| 1. |
- Franciskovic, Eric, et al.
(författare)
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Linear epitopes of bony fish β-parvalbumins
- 2024
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Ingår i: Frontiers in Immunology. - 1664-3224. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Fish β-parvalbumins are common targets of allergy-causing immunity. The nature of antibody responses to such allergens determines the biological outcome following exposure to fish. Specific epitopes on these allergens recognised by antibodies are incompletely characterised. Methods: High-content peptide microarrays offer a solution to the identification of linear epitopes recognised by antibodies. We characterized IgG and IgG4 recognition of linear epitopes of fish β-parvalbumins defined in the WHO/IUIS allergen database as such responses hold the potential to counter an allergic reaction to these allergens. Peripheral blood samples, collected over three years, of 15 atopic but not fish-allergic subjects were investigated using a microarray platform that carried every possible 16-mer peptide of known isoforms and isoallergens of these and other allergens. Results: Interindividual differences in epitope recognition patterns were observed. In contrast, reactivity patterns in a given individual were by comparison more stable during the 3 years-course of the study. Nevertheless, evidence of the induction of novel specificities over time was identified across multiple regions of the allergens. Particularly reactive epitopes were identified in the D helix of Cyp c 1 and in the C-terminus of Gad c 1 and Gad m 1.02. Residues important for the recognition of certain linear epitopes were identified. Patterns of differential recognition of isoallergens were observed in some subjects. Conclusions: Altogether, comprehensive analysis of antibody recognition of linear epitopes of multiple allergens enables characterisation of the nature of the antibody responses targeting this important set of food allergens.
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| 2. |
- Hoh, Ramona A., et al.
(författare)
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Clonal evolution and stereotyped sequences of human IgE lineages in aeroallergen-specific immunotherapy
- 2023
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Ingår i: Journal of Allergy and Clinical Immunology. - : Elsevier BV. - 0091-6749. ; 152:1, s. 214-229
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Tidskriftsartikel (refereegranskat)abstract
- Background: Allergic disease reflects specific inflammatory processes initiated by interaction between allergen and allergen-specific IgE. Specific immunotherapy (SIT) is an effective long-term treatment option, but the mechanisms by which SIT provides desensitization are not well understood. Objective: Our aim was to characterize IgE sequences expressed by allergen-specific B cells over a 3-year longitudinal study of patients with aeroallergies who were undergoing SIT. Methods: Allergen-specific IgE–expressing clones were identified by using combinatorial single-chain variable fragment libraries and tracked in PBMCs and nasal biopsy samples over a 3-year period with antibody gene repertoire sequencing. The characteristics of private IgE-expressing clones were compared with those of stereotyped or “public” IgE responses to the grass pollen allergen Phleum pratense (Phl p) 2. Result: Members of the same allergen-specific IgE lineages were observed in nasal biopsy samples and blood, and lineages detected at baseline persisted in blood and nasal biopsy samples after 3 years of SIT, including B cells that express IgE. Evidence of progressive class switch recombination to IgG subclasses was observed after 3 years of SIT. A common stereotyped Phl p 2–specific antibody heavy chain sequence was detected in multiple donors. The amino acid residues enriched in IgE-stereotyped sequences from seropositive donors were analyzed with machine learning and k-mer motif discovery. Stereotyped IgE sequences had lower overall rates of somatic hypermutation and antigen selection than did single-chain variable fragment–derived allergen-specific sequences or IgE sequences of unknown specificity. Conclusion: Longitudinal tracking of rare circulating and tissue-resident allergen-specific IgE+ clones demonstrates persistence of allergen-specific IgE+ clones, progressive class switch recombination to IgG subtypes, and distinct maturation of a stereotyped Phl p 2 clonotype.
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| 3. |
- Huang, Yixun, et al.
(författare)
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Computational Inference, Validation, and Analysis of 5’UTR-Leader Sequences of Alleles of Immunoglobulin Heavy Chain Variable Genes
- 2021
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Upstream and downstream sequences of immunoglobulin genes may affect the expression of such genes. However, these sequences are rarely studied or characterized in most studies of immunoglobulin repertoires. Inference from large, rearranged immunoglobulin transcriptome data sets offers an opportunity to define the upstream regions (5’-untranslated regions and leader sequences). We have now established a new data pre-processing procedure to eliminate artifacts caused by a 5’-RACE library generation process, reanalyzed a previously studied data set defining human immunoglobulin heavy chain genes, and identified novel upstream regions, as well as previously identified upstream regions that may have been identified in error. Upstream sequences were also identified for a set of previously uncharacterized germline gene alleles. Several novel upstream region variants were validated, for instance by their segregation to a single haplotype in heterozygotic subjects. SNPs representing several sequence variants were identified from population data. Finally, based on the outcomes of the analysis, we define a set of testable hypotheses with respect to the placement of particular alleles in complex IGHV locus haplotypes, and discuss the evolutionary relatedness of particular heavy chain variable genes based on sequences of their upstream regions.
