RESUMEN
Because of its production and use in Vietnam, the most widely used oral cholera vaccine consists of heat- or formalin-killed Vibrio cholerae whole cells (WC). An earlier version of this type of vaccine called whole cell-recombinant B subunit vaccine (BS-WC) produced in Sweden also contained the B subunit of cholera toxin (CTB). Both WC and BS-WC vaccines produced moderate levels of protection in field trials designed to evaluate their cholera efficacy. V. cholerae cells in these vaccines induce antibacterial immunity, and CTB contributes to the vaccine's efficacy presumably by stimulating production of anti-toxin neutralizing antibody. Although more effective than the WC vaccine, the BS-WC vaccine has not been adopted for manufacture by developing world countries primarily because the CTB component is difficult to manufacture and include in the vaccine in the doses needed to induce significant immune responses. We reasoned this was a technical problem that might be solved by engineering strains of V. cholerae that express cell-associated CTB that would co-purify with the bacterial cell fraction during the manufacture of WC vaccine. Here we report that construction of a V. cholerae O1 classical strain, O395-N1-E1, that has been engineered to accumulate CTB in the periplasmic fraction by disrupting the epsE gene of type II secretion pathway. O395-N1-E1 induces anti-CTB IgG and vibriocidal antibodies in mice immunized with two doses of formalin killed whole cells. Intraperitoneal immunization of mice with O395-N1-E1 induced a significantly higher anti-CTB antibody response compared to that of the parental strain, O395-N1. Our results suggest that this prototype cholera vaccine candidate strain may assist in preparing improved and inexpensive oral BS-WC cholera vaccine without the need to purify CTB separately.
Asunto(s)
Toxina del Cólera/biosíntesis , Toxina del Cólera/inmunología , Vacunas contra el Cólera/biosíntesis , Vibrio cholerae/inmunología , Vibrio cholerae/metabolismo , Animales , Vacunas contra el Cólera/genética , ADN Bacteriano/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunoglobulina G/biosíntesis , Ratones , Ratones Endogámicos ICR , Mutación , Plásmidos , Vibrio cholerae/genéticaRESUMEN
The most widely used oral whole-cell-recombinant B subunit cholera vaccine contains the nontoxic cholera toxin B subunit (CTXB) and either heat- or formalin-killed Vibrio cholerae O1 strains. Vibrio cholerae O1 strains in the vaccine provide antibacterial immunity, and CTXB contributes to the vaccine's efficacy by stimulating production of anti-CTXB antibody. Various attempts have been made to increase CTXB production. In this study, the mariner-FRT transposon delivery system developed by Chiang and Mekalanos was used to place the ctxB gene under the control of a strong chromosomal promoter in a nontoxigenic V. cholerae El Tor strain, M7922. The expression level of CTXB in transposon insertion mutant clones was screened by ganglioside-dependent enzyme-linked immunosorbent assay. Among CTXB-producing V. cholerae clones that were isolated, M7922-C1 produced the highest amount of CTXB (3.17+/-1.69 microg mL(-1)). M7922-C1 harbors a single insertion of ctxB into VC0972, which encodes a putative porin protein. Although the level of CTXB expression in this strain was not exceptionally high, this study indicates the possibility of using this delivery system to construct vaccine strains that overexpress specific antigens.