title: Induction of neutralizing antibodies against diphtheria toxin by priming with recombinant Mycobacterium bovis BCG expressing CRM(197), a mutant diphtheria toxin.
abstract: BCG, the attenuated strain of Mycobacterium bovis, has been widely used as a vaccine against tuberculosis and is thus an important candidate as a live carrier for multiple antigens. With the aim of developing a recombinant BCG (rBCG) vaccine against diphtheria, pertussis, and tetanus (DPT), we analyzed the potential of CRM(197), a mutated nontoxic derivative of diphtheria toxin, as the recombinant antigen for a BCG-based vaccine against diphtheria. Expression of CRM(197) in rBCG was achieved using Escherichia coli-mycobacterium shuttle vectors under the control of pBlaF*, an upregulated beta-lactamase promoter from Mycobacterium fortuitum. Immunization of mice with rBCG-CRM(197) elicited an anti-diphtheria toxoid antibody response, but the sera of immunized mice were not able to neutralize diphtheria toxin (DTx) activity. On the other hand, a subimmunizing dose of the conventional diphtheria-tetanus vaccine, administered in order to mimic an infection, showed that rBCG-CRM(197) was able to prime the induction of a humoral response within shorter periods. Interestingly, the antibodies produced showed neutralizing activity only when the vaccines had been given as a mixture in combination with rBCG expressing tetanus toxin fragment C (FC), suggesting an adjuvant effect of rBCG-FC on the immune response induced by rBCG-CRM(197). Isotype analysis of the anti-diphtheria toxoid antibodies induced by the combined vaccines, but not rBCG-CRM(197) alone, showed an immunoglobulin G1-dominant profile, as did the conventional vaccine. Our results show that rBCG expressing CRM(197) can elicit a neutralizing humoral response and encourage further studies on the development of a DPT vaccine with rBCG.
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil. enmiyaji@uol.com.br
authors.forename: E N
authors.initials: EN
authors.lastname: Miyaji
authors.forename: R P
authors.initials: RP
authors.lastname: Mazzantini
authors.forename: W O
authors.initials: WO
authors.lastname: Dias
authors.forename: A L
authors.initials: AL
authors.lastname: Nascimento
authors.forename: R
authors.initials: R
authors.lastname: Marcovistz
authors.forename: D S
authors.initials: DS
authors.lastname: Matos
authors.forename: I
authors.initials: I
authors.lastname: Raw
authors.forename: N
authors.initials: N
authors.lastname: Winter
authors.forename: B
authors.initials: B
authors.lastname: Gicquel
authors.forename: R
authors.initials: R
authors.lastname: Rappuoli
authors.forename: L C
authors.initials: LC
authors.lastname: Leite
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis*; D001500:BCG Vaccine / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology*; D004167:Diphtheria Toxin / Q000276:immunology*; D007114:Immunization; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D014614:Vaccines, Synthetic / Q000276:immunology*
chemical_list: D000907:Antibodies, Bacterial; D001500:BCG Vaccine; D001426:Bacterial Proteins; D004167:Diphtheria Toxin; D014614:Vaccines, Synthetic; C028581:CRM197 (non-toxic variant of diphtheria toxin)