W135 - Next-generation B Cell ImmunoSpot® Assays Permit In-depth Assessment of the B Cell Response Elicited by SARS-CoV-2 Infection And/or COVID-19 mRNA Vaccination

Noemi Becza – Research and Development – Cellular Technology Limited; Lingling Yao, PhD – Research and Development – Cellular Technology Limited; Zhigang Liu, PhD – Research and Development – Cellular Technology Limited; Jack Chepke – Research and Development – Cellular Technology Limited; Paul Lehmann, PhD – Cellular Technology Limited

Abstract Text: The affinity distribution of the antigen-specific memory B cell (Bmem) repertoire in the body is a critical variable that defines an individual’s ability to rapidly generate protective antibody specificities. Defining the Ig class and IgG subclass usage of such antigen-specific Bmem is also critical since it forecasts the anamnestic, recall response to be engaged upon antigen re-encounter. Here, we leveraged multiplexed B cell ImmunoSpot® assays to comprehensively define the magnitude and Ig class/IgG subclass usage of the SARS-CoV-2 Spike-specific Bmem repertoire present in convalescent subjects following PCR-verified infection, and/or after COVID-19 mRNA vaccination. Beyond evidencing past SARS-CoV-2 infection more reliably than assessment of plasma IgG reactivity against the intracellular nucleocapsid (NCAP) protein, direct assessment of the Bmem repertoire also provided invaluable insights into the affinity distribution and cross-reactivity profile of the Spike-specific Bmem repertoire. To do so, the frequency of receptor-binding domain (RBD)-specific antibody-secreting cells (ASC) in the test sample was first determined through quantification of individual spot-forming units (SFU) in wells seeded with decreasing cell inputs. Using additional aliquots of cryopreserved PBMC, and seeding the test sample in many replicate wells at the so-called “Goldilocks number” to generate ~50 SFU based on the previously calculated frequency, the affinity distribution of the RBD-specific ASC repertoire was evaluated against the prototype (Wuhan/1) and additional variant strains of SARS-CoV-2. Collectively, B cell ImmunoSpot® assays offer tremendous value for future B cell immune monitoring efforts owing to the ease of implementation, economical utilization of PBMC, high-throughput capacity, and suitability for regulated testing.