Immunologic and Virologic Strategies Directed Toward HIV

在人类免疫缺陷病毒（HIV）-1感染的个体中使用高效抗逆转录病毒疗法（HAART）显著改变了许多患者的临床结果，并导致获得性免疫缺陷综合征（AIDS）的发病率和AIDS相关死亡率的大幅下降。然而，现在很清楚，长期抑制血浆病毒血症的HAART是不可能根除艾滋病毒在大多数感染者。此外，长期HAART可能导致药物诱导的毒性，难以坚持药物治疗方案，并可能产生耐药病毒。总之，迫切需要开发旨在有效抑制感染个体中HIV的替代免疫学和病毒学策略。在过去一年，我们启动了三个基础和临床研究项目，以实现上述目标。 1.从HIV感染的长期非进展者中鉴定CD 8 + T细胞相关的抗病毒因子 对HIV感染者产生的免疫应答的研究表明，CD 8 + T细胞在控制病毒复制方面发挥重要作用。至少有两种类型的CD 8 + T细胞介导的抗病毒活性已被描述在HIV感染。第一种是对HIV的抑制活性，涉及以抗原特异性、HLA限制性方式裂解感染细胞，而第二种机制在不存在细胞杀伤的情况下抑制病毒复制。先前的研究表明，这些因素包括CC-趋化因子。尽管这些CC-趋化因子对HIV结合和最终进入CD 4 + T细胞和巨噬细胞发挥其抗病毒活性，但另一类目前未鉴定的抗病毒因子已显示通过下调HIV在感染细胞中的转录来抑制进入后病毒复制，并在感染的CD 4 + T细胞和CD 8 + T细胞之间的共培养环境中发挥最大水平的病毒抑制。我们已经从三个HIV感染的长期非进展者的新鲜分离的CD 8 + T细胞构建了cDNA文库，并且已经开始筛选过程以鉴定通过细胞与细胞接触参与抑制HIV的基因。在鉴定出这些抗病毒候选物后，我们计划在体外表征这些分子的生物学特性，然后设计临床策略以在体内利用这些CD 8 + T细胞衍生的抗病毒因子。 2.免疫抑制药物在HIV复制中的作用。 尽管开发了成功的治疗策略，但仍不可能根除感染个体中的HIV，主要是由于各种病毒储库的持续存在。特别是，在血浆病毒血症低于检测限的大多数感染个体中，已证实CD 4 + T细胞中存在有复制能力的病毒、HIV-1前病毒DNA（包括2个LTR环）、剪接和未剪接的HIV-1 RNA。这种持久的病毒储存库已成为阻止根除艾滋病毒的主要障碍。此外，我们最近的工作表明，在接受HAART治疗的病毒血症感染个体中，携带HIV-1前病毒DNA的CD 4 + T细胞频率与CD 4 +/CD 8 + T细胞比率之间存在统计学显著的负相关性，其中血浆病毒血症已被抑制至检测限以下较长时间（>2.5年）。上述观察结果表明，旨在最大限度地减少细胞活化的策略可能会进一步减少接受HAART的患者中残留的病毒复制。为了解决这个问题，我们已经开始了一项试点临床试验，以检查轻度免疫抑制剂daclizumab的安全性和耐受性。达克珠单抗是一种人源化IgG 1单克隆抗体，可与活化淋巴细胞表面表达的人高亲和力IL-2受体的α亚基（p55 α、CD 25或Tac亚基）特异性结合。考虑到免疫激活程度与HIV复制之间存在密切关系，我们希望证明daclizumab可以使免疫学特征正常化，并减少研究受试者的血浆病毒血症。 3.使用抗CCR 5单克隆抗体治疗恒河猴中的SIV感染。 目前抗逆转录病毒疗法的成功受到了艾滋病毒抗药性的出现、必须持续坚持复杂的治疗方案以及药物引起的副作用的可能性的阻碍。因此，需要开发新的安全有效的治疗HIV/AIDS的药物。靶向HIV进入CD 4 + T细胞的抗病毒药物为这一领域带来了希望。第一个被批准用于治疗HIV/AIDS的HIV进入抑制剂是恩夫韦肽，一种36个氨基酸的肽，其干扰病毒包膜蛋白与细胞膜的融合。此外，趋化因子受体CCR 5是大多数HIV毒株进入HIV的共受体，是临床开发中的几种小分子抑制剂的靶点。开发一种安全、耐受性良好、具有有效抗病毒活性的针对CCR 5的完全人源化单克隆抗体将进一步增强治疗选择。这样的HIV抑制剂可以提供额外的优点，包括减少的药物-药物相互作用、改善的耐药性特征以及由于抗体分子的血浆半衰期延长而在感染患者中不频繁施用的便利性。为了检查这种抗病毒分子的功效，我们开始了一项动物研究，其中将两种类型的抗CCR 5单克隆抗体沿着同种型对照注射到10只恒河猴中。我们希望证明抗CCR 5抗体可以在感染动物中有效地阻断病毒复制很长一段时间，并在不久的将来在HIV感染者中探索这种策略。

The use of highly active antiretroviral therapy (HAART) in human immunodeficiency virus (HIV)-1 infected individuals has dramatically changed the clinical outcome in many patients and has led to a substantial decline in the incidence of acquired immunodeficiency syndrome (AIDS) and in AIDS-related mortality. However, it is now clear that prolonged suppression of plasma viremia by HAART is not likely to eradicate HIV in most infected individuals. In addition, long-term HAART may lead to drug-induced toxicities, difficulties in adhering to drug regimens, and potential to develop drug resistant virus. Taken together, development of alternative immunologic and virologic strategies aimed at efficient suppression of HIV in infected individuals is urgently needed. Over the past year, we have initiated three basic and clinical research projects to address the above goal. 1. Identification of CD8+ T cell-associated antiviral factors from HIV-infected long-term non-progressors. Studies of the immune responses generated in HIV-infected individuals suggest that CD8+ T cells play an important role in controlling viral replication. At least two types of CD8+ T cell-mediated antiviral activities have been described in HIV infection. The first is a suppressive activity against HIV involving lysis of infected cells in an antigen-specific, HLA-restricted fashion, while the second mechanism inhibits viral replication in the absence of cell killing. Previous studies have demonstrated that these factors include CC-chemokines. Whereas these CC-chemokines exert their antiviral activity on the binding and ultimate entry of HIV into CD4+ T cells and macrophages, another class of currently unidentified antiviral factor (s) has been shown to suppress post-entry viral replication by down-regulating transcription of HIV in infected cells and exerts maximal levels of viral suppression in the setting of co-culture between infected CD4+ T cells and CD8+ T cells. We have constructed a cDNA library from freshly isolated CD8+ T cells