Background and Objective: The role of previous SARS-CoV-2 infection/vaccine-induced humoral immunity against protection of Omicron BA.5 infection is unknown. We examined the association between pre-infection anti-SARS-CoV-2 spike antibody titers and the effectiveness against Omicron BA.5 infection among staff of a medical and research center in Tokyo.  Methods: A total of 2610 staff participated in a serosurvey in June 2022 (baseline), were measured with anti-SARS-CoV-2 antibodies (spike and nucleocapsid [N] proteins; Abbott and Roche), and answered a questionnaire. Previous SARS-CoV-2 infection was defined according to a history of COVID-19 and anti-N seropositivity at baseline. Using in-house COVID-19 registry, we followed participants for SARS-CoV-2 infection from baseline to September 21, 2022, during the Omicron BA.5 epidemic in Japan. We used a Cox proportional hazard model to estimate the hazard ratio of Omicron BA.5 infection; and calculated effectiveness as (1 – hazard ratio)×100.   Results: At baseline, 92% have completed 3-dose vaccinations, whereas 16% had previous SARS-CoV-2 infection (mainly occurred during Omicron BA.1/BA.2 waves). Those with previous infection had higher anti-spike antibody titers than infection-naïve (median titer: 29,201 v.s. 4,849). After adjusting confounders, higher anti-spike antibody titers were associated with higher effectiveness (3.7% per 1000 titer [95% CI: 3.3–4.2]). The association appears stronger among those previously infected; 50% effectiveness was achieved at 20,000 and 28,000 AU/ml among those previously infected and infection-naïve participants, respectively, and 80% effectiveness was achieved only among the former at 54,000 AU/ml.   Conclusions: Among vaccine recipients (mainly 3-dose) who had experienced Omicron BA.1/BA.2 wave, we found that higher anti-spike antibody titers were associated with a lower risk of Omicron BA.5 infection, and the association was enhanced by previous infection. These data suggest that pre-infection spike antibody titers inform the risk of Omicron BA.5 infection and that high effectiveness can only be achieved with hybrid immunity from infection and 3-dose vaccination.