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 T淋巴细胞显示细胞毒性和非细胞毒性抗HIV活性。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)，并且在HIV感染的很早就出现。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。提出了蛋白质组学和分子研究。蛋白质组学方法包括从含CaF的液体中进行生化分离的蛋白质的质谱分析。多维色谱-串联质谱分析和培养中氨基酸的稳定同型标记(SILAC)将被用来识别相对于对照液体在CAF活性液体中独特或过度表达的多肽。如有需要，还将进行标准的蛋白质纯化程序，以进一步从其他分泌的CD8细胞蛋白质中分离出CAF。这些基因正在通过转导293T细胞进行进一步评估，然后评估产生抗HIV蛋白的情况。CAF的鉴定具有作为HIV感染的治疗剂和开发有效疫苗的突出潜力。

DESCRIPTION (provided by applicant): CD8+ T 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 asymptomatic infected individuals 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), and appears very early in HIV infection. It is associated with secretion of an unidentified CD8+ cell antiviral factor (CAF), a protein stable to heat and low pH that inhibits HIV transcription. 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 mediates CAF activity. Our ultimate goal is to clone/sequence and produce CAF for evaluation in therapeutic and diagnostic trials. Proteomics and molecular studies are proposed. The proteomics approach involves mass spectrometric analysis of biochemically fractionated proteins from CAF-containing fluids. For these protein purification studies, we propose to use the chromatographic procedures we have developed thus far to separate CAF from many extraneous CD8+ cell culture proteins. Multidimensional chromatography-tandem mass spectrometric analysis and a stable isotype labeling of amino acids in culture (SILAC) will be employed to identify peptides unique or overexpressed in CAF-active fluids relative to control fluids. Standard protein purification procedures will also be conducted, where necessary, to further resolve CAF from other secreted CD8+ cell proteins. By the molecular approach, DMA microarray studies, confirmed by RT-PCR analyses, have identified 21 of the 107 candidate gene(s) found associated with CNAR/CAF activity. These genes are being further evaluated by transduction into 293T cells that are then assessed for production of anti-HIV proteins. In addition, candidate genes will be expressed in human GD8+ cells and the ability of these cells to suppress HIV replication and to produce CAF-like proteins will be tested. Finally, the clinical relevance of the identified protein(s) to CNAR/CAF will be established by shRNA studies and anti-sense mRNA alternative splicing procedures. The identification of CAF has outstanding potential as a therapeutic agent for HIV infection and for the development of an effective vaccine.