新型冠状病毒和甲型流感病毒双价DNA疫苗的构建与免疫原性研究  

Construction of a novel bivalent DNA vaccine against SARS-CoV-2 and influenza A virus H3N2 and evaluation of its immunogenicity in mice

作　　者：翟程程 韩頔 邓瑶[2] 任皎[2] 王文[2] 王东红[2] 王文玲[2] 高龙 谭文杰[2] Zhai Chengcheng;Han Di;Deng Yao;Ren Jiao;Wang Wen;Wang Donghong;Wang Wenling;Gao Long;Tan Wenjie(School of Public Health,Baotou Medical College,Inner Mongolia University of Science and Technology,Baotou 014000,China;Key Laboratory of Biosafety,National Health Commission of the People′s Republic of China,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]内蒙古科技大学包头医学院公共卫生学院,包头014000 [2]中国疾病预防控制中心病毒病预防控制所,国家卫生健康委员会生物安全重点实验室,北京102206

出　　处：《中华微生物学和免疫学杂志》2022年第9期683-690,共8页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(82041041);国家重点研发计划(2021YFC0863300)。

摘　　要：目的构建新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)和甲型流感病毒双价DNA疫苗,并在小鼠模型中评价其免疫原性。方法将SARS-CoV-2 Beta突变株S1蛋白编码序列和甲型流感病毒Cambodia(H3N2)株HA蛋白编码序列进行密码子优化合成,采用Over-lapping PCR方法,将两个编码基因通过自裂解2A肽(self-cleaving 2A peptides)序列连接成S1-2A-HA片段,构建能同时表达S1蛋白和HA蛋白的双价DNA疫苗,命名为pVRC-S1-2A-HA,间接免疫荧光和Western blot验证S1蛋白和HA蛋白的表达。将DNA疫苗以肌肉注射加电转途径(IM+EP)免疫BALB/c小鼠,间接ELISA、假病毒中和试验和血凝抑制试验检测小鼠体液免疫应答,IFN-γELISPOT、胞内细胞因子染色(intracellular cytokine staining,ICS)和CBA法检测小鼠细胞免疫应答。结果双价DNA疫苗pVRC-S1-2A-HA可以同时表达S1蛋白和HA蛋白,初次免疫后能够显著诱导小鼠产生针对S1蛋白的特异性细胞免疫和针对HA蛋白的特异性IgG结合抗体;加强免疫后特异性免疫应答都显著增强,针对S1蛋白的特异性IgG结合抗体几何平均滴度(geometric mean titer,GMT)提高至3251,细胞免疫提高至1238 SFC/106个细胞,针对HA蛋白的特异性IgG结合抗体GMT达到45407。ICS法检测到疫苗加强免疫可诱导小鼠CD4+和CD8+T细胞产生IL-2、IFN-γ、TNF-α;CBA结果显示单针免疫后小鼠脾细胞分泌多种细胞因子(IL-2、IL-4、IL-6、IL-10、IFN-γ)。结论本研究成功构建能同时表达SARS-CoV-2 S1蛋白和甲型流感病毒H3N2亚型HA蛋白的双价DNA疫苗,该疫苗可诱导产生针对S1蛋白和HA蛋白的特异性体液免疫和细胞免疫应答,具有较好的研发与应用前景。Objective To construct a bivalent DNA vaccine against SARS-CoV-2 and influenza A virus H3N2 and to evaluate its immunogenicity in mice.Methods The coding sequences for spike 1(S1)protein of SARS-CoV-2 Beta variant and hemagglutinin(HA)of influenza A virus Cambodia(H3N2)strain were codon-optimized and synthesized.The two coding genes were ligated by the self-cleaving 2A peptide using over-lapping PCR to construct S1-2A-HA fragment,which was inserted into pVRC vector to construct the bivalent DNA vaccine,named as pVRC-S1-2A-HA.Indirect immunofluorescence assay(IFA)and Western blot were performed to detect the expression of S1 and HA proteins.BALB/c mice were immunized with pVRC-S1-2A-HA by intramuscular injection and electroporation.The humoral immune responses induced in mice were detected by indirect ELISA,pseudovirus neutralization assay and hemagglutination inhibition assay.Cellular immune responses were detected by IFN-γELISPOT,intracellular cytokine staining(ICS)and cytometric bead array(CBA).Results The bivalent DNA vaccine pVRC-S1-2A-HA could express S1 and HA proteins in vitro.Specific cellular immune responses against S1 protein and specific IgG antibody against HA protein were significantly induced in mice with single-dose immunization.The antigen-specific immunity was significantly enhanced after booster immunization.The geometric mean titer(GMT)of specific IgG antibody increased to 3251 for S1 protein and 45407 for HA protein after two-dose immunization.Moreover,the S1-specific T cells increased to 1238 SFC/106 cells.ICS results indicated that the booster vaccination induced CD4+T and CD8+T cells to produce IL-2,IFN-γand TNF-αin mice.The secretion of various cytokines including IL-2,IL-4,IL-6,IL-10 and IFN-γin mouse splenocytes was induced after single-dose immunization.Conclusions A bivalent DNA vaccine against SARS-CoV-2 and influenza A virus H3N2 was constructed and could induce S1-and HA-specific humoral and cellular immune responses in mice,suggesting the great potential of it for further deve

关 键 词：新型冠状病毒 甲型流感病毒 DNA疫苗 体液免疫 细胞免疫 

分 类 号：R392-33[医药卫生—免疫学]