Characterization of Vibrio cholerae outer membrane vesicles as a candidate vaccine for cholera

Abstract

Outer membrane vesicles (OMVs) offer a new approach for an effective cholera vaccine. We recently demonstrated that immunization of female mice with OMVs induces a long-lasting immune response and results in protection of their neonatal offspring from Vibrio cholerae intestinal colonization. This study investigates the induced protective immunity observed after immunization with OMVs in more detail. Analysis of the stomach contents and sera of the neonates revealed significant amounts of anti-OMV immunoglobulins (Igs). Swapping of litters born to immunized and nonvaccinated control mice allowed us to distinguish between prenatal and neonatal uptakes of Igs. Transfer of Igs to neonates via milk was sufficient for complete protection of the neonates from colonization with V. cholerae, while prenatal transfer alone reduced colonization only. Detection of IgA and IgG1 in the fecal pellets of intranasally immunized adult mice indicates an induced immune response at the mucosal surface in the gastrointestinal tract, which is the site of colonization by V. cholerae. When a protocol with three intranasal immunizations 14 days apart was used, the OMVs proved to be efficacious at doses as low as 0.025 microg per immunization. This is almost equivalent to OMV concentrations found naturally in the supernatants of LB-grown cultures of V. cholerae. Heterologous expression of the periplasmic alkaline phosphatase (PhoA) of Escherichia coli resulted in the incorporation of PhoA into OMVs derived from V. cholerae. Intranasal immunization with OMVs loaded with PhoA induced a specific immune response against this heterologous antigen in mice. The detection of an immune response against this heterologously expressed protein is a promising step toward the potential use of OMVs as antigen delivery vehicles in vaccine design.

FIG. 1.

Ig titers in sera of neonatal mice from dams immunized with OMVs. Shown are the IgG1 (A) and IgG2 (B) titers in sera collected from litters of i.n., i.g., and i.p. immunized female mice at the first or second challenge period. Each circle represents the specific isotype titer for one independent litter. The horizontal bars indicate the median of results from each data set.

FIG. 2.

Ig titers in the stomach contents of neonatal mice from dams immunized with OMVs. Shown are the IgA (A), IgG1 (B), IgG2 (C), and IgM (D) titers in the stomach contents collected from litters born from i.n., i.g., and i.p. immunized female mice at the first or second challenge period. Each circle represents the specific isotype titer for one independent litter. The horizontal bars indicate the median of results from each data set. One asterisk indicates that the result for one sample of the data set was below the detection limit, and two asterisks indicate that the results for all samples were below the limit of detection.

FIG. 3.

Ig titers and protection of neonatal mice after exchange of immunized and nonvaccinated dams. Shown are the IgA (A), IgG1 (B), and IgG2 (C) titers in sera collected on day 38 or day 78 from i.n. immunized female mice as well as the IgG1 (D) and IgG2 (E) titers in sera collected from litters born to i.n. immunized mice or nonvaccinated control mice (co). The neonatal mice were either kept with their dams (i.n. to i.n.) or swapped to a dam of the other group (i.n. to co or co to i.n.). Each circle represents the specific isotype titer for one immunized female mouse or independent litter. The horizontal bars indicate the median of results from each data set. Panel F shows the number of recovered CFU per small bowel after challenge with V. cholerae for neonatal mice born to the nonvaccinated control mice and kept with their dams (co to co), neonatal mice born to i.n. immunized mice and kept with their dams (i.n. to i.n.), or neonatal mice born to either nonvaccinated control or i.n. immunized mice and then swapped to a dam of the other group (co to i.n. or i.n. to co). Each circle represents the number of CFU for one neonatal mouse. Due to the nature of the experiment (explained in Materials and Methods), the exact number of CFU for the inoculum doses varied from day to day, ranging from 15,100 to 17,000. If no bacteria could be recovered, then the number of CFU was set to the limit of detection (10 CFU/small bowel). The number of mice with detectable colonization/total number of challenged mice is shown above the panel for each group of mice.

FIG. 4.

