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. The increasing frequency of pathogens resistant to antibiotics poses immense global challenges 1 and novel therapeutic strategies are crucial. Over millions of years, human ancestors have evolved numerous countermeasures to pathogen threats. Our hard-earned defense mechanisms are obvious assets to build on in development of effective therapeutics to combat pathogens. Intriguingly, one group of human antibodies, IgG antibodies against the carbohydrate structure terminal Galα3Gal (IgG anti-αGal), were more than twenty years ago reported to bind most of sepsis-causing Gram-negative bacterial pathogens, including Escherichia coli and species of Klebsiella, Enterobacter, Serratia, Citrobacter, Proteus, and Pseudomonas 2,3. This broad pathogen reactivity is of interest in the context of developing new therapeutic measures. IgG anti-αGal were originally reported to comprise 1% of plasma IgG in all humans 4 , corresponding to around 100 mg/L. According to later studies, the average concentration seems to be maybe 10-fold lower 5-7 and to vary considerably between individuals (>400-fold 7). IgG anti-αGal is mainly IgG subclass 2 (IgG2) 5 in accordance with the general finding for anti-carbohydrate antibodies 8-10. Human plasma also contains anti-αGal antibodies of immunoglobulin classes A and M 11-13. It is unclear why humans produce antibodies to Galα3Gal. The antigen, Galα3Gal, is not synthesized by simians of the Catarrhini subdivision (which includes humans, other apes, and old-world monkeys) because the gene encoding the essential α1,3-galactosyltransferase was silenced in our common ancestor 14 whereas all other mammals synthesize Galα3Gal. As Galα3Gal is non-self, Catarrhini are allowed production of IgG anti-αGal which is found naturally occurring in these animals 15. The prevailing theory is that Galα3Gal-containing antigens presented to our immune syste...

Complement activation by human IgG antibodies to galactose‐α‐1,3‐galactose

“…Highly purified IgG anti-αGal was prepared as previously described. 12 In brief, normal human IgG pool (nhIgG; Beriglobulin, CSL Behring, King of Prussia, PA) was diluted to 30 g/l and passed through a column containing Galα3Gal-derivatized beads. The preparation of the Galα3Gal-derivatized beads is described in the Supplementary material (Appendix S1).…”

“…13 Affinity-purified IgG anti-αGal efficiently binds pRBCs. 12 We applied IgG anti-αGal at a concentration of 20 mg/l and 1% HHS as a complement source to investigate the deposition of complement fragments on pRBCs by flow cytometry. IgG anti-αGal markedly triggered deposition of C3 and C4 fragments compared with HHS alone (Fig.…”

“…The Gram-negative bacterium EcO86 is often used as a model bacterium for IgG anti-αGal reactivity. 12,14,33 As IgG anti-αGal react more frequently with Gram-positive bacterial pathogens, 12 we also included Sp9V, which was identified as highly reactive in preliminary screening of 91 pneumococcal serotypes (data being prepared for publication).…”

“…4 This is of particular interest as IgG anti-αGal is abundant in essentially all humans (comprising on average 0Á1% of the total IgG [8][9][10] ) and reacts with a wide range of extracellular bacterial pathogens. 4,11,12 In this light, clarification of the role of IgG anti-αGal in complement activation is imperative.…”

“…4 Other groups also reported that IgG anti-αGal did not activate the classical pathway (CP) of complement 8,15,[17][18][19] and seemed to inhibit complement activation by occupying binding sites for potent complement activators. 8,18 IgG anti-αGal is mainly IgG2, 8,12 which was proposed to explain the antibody's claimed attenuation of complement activation. 8 Historically, IgG2 was believed to activate the CP very poorly compared with IgG1 and IgG3.…”