S. Sta� hl
Table 1 A comparison of some properties of different vaccine types
Vaccine type Advantages Drawbacks
Live vaccines (attenuated) One or few doses normally required Controlled attenuation normally required
Long-lasting protection Risk of reversion to pathogenicity
Both humoral and cellular responses Certain risk of transmission
Poorly defined composition
Killed vaccines No risk of reversion to pathogenicity Multiple doses typically required
No risk of transmission Poorly defined composition
Antigen must be produced by cultivation of a pathogen
Mainly humoral responses
Adjuvants normally needed
Subunit vaccines (non-recombinant) Defined composition
Various delivery systems available
Subunit vaccines (recombinant) No risk of pathogenicity since the pathogenic organism is
not present
Defined composition
Various delivery systems available
Simplified large-scale production
Further engineering possible
Administration (FDA) and World Health Organization
(WHO), for exact definitions of the vaccine preparations, it
will probably become increasingly difficult to get new
vaccines of this class accepted for human use.
Subunit vaccines take advantage of the possibility of
using only part of the infectious micro-organism to raise a
protective immune response, and since subunit vaccines
cannot replicate in the host, there is no risk of pathogenicity.
The composition of a subunit vaccine can normally be clearly
defined, which is a significant advantage in terms of safety
considerations and minimization of side-effects. In order to
elicit a vigorous immune response, subunit vaccines often
require multiple doses, as well as the use of adjuvants.
Subunit vaccines can be based on peptides, proteins or
polysaccharides that have been shown to contain protective
epitopes. Many of the cell-surface carbohydrates of pathogenic
bacteria, e.g. capsular polysaccharides, are important
antigenic determinants for vaccine development. Subunit
conjugate vaccines [2,3] based on such polysaccharides, e.g.
Haemophilus influenzae type b (Hib) vaccines, which are
commonly delivered coupled to protein carriers such as the
tetanus toxoid [4], will not be described further in this
review.
Immunogenic components can be isolated directly from
pathogenic organisms, e.g. bacterial polysaccharides are
often shed during growth and can be enriched from the
culture medium. However, the production of subunit
vaccines often requires purification of the immunogens from
large quantities of the pathogenic organism, which is not
without risk and significant cost.
Recombinant-DNA technology allows controlled production
of protein-subunit vaccines in heterologous hosts.
Such strategies have several advantages, e.g. safe and cost-
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Antigen must be produced and purified by cultivation of
a pathogen
Multiple doses typically required
Adjuvants needed
Multiple doses typically required
Adjuvants needed
Table 2 Recombinant subunit vaccines and examples of their advantages
(+) and drawbacks (�)
Recombinant vaccine Advantages/drawbacks
Protein immunogens + No risk of pathogenicity since the pathogenic
organism is not present
+ Efficient production systems available
� Multiple doses required
� Adjuvants needed
Live delivery systems + May induce both humoral and cellular
responses
+ Adjuvants normally not needed
� Risk of reversion when using attenuated
pathogens as carriers
Bacterial + Surface display of antigens possible
+ Mucosal administration possible
Viral + Efficient induction of cellular responses
Nucleic acid vaccines + No risk of pathogenicity
+ Simple production
+ May induce both humoral and cellular
responses
� Variable immune responses
� Inefficient transfection
DNA + Dedicated delivery systems exist
� Inefficient delivery
� Risk of integration into host chromosomes
must be considered
� Moderate and variable immune responses
RNA + No risk of integration into host chromosomes
+ No need to enter the nucleus for translation
+ In vivo amplification systems available
� Cumbersome large-scale production
� Unstable
efficient production systems can be used. Recombinant
strategies further offer the possibility of delivering protein
subunits with the help of live delivery systems, bacterial or
viral, or even as antigen-encoding genes, so-called nucleic
acid vaccines (Table 2).

Recombinant subunit vaccines
Recent advances in immunology and protein engineering
have allowed the design and production of recombinant
subunit vaccines [3,5–8]. The epitopes recognized by neutralizing
antibodies are usually found in just one or a few
proteins present on the surface of the pathogenic organism.
Isolation of the genes encoding such epitope-carrying protein
immunogens and their expression in heterologous hosts
form the basis of recombinant-subunit-vaccine development.
The main advantage of using single proteins displaying
immunodominant epitopes as vaccines is the possibility of
inducing protective immunity without having side effects and
immune reactions caused by other parts of the pathogenic
organism. Potential challenges in the development of subunit
vaccines are that they often are poorly immunogenic and
have short in vivo half-lifes. Another difficulty with subunit
vaccines is that they often elicit only strain-specific protection,
so, to evoke full protection to a disease caused by
several related strains, combinations of immunogens from
the different strains might be needed.
Protein immunogens
Protein production in heterologous hosts is a wellestablished
technique today, and recombinant strategies can
thus combine the benefits of subunit vaccines with efficient
production systems using non-pathogenic expression
vehicles [8,9]. As will be discussed further, the use of fusion
proteins can facilitate the downstream purification of
immunogens [8,10] and, in addition, fusion of the target
immunogens to carrier proteins can increase both the halflife
[11,12] and immunogenicity [13–15] of the immunogen.
