European Journal of Medical Research - Deutsche AIDS ...

June 27, 2007 EUROPEAN JOURNAL OF MEDICAL RESEARCH
129
Conclusion: Our results demonstrate that expression of tBid
under the control of the HIV-1 LTR promoter preferentially
induces apoptosis in Tat- and Rev-positive cells suggesting
that this suicide vector has the potential for effective use in a
gene therapy to exclusively eliminate HIV-1 infected cells.
F.25 (Poster)
Induction of immune responses by Human
Immunodeficiency Virus Type-1 (HIV-1) Pr55Gag
virus-like particles (VLP) and the impact of the
producer system upon activation of innate immune
responses
Bredl S. 1 , Sertl S. 1 , Wiesel M. 1 , Wild J. 1 , Deml L. 1 ,
Wagner R. 1
1 Universität Regensburg, Institut für Medizinsche
Mikrobiologie und Hygiene, Molekulare Mikrobiologie und
Gentherapie, Regensburg, Germany
Objective: HIV-1 Pr55Gag virus-like particles (VLP) produced
in the baculovirus expression system have been shown
to represent strong inducers of the humoral and CMI in mice
and non-human primates. This study aimed towards investigating
the molecular mechanisms underlying the strong immunogenicity
and adjuvanticity of such VLP.
Methods: Pr55Gag VLP were produced (i) using the baculovirus
(BV) expression system and (ii) mammalian 293T
cells coexpressing the BV envelope protein gp64. Cytokine
production (release of gIFN, IL5, others) from splenic cells of
naïve mice and human monocyte derived DC (huMDDC) and
the upregulation of costimulatory molecules (CD40, 80, 83,
HAL DR, CCR7) on the surface of huMDDC were used as a
measure for the activation of innate immunity by VLP antigens.
Results: Ex vivo studies on huMDDC clearly demonstrated
that VLP of BV origin, but not VLP produced in mammalian
cells triggered MDDC maturation, activation and cytokine secretion.
This lack of mammalian cell derived VLP to activate
huMDDC could not be rescued by pseudotyping these VLP
with BV gp64. Comparable results were obtained when
murine splenic cells were used for the ex vivo studies. However,
splenic cells from TLR9 knockout mice could be stimulated
neither by mammalian, nor by BV produced VLP.
Conclusion: These results clearly indicated that the potency of
VLP to induce innate immune responses is not an intrinsic
property of VLP rather than mediated by BV or insect cell derived
components. The BV Env protein gp64 is clearly not
contributing to TLR triggering, may however play a beneficial
role in uptake of VLP by APC.
F.26 (Poster)
HIV vaccine candidates in cross-validation on
dendritic cells
Köstler J. 1 , Hofmann H. 1 , Böckl K. 1 , Tischer K. 2 ,
Osterrieder N. 3 , Wild J. 1 , Wagner R. 1
1 Universität Regensburg, Institut für Medizinsche
Mikrobiologie und Hygiene, Molekulare Mikrobiologie und
Gentherapie, Regensburg, Germany, 2 University Medical
Center, Institut für Medizinische Mikrobiologie und Virologie,
Kiel, Germany, 3 Cornell University, Microbiology and
Immunology, Ithaca, United States of America
Objectives: Recently we have generated a new HIV Vaccine
candidate based on the Equine Herpesvirus I (EHV-1). The
primary goal of the current study is to compare a clinical trial
lot of a recombinant New-York-Vaccinia Virus based HIV
vaccine candidate (NYVAC-C; expressing Gag/Pol/Nef) with
a corresponding EHV-based vaccine construct (EHV-C) regarding
their capacity to induce maturation of monocyte derived
dendritic cells (MDDC).
Methods: A recombinant EHV-1 C-GagPolNef (EHV-C) was
generated using BAC-technology and RED-Recombination.
MDDCs were infected with EHV-C and NYVAC-C at different
MOIs. Expression of the transgenes was monitored by
FACS and Western Blot analysis. Maturation of MDDCs was
determined by FACS analysis of differentiation markers and
in ELISA assay measuring secreted proinflammatory cytokine
levels. Furthermore, early/late gene expression and antigen
expression of the HIV vaccine candidates was measured via
RNA quantification.
Results: Depending on the used MOI a substantial fraction
(~40%) of EHV-C infected MDDCs displayed expression of
significant amounts of GagPolNef immunogens. MDDCs infected
with EHV-C show various markers for DC maturation
and activation as monitored by release of cytokines and surface
expression of costimulatory signals. In contrast, MDDCs
infected with NYVAC-C only weakly express HIV transgenes
and do, upon direct infection, not support DC maturation or
activation. NYVAC-C expresses very low amounts of a late
gene F17R on MDDC, which could be interpreted as a block
in the viral replication and responsible for the low transgene
expression.
Conclusion: EHV derived vectors support efficient transgene
expression and provide signals required for DC maturation &
activation.
F.27 (Poster)
Detection of distinct mutants of TRIM5-alpha
mRNA derived from SIV infected macaques and
HIV-1 infected individuals
Jensen N. 1 , Stahmer I. 1 , Hoffmann D. 2 , Krepstakies M. 2 ,
Hauber I. 2 , Stahl-Hennig C. 3 , Jordan S. 1 , Hauber J. 2 ,
Van Lunzen J. 1
1 HIV Research Laboratory, University Medical Center
Hamburg-Eppendorf, Hamburg, Germany, 2 Heinrich-Pette-
Institute for Experimental Virology and Immunology,
Hamburg, Germany, 3 German Primate Center (DPZ), Dept. of
Immunology and Virology, Goettingen, Germany
Background: Recently the tripartite motif protein TRIM5a
was identified as an intracellular restriction factor against
HIV-1.Although unmodified human TRIM5-alpha is not active
against HIV-1, its restriction potential was significantly
increased by different modifications in the SPRY domain of
the protein, which was defined earlier as the region responsible
for inhibitory activity. Thus certain mutated TRIM5a variants
may exist, which play a role in the delayed disease progression
of so called Long-Term-Non-Progressors (LTNP).
Objective: To study the genetic sequence of human and simian
TRIM5a and its potential role in the restriction of HIV-1
and SIV infection.
Materials and methods: Cloning and sequencing of TRIM5a
cDNA was performed in blood PBL from 10 subjects of either
of the following groups:
a) LTNP or “slow progressors”, who maintain a viral load <
2000 copies/ml and CD4 counts > 500 cells/l without any
treatment for up to 10 years;