PDF(Pubmed)
Abstract：
Mucosal vaccines protect against respiratory virus infection by stimulating the production of IgA antibodies that protect against virus invasion of the mucosal epithelium. In this study, a novel protein subunit mucosal vaccine was constructed for protection against infection by the beta coronavirus SARS-CoV-2. The vaccine was assembled by linking a gene encoding the SARS-CoV-2 virus S1 angiotensin converting enzyme receptor binding domain (ACE-2-RBD) downstream from a DNA fragment encoding the cholera toxin B subunit (CTB), a mucosal adjuvant known to stimulate vaccine immunogenicity. A 42 kDa vaccine fusion protein was identified in homogenates of transformed E. coli BL-21 cells by acrylamide gel electrophoresis and by immunoblotting against anti-CTB and anti-ACE-2-RBD primary antibodies. The chimeric CTB-SARS-CoV-2-ACE-2-RBD vaccine fusion protein was partially purified from clarified bacterial homogenates by nickel affinity column chromatography. Further vaccine purification was accomplished by polyacrylamide gel electrophoresis and electro-elution of the 42 kDa chimeric vaccine protein. Vaccine protection against SARS-CoV-2 infection was assessed by oral, nasal, and parenteral immunization of BALB/c mice with the CTB-SARS-CoV-2-ACE-2-RBD protein. Vaccine-induced SARS-CoV-2 specific antibodies were quantified in immunized mouse serum by ELISA analysis. Serum from immunized mice contained IgG and IgA antibodies that neutralized SARS-CoV-2 infection in Vero E6 cell cultures. In contrast to unimmunized mice, cytological examination of cell necrosis in lung tissues excised from immunized mice revealed no detectable cellular abnormalities. Mouse behavior following vaccine immunization remained normal throughout the duration of the experiments. Together, our data show that a CTB-adjuvant-stimulated CTB-SARS-CoV-2-ACE-2-RBD chimeric mucosal vaccine protein synthesized in bacteria can produce durable and persistent IgA antibodies in mice that neutralize the SARS-CoV-2 subvariant Omicron BA.1.1.
摘要：
粘膜疫苗通过刺激IgA抗体的产生来防止呼吸道病毒感染,所述IgA抗体防止病毒侵入粘膜上皮。在这项研究中,构建了一种新型蛋白质亚单位粘膜疫苗,用于预防β冠状病毒SARS-CoV-2的感染.通过将编码SARS-CoV-2病毒S1血管紧张素转换酶受体结合域（ACE-2-RBD）的基因连接到编码霍乱毒素B亚基（CTB）的DNA片段的下游来组装疫苗，一种已知刺激疫苗免疫原性的粘膜佐剂。通过丙烯酰胺凝胶电泳和通过抗CTB和抗ACE-2-RBD初级抗体的免疫印迹,在转化的大肠杆菌BL-21细胞的匀浆中鉴定了42kDa疫苗融合蛋白。通过镍亲和柱色谱法从澄清的细菌匀浆中部分纯化嵌合CTB-SARS-CoV-2-ACE-2-RBD疫苗融合蛋白。通过聚丙烯酰胺凝胶电泳和42kDa嵌合疫苗蛋白的电洗脱来完成进一步的疫苗纯化。通过口服评估疫苗对SARS-CoV-2感染的保护作用，鼻部,并用CTB-SARS-CoV-2-ACE-2-RBD蛋白对BALB/c小鼠进行肠胃外免疫。通过ELISA分析在免疫小鼠血清中定量疫苗诱导的SARS-CoV-2特异性抗体。免疫小鼠的血清中含有IgG和IgA抗体，可中和VeroE6细胞培养物中的SARS-CoV-2感染。与未免疫的小鼠相反，从免疫小鼠切除的肺组织中的细胞坏死的细胞学检查显示没有可检测到的细胞异常。疫苗免疫后的小鼠行为在整个实验期间保持正常。一起,我们的数据表明，在细菌中合成的CTB-佐剂刺激的CTB-SARS-CoV-2-ACE-2-RBD嵌合粘膜疫苗蛋白可以在小鼠中产生持久和持久的IgA抗体，从而中和SARS-CoV-2亚变体OmicronBA.1.1。
