HIV Infection in Bone Marrow: A Reservoir for Viral Persistence

描述(申请人提供)：尽管高效抗逆转录病毒疗法(HAART)极大地改善了艾滋病毒感染者的寿命和生活质量，但几乎没有例外，但它无法永久消除感染。艾滋病毒的持久性和潜伏期仍然是艰巨的挑战，有关这些条件如何确立的许多方面仍然令人费解。显然，重要的是准确识别在HAART期间可作为病毒长期储存库的所有宿主细胞和组织。组织巨噬细胞是长寿细胞，能够支持艾滋病毒复制，并可能对艾滋病毒的持久性做出重大贡献。大多数关于巨噬细胞储存库的研究都集中在大脑上。众所周知，骨髓中含有传染性艾滋病毒，含有相当数量的巨噬细胞，然而，几乎没有公开发表的针对骨髓中艾滋病毒感染的研究包括对这些细胞的检查。相反，CD34+造血祖细胞通常是焦点。我们在HAART上对感染者的HIV蛋白酶和逆转录酶序列的研究表明，BM可以作为一个长期静止的储存库。基于这些和其他数据，我们假设巨噬细胞是BM中HIV感染和复制的主要宿主细胞。在这里，我们建议使用BM核心活检组织和从特征良好的感染者身上收集的吸入物来检验这一假设。该收集主要由HAART患者的标本组成。将检查骨髓巨噬细胞和CD4+T细胞的感染情况，重点是定量比较。(根据审查者的建议，与CD34+祖细胞感染有关的研究已从修订后的申请中删除。)表达HIVp24的细胞类型(S)将通过核心活检的双标记免疫染色来确定，这些样本没有血液污染，可能会影响与T细胞有关的结果。此外，每种细胞类型中存在的HIV DNA水平将被检测，并使用从同一患者同时收集的冷冻保存的骨髓抽吸物中提纯的细胞进行比较。还将进行实验，以确定体内感染的骨髓巨噬细胞和T细胞是否会产生传染性的、可传播的艾滋病毒。最后，为了明确确定骨髓中哪些巨噬细胞系细胞亚群对HIV感染易感，这些细胞中病毒表达的寿命和动力学，以及这种感染是否可以被抗逆转录病毒药物抑制，我们将使用从正常供体BM培养的巨噬细胞和具有复制能力的GFP+CCR5嗜性HIV毒株进行VITR感染实验。终生坚持艾滋病毒仍然是维持感染者健康的一项重大挑战。准确识别和确定所有促进艾滋病毒持久性和潜伏期的细胞和组织储存库的特征，对于制定旨在从体内彻底根除病毒的新治疗战略至关重要。这里提出的研究将确定骨髓巨噬细胞是否是维持这种持久性的重要参与者。

DESCRIPTION (provided by applicant): 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.