Human Antibody Cross-Reactivity in Non-Polio Enteroviruses

项目摘要 肠病毒感染的表现多种多样，有几种严重的疾病，如脑膜炎、急性 弛缓性心肌炎（AFM）、心肌炎和新生儿败血症。脊髓灰质炎病毒疫苗在预防脊髓灰质炎方面近乎完美 通过产生强烈的循环抗体反应而导致严重疾病。然而，脊髓灰质炎病毒仅代表 在100多种能够引起人类疾病的肠道病毒中，最近批准的疫苗， 肠道病毒A71证明了其他肠道病毒疾病，在这个例子中是手足口病， 也可以通过接种疫苗来预防。我们之前的工作表明，交叉反应的单克隆抗体 肠道病毒D68的所有分支都可以保护感染的小鼠免受AFM样疾病的影响， 发病与其尝试生产100种疫苗来覆盖每种肠病毒，我们建议了解 人类的抗体反应，可以提供交叉反应免疫许多肠道病毒。与 根据这一知识，可以设计出保留刺激交叉反应的B细胞表位的免疫原。 抗体的我们的第一个目标是确定肠道病毒抗体应答在B细胞上的交叉反应程度 水平我们将补充对数百名健康成年人的血清流行病学研究， 通过研究刺激，其中的人已经对四种肠道病毒自然产生了抗体反应 来自外周血单核细胞室的记忆B细胞。后者将使我们看到如何 健康成人的许多单个B细胞产生抗体，这些抗体在肠道病毒种类之间发生交叉反应， 知识差距，没有现有数据。其次，我们的目标是确定肠道病毒种的分子细节 交叉反应性。我们将创造出能产生单克隆抗体的人类杂交瘤 （mAb），筛选和选择结合多种肠道病毒的mAb。抗原结合片段 与所有免疫反应性肠道病毒的病毒体复合的mAb将用于冷冻电镜检查 研究获得抗体表位的原子分辨率图，在分子水平上详细说明 交叉反应性第三，我们的目标是利用人原代细胞感染来确定抗体作用的机制 模型当我们研究病毒生命周期的哪个阶段时，不同的mAb破坏发挥功能，使用前 体内分化的人上皮细胞培养物将保留病毒使用的天然细胞表面受体 在人体内感染。从这些研究中获得的知识将有助于详细了解 交叉反应抗体如何与肠道病毒结合以及它们如何发挥作用。反过来，mAb可用于 在功能上证实候选免疫原保留了这些交叉反应性B细胞表位。最后是一些 单克隆抗体以后可能有潜力直接用于人类，无论是作为预防或治疗剂。这是一 对于免疫功能低下的人群尤其重要，他们可能无法 在接种后产生强烈的抗体反应。

Project Summary Manifestations of enterovirus infections are variable, with several severe illnesses such as meningitis, acute flaccid myelitis (AFM), myocarditis, and neonatal sepsis. Poliovirus vaccines are near perfect at preventing severe illness by generating strong, circulating antibody responses. However, the polioviruses represent only three of the more than 100 enteroviruses capable of causing human disease. Recently approved vaccines for enterovirus A71 are demonstrating that other enterovirus diseases, in this case hand, foot, and mouth disease, can be prevented by vaccination as well. Our previous work shows that monoclonal antibodies that cross-react to all clades of enterovirus D68 can protect infected mice from AFM-like disease, and even treat disease after onset. Rather than try to make 100 vaccines to cover each enterovirus, we propose to understand the qualities of the human antibody response that could provide cross-reactive immunity across many enteroviruses. With this knowledge, immunogens can be designed that preserve the B cell epitopes that stimulate cross-reactive antibodies. We first aim to determine the degree of cross-reactivity of enterovirus antibody responses at a B cell level. We will complement seroepidemiology studies of hundreds of healthy adults, looking to see what proportion of them have naturally made antibody responses to the four species of enterovirus, with studies of stimulated memory B cells from the peripheral blood mononuclear cell compartment. The latter will allow us to see how many individual B cells in healthy adults produce antibodies that cross-react between enterovirus species, a knowledge gap with no existing data. Second, we aim to determine the molecular details of enterovirus species cross-reactivity using human mAbs. We will create human hybridomas that produce monoclonal antibodies (mAbs), screening and selecting for mAbs that bind to multiple enteroviruses. Antigen-binding fragments of the mAbs, in complex with virions from all immune-reactive enteroviruses, will be used for cryo-electron microscopy studies to obtain atomic resolution maps of antibody epitopes, detailing at a molecular level the determinants of cross-reactivity. Third, we aim to determine mechanisms of antibody action utilizing human primary cell infection models. As we investigate which stage of the viral life cycle different mAbs disrupt to exert function, using ex vivo differentiated human epithelial cell cultures will preserve the natural cell surface receptors used by viruses during in vivo human infection. The knowledge gained from these studies will allow detailed understanding of how cross-reactive antibodies bind to enteroviruses and how they function. In turn, the mAbs can be used to functionally validate that candidate immunogens preserve these cross-reactive B cell epitopes. Finally, some mAbs could later have potential for direct use in humans, either as prophylactic or therapeutic agents. This is an especially important consideration for populations such as the immunocompromised, who may not be able to generate strong antibody responses upon vaccination.