Although highly active anti-retroviral therapy (HAART) has dramatically improved the longevity and quality of life for HIV-infected individuals, with few exceptions, it has been unable to permanently eliminate the infection. HIV persistence and latency remain formidable challenges, and many aspects regarding how these conditions are established remain puzzling. Of obvious importance is accurate identification of all host cells and tissues that can serve as long-term reservoirs for the virus during HAART. Tissue macrophages are long-lived cells, capable of supporting HIV replication, and likely to be significant contributors to HIV persistence. Most studies of the macrophage reservoir have focused on brain. Bone marrow (BM), which is known to harbor infectious HIV, contains a sizeable population of macrophages and yet, almost no published investigations addressing HIV infection in marrow have included examination of these cells. Rather, CD34+ hematopoietic progenitor cells have typically been the focus. Our studies of HIV protease and reverse transcriptase sequences from infected individuals on HAART indicate that BM can serve as a long-term quiescent reservoir. Based on these and other data, we hypothesize that macrophages are the predominant host cells for HIV infection and replication in BM. We propose here, to test this hypothesis using BM core biopsies, and aspirates, collected ante-mortem, from well-characterized infected individuals. This collection is composed primarily of specimens from patients on HAART. Infection in BM macrophages and CD4+ T-cells will be examined, with emphasis on quantitative comparison. (Studies pertaining to infection in CD34+ progenitor cells have been deleted from this revised application, as per reviewers' suggestions.) The type(s) of cells expressing HIV p24 will be determined using double-label immunostaining of core biopsies, specimens that are free of blood contamination that can compromise results pertaining to T-cells. In addition, levels of HIV DNA present within each cell type will be determined and compared using cells purified from cryopreserved BM aspirates collected concomitantly from the same patients. Also included will be experiments to determine if BM macrophages and T-cells infected in vivo can produce infectious, transmissible HIV. Lastly, to definitively identify which subpopulations of macrophage lineage cells in BM are susceptible to HIV entry leading to productive infection, the longevity and kinetics of virus expression in these cells, and whether this infection can be inhibited by antiretroviral drugs, we will perform in vitr infection experiments using macrophages cultured from normal donor BM, and a replication-competent GFP+ CCR5-tropic strain of HIV. Lifelong HIV persistence continues to represent a major challenge to maintaining the health of infected individuals. Precise identification and characterization of all cell and tissue reservoirs that promote HIV persistence and latency is critical for the development of new therapeutic strategies directed towards complete eradication of the virus from the body. The research proposed here will determine if BM macrophages are significant participants in maintaining this persistence.
Although HIV typically persists indefinitely within the body, even in the presence of uninterrupted long-term highly active antiretroviral therapy (HAART), how this persistence develops and is maintained remains a mystery and a significant public health concern. Bone marrow appears to be an important site for HIV persistence, but there is considerable controversy regarding the type(s) of cells infected and moreover, macrophages, which are resident cells in bone marrow and known to be hosts for HIV infection, have scarcely been investigated. This application will (1) quantitatively compare levels of HIV infection in macrophages and T-cells using bone marrow specimens from infected individuals on HAART, and (2) examine mechanisms of HIV persistence in bone marrow macrophages.