from three HIV-infected long-term non-progressors and have begun the screening process to identify genes involved in inhibition of HIV via cell-to-cell contact. Upon identification of such antiviral candidates, we plan to characterize biological properties of such molecules in vitro followed by design of a clinical strategy to utilize these CD8+ T cell-derived antiviral factors in vivo. 2. Role of an immunosuppressive drug in HIV replication. Despite the development of successful therapeutic strategies, it has not been possible to eradicate HIV in infected individuals, mainly due to the persistence of various viral reservoirs. In particular, the presence of replication-competent virus, HIV-1 proviral DNA, including 2 LTR circles, spliced and unspliced HIV-1 RNA in CD4+ T cells has been demonstrated in the majority of infected individuals in whom plasma viremia has fallen below the limit of detection. This persistent viral reservoir has emerged as the major obstacle in preventing eradication of HIV. In addition, our recent work has shown a statistically significant inverse correlation between the frequency of CD4+ T cells carrying HIV-1 proviral DNA and the CD4+/CD8+ T cell ratios in aviremic infected individuals receiving HAART and in whom plasma viremia had been suppressed below the limit of detection for prolonged periods of time (>2.5 years). The above observations suggest that strategies aimed at minimizing cellular activation may further diminish residual viral replication in patients receiving HAART. In order to address this question, we have begun a pilot clinical trial to examine the safety and tolerability of a mildly immunosuppressive agent, daclizumab. Daclizumab is a humanized IgG1 monoclonal antibody that binds specifically to the alpha subunit (p55 alpha, CD25, or Tac subunit) of the human high-affinity IL-2 receptor expressed on the surface of activated lymphocytes. Considering the existence of an intimate relationship between the degree of immune activation and HIV replication, we hope to demonstrate that daclizumab can normalize immunologic profiles as well as reduce plasma viremia in study subjects. 3. Use of anti-CCR5 monoclonal antibodies to treat SIV infection in rhesus monkeys. The success of current antiretroviral therapies has been hampered by the emergence of drug resistant HIV, the necessity of sustained adherence to complex therapeutic regimens, and the potential for drug-induced side effects. Therefore, there is a need to develop novel classes of safe and effective agents for the treatment of HIV/AIDS. Antiviral agents that target the entry of HIV into CD4+ T cells offer hope in this area. The first HIV entry inhibitor that was approved for treatment of HIV/AIDS is Enfuvirtide, a 36-amino acid peptide that interferes with the fusion of viral Envelope protein with cellular membrane. In addition, the chemokine receptor CCR5, a co-receptor for HIV entry for the majority of HIV strains, is a target for several small molecule inhibitors that are in clinical development. The development of a fully humanized monoclonal antibody against CCR5 that is safe, well tolerated, and has potent antiviral activity will further enhance therapeutic options. Such an inhibitor of HIV may provide additional advantages including reduced drug-drug interactions, improved resistance profile, and the convenience of infrequent administration in infected patients due to prolonged plasma half-life of antibody molecules. In order to examine the efficacy of such antiviral molecules, we initiated an animal study in which two types of anti-CCR5 monoclonal antibodies along with an isotype control were injected into 10 rhesus macaques. We hope to demonstrate that anti-CCR5 antibodies can effectively block viral replication for extended periods of time in infected animals and to explore such a strategy in HIV-infected individuals in the near future.