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170 Evaluation of the Immunogenicity of HIV-1 Vaccines in Baboons
A. Tang*1, D. R. Casimiro1, M. Davies1, T. Fu1, L. Chen1, K. Wilson1, K. Murthy2, K. Rice2, D. Freed1, D. Kaslow1, E. Emini1, and J. W. Shiver1
1Merck Res. Labs, West Point, PA, USA and 2Southwest Fndn. for Biomedical Res., San Antonio, TX, USA
Background: Nonhuman primates are excellent model systems for evaluating the efficacy of HIV-1 vaccines. Rhesus macaques are most widely utilized, yet they are considerably small compared with an adult human. Although chimpanzees are appropriately sized, they are not readily available. Adult baboons (25(40 kg) provide an attractive alternative to the Great Apes; however, quantitative assays for cell-mediated immunity are limited.
Methods: Peripheral blood mononuclear cells (PBMCs) were prepared from untreated baboons and baboons immunized with high (1011 particles) doses of replication-incompetent adenovirus type 5 (Ad5) vector expressing HIV-1 gag. Several commercial anti-human IFN-gamma and TNF-alpha clones were tested for cross-reactivity with baboon cytokines in an ELISpot and/or flow-cytometric assays. Numerous anti-human Abs for lymphocyte (CD3, CD4, and CD8) and natural killer cell (CD16) markers were analyzed for staining of baboon PBMCs.
Results: An IFN-gamma ELISpot assay using anti-human IFN-gamma 1D1K clone (Mabtech) and anti-human IFN-gamma polyclonal Abs from the same vendor provided excellent spot quality and reduced background counts for baboon PBMCs. Analyses of baboons immunized with multiple doses of Ad5-HIV-1 gag indicate levels of gag-specific T cells as high as 0.2% of circulating lymphocytes. An intracellular staining protocol for IFN-gamma or TNF-alpha was developed using anti-CD16 marker primarily to eliminate natural killer cell contributions and anti-CD4/CD8 markers to identify the T-cell subset(s) responsible for the responses. Characterization of gag-specific T cells from Ad5 vaccinees reveals a mixed CD4+/CD8+ population with a noticeable bias toward CD8+ T cells. Consistent with these observations, the vaccinees exhibited strong CTL-mediated killing of peptide-pulsed autologous cell lines.
Conclusions: ELISpot and intracellular staining methods have been developed for quantitative assessment of virus-specific T-cell responses in baboons. We show that Ad5 HIV-1 vaccines are immunogenic in this primate species; hence, baboons serve as a useful model for continuing development of T-cell-immunity-based AIDS vaccines.
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