CTB增强PsaA疫苗对肺炎链球菌急性中耳炎的保护作用

Intranasal Immunization with PsaA/CTB Enhances Adaptive Immune Responses and Protection against Pneumococcal Acute Otitis Media

目的研究以霍乱毒素B(CTB)作为PsaA蛋白疫苗黏膜佐剂经鼻腔黏膜途径免疫在BALB/c小鼠体内诱导的特异性免疫应答能力以及对肺炎链球菌急性中耳炎的保护作用。方法 6~8周龄的BALB/c小鼠随机分为3组,分别接受PsaA蛋白免疫(PsaA组,15μg PsaA蛋白),PsaA蛋白及CTB免疫(PsaA/CTB组,15μg PsaA/4μg CTB),CTB免疫(CTB组,4μg CTB),经鼻腔途径免疫,相同剂量和方法每周免疫两次,连续三周,末次免疫后两周收集鼻腔灌洗液、中耳灌洗液及支气管肺泡灌洗液检测特异性IgA抗体反应;收集血清,检测特异性IgG、IgG1及IgG2a抗体反应;末次免疫后2周,小鼠经鼓膜注射14型肺炎链球菌,攻毒后5天组织病理学评估中耳炎症程度。结果PsaA/CTB组鼻腔黏膜免疫获得了较高水平的全面免疫应答:鼻腔、中耳和支气管肺泡灌洗液中特异性IgA抗体水平明显高于PsaA组和CTB组(P<0.05);血清中特异性IgG、IgG1、IgG2a抗体水平明显高于PsaA组和CTB组(P<0.05);攻毒后5天PsaA/CTB组中耳炎症细胞数少于PsaA和CTB组(P<0.05)。结论 CTB是肺炎链球菌蛋白疫苗PsaA有效的黏膜免疫佐剂,黏膜免疫也是PsaA有效的免疫途径,该免疫策略研究为新一代肺炎链球菌疫苗的设计提供了实验基础。

Objective To study specific immune responses and protective effects against pneumococcal acute otitis media in BALB/c mice immunized intranasally with PsaA protein vaccine and cholera toxin B(CTB) as a mucosal adjuvant. Methods BALB/c mice aged 6 to 8 weeks old were randomly divided into 3 groups and were respectively immunized intranasally with PsaA protein(PsaA group, 15 μg PsaA protein), PsaA protein and CTB immunization(PsaA/CTB group, 15μg PsaA/4μg CTB), or CTB(CTB group, 4μg CTB) at the same dose two times weekly for three weeks.Two weeks after the last immunization, nasal lavage fluid(NW), middle ear lavage fluid(MEL) and bronchoalveolar lavage fluid(BALF) were collected for detection of specific IgA antibody. Serum was collected for detection of specific IgG, IgG1 and IgG2 a antibodies. Mice were challenged with type 14 streptococcus pneumoniae by the tympanic membrane route. Middle ear inflammation was evaluated by histopathology 5 days after challenging. Results Compared to mice immunized with PsaA or CTB, anti-PsaA specific IgA in mucosal lavages were elevated significantly in mice immunized with PsaA/CTB(P<0.05); and specific IgG, IgG1 and IgG2 a antibody levels in serum with PsaA/CTB immunization were also significantly higher(P<0.05). Significantly more inflammatory cells in the middle ear cavity were found in PsaA or CTB groups than in the PsaA/CTB group on day 5 after pneumococcal challenge(P<0.05). Conclusions Mucosal immunization with PsaA/CTB is efficacious in generating mucosal and systemic immune responses and in protecting against acute otitis media. CTB is an effective mucosal immune adjuvant for PsaA. The study provides an experimental basis for designinh a new generation of pneumococcal vaccine.