Construction and characterisation of O139 cholera vaccine candidates

Parenteral Immunization and Protection from Mucosal Infection

“…Most effort at developing vaccines against cholera has focused on the development of safe and effective vaccines for oral administration (Ledón et al, 2003;Liang et al, 2003). Earlier work with either killed whole-cell or liveattenuated V. cholerae showed that killed whole-cell vaccines administered orally were approximately 50% effective, similar to the efficacy observed previously with parenteral administration.…”

TLP01, an mshA mutant of Vibrio cholerae O139 as vaccine candidate against cholera

“…Southern blotting analyses were performed with the following digoxigenin (DIG)-labeled probes (Roche Diagnostics GmbH): a 1.5 kb PCR product spanning the region between gIII CTX and zot genes was used as CTX4 core-specific probe [28], a 643 bp fragment containing part of ctxAB genes amplified by PCR with the primer pairs 5 0 -ATG ATCATGCAA-GAGGAACTC-3 0 and 5 0 -AGGTGTTCCATGTGCATATG C-3 0 was used as the ctxAB-specific probe, the 2.9 kb EcoRI-PstI fragment containing the RS1 element from pURS1 was used as the RS-specific probe [29], the 3.2 kb HindIII insert of pCH 2 [30], containing hapA gene, was used as a hapA-specific probe and, finally, a 2.6 kb-fragment containing part of mshA gene and its flanking regions was used as mshA-specific probe [31].…”

“…1 Endoglucanase A activity in V. cholerae was detected as previously described [17]. For motility determination were used swarm agar plates as described elsewhere [31]. Bacterial strains were tested for their susceptibility to streptomycin at 100 mg/ml on LB plates and to sulfamethoxazole (23.75 mg) and trimethoprim (1.25 mg) (SXT) by the disc diffusion technique.…”

“…In both cases the inoculation with a dose of about 10 6 CFU was performed as in the virulence assay. The traditional short-term variant was performed as described previously [31]. In the long-term variant, 4 h after challenged the mothers were returned to their breeding and mice were euthanized after 24, 72 and 120 h.…”

“…Five groups of rabbits, comprising of four animals per group were intraduodenally inoculated with about 10 9 CFU of TLP13, TLP132, TLP01, TLP05 or WT, respectively. Blood samples were taken at days 0, 7, 14, 21 and 28 from the central vein of the ear [31].…”