Inhibition of HIV 1 Replication by Cytotoxic Lymphocytes

描述（由申请人提供）：在第一批艾滋病病例被描述后的四分之一个多世纪，控制HIV-1的基本免疫机制仍然未知。诱导hiv -1特异性CD8 T细胞与急性感染后血浆病毒血症的下降有关，但通过IFN-3表达测量的这些反应的幅度和广度与疾病控制无关。这些试验不能评估CTL识别和杀死病毒感染细胞的能力。事实上，目前测量的特性可能只是反映了病毒血症的水平，而不是揭示免疫控制的原因。我们假设，鉴定CTL中的关键抗病毒效应物功能，以及这些活性中的潜在缺陷，将需要直接检查T细胞与活的hiv -1感染细胞之间的相互作用，这一点从未被全面分析过。在之前的资助期间，我们开发了一种强大的体外病毒抑制试验，可以识别控制HIV-1复制的T细胞。我们的数据表明，这种短期共培养试验很容易区分功能性和非功能性CTL：即使在单个个体中，我们也观察到具有不同特异性的T细胞之间存在1000倍的差异。这个强大的工具允许详细的细胞和分子分析，因为系统的每个组成部分（CTL，病毒和靶细胞）可以单独操作。我们现在建议确定体外抑制HIV-1感染的CTL群体，并直接评估参与抗病毒控制和CTL功能丧失的分子途径。具体来说，我们建议：1)在体外鉴定最能控制HIV-1复制的CTL表位特异性；2)确定TCR和病毒序列变异对CTL亲和力、细胞信号传导和抗病毒抑制能力的影响；3)利用基因表达分析、过表达载体和RNAi技术确定CTL中必需的抗病毒效应分子和重要的调控途径。这些研究与疫苗设计和测试高度相关，因为它们将提供关键的新数据，以增强我们对抗病毒CTL功能的理解，并将确定宿主CTL有效靶向的病毒区域，这些区域可以纳入新的免疫原。公共卫生相关性：人类免疫缺陷病毒1型（HIV-1）是获得性免疫缺陷综合征（艾滋病）的病因，在全世界已造成约2500万人死亡。目前有3 300多万人受到感染，遏制这一流行病的最大希望是开发有效的疫苗。该项目的目的是了解免疫系统的特定细胞，称为细胞毒性T淋巴细胞（CTL），如何识别和杀死病毒感染的细胞，并确定这些细胞可以靶向的病毒最脆弱的蛋白质。这些努力对于理解为什么一些CTL在抗病毒功能上更有效而另一些更低效至关重要，并且对于开发有效的疫苗以产生针对HIV-1的最佳免疫反应是必要的。

DESCRIPTION (provided by applicant): More than a quarter of a century after the first AIDS cases were described, the basic immunologic mechanisms that control HIV-1 remain unknown. Induction of HIV-1-specific CD8 T cells is associated with a decline in plasma viremia following acute infection, but neither the magnitude nor breadth of these responses, as measured by IFN-3 expression, is correlated with disease control. These assays do not assess the ability of CTL to recognize and kill virus- infected cells. Indeed, the properties currently measured may simply reflect the level of viremia, rather than reveal causes of immune control. We hypothesize that identifying key antiviral effector functions in CTL, and potential defects in these activities, will require direct examination of the interaction between T cells and live HIV-1-infected cells, which has never been comprehensively analyzed. During previous funding periods of this grant, we developed a robust in vitro viral inhibition assay that allows identification of T cells that control HIV-1 replication. Our data indicate that this short-term co-culture assay readily distinguishes functional and non-functional CTL: even within a single individual, we observed 1,000-fold differences between T cells with differing specificities. This powerful tool allows for detailed cellular and molecular analyses, since each component of the system (CTL, virus, and target cell) can be manipulated individually. We now propose to define CTL populations that inhibit HIV-1 infection in vitro and to directly assess the molecular pathways involved in antiviral control and loss of CTL function. Specifically we propose to: 1) Identify CTL epitope specificities that are best able to control HIV-1 replication in vitro; 2) Determine the impact of TCR and viral sequence variation on CTL avidity, cell signaling, and antiviral inhibition capacity; and 3) Define essential antiviral CTL effector molecules and important regulatory pathways in CTL using gene expression analysis, over-expression vectors, and RNAi technology. These studies are highly relevant to vaccine design and testing since they will provide critical new data to enhance our understanding of antiviral CTL function and they will identify viral regions that are efficiently targeted by host CTL that can be incorporated into novel immunogens. PUBLIC HEALTH RELEVANCE: Human immunodeficiency virus type 1 (HIV-1) is the cause of acquired immuno-deficiency syndrome (AIDS), and has already resulted in an estimated 25 million deaths worldwide. With over 33 million persons currently infected, the greatest hope for stemming the epidemic is through the development of an effective vaccine. The aim of this project is to understand how specific cells of the immune system, called cytotoxic T lymphocytes (CTL), identify and kill virus- infected cells, and to define the most vulnerable proteins of the virus that can be targeted by these cells. These efforts are essential to understand why some CTL are more efficient and others less efficient in their antiviral function and are necessary to develop effective vaccines that generate optimal immune responses against HIV-1.