Atina Vakili; Seyed Latif Mousavi Gargari; Shahram Nazarian; Jafar Amani
Abstract
Background: Cholera disease caused by Vibrio cholerae remains a major cause of morbidity and mortality throughout the world. Various strategies with different proteins as immunogens have been tried for vaccine development, none of which have been sufficiently effective to preclude cholera. Chimeric proteins, ...
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Background: Cholera disease caused by Vibrio cholerae remains a major cause of morbidity and mortality throughout the world. Various strategies with different proteins as immunogens have been tried for vaccine development, none of which have been sufficiently effective to preclude cholera. Chimeric proteins, with their ability to present multiple antigens at the same time, can play important roles in immunization. Objective: To evaluate the immunogenicity of a chimeric construct, comprised of OmpW and CtxB as immunogenic proteins of Vibrio cholera, in BALB/c mice. Methods: The construct was designed after bioinformatics assessments and then expressed in E.coli. Chimeric protein, OmpW, and CtxB were purified with Ni-NTA chromatography and confirmed by Western blotting. Mice were immunized with purified recombinant proteins. The antibody titers and specificity of the immune sera were then analyzed by ELISA and challenged on the pups of immunized mice with 1, 5 and 10 LD50. Mice ileal loop assay was also performed. Results: Significant differences were observed in antibody titers in immunized mice compared to the control groups. Infant mouse challenge was performed so as to compare the protective efficacies of the selected immunogen regimens. Of the Pups from dams immunized with chimeric protein which received 1 LD50, 75% survived. Pups belonging to PBS-immunized dams, experienced 100% mortality. The serum raised toward immunogenic construct, inhibited cholera toxin activity in ileal loop test up to 68%. Conclusion: Chimeric construct is able to induce the immune system and provide up to 75% inhibition of toxin activity against 1 LD50 of Vibrio cholerae.
Babak Barati; Firouz Ebrahimi; Shahram Nazarian
Volume 15, Issue 1 , March 2018, , Pages 47-58
Abstract
Background: Cholera toxin (CT), responsible for the harmful effects of cholera infection, is made up of one A subunit (enzymatic), and five B subunits (cell binding). The release of cholera toxin is the main reason for the debilitating loss of intestinal fluid. Inhibition of the B subunit (CTB) may block ...
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Background: Cholera toxin (CT), responsible for the harmful effects of cholera infection, is made up of one A subunit (enzymatic), and five B subunits (cell binding). The release of cholera toxin is the main reason for the debilitating loss of intestinal fluid. Inhibition of the B subunit (CTB) may block CT activity. Objective: To determine the effect of anti CTB-IgY against oral challenge with V. cholera in suckling infant mice. Methods: The binding domain of cholera toxin was amplified and ligated into pET28a vector. The pET28a (+)/ctb expression vector was confirmed by endonuclease digestion and sequence analysis. The expression of recombinant CTB in E. coli was performed by induction with IPTG. After immunizing the chickens with recombinant CTB, IgY was purified by water dilution method and NaCl precipitation and analyzed by SDS-PAGE. Moreover, the activity and specificity of the IgY antibody were assessed by ELISA. Results: The SDS-PAGE and western blot techniques showed that CTB protein was successfully expressed and specifically recognized by polyclonal antibodies against the cholera toxin. The oral administration of anti- (V. cholera+CTB) in infant mice in challenge with active V. cholera bacterium demonstrated high rate of survival. Conclusion: The increase in the number of antibiotic resistant bacteria implies the necessity of finding novel antibiotics. Our results suggest the possibility of passive protection from purified IgY, hence implying that anti CTB-IgY may be useful in the treatment of cholera infections.
Fatemeh Malaei; Mahdi Hesaraki; Mojtaba Saadati; Ali Mohammad Ahdi; Mohammad Sadraeian; Hussein Honari; Shahram Nazarian
Volume 10, Issue 2 , June 2013, , Pages 110-117
Abstract
Background: Recombinant vaccine technology is one of the most developed means in controlling infectious diseases. However, an effective vaccine against Shigella is still missing. Objective: To evaluate recombinant IpaC protein of Shigella as a vaccine candidate. Methods: In this study we cloned IpaC ...
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Background: Recombinant vaccine technology is one of the most developed means in controlling infectious diseases. However, an effective vaccine against Shigella is still missing. Objective: To evaluate recombinant IpaC protein of Shigella as a vaccine candidate. Methods: In this study we cloned IpaC gene into an expression vector in prokaryotic system. The protein expression was evaluated by SDS-PAGE and Western-Blotting analysis. The recombinant protein was purified using Ni–NTA affinity chromatography. Guinea pigs were immunized with the recombinant protein and the level of immunogenicity was examined by ELISA and Western blotting of IpaC. Challenge test was done through the intraoculary injection of Shigella dysenteriae (6×108 CFU/eye) and after 48 hours was scored for keratoconjunctivitis. Results: The results showed a remarkable level of immunogenicity in terms of antibody response and protection against keratoconjunctivitis in tested animals. The recombinant IpaC protein provided a protective system against Shigella dysenteriae type I during the challenge test. Conclusion: The results showed the potential of using recombinant IpaC in preparation of vaccine in perspective studies.