Live delivery systems
Besides the possibility of producing recombinant protein
immunogens in heterologous hosts, technologies to construct
recombinant live viral and bacterial vaccine-delivery
vectors carrying foreign immunogens have been developed.
Several viruses have been investigated for their possible use
as recombinant vaccines (for extensive reviews see [3,8,
16,17]). The vaccinia vector is perhaps the most wellcharacterized
viral vector, and a vaccinia vector encoding a
rabies glycoprotein has been used for oral immunization of
wild animals [18,19]. Due to safety concerns, non-attenuated
recombinant vaccinia virus is not likely to be used in humans.
As alternatives, a highly attenuated recombinant vaccinia
virus vector (NYVAC), which has been constructed through
deletion of 18 open reading frames [20], as well as an avianpoxvirus
vector (ALVAC) [21], have been developed. A
NYVAC vector expressing seven different malarial antigens
has been constructed and demonstrated to induce a
Recombinant subunit vaccines 97
Plasmodium-specific antibody response in rhesus macaques
[22]. Adenoviruses have also been investigated as live vectors
[17], and are especially interesting, since they are not
particularly pathogenic in humans. The major disadvantage
with adenoviruses is the limited cloning capacity compared
with, for example, the vaccinia virus. Several other viruses
have also been studied for the possible use as live vectors
[17], but no virus-based vaccine, employed for live heterologous
vaccine delivery, is yet accepted for human use.
The use of live bacteria as mucosal vaccines has been
studied extensively, both against the corresponding disease
and also as delivery systems for heterologous diseases.
Mucosal vaccines are easy to administer, e.g. by the oral or
nasal route, and using bacteria as a delivery vehicle makes the
vaccine comparatively inexpensive to produce [23]. The
best-studied bacterial delivery systems are based on
attenuated Salmonella spp. as live vectors expressing heterologous
antigens and numerous studies have been performed
for development of oral vaccines against bacterial,
viral or parasitic diseases [24–26]. Live Salmonella, being an
intracellular pathogen, is generally capable of eliciting cellular
immune responses to the antigen delivered, a desired
property of the immune responses protecting against viral or
parasitic diseases.
The possibility of expressing foreign proteins on the
surface of bacteria makes live bacterial vectors highly
interesting. Surface display of heterologous proteins for the
construction of live vectors was first described for Gramnegative
bacteria (for reviews see [26,27]) and more recently
systems for surface display on Gram-positive bacteria have
also been described [28,29]. Surface display for the development
of live bacterial vectors is described in more
detail below.
Nucleic acid vaccines
Nucleic acid vaccines constitute a new class of recombinant
subunit vaccine, consisting of, for example, plasmid DNA
containing the gene encoding the antigen of interest under
the control of a strong mammalian viral promoter. The
antigen-encoding gene will be expressed by the vaccinee
upon delivery of the plasmid DNA. DNA vaccines have been
shown to generate both humoral and cellular immune
responses [30] and the first report of protective efficacy of
DNA immunization was against influenza [31]. Immunization
of BALB�c mice with plasmid DNA encoding influenza A
nucleoprotein resulted in the induction of nucleoproteinspecific
antibodies, and protection from a subsequent
challenge with a heterologous strain of influenza A virus [31].
In addition to the advantage of a subunit vaccine
including only the antigen (or antigens) required for protective
immunization, nucleic acid vaccines present several
other advantages. Nucleic acid vaccines are relatively easy to
construct and produce, and considering the post-trans-
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98 M. Hansson, P.-A� . Nygren and S. Sta� hl
lational modifications and the presentation of the antigen to
the host’s immune system, they provide antigen synthesis in
the host in a similar way to that which occurs during a natural
infection.
One drawback of DNA vaccines that has been extensively
discussed is the potential risk for integration into
the host’s genome. This risk could be minimized by using
RNA instead of DNA for immunization. Due to the low
stability of mRNA, preparation and administration of RNA
vaccines are not entirely simple. These limitations could be
circumvented by constructing RNA or DNA vectors based
on parts of alphavirus genomes [32,33], carrying a gene
encoding the foreign antigen and a gene encoding an
alphaviral replicase. Upon transfection of such a construct,
the replicase gene will be translated and the produced
replicase will mass-replicate the antigen-encoding RNA. The
transfected cell will express large amounts of the foreign
protein for a short period of time, followed by cell death
[32]. In a recent study, mice immunized with an alphaviral
DNA vector encoding influenza antigens developed humoral
and cellular responses at higher levels than mice that
received a conventional DNA-vaccine vector [33]. In addition,
protective immunity against influenza challenge was
elicited in the immunized mice. Nucleic acid vaccines, based
on either DNA or self-replicating RNA, will without doubt
find a role in the future of vaccine development (for reviews
see [30,34]).
Recombinant production of protein
immunogens
Gene construction
Recombinant-DNA techniques offer several ways to construct
genes coding for the immunogens to be produced.
