Abstract Book Vol. 2 [International Conference on AIDS (14th: 2002: Barcelona, Spain)]

14 Abstracts WePeA5747-WePeA5750

XIV International AIDS Conference

tropic HIV-1 strain. The resulting virus termed SIVagm3-X4mc exclusively used
CD4 and CXCR4 for cell entry Replication of SIVagm3-X4 was inhibited by SDF1, the natural ligand of CXCR4, in a dose-dependent manner as expected for a
CD4/CXCR4-using lentivirus. Surprisingly, SIVagm3-X4mc was able to replicate
in vitro not only in IL2/PHA-stimulated, but also in non-stimulated peripheral blood
mononuclear cells from pig-tailed macaques and from African green monkeys.
After experimental infection of two pig-tailed macaques either with SIVagm3 -X4mc or SIVagm3mc, the co-receptor usage of the viruses maintained during
in vivo replication. Cell-associated and plasma viral loads as well as viral DNA
copy numbers were comparable, and no pathological changes were observed
up to 14 months post infection. Interestingly, HIV specific antibodies were detectable in SIVagm3-X4 infected macaque. Moreover, this V3 loop exchange rendered SIVagm3-X4mc susceptible to neutralizing antibodies present in sera of
SIVagm3-X4mc and SIVagm3mc infected macaques. Our study describes for the
first time a successful exchange of a V3 loop in non-pathogenic SIVagm resulting
in CD4/CXCR4-usage and modulation of virus replication in resting PBMCs as
well as sensitivity towards neutralization.
Presenting author: Klaus Cichutek, Paul-Ehrlich-lnstitut, Dept. Medical Biotechnology, Paul-Ehrlich-Str. 51-59, 63225 Langen, Germany, Tel.: [phone],
Fax: [phone], E-mail: [email protected]
WePeA5747 Spontaneous T cell apoptosis in FIV-infected cats
is blocked by the addition of interleukin-2 via a
caspase-independent mechanism
M.E. Bull, T.W. Vahlenkamp, M.B. Tompkins, W.A.F Tompkins. Immunology
Program, North Carolina State University, Raleigh, NC, United States
Background: Human immunodeficiency virus (HIV) and feline immunodeficiency
virus (FIV) cause lymph node (LN) T cell apoptosis that correlates with development of immunodeficiency. The mechanism(s) mediating T cell apoptosis and immunodeficiency remain controversial. FIV-infected domestic cats provide a useful
animal model to study the pathogenesis of HIV. B7 costimulatory molecules on
antigen presenting cells bind CD28 on T cells to provide the second signal for T
cell activation. Conversely, ligation of B7 with CTLA4 on activated T cells terminates the immune response by suppressing IL2 and driving the cell into apoptosis.
Chronic immune stimulation upregulates B7 on T cells enabling them to interact
with CTLA4 on neighboring T cells with subsequent induction of apoptosis. Previous work in FIV-infected cats demonstrated an increased percentage of LN T
cells that co-express B7 and CTLA4 molecules. When apoptosis was examined
using three-color flow cytometry with TUNEL staining the apoptosis in CD4 and
CD8 lymph node T cells from FIV+ cats was associated with cells expressing
B7.1, B7.2 and CTLA4.
Hypothesis: Interactions between B7.1/B7.2 and CTLA4 expressing T cells suppress IL2 resulting in non-responsiveness and increased apoptosis. Exogenous
IL2 or anti-CTLA4 can inhibit this interaction and decrease apoptosis.
Methods: LN cells from FIV- and FIV+ cats were cultured with anti-CTLA4 or IL2
then examined by flow cytometry and TUNEL staining.
Results: Addition of anti-CTLA4 or exogenous IL2 to LN cell cultures inhibited T
cell apoptosis. However, the combination of CTLA4 and IL2 did not significantly
differ from either treatment alone. Spontaneous apoptosis observed after 24 hrs
is not inhibited by the pan-caspase inhibitor Z-VAD-fmk.
