Rapid identification and fine-mapping of HIV-1-specific CTL epitopes using the elispot assay.
Int Conf AIDS 2000 Jul 9-14; 13:(abstract no. MoOrA222)
Altfeld M, Rosenberg ES, Eldridge RL, Brander C, Trocha AK, Addo MM, Kalams S, Walker BD, Goulder PJ M. Altfeld, Partners AIDS Research Center, 149 13th Street, ID Unit, 5th floor, Charlestown, MA, United States, Tel.: +1 617 724 2461, Fax: +1 617 726 5411, E-mail: maltfeld@partners.org
Given the important role of CTL responses in controlling HIV-infection, the characterization of virus specific epitopes targeted by CTL may provide the basis for the development of more effective HIV vaccines. The detection and fine-mapping of CTL responses are traditionally done with CTL clones and Cr51 release assays (CRA). These assays are both labor intensive and costly. Here we compare the Interferon gamma Elispot assay to the classic CRA approach. Using overlapping peptides (15-20mers) spanning the HIV-1 sequence of Gag, RT, Env and Nef, we identified 5 new HIV-1 specific CTL responses. These responses were fine-mapped using serial dilutions of truncated peptides in the Elispot assay with fresh PBMC. CTL clones specific for the corresponding epitopes were generated and the sequences of the optimal epitopes predicted by Elispot were confirmed using the CRA. CRA was also used to define the HLA restriction of these epitopes. Determination of the HLA restriction of the novel CTL responses was also performed and reproduced in the Elispot assay using partially HLA-matched B cell lines. This assay required approximately 10 fold less effector cells and was much less cost and labor intensive than the CRA. These data demonstrate that the amino acid sequence of optimal CTL epitopes can be rapidly and reliably determined by using truncations of overlapping 15-20mer peptides in the Elispot assay. Furthermore, the Elispot assay allowed fine-mapping, HLA restriction analysis and determination of novel optimal CTL epitopes for HIV-1. These findings have practical applications in the rapid identification of novel CTL responses under conditions that do not allow for long-term expansion of CTL clones and extensive use of radioactive labels as is needed by CRA. This approach might facilitate the identification of new CTL epitopes in non-B clade HI-Viruses in the countries of high prevalence.
Keywords: AEGIS, HIV-1, Epitopes, AIDS Vaccines, HIV Infections, HLA-B Antigens, Enzyme-Linked Immunosorbent Assay, HLA Antigens, Case-Control Studies, immunology 000709
MoOrA222
