Abstract <p>Monkeypox (Mpox) remains a significant public health concern due to the emergence of rapidly evolving strains and the suboptimal efficacy of existing smallpox vaccines. Recombinant subunit vaccines offer a promising and safe alternative. The A29L protein of the Mpox virus (MPXV) is essential for viral attachment and entry. The N-terminal residues 1–34 of A29L, which harbor the heparin-binding domain, are critical targets for neutralizing antibodies. Based on this, we hypothesized that this truncated 34-residue peptide of A29L could serve as a potent subunit vaccine candidate. To enhance immunogenicity, the recombinant antigen comprising three tandem repeats of the 34-residue peptide (designated as Tri(1–34)) was engineered. Following the immunization of BALB/c mice, Tri(1–34) elicited robust A29L-specific antibody titers comparable to those induced by the full-length protein, demonstrating equivalent immunogenicity. Furthermore, antisera collected from Tri(1–34)-immunized mice specifically recognized the A29L N-terminus and effectively neutralized MPXV infection in Vero E6 cells, as evidenced by the plaque reduction neutralization test (PRNT) assays. Immunization with Tri(1–34) also triggered strong Th1-biased cellular immune responses, characterized by significant interferon-gamma (IFN-γ) production. In conclusion, the engineered Tri(1–34) antigen represents a safe and effective subunit vaccine candidate, offering a strategic framework for future Mpox vaccine development.</p> Key points <p>• <i>Engineered Tri(1–34) targets the critical A29L N-terminus of Mpox virus</i></p> <p>• <i>Tri(1–34) elicits potent neutralizing antibodies against Mpox virus</i></p> <p>• <i>Tri(1–34) induces a robust Th1-biased response with high IFN-γ levels</i></p>