78 Co-occurrence of peptides identifies multiple epitopes for the same antigen, antibody cross-reactivity in related structures and co-occurrence of antibodies against unrelated structures To understand the relationbetween antibody-boundpeptides, we computed their correlation and built a network using weighted gene co-expression network analysis by computing correlation coefficients from the binary profile of all selected peptides without missing values. In total, 435 peptides could be assigned to 22 modules of at least 10 highly correlated peptides (Figure 2, Supplementary Table 1.1). Assessing antibody-bound peptides within each of the modules (denoted by the number of peptides per module, 1 to 22) and the sequence similarity between them allowed us to identify three main types of modules: (1) modules driven by antigens from the same biological source, (2) modules driven by antigens of similar peptide sequence and (3) modules that include peptides that are not taxonomically or structurally related, but do correlate strongly with each other (Supplementary Table 1.2). We observed five category (1) modules (Figure 2). For example, module 16 was composed of two different Epstein-Barr virus (EBV) proteins, including capsid protein VP26 and nuclear antigen 1 (EBNA-1); module 20 was composed of high-identity peptides belonging to different strains of Influenza B viruses and module 1 is mainly driven by CMV peptides, while also including some EBV and other peptides. All modules are described in (Supplementary Table 1.2). Category (2) modules, driven by similar sequences in different peptides, highlight the cross-reactivity of antibody response (Figure 2). For example, module 21 is composed of plant thionins, small cytotoxic plant compounds produced by many species, but here mainly derived from common wheat (Triticumaestivum), barley (Hordeumvulgare) and rye (Secale cereale). Module 9 contained related antigens from wheat, Asian rice (Oryza sativa), rye, barley and grass (Setaria italica) that represent plant granule–bound starch synthase peptides. Modules 14, 17 and 18 were characterized by antibody-bound peptides representing genome polyproteins from a series of viruses, including Enterovirus A71, B and C; Rhinovirus B and serotype 2; Coxsackievirus (type A9) and Poliovirus. Module 3 was dominated by allergen peptides, including antigens involved in common insect and seafood allergies, e.g. Artemia franciscana (shrimp), Octopus vulgaris (octopus), Blattella germanica (German cockroach), Dermatophagoides farinae (house dust mite), Portunus trituberculatus (gazami crab), Bombus hypocrita (bumble bee) and Ctenocephalides felis (cat flea). Examples from category (3), where no structural or taxonomic relation is seen, are harder to interpret (Figure 2). While some members in this category have a majority of peptides belonging to category (1) or (2), others do not show major structural relations and are mainly composed of bacterial peptides or bacterial and autoimmune peptides clustering together. Chapter 3
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