The use of the PCR enables direct isolation of the gene from
its natural source, but requires knowledge of the target
DNA sequence. In the PCR amplification, suitable restriction
sites can be introduced for direct cloning into a desired
expression vector. Building up genes de novo using synthetic
oligonucleotides can sometimes be preferable [35–37], e.g. if
only the protein sequence is known, or if the GC content of
the original gene would differ significantly from that of the
host selected for expression. It may also be desirable to
adapt the codons used to the chosen host [37], since
heterologous genes rich in codons that are used rarely by,
for example, Escherichia coli, may not be expressed efficiently
in E. coli [38].
Host vector systems
The choice of expression system depends upon many
factors, including (i) the requirements for post-translational
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modification, (ii) the proteolytic stability of the target
protein, (iii) whether the protein is secretable, (iv) the
possibility of renaturation of a protein produced in a
misfolded form and (v) the acceptable costs for the final
product. There are four major expression hosts that are
commonly used to produce vaccine antigens; bacterial,
yeast, insect and mammalian expression systems. In addition,
transgenic plant expression systems have started to emerge,
with the aim of utilizing the plant both for production of the
subunit vaccine and for vaccine delivery via the edible plant
[39]. A plant-based subunit vaccine has indeed been tested in
human volunteers. Transgenic potatoes expressing LTB, the
binding subunit of the heat-labile toxin of enterotoxigenic E.
coli, was consumed by volunteers who then developed
significant LTB-specific antibody (IgG and IgA) responses
[40]. Despite a costly development phase, transgenic plants
might become interesting as future vaccine production�
delivery vehicles, but their efficacy needs to be proven
further.
Bacterial systems can express antigens at very high
levels and are suitable for expressing vaccine antigens that do
not require significant post-translational modifications. E. coli
is the most commonly used bacterium for production of
heterologous proteins, being easy to manipulate, genetically
and physiologically well defined, and yielding high expression
levels [41]. A multitude of vectors and strains are available,
making it possible to design a suitable expression system.
Expression of recombinant antigens in bacterial systems
other than E. coli may sometimes be advantageous [42].
Salmonella typhimurium [43,44], Vibrio cholerae [45] and
Bacillus brevis [46,47] are some examples of other bacteria
that have been used for expression of antigens for vaccine
production purposes. One particular feature in favour of
Gram-positive bacteria is that the risk of contaminating
lipopolysaccharides is avoided.
Among the eukaryotic expression systems, the baker’s
yeast Saccharomyces cerevisiae is the most commonly used
[48]. Yeast may not express proteins at the levels that can be
obtained in E. coli, but Saccharomyces can easily be grown to
high cell densities. The first human subunit vaccine produced
by recombinant means was the hepatitis B vaccine, which
consists of a major surface antigen (HBsAg) and which is
produced in Sacc. cerevisiae [49]. In later years the yeast
Pichia pastoris became a very promising production host due
to high production levels [48]. Yeast cells possess some of
the eukaryotic possibilities for post-translationally modifying
proteins, e.g. phosphorylation and glycosylation, but, since
the glycosylations differ from the glycosylations in mammalian
cells, it can potentially also be disadvantageous [50].
Baculovirus-based expression systems take advantage
of the ability of a virus to infect arthropod cells, and
heterologous proteins can be efficiently produced in both
insect cells and larvae [51], but to a significantly higher cost

than in microbial expression systems. Advantages of baculovirus
over other expression systems are that proteins are
expressed at very high levels, and since these cells are
eukaryotic, they are able to make many of the posttranslational
modifications of mammalian cells, even though
the glycosylations are not the same as in mammalian systems.
Several mammalian expression systems for production
of recombinant proteins have been developed [51], but most
vaccines requiring eukaryotic post-translational modifications
are still being expressed in yeast. The main reasons
are the higher product yield in yeast and the ease with which
yeast can be manipulated and grown. The major advantage of
a mammalian expression system is that it will post-translationally
modify the expressed product with the highest
degree of fidelity compared with the other expression
systems, and expressed viral proteins will thus be glycosylated
in a human-like fashion. Drawbacks of mammalian
expression systems are the laborious and time-consuming
generation of stable cell lines, the high cultivation costs, and
the problems associated with viral and bacterial (e.g.
Mycoplasma) infections.
The choice of a suitable production system thus
depends on several factors, e.g. the biochemical and biological
properties of the protein of interest, the amount of
recombinant protein needed and the requirements for posttranslational
modification. Concerning the latter point,
bacterial antigens do not normally need such modifications
and would thus be likely to be expressed successfully in
bacteria. Parasitic antigens originating from eukaryotic
organisms may have such requirements, and a eukaryotic
expression system might be needed in cases where the posttranslational
modifications are necessary for eliciting protective
immune responses. Nevertheless, a normally highly
glycosylated antigen can be found to be perfectly immunogenic
when produced in bacteria [50]. Normally when
setting up a production scheme for a target subunit
immunogen, the least-complicated bacterial expression
systems are carefully investigated before more complicated
and more costly host�vector systems are evaluated.
Strategies for improved bioprocesses
Recombinant-DNA techniques and the expression of heterologous
proteins in E. coli and other hosts have provided a
source of proteins of a quantity and quality that, previously,
was difficult or impossible to achieve through their isolation
from natural sources. In addition, gene technology can be
used to engineer proteins to facilitate bioprocessing. Geneproduct
design can influence the yield and the localization of
the product, as well as adapting the gene product by fusion
technology to specific unit operations suitable for large-scale
downstream processing. Since cost-efficient production of
subunit vaccines is of utmost importance, it is advisable to
Recombinant subunit vaccines 99
use careful genetic design, when possible, to simplify and
improve the overall production process.
