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

362 Abstracts ThPeA7111-ThPeA7114

XIV International AIDS Conference

dividuals, including the seronegative partners of infected persons. While in vitro
peptide-stimulation increased the number of HIV-1-specific T cells up to 20 fold
in the infected individuals, no HIV-1-specific responses were detectable after
peptide-stimulation in the uninfected subjects. In the HIV+ subjects, HIV-1 clade
C specific CD8 T cell responses were strong and broadly directed against p24 -Gag (100% of individuals), Nef (94%), p17-Gag (50%), Rev (22%) and Tat (22%).
Several novel epitopes within these proteins were identified and characterized.
Conclusions: In this blinded study using sensitive techniques, HIV-1 specific
CD8+ T cell responses were readily detectable ex vivo in HIV-1 infected individuals, but not in their exposed uninfected partners.
Presenting author: Marylyn Addo, Partners AIDS Research Center/MGH, 149
13th street, MGH-East, 5th floor, Charlestown, MA 02129-2000, United States,
Tel.: [phone], Fax: [phone], E-mail: addo@ helix.mgh.harvard.
edu
ThPeA7111 Computer-based design of a CTL assay for
monitoring cell-mediated HIV-specific immunity
independently of patient HLA-haplotypes and
HIV-clades
M. Amicosante1, C. Gioia1, C. Montesano', R. Casettil, S. Topinol,
G. D'Offizil, G. Cappelli2, G. Ippolito1, V. Colizzi3, E Poccia1, L.P. Pucillo1.
1INMI - "L. Spallanzani" IRCCS, Lab. Clinical Pathology INMI - Via Portuense
292, 00149 - Rome, Italy; 2CIRBA, Abidjan, Cote d7voire; 3University "Tor
Vergata", Rome, Italy
Background: HIV-specific CD8-positive cytotoxic T-lymphocytes (CTL) play a key
role in controlling HIV-infection, monitoring CTL-response could be clinically relevant during stucturated therapy interruption (STI), HIV exposure and vaccine trials. However, HLA patients' restriction and HIV variability limited the development
of a CTL assay with broad specificity
Methods: We designed an HLA-class I/HIV-1 clade independent assay for assessing HIV-specific CTL by using a computer-assisted selection of the CTL
epitopes. Twenty-eight 15-mers on the consensus sequences of GAG protein
were selected by peptide-binding motifs analysis using different database (HIVImmunology Database, SYFPEITHI, BIMAS).
Results: Altogether the peptides putatively bind to more than 90% of HLAhaplotypes in different populations, with an overall HIV-1 variability below 9%.
The peptide pool was used as antigen in an intracellular cytokine staining (ICS)
assay for quantifying HIV-specific CTL-response. The test can be performed using
both fresh and cryopreserved peripheral blood mononuclear cells (PBMC), while
GAG protein as antigen works only on fresh PBMC. A significative higher CTLresponse respect to HIV-negative controls was detected in all HIV-1 infected subjects of two groups of patients with different ethnicity (Caucasians and Africans)
and coming from area with different HIV-1 clade prevalence (clade B and A/G
respectively). In Caucasian patients after one month of STI the number of HIV-1
specific CTL (2896~2780 IFN-y specific CD8 cells/mi) was significative higher
than at the enrolment (2125~4426 IFN-y specific CD8 cells/ml, p<0.05).
Conclusions: These data indicate that this CTL assay is broadly specific and
could represent a useful clinical tool for HIV-immunodiagnostic independently of
HLA-haplotype and HIV-clade variability.
Presenting author: Massimo Amicosante, Lab. Clinical Pathology, INMI - Via
Portuense 292, 00149 - Rome, Italy, Tel.: [phone], Fax: [phone]
904, E-mail: amicosan @ uniroma2.it
ThPeA7112 Preferential targeting of regions in p24 Gag by
cytotoxic T lymphocytes in subtype C HIV-1
infected individuals from southern Africa
C.M. Gray1, G. Khoury2, A. Masemola2, T. Mashishi2, P. Mohube2, A.J. Puren2,
L. Morris2, H. Bredell3, C. Williamson3, G. Ferrari4, H. Cao5, H.W. Sheppard5.
1 National Institute for Communicable Diseases, private bag x4, sandringham,
2131, South Africa; 2National Institute for Communicable Diseasesirology
Johannesburg, South Africa; 3 University of Cape Town, Cape Town, South
Africa; 4Duke University, North Carolina, United States; 5California Department
of Health Services, Richmond, United States
Background: identifying the relationship between control of subtype C HIV-1 and
patterns of CTL epitope responses is important for subsequent interpretation of
vaccine-induced CTL. We have studied the association between HIV-1 viral load
and anti-Gag CTL responses in subtype C HIV-1 infected individuals from southern Africa prior to commencing a subtype C Gag HIV-1 vaccine trial in the region.
Methods: 66 HIV-1 infected individuals were recruited into a natural history study
(HIVNET 028) within the first year of infection and followed over one year. Individuals were recruited from South Africa (Johannesburg and Durban), Zambia (Lusaka), Malawi (Blantyre) and Zimbabwe (Harare). Gag-specific CTL responses were measured at one time point using a combination of the IFNg
ELISPOT assay, intracytoplasmic cytokine staining and 51-Cr-release assays.

