Coronavirus vaccine development

当中东呼吸综合征冠状病毒爆发引起全球健康担忧时，我们启动了开发候选疫苗的计划。从Spike糖蛋白(S)序列开始，我们开发了一种由全长S DNA引物和S1亚基蛋白增强组成的免疫策略，该策略可引发针对8种不同MERS-CoV菌株的高滴度中和抗体。免疫血清中含有针对受体结合域（RBD）、S1非RBD部分和S2亚基的强效中和抗体。从免疫小鼠中，我们产生了一组杂交瘤，并产生了单克隆抗体，从中选择了具有高中和活性的品种进行进一步表征。单克隆抗体D12与RBD复合物的原子结构揭示了它们阻断与MERS-CoV受体DPP4结合的两种不同机制。此外，在非人灵长类动物中测定了免疫原性。因此，从S序列设计的疫苗免疫原诱导了多种中和抗体，证明了一种有效的疫苗设计方法，可能适用于其他新兴病毒。对HKU1 β -冠状病毒刺突糖蛋白的结构研究也开始了，以探索结构和受体发现，并产生了刺突三聚体结构解决方案。建立人气道上皮细胞培养系统，启动HKU1的进入和发病机制研究。通过合作，我们已经解决了MERS S蛋白的结构，并基于该结构设计了稳定突变，使S蛋白稳定在其预融合构象中。我们目前正在评估稳定的MERS蛋白的免疫原性，并评估其他冠状病毒刺突蛋白的稳定突变。

When the MERS CoV outbreak raised global health concerns, we initiated a program to develop a candidate vaccine. Starting from Spike glycoprotein (S) sequences, we developed an immunization strategy consisting of a full-length S DNA prime and a S1 subunit protein boost that elicited high titers of neutralizing antibodies against eight different MERS-CoV strains. Immune sera contained potent neutralizing antibodies targeting the receptor binding domain (RBD), non-RBD portions of S1, and the S2 subunit. From the immunized mice, we produced a panel of hybridomas and produced monoclonal antibodies from which a variety with high neutralizing activity were selected for further characterization. The atomic structure of a monoclonal antibody, D12, in complex with the RBD revealed two distinct mechanisms by which they block binding to the MERS-CoV receptor, DPP4. In addition, immunogenicity was measured in nonhuman primates. Thus, vaccine immunogens designed from S sequences induced a diverse repertoire of neutralizing antibodies, demonstrating an efficient approach to vaccine design that may be applicable to other emerging viruses. Structural studies were also initiated with the spike glycoprotein of the HKU1 beta-coronavirus, to explore structure and for receptor discovery and led to a spike trimer structure solution. Human airway epithelial cell culture system was established to initiate entry and pathogenesis studies of HKU1. Through collaborations, we have solved the structure of the MERS S protein and based on this structure, designed stabilizing mutations which stabilize the S protein in its prefusion conformation. We are currently assessing immunogenicity of the stabilized MERS S protein, and evaluating stabilizing mutations in other coronavirus spike proteins.