In July 2022, the ongoing monkeypox (MPX) outbreak was declared a public health emergency of international concern by the World Health Organization. Modified vaccinia virus Ankara (MVA-BN, also known as Imvamune, Jynneos, or Imvanex) is a 3rd generation smallpox vaccine that was generated by serial passaging of the more pathogenic parental vaccinia virus (VACV), and is authorized as a vaccine against MPX in humans in a two-shot regimen. Up to now, there is a lack of data that demonstrate MPX virus (MPXV)-neutralizing antibodies in vaccinated individuals and vaccine efficacy data against MPXV infection. Here, we measure MVA-, VACV-, and MPXV-reactive binding and neutralizing antibodies with validated in-house assays in cohorts of historically smallpox-vaccinated, MPXV PCR-positive, and recently MVA-BN-vaccinated individuals. We show that MPXV neutralizing antibodies were detected across all cohorts in individuals with MPXV exposure as well as those who received historic (VACV) vaccination. However, a primary MVA…