Secretion By taking advantage of a signal peptide (secretory
leader), normally fused to the N-terminus of the target
protein, efficient secretion of the protein can be achieved.
For example, export of a protein from the cytoplasm into
the periplasm of E. coli offers several advantages [52,53].
Secretion of the target protein to the periplasm functions as
an initial purification step and simplifies the downstream
purification considerably, since the periplasm contains
approx. 100 host proteins [54] compared with more than
4000 proteins in the cytoplasm [55]. The number of
proteases in the periplasm is also drastically fewer [56],
which reduces the risk of degradation, and the oxidizing
environment in the periplasm favours disulphide-bond
formation, which thus stimulates a potentially correct folding
of cysteine-containing target proteins. In addition to
secretion into the periplasmic space of the E. coli cell, the
protein can in some cases be recovered from the culture
medium [57–59]. The mechanism for the release of proteins
into culture medium by E. coli cells is not known in detail, but
is probably due to non-specific leakage from the cells [60,61].
Recovery of proteins from the culture medium has the
advantages that cell disruption is not needed and that there
is a low number of host proteins present [59]. A potential
drawback may be the dilution of the protein, which could
potentially complicate the purification in large-scale bioprocesses.
Improved recovery by engineering of protein properties The
addition of charged amino acid residues or mutation of
amino acids of a protein to alter the isolectric point (pI), can
improve the purification by allowing ion-exchange chromatography
[62]. Fusion of hydrophobic tails to the target
protein has been shown to alter the partitioning of the fusion
protein in aqueous two-phase systems, enabling the use of
such a system as a primary purification step for efficient
recovery of the target protein [63,64]. Hydrophobic tagging
of protein immunogens has the additional advantage, as will
be discussed further below, that it could allow direct adjuvant
incorporation of purified subunit vaccines through hydrophobic
interaction [65,66].
An illustrative example of how genetic design can be
used to achieve integration of unit operations was presented
in the process design for production of a malaria subunitvaccine
candidate [59,67]. The fusion protein ZZ-M5,
constituting the malaria subunit-vaccine candidate, was
produced in E. coli using a secretion strategy. The M5
polypeptide is derived from the central repeat region of the
Plasmodium falciparum blood-stage antigen Pf155�RESA, and
the ZZ region represents an IgG-binding affinity tag derived
from Staphylococcus aureus Protein A (SpA). More than 65%
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100 M. Hansson, P.-A� . Nygren and S. Sta� hl
of the recombinant gene product was found to be excreted
into the culture medium, from where it could be recovered
in a single step directly from the crude fermentor broth
without prior cell removal by expanded-bed anion-exchange
adsorption. The fusion protein had a relatively low pI that
allowed anionic-exchange adsorption at pH 5.5, at which
most E. coli host proteins are not adsorbed. This strategy
allowed an integration of the cell-separation step with the
ion-exchange adsorption of the gene product with simultaneous
volume reduction, which resulted in a highly
condensed, but still efficient, recovery process [59]. Since
the ZZ-M5 protein was to be used in a preclinical malaria
vaccine trial in Aotes monkeys [67], a ‘polishing’ step was
included in which the IgG-binding capacity of the fusion
protein was employed. After affinity chromatography on
IgG–Sepharose, contaminating DNA and endotoxin levels
were well below the demands set by regulatory authorities.
The overall yield of the process, performed in pilot scale,
exceeded 90%, resulting in 550 mg of product�l of culture
[59].
Solubility and proteolytic stability Upon expression in bacterial
expression systems, many proteins will precipitate and
form inclusion bodies [68]. If the target protein is sensitive to
proteolytic degradation, this could be advantageous, since
the formation of inclusion bodies normally protects unstable
proteins from degradation. However, renaturation in vitro
has to be employed to regain a native structure. This used to
be a significant drawback in setting up a large-scale bioprocess,
but today refolding can often be performed
efficiently even on an industrial scale [68]. For many proteins
that are not too proteolytically labile, improved solubility
can be of significant importance, since higher in vivoexpression
levels can be obtained without product precipitation
and eventual in vitro refolding can be performed at
significantly higher protein concentrations [69–72]. The
solubility of a recombinant protein can be increased by fusing
a target protein, at the level of the gene, to a highly soluble
fusion partner, to thereby increase the overall solubility of
the fusion protein [37,68,70–72]. The SpA-derived ZZ, and
BB derived from streptococcal protein G (SpG), constitute
relevant examples of fusion partners that have proven to be
suitable in this context (for reviews see [73,74]). In addition,
the proteolytic stability of a fusion protein can also be
improved by single or double (N- and C-terminal) fusions to
proteolytically stable fusion partners, such as the ZZ and BB
tags [56,69,75].
