Oral Abstract Session 01 - Global HIV Vaccine Enterprise

Topic 2: Animal Models and Preclinical Trials
P02.13
Differential Induction of Activation and Apoptosis
by TCR Signaling in Sooty Mangabeys and Rhesus
Macaques
S. Yu 1 , K. Rogers 2 , F. Villinger 2 , A. Kaur 1
1 Harvard Medical School, Southborough, USA; 2 Emory
University, Atlanta, GA, USA
Background: We previously showed that an increase in CD4+ T
lymphocyte apoptosis and elevation of plasma tumor necrosisreceptor
associated apoptosis-inducing ligand (TRAIL) occurs in
rhesus macaques (RM) but not sooty mangabeys (SM) during
acute SIV infection.
Methods: To further examine the mechanisms underlying
differential apoptosis in SIV-infected RM and SM, we compared
the in vitro responses to TCR signaling in seven SIV-negative SM
and seven SIV-negative RM. PBMC were cultured for 18 hours
with cross-linked CD3 and CD28 and subsequently analyzed
by flow cytometry for upregulation of CD69, TRAIL and active
Caspase 3, and for production of cytokines.
Results: Following TCR stimulation, there was a significant
increase in TRAIL on T cell subsets, NK cells and myeloid DC cells
(mDCs) in both species. However, levels of membrane TRAIL
were significantly higher in CD8+ T lymphocytes and NK cells of
SM compared to RM suggesting that they may be more cytotoxic
on activation. TCR stimulation also resulted in an upregulation
of CD69 and production of IFNγ, IL-2 and TNFα by T cell subsets
in both species with greater levels being observed in SM. In
contrast to SM, RM showed significantly higher frequencies of
apoptotic mDCs both ex vivo and following TCR stimulation. In
vivo inoculation of RM with SIVmac239 resulted in increased
frequency of ex vivo apoptotic mDCs at 2-3 weeks post-infection.
Increased apoptosis was also observed after overnight culture in
medium but was abrogated by addition of soluble death receptor
5 indicating that it was TRAIL-mediated.
Conclusion: Overall, these data show an increased susceptibility
to apoptosis of mDCs in RM, and a disconnect between T cell
activation and apoptosis in SM. Elucidating the mechanisms by
which SM are protected from apoptosis will be important for
understanding the basis of nonpathogenicity in natural hosts of
SIV infection.
P02.14
AIDS Vaccine 2012
Posters
Assessing the Protective Efficacy of Antibodies to the
HIV gp41 Region by Active Vaccination
S.K. Sharma 1 , J. Pokorski 2 , M.G. Finn 2 , J. Jacqueline 1 , E. Rakasz 3 ,
D. Burton 1 , R. Wyatt 1
1 International AIDS Vaccine Initiative, La Jolla, CA, USA; 2 The
Scripps Research Institute, La Jolla, CA, USA; 3 Wisconsin
National Primate Research Center, Univ. of Wisconsin-
Madison, WI, USA
Background: The gp41 cluster I is a conserved immunodominant
loop connecting the heptad repeat 1 (HR 1) and heptad
repeat 2 (HR2) of the HIV-1 the envelope glycoproteins (Env).
Following HIV-1 infection or vaccination with gp41-containing
Env immunogens, this region elicits relatively high titers of
antibodies generally considered to be non-neutralizing in
nature. However, in a recent passive immunization study using
a cluster 1 antibody, partial protection against SHIV SF162 P4
challenge was observed. In the present study, we sought to
determine if vaccine-elicited cluster 1 antibodies might afford
some protective capacity by active vaccination, presumably by
binding to non-functional spikes on the virus and slowing the
viral entry process in vivo.
Methods: To generate cluster 1-specific antibodies, we added
residues flanking the cluster I cysteine-loop region to allow it to
assume its preferred structural conformation. The resultant 20
residues peptides were expressed on the genetically modified
Q-beta bacteriophage particles and also chemically coupled to
KLH. Sera from rabbits immunized with these antigens were
analyzed by ELISA for the binding to cluster 1 peptides and by
cross-competition with the known cluster I antibodies.
Results: The cluster I region was found to be immunogenic and,
interestingly, a version of the epitope in which alanines were
substituted in place of the small cysteine-linked loop was found
to be more immunogenic than the wild-type cysteine-cysteine
motif. The sera from rabbits inoculated with either carrier crosscompeted
with the known cluster I antibodies such as F240.
Though the sera did not neutralize JR-FL viruses, they serum
antibodies were able to capture many different viruses in vitro.
Conclusion: We conclude that we have specifically elicited
antibodies directed to the cluster 1 region of gp41 possessing
properties similar to the known monoclonal antibodies. Active
immunization of non-human primates by both the intranasal and
intramuscular routes, followed by SHIV
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POSTERS