Key determinants of dengue virus neutralization by naturally occurring human mAbs

天然存在的人类单克隆抗体中和登革热病毒的关键决定因素

• 批准号: 8699505
• 负责人: Scott Alan Smith
• 金额: $ 12.61万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-13 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8699505
• 关键词: Affinity; Antibodies; Antibody Formation; Antibody-Dependent Enhancement; Antigens; Area; B-Lymphocytes; Binding; Biochemical; Biology; Biosensor; Blood Circulation; Cells; Clinical; Clinical Skills; Collaborations; Communicable Diseases; Complex; Cryoelectron Microscopy; Dengue; Dengue Virus; Development; Disease; Enhancing Antibodies; Environment; Enzyme-Linked Immunosorbent Assay; Epitopes; Escape Mutant; Exhibits; Family; Fc Receptor; Future; Generations; Genes; Genetic; Germ Lines; Goals; HCV Vaccine; Human; Hybridomas; Immune; Immune Sera; Immune response; Immunity; Immunoglobulin Somatic Hypermutation; Immunology; In Vitro; Individual; Infection; Influenza; Internal Medicine; Knowledge; Laboratories; Life; Maps; Mediating; Mediator of activation protein; Medicine; Membrane Proteins; Mentors; Mentorship; Modeling; Molecular; Monoclonal Antibodies; Mus; Mutate; Phylogeny; Physicians; Population; Poxviridae; Process; Production; Property; Proteins; Recombinant Antibody; Recombinants; Research Personnel; Risk; Science; Scientist; Serotyping; Severity of illness; Shock; Singapore; Site; Specialist; Specificity; Structure; Surface; Techniques; Technology; Training; Translational Research; Universities; Vaccine Design; Vaccines; Variant; Viral; Viral Pathogenesis; Virion; Virus; Virus Diseases; Work; Xenograft procedure; base; bioimaging; career; career development; cytokine; design; experience; functional group; human monoclonal antibodies; improved; neutralizing antibody; novel; peripheral blood; post-doctoral training; programs; pyrosequencing; response; secondary infection; skills; vaccine development; vaccinology; virology; Affinity; Antibodies; Antibody Formation; Antibody-Dependent Enhancement; Antigens; Area; B-Lymphocytes; Binding; Biochemical; Biology; Biosensor; Blood Circulation; Cells; Clinical; Clinical Skills; Collaborations; Communicable Diseases; Complex; Cryoelectron Microscopy; Dengue; Dengue Virus; Development; Disease; Enhancing Antibodies; Environment; Enzyme-Linked Immunosorbent Assay; Epitopes; Escape Mutant; Exhibits; Family; Fc Receptor; Future; Generations; Genes; Genetic; Germ Lines; Goals; HCV Vaccine; Human; Hybridomas; Immune; Immune Sera; Immune response; Immunity; Immunoglobulin Somatic Hypermutation; Immunology; In Vitro; Individual; Infection; Influenza; Internal Medicine; Knowledge; Laboratories; Life; Maps; Mediating; Mediator of activation protein; Medicine; Membrane Proteins; Mentors; Mentorship; Modeling; Molecular; Monoclonal Antibodies; Mus; Mutate; Phylogeny; Physicians; Population; Poxviridae; Process; Production; Property; Proteins; Recombinant Antibody; Recombinants; Research Personnel; Risk; Science; Scientist; Serotyping; Severity of illness; Shock; Singapore; Site; Specialist; Specificity; Structure; Surface; Techniques; Technology; Training; Translational Research; Universities; Vaccine Design; Vaccines; Variant; Viral; Viral Pathogenesis; Virion; Virus; Virus Diseases; Work; Xenograft procedure; base; bioimaging; career; career development; cytokine; design; experience; functional group; human monoclonal antibodies; improved; neutralizing antibody; novel; peripheral blood; post-doctoral training; programs; pyrosequencing; response; secondary infection; skills; vaccine development; vaccinology; virology

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.

描述（由申请人提供）：我在病毒发病机理和免疫力领域拥有超过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。我最近的工作构成了当前应用中科学的基础，重点是对登革热病毒的人类单克隆抗体的产生和研究。该信息及时重要，因为有效的登革热病毒疫苗的合理设计是必需的。我也接受过培训，并获得了内科医学和传染病临床专家的培训和董事会认证。我的长期职业目标是作为医生科学家继续学习学术医学，并在免疫病毒学领域进行转化研究，同时提高我的临床技能和感染性疾病的知识。通过利用我从这项提议的工作中获得的知识，我希望能够独立于导师的方向发展自己的职业，并开始从事乙型肝炎病毒疫苗的理性设计。我在范德比尔特（Vanderbilt）的周围环境非常适合我提议的项目和职业发展。智力环境再好不过了，我打算通过参加结构化课程的课程来充分利用这一点。我的导师的实验室具有巨大的隔离和研究人类单克隆抗体的经验，这是我的提议和理性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，在疫苗学，免疫学和病毒发病机理方面也拥有丰富的专业知识。在范德比尔特（Vanderbilt）和他的指导下，我的工作一直在迅速发展，以使我的职业生涯成为独立调查员。有症状的登革热病毒感染的疾病严重程度从类似流感的疾病到威胁生命的休克。严重疾病的基础机制的一种模型提出，在原发性感染期间诱导的弱交叉反应抗体有助于在随后的继发感染期间进入FC受体受体细胞。人们认为这会增加细胞因子和血管活性介质的释放，最终导致冲击。这种独特的过程被称为抗体依赖性感染的增强，从而显着阻碍疫苗的发育。人们担心必须对所有四种登革热病毒血清型产生有效的中和抗体，作为一种疫苗，可诱导弱交叉反应，非中和抗体，可能会增加重新暴露后发生严重疾病的可能性。我们对这一过程的大部分理解都来自使用小鼠mAB的研究。但是，小鼠的抗体反应通常比人类的抗体反应表现出更少的复杂性。非常需要更好地了解人类天然登革热病毒感染的体液免疫反应。现在使用在我们实验室开发的高效人类杂交瘤技术，现在可以从从感染中恢复过的个体的外周血中可靠地与B细胞可靠地产生人类杂交瘤。采用这项技术，我们已经产生了250多个杂交瘤，将人物mAB分泌到从初次或继发感染中恢复的受试者的登革热病毒中。这些抗体中的绝大多数都是宽性血清型交叉反应性，针对包膜或膜前蛋白，并且能够抗体介导的感染增强。有趣的是，几乎没有增强活性的非常罕见的血清型特异性，有效中和抗体也是由人类响应感染而产生的。理解这些天然抗体抗体的表位和活性对于疫苗的发育至关重要，因为诱导缺乏增强活性的高效力中和抗体的疫苗是可取的。理想情况下，表位的反应性由 在登革热疫苗的合理发展期间，应在候选抗原中降低或消除增强抗体，以通过体液免疫反应来阻止对这些抗原特征的这种主要识别。长期目标是在登革热疫苗的合理设计中使用这种分子信息，从而增强保护性中和抗体的诱导并降低严重疾病的发展风险。 叙述：这项指导的提案概述了雄心勃勃的研究和职业发展，其中我将有效中和人类单克隆抗体对登革热病毒，并确定其结合的生化，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，我将获得技能 病毒学，免疫学，遗传学和重组抗体技术领域。这些技能以及教学课程将帮助我发展自己的职业，作为医生科学家，独立于我的导师的方向。