Construction of a Vibrio cholerae Vaccine Candidate Strain O395-N1-E1 which Accumulates Cell-associated Cholera Toxin B Subunit

摘要

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 technical problem that might be solved simply by engineering strains of V. cholerae that express cell-associated CTB that would co-purify with the bacterial cell fraction during manufacture of WC vaccine. Here we report that construction of a V. cholerae O1 classical strain, 0395-N1-E1, that has been engineered to accumulate CTB in the periplasmic fraction by disrupting the epsE gene of type II secretion pathway. 0395-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, 0395-N1. Our results suggest that this type of cholera vaccine candidate strain may assist in preparing improved, effective, and inexpensive oral or parenteral cholera vaccine without the need to purify CTB separately.