IgM antibodies derived from memory B cells are potent cross-variant neutralizers of SARS-CoV-2

Abstract

Humoral immunity to SARS-CoV-2 can be supplemented with polyclonal sera from convalescent donors or an engineered monoclonal antibody (mAb) product. While pentameric IgM antibodies are responsible for much of convalescent sera's neutralizing capacity, all available mAbs are based on the monomeric IgG antibody subtype. We now show that IgM mAbs derived from immune memory B cell receptors are potent neutralizers of SARS-CoV-2. IgM mAbs outperformed clonally identical IgG antibodies across a range of affinities and SARS-CoV-2 receptor-binding domain epitopes. Strikingly, efficacy against SARS-CoV-2 viral variants was retained for IgM but not for clonally identical IgG. To investigate the biological role for IgM memory in SARS-CoV-2, we also generated IgM mAbs from antigen-experienced IgM+ memory B cells in convalescent donors, identifying a potent neutralizing antibody. Our results highlight the therapeutic potential of IgM mAbs and inform our understanding of the role for IgM memory against a rapidly mutating pathogen.

Graphical abstract

Figure 1.

Diverse IgG⁺ MBC-derived RBD-specific antibodies gain potency when expressed as IgM. (A) Panel of eight IgG⁺ MBC-derived IgG mAbs indicating affinity for RBD and NT₅₀ of WA-1 SARS-CoV-2 in PRNT (Rodda et al., 2021). Samples were analyzed in duplicate in at least two separate experiments. (B) Binding kinetics for individual mAbs as determined by BLI using sensor-bound IgG mAbs and serial dilution of soluble RBD protein. Each mAb was tested with six dilutions of RBD. (C and D) Summary of epitope-mapping experiments showing relative competition for RBD between the indicated MBC-derived IgG and well-characterized Fabs (C) or each other (D); individual data are provided in Figs. S1 A and S2 A. (E) Comparison of neutralizing potency of clonally identical IgG and IgM mAbs against pseudovirus. Bars indicate mean and SD for three independent experiments; individual symbols indicate the average of internal duplicates for each experiment. Average fold difference in NT₅₀ potency for IgG vs. IgM for each BCR clone is shown below the graph. (F) Representative results for 297 IgM vs. IgG in neutralization assays using the pseudovirus (left panel; average and SD are shown for three independent experiments, each performed in duplicate) or a WA-1 SARS-CoV-2 PRNT (middle panel; representative of three independent experiments) and summary of NT₅₀ data (right table).

Figure S1.

BLI competition assays. Competition with well-characterized mAbs for the panel of eight IgG⁺ MBC-derived IgG mAbs. The timing of the exposure to each antibody is indicated with a black arrow for the well-characterized mAb and a red arrow for the MBC-derived mAb.

Figure S2.

BLI competition assays and SEC-MALS demonstrating molecular weight of IgM pentamer. (A) Intrapanel competition BLI plots displaying sensor-detected RBD binding after addition of the indicated IgG⁺ MBC-derived IgG mAb. (B) Competition with well-characterized mAbs and the IgM⁺ MBC-derived 204 IgG mAb for RBD by BLI. (C) SEC-MALS traces for the purified 297 IgM mAb. The light scattering trace is in blue, and the molecular weight (MW) calculated from the combination of scattering, refractive index, and UV absorbance is shown in red across the peak elution window. The average calculated MW is shown in the inset, along with the predicted MW (MW_pred). The error is based on the SD from the 56 scans along the peak elution profile; however, the expected uncertainty can be as large as 10% due to assumptions about molar extinction coefficients and glycan occupancy. The hydrodynamic radius (Rh) was estimated from dynamic light scattering, which was also measured online and indicated under the peak. (D) Competition of mAb 204 against the panel of eight IgG⁺ MBC-derived mAbs.

Figure 2.

IgM MBCs encode RBD-specific antibodies, including a neutralizing mAb with improved activity when expressed as IgM vs. IgG. (A and B) Supernatants from cells transfected with plasmids encoding IgM⁺ MBC-derived mAbs as IgG vs. IgM screened by ELISA for anti-IgG (left) or -IgM (right), with untransfected supernatant as a negative control (blue; A); or binding to SARS-CoV-2 spike RBD protein (B). (C) Supernatants’ ability to block RBD from binding to plate-bound human ACE2. BCR clone 204 is highlighted (in red) in B and C. IgG⁺ MBC-derived antibody, 297 IgG, is included as a positive control (in pink) in C. Data are representative of two independent experiments performed in triplicate. (D) BLI data showing binding and dissociation kinetics of BCR clone 204 as purified IgG vs. IgM. (E) SARS-CoV-2 PRNT on Vero cells at the indicated concentrations of 204 IgG vs. IgM or an anti-malaria mAb (negative control; Thouvenel et al., 2021). (F and G) NT₅₀ titers are shown based on mass (F) or molar (G) concentrations. For conditions that did not approach an NT₅₀, an arbitrary NT₅₀ was assigned of 2× the maximum concentration tested (dashed line). The assay was performed in duplicate and repeated at least twice. (H) Epitope mapping of mAb 204 IgG by BLI against well-characterized Fabs/mAbs and the other mAbs in our panel; individual data are provided in Fig. S2, B and D.

Figure 3.

IgM antibodies retain activity against viral spike variants that escape clonally identical IgG. (A) Binding to Wuhan-Hu-1 (WH-1) or Beta variant RBD proteins by purified IgG mAbs in ELISAs, quantified as the area under the curve (AUC) for a 10-dilution series. Results for the panel of eight IgG⁺ MBC-derived mAbs are shown (left). For clarity, simultaneously performed ELISAs testing well-characterized IgG mAbs, or an ACE2-Fc fusion protein (ACE2), are shown separately (right). Data are representative of two independent experiments performed in triplicate. (B) Affinity for Beta RBD of representative antibodies with significantly reduced binding, 203 (top), and moderate reduced binding, 297 (bottom). (C) Neutralization potencies for each mAb as IgG vs. IgM against WA-1 (blue) or Beta (pink) spike pseudovirus. Dashed line illustrates the maximum antibody concentration tested (2 μg/ml). For antibodies that did not approach an NT₅₀, the NT₅₀ is graphed arbitrarily in the shaded area as 4 μg/ml. Each IgG/IgM pair was tested in duplicate against both viruses in three independent experiments. (D) Varying neutralization potency against WA-1, Beta, Delta, and Omicron BA.1 spike pseudoviruses for IgG (left) vs. IgM (right) mAbs. Error bars illustrate mean ± SD for three or more experiments with internal duplicates. (E) Summary neutralization potencies of IgG vs. IgM mAbs for the indicated variants in experiments described in D. Control, malaria-specific IgG or IgM (MaliA01 mAbs; Thouvenel et al., 2021). (F) Neutralization in a Vero cell plaque reduction assay of Delta SARS-CoV-2 for 297 IgG vs. IgM. Representative plot of five independent experiments.

Figure 4.

An anti-RBD IgM mAb protects against SARS-CoV-2 infection in differentiated human airway epithelia cultures. (A) Schematic of experimental design using airway epithelial cultures differentiated to an organotypic state at an ALI. (B and C) Quantification of viral RNA copy number in ALI cultures treated with the indicated antibody. Data represent independent experiments; cultures were generated from primary human bronchial epithelial cells derived from unique donors. Each condition was tested in independent duplicates. For B, duplicates were pooled as RNA before quantitative PCR.