67 Introduction The adaptive immune system encompasses an extremely complex group of biological processes that orchestrate responses to invading pathogens in all jawed vertebrates (gnathostomes), including humans.1 The adaptive immune system’s capacity to recognize, adapt to and remember a wide variety of threats is determined by highly polymorphic genetic structures that encode receptors able to interact with complex structures known as antigens that most commonly represent amino acid sequences (epitopes) from foreign proteins.1 Antibodies are the key effector molecules in the human adaptive immune system and are responsible for humoral immunity. Each individual’s antibody epitope repertoire is characterized by a high degree of versatility and adaptability and is continuously altered during lifetime, with host genetics and environmental factors being the main contributors. Antibody repertoires determine the fate of the immune response against pathogens and the development of autoimmunity or allergies, and they have garnered special attention because they can be used to study herd immunity acquisition.2 In an adult human, there are around 1010–1011 B-lymphocytes, each expressing a unique B-cell receptor (BCR) (a non-soluble antibody form) that identifies a molecular pattern.3 The antigenic diversity of BCRs is the net result of the high diversity and somatic rearrangements of V(D)J gene segments, insertion and deletion (indel) of nucleotides and subsequent somatic hypermutation (SHM) to increase antigen affinity and specificity.4 To gain more insight into antibody–antigen interaction, efforts have been made to directly sequence the BCR5,6 and to directly infer it from single-cell transcriptomic sequencing.7 Although this methodology provides information on the potential for generation of immune responses against yet unknown antigens, it does not directly link BCR sequences to the exact nature of antigenic epitopes. In addition, in terms of scaling, it is limited to just a small proportion of the immense number of these receptors.8 On the other hand, antibody-binding analysis, such as peptide microarrays9,10 or enzyme-linked immunosorbent assay (ELISAs), enable the determination of antibody seroprevalence against selected antigens. While easily implemented for a limited set of antigens, these methodologies have been difficult to scale up to thousands of antigens in a large population. Phage-display immunoprecipitation sequencing (PhIP-Seq) is an immunoprecipitation–based sequencing technique that enables quantification of antigen peptides that are displayed as phage libraries, which subsequently react with human antibodies, and antibody-bound phages are eventually sequenced to obtain an ‘immunological fingerprint’ of an individual’s antibody repertoire. PhIP-Seq has been described previously11,12 and has been successfully applied to characterize autoimmune antibody prevalence in patients with multiple sclerosis, type 1 diabetes and rheumatoid arthritis,13,14 especially regarding the human virome,15-19 the widespread presence of antibodies against virulence factors20,21 and the gut microbiome.21 However, no comprehensive study has been carried out to date that identifies the environmental, intrinsic, lifestyle and genetic factors that determine antibody generation against antigen exposures in the general population. In this work, we set out to uncover the antibody epitope repertoire in a deeply phenotyped population cohort from the northern part of the Netherlands, Lifelines-DEEP (LLD).22 We used the PhIP-Seq libraries described in21 and23 to characterize 344,000 peptide antigens related to: (1) microbes (including human gut microbiota, probiotic strains, pathobionts, antibody-coated Determinants of the human antibody epitope repertoire
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