AbstractLimited data exist on antibody responses to mixed vaccination strategies involving inactivated COVID-19 vaccines, particularly in the context of emerging variants. We conducted an open label trial and administered a third vaccine dose of an mRNA vaccine (BNT162b2, BioNTech/Fosun Pharma) in adults aged ≥30 years who had previously received two doses of an inactivated COVID-19 vaccine. We collected blood samples prior to administering the third dose and 28 days later, and tested for antibodies to the ancestral virus using a binding assay (ELISA), a surrogate virus neutralization test (sVNT) and a live virus plaque reduction neutralization test (PRNT), and to the Omicron variant using PRNT. A third dose of BNT162b2 substantially increased antibody titers on each assay. Mean ELISA levels increased from an optical density (OD) of 0.3 to 2.1 (p<0.01), and mean sVNT levels increased from an inhibition of 17% to 96% (p<0.01). In a random subset of 20 participants, the geometric mean PRNT50 titers rose very substantially by at least 27 fold from Day 0 to Day 28 against the ancestral virus (p<0.01) and rose by at least 14 fold against the Omicron variant (p<0.01). In daily monitoring, post-vaccination reactions subsided within 7 days for over 99% of participants. In conclusion, a third dose of COVID-19 vaccination with an mRNA vaccine substantially improved antibody levels against the ancestral virus and against the Omicron variant with well-tolerated safety profile, in adults who had received two doses of inactivated vaccine 6 months earlier.