A Compartmental Analysis of HIV Reservoirs and Immune Reconstitution

描述(由申请人提供)：认识到目前的治疗不可能根除艾滋病毒-1，这引发了关于艾滋病毒发病机制的两个基本问题：病毒如何以及在哪里作为终身持续感染维持自身？我们的初步数据表明，淋巴组织(LT)，即肠道相关淋巴组织(GALT)和次级淋巴结(LN)，是HIV持续存在的主要部位。我们发现，在HIV感染后不久，CD4细胞中的GALT和LN迅速耗尽，抗逆转录病毒治疗(ARV)后的CD4重建很差。在这些血浆中HIV RNA含量为400或50拷贝·ml的患者中，我们还发现：在GALT和LN中有2个LTRepisome(最近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细胞的枯竭可能与慢性潜伏的粘膜病原体如单纯疱疹病毒(HSV2)重新激活的频率有关。这些数据表明，这种隐蔽的HIV复制在LT中是一个潜在的重要储存库，并为3个相互关联的假设提供了基础：1)隐蔽复制发生在LT中，并在治疗中断时促进病毒复发；2)它是GALT和LN的CD4细胞中药物浓度不足的结果；3)它最终破坏了GALT和LN的免疫功能。检验这些假说及其推论的关键预测和关键测量方法是确定GALT和LN中隐蔽的HIV复制程度与活性药物水平的关系，并评估正在进行的复制对GALT和LN中免疫功能的影响。史蒂文森博士将从所有隔室中分离病毒和上体DNA，以确定LT储存库的存在，并进行序列分析，以确定在LT的隐蔽储存库治疗中断后出现在PB中的病毒的相关性。哈斯博士将使用原位杂交、激光捕获显微解剖和原位PER来确定每个隔室持续感染宿主的大小和位置。Fletcher博士将测定GalT和LN CD4细胞中的ARV水平，寻找导致细胞内药物浓度下降的外排机制的证据，并估计完全抑制隐蔽复制所需的LT中的药物浓度。Schacker博士将确定每个隔室中的CD4亚群的绝对大小，以及隐蔽复制水平、GALT中的CD4耗尽水平和粘膜病原体重新激活的指标之间的关系。杜埃克博士将进行流式细胞术，以测量抗原特异性反应，并从治疗中断前和治疗期间从每个隔室获得的CD4细胞中分离和培养病毒。如果我们的假设是正确的，它将为改进治疗指明方向，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.