Discovery of soluble HIV suppressor factors

可溶性 HIV 抑制因子的发现

• 批准号: 7006245
• 负责人: FIORENZA COCCHI
• 金额: $ 22.28万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7006245
• 关键词: SDS polyacrylamide gel electrophoresis; affinity chromatography; antibody neutralization test; antiviral agents; cell line; enzyme linked immunosorbent assay; growth inhibitors; helper T lymphocyte; high performance liquid chromatography; human immunodeficiency virus 1; human subject; protein sequence; protein structure function; suppressor T lymphocyte; tissue /cell culture; virus infection mechanism; virus replication

DESCRIPTION (provided by applicant): Human T cells produce soluble, noncytolytic factors (HIV suppressor factors) that suppress the replication of virtually all HIV strains. Numerous clinical studies have shown that an inborn capacity to release high levels of CD8+ T cell-derived factors correlates with better control of HIV infection, In particular, this correlation was observed in a subset of HIV-infected subjects (called long term non progressors or LTNPs) that do not develop AIDS for more than 10 years in the absence of antiretroviral therapy. Since these factors are noncytolytic, broadly active against HIV strains, and associated with non-progression to AIDS, they are potentially valuable resources for the development of new approaches to treat HIV infection. Moreover, the development of new therapeutic agents is now clearly linked to a sustained search for soluble suppressor factors. In 1996, our group identified the chemokines, RANTES, MIP1-alpha, and MIP1-beta as the CD8+ T cell factors responsible for the suppression of "R5" HIV isolates that use the CCR5 coreceptor for entry. This discovery, coupled with seminal studies on CCR5, has led to the development of a new generation of highly promising drugs that interfere with R5 HIV entry. The current goal is to identify the elusive HIV suppressor factors that selectively suppress "X4" HIV strains. This will require novel techniques that avoid the inherent problems of primary cell cultures and optimize the chances for success. Our hypothesis is that we can identify X4 suppressor factors by fractionating conditioned medium from HTLV-immortalized cell lines derived from the T cells of LTNPs. Aim 1 will be to purify and identify soluble X4 HIV suppressor factor(s) secreted by transformed CD4+ and CD8+ T cells from LTNPs. Our newly developed HTLV-1 transformed CD8+ T and CD4+ T cell lines from LTNPs, which consistently release high levels of noncytolytic X4 HIV suppressor activity, will be used to produce serum free conditioned media. This material will be fractionated by affinity chromatography and HPLC and screened for antiviral activity. Amino acid sequence analyses or peptide fragment analyses will used to identify the proteins associated with virus suppression. Aim 2 will be to determine the natural relevance of anti-X4 Factor(s) discovered in Aims 1 to the natural HIV suppressor activity released by primary cells. We will use antibody neutralization and/or depletion experiments to determine whether a candidate factor contributes to the natural suppressor activities of primary cells. Mixtures of cognate antibodies will be used in cases where we have evidence that several antiviral factors mediate redundant antiviral activity.

描述（由申请人提供）：人类 T 细胞产生可溶性非溶细胞因子（HIV 抑制因子），可抑制几乎所有 HIV 毒株的复制。大量临床研究表明，释放高水平 CD8+ T 细胞衍生因子的先天能力与更好地控制 HIV 感染相关，特别是，在 HIV 感染者的子集（称为长期非进展者或LTNP）在没有抗逆转录病毒治疗的情况下超过 10 年不会发展为艾滋病。由于这些因子是非溶细胞性的，对 HIV 病毒株具有广泛的活性，并且与艾滋病毒的不进展有关，因此它们是开发治疗 HIV 感染的新方法的潜在宝贵资源。此外，新治疗剂的开发现在显然与对可溶性抑制因子的持续探索有关。 1996年，我们的小组确定了趋化因子RANTES、MIP1-α和MIP1-β作为CD8+ T细胞因子，负责抑制使用CCR5辅助受体进入的“R5”HIV分离株。这一发现，加上对 CCR5 的开创性研究，导致了新一代干扰 R5 HIV 进入的非常有前途的药物的开发。当前的目标是确定选择性抑制“X4”HIV 毒株的难以捉摸的 HIV 抑制因子。这将需要新的技术来避免原代细胞培养的固有问题并优化成功的机会。我们的假设是，我们可以通过从 LTNP 的 T 细胞衍生的 HTLV 永生化细胞系中分离条件培养基来鉴定 X4 抑制因子。目标 1 是纯化并鉴定 LTNP 中转化的 CD4+ 和 CD8+ T 细胞分泌的可溶性 X4 HIV 抑制因子。我们新开发的来自 LTNP 的 HTLV-1 转化 CD8+ T 和 CD4+ T 细胞系，持续释放高水平的非溶细胞性 X4 HIV 抑制活性，将用于生产无血清条件培养基。该材料将通过亲和色谱和 HPLC 进行分级，并筛选抗病毒活性。氨基酸序列分析或肽片段分析将用于鉴定与病毒抑制相关的蛋白质。目标 2 将确定目标 1 中发现的抗 X4 因子与原代细胞释放的天然 HIV 抑制活性的天然相关性。我们将使用抗体中和和/或耗竭实验来确定候选因子是否有助于原代细胞的天然抑制活性。如果我们有证据表明多种抗病毒因子介导多余的抗病毒活性，则将使用同源抗体的混合物。