Role of Innate Immunity in Controlling HIV Infection

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。 我们的实验室专注于CD 8+细胞的非细胞毒性反应（CNAR），似乎是一部分， 先天免疫系统对HIV感染的反应。当来自无症状感染者的CD 8+细胞 与HIV急性感染的CD 4+细胞共培养，抑制病毒复制， 杀死CD 4+细胞。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HIV-1， HIV-2和SIV分离株测试），并出现在艾滋病毒感染的早期。它与一种 未鉴定的CD 8+细胞抗病毒因子（CAP），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几项保护个体的研究中得到证实 艾滋病感染和疾病进展。 本发明的具体目的是鉴定介导CAP活性的多肽。 我们的最终目标是克隆/测序和生产CAP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。蛋白质组学方法包括质谱分析 从含CAF的液体中生化分离蛋白质的分析。对于这些蛋白质纯化 研究，我们建议使用色谱程序，我们已经开发到目前为止，分离CAF从 许多外来的CD 8+细胞培养蛋白。多维色谱-串联质谱 将采用分析和培养物中氨基酸的稳定同种型标记（SILAC）来鉴定肽 相对于对照流体，在CAF活性流体中独特或过表达。标准蛋白纯化程序 在必要时，还将进行进一步从其他分泌的CD 8+细胞蛋白中分离CAF。 通过分子方法，动态RT-PCR程序将继续完成分析， 与CAF产生相关的候选基因。这些基因，由DMA微阵列研究确定，将被 通过转导到293 T细胞中进一步评估，然后评估抗HIV蛋白的产生。在 此外，候选基因将在人CD 8+细胞中表达，这些细胞抑制HIV的能力 复制和产生CAF样蛋白将被测试。最后，确定CAF的临床相关性 将通过shRNA研究来确定CNAR/CAF的蛋白质。

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 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 (CAP), 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 mediate CAP activity. Our ultimate goal is to clone/sequence and produce CAP 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 spectometric 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, kinetic RT-PCR procedures will be continued to complete the analysis of candidate gene(s) associated with CAF production. These genes, identified by DMA microarray studies, will be 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 CD8+ 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 CAF protein(s) to CNAR/CAF will be established by shRNA studies.