HIV Persistence During Suppressive Antiretroviral Therapy

Division Of Basic Sciences - Nci

Investigators

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Abstract

Combination antiretroviral therapy (ART) results in marked suppression of viremia in persons with HIV-1 infection. Therapy is not curative, however, and detectable viremia and replication-competent HIV-1 persist despite ART-induced suppression. The origin of persistent viremia on therapy is uncertain; potential sources include ongoing complete cycles of HIV-1 replication, long-lived reservoirs of chronically infected cells, sanctuary sites into which antiretrovirals have poor penetration, or a combination of these possibilities. Understanding the source and mechanisms of viral persistence on ART has critical implications for future therapeutic approaches and strategies for virus eradication. We began our investigation of the source of persistent HIV by developing an assay for viremia (HIV RNA) with single-copy sensitivity and by developing clinical protocols to determine the effects of ART intensification. These studies and others revealed no decrease in persistent viremia after drug intensification, suggesting that persistent viremia may be the product of long-lived reservoirs of chronically infected cells. Others, however, suggested ongoing replication of HIV is the source of persistent viremia. In the last year, we conducted a critical test of the question of whether ongoing HIV replication persists in sanctuary locations by analyzing HIV populations in blood and tissues from individuals after prolonged cART (8-16 y). In untreated individuals, HIV replication is rapid and error prone, leading to the accumulation of genetic changes that are readily detectable in the proviruses. Here we found that infected cells persist and clonally expand, but there was no detectable evidence of HIV evolution nor was there any evidence of compartmentalization of the HIV proviruses in blood or tissues in six individuals who were studied during effective combination ART for prolonged periods, cumulatively totaling over 60 patient-years of follow-up. These studies did not reveal any evidence for ongoing replication as a mechanism for HIV persistence during ART. Instead, we found that HIV is maintained during therapy through long-lived cells that persist and can undergo clonal expansion. 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 suppressed less than 50 copies who are undergoing colonoscopy at the NIH Clinical Center. We have performed these colonoscopies in collaboration with J. Kovacs (CCMD) in the protocol Virologic and Immunologic Evaluation of Lymph Node, Tonsillar and Intestinal Biopsies, and Bronchoalveolar Lavage Fluid and have used a new sampling strategy that will yield useful information regarding the distribution of HIV-infected cells in the gastrointestinal tract.___In collaboration with S. Hughes (HIV DRP), X. Wu (Leidos), J. Coffin (Tufts), M. Kearney (HIV DRP) , and J. Mellors (University of Pittsburgh), we have investigated HIV integration sites in vivo in these individuals. We are expanding these analyses to investigate HIV-infected cells in CD4 cell subsets in individuals undergoing long-term combination ART (supported by Intramural AIDS Targeted Antiviral Program funding). It is critical to learn how clonal expansion shapes the overall population of HIV-infected cells. It is also important to understand how HIV proviruses are expressed in these subsets. 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 also need to know whether clonal expansion of HIV-infected cells in blood and tissues reflects global T-cell expansion. We will therefore complement our studies of clonal expansions of HIV-infected cells by investigating the diversity and clonal expansion of the T-cell repertoire using next-generation sequencing to quantify the T-cell receptor diversity in these subsets (collaboration with E. Boritz and D. Douek). In this collaboration, we will study HIV-infected individuals undergoing combination ART for prolonged periods in our natural history study. Drs. Boritz and Douek will obtain highly purified subsets of CD4 cells, such as central and transitional, naive, and effector memory populations, by fluorescence-activated cell sorting (FACS) of PBMC from these participants and will determine T-cell receptor diversity and quantify the extent of T-cell clonal expansion in these T-cell subsets. We will quantify the levels of HIV proviruses in these highly purified subsets as well as characterize the genetic composition of HIV populations. With Dr. Hughes, we will determine the distribution of integration sites of the proviruses in these subsets and investigate the extent of clonal expansion. These studies will determine whether clonal expansion of HIV-infected cells reflects overall T-cell clonal expansion or represents a distinct process that takes place whether generalized T-cell clonal expansion occurs or not. In support of these studies, we recently reported new droplet digital PCR techniques to accurately measure relative amounts of defective proviruses and found that defective proviruses are enriched over time in most patients undergoing ART. The enrichment was progressive, and took place only after several years on therapy, suggesting the enrichment was due either to selective elimination of intact proviruses or to increases in defective proviruses, perhaps by clonal expansion. These new techniques will greatly assist the field in understanding the dynamics of HIV-infected cells during ART. We have also initiated several new studies of HIV persistence. Previously, in collaboration with Dr. Hughes, we identified relatively rare examples in which integration of a provirus in specific portions of particular genes (e.g., BACH2, MKL2, STAT5B) was implicated in persistence and clonal expansion. We suggested that the provirus affects the expression of these host genes, making it important to determine the structure of these proviruses. Most (over 98%) proviruses are defective, and it is not known whether the proviruses in these three genes are intact or highly defective. To resolve this issue, we are determining the structure of proviruses integrated in BACH2 and MKL2. Preliminary data suggest that proviruses integrated in these genes have large deletions, indicating that they are not infectious. By determining the structure of the proviruses, we will better understand how they affect gene expression and drive replication and persistence. 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.