Each dot represents the average of duplicate array proteins. (E) Representative microarray plots showing the z-scores given on a single human protein by the reference (Ref: mGO53, y axis) and test antibody (x axis). HEp-2-reactive anti-SARS-CoV-2 S antibody Cv2.3132 was also included for comparison. The negative (mGO53), low-positive (ED38), and kit's positive (Ctr+) controls were included in the experiment. (D) Microscopic images showing the reactivity of selected SARS-CoV-2-neutralizing mAbs (n = 5) to HEp2-expressing selfantigens assayed by indirect immunofluorescence assay. Ctr+ and Ctr− are the positive and negative control of the kit, respectively. Means ± SD of duplicate values are shown. (C) Bar graph showing the HEp-2 reactivity of selected SARS-CoV-2 antibodies as measured by ELISA. Darker blue colors indicate high binding while light colors show moderate binding (white = no binding). (B) Heatmap comparing the AUC values determined from the ELISA binding analyses shown in A.
Anti-SARS-CoV-2 S antibody Cv2.3132 showing HEp-2 reactivity in C was included for comparison. mGO53 (Wardemann, 2003) and ED38 (Meffre et al., 2004) are negative and positive control antibodies, respectively. (A) Representative ELISA graphs showing the reactivity of selected SARS-CoV-2-neutralizing antibodies (n = 5) against double-stranded DNA (DNA), flagellin (Fla), YU2 HIV-1 Env (gp140), insulin (INS), keyhole limpet hemocyanin (KLH), lipopolysaccharide (LPS), lysozyme (LZ), MAPK-14 (MAPK), proteoglycan (PG), and thyroglobulin (Tg). Off-target binding and Fc-effector functions of potent SARS-CoV-2-neutralizing antibodies. Cladogram (right) showing the distribution of individual shared clones between donors (n = 9). (J) Circos plot (left) showing the clonal variants shared between distinct donors with the size of the links proportional to the number of clones sharing 75% CDR H 3 amino acid identity. Numbers of mutations were compared across groups of antibodies using unpaired student t test with Welch's correction. The average number of mutations is indicated below. (I) Violin plots comparing the number of mutations in V H genes of SARS-CoV-2 S-specific (n = 101) and control IgG + memory B cells from unexposed healthy individuals (n = 72 Prigent et al., 2016). Gray and blue dots indicate statistically significant differences between both Ig gene repertoires. (H) Volcano plot analysis comparing the immunoglobulin gene repertoire of SARS-CoV-2 S-specific IgG + /IgA + B cells from convalescent donors and IgG + memory B cells from healthy individuals (IgG.mB, unexposed to SARS-CoV-2 Prigent et al., 2016). Spearman correlation coefficients with the corresponding P values are indicated. CXCR3 + cTfh, CXCR3 − cTfh, cTfh1, and cTfh2 cells. (G) Correlation plots showing the frequency of SARS-CoV-2 tri-S + IgG + RM B cells vs. (F) Violin plots comparing the frequency of PD1 +, PD1 hi, ICOS +, and ICOS + PD1 + cells among cTh1, cTfh2, and cTh17 cell subsets in the blood of convalescent COVID-19 individuals (n = 10). (E) Violin plots showing the frequency of total CD4 +, CD4 + CXCR5 + lymphocytes, and cTfh cell subsets in the blood of convalescent COVID-19 individuals (n = 10). Blue dots indicate potent neutralizing antibodies while the red dot is the ultra-potent neutralizer Cv2.1169 (red arrow). (D) Immunophenotyping flow cytometric plots showing the expression of B-cell surface markers on sorted SARS-CoV-2 tri-S-specific B cells (n = 101, black, blue, and red dots). CS mB, class-switched memory B cells in convalescent COVID-19 individuals (n = 10). (C) Violin plots showing the distribution of total and SARS-CoV-2 tri-S + IgG + and IgA + memory B-cell subset frequencies as depicted in B.
Black and pink dots indicate tri-S + and RBD + IgG + and IgA + B memory cells in the density maps. IM (intermediate memory, CD27 − CD21 + ), RM (resting memory CD27 + CD21 + ), AM (activated memory, CD27 + CD21 − ), and TLM (tissue-like memory CD27 − CD21 − ). Density maps presenting the staining intensity of CD27 and CD21 markers used to define memory B-cell subsets. (B) Pseudocolor plots showing the t-SNE analysis of concatenated Vivid − CD19 + CD10 − B cells in convalescent COVID-19 individuals (n = 10). (A) Violin plots showing the percentage of SARS-CoV-2 tri-S + cells among total IgG + and IgA + memory B cells (top) and of SARS-CoV-2 RBD + cells among tri-S + IgG + and IgA + memory B cells (bottom) in the blood of convalescent COVID-19 individuals (n = 10). Immunophenotyping and antibody gene repertoire of SARS-CoV-2 spike-specific memory B cells.
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