An Ultrasensitive in Vivo Latent HIV Viral Outgrowth Assay Using Humanized Mice

Sponsor: Colorado State University

Location(s): United States


A fully cured HIV patient named the Berlin patient's example instilled a lot of optimism in the HIV field and the current emphasis is to develop a complete cure. In this context it is important that reliable tests are developed that can verify if a patien is fully cured and free of the virus. Since the current existing tests are not found to be fully reliale, our goal here is to develop an improved and more sensitive test. To accomplish this goal, we will use humanized mice that are susceptible to HIV infection to develop the hmVOA test. Samples from patients undergoing prolonged intensive anti- HIV therapies in which virus could not be detectable by standard tests will be evaluated in this novel system. Experiments are planned to further increase the sensitivity of the hu- mouse hmVOA test by using virus awakening drugs. Results of these studies are expected to be of high importance to the HIV Cure field.

HIV-1 reservoirs continue to exist in latent form despite long-term suppression of circulating virus with antiretroviral therapy. The main challenge in achieving a cure for HIV-1 infection is the persistence of these latent viral reservoirs. To dat, there has only been one functional "cure" of HIV-1 infection in an individual who underwent myeloablative allogeneic hematopoietic stem cell transplant for acute myeloid leukemia with donor cells lacking functional CCR5, a co-receptor used by HIV-1 to enter host cells. Reduction of HIV-1 reservoirs with pre-transplant cytotoxic chemotherapy likely contributed to viral eradication; however, the effects of cytotoxic chemotherapy with or without hematopoietic stem cell transplant (HSCT) on the viral reservoir and host factors and immune responses are largely unknown. Our study will provide greater insight into the persistence and evolution of HIV-1 that will have practical implications for therapeutics aimed at eliminating HIV reservoirs. We propose to investigate the effect of cytotoxic chemotherapy and/or HSCT on viral reservoir size, evolution, and immune function in HIV-1 infected individuals with hematologic malignancies initiating systemic anti-neoplastic therapy. We hypothesize that cytotoxic chemotherapy or HSCT reduces pools of latently infected cells, and will be manifested as a long-term reduction in the amount of total and integrated HIV-1 DNA. We also hypothesize that there is no significant evolution during immune reconstitution following cytoreductive therapy, but reductions in HIV-1 DNA and subsequent re-expansion of CD4+ lymphocyte subsets and other tissue compartments may lead to changes in the diversity of the remaining viral reservoir. Specific aims of this proposal include: 1) investigate the effects of chemotherapy or HSCT for hematologic malignancy on peripheral blood and bone marrow reservoir size, 2) study the changes in residual viremia, lymphocyte activation, and HIV-specific immune responses before and after chemotherapy, and, 3) investigate the effects of cytotoxic chemotherapy on HIV-1 evolution, tissue compartmentalization, and reservoir diversity. This five-year study will utilize innovative approaches to investigate reservoir dynamics such as 454 deep sequencing and assays to detect and quantify low levels of HIV-1 DNA from host tissue and plasma viremia below the limit of detection of standard tests. Measures of lymphocyte activation, host-entry factors and HIV-specific immunity will also be integrated into the research plan in collaboration with immunologists and evolutionary virologists.