Key determinants of dengue virus neutralization by naturally occurring human mAbs

描述（由申请人提供）：我在病毒发病机理和免疫领域有超过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，形成了科学的基础，在当前的应用，是集中在产生和研究人类单克隆抗体的登革热病毒。这一信息具有及时的重要性，因为它对于合理设计有效的登革热病毒疫苗是必要的。我还接受过内科和传染病临床专家的培训和认证。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，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。我希望利用我在这项工作中获得的知识，在独立于导师的方向上发展我的事业，并开始从事丙型肝炎病毒疫苗的合理设计工作。我在范德比尔特的环境对我的项目和职业发展都很理想。这里的智力环境再好不过了，我打算充分利用这一点，参加一个结构化的课程项目。我导师的实验室在分离和研究人类单克隆抗体方面有丰富的经验，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理方面也很有专长。在范德比尔特大学期间，在他的指导下，我的工作得到了迅速发展，从而开启了我作为一名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不等。一种严重疾病机制的模型提出，在原发性感染期间诱导的弱交叉反应抗体在随后的继发性感染期间促进病毒进入携带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。这种被称为抗体依赖性感染增强的独特过程严重阻碍了疫苗的开发。令人关切的是，必须产生针对所有四种登革热病毒血清型的强效中和抗体，因为疫苗可诱导弱交叉反应的非中和抗体，可能增加再次接触后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于对小鼠单克隆抗体的研究。然而，小鼠的抗体反应通常没有人类的复杂。迫切需要更好地了解人类对天然登革热病毒感染的体液免疫反应。利用我们实验室开发的高效人类杂交瘤技术，现在可以用感染后恢复的个体外周血中的B细胞可靠地产生人类杂交瘤。利用这项技术，我们从原发性或继发性感染恢复的受试者中产生了250多个分泌登革热病毒单克隆抗体的杂交瘤。这些抗体绝大多数具有广泛的血清型交叉反应性，针对包膜蛋白或膜前蛋白，并且能够通过抗体介导增强感染。有趣的是，人类在应对感染时也会产生非常罕见的血清型特异性、强中和抗体，这些抗体几乎没有增强活性。了解这些天然抗体的表位和活性对疫苗开发至关重要，因为需要诱导缺乏增强活性的高效中和抗体的疫苗。理想情况下，表位结合的反应性

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.