Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
Primers used in this study with their sequences, annealing temperature and functions.
Figure 1.
Diagram of the auxotrophic strains, VCUSM20 and VCUSM21, construction.
The diagram is a schematic representation of mutation introduced at the rtx gene cluster on VCUSM14 (A) and VCUSM20 (B) chromosomes. It shows the double copies of Δctx operon, ΔhemA-hemM genes and rtxC::aphA genes in VCUSM20, and rtxC::aphA replaced with ΔrtxC/A in VCUSM21. Gene designations: ΔctxA operon, mutation in CT A subunit with complete deletion of accessory cholera enterotoxin (ace) and zonula occludens toxin (zot); repeat-in-toxin (rtx); bla, β-lactamase; sacB, Bacillus subtilis counterselectable marker sacB which encodes levan sucrase.
Figure 2.
Diagram of the prototrophic vaccine candidate, VCUSM21P, construction.
The diagram is a schematic representation of ΔhemA mutation removed from VCUSM21 chromosome, and replacing it with the native hemA gene from V. cholerae O139. It shows the double copies of Δctx operon, ΔrtxC/A, and hemA/M genes in VCUSM21P. Gene designations: as Figure 1.
Figure 3.
Inverted microscopy reveals morphological changes on HEp-2 cells after infection with WT (B) and VCUSM21P (C), at a magnification of ×40.
As control, HEp-2 cells in PBS (A) were included in the cytotoxic assay. HEp-2 cells were incubated for 1 hour with WT and VCUSM21P cells at multiplicity of infection (MOI) of 100. B shows rounded HEp-2 cells indicating cytotoxic effects. A and C show HEp-2 cells with normal morphology indicating absence of cytotoxicity.
Figure 4.
Cholera toxin production by vaccine candidate VCUSM21P and WT strain.
Values are expressed as mean± standard deviation.
Table 4.
Cholera toxin, CT A and CT B production by vaccine candidate VCUSM21P and WT strain.
Figure 5.
Fluid accumulation ratio in loops of unvaccinated rabbits.
Table 3.
Antibodies titers of rabbits immunized with VCUSM21P.