Understanding the Role of the Host Response in HIVSIV Infection

背景- CTLA-4阻断抗体MDX-010的短期治疗与淋巴结病毒RNA水平下降和siv特异性CD4+和CD8+ t细胞效应功能增加有关，我们对此结果感到鼓舞，我们采用这种方法并测试了长期CTLA-4阻断治疗与不接种疫苗或单独接种疫苗作为对照的效果。我们发现，长期的CTLA-4阻断显著增加了原发性SIVmac251感染中t细胞的活化和病毒复制，特别是在粘膜部位，并增加了IDO的表达和活性。因此，长期使用抗ctla -4抗体治疗慢性SIVmac251感染的RMs降低了对抗逆转录病毒治疗的反应性，并取消了治疗性t细胞疫苗降低病毒设定点的能力。这些数据提供了免疫激活驱动病毒复制的第一个直接证据，并建议在使用增加免疫激活的长期治疗方法治疗HIV感染时要谨慎。另一项研究表明，抗逆转录病毒治疗和病毒抑制足以减少siv感染猕猴淋巴组织中的死亡配体，但不能减少死亡受体。我们研究了抗逆转录病毒治疗前后纵向采集的siv感染rm血液和淋巴结中参与程序性细胞死亡的分子（TRAIL和FasL）及其受体（DR5和Fas）的mRNA表达。TRAIL、FasL、DR5和Fas在siv感染rm的循环CD4+ t细胞中的表达升高，抗逆转录病毒治疗降低了外周血中TRAIL和DR5的表达，但在同一动物的淋巴结中只有TRAIL而不是DR5。这些发现表明抗逆转录病毒治疗在降低淋巴组织中凋亡死亡受体的表达方面是无效的，并且仅限于血液白细胞的分析可能不会揭示这种缺陷。我们的研究结果强调了一种潜在的免疫状况的持久性，这种状况可能会阻止治疗诱导的淋巴组织中CD4+ t细胞的恢复。干扰素阻断-感染的早期阶段出现了病毒必须克服的巨大障碍以建立感染。了解和利用这些弱点可以提供一个机会来防止暴露宿主的生产性感染。生殖道传播的早期阶段无法在人类中进行研究，因此，生殖道内SIV感染是研究这些事件的最佳动物模型之一。我们将测试粘膜入口的早期先天IFN反应对rm中SIV感染的建立和传播率的贡献。我们将通过使用靶向所有这些干扰素受体的阻断抗体64G12治疗RMs来抑制ifn - α和ifn - β的生物活性。事实上，64G12抑制SIV-AT-2诱导的RM pDCs中ifn - α和tnf - α的产生。具体而言，我们希望测试64G12对以下方面的影响：1)RMs阴道内SIV感染的建立以及SIV向粘膜和外周LT传播的速度；2)病毒和CD4+ t细胞动力学以及SIV特异性免疫反应；3)粘膜和全身免疫激活水平以及疾病进展。感染的早期阶段存在巨大的障碍，病毒必须克服这些障碍才能建立感染。了解和利用这些弱点可以提供一个机会来防止暴露宿主的生产性感染。CQ治疗-早期的体外研究表明，CQ可以阻断HIV包膜糖基化和HIV复制。然而，两项小型临床试验报告血浆病毒仅适度减少0.4-0.6 log copies/ml。最近，CQ已被证明可以阻断hiv诱导的免疫激活，以及hiv激活的ifn - α产生和正常人类pbmc中浆细胞样树突状细胞（pDCs）的PDL-1表达。还应注意的是，最近有报道称CQ可增强星形胶质细胞对HIV感染的容错性，这增加了该药物并发症和有害作用的可能性。我们建议研究CQ给药对小鼠SIV感染的体内影响。抗PD-1治疗-我们发现SIV特异性t细胞具有增殖缺陷，这可能解释了疫苗无法消除感染细胞的原因。SIV特异性CD8+ t细胞表达高水平的PD-1和细胞因子，并且在急性和慢性SIVmac251感染中增殖能力受损。程序性死亡-1 （PD-1）是病毒特异性CD8+ t细胞衰竭的关键介质。我们检测了PD-1在siv特异性CD8+ t细胞上的表达及其可能参与细胞因子产生、增殖和这些细胞存活的调节。我们发现，在所有测试的组织中，大多数siv特异性CD8+ t细胞表达PD-1（高）表型，与它们的分化状态无关。PD-1在siv衍生表位发生突变逃逸的CD8+ t细胞上的表达逐渐下降，表明抗原特异性TCR刺激是PD-1表达的主要决定因素。siv特异性PD-1（高）CD8+ t细胞在同源肽刺激下产生ifn - γ、tnf - α和IL-2，但与PD-1（低）siv特异性CD8+ t细胞相比，其增殖能力降低。PD-1（高）siv特异性CD8+ t细胞在体外刺激后对细胞死亡非常敏感，导致细胞丢失。因此，PD-1可能对维持CD8+ t细胞对HIV/SIV的有效应答产生负面影响，操纵PD-1与其配体的相互作用可能导致SIV感染中t细胞应答的恢复。IL-21治疗-我们详细研究了SIV感染对受感染猕猴粘膜部位的免疫损伤，发现IL-17产生CD4+ t细胞（Th17），最近发现的一种效应CD4+ t辅助细胞谱系，在体外和体内被SIVmac251感染，并且在感染的几周内，其在粘膜和全身部位的频率显著降低。在进展为疾病的动物中，与Th17细胞相比，Th1细胞的比例过高，而在sivmac251感染的精英对照RMs中，Th17/Th1的正常平衡得以维持。事实上，粘膜部位Th17细胞的频率与血浆病毒水平的回归分析显示出负相关，表明它们在HIV/SIV发病机制中的重要性。由于Th17细胞在对细胞外细菌的先天和适应性免疫反应中起着核心作用，我们的发现可能解释了HIV感染中的慢性肠病。因此，重建Th1和Th17之间的适当平衡的治疗方法，如IL-21治疗，可能被认为可以改善HIV感染的临床过程。

Background- We were encouraged by the results that a short treatment with the CTLA-4 blocking antibody MDX-010 was associated with decreased viral RNA levels in lymph nodes and an increase in the effector function of both SIV-specific CD4+ and CD8+ T-cells, we pursued this approach and tested the efficacy of protracted CTLA-4 blockade treatment with and without vaccination or vaccination alone as a control. We found that protracted CTLA-4 blockade significantly increased T-cell activation and viral replication in primary SIVmac251 infection, particularly at mucosal sites, and increased IDO expression and activity. Accordingly, protracted treatment with anti-CTLA-4 antibody of RMs chronically infected with SIVmac251 decreased responsiveness to antiretroviral therapy