The impact of antigenic distance on Orthopoxvirus Vaccination and Mpox Infection for cross-protective immunity

Immunological memory mediates rapid protection following infection or vaccination including heterologous exposure. However, cross-reactive memory responses in humans remain poorly characterized. We explored the longevity and specificity of cross-protective responses to orthopoxviruses through smallpox vaccination and Mpox virus (MPXV) infection. Smallpox vaccination using Vaccinia virus (VACV)-based vaccines provides a unique opportunity to study long-term cross-protective immunity without antigen re-exposure. We assessed systemic and mucosal responses in four human cohorts, including first-(Dryvax) and/or third-generation (JYNNEOS) smallpox vaccine recipients (vaccinated 1 week-80 years ago), along with Mpox-infected individuals. First-and third-generation smallpox vaccines elicited strong VACV- and MPXV-specific antibodies. VACV-neutralizing antibodies persisted for decades in first-generation vaccine recipients and were further enhanced after JYNNEOS vaccination. However, despite the high levels of anti-MPXV-specific antibodies in the plasma, cross-neutralization activity was directly correlated with the antigenic distance. Higher neutralization was observed for the cowpox virus (CWPXV) than for MPXV, which showed lower antigenic conservation with VACV. Similarly, Mpox-infected patients had lower neutralization titers for VACV than for CWPXV. Individuals who received vaccination boosters showed more robust, diverse, and prolonged cross-neutralizing responses. Long-term memory analysis revealed an increase in neutralization capacity for VACV over decades, with 80-years-old displaying the most robust humoral response, although this trend was not observed for cross-reactive antigens. Finally, T-cell reactivity to VACV and MPXV epitopes was detected decades post-vaccination, suggesting a role of long-lasting cross-reactive T-cell memory responses in vaccine efficacy. Our findings underscore the pivotal influence of antigenic distance on vaccine effectiveness with implications for cross-protective vaccine design.