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

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Integrated reiterative pipeline for rapid epitope-based pan-alphavirus vaccines

Versiani, Alice F.; McCaffrey, Peter; Ribeiro-Filho, Helder V.; Silva, Natalia I. O.; Lopes-de-Oliveira, Paulo S.; Carrera, Jean-Paul; Nogueira, Mauricio L.; Marques, Rafael E.; Rossi, Shannan L.; Vasilakis, Nikos
Sci Adv.
Mar 2026
10.1126/sciadv.aeb2066
The vast diversity of the virosphere underscores the need for rapid, adaptable vaccine development infrastructures. Arthropod-borne zoonotic alphaviruses, in particular, continue to pose substantial threats to human and animal health. We present a fast, multitarget vaccine design pipeline integrating machine learning-based epitope prediction, protein modeling, and docking to prioritize viral peptides by immunogenicity, allele coverage, solubility, and stability. T cell epitopes were validated using peptide microarrays and molecular dynamics simulations, confirming receptor binding accuracy. Flow cytometry of murine and human peripheral blood mononuclear cells demonstrated robust T cell activation and cytokine secretion (IFN-γ, TNF-α, or IL-2), dependent on species and HLA allele. Final candidates were selected by composite immunogenicity scores. While this study primarily validates the T cell-specific arm of our predictive pipeline, complementary B cell epitope analyses are ongoing. Our findings support the development of broadly protective pan-alphaviral vaccines and the establishment of efficient, tunable processes for global vaccine development.

Anti-TRPV2 Autoantibody Linked to Sudden Infant Death Syndrome

Maguy, Ange; Tessier, Agnès; Mahendran, Yuvaraj; Denis, Manon; Lauzier, Benjamin; Charpentier, Flavien; Li, Jin
Jul 2025
10.1161/circulationaha.125.073748
As a leading cause of infant death, sudden infant death syndrome (SIDS) remains a perplexing diagnosis with no clear underlying biological substrate.1 In the past decade, studies have emerged demonstrating that circulating autoantibodies targeting cardiac antigens can underlie life-threatening arrhythmias.2 Because autoimmunity as a cause of SIDS has not yet been explored, we screened infant serum samples for the presence of autoantibodies targeting cardiac ion channels and examined how immunoglobulins may play a driving role in the pathogenesis of SIDS. Comparing cases of SIDS and accidental suffocation and strangulation in bed with healthy controls, we established the autoantibody profile of 47 serum samples using peptide microarray (Figure [A]), as previously described.2 Strikingly, only 1 single autoantibody targeting the transient receptor potential vanilloid 2 (TRPV2) channel (PTGPNATESVQPMEGQEDEG) was significantly associated with SIDS (P=0.028 versus controls, the default correction in limma). Collectively, we detected anti-TRPV2 autoantibodies in 84.6% of infants with SIDS compared with 50.0% in cases of accidental suffocation and strangulation in bed and 25.0% in controls.

Mapping autoantibody targets of full-length C-reactive protein in systemic lupus erythematosus: importance for neutrophil function and classical complement activation

Karlsson, Jesper; Wirestam, Lina; Duàn, Hanna; Ahmad, Suhana; Appelgren, Daniel; Enocsson, Helena; Wetterö, Jonas; Sjöwall, Christopher
Front. Immunol..
May 2025
10.3389/fimmu.2025.1578372
C-reactive protein (CRP) is an important pattern recognition molecule of innate immunity. Autoantibodies targeting CRP are common in patients with systemic lupus erythematosus (SLE) and the levels correlate with disease activity. The purpose of this study was to investigate binding sites of IgG autoantibodies on the full linear sequence of CRP and identify potential associations with clinical variables in well-characterized SLE patients; a secondary aim was to investigate the effect of an epitope-based synthesized peptide motif on neutrophil functions. The levels of anti-CRP and SLE-associated antibodies were assessed, and a microarray-based linear epitope mapping was performed to detect binding sites on the full CRP monomer. We observed that anti-CRP antibodies bind to a variety of linear epitopes with a higher prevalence in SLE compared to healthy blood donors. Eleven unique epitopes were identified, of which five were found exclusively in SLE. Furthermore, we show that patients with anticardiolipin IgG and/or anti-β2GPI IgG antibodies have a higher number of positive CRP epitopes, and some CRP autoantibody-specificities associate with antiphospholipid antibodies, disease activity, and classical complement activation. In addition, one identified motif was selected, synthesized, and used for studying neutrophil function. This peptide showed modulatory capacity on neutrophil oxidative burst and chemotaxis, but not on neutrophil extracellular trap formation. Our results implicate a wide variation of anti-CRP autoantibody binding motifs of the linear structure of CRP in SLE patients. Some epitopes have the potential to modify innate host responses of relevance to the pathogenesis of SLE.

