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

360 Abstracts ThPeA7103-ThPeA7106

XIV International AIDS Conference

T-cell responses were determined using IFN-y ELISPOT assay using recombinant
vaccinia constructs expressing the
HIV proteins: Gag, Pol, Env, Nef, Rev and Tat and the CMV matrix protein pp65.
Results There were no significant differences of the CD4+ and CD8+ T-cell
counts between the three groups. The breadth of the HIV response was significant higher in the group that had experienced viremic episodes compared to the
patients with controlled viremia. A similar tendency was seen comparing the magnitude of the responses. Interestingly, the CMV response was significantly higher
in the group of patients with viral blips compared to the group with controlled
viremia.
Conclusions Our data indicates that even a very low-grade HIV-1 replication is
sufficient to cause an overall immune activation, including HIV-1 and CMV specific
CD8+ T-cells, in chronically infected individuals receiving HAART Our data also
support an "antigenic threshold model" for the relationship between viral replication and the frequency of HIV-1 specific CD8+ T-cells
Presenting author: Annika Karlsson, Gladstone Institute of Virology and Immunology, P.O. Box 419100, San Francisco, CA 94141-9100, United States, Tel.:
[phone], Fax: [phone], E-mail: [email protected].
edu
I ThPeA7103 Differential gene expression in CD8+ cells from
HIV-1 infected subjects showing suppression of
HIV replication
B. Martinez-Mari io, J.A. Levy University of California, San Francisco, University
of California, San Francisco, Department of Medicine, 513 Parnassus, Box 1270,
San Francisco, CA 94143-1270, United States
Background: CD8+ cells from healthy HIV-1 infected individuals suppress virus
replication in CD4+ cells by a non-cytotoxic mechanism (CNAR). The differential gene expression between CD8+ cells from infected subjects with high CNAR
and CD8+ cells from uninfected controls which lack this activity was evaluated to
identify the gene(s) responsible for CNAR.
Methods: CD8+ cells were mixed at an input ratio of 1:1 with HIV-1 acutely infected CD4+ cells for 5 days. Anti-HIV activity was considered positive if >80%
of virus replication (measured by reverse transcriptase activity) was reduced in
comparison to control HIV-infected CD4+ cells cultured alone. Total RNA was
extracted from CD8+ cells, converted to cRNA, and hybridized to the Affymetrix
Human Genome U95 Set MicroArrays (-.60,000 mRNA transcripts from genes
and EST's). Data was analyzed using GeneChip Analysis Suite, MicoDBTM and
Data Mining Tool Software.
Results: Average CD8+ cell suppression of HIV replication was 87% for infected
subjects and 20% for controls. Expression analysis revealed that -10% of genes
were differentially expressed. Up-regulated genes (2%) were further analyzed.
Comparison Ranking using Affymetrix's Difference Call ("Increased") identified
345 genes with >2.0 fold difference in expression levels and >50% concordance
of difference call. These genes functioned in a wide variety of cellular processes
and included -190 expressed sequence tags (EST's). Stringent selection criteria (>68% concordance) narrowed down the list to 122 genes of which -50 are
EST's.
Conclusion: Sequences of certain known genes and potentially some ESTs may
be associated with CNAR. They are under further investigation.
Presenting author: Beatriz Martinez-Mari io, University of California, San Francisco, Department of Medicine, 513 Parnassus, Box 1270, San Francisco, CA
94143-1270, United States, Tel.: [phone], Fax: [phone], E-mail:
[email protected]
ThPeA7104 Consistent pattern in the development and
immunodominance of epitope-specific CTL
responses during acute HIV-1 infection
X.G. Yu, M.M. Addo, W.R. Rodriguez, C. Fitzpatrick, P.K. Lee, D.A. Strick,
M.N. Johnston, E.S. Rosenberg, P.J.R. Goulder, B.D. Walker, M. Altfeld. Partners
AIDS Research Center, Massachusetts General Hospital/Division of AIDS,
Harvard Medical School Partners AIDS Research Center, 149, 13th street, 5th
floor, Charlestown, MA, 02129, United States
Background: HIV-specific CTL generated during acute infection play a critical
role in the initial control of viremia. However, the hierarchy in appearance of
epitope-specific responses has never been comprehensively characterized.
Methods: HIV-specific CTL responses in an acutely infected individual (ACO6)
expressing HLA-A3 and B7 were characterized using 505 overlapping peptides
spanning the entire expressed HIV sequence. All optimal CTL epitopes targeted
were defined using the Elispot assay and an IFN-y capture assay and responses
were studied longitudinally at 11 different timepoints following acute infection. The
hierarchy in the appearance of responses to individual CTL epitope was studied
in an additional 17 acute infected individuals expressing HLA-A3 and/or B7.
Results: Longitudinal studies in ACO6 demonstrated that only 2 epitopes were

targeted during symptomatic acute infection, but that CTL responses broadened
significantly during subsequent exposure to virus, ultimately targeting 27 distinct
CTL epitopes. These included 13 novel epitopes described here for the first time.
16 different CTL epitopes were restricted by a single HLA class I allele (HLAA3). The same pattern of development and immunodominance of responses to
HIV-specific CTL epitopes restricted by A3 and B7 in ACO6 was observed during
acute HIV-1 infection in the additional 17 individuals studied.

