Potent protective ability of vaccinia virus LC16 strains lacking the B5R gene

We constructed two novel LC16-derived vaccinia viruses as candidate mpox vaccines to clarify the contribution of B5 to vaccine-induced protection. LC16m8Δ2 is a B5R-null mutant derived from LC16mO that contains no artificial sequences. LC16mOB5RΔTM expresses a soluble B5 ectodomain lacking the transmembrane and cytoplasmic domains, enabling evaluation of B5-specific immunity. A single low-dose vaccination (10² PFU) with either virus robustly protected BALB/c mice against an intranasal lethal challenge with 2 × 10⁷ PFU of the pathogenic WR strain. Notably, protection was observed at doses that induced no detectable neutralizing antibodies against either extracellular enveloped viruses (EEV) or intracellular mature viruses (IMV), suggesting that rapid recall responses and/or cellular and innate immune responses may contribute substantially to protection at low doses. Expression of the B5 ectodomain did not enhance anti-EEV neutralizing antibody titers or protective efficacy compared with the B5R-null mutant. These findings indicate that functional B5 is not required for potent protection by LC16-based vaccines, particularly at very low immunization doses.