Preclinical characterization of an active immunotherapy targeting calcitonin gene-related peptide

Boyd, Justin D.; Wang, Shixia; Lin, Hsiao-Wen; Hsieh, Yueh-Ting; Sun, Yu Shuang; Thibodeaux, Brett A.; Lu, Hanxin; Sahni, Jaya; Wiggins, Jonathan; Longo, Matthew S.; Brooks, Jeanne K.; Vroom, Madeline M.; Chang, Yi-Pin; Liu, Zhi; Ding, Shuang; Dodart, Jean-Cosme
Commun Med.
Apr 2025
10.1038/s43856-025-00870-2
Abstract **Background** The success of passive immunotherapies targeting Calcitonin gene-related peptide (CGRP) for managing migraine has prompted our efforts towards developing an active immunotherapy that induces the production of endogenous antibodies against CGRP. Achieving efficacious antibody titers via immunization could provide a more convenient and cost-effective treatment alternative to anti-CGRP monoclonal antibody (mAb) therapies. However, immunization against endogenous CGRP faces multiple challenges such as breaking immune tolerance, inducing sufficient antibody titers, and avoiding immune response-associated toxicity. **Methods** Synthetic peptide immunogens formulated in adjuvants were delivered intramuscularly. Serum samples were collected post immunization and used to measure antibody titers as well as for the isolation of antibodies specific to CGRP. Antibodies were characterized for their binding affinities and specificities. The capsaicin-induced increase in dermal blood flow model was used in rats for the assessment of the pharmacodynamic effect of immunization. **Results** Here we demonstrate that a peptide-based active immunotherapy designed to induce antibodies against CGRP promotes robust antibody titers across preclinical species. Characterization of the immune response strongly suggests that this peptide immunogen primarily stimulates a humoral response and only induced CGRP-specific antibodies. Antibodies produced by immunization are primarily IgG1 and demonstrate binding and activity potencies similar to marketed monoclonal antibodies against CGRP. Finally, immunization demonstrates in vivo efficacy in a rat pharmacodynamic model. **Conclusion** Our results strongly suggest that a peptide-based active immunotherapy against CGRP could provide an affordable and convenient therapeutic for the prevention of migraine.

Paediatric autoimmune uveitis is associated with intraocular antibodies against Epstein–Barr virus Nuclear Antigen 1 (EBNA-1)

Hendrikse, Jytte; Bont, Louis J.; Schellekens, Peter A.W.J.F.; De Groot-Mijnes, Jolanda D.F.; De Boer, Joke H.; Kuiper, Jonas J.W.
eBioMedicine.
Mar 2025
10.1016/j.ebiom.2025.105681
**Background** Non-infectious uveitis is an immune-mediated disease characterized by vision-threatening inflammation within the eye. Increasing evidence indicates that microbial agents promote non-infectious uveitis, but the natural history of immune responses to pathogens in patients remains unexplored. We determined intraocular antibodies against pathogens in paediatric uveitis. **Methods** We used peptide microarrays containing 3760 linear B-cell epitopes from 196 human pathogens to profile IgG levels in eye fluid biopsies and paired serum samples from 18 Dutch paediatric patients and 6 age-matched controls. We compared intensities of single epitopes and clusters based on overlapping amino acid sequence of peptides. Next-generation sequencing data was obtained to determine the HLA-DRB1∗15:01 genotype. **Findings** Intraocular antibody profiles largely matched serum profiles and were characterized by high IgG against the conserved PALTAVET-motif of enterovirus family members, as well as broad epitope reactivity against Epstein–Barr virus (EBV). The aqueous humour of patients showed elevated levels of antibodies against peptides containing the RRPFFHPV-motif of Epstein–Barr Virus Nuclear Antigen 1 [EBNA-1]. Antibody levels against the RRPFFHPV-motif of EBNA1 were significantly higher in individuals that carry the HLA-DRB1∗15:01 risk allele of paediatric uveitis. **Interpretation** Intraocular antibodies against an immunogenic epitope of EBV showed an association with paediatric uveitis, particularly HLA-DRB1∗15:01 positive uveitis, indicating a potential link between EBV-specific immune responses and autoimmune uveitis. **Funding** Funding for this research was received from Fischer Stichting (UZ2022-3), ODAS (2021-02), LSBS and ANVVB.

HCV immunodominant peptide mapping reveals unique HLA-A*02-restricted signatures: insights for CD8+ T-cell-based vaccines and immunotherapies

Cardoso Corrêa-Dias, Laura; Lopes-Ribeiro, Ágata; Marques-Ferreira, Geovane; Gomes-de-Pontes, Letícia; Pereira-Santos, Thaiza Aline; De Sousa Reis, Erik Vinicius; Silva Moraes, Thaís De Fátima; Assis Martins-Filho, Olindo; Figueiredo Barbosa-Stancioli, Edel; Guimarães Da Fonseca, Flávio; Coelho-dos-Reis, Jordana Grazziela
Immunogenetics.
Jan 2025
10.1007/s00251-025-01370-2
Several barriers for the development of an HCV vaccine still exist, including the genetic diversity of the virus, and the shortage of assessable models for in vitro and in vivo assays. Therefore, in this study, HCV epitope mapping was performed for 59 polyprotein sequences from 7 HCV genotypes. Around 2,880 peptides were considered epitopes for CD8+ T cells. The peptide induction of cytokines from Th1 and/or Th2 axes of the cellular immune response was assessed, indicating a tendency for Th2 axis. In vitro evaluation was performed using peptide microarray and a recombinant HLA-A*02:01 molecule. A total of 615 peptides of high reactivity to HLA-A*02:01 were identified, with predominance of leucine and tryptophan residues, highlighting their importance for TCR-epitope binding and CD8+ T activation. Finally, HCV-derived peptide patterns restricted to HLA-A2*02:01 observed in this study provide important information for the development of a multi-epitope-based pan-genotypic vaccine against the virus.

