title: Adjuvant activity of Mycobacterium bovis BCG expressing CRM197 on the immune response induced by BCG expressing tetanus toxin fragment C.
abstract: In order to develop a combined recombinant Mycobacterium bovis BCG (rBCG) vaccine against diphtheria, pertussis and tetanus (DPT), we have constructed different strains of rBCG expressing tetanus toxin fragment C (FC), driven by the up-regulated M. fortuitum beta-lactamase promoter, pBlaF*. Tetanus toxin FC was expressed in comparable levels in native form or in fusion with the beta-lactamase exportation signal sequence; however, in both constructs it was localized to the cytosol. Immunization of mice with rBCG-FC or its combination with rBCG expressing CRM197, induced anti-tetanus toxin antibodies with a Th2 immunoglobulin profile. Administration of a subimmunizing dose of the diphtheria-tetanus toxoid vaccine showed that rBCG-FC primed mice for production of an intense humoral response. Interestingly, the combination of rBCG-FC and rBCG-CRM197 reduced the time required for maturation of the immune response and increased anti-tetanus toxin antibody levels, suggesting adjuvant properties for rBCG-CRM197; this combination induced 75% protection in mice challenged with 100 minimum lethal doses (MLD) of tetanus toxin. Antisera from guinea pigs immunized with this combination were shown to neutralize tetanus toxin and diphtheria toxin. Our results suggest reciprocal adjuvant effects of rBCG-FC and rBCG-CRM197, which may contribute to induction of a more effective immune response against both diseases.
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, Av Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil.
authors.forename: Rogerio P
authors.initials: RP
authors.lastname: Mazzantini
authors.forename: Eliane N
authors.initials: EN
authors.lastname: Miyaji
authors.forename: Waldely O
authors.initials: WO
authors.lastname: Dias
authors.forename: Dirce
authors.initials: D
authors.lastname: Sakauchi
authors.forename: Ana Lúcia T O
authors.initials: AL
authors.lastname: Nascimento
authors.forename: Isaías
authors.initials: I
authors.lastname: Raw
authors.forename: Nathalie
authors.initials: N
authors.lastname: Winter
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gicquel
authors.forename: Rino
authors.initials: R
authors.lastname: Rappuoli
authors.forename: Luciana C C
authors.initials: LC
authors.lastname: Leite
mesh_terms: D000276:Adjuvants, Immunologic; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000917:Antibody Formation / Q000276:immunology; D001500:BCG Vaccine / Q000276:immunology*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D004167:Diphtheria Toxin / Q000037:antagonists & inhibitors; D015721:Diphtheria-Tetanus-Pertussis Vaccine / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009163:Mycobacterium bovis / Q000235:genetics* / Q000254:growth & development / Q000276:immunology*; D009500:Neutralization Tests; D012703:Serotyping; D013744:Tetanus Toxin / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000276:immunology*; D017778:Vaccines, Combined; D014614:Vaccines, Synthetic / Q000276:immunology; D014709:Vero Cells
chemical_list: D000276:Adjuvants, Immunologic; D000907:Antibodies, Bacterial; D001500:BCG Vaccine; D001426:Bacterial Proteins; D004167:Diphtheria Toxin; D015721:Diphtheria-Tetanus-Pertussis Vaccine; D013744:Tetanus Toxin; D017778:Vaccines, Combined; D014614:Vaccines, Synthetic; C028581:CRM197 (non-toxic variant of diphtheria toxin)