Structural and mechanistic insights into antibody neutralization of human metapneumovirus

人类偏肺病毒(Hmpv)是儿童急性呼吸道感染的主要原因，目前正在被 近年来更多地被认为是免疫受损疾病的重要病因，并在 成人慢性阻塞性肺病的加重。尽管发现了近两种人乳头瘤病毒 几十年前，目前还没有疫苗或治疗方法可用于预防或治疗hMPV疾病，以及 我们对自然感染时产生的保护性免疫反应知之甚少。这个 中和抗体的唯一靶点是hMPV融合(F)蛋白。这项研究的总体目标是 项目资助是为了确定人类对hMPV F蛋白的体液免疫反应。生成的数据 这个研究项目解决了我们的总体假设，即有效的中和抗体活性 HMPV是(1)广泛的抗体体细胞突变和(2)F表面特定表位识别的结果 抑制病毒附着和融合过程中关键的结构转变的蛋白质。利用血液 来自人类受试者的样本，人类记忆B细胞将被筛选针对前- 融合和融合后hMPV F蛋白的形式，在这一点上，B细胞将与骨髓瘤细胞融合，以 产生人类杂交瘤。针对hMPV F蛋白的分离的人单抗将被鉴定为 确定亲和力、良好的表位特异性、中和效力，对于选定的单抗，我们将确定 与hMPV F蛋白形成复合体的三维结构。这些数据将为合理的疫苗设计提供依据 一种有效的hMPV疫苗。确定hMPV F蛋白的主要中和表位将为 通过识别对hMPV F免疫重要的氨基酸区域来设计疫苗，这些氨基酸区域可以并入 亚单位或支架疫苗，以诱导靶向抗体。此外，我们将检查这些单抗是否可以预防 体内的hMPV病，这将是临床使用的前奏。在具体目标1中，我们将确定 几种hMPV F-的个体发育、中和机制、亚群反应性和构象特异性 成人和儿童体内的特异性人类单抗。在具体目标2中，我们将确定结构相关性 能中和hMPV的强效抗体。在具体目标3中，我们将确定我们的治疗效果 预防和治疗小鼠和大鼠hMPV病的首选人源单抗 感染的可能性。

Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infection in children, and is being recognized more in recent years as a significant cause of disease in the immunocompromised, and in exacerbations of chronic obstruction pulmonary disease in adults. Although hMPV was discovered nearly two decades ago, no vaccine or therapeutic is currently available for prevention or treatment of hMPV disease, and we have a poor understanding of protective immune responses generated in response to natural infection. The sole target of neutralizing antibodies is the hMPV fusion (F) protein. The overall objective of this research project grant is to determine the human humoral immune response to the hMPV F protein. The data generated from this research project addresses our overall hypothesis that potent neutralizing antibody activity against hMPV is a result of (1) extensive antibody somatic mutation, and of (2) recognition of specific epitopes on the F protein that inhibit structural transitions critical for the viral attachment and fusion processes. Utilizing blood samples from human subjects, human memory B cells will be screened for antibody-specificity targeting the pre- fusion and post-fusion forms of the hMPV F protein, at which point the B cells will be fused to myeloma cells to generate human hybridomas. Isolated human mAbs targeting the hMPV F protein will be characterized to determine affinity, fine epitope specificity, neutralization potency, and for select mAbs we will determine the three-dimensional structure in complex with the hMPV F protein. These data will inform rational vaccine design of an effective hMPV vaccine. Determining the major neutralizing epitopes on the hMPV F protein will inform vaccine design by identifying amino acid regions important for hMPV F immunity, which can be incorporated into subunit or scaffolded vaccines to elicit target antibodies. Additionally, we will examine if these mAbs can prevent hMPV disease in vivo, which will be a prelude to use in the clinic. In Specific Aim 1, we will determine the ontogeny, neutralizing mechanisms, subgroup-reactivity, and conformational specificity of several hMPV F- specific human mAbs in both adults and children. In Specific Aim 2, we will determine the structural correlates of potent antibody neutralization of hMPV. In Specific Aim 3, we will determine the therapeutic efficacy of our top candidate human mAbs for prevention and treatment of hMPV disease in both mice and cotton rat models of infection.