A major obstacle to the development of an effective HIV vaccine is our lack of understanding of the critical components that constitute a protective, HIV-specific immune response.
The aim of this proposal is to investigate, more comprehensively than has been done to date, the potential immunoprotective roles of a broad spectrum of different HIV epitope-specific T cell functions in controlling HIV replication among untreated patients with early HIV infection. The epitope-specific T cell functions that will be measured include CD4+ and CD8+ T cell IFN-g, TNF-a and IL-2 responses (and the maturational state of these responding cells), CD4+ and CD8+ T cell proliferation and CD8+ T cell perforin degranulation. All these functions can be measured by multiparameter flow cytometry after stimulation of PMBC with overlapping peptides that span relevant HIV antigenic proteins. We propose to begin this work by focusing on HIV Gag and Nef epitopes. However, since the measurement of all these T cell responses just these epitopes would require prohibitively large quantities of blood, we will focus on peptide motifs within an individual's autologous Gag/Nef sequence that are known to be HLA-restricted for that individual's haplotype. After sequencing autologous Gag and Nef sequences from stored plasma samples obtained just before untreated patients with early HIV infection established good control of HIV replication, and then again from subsequent timepoints when these same patients have lost that control, we will scan the sequences for HLA haplotype-appropriate motifs and then synthesize them as 9- and 15-mer peptides for Class I and II stimulation, respectively. These peptides will then be used in flow cytometry assays to identify autologous Gag/Nef epitope-specific CD4+ and CD8+ T cell proliferation and IFN-g, TNF-a and IL- 2 responses (and the maturational state of the cytokine-positive cells) and CD8+ T cell perforin degranulation responses. To control for changes in innate immune responses that could also affect control of HIV replication, we will also measure NK cells and function, plasmacytoid and myeloid dendritic cells and regulatory T cells in the same PBMC specimens. Understanding this pattern for clinically important, conserved HIV proteins such as Gag and Nef should improve understanding of the critical components of a protective, HIV-specific immune response and provide insight into the type of HIV epitope-specific T cell responses that may be useful to monitor in future trials of candidate HIV vaccines. ? ? ?