A mucosally targeted MERS-CoV vaccine produced in plants

 描述（由申请方提供）：中东呼吸综合征（MERS）冠状病毒（MERS-CoV）引起严重急性呼吸道疾病，在报告病例中具有高致死率。MERS于2012年首次报道，但该病毒已在骆驼中传播至少20年，骆驼被认为是动物宿主。迄今为止，世界卫生组织已收到1000多例MERS病例报告，其中400多人死亡。虽然大多数病例发生在沙特阿拉伯，并且在某些病例中涉及与骆驼或骆驼产品的接触，但也记录了人与人之间的传播，该病毒被认为具有流行潜力。目前还没有批准的MERS疫苗或治疗药物。 MERS-CoV刺突（S）蛋白介导病毒进入表达病毒受体二肽基肽酶4（DPP 4）的宿主细胞。在S1亚基内，已经鉴定了约200个氨基酸的受体结合结构域（RBD）。用含有S377-588与人IgG 1 Fc融合的重组融合蛋白（S377-588-Fc）免疫小鼠，在免疫动物中诱导高滴度的中和抗体。不幸的是，S377-588-Fc（或RBD-Fc）在哺乳动物细胞培养物中的表达较低，这可能是进一步开发这种有前途的候选疫苗的障碍。 我们将通过使用完善的植物表达系统生产MERS-CoV RBD-Fc及其修饰物来规避生产问题。我们可扩展的瞬时植物表达系统允许快速生产重组蛋白（一周内）;蛋白质生产成本不到哺乳动物细胞系统的10%。我们将生产与小鼠新生儿Fc受体（FcRn）结合增强的MERS-CoV RBD-Fc变体，以检验通过FcRn改善抗原呈递将导致增强免疫应答的假设。 我们将通过分析体液和细胞介导的免疫来评估小鼠中粘膜施用MERS-CoV RBD-Fc的免疫原性。我们将评估RBD特异性抗体的滴度，通过细胞内细胞因子染色分析T细胞应答，然后进行流式细胞术分析，并将测量免疫小鼠血清体外中和活MERS-CoV的能力。

 DESCRIPTION (provided by applicant): Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV) causes severe acute respiratory illness with a high fatality rate among reported cases. MERS was first reported in 2012, but the virus has been circulating in camels, which are thought to be an animal reservoir, for at least 20 years. To date over 1000 MERS cases have been reported to the World Health Organization, including over 400 deaths. While most cases have been in Saudi Arabia and contact with camels or camel products has been implicated in some cases, human-to-human transmission has been documented as well and the virus is thought to have epidemic potential. There are currently no approved vaccines or therapeutics for MERS. The MERS-CoV spike (S) protein mediates viral entry into host cells expressing the viral receptor dipeptidyl peptidase 4 (DPP4). Within the S1 subunit, a Receptor Binding Domain (RBD) of about 200 amino acids has been identified. Immunization of mice with a recombinant fusion protein containing S377-588 fused to human IgG1 Fc (S377-588-Fc) induced high titers of neutralizing antibodies in immunized animals. Unfortunately, expression of S377-588-Fc (or RBD-Fc) in mammalian cell culture is low, which may be an impediment for further development of this promising candidate vaccine. We will circumvent the production problem by producing MERS-CoV RBD-Fc, and modifications thereof, using a well-established plant-expression system. Our scalable transient plant-expression system allows rapid production of recombinant protein (within a week); with protein production costs less than 10% that of mammalian cell systems. We will produce variants of MERS-CoV RBD-Fc with enhanced binding to the mouse neonatal Fc receptor (FcRn), in order to test the hypothesis that improved antigen presentation via FcRn will result in an enhanced immune response. We will evaluate the immunogenicity of mucosally-administered MERS-CoV RBD-Fc in mice, by analyzing both humoral and cell-mediated immunity. We will evaluate titers of RBD-specific antibodies, analyze T cell responses by intracellular cytokine staining followed by flow cytometry analysis, and will measure the ability of sera from immunized mice to neutralize live MERS-CoV in vitro.