Mechanisms of Resistance in HIV-1 Exposed Sero-negative IV-drug Users: Induction of IFN-mediated Factors and S100 Proteins as Determinants of NK Cell-Mediated Clearance and Low CD4+ T Cell Infectivity

 描述（由申请人提供）：缺乏针对HIV-1的明确免疫相关性，突出了识别感染宿主抗性新途径的迫切需要。这项R21提案的总体目标是在一组尽管多年的高风险行为和暴露但仍保持血清阴性（HESN）的HIV暴露个体中确定新的保护机制。先前对通过静脉注射药物和共用针头（HESN-IDU）暴露于HIV-1的HESN受试者的研究已经确定了几种潜在的先天和内在保护机制，包括提高自然杀伤（NK）细胞功能和提高CD 4 + T细胞对HIV-1感染的抵抗力。我们的初步数据现在提供了一个基础，以测试一个创新的模型，这些先天和内在的耐药机制如何合作，以提供一个持久的屏障，对HIV-1感染的HESN-IDU受试者。使用来自费城的高风险HESN-IDU受试者的充分描述的队列，我们已经鉴定了来自HESN-IDU受试者的NK细胞上的独特蛋白质组特征，包括多种干扰素诱导的蛋白质如ISG-15、MHC-I类、粒酶、STAT 1/2、以及先前未在HESN受试者中鉴定的免疫调节蛋白S100家族的几个成员的表达增加。具体而言，我们确定了两种细胞质S100蛋白S100 A4和S100 A6的表达增加，这两种蛋白可以刺激NK细胞对病毒感染细胞的脱粒能力，以及分泌的S100蛋白S100 A14，这可能增强CD 4 + T细胞中的HIV-1抗性。这些结果表明，在受保护的HESN-IDU受试者中，高风险针头共用可能引发涉及分泌的干扰素和/或S100蛋白的抗病毒环境，这可能导致针对病毒感染细胞的NK活性增强，并增加CD 4+靶细胞对HIV-1的抗性。在具体目标1中，我们将使用HIV抑制试验测量来自HESN-IDU受试者和NS-IDU对照的NK细胞限制Autumn HIV-1感染的CD 4+原代T细胞复制能力的能力。我们还将研究来自HESN-IDU受试者的NK细胞是否具有针对HIV-1感染的异源SupT 1细胞的增加的CD 107 a脱粒，以及这是否与增强的S100 A4和S100 A6募集到免疫突触中相关。在特定目标2中，我们将检测来自HESN-IDU受试者的纯化CD 4 + T细胞对HIV-1感染的抗性，并研究分泌的S100 A14蛋白进一步限制HIV-1复制能力的能力。我们将通过蛋白质组分析研究HESN-IDU受试者的CD 4 + T细胞中已知和未表征的宿主限制性因子的表达，并将其与感染性相关联。总之，本R21中提出的特定目的将检验新假设，即HESN-IDU受试者中干扰素诱导因子和S100蛋白表达的增加通过增加NK介导的病毒感染细胞清除率和降低CD 4 + T细胞中HIV-1复制效率来增强先天和内在耐药机制。

 DESCRIPTION (provided by applicant): The absence of clear immune correlates for protection against HIV-1 highlight the critical need to identify new pathways of host-resistance from infection. The overall goal of this R21 proposal is to identify novel mechanism(s) of protection in a cohort of HIV-exposed individuals who remain sero-negative (HESN) despite many years of high-risk behavior and exposure. Previous studies of HESN subjects exposed to HIV-1 through IV-drug use and needle-sharing (HESN-IDU) have identified several potential innate and intrinsic mechanisms of protection, including heightened Natural Killer (NK) cell function and increased resistance of CD4+ T cells to HIV-1 infection. Our preliminary data now provide the basis to test an innovative model for how these innate and intrinsic mechanisms of resistance may cooperate to provide a sustained barrier against HIV-1 infection in HESN-IDU subjects. Using a well-described cohort of high-risk HESN-IDU subjects from Philadelphia, we have identified a unique proteomic signature on NK cells from HESN-IDU subjects including the elevated expression of multiple interferon-induced proteins such as ISG-15, MHC-Class I, Granzyme, STAT1/2, as well as the increased expression of several members of the S100 family of immuno-modulatory proteins not previously identified in HESN subjects. Specifically, we identified the increased expression of two cytoplasmic S100 proteins, S100A4 and S100A6, that may stimulate NK degranulation capacity against virally infected cells and a secreted S100 protein, S100A14, that may augment HIV-1 resistance in CD4+ T cells. These results indicate that high-risk needle sharing in protected HESN-IDU subjects may trigger an anti-viral environment involving secreted Interferon and/or S100 proteins that can lead to greater NK activity against virally infected cells and increased CD4+ target cell resistance to HIV-1. In Specific Aim 1, we will measure the ability of NK cells from HESN-IDU subjects and NS-IDU controls to limit the replication capacity of Autologous HIV-1 infected CD4+ primary T cells using an HIV Suppression Assay. We will also investigate if NK cells from HESN-IDU subjects possess increased CD107a degranulation against HIV-1 infected heterologous SupT1 cells and if this correlates with enhanced S100A4 and S100A6 recruitment into the immunological synapse. In Specific Aim 2, we will test the resistance of purified CD4+ T cells from HESN-IDU subjects to HIV-1 infection and investigate the ability of the secreted S100A14 protein to further limit HIV-1 replication capacity. We will investigate the expression of known and uncharacterized host restriction factors in CD4+ T cells from HESN-IDU subjects by proteome analysis and correlate them with infectivity. Together, the Specific Aims proposed in this R21 will test the novel hypothesis that increased expression of interferon-induced factors and S100 proteins in HESN-IDU subjects augment innate and intrinsic mechanisms of resistance by increasing NK-mediated clearance of virally infected cells and reducing the efficiency of HIV-1 replication in CD4+ T cells.