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Discover how PEPperPRINT Peptide Microarray products have been used in different fields of research.

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Analysis of Plasmablasts from Children with Kawasaki Disease Reveals Evidence of a Convergent Antibody Response to a Specific Protein Epitope

Rowley, Anne H; Arrollo, David; Shulman, Stanford T; Torres, Abigail; O’Brien, Amornrat; Wylie, Kristine; Kim, Kwang-Youn A; Baker, Susan C
Feb 2023
10.1093/infdis/jiad048
Abstract Background Kawasaki disease (KD) is a febrile illness of young childhood that can result in coronary artery aneurysms and death. COVID mitigation strategies resulted in a marked decrease in KD cases worldwide, supporting a transmissible respiratory agent as the cause. We previously reported a peptide epitope recognized by monoclonal antibodies (MAbs) derived from clonally expanded peripheral blood plasmablasts from 3 of 11 KD children, suggesting a common disease trigger in a subset of patients with KD. Methods We performed amino acid substitution scans to develop modified peptides with improved recognition by KD MAbs. We prepared additional MAbs from KD peripheral blood plasmablasts and assessed MAb characteristics that were associated with binding to the modified peptides. Results We report a modified peptide epitope that is recognized by 20 MAbs from 11 of 12 KD patients. These MAbs predominantly use heavy chain VH3-74; two-thirds of VH3-74 plasmablasts from these patients recognize the epitope. The MAbs were nonidentical between patients but share a common CDR3 motif. Conclusions These results demonstrate a convergent VH3-74 plasmablast response to a specific protein antigen in children with KD, supporting one predominant causative agent in the etiopathogenesis of the illness.

Anti-brolucizumab immune response as one prerequisite for rare retinal vasculitis/retinal vascular occlusion adverse events

Karle, Anette C.; Wrobel, Matthias B.; Koepke, Stephan; Gutknecht, Michael; Gottlieb, Sascha; Christen, Brigitte; Rubic-Schneider, Tina; Pruimboom-Brees, Ingrid; Leber, Xavier Charles; Scharenberg, Meike; Maciejewski, Benjamin; Turner, Oliver; Saravanan, Chandra; Huet, Francois; Littlewood-Evans, Amanda; Clemens, Andreas; Grosskreutz, Cynthia L.; Kearns, Jeffrey D.; Mehan, Pawan; Schmouder, Robert L.; Sasseville, Vito; Brees, Dominique
Sci. Transl. Med..
Feb 2023
10.1126/scitranslmed.abq5241
In October 2019, Novartis launched brolucizumab, a single-chain variable fragment molecule targeting vascular endothelial growth factor A, for the treatment of neovascular age-related macular degeneration. In 2020, rare cases of retinal vasculitis and/or retinal vascular occlusion (RV/RO) were reported, often during the first few months after treatment initiation, consistent with a possible immunologic pathobiology. This finding was inconsistent with preclinical studies in cynomolgus monkeys that demonstrated no drug-related intraocular inflammation, or RV/RO, despite the presence of preexisting and treatment-emergent antidrug antibodies (ADAs) in some animals. In this study, the immune response against brolucizumab in humans was assessed using samples from clinical trials and clinical practice. In the brolucizumab-naïve population, anti-brolucizumab ADA responses were detected before any treatment, which was supported by the finding that healthy donors can harbor brolucizumab-specific B cells. This suggested prior exposure of the immune system to proteins with structural similarity. Experiments on samples showed that naïve and brolucizumab-treated ADA-positive patients developed a class-switched, high-affinity immune response, with several linear epitopes being recognized by ADAs. Only patients with RV/RO showed a meaningful T cell response upon recall with brolucizumab. Further studies in cynomolgus monkeys preimmunized against brolucizumab with adjuvant followed by intravitreal brolucizumab challenge demonstrated that high ADA titers were required to generate ocular inflammation and vasculitis/vascular thrombosis, comparable to RV/RO in humans. Immunogenicity therefore seems to be a prerequisite to develop RV/RO. However, because only 2.1% of patients with ADA develop RV/RO, additional factors must play a role in the development of RV/RO.