Measurements were correlated with viral loads (bDNA and Amplicor).
Results: 45/66 (68%) individuals responded to pools of subtype C-based overlapping Gag peptides using the ELISPOT assay. Five immunodominant peptides were identified: two in p17, GKLDKWEKIRLRPGGKKRYM (38% of participants) and GTEELRSLYNTVATLYCVHE (29%); three in p24: MFTALSEGATPQDLNTMLNT (31%), IYKRWIILGLNKIVRMYSPV (20%) and EPFRDYVDRFFKTLRAEQAT (44%). There were no significant differences in the magnitude

between regions recognized (median range: 352-516 sfc/10e6 PBMC), although
EPFRDYVDRFFKTLRAEQAT appeared more immunodominant and exclusively
recognized by 1/5 of responders. There was no geographic difference in epitope patterns and individuals who displayed Gag responses showed significantly
(p<0.04) lower viral loads (11629 copies/ml) when compared to individuals showing no Gag responses (26725 copies/ml).
Conclusion: Five immunodominant epitope regions in Gag were identified in subtype C HIV-1 infected individuals from the southern Africa region. Three epitopes
were strongly targeted in p24 which were significantly associated with lower viral
load.
Presenting author: Clive Gray, private bag x4, sandringham, 2131, South Africa,
Tel.: [phone], Fax: [phone], E-mail: [email protected]
ThPeA7113 Immunodominance of HLA-B57-restricted
HIV-1-specific CTL responses during acute HIV-1
infection
M. Altfeld1, F.M. Hecht2, PK. Lee', M.M. Addo1, X.G. Yu1, R. Draenert1,
C. Fitzpatrick1, M.N. Johnston', J. Osksenberg2, P.J.R. Goulder1, J.A. Levy2,
E.S. Rosenberg1, B.D. Walker'. 'Partners AIDS Research Center,
Massachusetts General Hospital/Harvard Medical school, Boston, MA, United
States; 2UCSF San Francisco, United States
Background: HLA-B57, as well as CTL responses restricted by this allele, have
been strongly associated with long-term non-progressive HIV infection. However,
their impact on viral replication during acute HIV infection is not understood. This
study assessed clinical and immunlogical parameters during acute HIV infection
in B57 positive and negative individuals.
Methods: The allele frequency of B57 was compared between individuals with
symptomatic acute and chronic infection and HIV-negative controls. HIV-specific
CTL responses were comprehensively assessed using an IFN-g Elispot assay
and the contributions of CTL responses restricted by B57 and the other expressed
HLA alleles to the total HIV-specific T cell responses were determined using newly
adapted flow cytometric methods.
Results: Individuals expressing B57 presented significantly less frequently with
symptomatic acute HIV infection (4/116, 3,5%) than expected from the frequency
of chronically infected (43/446, 9,6%, p<0.01) or uninfected (11%) B57 positive
individuals. During acute infection, B57 positive subjects had stronger CTL responses than B57 negatives and more epitopes were presented by B57 than by
other expressed alleles. In B57 positive persons, B57-restricted responses were
significantly stronger than responses restricted by all 5 additional major HLA class
I alleles together (p<0.01) and B57 always restricted the immunodominant response.
Conclusion: HLA-B57 does not protect against HIV infection, but individuals expressing this allele present significantly less frequently with symptomatic acute
infection. Strong B57-restricted CTL responses during acute infection may allow
for rapid control of early viremia, limiting clinical symptoms and reducing the initial immunological damage. The association of HLA-B57 with strong HIV-specific
CTL responses in acute infection and better long-term outcome exemplifies the
crucial role of the early cellular immune response in the outcome of HIV infection.
Presenting author: marcus altfeld, aids research center, massachusetts general
hospital / harvard medical school, 149, 13th street, 5th floor, United States, Tel.:
[phone], Fax: [phone], E-mail: [email protected]
ThPeA7114 Nef is a major target of CTL in HIV-1 infected
patients
E. Harrer, S. Kastel, M. Bduerle, S. Bergmann, M. Hamacher, J.R. Kalden,
T. Harrer. Department of Medicine //III, University of Erlangen, Erlangen, Germany
Background: HIV-1 Nef and Tat are attractive targets for immunotherapy as they
are expressed early in the viral replication cycle and they play an important role in
the pathogenesis. To evaluate the immunogenicity of these molecules we started
a systematic study of recognition of Nef and Tat in a cohort of HIV infected patients.
Methods: The Net- and Tat -specific T-cell response was analysed in 41 HIV-1 infected patients in different stages of HIV-1 infection using a gamma-IFN ELISPOT
and a set of 20 amino acid long overlapping peptides derived from the HIV-1 Bru
variant.
Results: In 24/41 (58.5%) patients we could detect Net-specific T-cells. On average these patients recognized 3 epitopes (range 1 - 7). The median frequency
of Net-specific T-cells was 81/1 00 000 PBMC (range 5 - 590). One patient recognized one CD4-epitope. The major immunogenic part of Net was the region
spanning from aa 72 to aa 101, which was recognized by 50% of the patients. In
contrary, only 3/40 patients (7.5%) showed a TAT-specific CTL response with a
median recognition of 1 epitope (range 1 - 2).
Conclusions: Despite its immune evasive properties Nef is a very immunogenic
molecule recognized by the majority of HIV-infected patients in our cohort. There
fore, strategies for the development of preventive and therapeutic vaccines should
include immunisation against Nef.
Presenting author: Thomas Harrer, Department of Medicine III, University of
Erlangen, Krankenhausstrasse 12, 91054 Erlangen, Germany, Tel.: [phone], Fax: [phone], E-mail: [email protected]