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

描述(申请人提供)：我从小就对HIV-1感兴趣，当时我在父亲经营的针头交换诊所做志愿者。这种兴趣延续到了我的博士学习中，在那里我获得了流行病学博士学位，重点是艾滋病毒-1的遗传流行病学。从那时起，我作为华盛顿大学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感染中所起的作用。在我的博士后和博士后研究期间，我评估了人类白细胞抗原(HL A)I类等位基因对多种HIV-1结果的影响，发现了几个一致的结果，我在K01指导研究科学家发展奖提案的基础上建立了这些结果。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0.4log10拷贝/毫升，将HIV-1传播给婴儿的可能性几乎是前者的3倍。其次，在来自血清不一致夫妇的HIV-1感染者中，A*23也与高出0.4log10拷贝/毫升的HIV-1设定点有关，而B*53与高出0.7个设定点有关。流行病学证据表明，A*23在HIV-1感染过程中具有有害影响，这一证据得到了其他八项研究的进一步支持，这些研究表明，该等位基因与HIV-1控制较差有关。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。 以往的研究表明，某些人类白细胞抗原的等位基因，如人类白细胞抗原B*27和B*57，可以通过限制特定的CD8+T细胞表位来诱导更高幅度和更高功能的免疫反应，从而防止HIV-1疾病的进展；解释其他等位基因对HIV-1的影响的机制尚不清楚。解释人类白细胞抗原A*23和B*53与HIV-1设定点升高之间关系的一个假说是，这种有害影响是以T细胞表位依赖的方式引起的，导致对HIV-1感染的低效细胞毒性T淋巴细胞(CTL)反应。为了评估这一假设，我们建议1)使用干扰素？ELISPOT分析以确定针对由A*23或B*53呈现的特定T细胞表位的CTL反应是否与设定点的变化有关；以及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等位基因限制的T细胞反应的定性差异。了解为什么A*23与HIV-1的较差控制相关，可能有助于识别阻碍T细胞疫苗有效性的免疫原，并可能进一步阐明区分保护性和无效CTL的功能特征。此外，越来越多的研究表明，HLA等位基因也可以通过与树突状细胞(DC)上的免疫球蛋白样转录本4(ILT4)受体结合等替代机制来影响免疫反应性。与ILT4结合可以改变DC的成熟和耐受性T细胞反应的发展。因此，与其他等位基因相比，A*23和B*53等位基因可能通过与ILT4受体更强的结合而导致对HIV-1的较差控制。通过CTL或ILT4机制评估人类白细胞抗原A*23和B*53是否与较高的HIV-1设定点水平相关，是一种比较保护和有害等位基因机制的创新方法，并可能阐明导致保护的自然途径，可用于疫苗或治疗干预。 由于研究目标的跨学科性质，我组建了一个在艾滋病毒-1流行病学、全球卫生和艾滋病毒-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，并将就研究和培训计划的所有方面提供日常指导，特别是项目协调、数据解释和专业发展。Nicole Frahm博士(HIV疫苗试验网络实验室科学副主任兼华盛顿大学全球卫生助理教授)将提供病理生物学和免疫学方面的指导，并将为进行分析和实验提供实验室空间。按照审查员的建议，我还组建了一个咨询委员会，就职业发展提供评价和反馈。咨询委员会成员包括康妮·塞勒姆博士(全球健康、医学和流行病学教授)、吉姆·穆林斯博士(微生物学和医学教授)、朱莉·麦克拉斯博士(艾滋病毒疫苗试验网络实验室项目主任)和彼得·吉尔伯特博士(艾滋病毒疫苗试验网络统计数据管理中心联席主任和生物统计学研究教授)。除了具体的指导，我还将完成有关HIV-1分子生物学的正式课程，并将参加会议和其他会议，在那里我将介绍我的研究。总体而言，这项研究和培训计划将把我在流行病学和生物统计学方面的专业知识与直接使用基础科学数据的新经验结合起来，使我能够通过应用跨学科方法为HIV-1研究做出有用的贡献，并实现我的短期和长期职业目标--获得独立的R01资金并成为一名跨学科研究教员。

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-? 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.