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Array-based Autoantibody Profiling and Epitope Mapping
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0003-1242-0873
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antibodies are a class of proteins that are made by the immune system to recognize harmful organisms and molecules. Their exceptional capability of specifically recognizing molecules has been investigated for over a century and information thereof has been utilized for a variety of applications including vaccine and generation of therapeutic antibodies. Occasionally, instead of protecting the host against pathogens, antibodies can recognize constituents of the host and thereby cause an autoimmune reaction that eventually can lead to a disease. Therefore, it is of great interest to understand what the antibodies bind to and their specificities.

 

The last decades of technical development and availability of protein and peptide microarrays have enabled large-scale profiling of antibodies and precise determination of their specificities through epitope mapping. In this thesis the aim was to use affinity proteomics tools to profile antibodies, determine their specificities, and discover potential associations of autoantigens to disease by analyzing blood-derived samples with microarray-based methods.

 

In Paper I, 57 serum samples from patients with the suggested autoimmune disease narcolepsy, were analyzed on planar antigen microarrays with 10,846 human protein fragments. Verification on an independent sample collection consisting of serum samples from 176 individuals, revealed METTL22 and NT5C1A as two potential autoantigens. In Paper II, antibodies from 53 plasma samples from patients with first-episode psychosis, a condition suggested to have a partial autoimmune component, were analyzed on planar antigen microarrays with 2,304 human protein fragments. After a follow-up study of the patients, antibodies toward an antigen representing the three proteins, PAGE2, PAGE2B, PAGE5, was found associated to an increased risk of developing schizophrenia. In Paper III, serum and plasma samples from patients with the autoimmune diseases multiple sclerosis and narcolepsy, were epitope mapped on high-density peptide microarrays with approximately 2.2 million peptides. Technical and biological verification, by using other microarray technology and analyzing  samples from 448 patients, revealed one peptide for multiple sclerosis and narcolepsy, representing the proteins MAP3K7 and NRXN1, with higher antibody reactivity towards in each group, respectively. In Paper IV, purified polyclonal antibodies raised against a surface antigen found on malaria-infected erythrocytes, were profiled on the peptide microarrays representing all proteins found on malaria-infected erythrocytes derived from Plasmodium falciparum. Then, different Plasmodium falciparum strains were analyzed by immunofluorescence microscopy and western blots, using the epitope mapped antibodies. The performance of the immunoassays were compared to the identified epitopes, and validated by RNA sequencing.

 

In conclusion, these investigations describe multiplex methods to identify and characterize antibodies, their disease association and epitopes. Follow-up studies are needed to determine their potential use and clinical value.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. , p. 89
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:19
Keywords [en]
antibody, antigens, peptide, epitope mapping, autoimmunity, autoantibodies, microarrays
National Category
Medical Biotechnology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-213689ISBN: 978-91-7729-499-3 (print)OAI: oai:DiVA.org:kth-213689DiVA, id: diva2:1138286
Public defence
2017-10-06, Air & Fire, Tomtebodavägen 23A, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20170905

Available from: 2017-09-05 Created: 2017-09-04 Last updated: 2017-09-05Bibliographically approved
List of papers
1. Autoantibody targets in vaccine-associated narcolepsy
Open this publication in new window or tab >>Autoantibody targets in vaccine-associated narcolepsy
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2016 (English)In: Autoimmunity, ISSN 0891-6934, E-ISSN 1607-842X, Vol. 49, no 6, p. 421-433Article in journal (Refereed) Published
Abstract [en]

