Background: The COVID-19 pandemic provided a unique opportunity to evaluate how the human immune system responded to a novel pathogen and to determine whether immune responses initiated through natural infection differ from those elicited by vaccination against the same antigen. Here, we provide a comprehensive analysis of SARS-CoV-2 Spike (S-antigen)-reactive memory B cells (B_mem) elicited in previously immunologically naïve subjects following their first infection with the original Wuhan-Hu-1 (WH1)-like strain or their initial COVID-19 mRNA prime-boost regimen encoding the same WH1-S-antigen. In particular, we tested the hypothesis that the primary encounter of SARS-CoV-2 S-antigen in lung mucosal tissues during infection vs. intramuscular COVID-19 mRNA injection would elicit different B_mem responses. Methods: Cryopreserved peripheral blood mononuclear cell (PBMC) samples collected following primary infection with the WH1 strain or completion of the initial prime-boost vaccination regimen were tested in ImmunoSpot^® assays to assess the frequency, Ig class/subclass usage, and cross-reactivity of the S-antigen-reactive B_mem compartment; pre-pandemic blood draws served as naïve controls. Results: The B_mem repertoires generated post-infection vs. post-vaccination were found to be quite similar but with some subtle differences. In both cases, the prevalent induction of IgG1-expressing B_mem in similar frequencies was seen, ~30% of which targeted the receptor binding domain (RBD) of the WH1-S-antigen. Also, the extent of cross-reactivity with the future Omicron (BA.1) RBD was found to be similar for both cohorts. However, IgA⁺ B_mem were preferentially induced after infection, while IgG4⁺ B_mem were detected only after vaccination. Conclusions: B_mem elicited in naïve human subjects following SARS-CoV-2 infection or after WH1-S encoding mRNA vaccination were only subtly different, although the relevance of these differences as it relates to immune protection warrants further investigation. Our findings serve to illustrate the usefulness and feasibility of performing comprehensive monitoring of antigen-specific B cell memory in larger cohorts using the ImmunoSpot^® technique.

The measurement of serum antibodies that specifically recognize self-antigens is a critical diagnostic in autoimmunity. A limitation of such an approach is sensitivity to detect the antibody, particularly when abundant self-antigens in the body may bind and sequester circulating specific antibodies. The presence of specific memory B cells (B_mem) may provide a more sensitive and robust indicator of an autoimmune response, as is suggested for certain anti-viral responses. B cell enzyme-linked ImmunoSpot (ELISPOT) is capable of detecting antigen-specific B_mem cells in blood at the single cell level, following stimulation of peripheral blood mononuclear cells (PBMCs) to expand and differentiate the B_mem cells into functional antibody-secreting cells (ASCs). While this assay has been widely utilized in infectious diseases and vaccination, detection is more difficult for autoantigens due to self-tolerance and specific tissue compartmentalization of immune responses, making autoantigen-specific B cells rare in the circulation. The cycles of re-activation of B_mem cells to become ASCs, that may reflect disease flare-ups in autoimmunity, are not well defined. For several autoimmune diseases (ADs), the targeting of B cells via depleting monoclonal antibodies has proven to be an effective treatment, where B_mem cells are likely being targeted. The measurement of autoantigen-reactive B_mem cells may aid in diagnosis and staging of clinical severity, or be a metric for efficacious treatments, thus providing an additional informative biomarker of ADs. How B cell ELISPOT has been utilized to characterize B_mem cells in human ADs is described here, including the advantages and disadvantages of the assay.

Memory B cells (Bmem) facilitate the generation of renewed and rapid antigen-specific antibody responses long after the initial antigen exposure, at a time when circulating serum antibodies may have declined. As the generation and/or recruitment of Bmem is at the core of most vaccination strategies, the assessment of antigen-specific Bmem is highly informative for forecasting and profiling the elicited B cell immune response. The two prevalent techniques used to detect antigen-specific Bmem cells at single cell resolution are probe-based flow cytometry and B cell ImmunoSpot, while the measurement of B cell-derived antibodies in culture supernatants of stimulated B cells offers a semi-quantitative alternative. To the best of our knowledge, a direct side-by-side comparison of these test systems has not yet been reported. Hence, in a blinded fashion, we compared these three test systems in parallel, aiming to detect SARS-CoV-2 Spike protein- (S-antigen) specific IgG+ Bmem in peripheral blood mononuclear cells (PBMC) samples obtained from well defined cohorts comprising: pre-COVID era “naïve” individuals (negative controls), individuals shortly after recovery from a PCR-verified SARS-CoV-2 infection (positive controls), and a cohort of donor PBMC isolated in 2024 (the experimental group). Our results demonstrate that all three assays are suited for the detection of specific Bmem in antigen-primed individuals when such Bmem occur in the mid- to high frequency range; strengths and weaknesses of the three test systems are discussed.

Background: Memory B cells (B_mem) facilitate the generation of renewed and rapid antigen-specific antibody responses long after the initial antigen exposure, at a time when circulating serum antibodies may have declined. As the generation and/or recruitment of B_mem is at the core of most vaccination strategies, the assessment of antigen-specific B_mem is highly informative for forecasting and profiling the elicited B cell immune response. Methods: The two prevalent techniques used to detect antigen-specific B_mem cells at single-cell resolution are probe-based flow cytometry and B cell ImmunoSpot, while the measurement of B cell-derived antibodies in culture supernatants of stimulated B cells offers a semi-quantitative alternative. To the best of our knowledge, a direct side-by-side comparison of these assay systems has not yet been reported using the same starting PBMC material in a blinded fashion to test all three assays simultaneously. Results: These three assay systems were run in parallel to detect SARS-CoV-2 Wuhan-1 strain Spike-specific IgG⁺ B_mem in peripheral blood mononuclear cell (PBMC) samples obtained from well-defined cohorts comprising pre-COVID-19 era “naïve” individuals (negative controls), individuals shortly after recovery from a PCR-verified SARS-CoV-2 infection (positive controls), and a cohort of donor PBMCs isolated in 2024 (the experimental group). Each assay was able to discern Spike-exposed individuals from naïve , with ImmunoSpot suggesting superior sensitivity and specificity. ImmunoSpot and flow cytometry results were closely correlated. Conclusions: The study demonstrates that all three assays are suited for the detection of specific B_mem in antigen-primed individuals when such B_mem occur in the mid- to high-frequency range, and that they broadly concur. Strengths and weaknesses of the three test systems are discussed.