Immunologic Strategies Directed Toward HIV Infection

使用有效的抗逆转录病毒疗法(ART)治疗艾滋病毒感染者，极大地改变了许多患者的临床结果，并导致艾滋病发病率和艾滋病相关死亡率大幅下降。然而，现在很清楚的是，通过ART长期抑制血浆病毒血症不太可能根除大多数感染者的艾滋病毒。此外，长期的抗逆转录病毒治疗可能会导致药物毒性，难以坚持药物治疗方案，并产生耐药病毒。因此，迫切需要开发旨在有效抑制感染者体内艾滋病毒的替代免疫策略。在过去的一年里，我们进行了三个研究项目，两个基础项目和一个临床项目，以更好地了解宿主免疫系统如何控制艾滋病毒复制，并开发替代治疗策略。 HIV感染长期无进展患者CD8+T细胞相关抗病毒因子的特征及鉴定 至少有两种类型的CD8+T细胞介导的抗病毒活性在控制HIV感染中发挥着重要作用。第一种是对HIV的抑制活性，包括以抗原特异性的、HLA限制性的方式裂解受感染的细胞，而第二种机制是在包括CC趋化因子的过程中，在没有细胞杀伤的情况下抑制病毒复制。这些CC趋化因子在HIV结合并最终进入CD_4~+T细胞和巨噬细胞时发挥抗病毒活性，而另一类目前未知的抗病毒因子(S)通过下调HIV在感染细胞中的转录来抑制进入后的病毒复制。我们已经从三个HIV感染的长期非进展者中分离出CD8+T细胞，并构建了一个cDNA文库，并开始筛选参与抑制HIV复制的基因。在确定了这些候选抗病毒药物后，我们计划在体外鉴定它们的生物学特性，并在体内利用这些CD8+T细胞衍生的抗病毒因子进行临床治疗。 浆细胞样树突状细胞的抗HIV活性分析 浆细胞样树突状细胞(PDC)只占总血细胞的一小部分。然而，它们表现出非常强大的抗病毒活性，部分是通过产生大量的抗病毒化合物干扰素(干扰素)-a。已观察到艾滋病毒感染者血液中pDC数量的严重减少，在开始有效的抗病毒治疗后，pDC水平并未恢复到正常水平。以前的体外研究表明，pDC可能发挥抗HIV的作用，但一直缺乏解决患者来源的pDC抑制HIV复制的能力的工作。在过去的一年里，我们开始检测来自HIV感染者的pDC在体外抑制自体CD4+T细胞内源性病毒复制的能力。我们正在研究1)pDC抑制HIV复制的机制，2)病毒活跃复制对pDC功能的影响，3)血浆病毒血症对pDC基因差异表达的影响，4)HIV活跃复制在体外诱导pDC凋亡和坏死的水平。 免疫抑制药物在HIV复制中的作用 尽管制定了成功的治疗策略，但主要由于各种病毒库的持续存在，仍不可能根除感染者中的艾滋病毒。特别是，在大多数血浆病毒血症低于检测范围的感染者中，发现了具有复制能力的病毒、包括两个LTR环的HIV-1前病毒DNA以及CD4+T细胞中的剪接和非剪接的HIV-1RNA。这一持久的病毒库已成为阻碍根除艾滋病毒的主要障碍。此外，我们发现，在接受抗逆转录病毒治疗(血浆病毒水平长时间低于检测极限[&gt；2.5年])的无核型感染者中，携带HIV-1前病毒DNA的CD4+T细胞频率与CD4+/CD8+T细胞比率之间存在统计上的显著负相关。旨在最小化细胞激活的策略可能会进一步减少接受ART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，以检查一种温和的免疫抑制剂Daclizumab的安全性和耐受性，Daclizumab是一种人源化的IgG1单抗，专门与表达在激活的淋巴细胞表面的人类高亲和力IL-2受体的阿尔法亚单位结合。考虑到免疫激活程度与HIV复制之间的密切关系，我们的目的是确定Daclizumab是否能使研究对象的免疫学特征正常化，降低血浆病毒血症。

The use of effective antiretroviral therapy (ART) to treat HIV-infected individuals has dramatically changed the clinical outcome in many patients and has led to a substantial decline in the incidence of AIDS and in AIDS-related mortality. However, it is now clear that prolonged suppression of plasma viremia by ART is not likely to eradicate HIV in most infected individuals. In addition, long-term ART may lead to drug-induced toxicities, difficulties in adhering to drug regimens, and development of drug-resistant virus. Thus, development of alternative immunologic strategies aimed at efficient suppression of HIV in infected individuals is urgently needed. Over the past year, we conducted three research projects, two basic and one clinical, to better understand how the host immune system controls HIV replication and to develop alternative treatment strategies. Characterization and identification of CD8+ T cell-associated antiviral factors in HIV-infected long-term non-progressors At least two types of CD8+ T cell-mediated antiviral activities play an important role in controlling 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, in a process that includes 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) suppresses post-entry viral replication by down-regulating transcription of HIV in infected cells. We have constructed a cDNA library from CD8+ T cells isolated from three HIV-infected long-term non-progressors and have begun screening for genes involved in the inhibition of HIV replication. Upon identification of such antiviral candidates, we plan to characterize their biological properties in vitro and pursue a clinical strategy aimed at utilizing these CD8+ T cell-derived antiviral factors in vivo. Analysis of anti-HIV activities of plasmacytoid dendritic cells Plasmacytoid dendritic cells (pDCs) represent a small fraction of total blood cells. However, they exhibit very potent antiviral activities, in part through the production of large quantities of the antiviral compound interferon (IFN)-a. A severe reduction in numbers of blood pDCs has been observed in HIV-infected individuals, and the levels of pDCs do not return to normal following initiation of effective antiviral therapy. Previous in vitro studies have suggested that pDCs may exert an anti-HIV effect, but work addressing the ability of patient-derived pDCs to suppress HIV replication has been lacking. During the past year, we began to examine the ability of pDCs from HIV-infected individuals to suppress endogenous viral replication in autologous CD4+ T cells ex vivo. We are investigating 1) the mechanism by which pDCs suppress HIV replication, 2) the effect of active viral replication on pDC functions, 3) the effect of plasma viremia on differential expression of genes in pDCs, and 4) the level of apoptosis and necrosis in pDCs induced by active HIV replication in vitro. 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, replication-competent virus, HIV-1 proviral DNA including 2 LTR circles, and spliced and unspliced HIV-1 RNA in CD4+ T cells have been found in the majority of infected individuals in whom plasma viremia is below the limit of detection. This persistent viral reservoir has emerged as the major obstacle preventing the eradication of HIV. In addition, we find 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 ART (with plasma virus levels below the limit of detection for prolonged periods of time [>2.5 years]). Strategies aimed at minimizing cellular activation may further diminish residual viral replication in patients receiving ART. To address this question, we have begun a pilot clinical trial to examine the safety and tolerability of a mildly immunosuppressive agent, daclizumab, a humanized IgG1 monoclonal antibody that binds specifically to the alpha subunit of the human high-affinity IL-2 receptor expressed on the surface of activated lymphocytes. Considering the intimate relationship between the degree of immune activation and HIV replication, we aim to determine whether daclizumab can normalize immunologic profiles and reduce plasma viremia in study subjects.