Isotype conversion of Staphylococcal-specific IgG into IgM broadens the reactivity to other bacterial pathogens

Abstract

Therapeutic antibodies are actively explored as alternative to treat or prevent bacterial infections. However, the narrow antigen specificity of IgG in combination with broad diversity in bacterial surface structures currently hampers the development of therapeutic antibodies against bacteria. Here we reveal that isotype conversion of three highly specific anti-staphylococcal antibodies from IgG into IgM does not only affect Fc effector functions but also modifies the interaction of Fab domains with bacterial surface antigens. These converted IgMs gain cross-reactivity for a broad range of bacterial species, including Gram-negatives such as Escherichia coli and Neisseria meningitidis and even protect against invasive infection with Streptococcus pyogenes in vivo. Mechanistic studies show that enhanced cross-specificity by IgM is conferred by changed ligand specificity and multivalent binding to high-density antigens. Altogether, these findings provide important insights for the development of antibody therapy for bacterial infections.

Keywords: IgM; antibody therapies; bacteria; infection; species-specificity.

Graphical abstract

Figure 1

Conversion of anti-WTA IgG1 (4461) to IgM broadens its antigen reactivity with S. aureus and WTA (A–C) (A) Schematic representation of S. aureus and WTA structure and glycosylation. Concentration-dependent binding of anti-WTA (4461) IgG1 and IgM to (B) S. aureus LAC ΔSpA, sbi::Tn or to (C) S. aureus Wood46. (D) Phase-contrast and widefield fluorescence microscopy images of 1 μg/mL 4461-IgG1 or IgM binding to S. aureus Wood46. The white scale bar indicates 5 μm. Images for 4461-IgM have a three times lower exposure time for the GFP channel than the IgG1 images. Microscopy images are representative 20 by 20 μm cutouts of multiple larger images from two independent experiments. (E) Phagocytosis of GFP-expressing S. aureus Wood46 by neutrophils induced by a concentration range of anti-WTA (4461) IgG1 and IgM in 1% ΔIgG/M-serum. As isotype controls, 1 μg/mL anti-TNP IgG1 and IgM were included. (F) Binding of 1 μg/mL anti-WTA (4461) IgG1 and IgM to synthetic WTA beads with either only the ribitol phosphate (RboP) backbone or treated with TarM or TarS enzymes to add modifications of α-1,4-GlcNAc or β-1,4-GlcNAc respectively. All data represent mean ± SD of three (or four for B) independent experiments. A multiple unpaired t test was used to determine significant difference between the indicated samples in f), of which p values are indicated with ∗∗p < 0.01.

Figure 2

Three anti-staphylococcal IgMs cross-react with E. coli and induce complement-dependent lysis (A–C) (A) Schematic representation of Gram-negative E. coli MG1655 lipooligosaccharide (LOS) composition. The inset represents the E. coli K-12 LOS structure containing a GlcNAc moiety, depicted as a blue square. The waa mutants with varying sugar structures are indicated with arrows. The O-antigen is not present in any of the strains except wbbL+. Binding of a selection of IgG1 and IgM-converted mAbs to E. coli MG1655 (1 μg/mL) as measured by (B) flow cytometry and (C) phase-contrast and widefield fluorescence microscopy. The white scale bar in (C) indicates 5 μm. Image for 4461-, 4497-, and rF1-IgM indicated with an ∗ have a three times lower exposure time for the GFP channel than the other conditions. Microscopy images are a representative 25 by 25 μm cutout of multiple larger images from two independent experiments. (D) Binding of IgM-converted 4461, 4497, rF1, anti-TNP, and anti-StrepTagII to either E. coli BW25113 (Keio) Wt, ΔwaaR, or ΔwaaB, or E. coli CGSC7740 wbbL− or wbbL+ (1 μg/mL). (E) Complement-mediated killing, as measured by Sytox influx, of E. coli MG1655 using a concentration range of 4461, 4497, and rF1 IgG1 and IgM (four experiments) or anti-TNP isotype controls (two experiments) in a purified classical pathway assay. All other data represents mean ± SD of three independent experiments. A multiple unpaired t test was used to determine significant difference between the indicated bar graphs in which p values are indicated with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Figure 3

