Adenovirus-based vaccination against Clostridium difficile toxin A allows for rapid humoral immunity and complete protection from toxin A lethal challenge in mice

Abstract

Clostridium difficile associated diarrhea (CDAD) is a critical public health problem worldwide with over 300,000 cases every year in the United States alone. Clearly, a potent vaccine preventing the morbidity and mortality caused by this detrimental pathogen is urgently required. However, vaccine efforts to combat C. difficile infections have been limited both in scope as well as to efficacy, as such there is not a vaccine approved for use against C. difficile to date. In this study, we have used a highly potent Adenovirus (Ad) based platform to create a vaccine against C. difficile. The Ad-based vaccine was able to generate rapid and robust humoral as well as cellular (T-cell) immune responses in mice that correlated with provision of 100% protection from lethal challenge with C. difficile toxin A. Most relevant to the clinical utility of this vaccine formulation was our result that toxin A specific IgGs were readily detected in plasma of Ad immunized mice as early as 3 days post vaccination. In addition, we found that several major immuno-dominant T cell epitopes were identified in toxin A, suggesting that the role of the cellular arm in protection from C. difficile infections may be more significant than previously appreciated. Therefore, our studies confirm that an Adenovirus based-C. difficile vaccine could be a promising candidate for prophylactic vaccination both for use in high risk patients and in high-risk environments.

Fig. 1

Adenovirus based vaccine against Clostridium difficile toxin A is able to induce rapid and robust TA-specific humoral responses in mice. WT BALB/c mice were IM injected (10¹⁰ vp/mouse) with Ad5-Null (n = 5) or Ad5-C. difficile-TA (n = 5). At 3 dpi (A), 7 dpi (B) and 14 dpi (C) plasma samples were collected and total TA-specific IgG measured by ELISA as described in Section 2. Naïve mice (n = 5) were utilized as baseline control and these values were subtracted Ad-injected values. The error bars represent ±SD. Statistical analysis was completed using two-tailed Student’s t-test to compare 2 groups of virus-injected animals (*p < 0.05). One of two representative experiments shown.

Fig. 2

Adenovirus based vaccine against Clostridium difficile toxin A is able to induce robust TA-specific T cell responses, several clusters of immunogenic T cell epitopes revealed in non-enzymatic TA domain. (A) WT BALB/c mice were IM injected (10¹⁰ vp/mouse) with Ad5-C. difficile-TA (n = 3). Mice were sacrificed at 14 dpi, splenocytes prepared, pooled and stimulated with 2 μg/well of single peptides from a 15-mer-peptide library, spanning the C. difficile TA region, encoded by Ad-TA vaccine, followed by IFNγ ELISPOT, performed as described in Section 2. Naïve (n = 3) pooled mice were stimulated with same individual peptides as baseline control. Six clusters of immunogenic T cell epitopes was identified (grey) in non-enzymatic TA domain, two major immunodominant epitopes determined were #13 and #63. One of two representative experiments shown. (B) Representative pictures of wells from ELISpot are shown.

Fig. 3

Adenovirus based vaccine against Clostridium difficile toxin A is able to induce robust TA-specific T cell responses. WT BALB/c mice were IM injected (10¹⁰ vp/mouse) with Ad5-C. difficile-TA (n = 5). Mice were sacrificed at 14 dpi splenocytes prepared, and individually stimulated with pool of 12 peptides from the TA peptide library (each peptide 0.2 μg/well) and (A) IFNγ or (B) IL-2 ELISPOT was performed as described in Section 2. Naïve (n = 5) mice were stimulated with same individual peptides as baseline control. One of two representative experiments shown. Bars represent mean ± SD. Statistical analysis was completed using Two-Way ANOVA with a Bonferroni post hoc test (stimulations × treatments), p < 0.05 was deemed a statistically significant difference. *, **Statistically different from those in naïve mice (for the same stimulation), p < 0.05, p < 0.001, respectively.

Fig. 4

Ad5-Clostridium difficile-TA vaccine induces robust TA-specific T cell responses in contrast to Ad5-Null. WT BALB/c mice were IM injected (10¹⁰ vp/mouse) with Ad5-C. difficile-TA (n = 5) or Ad5-Null (n = 5). Mice were sacrificed at 14 dpi splenocytes prepared, and individually stimulated with 0.2 μg/well of single most immunogenic peptides from the TA library (or with inactivated Ad5 vector). IFNγ ELISPOT was performed as described in Section 2. Naïve (n = 3) mice were stimulated with same peptides as baseline control. Bars represent mean ± SD. Statistical analysis was completed using Two-Way ANOVA with a Bonferroni post hoc test (stimulations × treatments), p < 0.05 was deemed a statistically significant difference. *, **Statistically different from those in naïve mice (for the same stimulation), p < 0.05, p < 0.001, respectively; ^{#, ##}Significant inductions over Ad5-Null group within the same stimulation, p < 0.05, p < 0.001, respectively. One of two representative experiments shown.

