Key determinants of dengue virus neutralization by naturally occurring human mAbs

简介(申请人提供)：本人在病毒致病和免疫领域有超过12年的经验。我的博士学习和博士后培训使我对痘病毒生物学和异种移植生物学有了广泛的了解。我最近的工作是在目前的应用中形成这门科学的基础，重点是人类登革热病毒单抗的产生和研究。这一信息具有及时的重要性，因为它是合理设计有效的登革热病毒疫苗所必需的。我还接受过内科方面的培训，并获得了董事会认证，是一名传染病临床专家。我的长期职业目标是作为一名内科科学家留在学术医学领域，进行免疫病毒学的转化研究，同时增长我的临床技能和传染病知识。通过利用我从这项拟议的工作中获得的知识，我希望在独立于导师的方向上发展我的职业生涯，并开始致力于丙型肝炎病毒疫苗的合理设计。我在范德比尔特的环境非常适合我提出的项目和我的职业发展。这里的知识环境再好不过了，我打算通过参加一个有组织的课程项目来充分利用这一点。我导师的实验室有大量分离和研究人类单抗的经验，这是我的建议和合理疫苗设计概念的核心。我的导师是范德比尔特疫苗中心的主任，他在疫苗学、免疫学和病毒发病机制方面也有很强的专业知识。在范德比尔特工作期间，在他的指导下，我的工作迅速发展，开始了我作为独立调查员的职业生涯。有症状的登革热病毒感染的疾病严重程度从流感样疾病到危及生命的休克。一种导致严重疾病的机制的模型提出，在初次感染期间诱导的弱交叉反应抗体有助于病毒在随后的二次感染期间进入Fc受体携带细胞。这被认为增加了病毒的复制和细胞因子和血管活性介质的释放，最终导致休克。这种被称为抗体依赖的感染增强的独特过程严重阻碍了疫苗的开发。有人担心，必须针对所有四种登革热病毒血清型产生有效的中和抗体，因为诱导弱交叉反应的非中和抗体的疫苗可能会增加再次暴露时发展为严重疾病的可能性。我们对这一过程的大部分理解都来自于使用老鼠单抗的研究。然而，小鼠的抗体反应通常比人类的复杂。更好地了解人类对自然登革热病毒感染的体液免疫反应是迫切需要的。使用我们实验室开发的高效人类杂交瘤技术，现在可以可靠地从感染康复者的外周血液中产生人类杂交瘤。利用这项技术，我们已经从初发或继发感染康复的受试者身上产生了250多株分泌登革热病毒人单抗的杂交瘤细胞。这些抗体中的绝大多数是广泛的血清型交叉反应，针对包膜或膜前蛋白，并能够通过抗体介导的感染增强。有趣的是，非常罕见的血清型特异的、有效的中和抗体，几乎没有增强活性，也是由人类在感染时产生的。了解这些自然产生的抗体的表位和活性对于疫苗开发至关重要，因为诱导缺乏增强活性的高效中和抗体的疫苗是可取的。理想情况下，表位的反应性受 在合理开发登革热疫苗的过程中，应减少或消除候选抗原中的增强抗体，以阻止体液免疫反应对这些抗原特征的显性识别。长期目标是将这些分子信息用于合理设计登革热疫苗，以增强保护性中和抗体的诱导，并降低发生严重疾病的风险。 叙述：这份有指导意义的提案概述了雄心勃勃的研究和职业发展，在这些研究中，我将有效地分离中和登革热病毒的人类单抗，并确定它们结合的生化、结构和遗传基础。在确定登革热病毒中和免疫反应的细节的过程中，我将获得 病毒学、免疫学、遗传学和重组抗体技术领域。这些技能，加上教学课程，将帮助我发展我的职业生涯，作为一名内科科学家，朝着独立于导师的方向发展。

DESCRIPTION (provided by applicant): I have over 12 years of experience in the area of viral pathogenesis and immunity. My doctoral studies and postdoctoral training gave me a broad understanding of poxvirus biology, and of the biology of xenotransplantation. My more recent work, which forms the basis of the science in the current application, is focused on generation and study of human monoclonal antibodies to dengue viruses. This information is of timely importance, as it is needed for the rational design of an effective dengue virus vaccine. I am also trained, and board certified, in Internal Medicine and as an Infectious Diseases clinical specialist. My long-term career goal is to remain in academic medicine as a physician-scientist and conduct translational research in immunovirology while growing my clinical skills and knowledge of infectious diseases. By utilizing the knowledge that I gain from this proposed work, I hope to develop my career in a direction independently of my mentor and begin to work on the rational design of a hepatitis C virus vaccine. My surroundings at Vanderbilt are ideal for my proposed project and my career development. The intellectual environment could not be better, and I intend to take full advantage of this by attending a structured program of coursework. My mentor's laboratory has a tremendous amount of experience isolating and studying human monoclonal antibodies, central to my proposal and to the concept of rational vaccine design. My mentor, who is the director of the Vanderbilt Vaccine Center, also has great expertise in vaccinology, immunology, and viral pathogenesis. While at Vanderbilt and under his mentorship, my work has been rapidly evolving so as to launch my career as an independent investigator. Symptomatic dengue virus infection ranges in disease severity from an influenza-like illness to life-threatening shock. One model of the mechanism underlying severe disease proposes that weakly cross-reactive antibodies induced during a primary infection facilitate virus entry into Fc receptor-bearing cells during a subsequent secondary infection. This is thought to increase viral replication and release of cytokines and vasoactive mediators, culminating in shock. This unique process, known as antibody-dependent enhancement of infection, has significantly hindered vaccine development. There is a concern that potent neutralizing antibodies must be generated to all four dengue virus serotypes, as a vaccine that induces weakly cross-reactive, non-neutralizing antibodies may increase the likelihood of developing severe disease upon re-exposure. Much of our understanding of this process has come from studies using mouse mAbs. However, antibody responses in mice typically exhibit less complexity than those in humans. A better understanding of the humoral immune response to natural dengue virus infection in humans is sorely needed. Using a high-efficiency human hybridoma technology developed in our laboratory, it is now possible to generate human hybridomas reliably with B cells from the peripheral blood of individuals who have recovered from an infection. Employing this technology, we have generated over 250 hybridomas secreting human mAbs to dengue virus from subjects who had recovered from primary or secondary infection. The vast majority of these antibodies are broadly serotype cross-reactive, directed against either envelope or pre-membrane protein, and capable of antibody-mediated enhancement of infection. Interestingly, very rare serotype-specific, potently neutralizing antibodies, which are nearly devoid of enhancing activity, are also produced by humans in response to infection. Understanding the epitopes and activity of these naturally-occurring antibodies is critical for vaccine development, as vaccines that induce high potency neutralizing antibodies that lack enhancing activity are desirable. Ideally, the reactivity of epitopes bound by enhancing antibodies should be reduced or eliminated in candidate antigens during the rational development of a dengue vaccine, so as to discourage such dominant recognition of these antigenic features by the humoral immune response. The long-term goal is to use such molecular information in the rational design of dengue vaccines that enhance the induction of protective neutralizing antibodies and reduce the risk of development of severe disease. NARRATIVE: This mentored proposal outlines ambitious studies and career development, in which I will isolate potently neutralization human monoclonal antibodies to dengue viruses and determine the biochemical, structural, and genetic basis for their binding. In the process of determining fine details of the dengue virus neutralizing immune response, I will acquire skills in the areas of virology, immunology, genetics and recombinant antibody techniques. These skills, along with didactic coursework, will help me to develop my career, as a physician-scientist, in a direction independently of my mentor.