and abrogated the ability of therapeutic T-cell vaccines to decrease viral set point. These data provide the first direct evidence that immune activation drives viral replication and suggest caution in the use of long term therapeutic approaches that increase immune activation in the treatment of HIV infection. Another study showed that antiretroviral therapy treatment and virus suppression is sufficient to reduce death ligand but not death receptors in lymphoid tissue of SIV-infected macaques. We studied mRNA expression of molecules involved in programmed cell death (TRAIL and FasL) and their receptors (DR5 and Fas) in blood and lymph nodes collected longitudinally from SIV-infected RMs before and after antiretroviral therapy. TRAIL, FasL, DR5, and Fas expression were elevated in circulating CD4+ T-cells from SIV-infected RMs and antiretroviral therapy reduced both TRAIL and DR5 expression in peripheral blood, but only TRAIL and not DR5, in lymph nodes from the same animals. These findings suggest that antiretroviral therapy is ineffective in reducing expression of apoptotic death receptors in lymphoid tissue and that analysis limited to blood leukocytes may not reveal such a defect. Our results highlight the persistence of an underlying immunologic condition that may prevent therapy-induced restoration of CD4+ T-cells in lymphoid tissues. Interferon alfa Blockade- The early stages of infection present formidable obstacles that the virus must overcome to establish infection. Understanding and exploiting these weaknesses may provide a chance to prevent productive infection in an exposed host. The early stages of genital transmission can not be studied in humans, thus, intra-vaginal SIV infection of RMs represents one of the best animal models in which to study these events. We will test the contribution of the early innate IFN response at the mucosal portal of entry to the establishment and rate of dissemination of SIV infection in RMs. We will inhibit the biological activity of IFN-alpha and IFN-beta by treating RMs with a blocking antibody, 64G12, that targets the receptor for all these interferons. Indeed 64G12 inhibits SIV-AT-2 induced IFN-alpha and TNF-alpha production in pDCs of RM. Specifically, we wish to test the effect of 64G12 on 1) The establishment of intravaginal SIV infection in RMs and the rate of SIV dissemination to mucosal and peripheral LT, 2) viral and CD4+ T-cell dynamics and the SIV-specific immune responses, and 3) the levels of mucosal and systemic immune activation, and disease progression. The early stages of infection present formidable obstacles that the virus must overcome to establish infection. Understanding and exploiting these weaknesses may provide a chance to prevent productive infection in an exposed host. CQ treatment- Earlier in vitro studies indicated that CQ can block HIV envelope glycosylation and HIV replication. However, two small clinical trials reported only modest reductions in plasma virus of 0.4-0.6 log copies/ml. More recently, CQ has been shown to block