Alireza Felegary1, Shahram Nazarian2, Emad Kordbacheh1, Javad Fathi3, Mohamad Ebrahim Minae1. 1. Department of Biological Sciences, Faculty of Science, Imam Hossein University, Tehran, Iran. 2. Department of Biological Sciences, Faculty of Science, Imam Hossein University, Tehran, Iran. Electronic address: nazarian56@gmail.com. 3. Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
Abstract
OBJECTIVES: Shigellosis is one of the infectious diseases causing severe intestinal illness in human beings. Development of an effective vaccine against Shigella is a key to deal with this bacterium. The present study aimed at evaluation of the antibody response as well as the protection of the recombinant chimeric protein containing IpaD, IpaB, StxB, and VirG against Shigella dysentery and flexneri. METHODS: Chimeric protein was expressed and purified by Ni-NTA resin. The identity of the protein was determined by Western blot analysis. Mouse groups were immunized with the recombinant protein and the humoral immune response was measured by Enzyme-Linked Immunosorbent Assay (ELISA). Additionally, neutralization of the bacterial toxin by antibody was assessed by MTT assay. Animal challenge against S.dysentery and S. flexneri was evaluated, as well. RESULTS: Protein expression and purification were confirmed by SDS-PAGE and western blotting. Analysis of the immune responses demonstrated that the antibody responses were higher in the sera of the subcutaneously immunized mice compared to those immunized intraperitoneally. In vitro neutralization analysis indicated that the 1:10000 dilution of the sera had a high ability to neutralize 0.25 ng/µl (CD50) of the toxin on the Vero cell line. Furthermore, the results of the animal challenge showed that the immunized mice were completely protected against 50 LD50 of the bacterial toxin. Immunization also protected 80% of the mice from 10 LD50 by S. flexneri and S.dysentery. In addition, passive immunization conferred 60% protection in the mice against S. flexneri and S.dysentery. Organ burden studies also revealed a significant reduction in infection among the immunized mice. CONCLUSION: This study revealed that the chimeric protein produced inE. colicould be a promising chimeric immunogen candidate against Shigella.
OBJECTIVES: Shigellosis is one of the infectious diseases causing severe intestinal illness in human beings. Development of an effective vaccine against Shigella is a key to deal with this bacterium. The present study aimed at evaluation of the antibody response as well as the protection of the recombinant chimeric protein containing IpaD, IpaB, StxB, and VirG against Shigella dysentery and flexneri. METHODS: Chimeric protein was expressed and purified by Ni-NTA resin. The identity of the protein was determined by Western blot analysis. Mouse groups were immunized with the recombinant protein and the humoral immune response was measured by Enzyme-Linked Immunosorbent Assay (ELISA). Additionally, neutralization of the bacterial toxin by antibody was assessed by MTT assay. Animal challenge against S.dysentery and S. flexneri was evaluated, as well. RESULTS: Protein expression and purification were confirmed by SDS-PAGE and western blotting. Analysis of the immune responses demonstrated that the antibody responses were higher in the sera of the subcutaneously immunized mice compared to those immunized intraperitoneally. In vitro neutralization analysis indicated that the 1:10000 dilution of the sera had a high ability to neutralize 0.25 ng/µl (CD50) of the toxin on the Vero cell line. Furthermore, the results of the animal challenge showed that the immunized mice were completely protected against 50 LD50 of the bacterial toxin. Immunization also protected 80% of the mice from 10 LD50 by S. flexneri and S.dysentery. In addition, passive immunization conferred 60% protection in the mice against S. flexneri and S.dysentery. Organ burden studies also revealed a significant reduction in infection among the immunized mice. CONCLUSION: This study revealed that the chimeric protein produced inE. colicould be a promising chimeric immunogen candidate against Shigella.