Background: The immunogenicity of different COVID-19 vaccine regimens and combinations in naïve and convalescent individuals has not been formally tested in controlled studies, and real-life observational studies are scarce. Methods: We assessed the SARS-CoV-2 infection and COVID-19 vaccination-induced immunity of 697 hospital workers at the University Medical Center Hamburg-Eppendorf between 17 and 31 January 2022. Results: The overall prevalence of anti-NC-SARS-CoV-2 antibodies indicating prior infection was 9.8% (n = 68) and thus lower than the seroprevalence in the general population. All vaccinated individuals had detectable anti-S1-RBD-SARS-CoV-2 antibodies (median AU/mL [IQR]: 13,891 [8505–23,543]), indicating strong protection against severe COVID-19. Individuals who received three COVID-19 vaccine doses (median AU/mL [IQR]: 13,856 [8635–22,705]) and those who resolved a prior SARS-CoV-2 infection and had received two COVID-19 vaccine doses (median AU/mL [IQR] 13,409 [6934–25,000]) exhibited the strongest humoral immune responses. Conclusions: The current study indicates that three exposures to the viral spike protein by either SARS-CoV-2 infection or COVID-19 vaccination are necessary to elicit particularly strong humoral immune responses, which supports current vaccination recommendations.