HIV Infection in Bone Marrow: A Reservoir for Viral Persistence

描述（由申请人提供）：尽管高效抗逆转录病毒疗法（HAART）极大地改善了HIV感染者的寿命和生活质量，但除少数例外，它无法永久消除感染。艾滋病毒的持久性和潜伏性仍然是巨大的挑战，关于这些条件是如何形成的许多方面仍然令人困惑。显然，重要的是准确鉴定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的细胞，能够支持HIV复制，并且可能是HIV持续存在的重要贡献者。大多数关于巨噬细胞储库的研究都集中在大脑。骨髓（BM），这是已知的窝藏传染性艾滋病毒，含有相当大的群体的巨噬细胞，然而，几乎没有发表的研究解决艾滋病毒感染的骨髓已包括检查这些细胞。相反，CD34+造血祖细胞通常是焦点。我们对HAART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水库。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。我们建议在这里，以测试这一假设使用骨髓芯活检，和吸出物，收集死前，从良好的特点感染的个人。该标本集主要由HAART患者的标本组成。将检查BM巨噬细胞和CD4+ T细胞中的感染，重点是定量比较。（根据评审员的建议，与CD34+祖细胞感染相关的研究已从本修订申请中删除。）表达HIV p24的细胞类型将使用核心活检组织的双标记免疫染色来确定，样本不含可能影响T细胞相关结果的血液污染。此外，将使用从同一患者同时采集的冻存BM抽吸物中纯化的细胞，测定并比较每种细胞类型中存在的HIV DNA水平。还包括确定体内感染的BM巨噬细胞和T细胞是否可以产生传染性、可传播的HIV的实验。最后，为了明确鉴定BM中巨噬细胞谱系细胞的哪些亚群对HIV进入敏感，从而导致生产性感染，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+ CCR 5嗜性HIV株进行体外感染实验。艾滋病毒的终身持续性仍然是维持受感染者健康的一个重大挑战。精确鉴定和表征促进HIV持续存在和潜伏的所有细胞和组织储库对于开发旨在从体内完全根除病毒的新治疗策略至关重要。这里提出的研究将确定BM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.