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| 4. |
- Ohlin, Mats, et al.
(författare)
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Inferred Allelic Variants of Immunoglobulin Receptor Genes : A System for Their Evaluation, Documentation, and Naming
- 2019
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 10
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Forskningsöversikt (refereegranskat)abstract
- Immunoglobulins or antibodies are the main effector molecules of the B-cell lineage and are encoded by hundreds of variable (V), diversity (D), and joining (J) germline genes, which recombine to generate enormous IG diversity. Recently, high-throughput adaptive immune receptor repertoire sequencing (AIRR-seq) of recombined V-(D)-J genes has offered unprecedented insights into the dynamics of IG repertoires in health and disease. Faithful biological interpretation of AIRR-seq studies depends upon the annotation of raw AIRR-seq data, using reference germline gene databases to identify the germline genes within each rearrangement. Existing reference databases are incomplete, as shown by recent AIRR-seq studies that have inferred the existence of many previously unreported polymorphisms. Completing the documentation of genetic variation in germline gene databases is therefore of crucial importance. Lymphocyte receptor genes and alleles are currently assigned by the Immunoglobulins, T cell Receptors and Major Histocompatibility Nomenclature Subcommittee of the International Union of Immunological Societies (IUIS) and managed in IMGT®, the international ImMunoGeneTics information system® (IMGT). In 2017, the IMGT Group reached agreement with a group of AIRR-seq researchers on the principles of a streamlined process for identifying and naming inferred allelic sequences, for their incorporation into IMGT®. These researchers represented the AIRR Community, a network of over 300 researchers whose objective is to promote all aspects of immunoglobulin and T-cell receptor repertoire studies, including the standardization of experimental and computational aspects of AIRR-seq data generation and analysis. The Inferred Allele Review Committee (IARC) was established by the AIRR Community to devise policies, criteria, and procedures to perform this function. Formalized evaluations of novel inferred sequences have now begun and submissions are invited via a new dedicated portal (https://ogrdb.airr-community.org). Here, we summarize recommendations developed by the IARC-focusing, to begin with, on human IGHV genes-with the goal of facilitating the acceptance of inferred allelic variants of germline IGHV genes. We believe that this initiative will improve the quality of AIRR-seq studies by facilitating the description of human IG germline gene variation, and that in time, it will expand to the documentation of TR and IG genes in many vertebrate species.
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| 5. |
- Persson, Helena, et al.
(författare)
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In Vitro Evolution of Antibodies Inspired by In Vivo Evolution
- 2018
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- In vitro generation of antibodies often requires variable domain sequence evolution to adapt the protein in terms of affinity, specificity, or developability. Such antibodies, including those that are of interest for clinical development, may have their origins in a diversity of immunoglobulin germline genes. Others and we have previously shown that antibodies of different origins tend to evolve along different, preferred trajectories. Apart from substitutions within the complementary determining regions, evolution may also, in a germline gene-origin-defined manner, be focused to residues in the framework regions, and even to residues within the protein core, in many instances at a substantial distance from the antibody's antigen-binding site. Examples of such germline origin-defined patterns of evolution are described. We propose that germline gene-preferred substitution patterns offer attractive alternatives that should be considered in efforts to evolve antibodies intended for therapeutic use with respect to appropriate affinity, specificity, and product developability. We also hypothesize that such germline gene-origin-defined in vitro evolution hold potential to result in products with limited immunogenicity, as similarly evolved antibodies will be parts of conventional, in vivo-generated antibody responses and thus are likely to have been seen by the immune system in the past.
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| 6. |
- Thörnqvist, Linnea
(författare)
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Antibody repertoires : Exploration of their role in allergic disease and facilitation of reliable analyses thereof
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 7. |
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| 8. |
- Thörnqvist, Linnea, et al.
(författare)
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Critical steps for computational inference of the 3′-end of novel alleles of immunoglobulin heavy chain variable genes - illustrated by an allele of IGHV3-7
- 2018
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Ingår i: Molecular Immunology. - : Elsevier BV. - 0161-5890. ; 103, s. 1-6
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Tidskriftsartikel (refereegranskat)abstract
- Sequencing of immunoglobulin germline gene loci is a challenging process, e.g. due to their repetitiveness and complexity, hence limiting the insight in the germline gene repertoire of humans and other species. Through next generation sequencing technology, it is possible to generate immunoglobulin transcript data sets large enough to computationally infer the germline genes from which the transcripts originate. Multiple tools for such inference have been developed and they can be used for construction of individual germline gene databases, and for discovery of new immunoglobulin germline genes and alleles. However, there are challenges associated with these methods, many of them related to the biological process through which immunoglobulin coding genes are generated. The junctional diversity introduced during rearrangement of the immunoglobulin heavy chain variable (IGHV), diversity and joining genes specifically complicates the inference of the junction regions, with implications for inference of the 3′-end of IGHV genes. With the aim of coping with such diversity, an inference software package may not be able to identify novel alleles harbouring a difference in these regions compared to their closest relatives in the starting database. In this study, we were able to computationally infer one such previously uncharacterized allele, IGHV3-7*02 A318G. However, this was possible only if a strategy was used in which different variants of IGHV3-7*02 were included in the inference-initiating database. Importantly, the presence of the novel allele, but not the standard IGHV3-7*02 sequence, in the genotype was strongly supported by the actual sequences that were assigned to the allele. We thus showed that the starting database used will impact the germline gene inference process, and that difference in the 3′-end of IGHV genes may remain undetected unless specific, non-standard procedures are used to address this matter. We suggest that inferred genes/alleles should be confirmed e.g. by examination of the nucleotide composition of the 3′-bases of the inference-supporting sequence reads.
