Introduction. Cholera is an acute enteric infection caused by Vibrio cholerae, particularly in areas lacking access to clean water. Despite the global effort to improve water quality in these regions, the burden of cholera in recent years has not yet declined. Interest has therefore extended in the use of bicistronic DNA vaccine encoding ctxB and tcpA genes of V. cholerae as a potential vaccine.Hypothesis/Gap Statement. The potential of a bicistronic DNA vaccine, pVAX-ctxB-tcpA has not been determined in vitro and in vivo.Aim. The goal of present study was to evaluate in vitro expression and in vivo potential of pVAX-ctxB-tcpA vaccine against V. cholerae.Methodology. The pVAX-ctxB-tcpA was transiently transfected into mammalian COS-7 cells, and the in vitro expression was assessed using fluorescence and Western blot analyses. Next, the vaccine was encapsulated into sodium alginate using water-in-oil emulsification and evaluated for its efficiency in different pH conditions. Subsequently, oral vaccination using en(pVAX-ctxB-tcpA) was performed in vivo. The animals were challenged with V. cholerae O1 El Tor after 2 weeks of vaccination using the Removable Intestinal Tie-Adult Rabbit Diarrhoea (RITARD) model. Following the infection challenge, the rabbits were monitored for evidence of symptoms, and analysed for systemic cytokine expression level (TNF-α, IFN-γ, IL-6 and IL-10) using quantitative real-time polymerase chain reaction.Results. The in vitro expression of pVAX-ctxB-tcpA was successfully verified via fluorescence and Western blot analyses. Meanwhile, in vivo analysis demonstrated that the en(pVAX-ctxB-tcpA) was able to protect the RITARD model against V. cholerae infection due to a lack of evidence on the clinical manifestations of cholera following bacterial challenge. Furthermore, the bicistronic group showed an upregulation of systemic IFN-γ and IL-10 following 12 days of vaccination, though not significant, suggesting the possible activation of both T-helper 1 and 2 types of response. However, upon bacterial challenge, the gene expression of all cytokines did not change.Conclusion. Our findings suggest that the bicistronic plasmid DNA vaccine, pVAX-ctxB-tcpA, showed a potential role in inducing immune response against cholera through upregulation of in vitro gene and protein expression as well as in vivo cytokine gene expression, particularly IFN-γ and IL-10.
Keywords: DNA vaccine; RITARD; alginate; bicistronic construct; cholera; microencapsulation.