Evaluation of formalin-inactivated Clostridium difficile vaccines administered by parenteral and mucosal routes of immunization in hamsters

Abstract

Clostridium difficile produces toxins that cause inflammation, necrosis, and fluid in the intestine and is the most important cause of nosocomial antibiotic-associated diarrhea and colitis. We evaluated C. difficile antigens as vaccines to protect against systemic and intestinal disease in a hamster model of clindamycin colitis. Formalin-inactivated culture filtrates from a highly toxigenic strain were administered by mucosal routes (intranasal, intragastric, and rectal) with cholera toxin as a mucosal adjuvant. A preparation of culture filtrate and killed whole cells was also tested rectally. The toxoid was also tested parenterally (subcutaneously and intraperitoneally) and by a combination of three intranasal immunizations followed by a combined intranasalintraperitoneal boost. Serum antibodies against toxins A and B and whole-cell antigen were measured by enzyme-linked immunosorbent assay, neutralization of cytotoxic activity, and bacterial agglutination. The two rectal immunization regimens induced low antibody responses and protected only 20% of hamsters against death and 0% against diarrhea. The intragastric regimen induced high antibody responses but low protection, 40% against death and 0% against diarrhea. Hamsters immunized by the intranasal, intraperitoneal, and subcutaneous routes were 100% protected against death and partially protected (40, 40, and 20%, respectively) against diarrhea. Among the latter groups, intraperitoneally immunized animals had the highest serum anticytotoxic activity and the highest agglutinating antibody responses. Hamsters immunized intranasally and revaccinated intraperitoneally were 100% protected against both death and diarrhea. Protection against death and diarrhea correlated with antibody responses to all antigens tested. The results indicate that optimal protection against C. difficile disease can be achieved with combined parenteral and mucosal immunization.

Physical Characterization of Clostridium Difficile Toxins and Toxoids: Effect of the Formaldehyde Crosslinking on Thermal Stability

“…15 Thus, vaccination using toxoids A and B appears as a promising approach to prevent CDAD and to control reccurences. [10][11][12][13][14][15] C. difficile toxins A and B exhibit 49% amino acid identity and belong to the large clostrodial cytotoxin (LCT) family, which includes the Clostridium sordelii toxins and Clostridium novyi alpha toxin. Toxins A and B possess high molecular weight, an amino-terminal enzymatic domain (e.g., residues 1-543), a central hydrophobic region (e.g., residues 900-1200) and a carboxy-terminal domain carrying carbohydrate recognition sequence repeats (e.g., residues 1750-2710 or 2366).…”

The protective effect of recombinant Lactococcus lactis oral vaccine on a Clostridium difficile-infected animal model

“…Thereafter, the animals were observed 4 times a day on the feces, perianal area, activity, fur morphology, and gloss and mental status. The animals were scored based on the observations as follows [3,4]: 0, normal; 1, light stool, normal activity; 2, wet perianal and tail, close to normal activity; 3, less activity but respond to stimuli; soft abdomen; and 4, wet tail, claws and lower abdomen; curled; no activity; soft abdomen; balance impaired; fur shrug. Animals were killed when the score was 4, and the surviving animals were sacrificed at the end of the experiment on day 35, i.e., 14 days after Clostridium exposure ( Figure 1).…”

“…200 ng/ml) followed by 100 μl of PBS 1-h later; [3] anti-TcdA serum pre-incubation, cells received 100 μl of anti-TcdA serum followed by 100 μl of TcdA (final conc. 200 ng/ml) 1-h later; and [4] anti-TcdA serum treatment, cells received 100 μl of TcdA (final conc. 200 ng/ml) followed by 100 μl of anti-TcdA serum 30-min later.…”

“…Vaccine development involves the establishment of an appropriate animal model and further evaluation of the efficacy and safety of the vaccine. In previous research of C. difficile vaccines, Kink et al [3] successfully established CDI model in hamster by intragastric gavage of 10 5 CFU C. difficile 18-24 h after subcutaneous injection of clindamycin (CLDM, 1 mg per 100 g body weight), and Torres et al [4] administered the animals with 10 5 CFU C. difficile 3-h after CLDM gavage (0.5 mg per 100 g body weight). In general, golden hamster is considered ideal for establishing CDI model, because C. difficile-produced toxins can be neutralized by anti-C. difficile antibodies and CLDM-induced colitis model can be used as animal model of human CDIs.…”

“…Regarding the development of vaccines for C. difficile, great research progress has been made over the last two decades [1]. For example, Torres et al [4] reported that formalin-inactivated C. difficile culture filtrate has a protective effect on hamsters by nasal, peritoneal, or subcutaneous administration. Ryan et al [5] and Ward et al [6] developed recombinant vaccines for C. difficile by engineering a plasmid to express recombinant toxin A proteins from the nontoxic C-terminal receptor binding region of C. difficile toxin A (TcdA) covalently bonded to polysaccharide of other bacterium, and then introducing this plasmid into attenuated Salmonella typhimuriu or Vibrio cholerae.…”