A Strategy for the Rapid Development of a Safe Candidate Vaccine Strain.

Approximately 1/6 of humanity is at high risk of experiencing cholera epidemics. The development of effective and safe vaccines against the primary cause of cholera, is part of the public health measures to prevent cholera epidemics. Natural nontoxigenic isolates represent a source of new genetically improved and relatively safe vaccine strains. However, the genomic engineering of wild-type strains is difficult, and these strains are genetically unstable due to their high homologous recombination activity. We comprehensively characterized two isolates using genome sequencing, bioinformatic analysis, and microscopic, physiological, and biochemical tests. Genetic constructs were Gibson assembled and electrotransformed into . Bacterial colonies were assessed using standard microbiological and immunological techniques. As a result, we created a synthetic chromoprotein-expressing reporter operon. This operon was used to improve the genome engineering approach and monitor the stability of the genetic constructs. Finally, we created a stable candidate vaccine strain bearing a deletion and expressing the β-subunit of cholera toxin. Thus, we developed a strategy for the rapid creation of genetically stable and relatively safe candidate vaccine strains. This strategy can be applied not only to but also to other important human bacterial pathogens.