Fig. 5

Ad5-Clostridium difficile-TA vaccine induces pleiotropic TA-specific T cell responses in contrast to Ad5-Null. WT BALB/c mice were IM injected (10¹⁰ vp/mouse) with Ad5-C. difficile-TA (n = 5) or Ad5-Null (n = 5). Mice were sacrificed at 14 dpi splenocytes prepared, and individually stimulated with 0.2 μg/well of single most immunogenic peptides from the TA library (or with inactivated Ad5 vector). (A) IL-4 and (B) IL-2 ELISPOT was performed as described in Section 2. Naïve (n = 3) mice were stimulated with same peptides as baseline control. Bars represent mean ± SD. Statistical analysis was completed using Two-Way ANOVA with a Bonferroni post hoc test (stimulations × treatments), p < 0.05 was deemed a statistically significant difference. *, **Statistically different from those in naïve mice (for the same stimulation), p < 0.05, p < 0.001, respectively; ^{#, ##}Significant inductions over Ad5-Null group within the same stimulation, p < 0.05, p < 0.001, respectively. One of two representative experiments shown.

Fig. 6

Ad5-Clostridium difficile-TA vaccine induces robust TA-specific CD8 T cell specific responses in contrast to Ad5-Null. WT BALB/c mice were IM injected (10¹⁰ vp/mouse) with Ad5-C. difficile-TA (n = 3) or Ad5-Null (n = 3). Mice were sacrificed at 14 dpi splenocytes prepared, and individually stimulated with 2 μg/well of peptide #63 or with mixture of peptides (#9, #13, #51, #55, #63, all 0.4 μg/well), stained and FACS sorted as described in Section 2. The bars represent mean ± SEM. Statistical analysis was completed using a two tailed homoscedatic Student’s t-test.

Fig. 7

Ad5-Clostridium difficile-TA vaccination completely protects mice from lethal TA challenge. (A) WT BALB/c mice were IM injected (10¹⁰ vp/mouse) with Ad5-Null (n = 5) or Ad5-C. difficile-TA (n = 5). At 14 dpi mice were IP challenged with 300 ng (6 × LD₅₀ ) of purified toxin A, purchased from Calbiochem. Kaplan–Meyer survival curves are shown. Curves were compared by log-rank analysis and were found to be significantly (p < 0.05) different. (B) WT BALB/c mice were left uninjected (naïve, n = 11) or were IM injected (10¹⁰ vp/mouse) with Ad5-Null (n = 8) or Ad5-C. difficile-TA (n = 7). At 14 dpi mice were IP challenged with 300 ng (6 × LD₅₀ ) of purified toxin A, purchased from List Biological Laboratories Inc. Kaplan–Meyer survival curves are shown. Curves were pair-wise compared by log-rank analysis and the following results were obtained: naïve versus Ad5-TA curve were significantly (p = 0.0043) different, Ad5-Null versus Ad5-TA curves were significantly (p = 0.036) different, however naïve versus Ad5-Null curves were not significantly different (p = 0.486).

Fig. 8

Ad5-TA vaccinated mice had overall reduced portal, periportal and lobular hepatic inflammation as compared to unvaccinated mice, when challenged with toxin A. WT BALB/c mice were IM injected (10¹⁰ vp/mouse) with Ad5-Null or Ad5-C. difficile-TA. At 14 dpi mice were challenged with 300 ng (6 × LD₅₀ ) of purified toxin A (IP). At 14 dpi survivors from both groups were sacrificed, liver sections were stained with H&E and morphometric evaluation of these sections was performed as described in Section 2. (A) Representative sections from each treated animal were analyzed, scored and averaged for the levels of portal, periportal and lobular inflammation, as described in Section 2. The sum of averages for each category was computed to obtain a total inflammation index score. The error bars represent ±SD. Unvaccinated censored mice (n = 2) were used as positive control (high inflammation). Statistical analysis was completed using two-tailed Student’s t-test to compare 2 groups of virus-injected animals: Ad5-Null injected (n = 4) and Ad5-TA injected (n = 4). There was a trend of reduced inflammation in vaccinated mice, however, no significant differences was detected. (B) Representative liver sections for each group of mice are shown. Arrows indicate the following: (1) complete lobular disarray, with pooling of erythrocytes throughout the tissue subsequent to hemorrhage; (2) glassy eosinophilic hyalin deposition; (3) necrosis; (4) areas of cellular swelling indicating cell stress.