An illustrative example of how product engineering was
employed to obtain increased product solubility and stability
has been described in [37]. Site-directed mutagenesis was
employed to engineer the hydrophobic properties of a 101amino-acid
fragment from the human-respiratory-syncytial-
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virus (RSV) major glycoprotein [37]. Hydrophobic engineering
of four clustered phenylalanine residues, yielding
mutant variants with the four phenylalanine residues either
substituted for serine residues or deleted, increased the
fraction of soluble protein in vivo from 27 to 75%. Interestingly,
this effect was accompanied by a remarkable
increase in product stability. Protein engineering of hydrophobic
residues could obviously influence protein structure
but, in the reported example, CD analysis and the antigenicity
pattern suggested a retained structure [37]. These results
indicate that, in certain cases, single amino acid substitutions
and engineering of hydrophobic residues [76] (by substitution
for more hydrophilic residues) might be used as
tools to improve production processes for recombinant
subunit immunogens. Also other recombinant strategies can
be utilized to improve proteolytic stability (for a review see
[56]).
Product yield The product yield is dependent on many
factors, including mRNA and product stability, transcriptionand
translation-initiation efficiency, vector copy number and
stability [38,41]. In addition, a number of promoters of
different strengths and with different features are available
for use in E. coli [38]. Large-scale production of recombinant
proteins preferably employs cell growth to high densities and
minimal expression of the target protein, followed by
induction or de-repression of the promoter. Production of
heterologous proteins that might be toxic to the host
require tight regulation of the promoter, e.g. the T7 system
[77]. Constitutive promoters can preferably be used for
non-regulated expression of secreted, non-toxic proteins
[59]. Production of a target protein as a multicopy protein,
followed by specific cleavage to release several copies of the
native protein, is another way to increase product yield
[78,79].
Strategies to improve potency of
protein-subunit vaccines
The construction of gene fusions allows the generation of
fusion proteins potentially carrying the combined properties
of each parental protein. The reasons for fusing a target
protein to another protein by genetic means are several
[74,80], e.g. to enable affinity purification, increase solubility,
decrease proteolysis or to target the protein to a different
compartment of the host cell. The fusion partner can also be
used as a reporter molecule, to increase the half-life of
therapeutic proteins, provide carrier-related properties, or
be used to enable display of the target protein on the
surfaces of cells or phages. Gene-fusion strategies can also

e used to combine B- and T-cell epitopes in composite
immunogens.
Immunopotentiating fusion partners
Gene-fusion strategies can be used as a powerful method to
improve the immune response of an immunogen. For
example, the fusion partner BB, derived from SpG [81],
has been shown to possess immunopotentiating properties.
Immunization of mice with a malarial antigen fused to the BB
protein elicited an antibody response in mice strains that
were non-responders to the malarial antigen alone [14].
Furthermore, a candidate RSV subunit vaccine, BB-G2N,
comprising the BB protein fused to a 101-amino-acid
sequence from the human RSV G protein, was shown to
induce protective immunity in mice to RSV [15,50,82–85]. It
was shown that, by inclusion of the BB part, a more potent
G2N-specific B-cell memory response was evoked [15]. This
indicates that the SpG-derived BB can function both as an
affinity tag to facilitate purification and as a carrier protein
with immunopotentiating properties. To date, it has not
been fully elucidated whether this capacity is due to strong
T-cell epitopes [14] or related to the serum-albumin-binding
activity resulting in a prolonged exposure [11,12] of the
immunogen to the immune system, or a combination of
both.
Targeting of protein immunogens
Targeting of chimaeric antigens to immunoreactive sites can
be achieved, for example, by the use of adhesion factors,
monoclonal antibodies or other molecules capable of
specifically binding to eukaryotic cell receptors or polysaccharides
[8]. The cholera toxin B (CTB) subunit has been
evaluated extensively as a fusion partner to various antigens
for its immunopotentiating properties. Its capacity for
binding to the ganglioside G M1 present on mucosal epithelial
cells has been used for targeting of mucosal vaccines [13].
Intracellular targeting of antigens is an elegant variation of
the theme. Fusions to the N-terminal catalytic domain of
adenylate cyclase toxin (CyaA) of Bordetella pertussis resulted
in delivery of the foreign viral epitope to the cytosol of cells
by the detoxified invasive toxin. Protective cytotoxic
T-lymphocyte responses against challenge with intracerebral
lymphocytic choriomeningitis virus (LCMV) challenge was
obtained by this targeting strategy [86]. Chimaeric composite
immunogens can also be created by fusion of different
antigens, such as the hybrid CTB–LTB molecules, which are
candidate oral vaccines against both enterotoxic E. coli
infections and cholera [87]. Such targeting strategies have
also been utilized in efforts to improve live delivery vehicles
(see below).
Recombinant subunit vaccines 101
Adjuvant incorporation
Since single-protein antigens are normally rather weak
immunogens, requiring co-administration of an adjuvant to
increase the immune response, the development of novel
adjuvants [88,89] and strategies for efficient antigen association
are important topics in present vaccinology research.
For several adjuvants, such as liposomes, cochleates,
non-ionic block polymers and immunostimulating complexes
(iscoms), the protein antigens are normally incorporated by
means of hydrophobic interactions [88], and the physical
association has been demonstrated to be essential for the
adjuvant to exert its immunostimulating capacity. For iscoms,
it has been shown that membrane antigens containing
hydrophobic transmembrane sequences are efficiently incorporated.