Ig titers from sera of mice immunized with OMVs. Shown are the median titers over time of IgA (A), IgG1 (B), IgG2 (C), and IgM (D) Igs to OMVs in sera from mice immunized i.n. with 25 μg (solid line), 2.5 μg (dashed line), 0.25 μg (dotted line), or 0.025 μg (dashed and dotted line) OMVs per immunization (n ≥ 5 for each group). The error bars indicate the interquartile range of results from each data set for each time point.

FIG. 5.

Half-maximum total Ig titers in sera of mice immunized with OMVs derived from V. cholerae and the control group. Results are shown for sera taken on day 38 (A) and days 125 to 135 (B) from mice i.n. immunized with different quantities of OMVs. The OMV dose per immunization is given for each group on the x axis. The nonvaccinated control group is indicated by “co.” Each circle represents the half-maximum total Ig titer for one mouse. The horizontal bars indicate the median of results from each data set.

FIG. 6.

Ig titers in fecal pellets of mice immunized with different quantities of OMVs derived from V. cholerae. Shown are the titers IgA (A) and IgG1 (B) extracted from fecal pellets collected on day 38 from mice i.n. immunized with different quantities of OMVs. The OMV dose per immunization is given for each group on the x axis. The nonvaccinated control group is indicated by “co.” Each circle represents the specific isotype titer for one mouse. The horizontal bars indicate the median of results from each data set. The number of samples with detectable Ig titers/total number of samples is shown above the panel for each data set. The values for samples with no detectable isotype titers were set to the respective limits of detection, which were 5 μg/g fecal pellet in the case of IgA and 0.05 μg/g fecal pellet in the case of IgG1.

FIG. 7.

Ig titers in the stomach contents and sera of neonatal mice from dams immunized with OMVs. Shown are the IgA (A) and IgG1 (B) titers in the stomach contents as well as the IgG1 (C) and IgG2 (D) titers in sera collected from litters born to mice i.n. immunized with different quantities of OMVs. The OMV dose per immunization is given for each group on the x axis. The nonvaccinated control group is indicated by “co.” Each circle represents the Ig titer in the pooled sample from neonates of one independent litter. The horizontal bars indicate the median of results from each data set. Values for samples with no detectable isotype titers were set to the limit of detection (0.5 μg/100 mg stomach content or 0.5 μg/ml serum). The number of litters with detectable Igs/total number of independent litters is shown above the panel for the respective group.

FIG. 8.

Protection against colonization with V. cholerae for neonatal mice born to dams immunized with different quantities of OMVs. The numbers of recovered CFU per small bowel are shown on the y axis versus the respective inoculum on the x axis. Results are shown for neonatal mice born to dams of the nonvaccinated control group (A) and groups i.n. immunized with 0.0025 μg (B), 0.025 μg (C), 0.25 μg (D), 2.5 μg (E), and 25 μg (F) OMVs per immunization. Each circle represents the number of CFU for one neonatal mouse. Due to the nature of the experiment (explained in Materials and Methods), the exact number of CFU for each inoculum varied from day to day, ranging from 1,030 to 1,950 and 7,600 to 19,500 for the individual 10-fold dilutions of the inoculation mixture. If no bacteria were recovered, then the number of CFU was set to the limit of detection (10 CFU/small bowel). The number of mice with detectable colonization/total number of challenged mice is shown above the panel for the respective inoculum.

FIG. 9.

Heterologous proteins can be loaded into OMVs derived from V. cholerae and serve as antigens. Panel A shows an immunoblot against PhoA and Coomassie-stained sodium dodecyl sulfate gel (below), loaded with whole-cell lysates of AC108 (lane 1) or AC53 (lane 2) and OMVs derived from AC108 (lane 3) or AC53 (lane 4). Values at left indicate the molecular masses of the protein standards in kilodaltons. The PhoA activities of the individual samples are given in panel B. Furthermore, the half-maximum total Ig titers against OMVs (C) or PhoA (D) in sera of mice i.n. immunized with OMVs derived from AC108 are shown. The nonvaccinated control group is indicated by “co.” Each circle represents the half-maximum total Ig titer for one mouse. The horizontal bars indicate the median of results from each data set.