论文部分内容阅读
目的以冷适应、减毒流感活疫苗株为载体,构建并拯救喷鼻重组HAd V疫苗候选株,并评价其免疫效果,为腺病毒候选疫苗研制提供实验数据。方法应用反向遗传学技术,以冷适应、减毒流感活疫苗A/Ann Arbor/6/60ca(H2N2)株为骨架,与季节性流感病毒A/California/07/209疫苗株的血凝素(haemagglutination,HA)及插入HAd V六邻体蛋白抗优势原表位的2个重复序列经改造的神经氨酸酶(neuraminidase,NA)基因构建流感病毒八质粒系统,转染COS-1/MDCK细胞筛选疫苗候选株并鉴定;制备的候选疫苗株滴鼻免疫Balb/c小鼠,通过中和抗体、s Ig A黏膜抗体及细胞因子的测定评价其免疫原性,并用野生HAd V-7病毒株攻击评价免疫保护效果。结果成功拯救获得重组HAd V疫苗候选株,命名为rg Flu/HAd V-Ca,其形态符合流感病毒典型特征,可有效表达HAd V病毒Hexon优势表位,且具有冷适应、减毒表型。小鼠免疫后可产生针对HAd V-7及H1N1流感病毒的特异性中和抗体,肺鼻灌洗液中可检测到针对HAd V-7中和抗体s Ig A抗体,并可检测到脾淋巴细胞分泌HAd V-7特异性细胞因子IFN-γ、IL-4;攻毒实验显示,rg Flu/HAd V-Ca疫苗候选株可有效降低小鼠肺病毒载量。结论重组冷适应、减毒HAd V活疫苗株rg Flu/HAd V-Ca具有良好的免疫效果,为腺病毒候选疫苗的研制提供了新思路。
Objective To construct and rescue recombinant HAd V vaccine candidate strains with cold-adapted and attenuated live attenuated influenza virus strains as vaccine, and evaluate their immunogenicity to provide experimental data for the development of adenovirus vaccine candidates. Methods Reverse genetics technique was used to detect the hemagglutinin of seasonal influenza A / California / 07/209 vaccine strain with cold-adapted and attenuated influenza A / Ann Arbor / 6 / 60ca (H2N2) (HA) and two repeats inserted into the anti-predominant epitope of HAd V hexon protein were used to construct the eight-plasmid system of influenza virus by neuraminidase (NA) gene transfection and transfected into COS-1 / MDCK Cells were screened and identified as vaccine candidate strains. Balb / c mice were immunized intranasally with the candidate vaccine strains prepared and evaluated for immunogenicity by neutralizing antibodies, s Ig A mucosal antibodies and cytokines, and wild-type HAd V-7 virus Strains attack to evaluate immune protection effect. Results The recombinant vaccine of HAd V was successfully rescued and named as rg Flu / HAd V-Ca. Its morphology conformed to the typical characteristics of influenza virus, which effectively expressed Hexon predominant epitope of HAd V virus and had a cold-adapted and attenuated phenotype. The mice were immunized to produce specific neutralizing antibodies to HAd V-7 and H1N1 influenza virus. Anti-HAd V-7 neutralizing antibody s Ig A antibody could be detected in pulmonary and nasal lavage fluid and spleen lymph nodes could be detected The cells secreted HAd V-7 specific cytokines IFN-γ and IL-4. The challenge experiment showed that the rg Flu / HAd V-Ca vaccine candidate could effectively reduce the lung viral load in mice. Conclusion Recombinant cold-adapted and attenuated live attenuated HAd V vaccine strain rg Flu / HAd V-Ca has a good immune effect and provides a new idea for the development of adenovirus candidate vaccine.