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淋巴细胞显示细胞毒性和非细胞毒性抗hiv活性。我们的实验室专注于CD8+细胞非细胞毒性反应（CNAR），这似乎是先天免疫系统对HIV感染反应的一部分。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，在不杀死CD4+细胞的情况下抑制病毒复制。CNAR不受hla限制，不针对特定的逆转录病毒（抑制所有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是一种对高温和低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若干保护个体免受HIV感染和疾病进展的研究中得到证实。本提案的具体目标是确定介导CAF活性的多肽。我们的最终目标是克隆/测序并生产用于治疗和诊断试验评估的CAF。建议进行蛋白质组学和分子研究。蛋白质组学方法涉及对含钙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从许多外来CD8+细胞培养蛋白中分离出来。多维色谱-串联质谱分析和稳定的培养氨基酸同型标记（SILAC）将被用于鉴定相对于对照液，在cafo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CD8+细胞蛋白中分离CAF。通过分子方法，经RT-PCR分析证实的DMA微阵列研究已经鉴定出107个与CNAR/CAF活性相关的候选基因中的21个。这些基因正在通过转导到293T细胞中进行进一步评估，然后评估293T细胞是否产生抗hiv蛋白。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选择性剪接程序来确定鉴定的蛋白与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突出潜力。