CTL mechanisms underlying effects of HLA-A*2301 and B*5301 on HIV-1 set-point

描述（由申请人提供）：我对HIV-1的兴趣始于我还是个孩子的时候，当时我在我父亲经营的针头交换诊所做志愿者。这种兴趣延续到我的博士研究中，我获得了流行病学博士学位，重点是HIV-1的遗传流行病学。从那时起，我作为华盛顿大学（UW）国际临床研究中心的高级研究员，继续研究宿主基因组因素在HIV-1感染中的作用。在我的博士前和博士后研究期间，我评估了人类白细胞抗原（HLA）I类等位基因对多种HIV-1结果的影响，并发现了几个一致的结果，我在此基础上建立了K 01指导研究科学家发展奖提案。首先，在肯尼亚围产期HIV-1传播队列中，携带A*23等位基因的妇女血浆HIV-1 RNA水平高出0.4 log 10拷贝/mL，将HIV-1传播给婴儿的可能性几乎是其3倍。其次，在血清学一致性夫妇中的HIV-1感染者中，A*23也与0.4 log 10拷贝/mL的HIV-1设定值升高相关，B*53与0.7个设定值升高相关。流行病学证据表明，A*23在HIV-1感染期间具有有害影响，其他8项研究进一步支持该等位基因与HIV-1控制较差相关。然而，解释为什么A*23等位基因与这些有害结果相关的机制尚不清楚。 以前的研究已经证明，某些HLA等位基因，如HLA-B *27和B*57，通过限制引发更高强度和功能性免疫应答的特异性CD 8 + T细胞表位来防止HIV-1疾病进展;解释其他等位基因对HIV-1影响的机制尚未了解。解释HLA-A*23和B*53与增加的HIV-1设定点的关联的一个假设是，有害作用是以T细胞表位依赖性方式引起的，其导致响应HIV-1感染的无效细胞毒性T淋巴细胞（CTL）应答。为了评估这一假设，我们建议：1）使用IFN-γ ELISpot测定来确定对A*23或B*53呈递的特异性T细胞表位的CTL应答是否与设定点的变化相关; 2）使用细胞内细胞因子染色和流式细胞术比较受有害和保护性HLA等位基因限制的T细胞应答的定性差异。理解为什么A*23与HIV-1的较差控制相关可能有助于识别阻碍基于T细胞的疫苗有效性的免疫原，并可能进一步阐明区分保护性CTL和无效CTL的功能特征。另外，越来越多的研究表明，HLA等位基因也可以通过其他机制影响免疫应答，如与树突状细胞（DC）上的免疫球蛋白样转录本4（ILT 4）受体结合。HLA与ILT 4的结合可以改变DC成熟和致耐受性T细胞应答的发展。因此，A*23和B*53等位基因可能通过比其他等位基因更强地结合ILT 4受体而导致对HIV-1的更差控制。评估HLA-A*23和B*53是否通过CTL或ILT 4机制与更高的HIV-1设定点水平相关，这是一种创新的方法，可以预测保护性和有害HLA等位基因的机制，并可能阐明可用于疫苗或治疗干预的保护性天然途径。 由于研究目标的跨学科性质，我组建了一个在HIV-1流行病学和全球卫生以及HIV-1免疫学方面具有相当专业知识的指导团队。Jairam Lingappa博士（医学和全球健康，UW）将担任主要导师，并将提供有关研究和培训计划的各个方面的日常指导，特别关注项目协调，数据解释和专业发展。Nicole Frahm博士（艾滋病毒疫苗试验网络实验室科学副主任和华盛顿大学全球卫生助理教授）将提供有关病理生物学和免疫学的指导，并将提供实验室空间进行测定和实验。根据审查人员的建议，我还组建了一个咨询委员会，就职业进展提供评价和反馈。咨询委员会成员包括Connie Celum博士（全球卫生、医学和流行病学教授）、Jim Mullins博士（微生物学和医学教授）、Julie McElrath博士（HIV疫苗试验网络实验室项目主任）和Peter吉尔伯特博士（HIV疫苗试验网络统计数据管理中心联合主任和生物统计学研究教授）。除了具体的指导，我还将完成正式的课程，包括HIV-1分子生物学，并将参加会议和其他会议，我将介绍我的研究。总的来说，这项研究和培训计划将联合收割机我在流行病学和生物统计学的专业知识与直接与基础科学数据工作的新经验相结合，使我能够通过应用跨学科方法为HIV- 1研究做出有益的贡献，并实现我获得独立R 01资金和成为一名跨学科研究人员的短期和长期职业目标。

DESCRIPTION (provided by applicant): My interest in HIV-1 began as a child when I volunteered at needle-exchange clinic run by my father. This interest carried forward to my doctoral studies in which I earned a PhD in epidemiology with an emphasis on genetic epidemiology of HIV-1. Since then, I have continued to research the role that host genomic factors play in HIV-1 infection as a Senior Fellow with the International Clinical Research Center at the University of Washington (UW). During both my pre and post-doctoral research, I evaluated the impact of human leukocyte antigen (HLA) class I alleles on multiple HIV-1 outcomes and found several consistent results that I build upon for this K01 Mentored Research Scientist Development Award proposal. First, in a Kenyan perinatal HIV-1 transmission cohort, women who had the A*23 allele had 0.4 log10 copies/mL higher plasma HIV-1 RNA levels and were nearly 3-times as likely to transmit HIV-1 to their infants. Second, among HIV-1 infected individuals from seroidiscordant couples, A*23 was also associated with 0.4 log10 copies/mL higher HIV-1 set-points and B*53 was associated with 0.7 higher set-points. Epidemiologic evidence suggesting that A*23 has a harmful effect during HIV-1 infection is further supported by eight other studies showing that this allele is associated with worse control of HIV-1. However, mechanisms to explain why the A*23 allele is associated with these deleterious outcomes are unknown. Previous studies have demonstrated that certain HLA alleles such as HLA-B*27 and B*57 protect against HIV-1 disease progression by restricting specific CD8+ T-cell epitopes that elicit immune responses of higher magnitude and functionality; mechanisms to explain effects of other alleles