Conclusions: These studies demonstrate that HIV-specific CTL responses can
target a previously unexpected and unprecedented number of epitopes in a single
infected individual. The temporal hierarchy in the development of epitope-specific
responses restricted by the same HLA allele and their immunodominance during
acute infection was similar in all 18 individuals studied. These data indicate consistent early targeting of specific epitopes, which may be crucial for the design of
a highly immunogenic HIV-1 vaccine.
Presenting author: Xu Yu, Partners AIDS Research Center, 149, 13th street,
5th floor, Charlestown, MA, 02129, United States, Tel.: [phone], Fax: +1
617-726-5411, E-mail: [email protected]
ThPeA7105 T cell epitope mapping for HIV-1 subtype C gag
protein in Balb/c mice by ELISPOT
P. Chuqh1, P. Seth2. 1Ph.D. student, Department of Microbiology, AIIMS,
Department of Microbiology, All India Institute of Medical Sciences, Ansari
Nagar, New Delhi, India; 2Professor and Head, New Delhi, India
Background: The importance of cell mediated immunity in controlling HIV infection has been well established. Therefore, determination of T cell epitopes
while evaluating the immune responses in experimental animals is imperative.
ELISPOT assay has been adapted for direct ex-vivo quantification of peptide reactive T lymphocytes. Information on distribution of T-cell epitopes of HIV-1 subtype
B has been reported. Here we report mapping of T cell epitopes in gag region of
HIV-1 subtype C in Balb/c mice.
Methods: A set of forty-nine 20 mer overlapping peptides (by 10 residues) from
gag of subtype C was obtained from NIH AIDS Reference Reagent Program. A
matrix of 14 pools was generated. Each pool consisted of seven peptides. Splenocytes were taken from mice immunized with DNA vaccine construct pJWgagprotease49587. Each pool was incubated with one million splenocytes per well
overnight in precoated ELISPOT plates with anti murine IFN-y (R&D Systems)
followed by overnight incubation with biotinylated polyclonal antibody specific for
mouse IFN-y. The plates were developed and spots were read manually by using
gel documentation system.
Results: Five pools comprising 2 in columns and 3 in rows were found responding. From the matrix the peptides common to these columns and rows were determined.
8: GTEELRSLYNTVATLYCVHE, 10: GVEVRDTKEALDRIEEEQNK,
29: SILDIKQGPKEPFRDYVDRF, 31: FKTLRAEQATQEVKNWMTDT,
43: HQMKDCTERQANFLGKIWPS, 45: HKGRPGNFLQNRPEPTAR
Peptides 8 and 10 were identified from the p17 region, peptides 29 and 31 were
from p24 region and peptides 43 and 45 were from p15 region. Other workers
have shown presence of T cell epitopes (helper and cytotoxic) in these regions in
HIV subtype B.
Conclusions: These peptides can be used for evaluation of T cell responses in
mice studies with HIV-1 subtype C isolates.
Presenting author: Pradeep Seth, Department of Microbiology, All India Institute
of Medical Sciences, Ansari Nagar, New Delhi, India, Tel.: [phone], Fax:
[phone], E-mail: pseth @ aiims.aiims.ac.in
ThPeA7106 Distribution and functional analysis of memory
CD8 and CD4 T Lymphocytes in different anatomic
compartments
K. Ellefsen1, A. Harari1, P. Champagne2, R. Sekaly2, G. Pantaleo1. 'Lab of
AIDS Immunopathogenesis, Hopital Beaumont-CHUV, Lausanne, Switzerland;
2Lab d'Immunologie, Montreal, Canada
Background: Four subsets of memory CD8 T lymphocytes can be distinguished
on the basis of the expression of CD45RA and of CCR7 and a selective impairment of the differentiation of HIV-specific memory CD8+ T cells has been demonstrated. Virus-specific CD4 T lymphocytes can be quantified and analyzed on the
basis of the IFNg secretion following specific stimulation.
Methods: Peripheral blood mononuclear cells (PBMC) and lymph node mononuclear cells (LNMC) of 7 HIV-infected subjects with no ART were stained with mAbs
to CD8, CCR7, CD45RA and with HLA tetrameric complexes loaded with the relevant HIV and CMV peptides. The secretion of IFNg following specific stimulation
was determined on antigen-specific CD8 lymphocytes and was also used as a
tool to identify HIV- and CMV-specific CD4 effector cells. PBMC and LNMC obtained from 7 additional HIV-infected subjects were stimulated with HIV and CMV
antigens and were stained with mAbs to CD4, CCR7, CD69 and IFNg.
Results: The proportion of HIV-specific CD8 and CD4 memory lymphocytes was
similar in blood (CD8: blood 0.83%, N=6) and LN (0.69%, N=7; P=0.4); CD4:
blood 0.12%, N=7 and LN 0.11%, N=7, P=0.4). HIV-specific memory CD8 T cells
were mostly pre-terminally differentiated CD45RA-CCR7- T cells in both blood
and lymph node (PBMC: 79%, LNMC: 75%, N=7) while only a minor percentage
(<10%) were terminally differentiated memory CD8 T cells (CD45RA+CCR7-).

A higher proportion of CMV-specific CD8 memory lymphocytes belonged to the
terminally differentiated memory cell population (blood: 41%, LN: 27%), and both
memory CD8 and CD4 T cells accumulated in blood compared to lymph node
(CD8: blood 1.18%, N=7, LN: 0.08, N=6, P=0.003; CD4: blood 0.44%, N=7 and
LN 0.05%, N=6, P=0.00002).
Conclusions: These results demonstrate the selective impairment of the differentiation of HIV-specific memory CD8 T cells in both blood and LN and indicate a