MACROPHAGE ANTIGEN PROCESSING OF HIV SUBTYPES

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(Adapted from applicant?s abstract) CD8+, MHC-1 restricted cytotoxic T lymphocytes (CTL) are responsible for controlling viremia associated with human immunodeficiency virus (HIV) infection. CTL activity is directed against epitopes from lymphocytotropic (T-tropic) and monocytotropic (M-tropic) strains. T-tropic HIV strains do not cause a productive infection in human monocytes and macrophages. However, we have shown that macrophages exposed to T-tropic or M-tropic HIV are equally able to induce a primary CTL response, indicating that processing of viral antigens is occurring after exposure to both strains. Thus we hypothesize that macrophages can support both entry and processing of T-tropic HIV and subsequently elicit MHC class I-restricted cytotoxic T lymphocyte responses to specific viral epitopes in the absence of a productive infection. We will test this hypothesis by examining each of the steps involved in antigen processing using an in vitro fixed cell model of HIV infection. The following specific aims will be tested: 1) To determine the significance of various entry mechanisms by T-tropic and M-tropic HIV into alveolar macrophages and monocyte derived macrophages on subsequent CTL responses, 2) To determine whether MHC class I-restricted HIV epitopes are generated in the cytoplasmic compartment by proteasomes or in endosomes, 3) To determine if HIV peptide-MHC class I coupling requires synthesis of new MHC class I molecules or can occur with preexisting molecules via a regurgitant type pathway, 4) To determine specific epitopes recognized by CTL primed in vitro, and 5) To determine if specific HIV epitopes require transporter associated with antigen processing (TAP)-dependent MHC class I processing. Understanding these pathways may provide insight to novel therapies aimed at enhancing HIV antigen presentation and the subsequent cellular immune response in HIV. The candidate is currently a pulmonary fellow in the Department of Medicine at Indiana University. At the proposed start-up time the candidate will be a Lecturer on the faculty in the Pulmonary and Critical Care Division with 75 percent protected time allocated for research. To date the candidate has trained in the laboratory of Dr. Homer Twigg, acquiring basic immunologic knowledge and laboratory skills. This proposal is a logical mechanistic extension of this work designed to allow the candidate to develop a basic understanding of antigen processing pathways. Importantly, in this proposal the candidate will develop new research skills by working in the laboratories of Dr. Homer Twigg (CTL cloning, CTL generation and assays), Dr. Janice Blum (intracellular antigen processing pathways, transfection techniques using TAP-deficient cells), Dr. Randy Brukiewicz (working with vaccinia virus and constructs to study specific CTL epitopes), and Dr. Douglas Perry (liposome biology, phagocytic pathways). Each of these investigators have the expertise and resources (money and laboratory space) necessary to ensure successful completion of the training program. By acquiring the knowledge and skills outlined in this proposal, the candidate hopes to fulfill his career goal of becoming a full-time, funded researcher in an academic pulmonary division.