Chi-CpG NP的制备及其对rNMB0315蛋白的免疫效果

Preparation of Chi-CpG NP and its immune effects on the recombinant protein rNMB0315

目的制备黏膜免疫佐剂Chi-CpG NP,并检测其对重组B群脑膜炎奈瑟菌0315蛋白(rNMB0315)的免疫效果。方法用壳聚糖纳米颗粒(Chi NP)包裹免疫刺激分子(CpG-ODN)合成Chi-CpG NP,随后检测其理化性质和安全性。将不同黏膜免疫佐剂与rNMB0315充分混合、吸附,滴鼻免疫雌性BALB/c小鼠,检测PBS组、rNMB0315组、rNMB0315+CpG组、rNMB0315+Chi NP组、rNMB0315+Chi-CpG NP组小鼠免疫42天后的体液免疫(IgG、IgG1、IgG2a、sIgA)、细胞免疫水平[白细胞介素(IL)-4、γ干扰素(IFN-γ)、IL-17A]、免疫保护效果以及血清体外杀菌活性。结果成功制备的Chi-CpG NP呈球形,大小均一,有较好的吸附性能和良好的安全性。小鼠免疫42天后,体液免疫和细胞免疫水平rNMB0315+Chi-NP组、rNMB0315+Chi-CpG NP组高于rNMB0315组(P<0.05);rNMB0315+Chi-CpG NP组高于rNMB0315+CpG组和rNMB0315+Chi-NP组(P<0.05)。致死剂量B群脑膜炎奈瑟菌攻击各组小鼠后,rNMB0315+Chi-CpG NP组小鼠存活率最高;血清体外杀菌抗体滴度为1∶4。结论成功制备了Chi-CpG NP。Chi-CpG NP能增强rNMB0315蛋白的免疫效果,提高小鼠的存活率。

Aim To prepare the mucosal adjuvants of Chi-CpG NP,and subsequently immunize mice with recombinant Neisseria meningitides serogroup B 0315 protein(rNMB0315)by using Chi-CpG NP before the observation of immunostimulatory effects.Methods The physicochemical characteristics and safety of Chi-CpG NP were assessed by incorporating immunopotentiators(CpG-ODN)into Chitosan nanoparticles(Chi NP).Female BALB/c mice were immunized intranasally with various mucosal immune adjuvants containing the rNMB0315 protein.On days 42 days after immunization,the humoral(IgG,IgG1,IgG2a,sIgA),cellular(interleukin 4,interferon-γ,interleukin 17A)immunologic responses,as well as the serum bactericidal assay and immune protective effect,were evaluated in the PBS,rNMB0315,rNMB0315+CpG,rNMB0315+Chi NP,and rNMB0315+Chi-CpG NP groups.Results The Chi-CpG nanoparticles demonstrated favorable adsorption and safety properties and exhibited a spherical morphology.On days 42 days after immunization,the levels of humoral and cellular immunity in the rNMB0315+Chi NP group and rNMB0315+Chi-CpG NP group were higher than those in the rNMB0315 group(P<0.05).Moreover,the rNMB0315+Chi-CpG NP group elicited a higher level of humoral and cellular immunity than the rNMB0315+CpG,or the rNMB0315+Chi-NP group(P<0.05).After a lethal dose of Neiseria meningitids serogroup B infection,the survival rate of mice in the rNMB0315+Chi-CpG NP group was the highest,with serum bactericidal titers of 1∶4 against this strain.Conclusion Chi-CpG NP has been successfully prepared.Chi-CpG NP can enhance the immune effects of rNMB0315 protein and improve the survival rate of mice.