Fig 2: Spike and epitope binding by monoclonal antibodies Spike recognition by monoclonal antibodies assessed by flow cytometry. Each contour plot shows mCherry on the horizontal axis, so that spike‐mCherry expressing cells are on the right of the plot, and TE 0 cells on the left. Vertical axes show signal of secondary antibodies used to detect IgM, IgA, or IgG.Heatmap of antibody binding to spike protein subdomains (complete spike extracellular domain "spike", S1, S2, RBD and NTD) in ELISA. Color gradient correspond to the OD of each sample. The top three rows show binding of native IgM. The lower three rows show binding of the same antibodies, expressed as IgG1.

Fig 3: Spike‐capturing B cells from peripheral blood of convalescent donors Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike‐mCherry for 3 h, then retrieved and labeled with fluorescent antibodies shown in Appendix Table S4 and measured by flow cytometry. (A) UMAP algorithm (5,000 randomly selected cells/sample) was used to depict the major B cell subsets clustered according to marker expression. FlowSOM‐based B cell subpopulations are overlaid as a color dimension, and the colors of the clusters are shown on the left of heatmap (B). Cells falling in the MACAC gate (see below) are marked with red rhombi.Heatmap showing mean population expression levels of markers used for UMAP visualization and FlowSOM‐clustering. Colors shown in the legend on the left are also used in the UMAP representation in (A).Gating strategy to define CD69‐high, mCherry‐high, spike‐capturing B cells (i.e., membrane antigen capture activated cells, MACAC, red box). Dot plot shows CD69 and mCherry fluorescence for B cells from one of three donors gated by forward and side scatter, and negative for the dye used to mark the antigen donor cells.Relative fractions of different B cell subpopulations, from three donors, as shown in A‐B, in all B cells compared to within MACAC B cells. P values are based on two‐tailed t‐tests between the groups. Correlation coefficients (r) were calculated from the z‐statistic of the Wilcoxon–Mann–Whitney test. A black horizontal line represents the median. Boxplots represent the interquartile range (IQR). Whiskers extend to the farthest data point within a maximum of 1.5× IQR. Every point represents one donor.Gating strategy in MACAC sorting. Single cells are selected based on scatter, antigen‐expressing TE spike‐mCherry cells excluded on Cell Trace Violet label, and the spike‐capturing (mCherry‐high), activated (CD69‐high) B cells (population labeled “MACAC” in red on the right‐most plot) are sorted. The middle plot shows B cells that did not adhere to the TE‐spike‐cherry antigen‐expressing cells (putatively antigen‐irrelevant), and the right plot those that did (putatively antigen‐recognizing). Plots show data from one of five convalescent donors (those whose serum results are labeled with black triangles in Fig 1A). Cells in the MACAC gate were singly distributed into wells of 384‐well plates and cultured for 9 days with IL‐21 and CD40L, and then, the single well culture supernatants were screened for anti‐spike antibody binding and virus neutralization as described below.Results of single well supernatant screening for antibody binding to SARS‐CoV‐2 spike protein. Results from 3266 wells from 5 donors are shown for IgM (left), IgG (middle), and IgA (right). Screening and plotting methods are the same as in (Fig 1A).

Fig 4: Spike‐protein specific antibodies of classes M, G, and A in human serum Spike‐binding antibodies in serum. Binding by antibodies of classes M, G, and A is detected with secondary antibodies conjugated to different fluorochromes, and the results are shown on the left, middle, and right plots, respectively. Binding (geometric mean fluorescence intensity—GMFI—of corresponding secondary antibody) to TE cells expressing SARS‐CoV‐2 spike protein is plotted on the vertical axis, and binding to untransfected cells on the horizontal axis. Each point represents a value from one donor. Samples from donors with no known exposure to SARS‐CoV‐2 antigens (n = 86) are plotted with blue circles, recently infected donors (n = 34) with red circles, and vaccinated donors (n = 14) with green diamonds. Red circles with black triangles correspond to donors from whose B cells monoclonal antibodies were isolated (n = 5, Appendix Table S2). P values are derived from a two‐way analysis of variance followed by Tukey's test to compare the specific binding, that is (binding to spike‐expressing cells)/(binding to untransfected cells) between conditions (convalescent or vaccinated against unexposed) within each antibody class. The experiment was independently repeated three times, and results shown come from the third replicate.Change in antibody binding following vaccination against SARS‐CoV‐2. Binding of antibodies from sera assessed in the experiment shown in (A) is shown for two samples of serum from each of 7 donors. Data are plotted as in (A) with binding to spike‐expressing cells on the vertical axis, and to untransfected cells on the horizontal, using blue symbols for samples from before vaccination, and red symbols for samples from 15 to 29 days after the first vaccination (but before any second vaccination). Black lines connect points corresponding to pre/post pairs of samples from each donor. Left plot shows results for IgM and right plot for IgG.Comparison of post‐vaccination increase in binding to untransfected cells between IgG and IgM. The increase in binding to untransfected TE 0 cells between pre‐ and post‐vaccination samples is shown on the left with blue symbols for IgG and on the right with black symbols for IgM. The horizontal line at y = 0 is the expected result when no increased binding is observed. P value is derived from a two‐tailed, Wilcoxon matched pairs signed rank test.Comparison of spike‐specific IgM, as measured by flow cytometry or ELISA. The left two “FC” are the results of flow cytometry, and the left vertical axis shows the ratio of serum IgM binding to spike‐expressing cells, vs binding to non‐expressing control cells, as plotted in A. The right two columns show spike‐RBD‐specific IgM as measured by an IgM‐capture ELISA, and the right vertical axis shows the optical densities (OD). P values are calculated by two‐way analysis of variance, followed by Sidak's multiple comparisons test. The entire experiment was repeated three times, and the data shown are derived from the third replicate.

Fig 5: Spike binding and virus neutralization by single well supernatantsSupernatants shown in (B) that exhibited a ratio of fluorescent signal on spike‐expressing cells to signal on non‐expressing cells > 1.2 (n = 326: 79 IgM, 141 IgG 106 IgA) were assayed for ability to neutralize SARS‐CoV‐2 pseudotyped VSV. Supernatants were serially diluted from 20–1,960 fold, and mixed with 100 pfu of VSV*ΔG‐SΔ21, which was then used to infect Vero cells. Assay was performed once for each supernatant in quadruplicate, and the titer defined as the maximum dilution at which virus proliferation was completely inhibited. For each supernatant tested, the GMFI as shown in (B) is shown on the vertical axis, and the neutralization titer is shown on the horizontal axis, slightly staggered by class, in order to minimize overlap of points. IgM, IgG, and IgA are plotted with black, blue, and red points respectively. P values at the top of the plot were calculated by chi‐square test of the null hypothesis that the likelihood that supernatants show a titer of 20 (the lowest dilution tested) or greater is equal among the three antibody classes.