SARS-CoV-2 cross-reactive B-cells outnumber seasonal coronavirus spike-specific clones at the end of the COVID-19 pandemic

B-cell responses towards seasonal human coronaviruses (sHCoVs), particularly OC43, impacted those towards SARS-CoV-2 due to immune imprinting in severe COVID-19 patients. However, little is known on how widespread SARS-CoV-2 circulation and COVID-19 vaccination campaigns over the course of the pandemic affected immunity towards sHCoVs in the general population. To explore potential differences in immune recognition of sHCoVs in immunocompetent adults, we compared two cross-sectional cohorts: one sampled between 2018 and 2019 (pre-pandemic), the other at the end of the pandemic (February - March 2023). We compared serum IgG and IgA titers, antibody cross-reactivity patterns at the clonal level, and specificity towards Spike (S) domains for all sHCoVs and dominant SARS-CoV-2 variants by B-cell analysis. Subsequently, we determined the OC43 neutralization potential of sera and monoclonal antibodies targeting different S domains. In pre-pandemic individuals, SARS-CoV-2-reactive antibody and B-cell levels, and sHCoV/SARS-CoV-2 cross-reactivity were negligible. IgA and IgG reactivity against the S of sHCoVs was distributed over spike domain 1 and 2 (S1, S2). In end-pandemic donors, SARS-CoV-2-specific immune responses strongly dominated and the majority of sHCoV reactive clones cross-reacted with SARS-CoV-2. The SARS-CoV-2/sHCoVs cross-reactive clones accounted for higher NL63 S1- and OC43 S2-specific B-cell frequencies and matched higher serum antibody titers. For OC43, the immunodominance of SARS-CoV-2/OC43 cross-reactive IgG B-cells resulted in a strong bias towards S2. Serum OC43 neutralization titers were higher in end-pandemic donors and correlated with OC43 S1 and S2-specific IgG titers and reactive B-cells frequencies. However, the SARS-CoV-2/OC43 S2 cross-reactive IgG clones did not independently correlate with OC43 neutralization titers. We conclude that the establishment of SARS-CoV-2-specific immune responses altered responses to sHCoVs in our cohort, particularly for OC43 and NL63. This could have implications for the immune protection and offers insights for the development of pan-coronavirus treatments and vaccines.