Immunoinformatics‐Guided Whole Proteome‐Based Multi‐Epitope mRNA Vaccine Design Against <i>Nocardia asteroides</i> Using Surface Antigens—A Subtractive Proteomics and Reverse Vaccinology Approach

Nocardiosis is caused by the aerobic gram-positive bacterium Nocardia asteroides which is multidrug-resistant and primarily affects immunocompromised individuals. This study aims to design a broad-spectrum vaccine against Nocardiosis. We used subtractive proteomics and vaccinomics approaches to identify vaccine candidates to design an epitope-based vaccine against Nocardiosis. Four proteins in the outer membrane and extracellular regions were shortlisted on the basis of immunogenic parameters. T- and B-cell epitopes were predicted on the basis of IC₅₀ < 200 nM, and a multi-epitope vaccine construct was designed using six overlapping CTL, HTL, and B-cell epitopes conjugated by appropriate linkers. A highly immunogenic adjuvant was integrated at the N-terminus of the multi-epitope vaccine to stimulate a robust immune response. The tertiary structure of the vaccine construct was predicted and validated with the 96% residues in the favorite region of the Ramachandran plot and a Z-score of -4.76. The proposed vaccine exhibited strong immunological and physicochemical features. High docking scores and strong binding energies of approximately -1051.9 to -1274.5 kcal/mol with human immune receptors ensured that the designed vaccine construct could induce potential immunogenic responses in the host immune system. The efficacy of the vaccine was evaluated by the immune simulation to determine the potential of the vaccine to simulate innate and adaptive immunity with the development of long-lasting memory immune cells. The molecular dynamic simulation of a 100 ns study was conducted to determine the structural stability and molecular function of the designed vaccine in the cellular microenvironment. In silico restriction cloning analysis determined the successful expression of the engineered vaccine construct in Escherichia coli plasmid with a size of 6323 bp. On the basis of the current study, we assume that the proposed vaccine is worthy of further in vitro in vivo and in vivo validations to ensure the efficacy of the engineered vaccine in this study.