Immunization with Cholera Toxin B Subunit Induces High-Level Protection in the Suckling Mouse Model of Cholera

Gregory A Price,Randall K Holmes,Kim Mcfann,Luciana C C Leite

doi:10.1371/journal.pone.0057269

Gregory A Price, Randall K Holmes + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0057269

Copy DOI

Abstract

Cholera toxin (CT) is the primary virulence factor responsible for severe cholera. Vibrio cholerae strains unable to produce CT show severe attenuation of virulence in animals and humans. The pentameric B subunit of CT (CTB) contains the immunodominant epitopes recognized by antibodies that neutralize CT. Although CTB is a potent immunogen and a promising protective vaccine antigen in animal models, immunization of humans with detoxified CT failed to protect against cholera. We recently demonstrated however that pups reared from mice immunized intraperitoneally (IP) with 3 doses of recombinant CTB were well protected against a highly lethal challenge dose of V. cholerae N16961. The present study investigated how the route and number of immunizations with CTB could influence protective efficacy in the suckling mouse model of cholera. To this end female mice were immunized with CTB intranasally (IN), IP, and subcutaneously (SC). Serum and fecal extracts were analyzed for anti-CTB antibodies by quantitative ELISA, and pups born to immunized mothers were challenged orogastrically with a lethal dose of V. cholerae. Pups from all immunized groups were highly protected from death by 48 hours (64–100% survival). Cox regression showed that percent body weight loss at 24 hours predicted death by 48 hours, but we were unable to validate a specific amount of weight loss as a surrogate marker for protection. Although CTB was highly protective in all regimens, three parenteral immunizations showed trends toward higher survival and less weight loss at 24 hours post infection. These results demonstrate that immunization with CTB by any of several routes and dosing regimens can provide protection against live V. cholerae challenge in the suckling mouse model of cholera. Our data extend the results of previous studies and provide additional support for the inclusion of CTB in the development of a subunit vaccine against V. cholerae.

Highlights

There are over 200 serogroups of Vibrio cholerae only two, 01 and 0139, are known to cause epidemic/pandemic cholera, which is characterized by acute watery diarrhea [1]
We previously demonstrated that IP immunization with recombinant CTB (rCTB) elicited a high level of protection against challenge by the virulent V. cholerae El Tor strain N16961 in pups reared from CTB or CTB+TcpF immunized mice [34]
We demonstrated here that recombinant CTB is a potent immunogen and generated strong serum CTB-specific IgG responses in adult mice following SC, IP, or IN immunization

Summary

Introduction

There are over 200 serogroups of Vibrio cholerae only two, 01 and 0139, are known to cause epidemic/pandemic cholera, which is characterized by acute watery diarrhea [1]. The O1 serogroup contains two biotypes, the classical and El Tor, and two primary serotypes Inaba and Ogawa [2]. The first 6 cholera pandemics were thought to be caused by the classical biotype, and the current 7th pandemic has been primarily caused by the El Tor biotype with the appearance of the 0139 serogroup in 1992 contributing to the current pandemic [2]. Cholera toxin (CT) is the principal virulence factor responsible for the effusive diarrhea associated with severe cholera infection. The toxic A subunit is tethered non-covalently to the B subunit via the non-toxic A2 domain which passes through the central pore of CTB [3]. The CTB pentamer serves as the binding domain for CT and binds multivalently to cellular surface receptor

Methods

Results

Conclusion