Evaluating Memory B Cell Cross-Reactivity Between Ancestral and Future SARS-CoV-2 Variants—Evidence for Original Antigenic Sin

Background: Despite the circulation of evolutionarily related cold-causing coronaviruses (CCCs) in the pre-COVID era, most individuals lacked pre-existing serum IgG and/or class-switched memory B cell (Bmem) reactivity for the SARS-CoV-2 Spike (S) glycoprotein expressed by the ancestral Wuhan-Hu-1 (WH1) strain. Subsequent priming of the immune system through natural infection or prophylactic COVID-19 mRNA vaccination successfully generated robust Bmem responses against the WH1-S antigen, along with eliciting cross-reactivity for the future Omicron (BA.1) variant responsible for breakthrough infections (BTIs). However, to what extent immunological imprinting of Bmem towards the WH1-S antigen detrimentally constrains the elicitation of variant-specific antibody responses following subsequent booster vaccinations or BTIs—a phenomena referred to as “original antigenic sin”—remains an unresolved and open question. Methods: Using ImmunoSpot®, we evaluated peripheral blood mononuclear cells (PBMCs) from defined human cohorts for IgG+ ASC reactivity against Spike proteins representing CCCs and SARS-CoV-2. Additionally, we developed a novel dual-label inverted FluoroSpot assay to distinguish between strain-specific and cross-reactive IgG+ ASCs recognizing epitopes in the receptor binding domain (RBD) of SARS-CoV-2 Omicron variants. Results: Our data demonstrate a lack of appreciable back-boosting of IgG+ Bmem recognizing structurally conserved epitopes shared between CCCs and SARS-CoV-2. Moreover, we found evidence for immunological imprinting and the preferential expansion of Bmem recognizing cross-reactive epitopes in the RBD following BTI. Nevertheless, Omicron strain-specific Bmem were detected in PBMC donors collected in 2025. Conclusions: Our novel inverted dual-label FluoroSpot methodology evidenced preferential expansion of cross-reactive Bmem following breakthrough SARS-CoV-2 infection and supports the influence of original antigenic sin shaping the recall response. Moreover, the inverted dual-label assay provides a highly flexible and easily implementable technique for distinguishing between strain-specific and cross-reactive B cell responses and has broad applications in translational vaccine research against pathogens that undergo antigenic drift.