Immunogenicity and protective efficacy of a live, oral cholera vaccine formulation stored outside-the-cold-chain for 140 days

Abstract

Background: Cholera, an acute watery diarrhoeal disease caused by Vibrio cholerae serogroup O1 and O139 across the continents. Replacing the existing WHO licensed killed multiple-dose oral cholera vaccines that demand 'cold chain supply' at 2-8°C with a live, single-dose and cold chain-free vaccine would relieve the significant bottlenecks and cost determinants in cholera vaccination campaigns. In this direction, a prototype cold chain-free live attenuated cholera vaccine formulation (LACV) was developed against the toxigenic wild-type (WT) V. cholerae O139 serogroup. LACV was found stable and retained its viability (5 × 10 6 CFU/mL), purity and potency at room temperature (25°C ± 2°C, and 60% ± 5% relative humidity) for 140 days in contrast to all the existing WHO licensed cold-chain supply (2-8°C) dependent killed oral cholera vaccines. Results: The LACV was evaluated for its colonization potential, reactogenicity, immunogenicity and protective efficacy in animal models after its storage at room temperature for 140 days. In suckling mice colonization assay, the LACV recorded the highest recovery of (7.2 × 10 7 CFU/mL) compared to those of unformulated VCUSM14P (5.6 × 10 7 CFU/mL) and the WT O139 strain (3.5 × 10 7 CFU/mL). The LACV showed no reactogenicity even at an inoculation dose of 10 4-10 6 CFU/mL in a rabbit ileal loop model. The rabbits vaccinated with the LACV or unformulated VCUSM14P survived a challenge with WT O139 and showed no signs of diarrhoea or death in the reversible intestinal tie adult rabbit diarrhoea (RITARD) model. Vaccinated rabbits recorded a 275-fold increase in anti-CT IgG and a 15-fold increase in anti-CT IgA antibodies compared to those of rabbits vaccinated with unformulated VCUSM14P. Vibriocidal antibodies were increased by 31-fold with the LACV and 14-fold with unformulated VCUSM14P. Conclusion: The vaccine formulation mimics a natural infection, is non-reactogenic and highly immunogenic in vivo and protects animals from lethal wild-type V. cholerae O139 challenge. The single dose LACV formulation was found to be stable at room temperature (25 ± 2°C) for 140 days and it would result in significant cost savings during mass cholera vaccination campaigns.

Bacterial Ghosts of Pseudomonas aeruginosa as a Promising Candidate Vaccine and Its Application in Diabetic Rats

“…To generate protective immunity, a mucosal adjuvant can be added to these vaccination formulations [43]. Cimetidine (50 mg/kg) administered intravenously to lower gastric acid output in the stomach (in rabbit) and 15 mL of 5% sodium bicarbonate injected twice intragastrically to neutralize stomach acid were utilized by Xian et al (2020) [44]. For example, cellulose acetate phthalate (Eudragit) is frequently employed as a polymer film to protect a capsulated vaccination since it is insoluble at low pH in the stomach but dissolves rapidly at higher pH in the gut, depending on the type of Eudragit used [45,46].…”

Dual-Use Vaccine for Diarrhoeal Diseases: Cross-Protective Immunogenicity of a Cold-Chain-Free, Live-Attenuated, Oral Cholera Vaccine against Enterotoxigenic Escherichia coli (ETEC) Challenge in BALB/c Mice

“…Normal saline was used as a negative control for Group B unimmunised mice. Blood samples were collected from pre and post-immunised mice on days 0, 14, 28, and 42 by sinus orbital, as previously described [28,30]. The blood samples were kept at room temperature for 2 h to allow clotting, followed by centrifugation (3500 rpm, 15 min at 4 • C) to separate the serum.…”

“…Detection of Anti-Cholera Toxin (Anti-CT), Anti-Heat Labile (Anti-LT) and Anti-Subunit Antibodies Anti-CT or -B and anti-LT or -B serum antibody ELISAs were performed on mice serum samples with wells coated with 0.5 µg of antigen and quantified with an external mouse IgG or IgA standard, as previously described [28]. Briefly, the ELISA plates (MaxiSorp, Nunc, Roskilde, Denmark) were coated with 0.5 µg/well of antigen (CT/LT/CT-B/LT-B) in 60 mM carbonate buffer (pH 9.6) and incubated at 4 • C for 16 h. The plates were then blocked with 5% skim milk and incubated at 37 • C for 1 h. The wells were washed 3 times with wash buffer (PBS-Tween 20), and 100 µL of each sera sample (1:10-1:1280 diluted in PBS) was added and incubated at 37 • C for 2 h. The anti-cholera toxin, as a primary antibody, was used as the positive control, and PBS without any serum was used as the negative control.…”

“…A prototype coldchain-free live cholera vaccine formulation with VCUSM14P was developed (Patent filed: PI 2018700106, Malaysia). The vaccine formulation (MyChol TM ) is a liquid suspension consisting of 10 7 CFU/mL of the live-attenuated vaccine strain (VCUSM14P), and it retains its potency at room temperature (25 • C ± 2 • C and RH 60% ± 5%) for 140 days [27,28]. My-Chol™ mimics a natural infection, is non-reactogenic, immunogenic in vivo, and protects animals from a lethal wild-type V. cholerae O139 challenge [27,28].…”

Live, Genetically Attenuated, Cold-Chain-Free Cholera Vaccine—A Research and Development Journey: Light at the End of a Long Tunnel

“…The vaccine formulation mimicked a natural infection, was non-reactogenic and highly immunogenic in vivo and protected animals from a lethal WT V. cholerae O139 challenge. The single-dose LACV formulation was stable at room temperature (25 ± 2 °C) for 140 days, indicating that it would result in significant cost savings during mass cholera vaccination campaigns ( 9 ).…”