HIV-induced immune activation as well as HIV-activated IFN-alpha production and PDL-1 expression by plasmacytoid dendritic cells (pDCs) in normal human PBMCs. It should also be noted that CQ was recently reported to enhance astrocyte permissiveness to HIV infection, raising the possibility of complicating and detrimental effects of this drug. We propose to study the in vivo effects of CQ administration on SIV infection of RMs. Anti PD-1 treatment- We found that SIV specific T-cells have a proliferative defect that may explain the inability of vaccines to eliminate infected cells. SIV specific CD8+T-cells express high level of PD-1 and cytokines, and have impaired proliferative capacity in acute and chronic SIVmac251 infection. Programmed Death-1 (PD-1) is a critical mediator of virus-specific CD8+ T-cell exhaustion. We examined the expression of PD-1 on SIV-specific CD8+ T-cells and its possible involvement in the regulation of cytokine production, proliferation, and survival of these cells. We found that the majority of SIV-specific CD8+ T-cells expressed a PD-1(high) phenotype, independently of their differentiation status, in all tissues tested. PD-1 expression gradually declined on CD8+ T-cells specific for SIV-derived epitopes that had undergone mutational escape, indicating that antigen-specific TCR stimulation is the primary determinant of PD-1 expression. SIV-specific PD-1(high) CD8+ T-cells produced IFN-gamma, TNF-alpha, and IL-2 under cognate peptide stimulation but had a reduced proliferative capacity when compared to PD-1 (low) SIV-specific CD8+ T-cells. PD-1(high) SIV-specific CD8+ T-cells were highly susceptible to cell death leading to loss of such cells after in vitro stimulation. Thus, PD-1 may negatively affect the maintenance of effective CD8+ T-cell responses to HIV/SIV, and manipulation of the interaction of PD-1 with its ligands could result in restoration of the T-cell responses in SIV infection. IL-21 treatment- We have studied in detail the immunological damage induced by SIV infection at mucosal sites of infected macaques and found that IL-17 producing CD4+T-cells (Th17), a lineage of effector CD4+ T-helpers recently identified, are infected by SIVmac251 in vitro and in vivo, and their frequency significantly decreases at mucosal and systemic sites within a few weeks of infection. In animals that progress to disease, Th1 cells are over-represented compared to Th17 cells whereas, in SIVmac251-infected elite controller RMs, a normal Th17/Th1 balance is maintained. Indeed, regression analysis of the frequency of Th17 cells at mucosal sites and plasma virus level demonstrates a negative correlation, suggesting their importance in HIV/SIV pathogenesis. Because Th17 cells play a central role in innate and adaptive immune responses to extracellular bacteria, our finding may explain the chronic enteropathy in HIV infection. Thus, therapeutic approaches to reconstitute an adequate balance between Th1 and Th17, like IL-21 treatment, may be considered to ameliorate the clinical course of HIV infection.