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| 9. |
- Thörnqvist, Linnea, et al.
(författare)
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Data on the nucleotide composition of the first codons encoding the complementary determining region 3 (CDR3) in immunoglobulin heavy chains
- 2018
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Ingår i: Data in Brief. - : Elsevier BV. - 2352-3409. ; 19, s. 337-352
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Tidskriftsartikel (refereegranskat)abstract
- The highly variable complementary determining region 3 (CDR3) of antibodies is generated through recombination of immunoglobulin heavy chain variable (IGHV), diversity, and joining genes. The codons encoding the first residues of CDR3 may be derived directly from the IGHV germline gene but they may also be generated as part of the rearrangement process. Data of the nucleotide composition of these codons of rearranged genes, an indicator of the degree of contribution of the IGHV gene to CDR3 diversity, are presented in this article. Analyzed data are presented for two unrelated sets of raw sequence data. The raw data sets consisted of sequences of antibody heavy chain-encoding transcripts of six allergic subjects (European Nucleotide Archive accession number PRJEB18926), and paired antibody heavy and light chain variable region-encoding transcripts of memory B cells of three subjects (European Nucleotide Archive accession numbers SRX709625, SRX709626, and SRX709627). The nucleotide compositions of the corresponding 5'-ends of sequences encoding the CDR3 are presented for transcripts with an origin in 47 different IGHV alleles. These data have been used (Thörnqvist and Ohlin, 2018) [1] to demonstrate the extent of incorporation of the 3' most bases of IGHV germline genes into rearranged immunoglobulin encoding sequences, and the extent whereby any difference in incorporation affects the specificity of inference of the 3'-end of IGHV genes from immunoglobulin-encoding transcripts. They have also been used to assess the effect of observed gene differences on the composition of the ascending strand of CDR3 associated to antibodies with an origin in different IGHV genes (Thörnqvist and Ohlin, 2018) [1].
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| 10. |
- Thörnqvist, Linnea, et al.
(författare)
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Linear Epitope Binding Patterns of Grass Pollen-Specific Antibodies in Allergy and in Response to Allergen-Specific Immunotherapy
- 2022
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Ingår i: Frontiers in Allergy. - : Frontiers Media SA. - 2673-6101. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- Allergic diseases affect many individuals world-wide and are dependent on the interaction between allergens and antibodies of the IgE isotype. Allergen-specific immunotherapy (AIT) can alter the development of the disease, e.g., through induction of allergen-specific IgG that block allergen-IgE interactions. The knowledge of epitopes recognized by allergy-causing and protective antibodies are limited. Therefore, we developed an allergome-wide peptide microarray, aiming to track linear epitope binding patterns in allergic diseases and during AIT. Here, we focused on immune responses to grass pollen allergens and found that such epitopes were commonly recognized before initiation of AIT and that AIT commonly resulted in increased antibody production against additional epitopes already after 1 year of treatment. The linear epitope binding patterns were highly individual, both for subjects subjected to and for individuals not subjected to AIT. Still, antibodies against some linear epitopes were commonly developed during AIT. For example, the two rigid domains found in grass pollen group 5 allergens have previously been associated to a diversity of discontinuous epitopes. Here, we present evidence that also the flexible linker, connecting these domains, contains regions of linear epitopes against which antibodies are developed during AIT. We also describe some commonly recognized linear epitopes on Phl p 2 and suggest how antibodies against these epitopes may contribute to or prevent allergy in relation to a well-defined stereotyped/public IgE response against the same allergen. Finally, we identify epitopes that induce cross-reactive antibodies, but also antibodies that exclusively bind one of two highly similar variants of a linear epitope. Our findings highlight the complexity of antibody recognition of linear epitopes, with respect to both the studied individuals and the examined allergens. We expect that many of the findings in this study can be generalized also to discontinuous epitopes and that allergen peptide microarrays provide an important tool for enhancing the understanding of allergen-specific antibodies in allergic disease and during AIT.
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