A DNA vaccine targeting the receptor-binding domain of Clostridium difficile toxin A

Abstract

Clostridium difficile is a pathogen with increasing severity for which host antibody responses provide protection from disease. DNA vaccination has several advantages compared to traditional vaccine methods, however no study has examined this platform against C. difficile toxins. A synthetic gene was created encoding the receptor-binding domain (RBD) of C. difficile toxin A, optimized for expression in human cells. Gene expression was examined in vitro. Mice were inoculated and then challenged with parenteral toxin A. Vaccination provided high titer antibodies and protected mice from death. This represents the first report of DNA vaccine inducing neutralizing antibodies to C. difficile toxin A.

Figure 1. A schematic description of C. difficile toxin A and the vaccine vectors

A Linear depiction of the three major domains identified within C. difficile toxin A. (Modified from Voth and Ballard. Clin Micro Reviews, April 2005; p 247–263.) The region containing the vaccine sequence is indicated by the underline. B. Schematic depiction of the vaccine gene sequence as inserted into the eukaryotic expression vector, pVAX. These plasmids differ only in the presence or absence of a tissue plasminogen activator (tPA) signal peptide sequence.

Figure 2. Protein expression from vaccine vectors or control following transient transfection in vitro

Immunoblot of 293T cell lysates and supernatants following transient transfection with pVAX encoding green-fluorescent protein (Mock), TxA-RBD in pVAX, or tPA-TxA-RBD in pVAX for detection of expressed protein products. Supernatant was clarified at 20,000 × g for 30 minutes prior to the procedure. The expected size of the expressed product is 100kD. Identical preparations were examined for the presence of human β-actin using a monoclonal antibody specific for β-actin as a volume loading control.

Figure 3. Schematic representation of the vaccination sequence

AAnimal groups, vaccine doses, and vaccine delivery mechanism for subsequent immunological analysis for both BALB/c and CD-1 mice. B. Vaccine inoculation sequence, timing of blood harvest, and final toxin challenge for both BALB/c and CD-1 mice.

Figure 4. Toxin-specific antibody responses in BALB/c mice following the vaccination procedures

Serum anti-toxin IgG ELISA responses in BALB/c mice. The data is the result of two independent experiments. Values are representative of two independent ELISA procedures. Group-specific titers were compared using Kruskal-Wallis test for overall significance (p< 0.0001). Asterisk (*) represents statistical significance between group titers at p < 0.05 following pair-wise one-way ANOVA with Bonferroni correction for multiple comparisons.

Figure 5. Toxin-specific antibody responses in CD-1 mice following the vaccination procedures

Serum anti-toxin IgG ELISA responses in CD-1 mice representing data from of two independent experiments. Group-specific antibody titers were compared using a Kruskal-Wallis test for overall significance (p< 0.0001). Asterisk (*) represents statistical significance between group titers at p < 0.05 following pair-wise one-way ANOVA with Bonferroni correction for multiple comparisons. Symbol (§) indicates p> 0.05 for the indicated pairwise comparison.

Figure 6. Survival in vaccinated BALB/c mice following challenge with purified C. difficile toxin A

Kaplan Meier survival curve in vaccinated BALB/c mice following challenge with 300ng of freshly reconstituted toxin A from C. difficile in 100μl of sterile saline. The data is the result of two independent experiments. Statistical (p) value represents the likelihood of a significant difference between all groups following pair-wise log-rank analysis between groups.

Figure 7. Survival in vaccinated CD-1 mice following challenge with purified C. difficile toxin A

Kaplan Meyer survival curve in vaccinated CD-1 mice following challenge with 300ng of freshly reconstituted toxin A from C. difficile in 100μl of sterile saline. The data is the result of two independent experiments. Statistical (p) value represents the likelihood of a significant difference between all groups following pair-wise log-rank analysis between groups. “NS” indicates differences not significant p>0.05 following pair-wise log rank analysis.