重组人肠道病毒71型疫苗效价测定方法建立及免疫原性研究

目的：建立重组人肠道病毒71型（EV71）疫苗效价测定方法以及免疫原性研究。方法：将BALB/c小鼠按不同周龄、免疫途径和免疫剂量分组，共5组，分别于d天免疫，14、21、28 d采血，采用经典微量细胞病变法进行EV71中和抗体阳转率的检测。另外将6~8周龄小鼠分成6组（10只·组^-1），剂量范围（1~0.0312 μg），0 d免疫，28 d采血进行中和抗体效价测定，同时取脾淋巴细胞采用ELISPOT法测定经体外刺激分泌IFN-γ的水平，并取细胞上清液采用ELISA法测定分泌IFN-γ，IL-2和IL-4的水平。结果：通过比较各组血清中和抗体阳转率可以得出：效价测定最佳条件为：6~8周龄雌性BALB/c小鼠，免疫途径为腹腔注射，0 d免疫，28 d采血，剂量范围0.5~0.0039 μg。BALB/c小鼠经1次免疫后，EV71疫苗组小鼠血清中和抗体效价呈剂量效应关系，所有EV71疫苗组小鼠脾淋巴细胞分泌的IFN-γ SFC值均显著高于佐剂组（P<0.01）；EV71疫苗组分泌IFN-γ和IL-2水平显著高于佐剂组（P<0.05），并且EV71疫苗组也能分泌一定量的IL-4。经统计分析，ELISPOT法与ELISA法检测IFN-γ水平的结果高度相关（r=0.751）。结论：建立了一种重组肠道病毒71型疫苗效价测定的方法，EV71疫苗能够使BALB/c小鼠产生具有剂量效应关系的体液免疫反应，并且还能诱导BALB/c小鼠脾淋巴细胞产生特异性IFN-γ、IL-2和IL-4细胞免疫反应的应答，为后续的EV71疫苗研发及质量控制提供了一定基础。

Objective: To establish a potency assay method and evaluate immunogenicity of recombinant human enterovirus 71(EV71)vaccine. Methods: BALB/c mice were divided into 5 groups by age,immunity approach and immunization dose,and immunized at day 0. Serum samples were collected at day 14,21 and 28 after injection. The positive conversion rates of neutralizing antibody against EV71were tested by using classic micro-cytopathic method. Moreover,six-to eight-week-old female BALB/c mice were divided into six groups of 10 mice in each group. Dosage ranged from 1 μg to 0.0312 μg. Each mouse was vaccinated at day 0 and its blood was drawn at day 28 to test the neutralizing antibody titers. Meanwhile splenic lymphocytes were tested for their ability to secrete IFN-γ in response to in vitro stimulation,using the ELISPOT assay. The cell culture supernatant samples were analyzed for the secretion levels of IFN-γ,IL-2 and IL-4 by ELISA. Results: By comparing the serum neutralizing antibody seroconversion rates of all groups,it was demonstrated that the best condition for potency determination was achieved with 6-8 week old,female BALB/c mice immunized through intraperitoneal injection. Under this condition,mice were vaccinated at day 0 and their blood was drawn at day 28,dosage ranged from 0.5 μg to 0.0039 μg. The neutralizing antibody titers of BALB/c mice immunized once with EV71 vaccine were dose-dependent. The spot-forming cells(SFCs)of IFN-γ secreted by splenocytes in groups immunized with EV71 vaccine were all significantly higher than those with adjuvant(P<0.01). The secretion levels of IFN-γ and IL-2 in groups immunized by EV71 vaccine were significantly higher than those with adjuvant(P<0.05)and some groups of EV71 vaccine could secrete IL-4. The results of IFN-γ level by ELISPOT and ELISA were highly correlated(r=0.751). Conclusion: A potency assay for EV71 vaccine was developed. The EV71 vaccine could induce dose-dependent humoral immune response of BALB/c. Vaccination with EV71 vaccine induced a cellular immune response as demonstrated by IFN-γ,IL-2 and IL-4 production in splenocytes of BALB/c mice. The article provides a foundation for the further research and quality control of EV71 vaccine.