The antibody repertoire of autoimmune sensory neuronopathies targets pathways of the innate and adaptative immune system. An autoantigenomic approach.

Moritz, Christian P.; Tholance, Yannick; Boutahar, Nadia; Borowczyk, Coralie; Berger, Anne-Emmanuelle; Paul, Stéphane; Antoine, Jean-Christophe; Camdessanché, Jean-Philippe
Journal of Translational Autoimmunity.
Jan 2025
10.1016/j.jtauto.2025.100277
Sensory neuronopathies (SNN) encompasses diverse etiologies, with autoimmunity playing a major role through both cellular and humoral responses. To investigate the humoral autoantibody repertoire in autoimmune SNN, we conducted a retrospective cohort study using large Human Proteome-wide protein microarrays (HuProt 3.1, HuProt 4.0, ProtoArrays). We specifically focused on immune system pathways within the repertoire of targeted antigens (the autoantigenome). We included 131 participants: 44 patients with non-paraneoplastic autoimmune SNN (12 with anti-FGFR3 and/or anti-AGO antibodies), 8 with paraneoplastic SNN and 79 controls. Results were validated in an independent cohort of 16 SNN patients. Overrepresentation of immune-system-related proteins was assessed via the Reactome database, and serum levels of IFN-γ and IL-6 were measured using the Bio-Plex Pro™ Reagent Kit. Autoimmune SNN sera interact with more immune system proteins than healthy controls (ProtoArrays: 271/863 vs. 14/863, HuProt: 112/1694 vs. 39/1694, both p<0.0001). Overrepresentation was observed in all immune sub-pathways, including innate, adaptive immune responses, and cytokine signaling. Anti-FGFR3-positive SNN patients were more reactive with immune system proteins than negative ones. The independent SNN cohort validated the finding of overrepresentatively targeted immune system pathways. Validation with dot blot and ELISA confirmed reactivity to TRIM21 and IL-6, and identified anti-IFN-γ-positive SNN patients. IFN-γ levels correlated weakly with levels of anti-IFN-γ antibodies (Pearson’s r = 0.22, p=0.03). We conclude that the antibody repertoire of autoimmune SNN targets pathways of the innate and adaptative immune system, potentially reflecting key disease-related immune pathways and highlighting the systemic role of immune dysregulation in SNN.

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.

Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

Lee, Yeon-Ah; Hahm, Dae-Hyun; Kim, Jung Yeon; Sur, Bonjun; Lee, Hyun Min; Ryu, Chun Jeih; Yang, Hyung-In; Kim, Kyoung Soo
Arthritis Res Ther.
Oct 2018
10.1186/s13075-018-1736-3
Background Different adiponectin isoforms appear to be differentially involved in the pathogenesis of various diseases. The purpose of this study was to generate monoclonal antibodies (mAbs) specific to different adiponectin isoforms and investigate whether these mAbs have potential as therapeutic agents for such diseases. Methods Hybridoma cells producing monoclonal antibodies were generated and screened using enzyme-linked immunosorbent assay and Western blotting for the production of mAbs recognizing human adiponectin isoforms. Results The mAb from hybridoma clone KH7–41 recognized both the middle molecular weight (MMW) (hexamer) and low molecular weight (LMW) (trimer) isoforms of adiponectin in human serum, whereas the KH7–33 mAb detected only MMW (hexamer) adiponectin. The KH4–8 clone recognized both the high molecular weight (HMW) (multimer) and MMW adiponectin isoforms. However, in mouse and rat sera, the abovementioned antibodies recognized only the MMW isomer. These mAbs also recognized adiponectin in various human tissues, such as lung, kidney, and adipose tissues, although the three mAbs had different staining intensities. The mAb from clone KH4–8 effectively inhibited increases in interleukin-6 (IL-6) and IL-8 expression in recombinant adiponectin-stimulated human osteoblasts and human umbilical vein endothelial cells. Also, the mAbs KH7–33 and KH4–8 significantly ameliorated rheumatic symptoms in a collagen-induced arthritis mouse model. This result suggests that these mAb treatments may ameliorate adiponectin-mediated inflammatory response. Conclusions mAbs against human adiponectin isomers can potentially be developed as therapeutic antibodies to target specific detrimental isoforms of adiponectin while maintaining the functions of beneficial isoforms.

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