Designing an mRNA vaccine encoding multi-epitopes to combat Pipistrellus bat coronavirus BtHKU5-CoV-2 based on consensus sequence using an immunoinformatics approach

Abstract

The bat-derived Pipistrellus bat coronavirus BtHKU5-CoV-2, a merbecovirus lineage, is an emerging zoonotic risk due to its ability to utilize human ACE2 receptors and ongoing spike protein recombination events that enhance cross-species transmission potential. At the moment no authorized vaccines are available against this emerging threat which emphasizes the urgent need for proactive vaccine development. This study employed reverse vaccinology and immunoinformatics approaches to design a novel mRNA vaccine candidate encoding multiple epitopes, aiming at conserved regions across four structural proteins (E, M, N, and S) of the virus. Protein sequences from UniProt underwent BLAST analysis, multiple sequence alignment, and phylogenetic mapping to identify conserved regions. Twenty-four high-affinity epitopes were selected: eight CTL, eight HTL, and eight linear B lymphocyte epitopes, meeting strict criteria of antigenicity, non-allergenicity, non-toxicity, and conservation. The selected epitopes achieved 100% combined coverage worldwide. The L7/L12 adjuvant was fused at the N-terminus via EAAAK linker, with epitopes linked by AAY, GPGPG, and KK linkers, resulting in a 539-residue construct. The vaccine demonstrated excellent physicochemical stability, high solubility, and broad population coverage. Refined 3D structure (Z-score − 7.5, Ramachandran favored 92.6%) docked favorably with TLR-3/4 (− 1043/ − 1125 kJ/mol), preferring TLR-4 (RMSD 5.41 Å vs. 9.03 Å, RMSF 2.33 Å, Rg 34.15 Å, H-bonds 889-1006, MM-GBSA ΔGbind − 187.10 kcal/mol post-100 ns MD). Immune simulations predicted robust Th1-biased responses following three doses: persistent cytotoxic T-cells, NK/DC/macrophage activation, year-long IgM/IgG memory, and elevated IFN-γ/IL-12/TNF-α cytokines. Codon-optimized mRNA (CAI 0.982, GC 52.56%) cloned into pET-28a (+) showed stable folding. PbatCoV-mVax represents a promising, stable, and theoretically immunogenic prioritization candidate requiring extensive in-vitro and in-vivo validation to establish actual preventive protection against BtHKU5-CoV-2 spillover.