Phage-display immunoprecipitation sequencing of the antibody epitope repertoire in inflammatory bowel disease reveals distinct antibody signatures

Inflammatory bowel diseases (IBDs), e.g., Crohn's disease (CD) and ulcerative colitis (UC), are chronic immune-mediated inflammatory diseases. A comprehensive overview of an IBD-specific antibody epitope repertoire is, however, lacking. Using high-throughput phage-display immunoprecipitation sequencing (PhIP-Seq), we identified antibodies against 344,000 antimicrobial, immune, and food antigens in 497 individuals with IBD compared with 1,326 controls. IBD was characterized by 373 differentially abundant antibody responses (202 overrepresented and 171 underrepresented), with 17% shared by both IBDs, 55% unique to CD, and 28% unique to UC. Antibody reactivities against bacterial flagellins dominated in CD and were associated with ileal involvement, fibrostenotic disease, and anti-Saccharomyces cerevisiae antibody positivity, but not with fecal microbiome composition. Antibody epitope repertoires accurately discriminated CD from controls (area under the curve [AUC] = 0.89), and similar discrimination was achieved when using only ten antibodies (AUC = 0.87). Individuals with IBD thus show a distinct antibody repertoire against selected peptides, allowing clinical stratification and discovery of immunological targets.</p

In-depth characterisation of the serum antibody epitope repertoire in Inflammatory Bowel Disease by high-throughput phage-displayed immunoprecipitation sequencing

BackgroundPatients with IBD show distinct antibody responses, particularly against microbiota. However, a comprehensive overview of the antibody epitope repertoire in IBD is lacking. Here, we characterized serum antibody responses in patients with IBD and population controls using a high-throughput phage-displayed immunoprecipitation sequencing (PhIP-seq) workflow and associated these to disease phenotypes and the faecal microbiome.MethodsPhIP-seq was leveraged to characterise antibody responses against 344,000 rationally selected peptide antigens in 497 patients with IBD which were compared with 1,326 individuals from a population-based cohort (Fig. 1A-B). Antibody profiles were linked to 23 IBD-specific clinical features such as disease location and surgical history and to faecal microbiota composition (Fig. 1C).ResultsPatients with IBD demonstrated distinct antibody epitope repertoires compared with individuals from the general population, with 373 differentially abundant antibody-bound peptides (202 overrepresented, 171 underrepresented) belonging to bacterial flagellins (69), virulence factors (102), other antigens of both commensal and pathogenic bacteria (90) as well as viruses (67) and food proteins (24) (Figure 2). In particular, antibody responses against bacterial flagellins, many of which belong to Lachnospiraceae bacteria (e.g. Roseburia spp.), but also Eubacterium spp. and pathogens (e.g. Legionella, Clostridium, Burkholderia) dominated in patients with Crohn’s disease (CD), and were associated with ileal disease involvement and more complicated disease behaviour (e.g. fibrostenotic disease, surgical history) as well as anti-Saccharomyces cerevisiae antibody positivity. Furthermore, many other antigens were newly identified, e.g. decreased responses to E. coli virulence factors and genome polyproteins of enteroviruses, and increased responses to food antigens (wheat, barley) and autoantigens (particularly collagen type I and VI). Antibody epitope repertoires were able to accurately discriminate CD from population controls (area under the curve [AUC]=0.88, test set evaluation), showing very high discriminative performance (positive and negative predictive value of 72% and 93%, respectively, representing predicted classes in test set) (Fig. 3A-C), which was less accurate for ulcerative colitis (UC) (Fig. 3D-F).ConclusionThis study demonstrates the size, diversity and complexity of systemic antibody epitope repertoires in patients with IBD compared to controls, showing that distinct clinical phenotypes of IBD are characterized by unique antibody signatures. PhIP-seq is a powerful tool for identifying systemic immune-based biomarkers and exposing novel immunological targets in immune-mediated inflammatory diseases like IBD

Population-wide diversity and stability of serum antibody epitope repertoires against human microbiota

Serum antibodies can recognize both pathogens and commensal gut microbiota. However, our current understanding of antibody repertoires is largely based on DNA sequencing of the corresponding B-cell receptor genes, and actual bacterial antigen targets remain incompletely characterized. Here we have profiled the serum antibody responses of 997 healthy individuals against 244,000 rationally selected peptide antigens derived from gut microbiota and pathogenic and probiotic bacteria. Leveraging phage immunoprecipitation sequencing (PhIP-Seq) based on phage-displayed synthetic oligo libraries, we detect a wide breadth of individual-specific as well as shared antibody responses against microbiota that associate with age and gender. We also demonstrate that these antibody epitope repertoires are more longitudinally stable than gut microbiome species abundances. Serum samples of more than 200 individuals collected five years apart could be accurately matched and could serve as an immunologic fingerprint. Overall, our results suggest that systemic antibody responses provide a non-redundant layer of information about microbiota beyond gut microbial species composition. Phage immunoprecipitation sequencing illustrates the wide breadth of systemic microbiota-specific antibody responses, which are more longitudinally stable than gut microbiome species abundances in a cohort of healthy individuals

Phage display sequencing reveals that genetic, environmental, and intrinsic factors influence variation of human antibody epitope repertoire

Phage-displayed immunoprecipitation sequencing (PhIP-seq) has enabled high-throughput profiling of human antibody repertoires. However, a comprehensive overview of environmental and genetic determinants shaping human adaptive immunity is lacking. In this study, we investigated the effects of genetic, environmental, and intrinsic factors on the variation in human antibody repertoires. We characterized serological antibody repertoires against 344,000 peptides using PhIP-seq libraries from a wide range of microbial and environmental antigens in 1,443 participants from a population cohort. We detected individual-specificity, temporal consistency, and co-housing similarities in antibody repertoires. Genetic analyses showed the involvement of the HLA, IGHV, and FUT2 gene regions in antibody-bound peptide reactivity. Furthermore, we uncovered associations between phenotypic factors (including age, cell counts, sex, smoking behavior, and allergies, among others) and particular antibody-bound peptides. Our results indicate that human antibody epitope repertoires are shaped by both genetics and environmental exposures and highlight specific signatures of distinct phenotypes and genotypes.</p