Search

Shigella and Salmonella cause serious problems in many subjects, including young children and the elderly, especially in developing countries. Chimeric proteins carrying immunogens increase immune response. In-silico tools are applied to design vaccine candidates. Invasion plasmid antigens D (ipaD) gene is one of the Shigella virulence factors. The N-terminal region of the IpaD plays a significant role in invading the host cell. Invasion protein H (invH) gene plays important role in bacterial adherence and entry into epithelial cells. A recombinant chimeric construct, containing IpaD and InvH was designed and used as a vaccine candidate against Shigella and Salmonella enteritidis. After bioinformatics assessments, the construct was designed, synthesized, and expressed in E.coli. Chimeric protein, IpaD, and InvH were purified with Ni-NTA chromatography. Purified proteins were confirmed with western blotting and then were injected into separate mice groups. The antibody titer was estimated with an enzyme-linked immunosorbent assay (ELISA). Mice were challenged with 10, 100, and 1000 LD50 of Salmonella, and the sereny test was performed for Shigella. The Codon adaptation index of the chimeric gene was increased to 0.84. Validation results showed that 97.9% of residues lie in the favored or additional allowed region of the Ramachandran plot. A significant antibody rise was observed in all test groups. The immunized mice with chimer and InvH could tolerate 100 LD50 of Salmonella. In the sereny test, the application of bacteria treated with immunized mice sera of both antigens showed no infection in Guinea pigs' eyes. The recombinant protein could protect animal models against Salmonella and Shigella and therefore can be considered as a suitable vaccine candidate against these two pathogens. [ABSTRACT FROM AUTHOR]

Shigella and Salmonella cause serious problems in many subjects, including young children and the elderly, especially in developing countries. Chimeric proteins carrying immunogens increase immune response. In-silico tools are applied to design vaccine candidates. Invasion plasmid antigens D (ipaD) gene is one of the Shigella virulence factors. The N-terminal region of the IpaD plays a significant role in invading the host cell. Invasion protein H (invH) gene plays important role in bacterial adherence and entry into epithelial cells. A recombinant chimeric construct, containing IpaD and InvH was designed and used as a vaccine candidate against Shigella and Salmonella enteritidis. After bioinformatics assessments, the construct was designed, synthesized, and expressed in E.coli. Chimeric protein, IpaD, and InvH were purified with Ni-NTA chromatography. Purified proteins were confirmed with western blotting and then were injected into separate mice groups. The antibody titer was estimated with an enzyme-linked immunosorbent assay (ELISA). Mice were challenged with 10, 100, and 1000 LD50 of Salmonella, and the sereny test was performed for Shigella. The Codon adaptation index of the chimeric gene was increased to 0.84. Validation results showed that 97.9% of residues lie in the favored or additional allowed region of the Ramachandran plot. A significant antibody rise was observed in all test groups. The immunized mice with chimer and InvH could tolerate 100 LD50 of Salmonella. In the sereny test, the application of bacteria treated with immunized mice sera of both antigens showed no infection in Guinea pigs' eyes. The recombinant protein could protect animal models against Salmonella and Shigella and therefore can be considered as a suitable vaccine candidate against these two pathogens.

Background and purpose: The most common cause of diarrhea is Shigella and no vaccine has been found so far. IpaD and STxB proteins (B subunit of Shiga toxin) play an important role in invasion, infection and pathogenesis caused by Shigella. To evaluate the immunogenicity of each of the proteins IpaD and STxB can using of two animal models mice and guinea pigs and could be determined role of each recombinant protein IpaD-STxB for appropriate vaccine. Materials and methods: In this experimental study, the gene sequences of stxB & ipaD were obtained from gene bank and was purchased the synthetic gene. This gene transferred to E. coli BL21DE3 and produced the protein by the bacterium. The level of recombinant protein expression was evaluated and confirmed by techniques of Western bloting. Recombinant proteins were purificated using column chromatography, and were injected into mice four times consecutively. After the second injection (first booster), a week after each injection, were collected blood samples and was performed ELISA. Results: The results showed that was raised antibody titers against IpaD-STxB protein in the mice. The immunogenicity was evaluated using active toxin E. coli: O157:H7. The challenge results showed that immunized mice could endure 7.5 times the LD50 Shiga toxin E. coli O157: H7. Conclusion: The protein obtained of the fusion ipaD and stxB genes could protect mice against Shiga toxin. Consequently, STxB proteins were induced immunogenicity in an animal model mice and recombinant protein obtained can be a suitable candidate for the production of recombinant vaccine against Shigella types. [ABSTRACT FROM AUTHOR]

Shigella and Salmonella cause serious problems in many subjects, including young children and the elderly, especially in developing countries. Chimeric proteins carrying immunogens increase immune response. In-silico tools are applied to design vaccine candidates. Invasion plasmid antigens D (ipaD) gene is one of the Shigella virulence factors. The N-terminal region of the IpaD plays a significant role in invading the host cell. Invasion protein H (invH) gene plays important role in bacterial adherence and entry into epithelial cells. A recombinant chimeric construct, containing IpaD and InvH was designed and used as a vaccine candidate against Shigella and Salmonella enteritidis. After bioinformatics assessments, the construct was designed, synthesized, and expressed in E.coli. Chimeric protein, IpaD, and InvH were purified with Ni-NTA chromatography. Purified proteins were confirmed with western blotting and then were injected into separate mice groups. The antibody titer was estimated with an enzyme-linked immunosorbent assay (ELISA). Mice were challenged with 10, 100, and 1000 LD50 of Salmonella, and the sereny test was performed for Shigella. The Codon adaptation index of the chimeric gene was increased to 0.84. Validation results showed that 97.9% of residues lie in the favored or additional allowed region of the Ramachandran plot. A significant antibody rise was observed in all test groups. The immunized mice with chimer and InvH could tolerate 100 LD50 of Salmonella. In the sereny test, the application of bacteria treated with immunized mice sera of both antigens showed no infection in Guinea pigs' eyes. The recombinant protein could protect animal models against Salmonella and Shigella and therefore can be considered as a suitable vaccine candidate against these two pathogens.

Introduction: It is estimated that 50% of vaccines produced annually are wasted because effectivity is dependent on protein structure and heat exposure disrupts the intermolecular interactions that maintain this structure. Since 90% of vaccines require a temperature-controlled supply chain, it is necessary to create a cold chain system to minimize vaccine waste. We have developed a more sustainable technology via the adsorption of Invasion Plasmid Antigen D (IpaD) onto mesoporous silica gels, improving the thermal stability of protein-based therapeutics., Methods: The solution depletion method using UV-Vis was utilized to study the adsorption of IpaD onto silica gels. The silica-IpaD complex is heated above the denaturing temperature of the protein and then the IpaD is removed using N,N-Dimethyldodecylamine N-oxide (LDAO) and their secondary structure is tested using circular dichroism (CD)., Results: Pore diameter, pore volume and surface area were characterized for seven different silica gels. Silica gels designated as 6389, 6378, and 6375 had an adsorption percentage above 95% at pore volumes of 2.2, 2.8 and 3.8 cm 3 mg-1, respectively. CD analyses confirmed that the adsorbed IpaD after the heat treatment displayed a similar "W" shape CD signal as the native IpaD, indicating the conservation of α-helices. In contrast, the unprotected IpaD after being exposed to high temperature shows a flat CD signal, demonstrating the loss of secondary structure., Conclusion: We have successfully increased the thermo-tolerance for IpaD using mesoporous silica and continue to further optimize mesoporous silica's physiochemical properties to improve adsorption and desorption yields., (© 2020 The University of Kansas Medical Center.)