Kimura A, Mountzouros K T, Schad P A, Cieplak W, Cowell J L
Bacteriology Research Department, Praxis Biologics, Rochester, New York 14623.
Infect Immun. 1990 Oct;58(10):3337-47. doi: 10.1128/iai.58.10.3337-3347.1990.
Bordetella pertussis TOX3201 has a 12-base-pair insertion in the S1 subunit gene of pertussis toxin (PTX), which encodes for a 4-amino-acid insertion between residues 107 and 108 of the mature S1 subunit (Black et al., Science 240:656-659, 1988). This mutant strain has been shown to secrete a holotoxin analog of PTX, designated CRM3201, with reduced ADP-ribosyltransferase activity. In the present study, we evaluated the biochemical, biological, and immunoprotective activities of purified CRM3201. Assay of enzymatic activities showed that CRM3201 had 20 to 30% of the ADP-ribosyltransferase activity and 55 to 60% of the NAD glycohydrolase activity of native PTX. CRM3201, however, had only 2 to 6% of the activity of PTX in clustering CHO cells, promoting leukocytosis, inducing histamine sensitization, and potentiating an anaphylactic response to bovine serum albumin. In contrast, activities associated with the B oligomer (binding to fetuin, hemagglutination of goose erythrocytes, and lymphocyte mitogen activity) were comparable to those of native PTX. Injection of BALB/c mice with CRM3201 mixed with Al(OH)3 elicited high titers of antibody to PTX (as measured by enzyme-linked immunosorbent assay), which neutralized a leukocytosis-promoting dose of PTX in these mice and neutralized PTX in a CHO cell assay. Passive transfer of the anti-CRM3201 antibody protected 20-day-old Swiss-Webster mice against a lethal aerosol challenge with B. pertussis 18323. Active immunization with CRM3201 significantly reduced lung colonization in adult BALB/c mice with a B. pertussis respiratory infection. These results demonstrate (i) that the reduced ADP-ribosyltransferase activity of CRM3201 is associated with reductions in certain biological and toxic activities of PTX (the enzymatic and biological activities are not, however, totally concordant); (ii) that CRM3201 possesses a functional B oligomer; and (iii) that CRM3201 can induce toxin-neutralizing antibodies which protect mice against a respiratory challenge with B. pertussis. Our studies with CRM3201 show that recombinant analogs of PTX have the potential to be developed into safe, protective immunogens for use in new acellular pertussis vaccines.
百日咳博德特氏菌TOX3201在百日咳毒素（PTX）的S1亚基基因中有一个12个碱基对的插入，该插入导致在成熟S1亚基的第107和108位氨基酸之间编码插入4个氨基酸（Black等人，《科学》240:656 - 659，1988年）。已证明该突变菌株分泌一种PTX的全毒素类似物，命名为CRM3201，其ADP - 核糖基转移酶活性降低。在本研究中，我们评估了纯化的CRM3201的生化、生物学和免疫保护活性。酶活性测定表明，CRM3201具有天然PTX的ADP - 核糖基转移酶活性的20%至30%以及NAD糖水解酶活性的55%至60%。然而，在使CHO细胞聚集、促进白细胞增多、诱导组胺致敏以及增强对牛血清白蛋白的过敏反应方面，CRM3201仅具有PTX活性的2%至6%。相比之下，与B寡聚体相关的活性（与胎球蛋白结合、鹅红细胞血凝以及淋巴细胞促有丝分裂活性）与天然PTX的活性相当。用与氢氧化铝混合的CRM3201注射BALB/c小鼠可引发高滴度的抗PTX抗体（通过酶联免疫吸附测定法测量），该抗体在这些小鼠中中和了促白细胞增多剂量的PTX，并在CHO细胞测定中中和了PTX。抗CRM3201抗体的被动转移保护20日龄的瑞士 - 韦伯斯特小鼠免受百日咳博德特氏菌18323致死性气溶胶攻击。用CRM3201进行主动免疫显著减少了成年BALB/c小鼠因百日咳博德特氏菌呼吸道感染而导致的肺部定植。这些结果表明：（i）CRM3201降低的ADP - 核糖基转移酶活性与PTX的某些生物学和毒性活性降低相关（然而，酶活性和生物学活性并非完全一致）；（ii）CRM3201具有功能性B寡聚体；（iii）CRM3201可诱导毒素中和抗体，保护小鼠免受百日咳博德特氏菌的呼吸道攻击。我们对CRM3201的研究表明，PTX的重组类似物有潜力被开发成安全的保护性免疫原，用于新型无细胞百日咳疫苗。
