腺苷酸环化酶毒素在无细胞百日咳疫苗中的免疫保护效果评价

Evaluation of the immune protective effect of adenylate cyclase toxin in acellular pertussis vaccine

目的利用小鼠百日咳感染模型评估在无细胞百日咳疫苗中加入腺苷酸环化酶毒素的C端结构域(RTX_(751)),能否提升无细胞百日咳疫苗的免疫保护效果。方法 (1)用腺苷酸环化酶毒素的C端结构域与百日咳毒素(pertussis toxin, PT)、丝状血凝素(filamentous hemagglutinin, FHA)、百日咳黏附素(pertussis adhesin, PRN)、氢氧化铝(aluminum hydroxide, Alum)佐剂制备成试验疫苗,对C57BL/6小鼠行腹腔两针免疫。(2)第2针免疫后21 d,用浓度为1×10^(11) CPU/mL的百日咳鲍特菌对小鼠进行气雾攻毒。攻毒后分别在3、7、14、28 d进行取样分析。(3)分析第2次免疫后IgG抗体水平和IgG1/IgG2a比例变化及感染百日咳鲍特菌后不同时间点呼吸道组织细菌载量和细胞因子IFN-γ、IL-17的变化。结果 1/40aP+RTX和1/80aP+RTX的IgG抗体水平高于1/40aP和1/80aP,差异有统计学意义(P<0.05),1/80aP+RTX的IgG2a/IgG1高于1/80 aP,差异有统计学意义(P<0.05);攻毒后14 d, 1/80aP+RTX组肺细菌菌落数最少、气管细菌已清除完毕,IFN-γ、IL-17细胞因子分泌量最低,与Alum组相比差异有统计学意义(P<0.05);Alum组与RTX_(751)组差异无统计学意义(P>0.05)。结论单独的RTX_(751)不能作为保护性抗原;但在小鼠模型中,RTX_(751)加入到无细胞百日咳疫苗可使免疫应答向Th1偏向去增强无细胞百日咳疫苗的免疫保护效果。

Objective A mouse model of pertussis infection was used to evaluate whether adding adenylate cyclase toxin C-terminal domain(RTX751) to acellular pertussis vaccine could enhance the immune protective effect of acellular pertussis vaccine. Methods(1) The experimental vaccine was prepared with the C-terminal domain of adenylate cyclase toxin, pertussis toxin(PT), filamentous hemagglutinin(FHA), pertussis adhesin(PRN) and aluminum hydroxide(Alum) adjuvant. C57 BL/6 mice were immunized twice by intraperitoneal injection.(2) Mice were aerosol challenged with Bordetella pertussis at the concentration of 1×10^(11)CPU/mL 21 days after the second immunization. Samples were collected and analyzed on days 3, 7, 14 and 28 after challenge.(3) The IgG antibody level and IgG1/IgG2 a ratio following the second immunization were detected, and the bacterial load and the levels of IFN-γ and IL-17 in respiratory tract tissue at different time points after infection with Bordetella pertussis were analyzed. Results The IgG antibody level of 1/40 aP+RTX and 1/80 aP+RTX was significantly higher than that of 1/40 aP and 1/80 aP(P<0.05), and the IgG2 a/IgG1 ratio of 1/80 aP+RTX was significantly higher than that of 1/80 aP(P<0.05). The number of the bacteria in lung and trachea from 1/80 aP+RTX group was the lowest, and the levels of cytokines IFN-γ and IL-17 were the lowest at day 14 after challenge, which was significantly lower than that in Alum group(P < 0.05). There was no difference between Alum group and RTX751 group(P>0.05). Conclusion This study shows that RTX751 alone can not be used as a protective antigen;however, in the mouse model, the addition of RTX751 to acellular pertussis vaccine could bias the immune response to Th1 and enhance the immune protective effect of acellular pertussis vaccine.