Recombinant lipoprotein-based vaccine candidates against C. difficile infections

Abstract

Background: Opportunistically nosocomial infections in hospitalized patients are often related to Clostridium difficile infections (CDI) due to disruption of the intestinal micro-flora by antibiotic therapies during hospitalization. Clostridial exotoxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) in the host intestine, disrupt the intestinal barrier leading to acute inflammation and diarrhea. The C-terminal receptor binding domain of TcdA (A-rRBD) has been shown to elicit antibody responses that neutralize TcdA toxicity in Vero cell cytotoxicity assays, but not effectively protect hamsters against a lethal dose challenge of C. difficile spores. To develop an effective recombinant subunit vaccine against CDI, A-rRBD was lipidated (rlipoA-RBD) as a rational design to contain an intrinsic adjuvant, a toll-like receptor 2 agonist and expressed in Escherichia coli.

Results: The purified rlipoA-RBD was characterized immunologically and found to have the following properties: (a) mice, hamsters and rabbits vaccinated with 3 μg of rlipoA-RBD produced strong antibody responses that neutralized TcdA toxicity in Vero cell cytotoxicity assays; furthermore, the neutralization titer was comparable to those obtained from antisera immunized either with 10 μg of TcdA toxoid or 30 μg of A-rRBD; (b) rlipoA-RBD elicited immune responses and protected mice from TcdA challenge, but offered insignificant protection (10 to 20 %) against C. difficile spores challenge in hamster models; (c) only rlipoA-RBD formulated with B-rRBD consistently confers protection (90 to 100 %) in the hamster challenge model; and (d) rlipoA-RBD was found to be 10-fold more potent than A-rRBD as an adjuvant to enhancing immune responses against a poor antigen such as ovalbumin.

Conclusion: These results indicate that rlipoA-RBD formulated with B-rRBD could be an excellent vaccine candidate for preclinical studies and future clinical trials.

Fig. 1

The expression and purification of rlipoA-RBD. The expression and the purity of rlipoA-RBD were confirmed by SDS-PAGE (a) and western blot with TcdA-specific monoclonal antibody (b). The purification of rlipoA-RBD as shown in panel a, lanes 2 to 4 were crude extract loaded on the first Ni-affinity column, eluent of 200 and 500 mM imidazole, respectively. The purity of rlipoA-RBD obtained from IMAC were confirmed by SDS-PAGE (panel a, lane 5) and western blot with TcdA-specific monoclonal antibody (panel b, lane 7). The first lane in each panel was the molecular markers

Fig. 2

MALDI-TOF analysis of tryptic digested lipopeptide fragments of rlipoA-RBD. The N-ternminal tryptic digested fragments were analyzed by MLADI-TOF using the procedure described in previous report [32]

Fig. 3

Up-regulation of surface biomarkers of BMDC by rlipoA-RBD. BMDC from C57BL/6 was collected and treated with GM-CSF on days 0 and 3. A-rRBD and rlipoA-RBD were treated on day 6 for 18 h, then DC were collected to analyze their surface markers, including CD-40 (a), CD-86 (b), CD-80 (c), and MHC-II (d) by flow cytometry. All groups were divided into polymyxin B (PMB) treated (black-net bar) or without (white bar) to validate insignificant LPS contamination. All surface marker signaling was normalized by calculating the ratio of mean fluorescence intensity (MFI) between medium control and treatments. The experiments had been performed at least three times

Fig. 4

Cytokine secretion from BMDC treated with rlipoA-RBD. After BMDC was treated with rlipoA-RBD on day 6 for 18 h, the culture supernatants were collected and analyzed for cytokine profiles using specific cytokine ELISA: (a) IL-6, (b) IL12p40, and (c) TNF- α. The experiments had been performed at least three times

Fig. 5

Adjuvant effect of rlipoA-RBD. To demonstrate adjuvant effect of rlipoA-RBD, the enhancement of anti-OVA IgG response was evaluated by co-administration of rlipoA-RBD and OVA. BALB/c mice were immunized with 3 × 2 μg of OVA formulated either with or without various amounts of rlipoA-RBD (0.3 and 3 μg), 10 μg of A-rRBD, or alum as positive control. Serum titer was determined by RBD-specific ELISA

Fig. 6

Mouse anti-RBD antibody responses elicited by different dosages of A-rRBD or rlipoA-RBD. BALB/c mice were immunized three times with either 3, 10 or 30 μg doses of A-rRBD; or with 3, 10 or 30 μg doses of rlipoA-RBD. Anti-RBD titers of mouse sera obtained at 0, 2, 4, 6, and 16 weeks were determined by RBD-specific ELISA

Fig. 7

Mouse protection elicited by rlipoA-RBD against lethal TcdA challenge. Panel a, BALB/c mice (10 mice per group) were challenged with a lethal dose of TcdA after three immunizations of either rlipoA-RBD or A-rRBD (0.3 and 3 ug). PBS served as the negative control. The final survival rates were reported. Panel b, BALB/c mice (10 mice per group) were challenged with a lethal dose of TcdA after three immunizations of either rlipoA-RBD (0.3 and 3 μg) or 30 μg of B-rRBD

Fig. 8

C. difficile spore challenge in hamster model studies. Panel a, five groups of hamsters (n = 6) were gastrically inoculated with 100 CFU of C. difficile (the dose can kill >50 % of hamsters) at 2 weeks after the third immunization with either PBS, A-rRBD, rlipoA-RBD, or rlipoA-RBD + B-rRBD. The final survival rates were reported. Panel b, The number of C. difficile colonies (CFU) grown in TCCFA selective medium. Bacterial colonies obtained from the faeces (~100 mg) of survived hamsters were determined every two days after challenge. The number of CFU shown in the figure represented the geometric mean of CFU per 100 mg of feacal pellets obtained from survived hamsters in each group after the spore challenge

Fig. 9

Adjuvant effect in hamster challenge model studies. Panel a, five groups of hamsters (n = 6) were gastrically inoculated with 100 CFU. of C. difficile (the dose can kill >50 % of hamsters) at 2 weeks after the third immunization with either PBS, A-rRBD + B-rRBD, rlipoA-RBD + B-rRBD, or A-rRBD + B-rRBD formulated with alum (300 μg) or Pam3CSK4 (10 μg). The final survival rates were reported. Panel b, The number of C. difficile colonies (CFU) grown in TCCFA selective medium. Bacterial colonies obtained from the faeces (~100 mg) of survived hamsters were determined every two days after challenge. The number of CFU shown in the figure represented the geometric mean of CFU per 100 mg of feacal pellets obtained from survived hamsters in each group after the spore challenge