Mona Khoshbakht; Mohammad Mahdi Forghanifard; Hossein Aghamollaei; Jafar Amani
Abstract
Background: Developing effective targeted treatment approaches to overcome drug resistance remains a crucial goal in cancer research. Immunotoxins have dual functionality in cancer detection and targeted therapy.Objective: This study aimed to engineer a recombinant chimeric fusion protein by combining ...
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Background: Developing effective targeted treatment approaches to overcome drug resistance remains a crucial goal in cancer research. Immunotoxins have dual functionality in cancer detection and targeted therapy.Objective: This study aimed to engineer a recombinant chimeric fusion protein by combining a nanobody-targeting domain with an exotoxin effector domain. The chimeric protein was designed to bind surface-expressed GRP78 on cancer cells, facilitating internalization and inducing apoptosis to inhibit proliferation and survival.Methods: Using a flexible linker, we designed two constructs linking VHH nanobody domains to Pseudomonas exotoxin (PE) domains II, III, and Ib. These constructs were then optimized for expression in E. coli BL21 (DE3) using the pET28a vector. Following the expression of the recombinant proteins, we purified them and tested their binding capability, cytotoxicity, and ability to induce apoptosis in breast cancer cell lines MDA-MB-231 and MCF-7, as well as in control cell lines HEK-293 and MDA-MB-468. The binding affinity was measured using a cell-based ELISA, internalization was assessed through Western blotting, cytotoxicity was evaluated by an MTT assay, and apoptosis was determined using flow cytometry with an Annexin V kit.Results: The immunotoxin specifically bound to cancer cells expressing csGRP78. The results of the cytotoxicity test showed that the cytotoxic effect of two constructs, I and II, depended on concentration and time. With an increase in both components, the effect of recombinant proteins also increased. Both constructs were able to penetrate and induce apoptosis in csGRP78+ cells.Conclusion: These immunotoxin structures showed therapeutic potential against GRP78-expressing cancers, making them suitable candidates for targeted therapy pending in vivo studies.
Ali Saffaei; Jafar Amani; Jafar Salimian; Gholamhossein Alishiri; Hassan Abolghasemi
Ahmad Karimi Rahjerdi; Mahyat Jafari; Mohammad Javad Motamedi; Jafar Amani; Ali Hatef Salmanian
Abstract
Background: Caused by bacterial, viral, and parasitic pathogens, diarrhea is the second leading cause of death among children under five. Two strains of E. coli, namely Enterotoxigenic, ETEC and Enterohemorrhagic EHEC are the most important causes of this disease in developing countries. EHEC is a major ...
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Background: Caused by bacterial, viral, and parasitic pathogens, diarrhea is the second leading cause of death among children under five. Two strains of E. coli, namely Enterotoxigenic, ETEC and Enterohemorrhagic EHEC are the most important causes of this disease in developing countries. EHEC is a major causative agent of bloody diarrhea and hemorrhagic uremic syndrome, while ETEC is the most important cause of diarrhea in neonates and travelers. Objective: To evaluate the immunologic properties of a subunit vaccine candidate comprising the main immunogenic epitopes from these two bacterial strains. Methods: The construct comprised of LTB and CfaB antigens from ETEC, and Intimin and Stx2B antigens from EHEC, was designed, analyzed and synthesized using bioinformatics methods. The chimeric gene was sub-cloned in the expression vector and expressed in E. coli host. The purified chimera protein was injected subcutaneously into the experimental animals. The production of specific antibodies was confirmed by immunological methods, and the protection capacity was evaluated by the challenge of immunized mice with the pathogenic bacteria. Results: Chimeric recombinant protein was able to increase IgG titer. Neutralization assay indicated that the antibodies generated against LtB moiety were able to neutralize ETEC toxin. In animal challenge study, all non-immune mice died within 3 days after the injection of toxin, but all immunized mice survived from Stx toxin. Conclusion: The immunity to both ETEC and EHEC bacteria is significant, and this structure can be considered as a candidate for vaccine production against these bacterial strains.
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.
