The overall goal of this proposal is to define the immunologic and virologic characteristics of the persistent cellular reservoirs for HIV-1, the extent of immune damage and potential for reconstitutions, and the evolution of anti-HIV-1 T infection before and after therapeutic intervention.
Our specific aims are to: 1) Determine the proportion and types of HIV-1 infected cells circulating in the blood stream responsible for maintaining the long-lived reservoir of viral infection, 2) Determine the extent of lymphocyte regeneration by thymus-dependent and - independent pathways and the capacity for immune reconstitution in patients receiving potent anti-retroviral therapy, and 3) Characterize the cellular immune response in HIV-1 infected patients, the impact over time of chronic viral replication on the maintenance of these responses, and the impact of short and long-term suppression of viral replication on the maintenance of these responses, and the impact of short and long term suppression of viral replication on the persistence of MHC-class I- restricted CD8+ and MHC-class II-restricted CD4+ memory T cell responses. These studies will include determining the viral variables associated with persistent cellular reservoirs following treatment of HIV- 1 infection using new molecular techniques to detect and quantify viral transcripts and viral evolution in FACS-sorted T cell subpopulations. Studies will also include elucidating the mechanism and extent of T cell reconstitution using kinetic PCR-based to quantify T cell excision circles and TG cell V-beta genes in specific phenotypically defined T cell subsets. Additionally, we will determine the dimensions of the virus- specific CD8+ T cell response in HIV-1 infected patients over time by flow cytometry using MHC class I/peptide tetramer staining, antigen- specific intracellular cytokine staining and classical T lymphocyte cytotoxicity assays. We will measure virus-specific CD4+ T cell responses in patients over time using stimulation by recombinant HIV-1 p24 and gp120 proteins and measurement of in vitro proliferation and intracellular cytokine staining detected by flow cytometry. From such data, detailed mathematical analyses can be done that provide not only a kinetic picture of viral pathogenesis, but also help establish theoretical principles to guide the rational approach to the development of efficacious treatment strategies.
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