MACROPHAGES: AN OVERLOOKED LATENT VIRAL RESERVOIR IN SIV INFECTION

HIV infection remains a worldwide epidemic without prospects of a cure. While antiretroviral therapy (ART) successfully decreases plasma viral load and prevents new cells from becoming infected, long-lived latently infected cells remain in blood and tissues. CD4+ T cells have been extensively studied and characterized as a major latent reservoir; however less is known about macrophages, another cell infected by HIV. A better understanding of all the latent reservoirs is needed in order to devise strategies for eradication. HIV infects both CD4+ T cells and cells from the myeloid lineage such as blood monocytes and tissue macrophages early during infection. While most CD4+ T cells become activated and die during the infection, some become memory cells and are able to persist in the body from months to years. Recent findings suggest that macrophages behave in a similar manner and have been shown to proliferate in situ contributing to new cells. Furthermore, tissue macrophages are found throughout the body in organs such as lungs, spleen, and brain among others. Infection of macrophages in these tissues has not been characterized nor the frequency of these cells compared to infected resting CD4+ T cells. Therefore we developed methods, which quantitated and compared both major cellular reservoirs (CD4+ T cells and macrophages). CD4+ T cells from the blood and spleen were assayed using the quantitative viral outgrowth assay (QVOA) while monocytes in the blood and macrophages in the bronchoalveolar lavage fluid, lungs, spleen and brain of SIV-infected pigtailed macaques were assayed using a similar assay, the macrophage quantitative viral outgrowth assay (MΦ-QVOA). The era of ART led to a decline in the frequency of HIV associated pathologies such as HIV-associated neurocognitive disorders (HAND). However, mild to moderate forms of HAND are still observed in viral controlled patients on successful ART. Markers of inflammation in the CSF are often reported in ART-treated patients showing signs of neurological impairment; therefore, low levels of macrophage activation and viral production in the brain are presumed to contribute to HAND. A debate still exists as to whether macrophages fit the definition of a latent reservoir. Replication-competent virus has not been recovered from latently infected macrophages in SIV or HIV-infected individuals on long-term suppression. Therefore, we used our MΦ-QVOA assay to quantitate the frequency of infection of macrophages in brain of SIV-infected ART-treated macaques. Despite long-term ART treatment of SIV-infected macaques, brain macrophages were shown to contain replication competent virus demonstrating for the first time latently infected microglia. In addition, markers of macrophage activation and neuronal damage were detected in the CSF of the ART suppressed macaques, suggesting in part that latently infected macrophages contribute to CNS inflammation. In situ hybridization from two brain regions confirmed the presence of rare SIV RNA positive cells but underestimated the size of the reservoir, as did tissue RNA and DNA measurements. The virus obtained in the MΦ-QVOA cultures caused productive infection in peripheral blood mononuclear cells de novo, thus demonstrating that the macrophage reservoir contained functional viral genomes and could spread from the brain to the periphery upon ART interruption. Finally, monocytes in the blood and macrophages in the bronchoalveolar lavage fluid, lungs, and spleen were assayed using the MΦ-QVOA and compared to the frequency of latently infected resting CD4+ T cells in the blood and spleen of SIV-infected ART-treated pigtailed macaques. Our findings confirmed that macrophages are latently infected in these peripheral tissues and the size of the reservoir was equivalent to the resting CD4+ T cell reservoir. The data presented here demonstrated that macrophages are yet another obstacle to HIV eradication, and suggest that macrophages contribute to the ongoing viral replication in tissues, thus playing a pivotal role in long-term tissue inflammation. Thesis Advisor: Janice Clements, Ph.D. Thesis Reader: Alan Scott, Ph.D