Antibody repertoires : Exploration of their role in allergic disease and facilitation of reliable analyses thereof

Sammanfattning: Allergic disease affects millions of individuals worldwide and leads to both impaired life quality of the affected individuals and large economic costs for the society. Allergen-specific antibodies of IgE type act as one of the key players in the disease, by activating effector cells upon binding of its allergen. Allergen-specific antibodies of other isotypes, such as IgG, can instead act protectively by blocking the binding of IgE antibodies. Today, specific immunotherapy (SIT) poses a treatment option that, in contrast to most anti-allergy drugs, can alter the disease progression or even cure the patient, rather than just ease the symptoms of the disease. The development of protective IgG antibodies is one of the mechanisms associated with successful SIT.In Paper I-II of this thesis, we have analysed how antibody repertoires are constituted in allergic subjects and how they are affected by SIT. Allergen-specific IgE clones and epitope specificity profiles of antibodies in serum were shown to be persistent during three years of SIT and an increased development of allergen-specific, supposedly protective, IgG antibodies were found in allergic donors subjected to SIT compared to allergic control donors. Analysis of a previously identified stereotyped Phl p 2-specific antibody response allowed us to identify properties in the maturation pattern that were distinct for these antibodies compared to other allergen-specific antibodies. Additionally, a machine learning approach were used to refine the previous definition of such antibodies and to identify additional residues in the antigen-binding regions that typically are conserved in these stereotyped antibodies.Studies of antibody repertoires, for example in relation to allergy, are dependent on robust and reliable bioinformatic analysis pipelines to ensure trustworthy conclusions. For example, correct annotations of the gene origin of antibody transcripts are important to not inflict the results of downstream analyses. Such annotations are however complicated by many factors, including the complexity of the immunoglobulin germline gene loci. In paper III-V of this thesis, we explored various aspects of immunoglobulin germline gene analyses that can impair antibody repertoire studies and suggested strategies that can be applied to ensure reliable results. For example, these papers highlight the importance of the reference database used as starting point for germline gene annotations and/or inferences as well as difficulties associated with the annotation/inference of the 3’ most bases of immunoglobulin heavy chain variable (IGHV) genes. We also show how the 5′ untranslated region and leader sequence found in heavy chain antibody transcripts can be exploited to support novel identified IGHV genes and for analyses of IGHV loci. For example, we use the sequences of these regions to identify IGHV duplications and to evaluate genotypic organisation of some IGHV genes.Altogether, this thesis expands our understanding of the role of antibody repertoires in allergy and during SIT, and provides insights on how to ensure reliable analyses of antibody repertoires in health, allergy, and/or other diseases.