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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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Analysis of average weight loss at 24 hours, at 48 hours, and during the 24- to 48-hour interval post infection with V. cholerae.Individual mouse pups were weighed at 24 and 48 hours post-infection and their weight loss was compared to their initial weight at T = 0 (A and B) or 24 hours (C). Error bars represent the SEM. Statistical differences between groups were analyzed by mixed models repeated measures analysis with Tukey-Kramer post-hoc test analysis. Symbols 4 A+B, # and % see Table 2 for statistical comparisons, and @ P<0.0001 vs IP3X and P = 0.0022 vs SC3X groups.
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pone-0057269-g004: Analysis of average weight loss at 24 hours, at 48 hours, and during the 24- to 48-hour interval post infection with V. cholerae.Individual mouse pups were weighed at 24 and 48 hours post-infection and their weight loss was compared to their initial weight at T = 0 (A and B) or 24 hours (C). Error bars represent the SEM. Statistical differences between groups were analyzed by mixed models repeated measures analysis with Tukey-Kramer post-hoc test analysis. Symbols 4 A+B, # and % see Table 2 for statistical comparisons, and @ P<0.0001 vs IP3X and P = 0.0022 vs SC3X groups.

Mentions: As can be seen in figure 4A, there was a striking difference in weight loss at 24 hours between the sham-infected group and the PBS immunized controls, demonstrating that the V. cholerae inoculum was virulent and caused rapid weight loss in the unprotected PBS-immunized group (P<0.0001; Table 2). Though protection from death by 48 hours was high in pups from all CTB immunized groups (Table 1), only pups from the IP3X and SC3X groups exhibited weight losses at 24 hours that did not differ significantly from the sham-infected group (P>0.05; Table 2). Furthermore, the IP3X and SC3X dams had the highest mean anti-CTB serum IgG concentrations, and their pups exhibited the highest survival rates at 48 hours (Fig. 2 and Table 1). Conversely, pups from the IN3X immunization group had the lowest survival at 48 hours (Table 1) and exhibited the greatest weight losses at 24 hours among all of the CTB immunization groups (Fig. 4A). At 24 hours the IN3X group showed significantly greater weight loss than both the IP3X and SC3X groups (P<0.0001 and 0.0022 respectively). These findings were influenced by excessive weight losses and 100% mortality among the pups reared from one mouse as noted above. Removal of these pups from the data analysis demonstrated that the rest of the pups from the IN immunized cohort had weight losses similar to the IP2X and IP1X immunized groups (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)

Analysis of average weight loss at 24 hours, at 48 hours, and during the 24- to 48-hour interval post infection with V. cholerae.Individual mouse pups were weighed at 24 and 48 hours post-infection and their weight loss was compared to their initial weight at T = 0 (A and B) or 24 hours (C). Error bars represent the SEM. Statistical differences between groups were analyzed by mixed models repeated measures analysis with Tukey-Kramer post-hoc test analysis. Symbols 4 A+B, # and % see Table 2 for statistical comparisons, and @ P<0.0001 vs IP3X and P = 0.0022 vs SC3X groups.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585264&req=5

pone-0057269-g004: Analysis of average weight loss at 24 hours, at 48 hours, and during the 24- to 48-hour interval post infection with V. cholerae.Individual mouse pups were weighed at 24 and 48 hours post-infection and their weight loss was compared to their initial weight at T = 0 (A and B) or 24 hours (C). Error bars represent the SEM. Statistical differences between groups were analyzed by mixed models repeated measures analysis with Tukey-Kramer post-hoc test analysis. Symbols 4 A+B, # and % see Table 2 for statistical comparisons, and @ P<0.0001 vs IP3X and P = 0.0022 vs SC3X groups.
Mentions: As can be seen in figure 4A, there was a striking difference in weight loss at 24 hours between the sham-infected group and the PBS immunized controls, demonstrating that the V. cholerae inoculum was virulent and caused rapid weight loss in the unprotected PBS-immunized group (P<0.0001; Table 2). Though protection from death by 48 hours was high in pups from all CTB immunized groups (Table 1), only pups from the IP3X and SC3X groups exhibited weight losses at 24 hours that did not differ significantly from the sham-infected group (P>0.05; Table 2). Furthermore, the IP3X and SC3X dams had the highest mean anti-CTB serum IgG concentrations, and their pups exhibited the highest survival rates at 48 hours (Fig. 2 and Table 1). Conversely, pups from the IN3X immunization group had the lowest survival at 48 hours (Table 1) and exhibited the greatest weight losses at 24 hours among all of the CTB immunization groups (Fig. 4A). At 24 hours the IN3X group showed significantly greater weight loss than both the IP3X and SC3X groups (P<0.0001 and 0.0022 respectively). These findings were influenced by excessive weight losses and 100% mortality among the pups reared from one mouse as noted above. Removal of these pups from the data analysis demonstrated that the rest of the pups from the IN immunized cohort had weight losses similar to the IP2X and IP1X immunized groups (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