Role of Innate Immunity in Controlling HIV Infection

描述（由申请方提供）：来自健康HIV感染个体的CD 8 + T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），并且在HIV感染的早期出现。它与一种未鉴定的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，可抑制HIV转录。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。提出了蛋白质组学和分子生物学研究。蛋白质组学方法涉及从含CAF的流体中生物化学分级分离的蛋白质的质谱分析。对于这些蛋白质纯化研究，我们建议使用我们迄今为止开发的色谱方法将CAF与许多外源性CD 8+细胞培养蛋白分离。将采用多维色谱-串联质谱分析和培养物中氨基酸的稳定同种型标记（SILAC）来鉴定相对于对照液，在CAF活性液中独特或过表达的肽。必要时，还将进行标准蛋白纯化程序，以进一步从其他分泌的CD 8+细胞蛋白中分离CAF。通过分子生物学方法，DNA微阵列研究，经RT-PCR分析证实，已经确定了与CNAR/CAF活性相关的107个候选基因中的21个。这些基因正在通过转导到293 T细胞中进行进一步评估，然后评估抗HIV蛋白的产生。此外，候选基因将在人GD 8+细胞中表达，并将测试这些细胞抑制HIV复制和产生CAF样蛋白的能力。最后，将通过shRNA研究和反义mRNA选择性剪接程序确定所鉴定的蛋白质与CNAR/CAF的临床相关性。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.