Peptide microarray IgM and IgG screening of pre-SARS-CoV-2 human serum samples from Zimbabwe for reactivity with peptides from all seven human coronaviruses: a cross-sectional study

Ashworth, Jordan; Mathie, Dayna; Scott, Fiona; Mahendran, Yuvaraj; Woolhouse, Mark; Stoevesandt, Oda; Mduluza, Takafira; Mutapi, Francisca
The Lancet Microbe.
Feb 2023
10.1016/S2666-5247(22)00295-6

Human antibody profiling technologies for autoimmune disease

Carlton, Lauren H.; McGregor, Reuben; Moreland, Nicole J.
Immunol Res.
Jan 2023
10.1007/s12026-023-09362-8
Abstract Autoimmune diseases are caused by the break-down in self-tolerance mechanisms and can result in the generation of autoantibodies specific to human antigens. Human autoantigen profiling technologies such as solid surface arrays and display technologies are powerful high-throughput technologies utilised to discover and map novel autoantigens associated with disease. This review compares human autoantigen profiling technologies including the application of these approaches in chronic and post-infectious autoimmune disease. Each technology has advantages and limitations that should be considered when designing new projects to profile autoantibodies. Recent studies that have utilised these technologies across a range of diseases have highlighted marked heterogeneity in autoantibody specificity between individuals as a frequent feature. This individual heterogeneity suggests that epitope spreading maybe an important mechanism in the pathogenesis of autoimmune disease in general and likely contributes to inflammatory tissue damage and symptoms. Studies focused on identifying autoantibody biomarkers for diagnosis should use targeted data analysis to identify the rarer public epitopes and antigens, common between individuals. Thus, utilisation of human autoantigen profiling technology, combined with different analysis approaches, can illuminate both pathogenesis and biomarker discovery.

Antibody isotype epitope mapping of SARS‐CoV‐2 Spike RBD protein: targets for COVID‐19 symptomatology and disease control

Contreras, Marinela; Vicente, Joaquín; Cerón, José Joaquín; Subiela, Silvia Martinez; Urra, José Miguel; Rodríguez‐del‐Río, Francisco J.; Ferreras‐Colino, Elisa; Vaz‐Rodrigues, Rita; de Mera, Isabel G. Fernández; Antunes, Sandra; Domingos, Ana; Gortázar, Christian; de la Fuente, José
Eur J Immunol.
Jan 2023
10.1002/eji.202250206

Antibody Properties Associate with Clinical Phenotype in LGI1 Encephalitis

Ludewig, Susann; Salzburger, Leonie; Goihl, Alexander; Rohne, Jana; Leypoldt, Frank; Bittner, Daniel; Düzel, Emrah; Schraven, Burkhart; Reinhold, Dirk; Korte, Martin; Körtvélyessy, Péter
Cells.
Jan 2023
10.3390/cells12020282
Autoimmune encephalitis (AE) associated with autoantibodies against leucine-rich glioma-inactivated protein-1 (LGI1) can present with faciobrachial dystonic seizures (FBDS) and/or limbic encephalitis (LE). The reasons for this heterogeneity in phenotypes are unclear. We performed autoantibody (abs) characterization per patient, two patients suffering from LE and two from FBDS, using isolated antibodies specified with single amino acid epitope mapping. Electrophysiological slice recordings were conducted alongside spine density measurements, postsynaptic Alpha-amino-3-hydoxy-5-methyl-4-isoaxole-proprionate-receptors (AMPA-R) and N-methyl-D-aspartate-receptors receptor (NMDA-R) cluster counting. These results were correlated with the symptoms of each patient. While LGI1 abs from LE patients mainly interacted with the Leucine-rich repeat section of LGI1, abs from both FBDS patients also recognized the Epitempin section as well. Six-hour incubation of mouse hippocampal slices with LE patients autoantibodies but not from the FBDS patients resulted in a significant decline in long-term potentiation (p = 0.0015) or short-term plasticity at CA3-CA1 neurons and in decreased hippocampal synaptic density. Cluster differentiation showed no decrease in postsynaptic AMPA-R and NMDA-R. LGI1 autoantibodies selected by phenotype show an almost distinct epitope pattern, elicit disparate functional effects on hippocampal neurons, and cause divergent effects on spine density. This data illuminates potential pathomechanisms for disease heterogeneity in LGI1 AE.

Immunodominant antibody responses directed to SARS-CoV-2 hotspot mutation sites and risk of immune escape

