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，这提出了关于HIV发病机制的两个基本问题：病毒如何以及在何处维持自身作为终身持续感染？我们的初步数据表明淋巴组织（LT），即，肠相关淋巴组织（GALT）和次级淋巴结（LN）是HIV持续存在的主要部位。我们发现，CD 4+细胞迅速耗尽GALT和LN后不久，艾滋病毒收购和有穷人的CD 4重建与抗逆转录病毒治疗（ARV）。在这些血浆中HIV RNA < 400或50的患者中copies.ml，我们还发现：GALT和LN中的2 LTR游离体（近期HIV复制的标志物），GALT中HIV DNA+细胞的频率增加，药物渗透到LN和GALT中受损，GALT中中央记忆CD 4细胞的耗竭可能与慢性潜伏性粘膜病原体如单纯疱疹2型（HSV 2）的再活化频率相关。这些数据表明LT中的这种隐蔽HIV复制是重要的潜在储存库，并为3个相互关联的假设提供了基础：1）LT中发生隐蔽复制，并在治疗中断时促进病毒复发，2）这是GALT和LN的CD 4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。关键的预测，因此关键的测量，以测试这些假设及其推论是，以确定隐蔽的HIV复制在GALT和LN的活性药物水平的浓度相关的程度，并评估正在进行的复制对免疫功能的影响GALT和LN。Dr.史蒂文森将从所有隔室中分离病毒和附加体DNA，以确定LT储库的存在，并进行序列分析，以确定治疗中断后PB中出现的病毒与LT中隐蔽储库的相关性。Dr. Haase将使用原位杂交、激光捕获显微切割和原位per确定每个隔室中持续感染储库的大小和位置。弗莱彻博士将测定GALT和LN CD 4+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，并估计完全抑制隐蔽复制所需的LT中药物浓度。Schacker博士将确定每个隔室中CD 4亚群的绝对大小，以及隐蔽复制水平、GALT中CD 4耗竭和粘膜病原体粘膜再活化指标之间的关系。Dr. Douek将进行流式细胞术，以测量抗原特异性应答和细胞分离，并在治疗中断前和治疗中断期间从每个隔室获得的CD 4细胞中培养病毒。如果我们的假设是正确的，它将指向改进的治疗方法，其影响将通过改善对病毒复制的控制和保护相关LT储库中的免疫功能来衡量。 项目1：隐蔽复制的病毒学测量 （史蒂文森，男） 项目1描述（由申请人提供）：高效抗逆转录病毒疗法（HAART）能够持续抑制HIV-1感染者的病毒复制。然而，如果治疗中断，病毒复制迅速反弹。流行的观点是，长寿命的静止记忆CD 4 +T淋巴细胞含有潜伏的前病毒，如果治疗中断，它可以重新点燃病毒复制。然而，我们有证据表明，HIV-1复制持续存在于HAART感染的个体中，并且在大多数HAART患者中可以检测到附加型病毒cDNA。我们证明了游离型cDNA在体外是不稳定的，在体内是动态的，因此，是正在进行的复制的指标。HAART患者的肠相关淋巴组织（GALT）和淋巴结（LN）中存在游离型cDNA，这表明淋巴组织（LT）是HIV-1在治疗过程中持续存在的储存库。假设1：HIV-1复制持续存在于HAART患者的淋巴组织中，这种隐蔽复制的储存库在治疗中断时促进病毒复发。假设2：隐蔽复制是GALT和LN CD 4+细胞中药物浓度低于抑制浓度的结果。假设3：隐性复制损害HAART患者GALT和LN的免疫功能。为了研究这些假设，我们建议：目的1：确定是否隐藏复制持续在淋巴组织中面对HAART和是否这种复制燃料病毒血症治疗中断。将对GALT和LN CD 4 + T细胞和巨噬细胞中未整合（游离型）和前病毒cDNA内的病毒包膜序列进行克隆和测序。这些包膜序列与治疗中断后复发病毒RNA中类似序列的系统发育比较将用于确定GALT和LN是否作为HAART患者中隐蔽复制的储存库。目标二：定义隐藏的病毒复制如何在HAART面前持续存在，以及隐藏的复制是否会破坏HAART患者的免疫功能。我们将确定HAART患者淋巴组织中隐匿病毒复制水平和药物浓度之间是否存在相关性，以及HAART患者淋巴组织中隐匿病毒复制程度是否与免疫缺陷程度（CD 4细胞数量和HSV 2脱落率）相关。我们相信这些研究将确定HAART期间病毒持续存在的机制，并为新的治疗策略提供理论基础，以更有效地截断正在进行的病毒复制。

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.