Dissection of functional structures of pertussis toxin and its application to prophylaxis and medication of whooping cough

百日咳毒素（Pertussis toxin，PT）是百日咳疫苗中最重要的保护性抗原，具有非常复杂的结构和活性。为了确定PT与其致病性和免疫保护性相关的重要结构和功能特征，对PT不同部分的20种单克隆抗体进行了表征。针对亚基1（S1）和亚基2/3（S2/3）的单克隆抗体在气溶胶攻击系统中显示出高的小鼠保护性，但识别S2和S3之间的共同结构的后者抗体对百日咳博德特氏菌脑内攻击显示出很小的保护性。这两种保护性抗体对感染该生物体的小鼠显示出显著的治疗效果。其他抗体在体外和体内测定系统中也显示出不同的抗毒性活性。这些抗体的毒素中和活性的多样性取决于每种抗体在PT上的识别位点。大多数抗S_1单克隆抗体对CHO细胞凝集和PT的组胺致敏活性无明显中和作用，而抗S_2和/或S_3单克隆抗体对PT的组胺致敏活性有明显中和作用。coli重组子进行分析。本文报道了一株百日咳突变株79 G，它能产生稳定的保护性抗体S234，而不含S1。S1必须是不稳定的，因为它用Tyr取代Cys-41。在E. coli和B.通过定点突变将S1的Arg-9变为Lys和/或Glu-129变为Gly，从而获得百日咳疫苗。这些突变型PT抗原的产生提示S1是PT毒性最强的功能结构，应进一步开展PT致小鼠脑病的研究，以建立人类百日咳脑病动物模型。

Pertussis toxin (PT) having very complicated structure and activities is the most important protective antigen in a pertussis vaccine. In order to define the important structural and functional features of PT associated with its pathogenicity and immunoprotectivity, twenty monoclonal antibodies against different parts of PT were characterized. Monoclonal antibodies against subunit 1 (Sl) and subunit 2/3 (S2/3) showed high mouse protectivity in aerosol challenge system but the latter antibodies which recognize common structure between S2 and S3 showed little protection against intracerebral challenge with Bordetelia pertussis. Both protective antibodies showed a significant therapeutic effect on the mice respiratory-infected with the organisms. The other antibodies also showed diverse anti-toxic activities in vitro and in vivo assay systems. The variety of the toxin-neutralizing activities of these antibodies depends on the recognition site of each antibody on PT. Most of anti-S1 antibodies did not show so high neutralization to CHO- cell clustering or histamine-sensitizing activities of PT although anti-S2 and/or S3 did.Using these monoclonal antibodies, protective antigenicity of various non-toxic PT produced by pertussis mutants or E. coli recombinants were analyzed. Pertussis mutants, 79G, producing stable protective antibens S234 without S1 was developed. The S1 must be unstable because of its replacement of Cys-41 with Tyr. Other genetic toxoids were also developed in E. coli and B. pertussis by change of Arg-9 to Lys and/or Glu-129 to Gly of Sl by site directed mutagenesis. The development of these mutant PT antigens suggests that S1 has the most functional structure for the toxicities of PT.The study on mouse-encephalopathy caused with PT should be continued further more to develop an animal model for pertussis encephalopathy in human.