In contrast, hydrophilic antigens cannot be
efficiently introduced into iscoms. Instead, such antigens
could be incorporated by partial denaturation to expose
hydrophobic sequences, or by chemical conjugation to
preformed iscoms or iscom-matrix alone [88].
In an attempt to simplify incorporation of hydrophilic
protein immunogens into adjuvant systems, a recombinant
strategy was presented for production of immunogens
furnished with a hydrophobic tag to improve incorporation
into iscoms [65]. The benefits of two fusion partners were
combined; an affinity tag, to enable efficient affinity purification
of the target fusion protein, and a hydrophobic tag, to
make direct incorporation into iscoms possible. Two of the
evaluated hydrophobic tags were found to mediate efficient
iscom association; a tryptophan�isoleucine-rich tag and a tag
derived from the membrane-integration sequence from
influenza haemagglutinin, and the formed iscoms were
capable of efficiently eliciting antibody responses to the
model antigen, a malaria peptide M5, derived from the
central repeat region of the P. falciparum blood-stage antigen
Pf155�RESA [65]. In a recent follow-up study, significant
improvements in the production systems were presented
[66], allowing high expression levels, efficient recovery and
adjuvant incorporation of hydrophobically tagged protein
immunogens.
Live bacterial and yeast vaccinedelivery
systems
Compared with proteinaceous subunit vaccines, recombinant
live vaccines would potentially be easier and less
costly to produce, since they do not require extensive
purification processes, and since they may be able to elicit
long-lasting immunity without the need for adjuvants.
Therefore, there is considerable interest in developing
systems for heterologous protein expression in non-pathogenic
micro-organisms to be used both as production
� 2000 Portland Press Ltd

102 M. Hansson, P.-A� . Nygren and S. Sta� hl
Table 3 Selected examples of immunization studies in which the antigens have been surface-displayed on heterologous bacteria or yeast
i.p., intraperitoneally; i.v., intravenously.
Micro-organism Display system Displayed antigen Animal model Result Reference
Gram-negative
E. coli E. coli LamB Hepatitis B virus fragment Rabbits/mice (i.v.) Antibodies recognizing viral fragments [93]
S. typhimurium E. coli OmpA Malaria blood-stage antigen fragments Mice (orally) Antigen-specific antibody response [94]
S. typhimurium
Gram-positive
E. coli/Shigella dysenteriae OmpA Malaria blood-stage antigen fragments Mice (i.p.) Antigen-specific antibody response [95]
Streptococcus gordonii Strep. pyogenes M6 protein Hornet venom allergen Mice (orally, intranasally) Antigen-specific antibody response [96]
Strep. gordonii Strep. pyogenes M6 protein Human papilloma virus protein Mice (orally, intranasally) Antigen-specific antibody response [97]
Strep. gordonii Strep. pyogenes M6 protein Human papilloma virus protein Mice (vaginally) Antigen-specific antibody response [98]
Strep. gordonii Strep. pyogenes M6 protein HIV-1 gp120 fragment human papilloma Cynomolgus monkeys Antigen-specific antibody response and [99]
virus protein
(vaginally)
T-cell response
Staphylococcus xylosus Staph. aureus protein A RSV protein fragment Mice (orally) Antigen-specific antibody response [100]
Staph. carnosus Staph. aureus protein A Streptococcal model protein Mice (orally) Antigen-specific antibody response [101]
Staph. carnosus Staph. aureus protein A RSV/CTB Mice (intranasally) Partial protection to RSV challenge [102]
Bacillus subtilis spores
Yeast
B. subtilis cell wall (CwbA) protein Yersinia pseudotuberculosis invasin protein
fragment
Mice (orally) Antigen-specific antibody response [103]
Sacc. cerevisiae Yeast α-agglutinin Hepatitis B virus protein fragments Mice (i.p.) Antigen-specific antibody response [104]
systems and as vehicles for live delivery of subunit vaccines.
For efficient presentation of the antigen to the immune
system several techniques for displaying heterologous proteins
on the surface of bacteria have been developed, since
cell-surface display of antigens has been shown to be
advantageous for the induction of an antigen-specific immune
response when using live recombinant bacteria for immunization
[76,90].
Two basic strategies exist for the generation of bacterial
live-delivery vehicles. One type of vehicle is based on
colonizing normally pathogenic bacteria, and attenuated or
non-pathogenic variants of that bacterium are employed in
order to obtain suitable vehicles, e.g. Salmonella [26,29] and
mycobacterial strains [91]. A second type of vehicle is based
on non-pathogenic commensal or food-grade bacteria, such
as Streptococcus gordonii and certain staphylococcal or
lactococcal strains. These approaches have been described
in recent reviews [26,28,29,92].
Gram-negative bacteria
The first described examples of heterologous display on
bacteria were with Gram-negative bacteria (Table 3), in
which short peptides were displayed on the bacterial surface
by genetic insertion into the exposed loops of E. coli outermembrane
proteins LamB [105], OmpA [106] and PhoE
[107]. The technique of inserting peptides into surfaceexposed
loops of outer-membrane proteins has been used
for other Gram-negative bacteria as well, e.g. S. typhimurium
[108] and Pseudomonas aeruginosa [109]. The drawback of
using exposed loops of outer-membrane proteins is the
limitation in the size of the peptide that can be inserted.
