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

为了改变几种疫苗的免疫程序，从长期以来的皮下注射到鼻内给药，我们研究了重组霍乱毒素B亚单位（rCTB）鼻内接种的一些疫苗对小鼠的全身和粘膜免疫反应。rCTB与破伤风类毒素（TT）、白喉类毒素（DT）、百日咳成分疫苗或乙型肝炎疫苗（HBV）鼻内共给药可诱导高水平的疫苗特异性血清IgG和粘膜IgA抗体滴度。此外，rCTB联合TT和DT对破伤风毒素攻击和白喉抗毒素滴度分别高于0.1 IU/ml的保护水平提供了完全的保护。用rCTB鼻内免疫HBV也可为高危人群或表现出高危行为的人群提供高于100 mIU/ml的血清抗HBV抗体滴度保护。在不含rCTB的情况下，更多大肠杆菌（ETEC）被纳入鼻黏膜上皮，并诱导了CFA/III特异性血清IgG和粘膜IgA抗体，提示鼻内疫苗预防ETEC产生的疾病的可能性。经5次鼻内接种rCTB和TT，以及随后5次鼻内接种rCTB加DT，前一次接种诱导的抗rCTB抗体对抗DT抗体的产生没有任何影响，特别是在两次接种间隔较长的时间内。重组CTB对巨噬细胞没有毒性作用，也没有增加血管通透性的作用。此外，在给予rCTB的鼻腔、小肠袢或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是一种安全有效的粘膜疫苗佐剂。我们利用短芽孢杆菌构建了肠出血性大肠杆菌（EHEC）志贺毒素I和II B亚基的高效合成和分泌系统，并对其进行纯化，用于研制预防肠出血性大肠杆菌（EHEC）引起的出血性结肠炎和溶血性尿毒症综合征的鼻内或口服疫苗。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