Role of Innate Immunity in Controlling HIV Infection

先天免疫在控制 HIV 感染中的作用

• 批准号: 7894208
• 负责人: JAY A LEVY
• 金额: $ 15万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-14 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7894208
• 关键词: Alternative Splicing; Amino Acids; Antiviral Agents; Antiviral Response; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Candidate Disease Gene; Cell Culture Techniques; Cells; Characteristics; Chromatography; Clinical; Complex Mixtures; Development; Diagnostic; Diagnostic Trial; Disease Progression; Evaluation; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; HIV; HIV Infections; HIV-1; HIV-2; Heating; Human; Immune response; Immune system; Individual; Kinetics; Label; Laboratories; Liquid substance; Mass Spectrum Analysis; Mediating; Messenger RNA; Molecular; Natural Immunity; Peptides; Procedures; Production; Protein Analysis; Proteins; Proteomics; Relative (related person); Research; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Role; SIV; Testing; Therapeutic; Therapeutic Agents; Vaccines; Virus Replication; clinically relevant; cytotoxic; kidney cell; killings; overexpression; polypeptide; protein purification; response; small hairpin RNA

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.

描述（由申请人提供）：来自健康 HIV 感染者的 CD8+ T 淋巴细胞显示出细胞毒性和非细胞毒性抗 HIV 活性。我们的实验室重点研究 CD8+ 细胞非细胞毒性反应 (CNAR)，它似乎是响应 HIV 感染的先天免疫系统的一部分。当来自无症状感染者的 CD8+ 细胞与 HIV 急性感染的 CD4+ 细胞共培养时，会在不杀死 CD4+ 细胞的情况下抑制病毒复制。 CNAR 不受 HLA 限制，对特定逆转录病毒不具有特异性（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。它与一种未知的 CD8+ 细胞抗病毒因子 (CAF) 的分泌有关，CAF 是一种对热和低 pH 值稳定的蛋白质，可抑制 HIV 转录。 CNAR/CAF 活性的临床重要性已在多项保护个体免受 HIV 感染和疾病进展的研究中得到证明。本提案的具体目标是鉴定介导 CAF 活性的多肽。我们的最终目标是克隆/测序并生成 CAF，用于治疗和诊断试验中的评估。提出了蛋白质组学和分子研究。蛋白质组学方法涉及对含有 CAF 的液体中生化分离的蛋白质进行质谱分析。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程序将 CAF 与许多外来 CD8+ 细胞培养蛋白分开。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 CAF 活性液体中相对于对照液体独特或过度表达的肽。如有必要，还将进行标准蛋白质纯化程序，以进一步从其他分泌的 CD8+ 细胞蛋白质中分离出 CAF。通过分子方法，经 RT-PCR 分析证实的 DMA 微阵列研究已鉴定出与 CNAR/CAF 活性相关的 107 个候选基因中的 21 个。这些基因通过转导到 293T 细胞中进行进一步评估，然后评估其抗 HIV 蛋白的产生。此外，候选基因将在人类GD8+细胞中表达，并测试这些细胞抑制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已识别蛋白质与 CNAR/CAF 的临床相关性。 CAF 的鉴定具有作为 HIV 感染治疗剂和开发有效疫苗的巨大潜力。