Key determinants of dengue virus neutralization by naturally occurring human mAbs

描述（由申请人提供）：我在病毒发病机制和免疫领域有超过12年的经验。我的博士研究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，形成了当前应用的科学基础，主要集中在登革热病毒的人单克隆抗体的产生和研究。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。我还接受过内科和传染病临床专家的培训并获得委员会认证。我的长期职业目标是作为一名医生科学家留在学术医学领域，并在免疫病毒学方面进行转化研究，同时提高我的临床技能和传染病知识。通过利用我从这项拟议工作中获得的知识，我希望独立于我的导师发展我的职业生涯，并开始研究丙型肝炎病毒疫苗的合理设计。我在范德比尔特的环境对我提出的项目和我的职业发展都是理想的。智力环境不能更好，我打算充分利用这一点，参加课程的结构化计划。我导师的实验室在分离和研究人类单克隆抗体方面有着丰富的经验，这对我的建议和合理疫苗设计的概念至关重要。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好的专业知识。在范德比尔特工作期间，在他的指导下，我的工作迅速发展，从而开始了我作为独立调查员的职业生涯。症状性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不等。严重疾病的潜在机制的一个模型提出，在原发性感染期间诱导的弱交叉反应性抗体促进病毒在随后的继发性感染期间进入携带Fc受体的细胞。这被认为会增加病毒复制和细胞因子和血管活性介质的释放，最终导致休克。这种独特的过程被称为抗体依赖性感染增强，严重阻碍了疫苗的开发。令人担忧的是，必须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性、非中和抗体的疫苗可能会增加再次接触时发生严重疾病的可能性。我们对这一过程的理解大部分来自使用小鼠mAb的研究。然而，小鼠中的抗体应答通常表现出比人类中的抗体应答更低的复杂性。我们迫切需要更好地了解人类对自然登革病毒感染的体液免疫反应。使用我们实验室开发的高效人杂交瘤技术，现在可以用从感染中恢复的个体的外周血中的B细胞可靠地产生人杂交瘤。采用这种技术，我们已经产生了超过250个分泌人单克隆抗体的杂交瘤登革病毒从谁已经从原发性或继发性感染恢复的主题。这些抗体中的绝大多数具有广泛的血清型交叉反应性，直接针对包膜或前膜蛋白，并且能够抗体介导的感染增强。有趣的是，人类在感染后也会产生非常罕见的特异性、强效中和抗体，这些抗体几乎没有增强活性。了解这些天然存在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力中和抗体的疫苗是期望的。理想情况下，与表位结合的表位的反应性 在登革热疫苗的合理开发过程中，候选抗原中的增强抗体应当减少或消除，以阻止体液免疫应答对这些抗原特征的这种显性识别。长期目标是利用这些分子信息合理设计登革热疫苗，增强保护性中和抗体的诱导，降低严重疾病的发展风险。 说明：这个指导建议概述了雄心勃勃的研究和职业发展，其中我将分离出有效中和登革热病毒的人单克隆抗体，并确定其结合的生物化学，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，我将获得以下技能： 病毒学、免疫学、遗传学和重组抗体技术领域。这些技能，沿着教学课程，将帮助我发展我的职业生涯，作为一个医生，科学家，在一个独立的方向，我的导师。

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.