Sequences of up to 70 amino acids have been reported as
possible for insertion into the MalE protein [110]. Insertion
of larger inserts (189 and 451 amino acids) of malarial
antigens in E. coli OmpA and subsequent surface exposure
on S. typhimurium has also been described [94]. Fusions to
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fimbriae proteins [111–113] and flagellar proteins [114–116]
have also been used to achieve surface display on Gramnegative
bacteria. However, the use of cell appendages is
limited by the size of the peptides that can be displayed.
To allow display of larger protein fragments as well as
entire proteins, different systems to anchor heterologous
protein to lipoproteins have been used. Fused to a major
outer-membrane protein, the TraT protein, a coat-protein
fragment of type-1 poliovirus was demonstrated to be
presented on the cell surface of E. coli [117]. Fusion of a
lysozyme-binding single-chain fragment to E. coli peptidoglycan-associated
lipoprotein (PAL) resulted in surface
exposure of functional antibody fragments [118], and the
entire Leishmania major glycoprotein gp63 has been displayed
on the surface of E. coli by fusion to the P. aeruginosa major
outer-membrane lipoprotein (opr1) [119]. Recently, a display
system based on the ice-nucleation protein (Inp) from
Pseudomonas syringae was described [120]. By fusion of
levansucrase to the C-terminus of Inp, an active enzyme
could be displayed on the surface of E. coli.
Taking advantage of the signal sequence and the first
nine N-terminal amino acids of the E. coli major lipoprotein
(Lpp) and a region of the E. coli OmpA, the entire βlactamase
protein could be displayed in an active form on the
surface of E. coli when fused C-terminally to the Lpp–OmpA
complex [121]. This Lpp–OmpA display system has been
shown to give efficient translocation and surface anchoring
of fused gene products, resulting in a high number of
chimaeric surface proteins present in an accessible form on
E. coli cells. Both recombinant enzymes and antibody
fragments [122,123] have been surface-displayed on E. coli
using this system. Reviews describing this system and other
systems for surface display on Gram-negative bacteria have
been published [26,27,124].
Many of the E. coli surface-expression systems for the
delivery of heterologous antigens to immunized animals have

een used for the surface-display of immunogens in Salmonella
live-vaccine candidates [26,29]. For example, malarial
antigens have been surface exposed on S. typhimurium by
genetic fusion to the ompA gene [94,95], and fusions to the E.
coli LamB and MalE proteins have been used for the surface
expression or periplasmic targeting, respectively, of viral
antigens [125] and the Shiga toxin B subunit [126]. Shiga-like
toxin IIe was exported by the use of an E. coli haemolysin
transport system and fusion to the outer-membrane protein
TolC [127]. Also, naturally existing Salmonella surface
proteins or appendages (flagellae and fimbriae) have been
used for surface display [128–130]. Despite extensive
research efforts and the existence of numerous expression
systems, the early optimism in using Salmonella spp. as
delivery systems for foreign antigens has been hampered
somewhat. The main reason is the risk of side effects due to
potential reversion into virulence in immunocompromised
humans, and lack of efficiency in human trials. Nevertheless,
Salmonella provides an excellent tool for research into
experimental vaccines, stimulating both the humoral and
cellular branches of the immune system.
Gram-positive bacteria
Several systems for heterologous surface display on Grampositive
bacteria (Table 3) have also been described [28,29].
The fibrillar surface M protein of Strep. pyogenes has been
used to express a foreign antigen on the surface of the
commensal bacterium Strep. gordonii [131]. The antigen was
fused to the C-terminal attachment motif of the M protein
and replaced nearly all of the surface-exposed region of the
M protein. Using this system, a number of proteins have been
displayed successfully on the surface of Strep. gordonii [92].
The anchoring region of the surface-exposed SpA has
also been used to display different heterologous immunogens
on the non-pathogenic Staph. xylosus and Staph. carnosus
[29,100,132–134]. An attempt to display tetanus toxin
fragment C (TTFC) on the surface of Lactococcus lactis by
fusion to the C-terminal anchoring domain of lactococcal
cell-surface-associated proteinase (PrtP) did not result in a
detectable surface exposure [135]. An interesting approach
to making heat-resistant live bacterial vectors by employing
sporulating Bacillus subtilis for surface display of an antigen,
has been described by Acheson and co-workers [103].
Antigenic domains of the Yersinia pseudotuberculosis invasin
protein (Inv) were fused to B. subtilis cell wall (CwbA) protein
and subsequently expressed on the surface. Mice
immunized orally with spores from the recombinant B.
subtilis developed an immune response to Inv [103].
Yeast
Surface display of an antigen on Sacc. cerevisiae has also been
demonstrated [104]. The C-terminal cell-wall-anchoring
Recombinant subunit vaccines 103
sequence of the mannoprotein α-agglutinin was utilized to
attach the antigen at the cell surface of yeast. Oral
immunizations of mice with yeast cells expressing the HBsAg
at their cell surface resulted in antigen-specific antibodies
(IgG), and so baker’s yeast can thus be seen as a potential
vaccine-delivery vehicle [104].
