中文摘要：

目的 研发高效广谱的人高致病性禽流感病毒H5N1实验疫苗.方法 首先构建了含H5N1（安徽株）结构基因[血凝素（HA）、神经氨酸酶（NA）、基质蛋白M1与M2]的两个双顺反子（HAop/M2,NAop/M1）重组痘苗病毒（rTTV天坛株）疫苗,采用不同剂量（104 PFU或107PFU）或组合（疫苗单独或联合）方式于0、4周二次免疫BALB/c小鼠,初步比较分析抗原特异的体液（HA血凝抑制抗体、NA特异性抗体、中和抗体）与细胞免疫应答（IFN-γ ELISPOT）特点.结果 重组痘苗病毒疫苗可有效表达H5N1靶抗原;高剂量组的重组痘苗病毒疫苗可快速激发较强的针对各个抗原的抗体与针对血凝素与神经氨酸酶蛋白的细胞免疫应答,含血凝素蛋白的重组痘苗病毒疫苗亦可诱导明显的中和抗体;但各组重组痘苗病毒疫苗所激发的针对基质蛋白（M1,M2）的细胞免疫应答均较弱;两个双顺反子（HAop/M2,NAop/M1）重组痘苗病毒疫苗联合应用所激发的针对基质蛋白2（M2）的体液免疫应答明显强于单双顺反子（HAop/M2）疫苗单独应用.结论 本研究中制备的各组重组痘苗病毒疫苗可诱导多个抗原特异的体液与细胞免疫应答,该研究为新型H5N1疫苗的研发及免疫方案的优化奠定了基础.

英文摘要：

Objective To develop an effective and broad immune protective H5N1 vaccine.Methods We first developed two recombinant vaccinia （ Tiantan strain） virus （ rTTV ） based H5N1 vaccines, which consisted of bicistron expressing the hemagglutinin（HA） and matrix protein 2（M2）, or bicistron expressing the neuraminidase（NA） and matrix protein 1 （M1）. The expression of H5N1 protein in rTTVs was confirmed. We immunized the BALB/c mice twice with two kind of dose （ 104 PFU, 107 PFU）using different combination. Subsequently, we assessed the humoral and cellular immune response in vaccinated mice. Results Our data showed that rTTV-based H5N1 vaccine induced rapidly robust HA- and NAspecific antibody level and IFN-γ secreting form cell（SFC） with either single dose of 107 PFU or twice dose of 104 PFU or 107 PFU. We also detected significant neutralizing antibody and matrix-specific immune response. In addition, we found that immunization with two kind of rTTV-based H5N1 vaccines induced much high level of M2-specific antibody than that with single of rTTV-based H5N1 vaccine. Conclusion rTTVbased H5N1 vaccines in this study elicited board array of immunity and our study offers a promising alternative H5N1 vaccine candidates with favorable potential to prevent various H5N1 pandemic.