HIV-1 infection is associated with progressive immunosuppression, associated with a progressive decline in CD4 lymphocyte number and function, and resulting ultimately in the development of opportunistic infections and neoplasms. Because CD4 cells are central to the proper functioning of a myriad of immune responses, loss or dysfunction of these cells is thought to be a key component contributing to immunodeficiency. Therapeutic trials with antiretroviral agents have resulted in transient and relatively short-lived boosts in CD4 number, and ex vivo expansion of these cells has not possible because stimulation and proliferation of Cd4 cells results in activation of HIV-1 replication. Strategies to protect CD4 cells for ex vivo expansion should facilitate not only adoptive therapy trials of these cells, but would also provide a source of CD4 cells for genetic manipulation. The purpose of this IRPG is to establish a collaborative interaction among groups of investigators with the express purpose of developing and implementing strategies for immune reconstitution in HIV-1 infection. Genetic manipulation of both mature lymphocytes as well as bone marrow stem cells will be explored through the implementation of closely interrelated researched projects, utilizing an adeno-associated virus vector system to explore intracellular immunization strategies. In preliminary studies in our laboratory, we have devised a strategy which enables the ex vivo expansion of uninfected CD4 lymphocytes from seropositive persons. PBMC are expanded in the presence of a bispecific monoclonal antibody which results in the selective polyclonal expansion of CD4 lymphocytes, which can be successfully protected from HIV-1 infection by the addition of combination antiretroviral therapies during the ex vivo expansion. In a number of infected persons, CD4 lymphocytes have been expanded greater than 10 5-fold, and the resultant cultures are negative for infection as evidenced by negative coculture and PCR negativity. We now proposed to expand upon these preliminary studies with the ultimate aim of adoptive therapy with ex vivo-expanded CD4 cells in infected persons. We also propose to adapt these studies to the rhesus macaque animal model in order to experimentally address critical questions regarding adoptive cell therapy of viral infections. Specifically, we propose to: a) expand CD4-positive lymphocytes from infected persons ex vivo in the absence of ongoing viral replication; b) expand populations of antigen-specific CD4 lymphocytes from infected persons ex vivo; c) protect expanded CD4 cells from HIV-1 infection by genetic manipulation using adenovirus-associated viral vectors and/or retroviral vectors; d) expand and genetically modify lymphocytes from normal and SIV-infected macaques, e) isolate and genetically modify pluripotent stem cells from macaques. These studies will contribute to the development of adoptive immunotherapeutic interventions for HIV-1 infection, and should contribute to the development of gene therapy for this disease.
