Genetic & structural determinants of neutralization of human mAbs to dengue virus

本项目将研究人类抗体在预防或增强感染方面的作用 由血清型3登革热病毒引起。先前存在的异型登革热抗体是危及生命的危险因素 严重的疾病，称为登革出血热（DHF）。尽管众所周知， 异型登革热抗体的诱导可使受试者易患登革出血热， 这个问题没有得到很好的理解。这一问题是有效开发登革热疫苗的主要障碍 因为不清楚四价疫苗制剂是否能够始终保留 所有四种血清型的免疫原性。更好地了解抗体介导的 需要中和感染。 在本申请中提出的工作中，我们将获得大量的人单克隆抗体， 从先前感染登革热病毒血清型3的受试者的B细胞中分离出该病毒。在以前的工作中 在RCE的资助下，我们开发了一种非常可靠和强大的方法来产生人单克隆抗体， 抗体分泌杂交瘤细胞系。我们将确定遗传和结构基础 为了通过抗体的序列分析有效中和登革病毒，产生重组 研究自然发生的体细胞突变的抗体，逃逸突变病毒的产生，定义 亲和力组分的决定因素（开和关速率），以及 免疫显性抗体结合病毒抗原。

This project will investigate the role of human antibodies in protection against or enhancement of infection caused by serotype 3 dengue viruses. Pre-existing heterotypic dengue antibody is a risk factor for lifethreatening severe illness, designated dengue hemorrhagic fever (DHF). Although it is well established that the induction of heterotypic dengue antibodies can predispose subjects to DHF, the molecular basis for this problem is not well understood. This issue is a major obstacle to effective development of dengue vaccines as it is not clear that tetravalent vaccine formulations can be established that always retain the immunogenicity of all four serotypes. Better knowledge of the molecular basis of antibody-mediated neutralization of infection is needed. In the work proposed in this application, we will derive large panels of human monoclonal antibodies directed to dengue virus serotype 3 from the B cells of subjects previously infected with that virus. In previous work funded by the RCE, we have developed a very reliable and robust method for generating human monoclonal antibody secretign hybridoma lines from immune donors. We will determine the genetic and structural basis for effective neutralization of dengue viruses by sequence analysis of antibodies, generation of recombinant antibodies for study of naturally occurring somatic mutations, generation of escape mutant viruses, definition of determinants of components of affinity (on and off rates), and determination of structures of immunodominant antibodies bound to viral antigens.