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.

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In low- and middle-income countries, diarrhoeal diseases are the second most common cause of mortality in children, mainly caused by enterotoxin-producing bacteria, such as Shigella, Vibrio, Salmonella, and Escherichia coli. Cholera and traveller’s diarrhoea are caused by Vibrio cholerae (O1 and O139 serogroups) and enterotoxigenic Escherichia coli (ETEC), respectively. The cholera toxin (CT) produced by V. cholerae and the heat-labile enterotoxin (LT) of ETEC are closely related by structure, function, and the immunological response to them. There is no exclusive vaccine for ETEC; however, cholera vaccines based on the CT-B component elicit a short-term cross-protection against ETEC infection. In this context, the cross-protective efficacy of MyCholTM, a prototype cold-chain-free, live-attenuated, oral cholera vaccine against V. cholerae O139 was evaluated in BALB/c mice. The 100% lethal dose (LD100) of 109 CFU/mL of the ETEC H10407 strain was used for the challenge studies. The mice immunised with MyChol™ survived the challenge by producing anti-CT antibodies, which cross-neutralised the LT toxin with no body weight loss and no sign of diarrhoea. Compared to unimmunised mice, the immunised mice elicited the neutralising antitoxin that markedly decreased ETEC colonisation and fluid accumulation caused by ETEC H10407 in the intestines. The immunised mice recorded higher antibody titres, including anti-CT IgG, anti-LT IgG, anti-CT-B IgG, and anti-LTB IgG. Only a two-fold rise in anti-CT/CT-B/LT/LT-B IgA was recorded in serum samples from immunised mice. No bactericidal antibodies against ETEC H10407 were detected. This investigation demonstrates the safety, immunogenicity, and cross-protective efficacy of MyCholTM against the ETEC H10407 challenge in BALB/c mice.

Cholera, a diarrheal disease caused by Vibrio cholerae (V. cholerae) O139 and O1 strains, remains a public health problem. The existing World Health Organization (WHO)- licenced, killed, multiple-dose oral cholera vaccines demand ‘cold-chain supply’ at 2 °C–8 °C. Therefore, a live, single-dose, cold-chain-free vaccine would relieve significant bottlenecks and costs of cholera vaccination campaigns. Our cholera vaccine development journey started in 2000 at Universiti Sains Malaysia with isolation of the hemA gene from V. cholerae, followed by development of a gene mutant vaccine candidate VCUSM2 against V. cholerae O139 in 2006. In 2010, VCUSM2 reactogenicity was reduced by replacing its two wild-type ctxA gene copies with mutated ctxA to produce strain VCUSM14. Introducing the hemA gene into VCUSM14 created VCUSM14P, a strain with the 5- aminolaevulinic acid (ALA) prototrophic trait and excellent colonisation and immunological properties (100% protection to wild-type challenged rabbits). It was further refined in Asian Institute of Medicine, Science and Technology (AIMST University), with completion of single- and repeated-dose toxicity evaluations in 2019 in Sprague Dawley (SD) rats, followed by development of a novel cold-chain-free VCUSM14P formulation in 2020. VCUSM14P is unique for its intact cholera toxin B, a known mucosal adjuvant. The built-in adjuvant makes VCUSM14P an ideal vaccine delivery platform for emerging diseases (e.g. severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] and tuberculosis). Our vaccine formulation mimics natural infection, remains non-reactogenic and immunogenic in vivo, and protects against infection and disease. It will also cost less and be less cumbersome to distribute due to its stability at room temperature. These features could revolutionise the outreach of this and other vaccines to meet global immunisation programmes, particularly in low-resourced areas. The next stage of our journey will be meeting the requisite regulatory requirements to produce the vaccine for rollout to countries where it is most needed.