Conclusions: Blockade of CTLA4 on T cells inhibits apoptosis, as does addition of exogenous IL2. Thus B7-CTLA4 signaling may results in inhibition of IL2
production that is corrected by exogenous IL2 or anti-CTLA4.
Presenting author: Marta Bull, 617 Hutton St, Raleigh, NC, 27606, United
States, Tel.: +[phone], Fax: +[phone], E-mail: MartaBull@ncsu.
edu
WePeA5748 Simian AIDS lymphomagenesis
P. Biberfeld1, E. Castanos-Velez1, T. Heiden1, C. Lindvall2, A. Sandlund 3,
G. Biberfeld4. 1lmmunopathology Lab., Karolinska Institutet, Immunopathology
Lab., Building Z6:03, Karolinska Hospital, S-17176 Stockholm, Sweden; 2Center
for Molecular Medicine - CMM, Karoinska Institutet, Stockholm, Sweden;
3Microbiology and Tumor Biology Center - MTC, Karolinska Inst itutet,
Stockholm, Sweden; 4Swedish Institute for Infectious Disease Control - SM/,
Karolinska Institutet, Stockholm, Sweden
Background: Next to Kaposi's sarcoma, malignant lymphoma is the most frequent malignancy of AIDS patients (ARL). In our Primate Center we have observed a high frequency of lymphomas (sARL) in non-human primates developing simian AIDS after SIV infection. The characteristics of 32 such sARL are
summarized here.
Methods: Asian cynomolgus monkeys were infected with varying doses of SIVsm
and were followed longitudinally for clinical and immune status, by virus isolation
and in some cases by consecutive LN biopsies. When disease progression or
lymphoma development became evident the animals were euthanized and sub
jected to necropsy. Biopsies and established cell lines were analyzed by morphology, immunochemistry, DNA-fluorometry, PCR, in situ hybridization, tumorigenesis in SCID mice and gene profile expression (DNA-chip array).
Results: All lymphomas presented at extranodal sites, were B-cell derived with
features of diffuse, large cell lymphoma (DLCL), with prominent infiltration of TIA+
T-cells and macrophages, some of which showed SIV infection. A simian herpes
virus (HVMF-1) with homologies to EBV was associated with all lymphomas. Most

showed moderate to high cell proliferative activity and low levels of apoptosis, related to Bcl-2 expression. Six primary tumors tested showed a normal karyotype
but random mutations were seen in some cases. Primary tumor cell cultures were
diploid and tumorigenic in SCID mice. Primary tumors and established cell lines
showed expression of various cytokines (IL-10, TNF, IL-15) which possibly could
stimulate (autocrine) tumor growth and/or down regulated cell mediated immunity.
Gene-expression assays with Clontech slides were not reproducible and are now
being repeated with Affymetrix.
Conclusions: Lymphomagenesis in non-human primates appears highly relevant in evaluating virological, cell biological and clinical aspects of AIDS related
lymphomas in humans and possibly useful for the development of various therapeutic modalities.
Presenting author: Peter Biberfeld, Immunopathology Lab., Building Z6:03,
Karolinska Hospital, S-17176 Stockholm, Sweden, Tel.: [phone], Fax:
[phone], E-mail: [email protected]
WePeA5749I Dynamics of ex vivo lymphocyte proliferation and
activation during primary infection in macaques
infected with SIVmac251 and treated with HAART
K. Benlhassan-Chahourl, C. Penit2, B. Vaslin1, V. Dioszeghy1,
N. Dereudre-Bosquet3, B. Delache', C. Feuillat-Aubenque3, F Vasseur2,
D. Dormont1, R. Le Grand'. 'Commissariat I'Energie Atomique,
Fontenay-aux-Roses, France; 2H6pital Necker, Paris, France; 3SPI-BIO, Massy,
France
Objectives: To study the impact of post-exposure prophylaxis with HAART
on T-cell proliferation and activation during primary infection of macaques with
pathogenic SIV.