Narcolepsy is a chronic sleep disorder with a yet unknown cause, but the specific loss of hypocretin-producing neurons together with a strong human leukocyte antigen (HLA) association has led to the hypothesis that autoimmune mechanisms might be involved. Here, we describe an extensive effort to profile autoimmunity repertoires in serum with the aim to find disease-related autoantigens. Initially, 57 serum samples from vaccine-associated and sporadic narcolepsy patients and controls were screened for IgG reactivity towards 10 846 fragments of human proteins using planar microarrays. The discovered differential reactivities were verified on suspension bead arrays in the same sample collection followed by further investigation of 14 antigens in 176 independent samples, including 57 narcolepsy patients. Among these 14 antigens, methyltransferase-like 22 (METTL22) and 5'-nucleotidase cytosolic IA (NT5C1A) were recognized at a higher frequency in narcolepsy patients of both sample sets. Upon sequence analysis of the 14 proteins, polymerase family, member 3 (PARP3), acyl-CoA-binding domain containing 7 (ARID4B), glutaminase 2 (GLS2) and cyclin-dependent kinase-like 1 (CDKL1) were found to contain amino acid sequences with homology to proteins found in the H1N1 vaccine. These findings could become useful elements of further clinical assays that aim towards a better phenotypic understanding of narcolepsy and its triggers.

Place, publisher, year, edition, pages
Taylor & Francis, 2016
Keywords
Narcolepsy, autoantibody, vaccine-associated, protein microarray, serum
National Category
Immunology
Identifiers
urn:nbn:se:kth:diva-198898 (URN)10.1080/08916934.2016.1183655 (DOI)000388594400009 ()27206786 (PubMedID)2-s2.0-84969988196 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceVINNOVAKnut and Alice Wallenberg Foundation
Note

QC 20170109

Available from: 2017-01-09 Created: 2016-12-22 Last updated: 2017-11-29Bibliographically approved
2. Untargeted screening for novel autoantibodies with prognostic value in first-episode psychosis
Open this publication in new window or tab >>Untargeted screening for novel autoantibodies with prognostic value in first-episode psychosis
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2017 (English)In: Translational Psychiatry, ISSN 2158-3188, E-ISSN 2158-3188, Vol. 7, article id e1177Article in journal (Refereed) Published
Abstract [en]

Immunological and inflammatory reactions have been suggested to have a role in the development of schizophrenia, a hypothesis that has recently been supported by genetic data. The aim of our study was to perform an unbiased search for autoantibodies in patients with a first psychotic episode, and to explore the association between any seroreactivity and the development of a Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) disorder characterized by chronic or relapsing psychotic symptoms. We collected plasma samples from 53 patients when they were treated for their first-episode psychosis, and 41 non-psychotic controls, after which the patients were followed for a mean duration of 7 years. Thirty patients were diagnosed with schizophrenia, delusional disorder, schizoaffective disorder, bipolar disorder or a long-term unspecified nonorganic psychosis during follow-up, whereas 23 patients achieved complete remission. At the end of follow-up, plasma samples were analyzed for IgG reactivity to 2304 fragments of human proteins using a multiplexed affinity proteomic technique. Eight patient samples showed autoreactivity to the N-terminal fragment of the PAGE (P antigen) protein family (PAGE2B/PAGE2/PAGE5), whereas no such autoreactivity was seen among the controls. PAGE autoreactivity was associated with a significantly increased risk of being diagnosed with schizophrenia during follow-up (odds ratio 6.7, relative risk 4.6). An immunohistochemistry analysis using antisera raised against the N-terminal fragment stained an unknown extracellular target in human cortical brain tissue. Our findings suggest that autoreactivity to the N-terminal portion of the PAGE protein family is associated with schizophrenia in a subset of patients with first-episode psychosis.

