A Compartmental Analysis of HIV Reservoirs and Immune Reconstitution

描述（由申请人提供）：认识到目前的治疗方法不可能根除HIV-1，这就提出了关于HIV发病机制的两个基本问题：病毒如何以及在哪里维持自己作为终身持续感染？我们的初步数据表明，淋巴组织（LT），即肠道相关淋巴组织（GALT）和继发淋巴结（LN）是HIV持续存在的主要部位。我们发现，在HIV感染后不久，GALT和LN中的CD4+细胞迅速耗尽，抗逆转录病毒治疗（ARV）的CD4重构很差。这些患者血浆中HIV RNA < 400或50拷贝。ml，我们还发现：在GALT和LN中有2次LTR发作（近期HIV复制的标志），GALT中HIV DNA+细胞的频率增加，药物对LN和GALT的渗透受损，GALT中中央记忆CD4细胞的消耗可能与慢性潜伏粘膜病原体如单纯疱疹2 （HSV2）的再激活频率相关。这些数据表明，LT中的隐性HIV复制是一个潜在的重要储库，并为3个相互关联的假设提供了基础：1)LT中发生隐性复制，并在治疗中断时引发病毒复发；2)这是GALT和LN的CD4细胞中药物浓度不足的结果；3)它最终破坏GALT和LN的免疫功能。测试这些假设及其推论的关键预测和关键测量是确定GALT和LN中与活性药物水平浓度相关的隐性HIV复制程度，并评估GALT和LN中持续复制对免疫功能的影响。Stevenson博士将从所有隔室中分离病毒和外泌体DNA，以确定LT储存库的存在，并进行序列分析，以确定治疗中断后出现在PB中的病毒与LT中隐性储存库的关系。Haase博士将使用原位杂交、激光捕获显微解剖和原位per来确定每个隔室中持续感染储存库的大小和位置。Fletcher博士将确定GALT和LN CD4+细胞中的ARV水平，寻找导致细胞内药物浓度降低的外排机制的证据，并估计完全抑制隐性复制所需的LT药物浓度。Schacker博士将确定每个隔室中CD4亚群的绝对大小，以及隐性复制水平、GALT中CD4耗竭和粘膜病原体粘膜再激活测量之间的关系。Douek博士将进行流式细胞术，以测量抗原特异性反应和细胞分离，并在治疗中断之前和期间从每个隔室获得CD4细胞培养病毒。如果我们的假设是正确的，它将为改进治疗指明道路，其影响将通过改善对病毒复制的控制和相关LT储存库中免疫功能的保存来衡量。

DESCRIPTION (provided by applicant): The realization that HIV-1 eradication is impossible with current treatment raises two fundamental questions about HIV pathogenesis: how and where does the virus sustain itself as a life-long persistent infection? Our preliminary data suggest lymphoid tissues (LT), i.e., gut associated lymphoid tissue (GALT) and secondary lymph nodes (LN), are a major site of HIV persistence. We found that CD4+ cells are rapidly depleted in GALT and LN soon after HIV acquisition and there is poor CD4 reconstitution with antiretroviral therapy (ARV). In these patients with HIV RNA in plasma < 400 or 50 copies.ml, we have also found: 2 LTR episomes (a marker for recent HIV replication) in GALT and LN, frequencies of HIV DNA+ cells in GALT increase, drug penetration into LN and GALT is impaired, and depletion of central memory CD4 cells in GALT may correlate to frequency of reactivation of chronic latent mucosal pathogens like herpes simplex 2 (HSV2). These data suggest this cryptic HIV replication in LT is a potential reservoir of importance and provide the basis for 3 inter-related hypotheses: 1) cryptic replication occurs in LT and fuels viral recrudescence with treatment interruption, 2) it is a consequence of inadequate concentration of drug in CD4 cells of GALT and LN, and 3) it eventually undermines immune function in GALT and LN. The critical predictions and thus key measurements to test these hypotheses and their corollaries are to determine the extent of cryptic HIV replication in GALT and LN in relation to concentrations of active drug levels and assess the impact of ongoing replication on immune function in GALT and LN. Dr. Stevenson will isolate viral and episomal DNA from all compartments to establish the existence of a LT reservoir and perform sequence analysis to determine relatedness of virus appearing in PB after treatment interruption to the cryptic reservoir in LT. Dr. Haase will use in situ hybridization, laser capture microdissection, and in situ per to establish the size and location of persistent reservoirs of infection in each compartment. Dr. Fletcher will determine ARV levels in GALT and LN CD4+ cells, look for evidence of efflux mechanisms causing decreased intracellular concentration of drug, and estimate the concentration of drug in LT required to fully suppress cryptic replication. Dr. Schacker will determine the absolute size of CD4 subpopulations in each compartment and relationships between levels of cryptic replication, CD4 depletion in GALT, and measures of mucosal reactivation of mucosal pathogens. Dr. Douek will perform flow cytometry to measure antigen specific responses and cell separation and culture virus from CD4 cells obtained from each compartment before and during treatment interruption. If our hypotheses are correct it will point the way toward improved therapies whose impact will be measured by improved control of viral replication and preservation of immune function in the relevant LT reservoirs. PROJECT 1: Virological Measurements of Cryptic Replication (Stevenson, M) PROJECT 1 DESCRIPTION (provided by applicant): Highly active antiretroviral therapy (HAART) is able to sustain suppression of viral replication in HIV-1 infected individuals. Nevertheless, viral replication rapidly rebounds if therapy is interrupted. The prevailing view is that long-lived, quiescent memory CD4+T lymphocytes contain latent proviruses which can rekindle viral replication if therapy is interrupted. However, we have evidence that HIV-1 replication persists in infected individuals on HAART and that episomal viral cDNA can be detected in the majority of patients on HAART. We demonstrate that episomal cDNAs are unstable in vitro and dynamic in vivo and, as such, are indicators of ongoing replication. The presence of episomal cDNA in gut associated lymphoid tissue (GALT) and lymph node (LN) of patients on HAART implicates lymphoid tissue (LT) as a reservoir for HIV-1 persistence in the face of therapy. This application is built on the following hypotheses: Hypothesis 1: HIV-1 replication persists in lymphoid tissue in patients on HAART and this reservoir of cryptic replication fuels viral recrudescence upon therapy interruption. Hypothesis 2: Cryptic replication is a consequence of subinhibitory drug concentrations in GALT and LN CD4+cells. Hypothesis 3: Cryptic replication compromises immune function in GALT and LN of patients on HAART. To investigate these hypotheses, we propose to: Aim 1: Determine whether covert replication persists in lymphoid tissue in the face of HAART and whether this replication fuels viremia upon treatment interruption. Viral envelope sequences within unintegrated (episomal) and proviral cDNA in GALT and LN CD4+ T cells and macrophages will be cloned and sequenced. Phylogenetic comparison of these envelope sequences with analogous sequences in recrudescing viral RNA following treatment interruption will be used to establish whether GALT and LN serve as reservoirs of cryptic replication in patients on HAART. Aim 2: Define how cryptic viral replication persists in the face of HAART and whether cryptic replication undermines immune function in patients on HAART. We will determine whether a correlation exists between the level of cryptic viral replication and concentrations of drug in lymphoid tissue of patients on HAART and whether the extent of cryptic viral replication correlates with the magnitude of the immune defect (CD4 cell number and HSV2 shed rates) in lymphoid tissues in patients on HAART. We believe that these studies will identify the mechanism of viral persistence during HAART and provide the rationale for new treatment strategies to more effectively truncate ongoing viral replication.