Basic research for the development of mucosal vaccines using cholera toxin B subunit as an adjuvant

为了改变长期实行的几种疫苗的皮下注射免疫程序，我们检测了一些重组霍乱毒素B亚单位(RCTB)鼻内接种疫苗对小鼠的全身和粘膜免疫应答。RCTB与破伤风类毒素(TT)、白喉类毒素(DT)、百日咳成分疫苗或乙肝疫苗(乙肝疫苗)鼻腔联合免疫可诱导高水平的疫苗特异性血清免疫球蛋白G和粘膜免疫球蛋白A抗体滴度。此外，rCTB与TT和DT联合使用，对破伤风毒素攻击和白喉抗毒素滴度分别高于0.1IU/ml的保护水平具有完全保护作用。用rCTB鼻内接种乙肝病毒也能使血清中的抗乙肝病毒抗体效价在100mIU/ml以上，对高危人群或表现出危险行为的人群具有保护作用。人肠毒素ES-…菌毛定植因子抗原在无rCTB的鼻黏膜上皮细胞中掺入更多的大肠埃希菌(ETEC)，并诱导出CFA/III特异性血清免疫球蛋白G和粘膜免疫球蛋白A抗体，表明ETEC鼻腔免疫预防该病的可能性较大。用rCTB和TT鼻腔免疫5次，然后用rCTB+DT鼻腔免疫5次，前一次免疫诱导的抗rCTB抗体对抗DT抗体的产生没有任何影响，尤其是在两次免疫间隔较长的时间。重组CTB对巨噬细胞无毒性作用，对血管通透性无增加作用。此外，rCTB在鼻腔、小肠环和肌肉中未观察到明显的局部组织病理学反应。这些事实表明，rCTB是一种安全有效的黏膜佐剂候选疫苗。我们利用短杆菌构建了肠出血性大肠杆菌志贺毒素I和II B亚单位的高效合成和分泌系统，并对其进行了纯化，以开发预防出血性结肠炎和溶血性尿毒症综合征的鼻内或口服疫苗。较少

For the purpose of changing the immunization procedure of several vaccines from subcutaneous injection, which have been in practice for a long time, to intranasal administration, we have been examining systemic and mucosal immune responses of mice to some vaccines inoculated intranasally with recombinant cholera toxin B subunit (rCTB). Intranasal co-administration of rCTB with tetanus toxoid (TT), diphtheria toxoid (DT), pertussis component vaccine or hepatitis B vaccine (HBV) induced high levels of vaccine-specific serum IgG and mucosal IgA antibody titers. Moreover, a combination of rCTB with TT and DT provided complete protection against tetanus toxin challenge and diphtheria antitoxin titers above a protective level of 0.1 IU/ml, respectively. Intranasal immunization of HBV with rCTB also provided serum anti-HBV antibody titers above 100 mIU/ml protective in people at increased risk or displaying risk behavior. Pilus colonization factor antigen (CFA/III) of human enterotoxigenic Es … More cherichia coli (ETEC) was incorporated into the nasal mucosal epithelium without rCTB and induced CFA/ III-specific serum IgG and mucosal IgA antibody, suggesting the possibility of intranasal vaccine for preventing the disease produced by the ETEC.When five intranasal immunizations of rCTB and TT and subsequent five intranasal administrations with rCTB plus DT were performed, anti-rCTB antibodies induced by the previous vaccination did not have any effects upon the production of anti-DT antibodies, especially, at long intervals between two kinds of inoculations. Recombinant CTB elicited no toxic effects to macrophages and no vascular permeability-increasing effects. Moreover, no distinct local histopathological reactions were observed in the nasal cavity, the small-intestinal loop or the muscle given rCTB.These facts indicate that rCTB is a safe and useful candidate as a mucosal vaccine adjuvant.We finished constructing an efficient synthesis and secretion system for Shiga toxin I and II B subunits of enterohemorrhagic E.coli (EHEC) usins Bacillus brevis and have been purifing them to develop intranasal or oral vaccine for preventing hemorrhagic colitis and hemolytic-uremic syndrome produced by EHEC. Less