HIV Persistence During Suppressive Antiretroviral Therapy

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Previously we and others demonstrated that the inability to eradicate HIV during antiretroviral therapy (ART) is the product of persistence and clonal expansion of cells infected with HIV prior to the initiation of ART . Host characteristics (e.g., immune selective pressures, age, sex) have been proposed as driving forces shaping the proviral landscape, but their relative contribution to shaping the proviral landscape is not well characterized, and new studies are essential. The role of sex in persistence is unclear, as females remain understudied in cure research; we also need to study older (50 y) PWH, who now comprise the majority of PWH in the US. We found significant sex differences in HIV populations: females had higher ratios of HIV LTR DNA /gag DNA, suggesting elimination of gag-containing proviruses was more effective in females. In addition, levels of HIV gag RNA (normalized to gag DNA) were 2.5 fold lower in males (P=0.0006, Mann-Whitney). Lower expression of HIV gag RNA in males may result in reduced immune selective pressure and explain why higher levels of gag containing proviruses persist in males. Levels of HIV DNA was strongly positively correlated with levels of CD8 cells, in both males and females but differences in other immune markers in males and females was evident. Age was an important positive correlate in linear regression models, with the strongest correlations in random effects models detected in PWH over 50 years of age. Our approach identifies important roles for chronic inflammation, sex, and aging in HIV persistence. We are now building on these studies to quantify and genetically characterize virus in plasma to investigate HIV reservoirs in cellular compartments and expanding the range of our analyses by applying new single-cell methodologies and isolating specific cell subsets in blood and tissue from infected individuals. We are investigating HIV in plasma, PBMC, and cells derived from ileum and colon in infected individuals taking combination ART with HIV less than 50 copies who are undergoing colonoscopy at the NIH Clinical Center. We have integrated our molecular virology studies with analysis of the histopathologic effects of long term antiretroviral therapy in the gastrointestinal tract. In our colonoscopy protocol, we obtain samples for histopathologic evaluation, and we perform immunohistochemistry for CD4, CD8, CD163, and CD68 expressing cells in gut samples. We are quantifying the presence of these cell subsets using established methods (HALO) and analyzing the levels of HIV proviruses in these tissues in the context of populations of immune cells. These studies will yield detailed understanding of the presence of HIV infected cells in gastrointestinal tract. In collaboration with S. Hughes (HIV DRP), X. Wu (Leidos), J. Coffin (Tufts), we have investigated HIV integration sites in vivo in these individuals. We began these studies using blood-derived T cells, but we also need to understand the dynamics of HIV-infected T cells as they traffic through lymphoid tissue, where 98% of all lymphocytes are located. We will accomplish this using new sorting facilities that we have established with Drs, Lifson and Trubey; these are new state of the art capabilities with appropriate containment for infectious material on the Frederick campus, greatly facilitating our work. In addition, we previously demonstrated infected cells may contain proviruses consisting of HIV solo LTRs. Although such structures have been previously identified in gammaretroviruses, they had never been demonstrated in HIV infected individuals. Solo LTR forms are generated after integration by homologous recombination, and understanding the development of these proviruses will provide new insights on persistence of infected cells. We have developed new in vitro tools to study the formation of solo LTR forms. In an analysis of HIV infected cell lines, we identified the presence of solo LTR formation in vitro in the 8E5 cell line, We have now developed new in vitro systems to study HIVB solo LTR formation in mammalian and yeast systems. We are also investigating the persistence of HIV infected cells in anatomic compartments. In collaboration with NIH CC/CCMD investigators Drs. J. Kovacs and B. Chimukangara, we have initiated new studies to characterize HIV infected cell populations in pulmonary tissue (supported by OAR Innovation Program Grant). In collaboration with investigators in NCI Pathology ( Drs. S. Hewitt and D. Kleiner) CCR (Drs. R. Yarchoan, K. Lurain, R. Ramaswami, J. Kanakry), NIH CC/CCMD (Dr. D. Chertow, J. Kovacs), and NINDS (Dr. Avi Nath) we have conducted autopsies of HIV infected individuals (N=10) and have now established a repository of samples for study. We are quantifying the levels, characterizing the composition and determining the integration sites of populations of proviruses in these tissues. We are also investigating the distribution of HIV infected cells in individuals undergoing bone marrow transplant at the NIH Clinical Center. With Dr. Kanakry, we have recently analyzed the distribution of HIV infected cells in autopsy derived tissues from an individual undergoing bone marrow transplant. We recently found prolonged HIV persistence of HIV clones following HSCT in a PWH who received a non delta32 bone marrow transplant who had been treated extensively for anaplastic ALK-negative T cell lymphoma (HTLV-negative) received allograft HSCT, with complete donor chimerism in peripheral blood within 40 days of transplant; he unfortunately expired at day 206 from an intercurrent pulmonary process not related to the transplant. He maintained ART throught post transplant period, at autopsy, HIV proviruses were still present in a number of organs including in brain tissue. Levels of proviruses were c. 10-100 fold lower than that typically detected in individuals undergoing ART, as expected as a result of the conditioning and HSCT. Unexpectedly, however, brain tissue contained 1-4 copies HIV DNA/million cells, comparable to levels detected in brain tissue from PWH not undergoing HSCT; these data suggest little decay in proviral copy number brain within the first year post transplant. In phylogenetic analyses, my lab demonstrated that HIV proviruses remaining after transplant were genetically diverse, and not the result of persistence of limited number of HIV variants. In collaboration with Drs S. Patro and X. Wu (FNLCR), my lab analyzed integration sites recovered from pre-transplant PBMC, and from post-transplant gut tissue, analysis revealed proviruses were present in diverse sites, none in ZNF genes or other genes in which integration is associated with persistence (). We found two clones, one with HIV integrated in the MUTED-TXNDC5 and as second with HIV in SIK3 genes that were present both in PBMC prior to transplant and in ileum >200 days following transplant, demonstrating that HIV infected clones persist in tissues many months after 100% chimerism has been achieved in blood. These data have direct relevance in understanding requirements for eradication of HIV. As part of this transplant analysis group. I am actively engaged in ongoing studies analyzing blood and tissue derived samples. By accumulating data from increasing numbers of transplants, I will obtain important data regarding dynamics of HIV proviruses and identify optimum eradication strategies. These studies demonstrating the persistence of clones long after infected cells were eliminated in the blood compartment shed new light on the mechanisms of HIV persistence and on understanding of HIV cures that have been reported after transplant. These studies will shed light on specific pathways necessary for HIV persistence and identify testable strategies to interrupt expansion and persistence driven by specific integration events.