Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
J Virol. 2018 Apr 27;92(10). doi: 10.1128/JVI.02002-17. Print 2018 May 15.
Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ∼35% mortality. The potential for a future pandemic originating from animal reservoirs or health care-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell cloning strategy, we have identified and characterized multiple neutralizing monoclonal antibodies (MAbs) specifically binding to the receptor-binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific MAbs tended to have greater neutralizing potency than non-RBD S1-specific MAbs. Six RBD-specific and five S1-specific MAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined cocrystal structures for two MAbs targeting the RBD from different angles and show they can bind the RBD only in the "out" position. We then showed that selected RBD-specific, non-RBD S1-specific, and S2-specific MAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD MAbs delayed the emergence of escape mutations in a cell-based virus escape assay. These studies identify MAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization and for developing more effective interventions to prevent or treat MERS-CoV infections. MERS-CoV causes a highly lethal respiratory infection for which no vaccines or antiviral therapeutic options are currently available. Based on continuing exposure from established reservoirs in dromedary camels and bats, transmission of MERS-CoV into humans and future outbreaks are expected. Using structurally defined probes for the MERS-CoV spike glycoprotein (S), the target for neutralizing antibodies, single B cells were sorted from a convalescent human and immunized nonhuman primates (NHPs). MAbs produced from paired immunoglobulin gene sequences were mapped to multiple epitopes within and outside the receptor-binding domain (RBD) and protected against lethal MERS infection in a murine model following passive immunization. Importantly, combining MAbs targeting distinct epitopes prevented viral neutralization escape from RBD-directed MAbs. These data suggest that antibody responses to multiple domains on CoV spike protein may improve immunity and will guide future vaccine and therapeutic development efforts.
中东呼吸综合征冠状病毒（MERS-CoV）可引起高致死性肺部感染，死亡率约为 35%。源于动物宿主或与卫生保健相关事件的未来大流行的潜在风险是一个主要的公共卫生关切。目前尚无针对 MERS-CoV 的疫苗或治疗药物。我们采用基于探针的单个 B 细胞克隆策略，从一名康复的 MERS-CoV 感染患者和免疫接种的恒河猴中鉴定和表征了针对受体结合域（RBD）或 S1（非 RBD）区域的多种中和单克隆抗体（MAb）。RBD 特异性 MAb 的中和效力往往大于非 RBD S1 特异性 MAb。根据竞争测定、中和逃逸变异体的定位和结构分析，6 种 RBD 特异性和 5 种 S1 特异性 MAb 可分为 4 种 RBD 和 3 种非 RBD 不同的结合模式。我们从不同角度确定了针对 RBD 的两种 MAb 的共结晶结构，并表明它们只能在“伸出”位置结合 RBD。然后我们表明，预防性给予选定的 RBD 特异性、非 RBD S1 特异性和 S2 特异性 MAb 可预防 MERS-CoV 在肺部的复制，并保护小鼠免受致死性挑战。重要的是，在基于细胞的病毒逃逸试验中，组合使用 RBD 和非 RBD MAb 可延迟逃逸突变的出现。这些研究鉴定了针对 S 上不同抗原表位的 MAb，这将有助于确定 MERS-CoV 中和的机制，并开发更有效的干预措施来预防或治疗 MERS-CoV 感染。MERS-CoV 引起高致死性呼吸道感染，目前尚无疫苗或抗病毒治疗方法。鉴于在单峰骆驼和蝙蝠中已建立的宿主中持续存在的接触，预计 MERS-CoV 将传播给人类并引发未来的爆发。我们使用针对 MERS-CoV 刺突糖蛋白（S）的结构定义探针（中和抗体的靶标），从康复的人类和免疫接种的非人类灵长类动物（NHP）中分离出单个 B 细胞。从配对免疫球蛋白基因序列中产生的 MAb 被映射到 RBD 内和外部的多个表位，并在通过被动免疫接种保护感染致死性 MERS 的小鼠模型中提供保护。重要的是，组合针对不同表位的 MAb 可防止 RBD 定向 MAb 的病毒中和逃逸。这些数据表明，针对 CoV 刺突蛋白多个结构域的抗体反应可能会改善免疫，并且将指导未来的疫苗和治疗开发工作。