on HIV-1 are not yet understood. One hypothesis to explain associations of HLA-A*23 and B*53 with increased HIV-1 set-point is that the harmful effects are elicited in a T-cell epitope dependent manner that results in inefficient cytotoxic T-lymphocyte (CTL) responses in response to HIV-1 infection. To evaluate this hypothesis, we propose to 1) use IFN-γ ELISpot assays to determine if CTL responses to specific T-cell epitopes presented by A*23 or B*53 are associated with changes in set-point; and 2) compare qualitative differences in T-cell responses restricted by harmful and protective HLA alleles using intracellular cytokine staining and flow cytometry. Understanding why A*23 is associated with worse control of HIV-1 may help identify immunogens that hinder effectiveness of T-cell based vaccines and may further elucidate functional characteristics that differentiate protective from ineffective CTL. Alternatively, studis have increasingly demonstrated that HLA alleles can also influence immune responsiveness through alternative mechanisms such as binding to to the immunoglobulin-like transcript 4 (ILT4) receptor on dendritic cells (DC). HLA binding to ILT4 can alter DC maturation and development of tolerogenic T-cell responses. Thus, it is possible that the A*23 and B*53 alleles lead to worse control of HIV-1 by binding the ILT4 receptor more strongly than other alleles. Evaluating whether HLA-A*23 and B*53 associate with higher HIV-1 set-point levels via CTL or ILT4 mechanisms is an innovative way to constrast mechanisms for protective and harmful HLA alleles and may elucidate natural pathways leading to protection that could be used in vaccines or therapeutic interventions. Due to the interdisciplinary nature of the research objectives, I have assembled a mentoring team with considerable expertise in HIV-1 epidemiology and global health and HIV-1 immunology. Dr. Jairam Lingappa (Medicine and Global Health, UW) will serve as the primary mentor and will provide daily guidance regarding all aspects of the research and training plan with particular focus on project coordination, data interpretation, and professional development. Dr. Nicole Frahm (Associate Director for Laboratory Science at the HIV Vaccine Trials Network and Assistant Professor of Global Health, UW) will provide mentoring regarding pathobiology and immunology, and will provide laboratory space for conducting assays and experiments. As suggested by the reviewers, I have also assembled an Advisory Committee to provide evaluation and feedback regarding career progression. The Advisory Committee includes Drs. Connie Celum (Professor of Global Health, Medicine, and Epidemiology), Jim Mullins (Professor of Microbiology and Medicine), Julie McElrath (Director, HIV Vaccine Trials Network Laboratory Program) and Peter Gilbert (Co-Director of the Statistical Data Management Center of the HIV Vaccine Trials Network and Research Professor of Biostatistics). In addition to specific mentoring, I will also complete formal coursework covering HIV-1 molecular biology and will participate in conferences and other meetings where I will present my research. Overall, this research and training program will combine my expertise in epidemiology and biostatistics with new experiences working directly with basic science data, positioning me to make useful contributions to HIV- 1 research by applying interdisciplinary approaches and fulfill my short and long-term career objectives of obtaining independent R01 funding and becoming an interdisplinary research faculty member.