Humoral and Cell-Mediated Immunity Against SARS-CoV-2 in Healthcare Personnel Who Received Multiple mRNA Vaccines: A 4-Year Observational Study

<b>Background/Objectives:</b> The long-term effects of multiple updated vaccinations against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have not been clarified. Humoral or cellular immunity dynamics in healthcare workers for four years were analyzed. <b>Methods:</b> Blood samples were collected at five time points from April 2021 to January 2024. Humoral immunity was analyzed using the 50% neutralizing titer (NT₅₀) against the original Omicron XBB and Omicron BA.2.86 strains and cellular immunity were analyzed using the ELISpot interferon-gamma releasing assay. NT₅₀s and the spot-forming count (SFC) of the ELISpot assay were compared in the SARS-CoV-2 Omicron XBB-, Omicron-infected, and uninfected subjects. <b>Results</b>: 32 healthcare workers (median age, 47 years) who received 3-7 vaccine doses were enrolled. The NT₅₀s against the original strain decreased after the second vaccination but were maintained after the third vaccine dose. NT₅₀s against the Omicron XBB and BA.2.86 strains were detected before the Omicron vaccine was introduced and increased following the updated vaccination. The NT₅₀s against the Omicron XBB and BA.2.86 strains were elevated after natural infection by the Omicron strain, albeit without differences compared with the findings in uninfected subjects. Multivariate regression analysis revealed no confounder that affected the antibody titer against the BA.2.86 strain at the fifth blood sampling. The median number of SFCs ranged from 78 to 208 after the first two doses. <b>Conclusions</b>: Multiple vaccinations induced the production of antibodies with divergent activity against emerging mutant strains and enhanced protective effects against the original strain. This finding supported the importance of updated vaccination.