Human Antibody Cross-Reactivity in Non-Polio Enteroviruses

项目摘要 肠道病毒感染的表现是多种多样的，有几种严重的疾病，如脑膜炎、急性 松弛性脊髓炎(AFM)、心肌炎和新生儿败血症。脊髓灰质炎病毒疫苗在预防方面近乎完美 通过产生强烈的循环抗体反应而导致的严重疾病。然而，脊髓灰质炎病毒仅代表 100多种能够引起人类疾病的肠道病毒中的三种。最近批准的疫苗 肠道病毒A71表明，其他肠道病毒疾病，在这种情况下是手足口病， 也可以通过接种疫苗来预防。我们之前的工作表明，具有交叉反应的单抗 对肠道病毒D68的所有分支都能保护感染的小鼠免受AFM样疾病的影响，甚至可以治疗感染后的疾病 开始了。与其尝试制造100种疫苗来覆盖每种肠道病毒，我们建议了解这些疫苗的质量 人类抗体反应可以提供对许多肠道病毒的交叉反应免疫。使用 在这种情况下，免疫原可以被设计成保存刺激交叉反应的B细胞表位 抗体。我们的第一个目标是确定B细胞中肠道病毒抗体反应的交叉反应程度 水平。我们将补充数百名健康成年人的血清流行病学研究，看看 其中一些人对这四种肠道病毒自然产生了抗体反应，并进行了刺激研究 记忆B细胞来自外周血单个核细胞室。后者将让我们看到如何 健康成年人中的许多个体B细胞产生在肠道病毒物种之间交叉反应的抗体，a 没有现有数据的知识鸿沟。第二，我们的目标是确定肠道病毒物种的分子细节 使用人源单抗进行交叉反应。我们将创造产生单抗的人类杂交瘤 (单抗)，筛选与多种肠道病毒结合的单抗。病毒的抗原结合片段 单抗与所有免疫反应肠道病毒的病毒粒子形成复合体，将用于冷冻电子显微镜检查。 研究获得抗体表位的原子分辨率图，在分子水平上详细描述抗体表位的决定因素 交叉反应。第三，我们的目标是利用人类原代细胞感染来确定抗体作用的机制。 模特们。当我们研究不同的mAb干扰病毒生命周期的哪个阶段发挥作用时，使用EX 活体分化的人上皮细胞培养将保留病毒使用的天然细胞表面受体 在体内感染人类的过程中。从这些研究中获得的知识将使我们能够详细地了解 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，mAbs可用于 从功能上验证候选免疫原是否保留了这些交叉反应的B细胞表位。最后，一些 单抗以后可能有潜力直接用于人类，作为预防或治疗药物。这是一个 对于免疫受损人群尤其重要的考虑因素，他们可能无法 在接种疫苗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.