嵌合鞭毛蛋白和壳聚糖/三聚磷酸(CS/TPP)纳米凝胶封装对鼻接种幽门螺杆菌黏附素诱导的免疫应答的佐剂作用

Adjuvant effects of chimeric flagellin and chitosan/tripolyphosphate (CS/TPP) nanogel encapsulation on immune response induced by nasal vaccination of Helicobacter pylori adhesin

摘要:

[目的] 幽门螺杆菌黏附素A（HpaA）是幽门螺杆菌疫苗的一种有希望的抗原。探索嵌合鞭毛蛋白（cFLN）和壳聚糖/三聚磷酸（CS/TPP）纳米凝胶包封对鼻递送抗原HpaA诱导的免疫应答增强的佐剂作用。[方法] 通过将鼠伤寒沙门氏菌鞭毛蛋白FliC的D0、D1结构域与幽门螺杆菌鞭毛FlaA的D2、D3结构域相结合，构建嵌合鞭毛蛋白cFLN。作为分子内佐剂，嵌合鞭毛蛋白cFLN与黏附素（HpaA）连接构建复合抗原cFLN-HpaA。cFLN、HpaA、cFLN-HpaA在重组大肠杆菌中表达，并通过Ni-NTA预装色谱柱进行纯化。使用离子凝胶法分别将cFLN、HpaA、cFLN-HpaA包封到壳聚糖/三聚磷酸（CS/TPP）纳米凝胶中。[结果] 成功表达和纯化了cFLN、HpaA和cFLN-HpaA，并用离子凝胶法将这3种抗原包封在CS/TPP/纳米凝胶中，制备了包封cFLN的CS/TPP纳米凝胶（cFLN-HpaA NG）、包封HpaA的CS/TPP纳米凝胶（HpaA NG）、包封cFLN-HpaA的CS/TPP/纳米凝胶（cFLN-HpaA NG）。经鼻免疫小鼠后，与HpaA相比，cFLN-HpaA分别导致血清中IgG、IgG1和IgA含量增加2.09倍、1.4倍和2.62倍。与cFLN、HpaA和cFLN-HpaA相比，cFLN NG、HpaA NG和cFLN-HpaA NG分别使血清中IgA、IgG、IgG1和IgG2a的含量增加了1.28至1.71倍。[结论] cFLN和CS/TPP NG可以显著增强鼻腔输送的HpaA诱导的体液免疫。通过检测鼻腔免疫后胃黏膜中IFN-γ、IL-4、IL-17和SIgA的含量，与HpaA相比，cFLN-HpaA分别诱导IFN-γ、IL-4和SIgA的含量增加1.38、1.16和1.58倍。与cFLN-HpaA相比，cFLN-HpaA NG分别使小鼠胃黏膜中IFN-γ、IL-4、IL-17和SIgA的含量增加1.81、1.71、2.16和2.1倍。表明cFLN和CS/TPP NG可以显著增强Th1和Th17型免疫应答。小鼠体内免疫原性研究表明，分子内佐剂cFLN和纳米凝胶包封不仅可以有效增强鼻腔输送HpaA诱导的体液免疫应答，而且还可以增强小鼠胃黏膜中的黏膜免疫应答——Th1和Th17型免疫应答。因此，这些结果表明，cFLN-HpaA NG可能是有希望的经鼻输送的用于预防幽门螺杆菌感染的疫苗系统。

关键词:幽门螺杆菌疫苗;幽门螺杆菌黏附素A;嵌合鞭毛蛋白;壳聚糖基纳米凝胶;黏膜佐剂

Abstract:

[Objective] H. pylori adhesin A (HpaA) is a promising antigen for Helicobacter pylori vaccine. Adjuvant effects of chimeric flagellin cFLN and chitosan/tripolyphosphate (CS/TPP) nanogel encapsulation on humoral and gastric mucosal immune response induced by nasal vaccination of HpaA was investigated. [Methods] Chimeric flagellin cFLN was constructed by combining D0 and D1 domains of Salmonella typhimurium flagellin FliC with D2 and D3 domains of Helicobacter pylori flagellin FlaA. Chimeric flagellin cFLN was then linked with adhesin (HpaA) to construct a complex antigen cFLN-HpaA. cFLN, HpaA and cFLN-HpaA was expressed in recombinant E. coli and further purified by Ni-NTA Prepacked chromatographic column. cFLN, HpaA and cFLN-HpaA were further encapsulated into chitosan/tripolyphosphate (CS/TPP) nanogels to prepare cFLN-loaded CS/TPP nanogels (cFLN NG), HpaA-loaded CS/TPP nanogels (HpaA NG), and cFLN-HpaA-loaded CS/TPP/nanogels (cFLN-HpaA NG) by an ionic gelation method. In vivo immunogenicity studies were performed with mice. [Results] cFLN, HpaA and cFLN-HpaA was successfully prepared and encapsulated within CS/TPP/nanogels. After immunizing mice through the nose, compared with HpaA, cFLN-HpaA caused a 2.09, 1.4 and 2.62-fold increase in serum IgG, IgG1 and IgA levels, respectively. Compared with cFLN, HpaA and cFLN-HpaA, cFLN NG, HpaA NG and cFLN-HpaA NG increased the serum levels of IgA, IgG, IgG1 and IgG2a by 1.28 to 1.71 times. [Conclusion] cFLN and CS/TPP NG can significantly enhance the humoral immunity induced by HpaA delivered in the nasal cavity. The contents of IFN-γ, IL-4, IL-17 and SIgA in gastric mucosa after nasal immunization were detected. Compared with HpaA, cFLN-HpaA induced a 1.38, 1.16 and 1.58-fold increase in the content of IFN-γ, IL-4 and SIgA, respectively. Compared with cFLN-HpaA, cFLN-HpaA NG increased the contents of IFN-γ, IL-4, IL-17 and SIgA in mouse gastric mucosa by 1.81, 1.71, 2.16 and 2.10 times, respectively. It shows that cFLN and CS/TPP NG can significantly enhance Th1 and Th17 immune responses. In vivo immunogenicity studies in mouse indicated that cFLN and nanogel encapsulation could effectively not only enhance hurmoral immune responses, but also gastric mucosal immune response, Th1 and Th17 type immune response in gastric mucosa induced by nasally delivered HpaA. Therefore, these results suggested that cFLN-HpaA NG might be a promising nasally delivered vaccine system for protection against Helicobacter pylori infection.

Key words:Helicobacter pylori vaccine;H. pylori adhesin A;chimeric flagellin;chitosan-based nanogels;mucosal adjuvant