Abundant human anti-Galα3Gal antibodies display broad pathogen reactivity

Abstract

Antibodies of the IgG class to terminal Galα3Gal (IgG anti-αGal) is abundant in human plasma and are reported to bind most sepsis-causing Gram-negative bacteria. However, these seminal findings, made more than two decades ago, have not been reexamined. Our aim was to assess IgG anti-αGal´s pathogen reactivity. We affinity purified IgG anti-αGal from a therapeutic grade normal human IgG pool applying two rounds of positive selection with Galα3Gal-coupled beads and included removal of column matrix reactive antibodies. The purified antibodies were rigorously characterized in terms of specificity and purity in various solid-phase immunoassays. We used flow cytometry to study reactivity against 100 consecutive clinical isolates diagnosed as cause of sepsis in humans. We found that the purified IgG anti-αGal displays high specificity for Galα3Gal. Also, IgG anti-αGal at 5 mg/L bound 56 out of 100 pathogens with predilection for Gram-positive bacteria binding 39 out of 52 strains. We confirm that although IgG anti-αGal comprise a small fraction of the human antibody pool (~0.1%), these antibodies targets an impressively large part of pathogens causing invasive disease.

Figure 1

IgG subclass distribution in starting material, intermediate and final preparation of IgG anti-αGal. Distribution of IgG subclasses was determined by a commercial ELISA in the starting material comprised of normal human IgG pool (nhIgG) (A), an intermediate preparation acquired after the first affinity isolation (B), and in the final preparation of IgG anti-αGal acquired after an additional affinity isolation and removal of column reactive antibodies (C). Bars are mean with 95% CI and circles are repeated measurements. Significant differences are identifiable from non-overlapping 95% CIs.

Figure 2

Antibody recovery in the IgG anti-αGal preparation and flow-through. Recovery of different antibodies in the IgG anti-αGal preparation (A) and in the flow-through from the affinity column (B). Quantifications were made by solid-phase immunoassays (TRIFMA) and the use of standard curves. Bars are mean with 95% CI and circles are repeated measurements. Significant differences are identifiable from non-overlapping 95% CIs.

Figure 3

Properties of IgG anti-αGal in the final purified preparation and in the starting material All panels show TRIFMA of antibody binding to solid-phase Galα3Gal. (A) Concentration of IgG anti-αGal in purified IgG anti-αGal relative to the starting material (nhIgG) at equimolar total IgG concentration. The ratio was determined for the following subtypes of IgG anti-αGal: All IgG subclasses, IgG2 subclass, and λ-light chain bearing IgG. Bars are means with 95% CI and circles depict repeated tests. Relative concentrations of the three types of IgG anti-αGal were tested for difference by ANOVA (logarithmic transformed data). No difference was evident suggesting that the composition of IgG2 and λ-light chain bearing antibodies among IgG anti-αGal was similar in purified IgG anti-αGal and in the starting material. (B) Antibody binding as a function of IgG anti-αGal concentration. Mean and SD of three independent experiments, each analyzed as duplicates. (C) TRIFMA of IgG anti-αGal binding to solid-phase αGal challenged by each of six different compounds. Inhibitors´ structures and IC50 (with 95% confidence intervals in parenthesis) are displayed with each set of curves. Mean and SD of duplicates. Each curve is representative of minimum two independent experiments.

Figure 4

Binding of IgG anti-αGal to red blood cells. (A) Symbolic depictions of the major red blood cell (RBC) carbohydrate antigens. Structure key in the lower right. Data in panels (B–D) were acquired by flow cytometry using fluorescent-coupled secondary F(ab’)₂ against human IgG to detect RBC bound purified IgG anti-αGal. (B) IgG anti-αGal reactivity with RBC presenting different major surface antigens (depicted in previous panel). Mean and SD of minimum two independent experiments. (C) Reactivity of purified IgG anti-αGal at 5 mg/L with RBCs in the presence of soluble disaccharides (inhibitor) relative to no inhibition (control). Reactivity was calculated from relative MFIs by means of concomitant analyzed standard curves. Mean and SD of two independent experiments. (D) RBC reactivity of purified IgG anti-αGal after adsorption on RBCs (pig or human B). Mean and SD of two independent experiments.

Figure 5

Binding of IgG anti-αGal to Escherichia coli O86 examined by flow cytometry. Antibody bound on Escherichia coli O86 was detected using fluorescent-coupled F(ab’)₂ anti-IgG. (A) Binding of antibody of irrelevant specificity (anti-CD20, rituximab) at 10 mg/L and IgG anti-αGal at 5 mg/L. (B) Bacterial antibody binding as a function of incubation time. Mean and SD of two independent experiments. Black, dotted line: y = 1. (C) IgG anti-αGal binding as a function of IgG anti-αGal concentration for each of three concentrations of E. coli O86. Mean and SD of two independent experiments. Grey, dotted line: y = 1. (D) Bacterial reactivity of IgG anti-αGal at 5 mg/L in the presence of soluble disaccharides (inhibitor) relative to no inhibition (control). Reactivity was calculated from relative MFIs by means of concomitant analyzed standard curves. Mean and SD of two independent experiments. (E) Bacterial reactivity of IgG anti-αGal after adsorption on RBCs (pig or human B). Mean and SD of two independent experiments.

Figure 6

IgG anti-αGal targets most sepsis-causing pathogens. Frequency of antibody reactivity to pathogens. Pathogens were consecutive clinical isolates (n = 100) obtained from blood cultures and diagnosed as the cause of sepsis in patients. Reactivity determined by flow cytometry. Each isolate was tested twice. Anti-CD20 was included to reveal binding through bacterial IgG-binding molecules.