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的明确的免疫相关性，突出了确定新的宿主抵抗感染途径的迫切需要。这项R21提案的总体目标是在一组艾滋病毒感染者中确定新的保护机制(S)，这些人尽管有多年的高危行为和暴露，但仍保持血清阴性(HESN)。以前对通过静脉用药和共用针头暴露于HIV-1的HESN受试者(HESN-IDU)的研究已经确定了几种潜在的天生和内在的保护机制，包括增强自然杀伤(NK)细胞功能和增强CD4+T细胞对HIV-1感染的抵抗力。我们的初步数据现在为测试一种创新的模型提供了基础，该模型说明了这些先天和内在的耐药机制如何协同作用，在HESN-IDU受试者中提供了对抗HIV-1感染的持续屏障。利用来自费城的一组描述良好的高危HESN-IDU受试者，我们在HESN-IDU受试者的NK细胞上发现了一个独特的蛋白质组特征，包括多种干扰素诱导蛋白的表达增加，如ISG-15、MHC-Class I、颗粒酶、STAT1/2，以及S100免疫调节蛋白家族中几个以前在HESN受试者中未发现的成员的表达增加。具体地说，我们发现两个细胞质S100蛋白S100A4和S100A6的表达增加，可能刺激NK对病毒感染细胞的脱颗粒能力，以及分泌的S100蛋白S100A14可能增强CD4+T细胞对HIV-1的抵抗力。这些结果表明，在受保护的HESN-IDU受试者中，共用高危针头可能会触发一种涉及分泌干扰素和/或S100蛋白的抗病毒环境，这可能会导致更强的NK对病毒感染细胞的活性，并增加CD4+靶细胞对HIV-1的抵抗力。在具体目标1中，我们将使用HIV抑制试验来测量来自HESN-IDU受试者和NS-IDU对照的NK细胞限制自体HIV-1感染的CD4+初级T细胞的复制能力的能力。我们还将调查来自HESN-IDU受试者的NK细胞是否对HIV-1感染的异种SupT1细胞具有更多的CD107a脱颗粒，以及这是否与增强的S100A4和S100A6募集到免疫突触有关。在特定目标2中，我们将测试来自HESN-IDU受试者的纯化的CD4+T细胞对HIV-1感染的抵抗力，并研究分泌的S100A14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已知和未知的宿主限制因子在HESN-IDU受试者的CD4+T细胞中的表达，并将它们与感染性相关联。总之，R21中提出的特定目标将检验新的假设，即在HESN-IDU受试者中干扰素诱导因子和S100蛋白的表达增加，通过增加NK介导的病毒感染细胞的清除和降低CD4+T细胞中HIV-1复制的效率，增强先天和内在的耐药机制。

 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.