Targeting of live bacterial vectors
Although systemic antibody responses to surface-exposed
antigens after oral delivery with the Staph. carnosus system
have been reported [8,101], attempts have been made to
improve the system in terms of antibody responses elicited
to the surface-exposed antigenic determinants upon immunization
via the mucosal routes [8,134,136,137]. The rationale
has been to target the staphylococcal vaccine vehicles to the
mucosa through the co-display of proteins that have binding
partners present on the mucosal epithelium. The surface
expression of a bacterial adhesion factor, a fibronectinbinding
domain from Strep. dysgalactiae, on the surface of
Staph. carnosus, resulted in a 1.5 log 10 increase in the serum
IgG responses to a co-displayed antigen upon intranasal
immunization of mice [8,138]. In another approach, coexposure
of a peptide (CTBp) comprising amino acids 50–75
of the CTB subunit on the Staph. carnosus surface, was
investigated. CTB is of significant interest for mucosal
vaccine development, and its immunopotentiating capacity
as a carrier molecule is considered to be related to its ability
to bind to the monosialoganglioside G M1, present on mucosal
epithelial cells [134,136,137]. The CTBp subfragment has
been shown to have an immunopotentiating effect [139]. The
introduction of CTBp into the chimaeric surface proteins,
containing a model antigen, significantly increased (close to
2 log 10) serum IgG responses upon intranasal immunization
[137]. The same increase in immune responses was not
observed upon oral delivery, and it was furthermore
demonstrated that live delivery of the staphylococci was
required to obtain this effect [137].
In a subsequent vaccination study, the Staph. carnosus
system, improved by co-display of CTBp, was used for
delivery of peptides from the G glycoprotein of human RSV
[102]. Three peptides, corresponding to the RSV G-protein
amino acids 144–159 (denoted G5), 190–203 (G9) and
171–188 (G4S), were expressed separately by recombinant
means on the surfaces of three different cultures of Staph.
carnosus, and their surface accessibility on the bacteria was
verified by fluorescence-activated cell sorting (FACS). Intranasal
immunization of mice with the live recombinant
staphylococci elicited significant anti-peptide as well as antivirus
serum IgG responses, and upon viral challenge with 10 5
tissue-culture infectious doses 50 (TCID 50, the virus dose
infecting 50% of the cells), lung protection was demonstrated
for approximately half of the mice in the G9 and G4S
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104 M. Hansson, P.-A� . Nygren and S. Sta� hl
immunization groups. To our knowledge, this represents the
first study in which protective immunity to a viral pathogen
has been evoked using food-grade bacteria as vaccinedelivery
vehicles [102].
Concluding remarks
Recombinant strategies are of great interest in the
development of new vaccines, and in addition for the
improvement of existing vaccines. New methods for
construction, production and targeting of recombinant
immunogens have been presented. Several new and rational
techniques have recently become available in the search for
novel target antigens for use in new vaccines. The largescale
genome-sequencing projects have provided enormous
amounts of information to be deciphered. Vaccinology will
most probably benefit through the identification of new
target antigens and also from improved understanding of
the mechanisms of infection and immunity.
New target antigens can also be identified for evaluation
of immunogenicity using expression library immunization
(ELI) [140]. To create an expression library, the genome of
a pathogen is digested into fragments, which are ligated into
eukaryotic expression plasmids, forming the total library. By
immunizing animals with sublibraries containing a large
number of different plasmids, immune responses evoked in
the animals are the basis for selection of a sublibrary to be
studied further for the identification of the single protective
plasmid. Another approach to the identification of target
antigens have been envisioned by Jacobsson and co-workers
[141], who used the method called ‘shotgun phage-display’
for identifying genes encoding binding domains of bacterial
adhesins [142,143]. Staph. aureus chromosomal DNA was
fragmented by sonication and inserted into phagemid vectors
for expression on phages. After affinity selection of the
libraries against various human proteins, ligand-binding
phages were identified that contained DNA encoding
the bacterial binding protein fragment. Identified fragments
of bacterial adhesins could be of interest in a future vaccine
as candidate antigens or as fusion partners for vaccine
targeting.
In the area of antigen targeting, proteins binding to a
surface receptor or polysaccharide could potentially also be
selected from protein libraries based on combinatorial
randomization of the surface of a suitable protein used as a
scaffold [144,145]. After selection of a binder from the
library, this protein can be used as a fusion partner for the
antigen, for targeting of the antigen to certain tissues or cell
types. Combinatorial protein libraries have also been described
by others [146] as novel promising strategies in
vaccine development.
Taken together, it is evident that recombinant-DNA
� 2000 Portland Press Ltd
technology will have a major impact on future strategies for
the prevention of infectious diseases. The design, selection
and production of recombinant subunit vaccines will be the
basis of modern vaccinology. Various strategies to administer
the subunit vaccine, as a protein immunogen, via a live
delivery vehicle or as a nucleic acid construct, will be
available.
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Received 17 April 2000�2 June 2000; accepted 6 June 2000
� 2000 Portland Press Ltd