Persistence of Vaccinia Virus-Neutralizing Antibodies and Cross-Reaction Against Monkeypox Virus Decades After Smallpox Vaccination in an Italian Population

Background/Objectives: Routine smallpox vaccination ended after eradication, leaving uncertainty about the persistence of vaccinia virus (VACV)-neutralizing antibodies and their activity against the monkeypox virus (MPXV) responsible for the 2022 outbreak. We assessed both responses in an Italian serum-bank cohort. Methods: We analyzed sera collected in 2016–2022 from 287 individuals born in or before 1974, whose presumed vaccination status was inferred from birth year, and 100 individuals born in 1977–1982. Neutralization of VACV ACAM2000 and an Italian clade IIb MPXV isolate was measured using the 50% plaque reduction neutralization test (PRNT50). We compared paired titers and calculated the proportions with titers ≥1:20, a putative threshold for VACV applied to MPXV for homogeneity, lacking any specific information. Results: In the earlier birth cohort, VACV-neutralizing antibodies were detected at titers ≥1:10 in 207/287 samples (72.1%) and MPXV-neutralizing antibodies in 137/287 (47.7%). Titers ≥1:20 were found in 121/287 (42.16%; 95% CI 36.59–47.94) for VACV and 68/287 (23.69%; 95% CI 19.14–28.94) for MPXV. MPXV titers were lower than paired VACV titers (p < 0.0001), and titers against the two viruses were positively correlated (Spearman’s rs = 0.90, p < 0.0001). All 100 sera from the younger birth cohort were negative for both viruses. Conclusions: VACV-neutralizing antibodies remained detectable in many individuals from cohorts likely vaccinated in childhood, including some individuals presumed vaccinated more than 60 years ago. Cross-neutralization of MPXV was less frequent and weaker. Integrated prospective investigations combining functional serology with T-cell responses in representative populations might be helpful for preparedness in case of deliberate release of variola virus. Real-world vaccine effectiveness data are crucial for epidemic emergency management of mpox.