Altering the intracellular trafficking of Necator americanus GST-1 antigen yields novel hookworm mRNA vaccine candidates.

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. Author summary: Our work on Na-GST-1 mRNA vaccines addresses a critical need in the fight against hookworm infections, which affect more than 100 million people worldwide. Na-GST-1, a protein essential for the survival of hookworms, has been explored as a target for vaccine development. By using mRNA technology, we designed vaccine candidates that express and deliver Na-GST-1 to different locations in cells and tissues of immunized mice, including forms that are secreted, anchored to the plasma membrane, or retained within cells (the native form). Our findings reveal that the location of antigens within cells significantly impacts the immune response. Secreted and membrane-anchored forms of Na-GST-1 generated strong antibody responses, which are important for neutralizing Na-GST-1 in the hookworm. Meanwhile, the intracellular and membrane anchored forms elicited a robust cytotoxic cell response, which would be more important for targeting intracellular pathogens. This research not only advances our understanding of how protein localization affects immune responses but also highlights the versatility and potential of mRNA vaccines to target various pathogens by manipulating how and where vaccine antigens are presented to the immune system. [ABSTRACT FROM AUTHOR]