Transdermal Delivery of an Adjuvanted SARS-CoV-2 Triple RBD Subunit Vaccine in Mice Using Dissolving Microneedle Array Patch

Background: The continuous emergence of antigenically distinct SARS-CoV-2 variants of concern (VOCs) necessitates the development of versatile and effective vaccine platforms capable of inducing broad and sustained immunity. Methods: We designed a chimeric trivalent recombinant protein, TriRBD, incorporating the receptor-binding domains (RBDs) of the Delta, Omicron BA.1, and XBB.1 subvariants. To enhance delivery efficiency and immunogenicity, TriRBD was formulated into a dissolving hyaluronic-acid-based microneedle array patch (MAP) using the droplet extension (DEN) technique. We investigated the synergistic effects of two squalene-based oil-in-water (O/W) emulsions, SqTS (Squalene/Tween-80/Span-85) and SqT (Squalene/Tween-80), as adjuvants within the MAP after gamma-irradiation sterilization, alongside safety evaluations via rabbit skin tests. Results: The SqTS-adjuvanted MAP formulation was found to be non-irritating. Immunogenicity studies in BALB/c mice revealed that MAP-delivered TriRBD elicited significantly higher and more durable IgG endpoint titers (EPT) compared to intramuscular injection, with robust antibody levels sustained for up to 52 weeks. Notably, the irradiated SqT-adjuvanted MAP group exhibited a highly balanced Th2/Th1 immune response (IgG1/IgG2a ratio), while the irradiated SqTS-adjuvanted MAP group shifted toward a Th2-preferred against TriRBD. Although the EPT against the Omicron BA.1 variant was relatively lower, the irradiated MAP-SqT-TriRBD group elicited significantly both of IgG1 and IgG2a, resulting in a balanced and broadened antibody repertoire. Conclusions: These findings demonstrate that combining a trivalent RBD antigen with a SqT-adjuvanted MAP platform provides a potent, safe, and thermostable strategy for broad-spectrum protection against evolving SARS-CoV-2 variants and future pandemic threats.