Intramuscular Non-replicating Tiantan Vaccinia Drives Superior Antibody Maturation and Sterilizing Protection Against Lethal Orthopoxvirus Challenge in Mice.

The global resurgence of orthopoxviruses, particularly MPXV, necessitates safe yet highly protective vaccines. Here, we systematically evaluated a non-replicating Tiantan vaccinia virus (NTV)-derived from the historically efficacious vaccinia Tiantan strain-across intramuscular (IM), subcutaneous (SC), and dose-sparing intradermal (ID) routes in lethal murine models. While all regimens elicited robust polyfunctional T cells, cross-neutralizing antibodies against MPXV, VTT, and ECTV, and protective immunity against standard lethal VACV-WR and ECTV challenges, under extreme viral exposure (70 LD50) and long-term challenge (16 week), only full-dose NTV-IM among the non-replicating regimens conferred complete sterilizing cross-protection, whereas the SC and dose-sparing ID regimens exhibited breakthrough virologic failure. Mechanistically, splenic transcriptomics revealed that NTV-IM uniquely enriched for a prominent germinal center (GC) transcriptional profile (marked by Aicda). This GC signature was multi-dimensionally validated by RT-qPCR, flow cytometry, and immunofluorescence, confirming robust expansion of GC B cells (CD95+GL7+) and highly organized follicular architecture in lymphoid organs. This optimized GC response closely correlated with superior affinity maturation and class-switch recombination, aligning with the production of the highest titers of IgG and cross-neutralizing antibodies in NTV-IM while without inducing systemic inflammation. Collectively, these findings highlight route-dependent immune programming, establishing NTV-IM as a scalable, next-generation orthopoxvirus vaccine candidate.