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Immunization with cholera toxin B subunit induces high-level protection in the suckling mouse model of cholera.

Price GA, McFann K, Holmes RK - PLoS ONE (2013)

Bottom Line: To this end female mice were immunized with CTB intranasally (IN), IP, and subcutaneously (SC).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Colorado School of Medicine, Aurora, CO, USA.

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.

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Related in: MedlinePlus

Serum CTB-specific IgG concentrations at 14 days following the final/only immunization via the intranasal (IN), subcutaneous (SC), or intraperitoneal (IP) route.Each symbol represents the serum anti-CTB antibody concentration for an individual mouse. The horizontal lines represent the geometric mean concentration per group (n = 5). Statistical differences between groups were analyzed using ANOVA with Tukey-Kramer post-test analysis (# P<0.001 and *P<0.05 versus the CTB/IP1X group).
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pone-0057269-g002: Serum CTB-specific IgG concentrations at 14 days following the final/only immunization via the intranasal (IN), subcutaneous (SC), or intraperitoneal (IP) route.Each symbol represents the serum anti-CTB antibody concentration for an individual mouse. The horizontal lines represent the geometric mean concentration per group (n = 5). Statistical differences between groups were analyzed using ANOVA with Tukey-Kramer post-test analysis (# P<0.001 and *P<0.05 versus the CTB/IP1X group).

Mentions: Groups of 5 female CD-1 mice were immunized by the IN, SC, or IP route with 30 µg of CTB on three occasions at 14-day intervals. Serum and fecal samples were collected one day prior to initial immunization and 14 days following the final/only immunization (See Fig. 1 for timetable). Furthermore, two additional groups of mice received IP primary immunizations on booster days to analyze antibody amounts following one or two doses of CTB (also shown in Fig. 1). Using quantitative ELISA, we analyzed serum and fecal samples for CTB-specific responses in all of these immunization groups. IP and SC immunizations with 3 doses of CTB gave the highest serum IgG anti-CTB antibody amounts with the geometric mean titers in these groups being 2.4 mg/ml and 1.6 mg/ml, respectively (Fig. 2). The IN3X and IP2X immunized groups had similar IgG anti-CTB geometric mean antibody titers of 0.84 mg/ml and 0.90 mg/ml, respectively. IP immunization with one dose of CTB gave an anti-CTB IgG geometric mean titer of 0.22 mg/ml. Immunization with three doses of CTB by the IP, SC, or IN route or two doses of CTB by the IP route generated serum anti-CTB IgG titers that were significantly higher than the group receiving only one dose of CTB IP (P<0.001 SC and IP3X; P<0.05 IN and IP2X; Fig. 2). All CTB immunized groups had significantly higher CTB-specific IgG antibodies compared to the PBS control group which had no measureable CTB-specific IgG (P<0.001; data not shown).


Immunization with cholera toxin B subunit induces high-level protection in the suckling mouse model of cholera.

Price GA, McFann K, Holmes RK - PLoS ONE (2013)

Serum CTB-specific IgG concentrations at 14 days following the final/only immunization via the intranasal (IN), subcutaneous (SC), or intraperitoneal (IP) route.Each symbol represents the serum anti-CTB antibody concentration for an individual mouse. The horizontal lines represent the geometric mean concentration per group (n = 5). Statistical differences between groups were analyzed using ANOVA with Tukey-Kramer post-test analysis (# P<0.001 and *P<0.05 versus the CTB/IP1X group).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585264&req=5

pone-0057269-g002: Serum CTB-specific IgG concentrations at 14 days following the final/only immunization via the intranasal (IN), subcutaneous (SC), or intraperitoneal (IP) route.Each symbol represents the serum anti-CTB antibody concentration for an individual mouse. The horizontal lines represent the geometric mean concentration per group (n = 5). Statistical differences between groups were analyzed using ANOVA with Tukey-Kramer post-test analysis (# P<0.001 and *P<0.05 versus the CTB/IP1X group).
Mentions: Groups of 5 female CD-1 mice were immunized by the IN, SC, or IP route with 30 µg of CTB on three occasions at 14-day intervals. Serum and fecal samples were collected one day prior to initial immunization and 14 days following the final/only immunization (See Fig. 1 for timetable). Furthermore, two additional groups of mice received IP primary immunizations on booster days to analyze antibody amounts following one or two doses of CTB (also shown in Fig. 1). Using quantitative ELISA, we analyzed serum and fecal samples for CTB-specific responses in all of these immunization groups. IP and SC immunizations with 3 doses of CTB gave the highest serum IgG anti-CTB antibody amounts with the geometric mean titers in these groups being 2.4 mg/ml and 1.6 mg/ml, respectively (Fig. 2). The IN3X and IP2X immunized groups had similar IgG anti-CTB geometric mean antibody titers of 0.84 mg/ml and 0.90 mg/ml, respectively. IP immunization with one dose of CTB gave an anti-CTB IgG geometric mean titer of 0.22 mg/ml. Immunization with three doses of CTB by the IP, SC, or IN route or two doses of CTB by the IP route generated serum anti-CTB IgG titers that were significantly higher than the group receiving only one dose of CTB IP (P<0.001 SC and IP3X; P<0.05 IN and IP2X; Fig. 2). All CTB immunized groups had significantly higher CTB-specific IgG antibodies compared to the PBS control group which had no measureable CTB-specific IgG (P<0.001; data not shown).

Bottom Line: To this end female mice were immunized with CTB intranasally (IN), IP, and subcutaneously (SC).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Colorado School of Medicine, Aurora, CO, USA.

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.

Show MeSH
Related in: MedlinePlus