Role of Innate Immunity in Controlling HIV Infection

描述(申请人提供)：来自健康HIV感染者的CD8+淋巴细胞显示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CD8+细胞非细胞毒性反应(CNAR)，这似乎是对HIV感染做出反应的先天性免疫系统的一部分。当来自无症状感染者的CD8+细胞与艾滋病毒急性感染的CD4+细胞共同培养时，病毒复制被抑制，而不会杀死CD4+细胞。CNAR不受人类白细胞抗原的限制，不是特定逆转录病毒的特异性(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，并与CD8+细胞抗病毒因子(CAF)的分泌有关，该因子尚未确定。CAF是一种对热和低pH值稳定的蛋白质，可以抑制HIV的转录。我们最近发现，蛋白水解酶抑制剂可以阻断CAF的活性，这表明一种蛋白水解酶可能参与了CNAR的调节。CNAR/CAF活性的临床重要性已经在几项保护个人免受艾滋病毒感染和疾病进展的研究中得到证明。本提案的具体目的是确定介导CAF活性的多肽(S)。我们的最终目标是克隆/测序并生产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。生化方法包括从含CaF的液体中对生化分离的蛋白质进行质谱分析。对于这些研究，我们建议使用：a)多维色谱-串联质谱分析和同位素编码亲和标签(ICAT)方法，以确定在CaF活性流体中相对于对照流体独特或过度表达的多肽；b)基于蛋白酶抑制剂的亲和层析；以及，必要时，c)标准蛋白质纯化程序。通过分子生物学方法，利用基因芯片和动力学RT-PCR法对编码CAF的基因(S)进行鉴定。对于抑制HIV复制的CD8+细胞和不抑制HIV复制的CD8+细胞，我们已经在CD8+细胞中鉴定出约42个差异表达基因，显示出CNAR。这些基因将被进一步评估，以验证它们在抑制HIV复制的CD8+细胞中上调。候选基因将在人类细胞中表达，并对分泌的蛋白质进行抗艾滋病毒活性评估。还将获得单抗，以建立CAF特异性的ELISA，并用于流式细胞术研究，以开发一种诊断试验，以衡量疾病的进展。

DESCRIPTION (provided by applicant): CD8+ lymphocytes from healthy HIV-infected individuals show cytotoxic and non-cytotoxic 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) multi dimensional chromatography-tandem mass spectrometric analysis and an isotope-coded affinity tag (ICAT) methodology to identify peptides unique or over expressed 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 42differentially 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.