Fundamental gaps in our understanding of HIV reservoirs preclude a precisely targeted approach to eradication. HIV is neither eliminated nor often controlled by the human immune system, and the immunologic defect(s) responsible for this lack of control are unknown. We are conducting several studies to investigate the effects of clinical events on HIV reservoirs. We have established useful collaborations to investigate the effects of clinical interventions on HIV reservoirs. We are collaborating with R. Yarchoan (HIV and AIDS Malignancy Branch, NCI) to study individuals with HIV infection and comorbid cancer treated at the NIH Clinical Center. These study patients receive antineoplastic cytotoxic and immune modulator therapy to treat cancers, treatment modalities that may also have significant effects on HIV-infected cells, and we are studying the effects of these therapies on HIV reservoirs. In collaboration with I. Sereti (NIAID), we are studying patients with advanced HIV/AIDS who initiate antiretroviral therapy (ART) and experience immune reconstitution inflammatory syndrome (IRIS) in response to coinfections such as tuberculosis cryptococcal meningitis, and progressive multifocal leukoencephalopathy (PML). IRIS results in a vigorous immune response to the comorbid infection with significant increases in CD4 T cells; as such, IRIS may have profound effects on HIV reservoirs. We have been studying individuals with IRIS and TB and found that HIV-infected cells are part of the robust immune response that occurs during IRIS. In fact, HIV-infected cells can specifically respond to TB antigens. We have also found that IRIS has long-term effects on the structure of HIV populations during therapy, suggesting IRIS may alter HIV reservoirs. In collaborations with Irini Sereti (NIAID) we have recently demonstrated the effects of PML-IRIS on HIV populations. In addition, we demonstrated that treating IRIS with anti-inflammatory agents can reduce the production of HIV from reservoirs over 100-fold. These studies point to new studies to manipulate the composition and production of HIV populations during therapy. Understanding additional forces that can affect HIV reservoirs have also been initiated. Antineoplastic chemotherapy may have profound effects on HIV populations and we are working with CCR investigators (Drs. K. Lurain, R. Ramaswami, R. Yarchoan) to investigate the effects of new chemotherapeutic regimens for Kaposi Sarcoma, Primary effucsion lymphoma, and Castlemans disease on HIV populations. In addition, we are now investigating the effects of the SARS-CoV-2 infection and vaccination. We have also initiated a new analytic treatment interruption study to determine the sources of rebound viremia when HIV therapy is interrupted (NIH protocol 000277). For these studies, we are conducting a multidisciplinary analysis of HIV reactivation. We use state of the art 18FDG positron emission tomography to characterize metabolic activity of lymphoid tissue, followed by site directed biopsy approach pioneered by our collaborators in NIH interventional radiology. We are quantifying the size and composition of HIV populations in biopsies and blood derived samples prior to and following a short (10 day) treatment interruption,and analyze these data in the context of detailed immunologic studies. As a result, we will obtain a comprehensive analysis of the earliest steps in HIV reactivation. We are also completing a clinical study (NIH protocol 13-I-0062) investigating generalized immune activation in the gastrointestinal-associated lymphoid tissue (GALT) on persistent HIV in individuals undergoing ART. We have been studying the effects of the nonabsorbable antibiotic rifaximin to specifically alter the gut microbiome and affect translocation of bacterial cell products, and the consequent levels of generalized immune activation and low-level HIV viremia. Although we identified changes in the gut microbiome, these changes did not result in downstream effects on levels of HIV viremia or levels of immune activation. These studies will further our understanding of the role of modifying the microbiome in immune activation. We are also investigating the role of innate immunity in HIV persistence by studying the effects of the innate immune modulator interferon alpha 2b. We have characterized the effects of exogenous interferon therapy in a series of HIV-infected individuals undergoing ART (NIH protocol 11-I-0057). We found no effect of interferon on levels of HIV in plasma or in peripheral blood lymphocytes, and are now characterizing interferon effects on the phylogenetic structure of the HIV populations. Our studies of HIV reservoirs indicate a critical role of anatomic locations, particularly lymphoid tissues, in HIV persistence. In a new studies, we are collaborating with Dr. S. Norberg in his CAR-T studies, which now have been expanded to include therapy for squamous cell carcinoma in HIV infected individuals. Dr. Norborg studies have been awarded additional support through CCR Flex award. Finally, we have initiated new collaboration with Dr. Genoveffa Franchini from NCI Vaccine Branch to evaluate new HIV vaccines based on highly successful candidates identified in primate studies. These studies are also expected to generate new studies of the effects of such agents in therapeutic vaccine studies as well.