Document Type : Original Articles
Authors
1
Department of Biology, Faculty of Sciences, Shahid Chamran University of Ahvaz, Ahvaz, Iran
2
Department of Microbiology, Faculty of Advanced Sciences and Technology, Islamic Azad University, Tehran Medical Branch, Tehran, Iran
3
Department of Microbiology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences and Health Services, Yazd, Iran
4
Department of Biology, Faculty of Sciences, Islamic Azad University, Damghan, Iran
5
Department of Microbiology, Faculty of Basic Sciences, Ayatollah Amoli Branch, Islamic Azad University, Amol, Iran
6
Department of Vector Biology and Control of Diseases, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Abstract
Introduction:
Methicillin-resistant Staphylococcus aureus (MRSA) is recognized as one of the most critical antibiotic-resistant pathogens worldwide and represents a major challenge to global public health systems. Its ability to resist multiple classes of antibiotics has led to increased morbidity, mortality, and healthcare costs. The limited effectiveness of current therapeutic options emphasizes the urgent need for alternative preventive strategies, among which vaccine development is considered a promising and sustainable approach.
Objective:
The present study aimed to design, construct, and evaluate a rational multi-epitope vaccine candidate against MRSA by integrating immunoinformatics-based prediction tools with experimental in vivo validation.
Materials and Methods:
Seven MRSA virulence-associated proteins were initially screened for antigenicity, immunogenicity, and allergenicity using established bioinformatics databases and servers. Highly immunogenic B-cell and T-cell epitopes were selected and assembled using suitable peptide linkers to ensure structural stability and optimal epitope presentation. An immunostimulatory adjuvant was fused to the N-terminal region of the construct to enhance immune responses. Physicochemical properties, including molecular weight, isoelectric point, stability index, and hydropathicity, were analyzed using ProtParam. Secondary and tertiary structures were predicted and refined using Prabi, Robetta, AlphaFold, and I-TASSER servers. Antigenicity and allergenicity were evaluated using VaxiJen and AllerTOP, respectively. Molecular docking analyses with Toll-like receptor 4 (TLR4) and selected human leukocyte antigen (HLA) alleles were performed using Molegro Virtual Docker to assess binding affinity and immune receptor interactions. The optimized gene sequence was cloned into the pcDNA3.1 expression vector and administered to BALB/c mice. The expression levels of immune-related genes, including TLR4 and interleukin-4 (IL-4), were quantified using real-time PCR.
Results:
Computational analyses demonstrated strong binding interactions between the vaccine construct and immune receptors, indicating a high potential for immune activation. The vaccine candidate was predicted to be non-allergenic and antigenic. In vivo experiments revealed a significant downregulation of TLR4 and IL-4 gene expression in immunized mice compared with control groups.
Conclusion:
The designed multi-epitope vaccine candidate exhibits favorable immunological and structural characteristics against MRSA. Although these findings are promising, further comprehensive in vitro and in vivo investigations are required to confirm its protective efficacy, immunogenicity, and long-term safety.
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