Subtractive proteomic analysis to identify highly antigenic proteins and reverse vaccinology based novel vaccine development against Haemophillius parainfluenzae.

Haemophillius parainfluenzae (H. parainfluenzae) is increasingly involved in invasive infections disrupting normal skin or mucosal barriers, specifically in immunocompromised individuals. No specific therapeutics or licensed vaccines are available for H. parainfluenzae infections. In the present study, the genome-scan vaccinomics analysis ranked three potential vaccine candidate proteins based on high immunogenic parameters, which were further used to prioritize lead B- and T-cell epitopes. The top-ranked highly immunogenic epitopes were conjugated using suitable linkers (KK, AAY, and GPGPG) and adjuvant (beta-defensin 3) sequences to engineer a potential chimeric vaccine construct. The immunological and physiochemical properties of the designed H. parainfluenzae vaccine exhibited high immunogenicity with an antigenicity score of 0.8688, structural and thermodynamic stability, and significant solubility. The tertiary structural analysis revealed a high-quality and stable 3D structure with > 89% of residues in the favored region of the Ramachandran plot and a Z-score of -4.45. The molecular docking analysis and molecular dynamic simulation studies revealed robust and stable binding interaction between the H. parainfluenzae vaccine and the TLR4 immune receptor. Immune simulation analysis revealed that the H. parainfluenzae vaccine had the potential to trigger both innate and adaptive immune responses in the host. In-silico restriction cloning demonstrated a significant level of gene expression for the H. parainfluenzae vaccine construct in the bacterial expression vector yielding a recombinant plasmid of 6357 bp. Experimental validation via in vitro and in vivo assays of the predicted vaccine model may prove worthwhile against H. parainfluenzae with improved potency and safety.