Immunogenicity and protective efficacy of an inactivated cluster 2.1 duck Tembusu virus vaccine in ducks: Evidence of cross-genotype immune responses.

Duck Tembusu virus (DTMUV) is an emerging flavivirus causing neurological disease and severe egg drop syndrome in ducks, resulting in significant economic losses across Asia. Most current DTMUV vaccine candidates are derived from cluster 2.2 Chinese strains, while vaccines targeting other circulating clusters, particularly those prevalent in Thailand, remain unavailable. Given the marked antigenic differences among DTMUV clusters, which may limit cross-protection, the development of region-specific vaccines is urgently needed. Here, we evaluated the immunogenicity and protective efficacy of a newly developed inactivated cluster 2.1 DTMUV vaccine using one- and two-dose regimens in ducks and assessed its ability to induce cross-genotype immune responses by measuring cross-neutralizing antibody responses and T-cell cross-reactivity against heterologous cluster 1 and/or cluster 3 viruses. Our results demonstrated that both vaccination regimens provided complete clinical protection against morbidity and mortality following lethal homologous cluster 2.1 DTMUV challenge. However, the two-dose regimen elicited stronger and more rapid anamnestic adaptive immune responses, conferred superior protection against pathological lesions, and more effectively reduced viral loads in target tissues and viral shedding compared with the one-dose regimen. Furthermore, the vaccine, particularly under the two-dose regimen, induced cross-reactive humoral and cellular immune responses to clusters 1 and/or 3; however, its ability to confer heterologous protection remains unknown and warrants further investigation through in vivo challenge studies. Taken together, these results highlight the inactivated cluster 2.1 DTMUV vaccine as a promising and effective candidate for homologous protection against cluster 2.1 DTMUV in ducks.