Oliveira, Jamille Ramos; Ruiz, Cesar Manuel Remuzgo; Machado, Rafael Rahal Guaragna; Magawa, Jhosiene Yukari; Daher, Isabela Pazotti; Urbanski, Alysson Henrique; Schmitz, Gabriela Justamante Händel; Arcuri, Helen Andrade; Ferreira, Marcelo Alves; Sasahara, Greyce Luri; de Medeiros, Giuliana Xavier; Júnior, Roberto Carlos Vieira Silva; Durigon, Edison Luiz; Boscardin, Silvia Beatriz; Rosa, Daniela Santoro; Schechtman, Deborah; Nakaya, Helder I.; Cunha-Neto, Edecio; Gadermaier, Gabriele; Kalil, Jorge; Coelho, Verônica; Santos, Keity Souza
Front. Immunol..
Jan 2023
10.3389/fimmu.2022.1010105
Introduction Considering the likely need for the development of novel effective vaccines adapted to emerging relevant CoV-2 variants, the increasing knowledge of epitope recognition profile among convalescents and afterwards vaccinated with identification of immunodominant regions may provide important information. Methods We used an RBD peptide microarray to identify IgG and IgA binding regions in serum of 71 COVID-19 convalescents and 18 vaccinated individuals. Results We found a set of immunodominant RBD antibody epitopes, each recognized by more than 30% of the tested cohort, that differ among the two different groups and are within conserved regions among betacoronavirus. Of those, only one peptide, P44 (S415-429), recognized by 68% of convalescents, presented IgG and IgA antibody reactivity that positively correlated with nAb titers, suggesting that this is a relevant RBD region and a potential target of IgG/IgA neutralizing activity. Discussion This peptide is localized within the area of contact with ACE-2 and harbors the mutation hotspot site K417 present in gamma (K417T), beta (K417N), and omicron (K417N) variants of concern. The epitope profile of vaccinated individuals differed from convalescents, with a more diverse repertoire of immunodominant peptides, recognized by more than 30% of the cohort. Noteworthy, immunodominant regions of recognition by vaccinated coincide with mutation sites at Omicron BA.1, an important variant emerging after massive vaccination. Together, our data show that immune pressure induced by dominant antibody responses may favor hotspot mutation sites and the selection of variants capable of evading humoral response.

Deciphering the Autoantibody Response to the OJ Antigenic Complex

Fritzler, Marvin J.; Bentow, Chelsea; Satoh, Minoru; McHugh, Neil; Ghirardello, Anna; Mahler, Michael
Diagnostics.
Jan 2023
10.3390/diagnostics13010156
(1) Background: Myositis specific antibodies (MSA) are important diagnostic biomarkers. Among the rarest and most challenging MSA are anti-OJ antibodies which are associated with anti-synthetase syndrome (ASS). In contrast to the other tRNA synthetases that are targets of ASS autoantibodies (e.g Jo-1, PL-7, PL-12, EJ, KS, Zo), OJ represents a macromolecular complex with several ribonucleoprotein subunits. Therefore, the choice of the antigen in autoantibody assays can be challenging. (2) Methods: We collected two independent cohorts with anti-OJ antibodies, one based on a commercial line immunoassay (LIA) (n = 39), the second based on protein immunoprecipitation (IP) (n = 15). Samples were tested using a particle-based multi-analyte technology (PMAT) system that allows for the simultaneous detection of antibodies to various autoantigens. For the detection of anti-OJ antibodies, two different antigens were deployed (KARS, IARS) on PMAT. The reactivity to the two antigens KARS and IARS was analyzed individually and combined in a score (sum of the median fluorescence intensities). (3) Results: In the cohort selection based on LIA, 3/39 (7.7%) samples were positive for anti-KARS and 7/39 (17.9%) for anti-IARS and 14/39 (35.9%) when the two antigens were combined. In contrast, in samples selected by IP the sensitivity of anti-KARS was higher: 6/15 (40.0%) samples were positive for anti-KARS, 4/15 (26.7%) for anti-IARS and 12/15 (80.0%) for the combination of the two antigens. 18/39 (46.2%) of the LIA samples generated a cytoplasmic IIF pattern (compatible with anti-synthetase antibodies), but there was no association with the antibody levels, neither with LIA nor with PMAT. (4) Conclusions: The combination of IARS and KARS might represent a promising approach for the detection of anti-OJ antibodies on a fully automated platform.

Molecular mimicry, genetic homology, and gene sharing proteomic “molecular fingerprints” using an EBV (Epstein-Barr virus)-derived microarray as a potential diagnostic method in autoimmune disease

Dreyfus, David H.; Farina, Antonella; Farina, Giuseppina Alessandra
Immunol Res.
Dec 2018
10.1007/s12026-018-9045-0
EBV (Epstein-Barr Virus) and other human DNA viruses are associated with autoimmune syndromes in epidemiologic studies. In this work, immunoglobulin G response to EBV-encoded proteins which share regions with human immune response proteins from the human host including ZEBRA (BZLF-1 encoded protein), BALF-2 recombinase expressed primarily during the viral lytic replication cycle, and EBNA-1 (Epstein-Barr Virus Nuclear Antigen) expressed during the viral latency cycle respectively were characterized using a laser-printed micro-array (PEPperprint.com). IgG response to conserved “A/T hooks” in EBV-encoded proteins such as EBNA-1 and the BALF-2 recombinase related to host DNA-binding proteins including RAG-1 recombinase and histones, and EBV-encoded virokines such as the IL-10 homologue BCRF-1 suggest further directions for clinical research. The author suggests that proteomic “molecular fingerprints” of the immune response to viral proteins shared with human immune response genes are potentially useful in early diagnosis and monitoring of autoantibody production and response to therapy in EBV-related autoimmune syndromes.

