Bioinformatics-Driven Design and Evaluation of Recombinant Multi-Epitope Immunogens Derived From Snake Venom Toxins as Potential Antivenom Candidates

Snakebite envenomation is a major public health concern, particularly in low- and middle-income regions where access to safe and effective antivenoms is limited. Traditional antivenoms, derived from immunization with crude venom, often trigger adverse reactions and lack specificity against key venom components. This study presents a bioinformatics-driven approach to design, construct, and evaluate a novel panel of recombinant multi-epitope toxin-derived immunogens targeting the most clinically significant snake venom toxin families. Five principal toxin families—three-finger toxins (3FT), phospholipase A2 (PLA₂), snake venom metalloproteinases (SVMP), snake venom serine proteases (SVSP), and dendrotoxins (DDT)—were computationally analyzed to identify conserved, antigenic, non-toxic, and non-allergenic B-cell and T-cell epitopes. Multi-epitope recombinant constructs were designed using linker systems, a TLR4 agonist adjuvant, and an MITD-signal sequence to enhance immunogenicity. Structural modeling, refinement, and validation were performed using AlphaFold 3 and GalaxyRefine. Protein–TLR interactions were assessed using molecular docking with ClusPro, selecting the top-ranked pose based on the lowest energy score for initial analysis, as these often correspond to the most stable configurations. Normal mode analysis (NMA), molecular dynamics simulation (MDS), and post-simulation analysis were used to evaluate the stability of the complexes. While in silico immune simulations evaluated the immunogenic potential of the constructs. The designed multi-epitope toxin-derived immunogens were predicted to have favorable physicochemical properties, including molecular weights ranging from 33.7 to 56.37 kDa, basic isoelectric points, high thermostability (aliphatic index: 66.72–77.08), and hydrophilicity (negative GRAVY scores). Refined 3D models exhibited more than 93% residues in Ramachandran favored regions, suggesting structural reliability. Molecular docking revealed strong and stable interactions with TLR2 and TLR4, particularly in the SVMP–TLR4 complex (ΔG = −21.0 kcal/mol; K_d = 3.7 × 10⁻¹⁶ M). NMA, MDS, and post-simulation analysis collectively showed that 3FT immunogen complexes with TLR2/4 were the most stable and compact with coordinated motions, PLA₂ and DDT displayed moderate flexibility with maintained integrity, SVSP showed intermediate instability, and SVMP, particularly with TLR2, exhibited pronounced conformational instability and dynamic disorder, highlighting clear receptor- and toxin-dependent differences in stability and collective behavior. Immune simulations theoretically predicted robust humoral and cellular immune responses, with early IgM/IgG production, expansion of B-cell and T-cell populations, and balanced cytokine profiles indicative of a safe immunogenic response. This study provides in silico evidence suggesting the potential of recombinant multi-epitope toxin-derived immunogens as a next-generation therapeutic strategy for snakebite management. The designed constructs may offer improvements in specificity, safety, and manufacturability over traditional antivenoms, providing a promising foundation for further experimental validation and clinical translation. Future refinements could incorporate cluster overlap evaluation to mitigate bias from single-pose selection, particularly for poses with overlapping scores.