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。

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.