Construction And Evaluation Of Multigene Mutants Of Vibrio Cholerae 0139 As Vaccine Candidates
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Date
2011-09
Authors
Murugaiah, Chandrika
Journal Title
Journal ISSN
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Publisher
Universiti Sains Malaysia
Abstract
Cholera is a major health issue, affecting millions of lives annually. In light of the recurrent outbreaks of cholera, there is a pressing need for the development of vaccines that allow rapid mass vaccination. In this study, genetically modified vaccine candidates, VCUSM21P and VCUSM22P, were designed. VCUSM21P is a prototrophic vaccine which encodes non-toxic cholera toxin A (ctxA) subunit immunogen and has accessory cholera enterotoxin (ace), zonula occludens toxin (zot) and repeats-in-toxin C/A (rtxC/A) mutations. On the other hand, VCUSM22P is ace, zot, ctxA, rtxC/A and haemagglutinin/protease (hap) mutant. Both mutants were found not to disassemble the actin of HEp2 cells. Mouse colonization assay was used to determine VCUSM21P and VCUSM22P colonization ability in vivo. Rabbit ileal loop assay was performed to evaluate the reactogenicity caused by them. The immune responses provoked by the two vaccine candidates and their protective function against cholera were evaluated in a rabbit model. The mutants were found to be good colonizer of the mouse intestine. In the ileal loop assay using non-immunized rabbits, fluid accumulation was found in loops injected with 1x106 and 1x108 colony forming unit (CFU) of Wild Type (WT) V. cholerae. Unlike the WT V. cholerae challenge, 1x106 and 1x108 colony forming unit (CFU) of the mutants did not cause any reactogenicity in non-immunized rabbits. Immunization using 1x1010 CFU of the mutants induced both IgA and IgG antibodies production against cholera toxin (CT) and O139 lipopolysaccharides (LPS), as well as elevated vibriocidal antibody. The reactogenicity caused by the WT V. cholerae in rabbits immunized with 1x1010 CFU of the mutants was found to be reduced as evidenced by absence of fluid in loops administered with 1x102-1x107 CFU of WT V. cholerae. In the Removable Intestinal Tie Adult Rabbit Diarrhoea (RITARD) experiment, the non-
immunized rabbits were found unprotected against a lethal challenge with 1x109 CFU WT V. cholerae. However, 100% of rabbits immunized with the mutants survived the WT V. cholerae challenge. Immunohistochemical, histopathological and ultrastructural examination of non-immunized rabbits‟ ileum challenged with WT V. cholerae revealed severe ileal damages. But less severe damages were noted following the WT V. cholerae challenge in the ileum of rabbits immunized with VCUSM21P and VCUSM22P. The multigene mutants could be used for vaccination against potentially fatal V. cholerae O139.
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Keywords
Construction and evaluation of multigene mutants , of vibrio cholerae 0139 as vaccine candidates