Regulatory T-cell deficiency leads to pathogenic bullous pemphigoid antigen 230 autoantibody and autoimmune bullous disease

Haeberle, Stefanie; Wei, Xiaoying; Bieber, Katja; Goletz, Stephanie; Ludwig, Ralf J.; Schmidt, Enno; Enk, Alexander H.; Hadaschik, Eva N.
Journal of Allergy and Clinical Immunology.
Dec 2018
10.1016/j.jaci.2018.04.006
Background Autoimmune bullous diseases/dermatoses (AIBDs) are severe autoantibody-mediated skin diseases. The pathogenic relevance of autoreactive CD4+ T cells for the induction of autoantibody production remains to be fully evaluated. Scurfy mice lack functional regulatory T (Treg) cells, experience spontaneous activation of autoreactive CD4+ T cells, and display severe erosive skin lesions suggestive of AIBDs. Objective We sought to determine whether AIBDs develop in Treg cell–deficient scurfy mice. Methods Histology, indirect immunofluorescence (IF) microscopy, direct IF, and ELISA were used to prove the presence of AIBDs in scurfy mice. Monoclonal autoantibodies from sera of scurfy mice were screened by using indirect IF on murine skin, and immunoprecipitation and mass spectrometry were used for target antigen identification, followed by confirmation in modified human embryonic kidney cells and murine keratinocytes. Pathogenicity was determined by injecting the autoantibody into neonatal mice and transferring scurfy CD4+ T cells into nu/nu mice. Results Autoantibodies against different known autoantigens of AIBDs spontaneously develop in scurfy mice. Histology reveals subepidermal blisters, and direct IF of skin of scurfy mice shows a predominant linear staining pattern. The mAb 20B12 shows a linear staining pattern in indirect IF, recognizes the murine hemidesmosomal protein bullous pemphigoid antigen 230 (BP230) as the target antigen, and cross-reacts with human BP230. Purified mAb 20B12 induces subepidermal blisters in neonatal mice. Transfer of scurfy CD4+ T cells is sufficient to induce antibodies with reactivity to AIBD autoantigens and subepidermal blisters in the skin of recipient T cell–deficient nu/nu mice. Conclusion We show that the absence of Treg cells leads to AIBDs by pathogenic autoantibodies targeting BP230.

Soybean Allergy Related Epitopes

Kern, Karolin; Spiegel, Holger; Havenith, Heide; Szardenings, Michael
Nov 2018
The invention relates to a compilation comprising at least five different peptides, each peptide comprising at least one sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented. The invention further relates to an in vitro method for determining a patient’s immune status to soybean allergens, to a method for detecting at least one soybean allergen in a substance and to a method for determining the allergenicity of a soybean variety. Additionally, the invention relates to a kit comprising at least one composition containing a compound comprising at least five different sequence elements each corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented. Furthermore, the invention relates to the use of a peptide comprising a sequence element corresponding to an epitope for providing a molecule binding to a protein or peptide comprising the epitope.

Linear epitopes in Onchocerca volvulus vaccine candidate proteins and excretory-secretory proteins

Lagatie, Ole; Verheyen, Ann; Van Dorst, Bieke; Batsa Debrah, Linda; Debrah, Alex; Stuyver, Lieven J.
Parasite Immunol.
Nov 2018
10.1111/pim.12587
In our previous study, a proteome-wide screen was conducted to identify linear epitopes in this parasite’s proteome, resulting in the discovery of three immunodominant motifs. Here, we investigated whether such antigenic peptides were found in proteins that were already known as vaccine candidates and excretome/secretome proteins for Onchocerca volvulus This approach led to the identification of 71 immunoreactive stretches in 46 proteins. A deep-dive into the immunoreactivity profiles of eight vaccine candidates that were chosen as most promising candidates for further development (Ov-CPI-2, Ov-ALT-1, Ov-RAL-2, Ov-ASP-1, Ov-103, Ov-RBP-1, Ov-CHI-1, and Ov-B20), resulted in the identification of a poly-glutamine stretch in Ov-RAL-2 that has properties for use as a serodiagnostic marker for O. volvulus infection. A peptide ELISA was developed, and the performance of this assay was evaluated. Based on this assessment, it was found that this assay has a sensitivity of 75.0% [95% CI: 64.9%-83.5%] and a specificity of 98.5% [95% CI: 94.6%-99.8%]. Furthermore, 8.7% reactivity in Asian parasite-infected individuals (8 out of 92) was observed. Besides this identification of a linear epitope marker, the information on the presence of linear epitopes in vaccine candidate proteins might be useful in the study of vaccines for river blindness.

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