呼吸道合胞病毒DNA疫苗的构建和免疫效果的初步观察

目的 构建呼吸道合胞病毒（RSV）的DNA疫苗并对其免疫效应进行观察，为RSV的免疫预防提供新思路。方法 构建表达RSV-F蛋白的质粒pcD-F，接种BALB/c小鼠后以RSV long株进行攻击，于攻击当日和攻击后第5、14天采用ELISA和ELISPOT方法分别检测小鼠RSV特异性IgG抗体和分泌RSV特异性IFN-γ的淋巴细胞，荧光定量PCR方法检测小鼠肺组织RSV RNA的含量。肺组织切片行苏木精-伊红染色，观察RSV long株攻击后肺组织病理改变。 结果 pcD-F免疫小鼠后，产生RSV特异性IgG抗体（滴度1∶60），RSV攻击后第14天， pcD-F免疫小鼠的特异性IgG抗体水平升至1∶250，明显高于对照组（P<0.05）。RSV攻击后，pcD-F免疫小鼠分泌RSV特异性IFN γ的淋巴细胞升至99个/1×105细胞，明显高于对照组的9个/1×105细胞（P<0.05）。 pcD-F免疫小鼠的肺部炎症反应明显轻于对照组，肺部RSV得到有效的清除。结论 成功构建的RSV-DNA疫苗具有良好的免疫原性。

Construction of respiratory syncytial virus DNA vaccine and study of immune efficacy

Objective Respiratory syncytial virus（RSV） is one of the principal causes of bronchiolitis and pneumonia in young children. There is no safe and effective vaccine. A DNA vaccine against RSV was constructed and its immune efficacy in mice was investigated.Methods The full length fusion genes of an RSV long strain were subcloned into pcDNA3.1(-) and the pcD-F constructs were transfected into Hela cells. Protein expressions were evaluated by Western blot analysis of lysed cells. Specific pathogen free, female BALB/c mice, 6- to 7- weeks old, were inoculated with pcD-F by intramuscular (im) immunization and challenged by RSV long strains. On 0, 5, and 14 days after RSV challenge, 6-7 mice were killed and blood and lung specimens were collected at each time point. ELISA and ELISPOT assays were applied respectively for determination of RSV specific antibody titers in serum and IFN-γ producing cells in spleens. RSV-RNA copies in lung tissues were detected by real time PCR. Sections stained with hemotoxylin and eosin were examined by microscopy for evidence of lung inflammation.Results The band recognized by the anti RSV-F monoclonal antibody was observed from pcD-F transfected Hela cells. The average geometric anti-RSV antibody titer was 1∶60 in mice vaccinated with pcD-F plasmid before challenge. A significantly higher titer (1∶250) was produced in mice immunized with pcD-F on 14 days post challenge compared with controls, P<0.05. The RSV specific IFN-γ producing cells in vaccinated mice were 99 spots/1×105 cells on 5 days post challenge, significantly higher than in the controls (9 spots/1×105 cells). Control mice also showed a slightly increased RSV specific cellular response on 5 days (9 spots/1×105 cells) and 14 days (18 spots/1×105 cells) after challenge, but this response was significantly lower than that in im immunization group. The vaccinated mice showed milder pulmonary inflammatory changes in lungs and effective RSV clearance compared with controls on 5 days post RSV challenge.Conclusions The study demonstrated that pcD-F was an effective DNA vaccine against RSV. Our study provides implication for the prevention strategy of RSV infection.