Most individuals maintain circulating antibodies against various pathogenic bacteria as a consequence of previous exposures. However, it remains unclear to what extent these antibodies contribute to host protection. This knowledge gap is linked to the need for better methods to characterize antimicrobial polyclonal antibodies, including their antigen and epitope repertoires, subclass distribution, glycosylation status, and effector functions. Here, we showcase a generic mass spectrometry-based strategy that couples systems antigenomics and systems serology to characterize human antibodies directly in clinical samples. The method is based on automated affinity purification workflows coupled to an integrated suite of high-resolution MS-based quantitative, structural- and glyco-proteomics readouts. We focused on Streptococcus pyogenes (Group A Streptococcus; GAS), a major human pathogen still awaiting an approved vaccine. Our methodology reveals that both healthy and GAS infected individuals have circulating Immunoglobulin G (IgG) against a subset of genomically conserved streptococcal proteins, including numerous toxins and virulence factors. The antigen repertoire targeted by these antibodies was relatively constant across healthy individuals, but considerably changed in GAS bacteremia. Detailed analysis of the antigen-specific IgG indicates inter-individual variation regarding titers, subclass distributions, and Fc-signaling capacity, but not in epitope and Fc-glycosylation patterns. Importantly, we show that the IgG subclass has a major impact on the ability of GAS-antibodies to trigger immune signaling, in an antigen- and Fc receptor- specific fashion. Overall, these results uncover exceeding complexity in the properties of GAS-specific IgG, and showcase our methodology as high-throughput and flexible workflow to understand adaptive immune responses to bacterial pathogens.
