A Multivalent Dengue Fusion Protein ΔcNS1–cEDIII–ΔnNS3 Confers Cross‐Serotype Protection and Durable Immunity in Mice

ABSTRACT Current dengue vaccines remain limited by serotype‐dependent efficacy and interference from preexisting anti‐dengue immunity. We developed a novel multivalent fusion protein vaccine composed of three engineered dengue virus (DENV) components: a C‐terminal truncated nonstructural protein 1 (ΔcNS1) to block NS1‐mediated pathologic effects without harmful cross‐reactivity, a consensus envelope protein domain III (cEDIII) to induce broad neutralizing antibodies, and an N‐terminal truncated NS3 (ΔnNS3) to enhance cellular immune responses. In a murine dengue disease model, three‐dose immunization with ΔcNS1–cEDIII–ΔnNS3 adjuvanted with Alum provides protection against all four DENV serotypes by significantly reducing viremia and prolonged bleeding time, with elicited robust antibody responses, enhanced cytotoxic activity of CD8 + T cells upon NS1/NS3 restimulation, and increased memory B and T cell populations. Notably, CpG oligodeoxynucleotides 1826 (CpG) plus Alum further enhanced immunogenicity, showing higher neutralizing activity, antigen‐specific plasmablast expansion, and enhanced T cell functional activity, which was associated with more consistent improvement in protection‐relevant outcomes compared with Alum alone. Importantly, two‐dose immunization with CpG plus Alum‐adjuvanted fusion protein conferred durable protection against the virulent DENV2 strain TW2015. These findings support this vaccine as a promising subunit candidate that addresses current limitations, offering both cross‐serotype coverage and potential long‐term efficacy.