Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused the global pandemic of the Coronavirus disease in late 2019 (COVID-19). Vaccine development efforts have predominantly been aimed at 'Extra-viral' Spike (S) protein as vaccine vehicles but there are concerns regarding 'viral immune escape' since multiple mutations may enable the mutated virus strains to escape from immunity against S protein. The 'Intra-viral' Nucleocapsid (N-protein) is relatively conserved among mutant strains of coronaviruses during spread and evolution. Herein, we demonstrate novel vaccine candidates against SARS-CoV-2 by using the whole conserved N-protein or its fragment/peptides. Using ELISA assay, we showed that high titers of specific anti-N antibodies (IgG, IgG1, IgG2a, IgM) were maintained for a reasonably long duration (> 5 months), suggesting that N-protein is an excellent immunogen to stimulate host immune system and robust B cell activation. We synthesized 3 peptides located at the conserved regions of N-protein among CoVs. One peptide showed as a good immunogen for vaccination as well. Cytokine arrays on post-vaccination mouse sera showed progressive upregulation of various cytokines such as IFN-γ and CCL5, suggesting that TH1 associated responses are also stimulated. Furthermore, vaccinated mice exhibited an elevated memory T cells population. Here, we propose an unconventional vaccine strategy targeting the conserved N-protein as an alternative vaccine target for coronaviruses. Moreover, we generated a mouse monoclonal antibody specifically against an epitope shared between SARS-CoV and SARS-CoV-2, and we are currently developing the First-in-Class humanized anti-N-protein antibody to potentially treat patients infected by various CoVs in the future.

中文翻译：

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针对病毒内保守核衣壳 (N) 蛋白作为针对 SARS-CoV 的新型疫苗。

严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) 于 2019 年底引起了冠状病毒病 (COVID-19) 的全球大流行。疫苗开发工作主要针对作为疫苗载体的“病毒外”刺突(S)蛋白，但人们担心“病毒免疫逃逸”，因为多种突变可能使突变的病毒株逃避针对S蛋白的免疫。在传播和进化过程中，“病毒内”核衣壳（N 蛋白）在冠状病毒突变株中相对保守。在此，我们通过使用整个保守的 N 蛋白或其片段/肽展示了针对 SARS-CoV-2 的新型候选疫苗。使用 ELISA 测定，我们发现高滴度的特异性抗 N 抗体（IgG、IgG1、IgG2a、IgM）可维持相当长的持续时间（> 5 个月），这表明 N 蛋白是刺激宿主免疫的极好免疫原系统和强大的 B 细胞激活。我们合成了 3 个位于 CoV 中 N 蛋白保守区域的肽。一种肽也显示出作为疫苗接种的良好免疫原。疫苗接种后小鼠血清上的细胞因子阵列显示各种细胞因子（例如 IFN-γ 和 CCL5）逐渐上调，表明 TH1 相关反应也受到刺激。此外，接种疫苗的小鼠表现出记忆 T 细胞数量增加。在这里，我们提出了一种非常规疫苗策略，以保守的 N 蛋白为目标，作为冠状病毒的替代疫苗靶点。此外，我们还生产了一种专门针对 SARS-CoV 和 SARS-CoV-2 之间共享表位的小鼠单克隆抗体，目前我们正在开发一流的人源化抗 N 蛋白抗体，以潜在治疗各种 CoV 感染的患者将来。