Place, publisher, year, edition, pages
Nature Publishing Group, 2017
National Category
Psychiatry
Identifiers
urn:nbn:se:kth:diva-212629 (URN)10.1038/tp.2017.160 (DOI)000406715200003 ()2-s2.0-85046053045 (Scopus ID)
Funder
Swedish Research Council, 521-2014-3857VINNOVAKnut and Alice Wallenberg FoundationEU, FP7, Seventh Framework Programme, FP7-PEOPLE-2013-ITN-607616Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20170824

Available from: 2017-08-24 Created: 2017-08-24 Last updated: 2019-01-07Bibliographically approved
3. Whole-Proteome Peptide Microarrays for Profiling Autoantibody Repertoires within Multiple Sclerosis and Narcolepsy
Open this publication in new window or tab >>Whole-Proteome Peptide Microarrays for Profiling Autoantibody Repertoires within Multiple Sclerosis and Narcolepsy
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2017 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 16, no 3, p. 1300-1314Article in journal (Refereed) Published
Abstract [en]

The underlying molecular mechanisms of autoimmune diseases are poorly understood. To unravel the autoimmune processes across diseases, comprehensive and unbiased analyses of proteins targets recognized by the adaptive immune system are needed. Here we present an approach starting from high-density peptide arrays to characterize autoantibody repertoires and to identify new autoantigens. A set of ten plasma and serum samples from subjects with multiple sclerosis, narcolepsy, and without any disease diagnosis were profiled on a peptide array representing the whole proteome, hosting 2.2 million 12-mer peptides with a six amino acid lateral shift. On the basis of the IgG reactivities found on these whole-proteome peptide micro arrays, a set of 23 samples was then studied on a targeted array with 174 000 12-mer peptides of single amino acid lateral shift. Finally, verification of IgG reactivities was conducted with a larger sample set (n = 448) using the bead-based peptide microarrays. The presented workflow employed three different peptide microarray formats to discover and resolve the epitopes of human autoantibodies and revealed two potentially new autoantigens: MAP3K7 in multiple sclerosis and NRXN1 in narcolepsy. The presented strategy provides insights into antibody repertoire reactivity at a peptide level and may accelerate the discovery and validation of autoantigens in human diseases.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017
Keywords
peptide microarrays, autoantibody profiling, epitope mapping narcolepsy, multiple sclerosis
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-205512 (URN)10.1021/acs.jproteome.6b00916 (DOI)000395726200017 ()28121444 (PubMedID)2-s2.0-85014691057 (Scopus ID)
Funder
VINNOVAKnut and Alice Wallenberg Foundation
Note

QC 20170524

Available from: 2017-05-24 Created: 2017-05-24 Last updated: 2017-09-05Bibliographically approved
4. Epitopes of anti-RIFIN antibodies and characterization of rif-expressing Plasmodium falciparum parasites by RNA sequencing
Open this publication in new window or tab >>Epitopes of anti-RIFIN antibodies and characterization of rif-expressing Plasmodium falciparum parasites by RNA sequencing
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 43190Article in journal (Refereed) Published
Abstract [en]

Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications. Coupled with its potential use as a vaccine candidate, the recent suggestion that the repetitive interspersed families of polypeptides (RIFINs) mediate blood group A rosetting and influence blood group distribution has raised the research profile of these adhesins. Nevertheless, detailed investigations into the functions of this highly diverse multigene family remain hampered by the limited number of validated reagents. In this study, we assess the specificities of three promising polyclonal anti-RIFIN antibodies that were IgG-purified from sera of immunized animals. Their epitope regions were mapped using a 175,000-peptide microarray holding overlapping peptides of the P. falciparum variable surface antigens. Through immunoblotting and immunofluorescence imaging, we show that different antibodies give varying results in different applications/assays. Finally, we authenticate the antibody-based detection of RIFINs in two previously uncharacterized non-rosetting parasite lines by identifying the dominant rif transcripts using RNA sequencing.

Place, publisher, year, edition, pages
Nature Publishing Group, 2017
National Category
Basic Medicine
Identifiers
urn:nbn:se:kth:diva-204077 (URN)10.1038/srep43190 (DOI)000394930400001 ()28233866 (PubMedID)2-s2.0-85013755282 (Scopus ID)
Note

QC 20170329

Available from: 2017-03-29 Created: 2017-03-29 Last updated: 2018-01-13Bibliographically approved

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