Immunoinformatics-Based Design of a Chimeric Vaccine Against Necator americanus Targeting the Na-APR-1, Na-GST-1, and Na-ASP-2 Vaccine Candidates

Hookworm infection, caused predominantly by Necator americanus, is a soil-transmitted helminthiasis that disproportionately affects impoverished communities across tropical and subtropical regions, with an estimated 406-480 million people infected globally. Control continues to rely largely on mass drug administration, an approach challenged by rapid reinfection, the absence of durable protective immunity, and growing concern over reduced drug efficacy. While the leading recombinant protein antigens Na-APR-1, Na-GST-1, and Na-ASP-2 have demonstrated clinical safety and immunogenicity, the withdrawal of Na-ASP-2 after triggering allergic reactions in individuals with prior natural exposure revealed both the promise and the risk of single-antigen approaches, underscoring the need for a rationally designed multi-antigen vaccine. We used an immunoinformatics approach to design a novel chimeric vaccine candidate by combining carefully selected B-cell and T-cell epitopes from the major clinical-stage hookworm protein antigens with an immunostimulatory adjuvant sequence. The candidate's 3D structure was predicted, refined, and validated using established bioinformatics approaches, and its interaction with the innate immune receptor TLR4 was assessed through molecular docking and normal-mode analyses. The selected antigens showed conservation across related hookworm species, suggesting the potential for broader cross-protection. The designed candidate was predicted to be antigenic, non-allergenic, and non-toxic, and immune simulation analyses indicated its capacity to elicit coordinated humoral and cellular immune responses. HLA coverage analysis indicated broad population applicability. Docking and normal-mode analyses of its interaction with TLR4 indicated stable and favourable binding. This study offers a rational, safety-driven approach to multi-epitope vaccine design against hookworm infection, integrating the strengths of clinically evaluated protein antigens while addressing the safety concerns raised by prior single-antigen candidates. Further experimental validation is required to confirm the safety and efficacy of the vaccine construct in vitro and in vivo. These findings lay the groundwork for future efforts toward an effective immunoprophylactic strategy against this neglected disease.