title: Expression and immunogenicity of a streptococcal M protein epitope inserted in Salmonella flagellin.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, California 94305.
authors.forename: S M
authors.initials: SM
authors.lastname: Newton
authors.forename: M
authors.initials: M
authors.lastname: Kotb
authors.forename: T P
authors.initials: TP
authors.lastname: Poirier
authors.forename: B A
authors.initials: BA
authors.lastname: Stocker
authors.forename: E H
authors.initials: EH
authors.lastname: Beachey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics*; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D002352:Carrier Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D005408:Flagellin / Q000235:genetics* / Q000276:immunology; D015870:Gene Expression; D007111:Immunity, Cellular; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D011817:Rabbits; D012475:Salmonella / Q000235:genetics*; D013290:Streptococcal Infections / Q000276:immunology; D013297:Streptococcus pyogenes / Q000276:immunology; D014613:Vaccines, Attenuated / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D009841:Oligonucleotides; D014613:Vaccines, Attenuated; C014689:streptococcal M protein; D005408:Flagellin