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

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.