Subtractive proteomics and reverse-vaccinology approaches for novel drug targets and designing a chimeric vaccine against Ruminococcus gnavus strain RJX1120

<i>Mediterraneibacter gnavus</i>, also known as <i>Ruminococcus gnavus</i>, is a Gram-positive anaerobic bacterium that resides in the human gut microbiota. Notably, this bacterium plays dual roles in health and disease. On one side it supports nutrient metabolism essential for bodily functions and on the other it contributes to the development of Inflammatory Bowel Disease (IBD) and other gastrointestinal disorders. <i>R. gnavus</i> strain RJX1120 is an encapsulated strain and has been linked to develop IBD. Despite the advances made on its role in gut homeostasis, limited information is available on strain-specific virulence factors, metabolic pathways, and regulatory mechanisms. The study of such aspects is crucial to make microbiota-targeted therapy and understand its implications in host health. A multi-epitope vaccine against <i>R. gnavus</i> strain RJX1120 was designed using reverse vaccinology-based subtractive proteomics approach. Among the 3,219 proteins identified in the <i>R. gnavus</i> strain RJX1120, two critical virulent and antigenic proteins, a Single-stranded DNA-binding protein SSB (A0A2N5PT08) and Cell division ATP-binding protein FtsE (A0A2N5NK05) were screened and identified as potential targets. The predicted B-cell and T-cell epitopes from these proteins were screened for essential immunological properties such as antigenicity, allergenicity, solubility, MHC binding affinity, and toxicity. Epitopes chosen were cross-linked using suitable spacers and an adjuvant to develop a multi-epitope vaccine. Structural refinement of the construct revealed that 95.7% of the amino acid residues were located in favored regions, indicating a high-quality structural model. Molecular docking analysis demonstrated a robust interaction between the vaccine construct and the human Toll-like receptor 4 (TLR4), with a binding energy of -1277.0 kcal/mol. The results of molecular dynamics simulations further confirmed the stability of the vaccine-receptor complex under physiological conditions. <i>In silico</i> cloning of the vaccine construct yielded a GC content of 48% and a Codon Adaptation Index (CAI) value of 1.0, indicating optimal expression in the host system. These results indicate the possibility of the designed vaccine construct as a candidate for the prevention of <i>R. gnavus</i>-associated diseases. However, experimental validation is required to confirm its immunogenicity and protective efficacy.