Background: Measuring frequencies of antigen-specific memory B cells (B_mem), their immunoglobulin (Ig) class and subclass usage, cross-reactivity, and affinity can provide insights into the efficacy of future antibody responses in case of antigen re-encounter. B cell ImmunoSpot^® assays can provide such information; however, like most cell-based tests, they require considerable amounts of blood to be drawn from the donor and this has hindered their inclusion in clinical trials and routine immune diagnostics. Methods: We introduce strategies for reducing the cell numbers required to 2–3 million peripheral blood mononuclear cells (PBMCs) per antigen, obtainable from 2–3 mL of blood from healthy adult donors. Results: Except when B_mem frequencies were very low, we found that testing PBMCs in singlet wells, but in serial dilution, enables as reliable B_mem frequency assessments as when testing replicate wells at a single fixed cell number. Additionally, B cell ImmunoSpot^® assays can be multiplexed for detecting four Ig classes, or IgG subclasses, simultaneously and without loss of sensitivity. The requirement for low cell numbers and the retention of B cell functionality by cryopreserved PBMCs equivalent to freshly isolated material implies that fewer than the standard 10 million PBMCs per vial can be frozen. This would reduce the number of individuals who could not be tested for B_mem due to insufficient availability of PBMCs, a common problem with such assays. Conclusions: The predictable need for and recovery of cryopreserved PBMCs facilitates planning of and optimal cell utilization in B cell ImmunoSpot^® assays and increases the practical feasibility of extensive B_mem characterization in larger cohorts.

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.

Background/Objectives: Despite clonal expansion during a primary immune response, or after subsequent antigen encounters, the frequency of memory B cells (B_mem) specific for an antigen remains low, making their detection difficult. However, unlike serum antibodies, which have a short half-life in vivo and thus require continuous replenishment to maintain stable titers, circulating B_mem are long-lived; they preserve immunological preparedness through their ability to rapidly engage in recall responses and differentiate into antibody-secreting cells (ASCs) upon antigen encounter. To this end, development of assays suited for the reliable detection of rare antigen-specific B_mem is critical and can provide insights into an individual’s antigen exposure history and immune status beyond that offered by traditional serum antibody measurements alone. Methods: ImmunoSpot^® has emerged as a suitable technique for the detection of individual antigen-specific B cells through visualizing their antibody-derived secretory footprints. Here, we report the theoretical and practical foundations for detecting rare antigen-specific B_mem in human peripheral blood mononuclear cells (PBMC). Leveraging the unique availability of verifiably naïve vs. antigen-experienced human samples, we used SARS-CoV-2 Spike (S-) and Nucleocapsid (NCAP) antigens to interrogate the presence of B_mem with these respective specificities. Results: While 100% diagnostic accuracy was achieved for both antigens, detection of NCAP-specific B_mem required reducing the lower detection limit of the standard assay. Specifically, this was achieved by testing a total of 2 million PBMC across multiple replicate assay wells and assessing the cumulative number of secretory footprints detected. Conclusion: The protocols described here should facilitate the reliable detection of ASCs present at varying precursor frequencies and serve as guidance for routine immune monitoring of rare B_mem with specificity for any antigen.

Background: Measuring frequencies of antigen-specific memory B cells (Bmem), their immunoglobulin (Ig) class and subclass usage, cross-reactivity and affinity can provide insights into the efficacy of future antibody responses in case of antigen re-encounter. B cell ImmunoSpot assays can provide such information; however, like most cell-based tests, they too require considerable amounts of blood to be drawn from the donor and this has hindered their inclusion into clinical trials and routine immune diagnostics. Methods: We introduce strategies for reducing the cell numbers required to 2-3 million peripheral blood mononuclear cells (PBMC) per antigen, obtainable from 2-3 mL of blood from healthy adult donors. Results: Except when Bmem frequencies were very low, we found that testing PBMC in singlet wells, but in serial dilution, enables as reliable Bmem frequency assessments as testing replicate wells at a single fixed cell number. Additionally, B cell ImmunoSpot assays can be multiplexed for detecting four Ig classes, or IgG subclasses, simultaneously and without loss of sensitivity. The requirement for low cell numbers and the retention of B cell functionality of cryopreserved PBMC equivalent to freshly isolated material implies that fewer than the standard 10 million PBMC per vial must be conserved. This would reduce the number of individuals who could not be tested for Bmem due to insufficient availability of PBMC, a common problem with such assays. Conclusions: The predictable need for, and recovery of cryopreserved PBMC facilitates planning of, and optimal cell utilization in B cell ImmunoSpot assays and increases the practical feasibility of extensive Bmem characterization in larger cohorts.

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 (Bmem) elicited in previously immunologically naïve subjects following their first infection with the original Wuhan-Hu-1 (WH1)-like strain, or their initial COVD-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 Bmem 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 Bmem compartment; pre-pandemic blood draws served as naïve controls. Results: The Bmem 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 Bmem 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+ Bmem were preferentially induced after infection, while IgG4+ Bmem were detected only after vaccination. Conclusions: Bmem elicited in naïve human subjects following SARS-CoV-2 infection or after WH1-S-antigen encoding mRNA vaccination were only subtly different. 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.

Despite clonal expansion during a primary immune response, or after sub-sequent antigen encounters, the frequency of memory B cells (Bmem) specific for an antigen remains low, making their detection difficult. However, unlike serum an-tibodies, which have a short half-life in vivo and thus require continuous replen-ishment to maintain stable titers, circulating Bmem are long-lived; they preserve immunological preparedness through their ability to rapidly engage in recall re-sponses and differentiate into antibody-secreting cells (ASCs) upon antigen en-counter. Consequently, developing means for the reliable detection of anti-gen-specific Bmem promises to offer a window into an individual’s exposure history and immune status beyond that gleaned from traditional serum antibody meas-urements alone. ImmunoSpot® has emerged as a suitable technique for the de-tection of individual antigen-specific B cells through visualizing their anti-body-derived secretory footprints. Here, we report the theoretical and practical foundations for detecting rare antigen-specific Bmem in human peripheral blood mononuclear cells (PBMC). Leveraging the unique availability of verifiably naïve vs. antigen-experienced human samples, we used SARS-CoV-2 Spike (S-) and Nucleocapsid (NCAP) antigens to interrogate the presence of Bmem with these re-spective specificities. While 100% diagnostic accuracy was achieved for both an-tigens, detection of NCAP-specific Bmem required extension of the lower detection limit (LOD) of the standard assay. This could be achieved using 2 million PBMC in aggregate. The protocols described here should facilitate reliable detection of ASC present at varying precursor frequencies and serve as guidance for routine im-mune monitoring of rare Bmem with specificity for any antigen.