Methods: 12 Macaques were infected iv with SIVmac251 (50 AID50) and were
then treated twice a day for a month (4h to 28 days postexposure). Group 1 received a placebo, group 2 received AZT (2x4.5mg/kg/day), 3TC(2x2.5mg/kg/day)
and Indinavir(2x20mg/kg/day) by the oral route, and group 3 received the same
doses of AZT and 3TC subcutaneously, and Indinavir (2x60mg/kg/day) orally. Kinetic of T-cell proliferation was analysed by overnight BrdU uptake and FACS
analysis.
Results: No protection from SIV infection was observed but HAART did strongly
impact on virus loads: reduction of viremia was observed during antiviral therapy
in group 2, with a better control after treatment was stopped; in group 3, PBMC
remained SIV negative(DNA PCR) during treatment, but control of virus load after
treatment interruption was not evidenced. An increase in circulating Brdu+ cells
was observed in placebo-treated animals, starting on day 11 p.i. A 12- to 20-fold
increase in Brdu+ memory CD8+ T-cells, and a 2- to 14- fold increase in Brdu+
memory CD4+ T-cells were observed at peak of plasma viremia. Proportion of
cycling CD3-CD8+ NK cells, naive CD4+ and CD8+ T-cells were also increased.
In contrast little changes occurred in PBMCs from group 2, and no changes were
noticed in the group treated with AZT and 3TC subcutaneously, until treatment
was stopped. Percentages of IFN-g+ CD8+ T-cells (CD28+ and CD28-) were increased on day 18 p.i. in groups 1, 2 and 3 with stronger responses in HAART
treated macaques.
Conclusion: Lymphocyte proliferation is associated with viral replication levels.
However percentage of circulating CD8+CD28+/CD28-cells expressing IFN-g appears to be increased in both groups of HAART treated animals.
Presenting author: KADIJA BENLHASSAN-CHAHOUR, COMMISSARIAT A
I'ENERGIE ATOMIQUE, SERVICE DE NEUROVIROLOGIE DSV/DRM, 18
ROUTE DU PANORAMA PB6, 92265 FONTENAY-AUX-ROSES, France, Tel.:
[phone], Fax: [phone], E-mail: [email protected]
WePeA5750 Env is a Cytopathic Determinant of R5-AIDS HIV-1
in SCID-hu Mice
R. Scoggins, K. Olivieri, A. Matthews, B. Brodrick, J. Taylor Jr., D. Chernauskas,
D. Camerini. University of Virginia, Charlottesville, VA, United States
Background: AIDS-associated R5 HIV-1 (R5-AIDS HIV-1) biological clones have
greater cytopathic effects (CPE) for CD4+ thymocytes and replicate to higher levels than pre-AIDS R5 biological clones in SCID mice bearing human thymus liver
grafts (SCID-hu mice). We hypothesize that the env and nef genes encode the
cytopathic determinants of R5-AIDS HIV-1.
Methods: We isolated and sequenced the gp120 V1 to V5 encoding region of env
and the nef-LTR region from R5-AIDS and R5-pre-AIDS biological clones from
the same patient. We created chimeric infectious molecular clones containing
the gp20 V1 to V5 encoding and nef-LTR regions of R5-AIDS and R5-pre-AIDS
HIV-I. Recombinant viruses were characterized in tissue culture and in SCID-hu
mice.
Results: Three to 8 molecular clones were derived from each biological clone.
Little sequence variation was seen among molecular clones derived from the
same biological clone pre or post culture in SCID-hu mice. In contrast, consis
tent differences were seen between the R5-AIDS and R5-pre-AIDS clones in env
and nef. The predicted R5-AIDS V1 and V2 regions were longer and contained
an additional glycosylation site, the V3 regions were more positively charged and
the Nef proteins had more characteristics associated with Nef proteins from rapid
progressors. All the env chimeric viruses were exclusively CCR5 tropic. The R5 -AIDS V1 to V5 chimeric virus was more cytopathic and replicated to higher levels
in SCID-hu mice than the pre-AIDS R5 chimeric virus.