The impact of orthopoxvirus vaccination and Mpox infection on cross-protective immunity: a multicohort observational study.

Background: Cross-reactive immune memory responses to orthopoxviruses in humans remain poorly characterised despite their relevance for vaccine design and outbreak control. We aimed to assess the magnitude, specificity, and durability of cross-reactive immune responses elicited by smallpox vaccines and mpox virus infection.

Methods: We did a multicohort observational study involving participants from the USA, Brazil, and Portugal across four groups: Dryvax (first-generation smallpox vaccine) recipients vaccinated 40-80 years ago, JYNNEOS (third-generation smallpox vaccine) recipients vaccinated within the past year, a cohort receiving both vaccines, and patients infected with clade IIb mpox. Samples were analysed for systemic and mucosal humoral responses, neutralising antibody titres, viral antigen structural analysis, and T-cell cross-reactivity to vaccina virus, cowpox virus, and mpox virus. Statistical analyses included correlation assessments and comparisons across cohorts to determine the magnitude, longevity, and breadth of immune responses.

Findings: Between July 7, 2022, and Aug 3, 2023, 262 participants were recruited, resulting in analysis of 378 samples. Both first-generation and third-generation smallpox vaccines elicited vaccinia virus-reactive and mpox virus-reactive antibodies, with the strongest responses targeting the less conserved extracellular virion antigens B5 and A33. Despite high concentrations of anti-mpox virus antibodies in the plasma, cross-neutralisation activity correlated with viral antigenic distance. Higher neutralisation was observed for cowpox virus than for mpox virus, which has lower antigenic conservation with vaccina virus. Complement-mediated neutralisation enhanced mpox virus neutralisation, overcoming the limitations of antigenic distance. Dryvax recipients sustained vaccina virus neutralisation titres for over 80 years, whereas cross-reactive responses did not show this durability. JYNNEOS-induced responses waned within a year. T-cell cross-reactivity was long-lasting, detected up to 70 years after vaccination. Booster vaccinations augmented the magnitude, breadth, and longevity of cross-neutralising responses.

Interpretation: Our findings highlight the potential combined role of antibody effector functions and T-cell memory in cross-protection against orthopoxviruses. Complement-mediated neutralisation enhances cross-protection, overcoming antigenic distance. These Fc-mediated functions, along with T-cell responses, contribute to effective and long-lasting immunity conferred by smallpox vaccines against other orthopoxviruses.

Funding: Yale University and Stavros Niarchos Foundation Institute for Global Infectious Disease.