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Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation
Published on: August 21, 2019
Design, purification, and biophysical characterization of a multi-epitope vaccine construct made from outer membrane
Yogeshwar Devarakonda1, Syed Mohammad Omer Ali1, Juan Edwin James2
1Genetics and Molecular Microbiology Laboratory, Department. of Biological Sciences, Institute of Eminence, Birla Institute of Technology and Sciences-Pilani, Hyderabad campus, Shamirpet-Mandal, Hyderabad, Telangana, 500078, India.
Abstract:
Pseudomonas aeruginosa is increasingly becoming resistant to multiple drugs and is held responsible for high rate of mortality and morbidity across the globe. This study aims to examine proteins from outer membrane such as OprE, OprF, OprC, and OprG, for the multi-epitope vaccine design. In this article, the prediction of epitopes for helper T-cells, cytotoxic T-cells, and B-cell epitopes was carried out by the application of various immune-informatics tools. All the predicted epitopes were aligned and assembled into a peptide sequence along with linkers and adjuvant sequences. Further, secondary structure and three-dimensional structure were predicted for the multiepitope construct. The vaccine construct designs were evaluated and validated for allergenicity, toxicity, and antigenicity. The validation of the predicted structure was carried out by determining its physiochemical properties by the application of ProtParam. Protein docking and molecular-dynamic simulation confirmed strong and stable interaction between the vaccine construct and Toll-like receptor-4. The vaccine construct was cloned into pET-29b vector and expressed in Escherichia coli. The designed multiepitope vaccine construct was overexpressed and purified by the application of Ni-NTA affinity chromatography and subjected to SDS-PAGE analysis. The circular dichroism spectroscopy analysis revealed it to be stable and structured. The hemolysis assay demonstrated minimal RBC toxicity suggesting that it was safe to use. The designed vaccine construct could activate both humoral and cellular immune responses as demonstrated by the advanced immunoinformatic approach making it a promising vaccine construct for protection against P. aeruginosa.
Insights
This study designed a multi-epitope vaccine against drug-resistant Pseudomonas aeruginosa using outer membrane proteins. Computational and experimental validation confirmed its stability, safety, and potential to elicit both humoral and cellular immune responses.
Area of Science:
- Microbiology
- Immunology
- Vaccine Design
Background:
- Pseudomonas aeruginosa exhibits increasing multidrug resistance, leading to significant global mortality and morbidity.
- Effective vaccines are crucial to combat infections caused by this opportunistic pathogen.
Purpose of the Study:
- To design and validate a multi-epitope vaccine targeting key outer membrane proteins of Pseudomonas aeruginosa.
- To computationally predict and experimentally verify the immunogenic and structural properties of the vaccine construct.
Main Methods:
- Immune-informatics tools were used to predict T-cell and B-cell epitopes from outer membrane proteins (OprE, OprF, OprC, OprG).
- Epitopes were assembled into a multi-epitope construct, followed by secondary and tertiary structure prediction.
- In silico analyses included allergenicity, toxicity, antigenicity, docking, and molecular dynamics simulations.
- Experimental validation involved cloning, expression in E. coli, purification, SDS-PAGE, circular dichroism, and hemolysis assays.
Main Results:
- A stable, non-allergenic, and non-toxic multi-epitope vaccine construct was designed.
- Computational modeling predicted strong interaction with Toll-like receptor-4.
- Experimental analysis confirmed the construct's stability, proper folding, and minimal toxicity.
- The construct demonstrated potential to activate both humoral and cellular immunity.
Conclusions:
- The designed multi-epitope vaccine construct shows promise for protecting against Pseudomonas aeruginosa infections.
- This approach offers a viable strategy for developing vaccines against multidrug-resistant bacteria.
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