Role of Innate Immunity in Controlling HIV Infection

超出所提供的空间。来自健康HIV感染者的CD 8+淋巴细胞显示出细胞毒性和非细胞毒性抗HIV活性。我们的实验室一直专注于CD 8+细胞非细胞毒性反应（CNAR），似乎是先天免疫系统对HIV感染的反应的一部分。当来自无症状感染个体的CD 8+细胞与HIV急性感染的CD 4+细胞共培养时，在不杀死CD 4+细胞的情况下抑制病毒复制。CNAR不是HLA限制性的，对特定的逆转录病毒没有特异性（抑制所有测试的HIV-1，HIV-2和SIV分离株），在HIV感染的早期出现，并且与尚未鉴定的CD 8+细胞抗病毒因子（CAF）的分泌有关。CAF是一种对热和低pH稳定的蛋白质，并抑制HIV转录。我们最近发现蛋白酶抑制剂阻断CAF活性，表明蛋白酶可能参与CNAR的介导。CNAR/CAF活性的临床重要性已在保护个体免受HIV感染和疾病进展的几项研究中显示。本发明的具体目的是鉴定介导CAF活性的多肽。我们的最终目标是克隆/测序和生产CAF用于治疗和诊断试验的评估。提出了生物化学和分子生物学方法。生物化学方法涉及质谱分析的生化分馏蛋白质从含CAF的流体。对于这些研究，我们建议使用：用途：a）多维色谱-串联质谱分析和同位素编码亲和标签（ICAT）方法，以鉴定相对于对照液，在CAF活性液中独特或过表达的肽; B）基于蛋白酶底物的亲和色谱;以及必要时，c）标准蛋白纯化程序。通过分子生物学方法，DNA芯片和动态RT-PCR方法将用于鉴定编码CAF的基因。用抑制HIV复制的CD 8+细胞和那些不抑制HIV复制的CD 8+细胞，我们已经确定了大约42个在CD 8+细胞中显示CNAR的差异表达基因。这些基因将被进一步评估，以验证它们在抑制HIV复制的CD 8+细胞中上调。候选基因将在人细胞中表达，并评价分泌蛋白的抗HIV活性。还将衍生单克隆抗体以建立CAF特异性ELISA，并用于流式细胞术研究，以开发诊断测试来测量疾病进展。性能现场=

EXCEED THE SPACE PROVIDED. CD8+ lymphocytes from healthy HIV-infected individuals show cytotoxic and noncytotoxic anti-HIV activities. Our laboratory has focused on the CD8+ cell noncytotoxic response (CNAR) that appears to be part of the innate immune system responding to HIV infection. When CD8+ cells from an asymptomatic infected individual are co-cultivated with HIV acutely infected CD4+ cells, suppression of virus replication takes place without killing the CD4+ cells. CNAR is not HLA-restricted, not specific for a particular retrovirus (inhibits all HIV-1, HIV-2 and SIV isolates tested), appears very early in HIV infection, and is associated with secretion of a CD8+ cell antiviral factor (CAF) that has yet to be identified. CAF is a protein stable to heat and low pH and inhibits HIV transcription. We have recently found that protease inhibitors block CAF activity suggesting that a protease may be involved in the mediation of CNAR. The clinical importance of CNAR/CAF activity has been shown in several studies of protection of individuals from HIV infection and disease progression. The specific objective of the present proposal is to identify the polypeptide(s) that mediate CAF activity. Our ultimate goal is to clone/sequence and produce CAF for evaluation in therapeutic and diagnostic trials. Biochemical and molecular approaches are proposed. The biochemical approach involves mass spectrometric analysis of biochemically fractionated proteins from CAF-containing fluids. For these studies we propose to use: a) multidimensional chromatography-tandem mass spectometric analysis and an isotope-coded affinity tag (ICAT) methodology to identify peptides unique or overexpressed in CAF-active fluids relative to control fluids; b) protease inhibitor-based affinity chromatography; and, when necessary, c) standard protein purification procedures. By the molecular approach, DNA microarray and kinetic RT-PCR procedures will be used to identify the gene(s) encoding CAF. With CD8+ cells that suppress HIV replication and those that do not, we have identified about 42 differentially expressed genes in CD8+ cells showing CNAR. These genes will be further evaluated to verify that they are upregulated in CD8+ cells that suppress HIV replication. Candidate genes will be expressed in human cells and the secreted proteins evaluated for anti-HIV activity. Monoclonal antibodies will also be derived to establish a CAF- specific ELISA and to use for flow cytometric studies in developing a diagnostic test to measure disease progression. PERFORMANCE SITE ========================================Section End===========================================