Investigation of HIV Levels, Latent Reservoirs, and Mechanisms of Latency in the Gut

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Though combined antiretroviral therapy (ART) has reduced both morbidity and mortality due to HIV, ART does not lead to eradication or restore normal health to HIV-infected individuals. Given the staggering health and financial costs of HIV, it seems imperative to explore new therapies that can possibly lead to viral eradication. Obstacles to eradication include latent infection in resting memory CD4+ T cells and the presence of other cell types or organs that serve as sanctuary sites for virus. Abundant evidence suggests that the gut is a site of persistence for HIV in ART-suppressed patients. We have shown that levels of HIV DNA and RNA are up to 10-fold higher in the gut compared to blood, that the gut may be a site of ongoing replication (in the ileum) as well as "latent" infection, and that different mechanisms may maintain HIV persistence in the gut and blood. This study seeks to quantify levels of latent or chronic productive infection in gut CD4+ T cells (or other cells) and to investigate the mechanisms that allow these cells to persist in the immunologically-activating environment of the gut. The specific aims are: 1) to measure levels of HIV in different cell populations in the gut; 2) to measure levels of inducible, replication-competent, or latent HIV in the gut; and 3) to investigate mechanisms of latency in vivo in the gut. The study has two main parts. Part 1 will make use of unused tissue from ART-suppressed HIV+ patients who are getting intestinal resections, while part 2 will utilize colonoscopic biopsies from the terminal ileum and rectum. Surgical and endoscopic gut tissue will be used for in situ studies, for measurement of bulk tissue HIV DNA and RNA, for short term culture of gut tissue (to measure inducible or replication-competent virus), and for isolation of large numbers of gut cells, which will then be FACS-sorted into various cell populations. The sorted cell populations will be used to measure levels of HIV DNA, RNA, and inducible, replication-competent, and/or latent HIV. Genomic DNA will also be used to investigate mechanisms of latency, such as epigenetic modification and integration site or orientation. The proposed study should greatly increase our knowledge of HIV reservoirs and may ultimately contribute to new and improved therapies aimed at eradication or lifelong coexistence with HIV.