Anti-staphylococcal IgMs do not cross-react with glycans on human cells or serum IgG (A) Binding of anti-WTA (4461), anti-WTA (4497), anti-SDR (rF1), and isotype control anti-TNP IgG1 and IgM to human PMNs, erythrocytes, and PBMCs (3 μg/mL). A positive control per cell type is indicated with green stars: for PMNs anti-CD32-FITC; for erythrocytes anti-CD35-PE; and for PBMCs anti-CD3-FITC. PMNs were incubated with 10 μg/mL FLIPr-like to block aspecific FcγRI-mediated binding of IgG. (B) Antibody binding of 4461, 4497, and rF1 IgM and the isotype controls anti-TNP and anti-StrepTagII IgM across a concentration range to ELISA-coated serum IgG. (C) Binding of anti-WTA (4461), anti-WTA (4497), and anti-SDR (rF1) IgM (1 μg/mL) to enzymatically glycosylated or synthetic RboP hexamers with β-1,4-GlcNAc modifications. (D) Binding of anti-WTA (4461), anti-WTA (4497), and anti-SDR (rF1) IgM (1 μg/mL) to WTA beads coated with a 10-fold and 100-fold dilution (compared to standard) of synthetic RboP hexamers with one terminal β-1,4-GlcNAc modification. (C and D) contain a schematic representation of the variation in the vertical and horizontal antigen density on WTA beads. Gray pentagons represent RboP monomers, and blue squares GlcNAc moieties. A multiple unpaired t test was used to determine significant differences between antigen densities in which p values are indicated with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Data represents mean ± SD of three independent experiments.

Figure 4

IgM cross-reactivity is due to multivalency (A and C) Binding of mAbs 4461, 4497, and rF1 across a concentration range and engineered with the following mutations: (A) IgM C575A or (C) IgG1 RGY and IgG1-μtp (C575S) to E. coli MG1655. (B) Sytox positivity of E. coli MG1655 after incubation with a concentration range of 4461, 4497, and rF1 IgM C575A in a purified classical pathway assay. (D) Sytox positivity of E. coli MG1655 after incubation with 4497-IgG1, IgG1 RGY, IgG1-μtp, and IgG1-μtp C575S (1 μg/mL) in a purified classical pathway assay. All data represents mean ± SD of three independent experiments. A multiple unpaired t test was used to determine significant difference between the indicated bar graphs in which p values are indicated with ∗∗∗∗p < 0.0001.

Figure 5

GlcNAc-dependent cross-reactivity of converted anti-staphylococcal IgMs with several human bacterial pathogens and 4497-IgM protection against invasive S. pyogenes infection in vivo (A) Heatmap of log transformed binding values of 1 μg/mL anti-WTA (4461), anti-WTA (4497), and anti-SDR (rF1) IgG1 and IgM to a range of bacterial species with described surface-exposed GlcNAc (the bar graph is presented in Figure S5D). (B) Schematic representation of S. pyogenes and the structure and glycosylation of its GAC. (C) Binding of anti-WTA (4461), anti-WTA (4497), and anti-SDR (rF1) IgG1 and IgM (all 1 μg/mL) to beads coated with synthetic rhamnose hexamers either without (left) or with (right) a β-1,3-GlcNAc modification per rhamnose dimer. Data represents mean ± SD of three independent experiments and a multiple unpaired t test was used to determine significant difference between the indicated bar graphs in which p values are indicated with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (D) Colony forming units (CFUs) in liver of mice (n = 15 per group) 24 h post infection with S. pyogenes 5448 (±5 × 10⁷ CFUs), passively immunized with 50 μg 4497-IgM, 4497-IgG1, anti-StrepTagII-IgM, or PBS. Three independent experiments with five mice per group were performed and pooled for a total of fifteen mice per group. Statistical analysis was performed using a two-way ANOVA comparison (∗p < 0.05).

Figure 6

Schematic overview of different bacterial surface structures containing exposed GlcNAc moieties IgGs targeting S. aureus WTA exclusively recognize GlcNAc in a specific anomeric configuration in the context of ribitol phosphate, whereas its IgM counterparts also bind to exposed GlcNAc moieties on other bacterial surface structures, such as LOS from E. coli, GAC from S. pyogenes, and LOS from N. meningitidis serogroup B.