BACKGROUND: The antigen Na-GST-1, expressed by the hookworm Necator americanus, plays crucial biochemical roles in parasite survival. This study explores the development of mRNA vaccine candidates based on Na-GST-1, building on the success of recombinant Na-GST-1 (rNa-GST-1) protein, currently assessed as a subunit vaccine candidate, which has shown promise in preclinical and clinical studies. METHODOLOGY/FINDINGS: By leveraging the flexible design of RNA vaccines and protein intracellular trafficking signal sequences, we developed three variants of Na-GST-1 as native (cytosolic), secretory, and plasma membrane-anchored (PM) antigens. After one immunization in mice, mRNA vaccines induced an earlier onset of antigen-specific antibodies compared to rNa-GST-1. Following two immunizations, mRNA vaccines induced similar or superior levels of antigen-specific antibodies compared to rNa-GST-1. Secretory Na-GST-1 was comparable to rNa-GST1 in producing neutralizing antibodies against Na-GST-1's thiol transferase activity, while native Na-GST-1 induced a more robust CD8+ T cell response due to its intracellular accumulation. Although PM Na-GST-1 elicited one of highest titers of antigen-specific antibody and a diverse set of memory T-cell populations, it resulted in a lower ratio of neutralizing antibodies after IgG purification compared to the other vaccine candidates. CONCLUSIONS/SIGNIFICANCE: These findings emphasize the importance of antigen localization in tailoring immune responses and suggest that extracellular antigens are more effective for inducing humoral responses, whereas cytosolic antigen accumulation enhances MHC-1 peptide presentation. Future studies will determine if these in vitro and immunogenicity findings translate to in vivo efficacy. Altogether, mRNA vaccines offer numerous possibilities in the development of multivalent vaccines with single or multiple antigens.