Residual Viremia, Cure Strategies, and Tissue Reservoirs in Human Immunodeficiency Virus Type 1 Infection

Human immunodeficiency virus-1 (HIV-1) is a retrovirus that primarily infects immune cells bearing CD4 and either CCR5 or CXCR4. It is transmitted horizontally through contact with infected bodily fluids via intravenous drug use, sexual contact, or blood transfusion and is transmitted vertically via mother-to-child transmission during birth or breastfeeding. Untreated infection in all but very rare populations of patients leads to depletion of CD4+ T cells and a resulting lack of immune function that results in acquired immunodeficiency syndrome (AIDS), in which the infected individual is increasingly susceptible to opportunistic infections that lead to death. Despite the insidious nature of the disease, effective treatment with antiretroviral therapy (ART) can substantially prolong the life of an individual living with HIV by blocking various stages in the HIV-1 replication cycle. Effective ART reduces viral loads to below the limit of detection of standard clinical assays by halting viral replication. Patients on an effective treatment regimen can therefore continue to live relatively normal lives thanks to ART. However, ART is not a cure. Using very sensitive laboratory assays, HIV can be detected in patients on ART even below the limit of detection of clinical assays; and using a quantitative viral outgrowth assay (QVOA) developed in our lab, replication-competent HIV can be measured from patients with clinically undetectable viral loads on ART. Because ART is not a cure, infected individuals must continue to take ART for the remainder of their lives; otherwise, their viral load will quickly rebound to the levels that occurred before treatment with ART, and the disease will progress to AIDS. The need to take ART for a lifetime results in an extremely high cost of care; additionally, many of the components of ART have undesirable side effects. Therefore, despite the effectiveness of ART at halting the disease’s progression to AIDS, a cure for HIV is still needed. Finding a cure for HIV will require both an understanding of the reservoirs that constitute the barrier to curing HIV and an understanding of treatments that will purge these reservoirs. In this work, we discuss a study investigating a small molecule, disulfiram, as a potential latency-reversing agent, the development of a streamlined assay for measuring the latent reservoir, and the investigation of two different tissue sites (hematopoietic progenitor cells and cells in the gut-associated lymphoid tissue) as anatomic sites that could contain cells with replication-competent HIV in patients on ART. Chapter 1 provides an introduction to HIV latency and the concepts that differentiate a reservoir from a compartment. Additionally, the introduction provides a discussion of various cell types and anatomic sites that have been considered potential barriers to HIV cure. Chapter 2 describes a study investigating the potential use of disulfiram as a latency-reversing agent. This clinical trial involved measuring the latent reservoir before and after treatment with disulfiram using the QVOA and measuring the amount of residual viremia before, during, and after treatment with disulfiram to detect changes in residual viremia that might indicate latency reversal. Although no significant change in the size of the latent reservoir was detected, some study participants exhibited transient but dramatic increases in residual viremia, suggesting that some latency reversal occurred but that this effect was not substantial enough to decrease the size of the latent reservoir. In Chapter 3, we investigate hematopoietic progenitor cells as a potential reservoir for HIV. Bone marrow aspirates were obtained from individuals on suppressive ART. Although HIV DNA was detected in CD4+ T cells from the peripheral blood and in bone marrow cellular fractions containing CD4+ T cells, no HIV DNA was detected in purified CD34+ cells (hematopoietic progenitor cells), and no replication-competent HIV was detected using the QVOA. In Chapter 4, we develop a streamlined QVOA for measuring the size of the latent reservoir more efficiently. Like the original assay, the streamlined QVOA uses a limiting- dilution coculture assay to measure the size of the latent reservoir. However, the streamlined assay uses the MOLT-4/CCR5 cell line in place of activated lymphoblasts from healthy donors, and it uses real-time PCR to detect full-length HIV RNA several days earlier than could be detected in the original assay using an ELISA for HIV p24. In Chapter 5, we investigate the gut-associated lymphoid tissue (GALT) as a tissue site that could serve as a barrier to HIV cure. We use aspects of the streamlined QVOA described in Chapter 4 to measure the size of the latent reservoir in circulating resting CD4+ T cells and in cells from the GALT of patients on suppressive ART; additionally, we use phylogenetic analysis to investigate whether GALT cells could contribute to residual viremia in these same patients. Although we detected replication- competent HIV in the circulating resting CD4+ T cells from most study participants, we detected replication-competent HIV in the GALT in only one participant. Despite the infrequency of this observation, however, our phylogenetic data are consistent with the concept that HIV in the GALT could contribute to residual viremia and therefore constitute a barrier to the cure of HIV in addition to circulating resting CD4+ T cells