Newton S M, Kotb M, Poirier T P, Stocker B A, Beachey E H
Department of Microbiology and Immunology, Stanford University School of Medicine, California 94305.
Infect Immun. 1991 Jun;59(6):2158-65. doi: 10.1128/iai.59.6.2158-2165.1991.
A synthetic 48-bp oligonucleotide specifying the N-terminal 15 amino acids of M protein of Streptococcus pyogenes type 5 (plus a CTA codon, to terminate translation of genes with the insert in reverse orientation) was inserted by blunt-end ligation at the site of the 48-bp EcoRV deletion in the Salmonella flagellin gene in plasmid pLS408 (S. M. C. Newton, C. O. Jacob, and B. A. D. Stocker, Science 244: 70-72, 1989). The resulting plasmid was transferred from Escherichia coli via a restriction-negative Salmonella typhimurium strain into an aromatic-compound-dependent, flagellin-negative live-vaccine strain of Salmonella dublin to produce strain SL7127, which was motile. Expression of the inserted epitope in flagellin and its exposure at the flagellar filament surface were shown by immunoblotting and by the reaction of flagellate bacteria (immobilization, immunogold labeling) with antibody raised by injection of the corresponding synthetic peptide, S-M5(1-15). Rabbits immunized by injection of the live-vaccine strain with flagella composed of the chimeric flagellin or by injection of concentrated flagella from such bacteria developed antibodies reactive in an enzyme-linked immunosorbent assay with peptide S-M5(1-15) and with the large peptic-digest peptide pepM5. These antibodies were opsonic for type 5 streptococci. Mice that were given parenteral live SL7127 (six doses, each 1 x 10(6) to 2 x 10(6), over 8 weeks) developed titers of ca. 12,800 for the M5-specific peptides and opsonizing activity for type 5 streptococci but not for type 24 streptococci. Sera from mice similarly immunized with a control live vaccine strain without an insert in the flagellin gene did not react with the M5-specific antigens. All of the five mice given the control strain, without an insert, died after challenge with type 5 streptococci or type 24 streptococci; by contrast, four of the five mice given strain SL7127, with an insert, survived the M5 challenge, but none of the five challenged with the type 24 strain survived. Therefore, our study shows that an M protein epitope can be expressed in the context of an unrelated protein and maintain its immunogenicity. Furthermore, we demonstrate that mice can be protected against a Streptococcus pyogenes type 5 challenge by immunization with a Salmonella live vaccine with flagella made of flagellin with an insert carrying a protective epitope of M5 protein but without the cross-reactive epitopes of the complete protein.
将一段合成的48个碱基对的寡核苷酸（其编码化脓性链球菌5型M蛋白的N端15个氨基酸，外加一个CTA密码子，用于终止反向插入基因的翻译）通过平端连接插入质粒pLS408中沙门氏菌鞭毛蛋白基因48个碱基对的EcoRV缺失位点（S.M.C.牛顿、C.O.雅各布和B.A.D.斯托克，《科学》244:70 - 72，1989年）。所得质粒通过一个限制阴性的鼠伤寒沙门氏菌菌株从大肠杆菌转移至一株对芳香化合物有依赖性、鞭毛蛋白阴性的都柏林沙门氏菌活疫苗株中，从而产生了有运动能力的SL7127菌株。通过免疫印迹以及鞭毛菌与注射相应合成肽S - M5(1 - 15)产生的抗体的反应（固定化、免疫金标记），证明了插入的表位在鞭毛蛋白中的表达及其在鞭毛丝表面的暴露。用由嵌合鞭毛蛋白组成的鞭毛的活疫苗株注射免疫的兔子，或用来自此类细菌的浓缩鞭毛注射免疫的兔子，产生了在酶联免疫吸附测定中与肽S - M5(1 - 15)以及大的胃蛋白酶消化肽pepM5发生反应的抗体。这些抗体对5型链球菌具有调理作用。经肠胃外给予活的SL7127（8周内分六剂，每剂1×10⁶至2×10⁶）的小鼠，针对M5特异性肽产生了约12,800的滴度，并且对5型链球菌具有调理活性，但对24型链球菌没有。用鞭毛蛋白基因中无插入片段的对照活疫苗株进行类似免疫的小鼠血清，不与M5特异性抗原发生反应。给予无插入片段的对照菌株的所有五只小鼠，在用5型链球菌或24型链球菌攻击后死亡；相比之下，给予有插入片段的SL7127菌株的五只小鼠中有四只在M5攻击中存活，但用24型菌株攻击的五只小鼠无一存活。因此，我们的研究表明，M蛋白表位可以在不相关蛋白的背景下表达并保持其免疫原性。此外，我们证明，用带有携带M5蛋白保护性表位但无完整蛋白交叉反应性表位的插入片段的鞭毛蛋白制成的鞭毛的沙门氏菌活疫苗免疫小鼠，可以使其免受5型化脓性链球菌的攻击。
