The overall goal of this project is to define the immunologic and virologic characteristics of the persistent cellular reservoirs for HIV-1, the extent of immune damage and potential for reconstitution, and the evolution of anti-HIV-1 T cell responses during the natural history of HIV-1 infection before and after therapeutic intervention. There are three specific aims: (1) The first is to 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) The next is to determine the extent of lymphocyte regeneration by thymus-dependent and -independent pathways and the capacity for immune reconstitution in patients receiving PART, and lastly, (3) to 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 persistence of major histocompatibility complex (MHC)-class I-restricted CD8+ and MHC-class II-restricted CD4+ memory T cell responses. These studies will include determination of 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 T cell subpopulations identified by fluorescence activated cell sorting (FACS). Studies will also include elucidating the mechanism and extent of T cell reconstitution using kinetic polymerase chain reaction (PCR)-based assays to quantify T cell excision circles and T cell V-beta genes in specific phenotypically defined T cell subsets. Additionally, the applicants 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. The investigators will measure virus-specific CD4+ T cell responses in patients over time using stimulation by recombinant HIV-1 p24 and gpl20 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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