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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.

HSP70iQ435A to subdue autoimmunity and support anti-tumor responses

Jaishankar, Dinesh; Cosgrove, Cormac; Ramesh, Prathyaya; Mahon, James; Shivde, Rohan; Dellacecca, Emilia R.; Yang, Shiayin F.; Mosenson, Jeffrey; Guevara-Patiño, José A.; Le Poole, I. Caroline
Cell Stress and Chaperones.
Sep 2021
10.1007/s12192-021-01229-x
Developing immunosuppressive therapies for autoimmune diseases comes with a caveat that immunosuppression may promote the risk of developing other conditions or diseases. We have previously shown that biolistic delivery of an expression construct encoding inducible HSP70 (HSP70i) with one amino acid modification in the dendritic cell (DC) activating moiety 435–445 (HSP70iQ435A) to mouse skin resulted in significant immunosuppressive activity of autoimmune vitiligo, associated with fewer tissue infiltrating T cells. To prepare HSP70iQ435A as a potential therapeutic for autoimmune vitiligo, in this study we evaluated whether and how biolistic delivery of HSP70iQ435A in mice affects anti-tumor responses. We found that HSP70iQ435A in fact supports anti-tumor responses in melanoma-challenged C57BL/6 mice. Biolistic delivery of the HSP70iQ435A-encoding construct to mice elicited significant anti-HSP70 titers, and anti-HSP70 IgG and IgM antibodies recognize surface-expressed and cytoplasmic HSP70i in human and mouse melanoma cells. A peptide scan revealed that the anti-HSP70 antibodies recognize a specific C-terminal motif within the HSP70i protein. The antibodies elicited surface CD107A expression among mouse NK cells, representative of antibody-mediated cellular cytotoxicity (ADCC), supporting the concept, that HSP70iQ435A-encoding DNA elicits a humoral response to the stress protein expressed selectively on the surface of melanoma cells. Thus, besides limiting autoimmunity and inflammation, HSP70iQ435A elicits humoral responses that limit tumor growth and may be used in conjunction with immune checkpoint inhibitors to not only control tumor but to also limit adverse events following tumor immunotherapy.

Location and expression kinetics of Tc24 in different life stages of Trypanosoma cruzi

Versteeg, Leroy; Adhikari, Rakesh; Poveda, Cristina; Villar-Mondragon, Maria Jose; Jones, Kathryn M.; Hotez, Peter J.; Bottazzi, Maria Elena; Tijhaar, Edwin; Pollet, Jeroen
PLoS Negl Trop Dis.
Sep 2021
10.1371/journal.pntd.0009689
Tc24-C4, a modified recombinant flagellar calcium-binding protein of Trypanosoma cruzi, is under development as a therapeutic subunit vaccine candidate to prevent or delay progression of chronic Chagasic cardiomyopathy. When combined with Toll-like receptor agonists, Tc24-C4 immunization reduces parasitemia, parasites in cardiac tissue, and cardiac fibrosis and inflammation in animal models. To support further research on the vaccine candidate and its mechanism of action, murine monoclonal antibodies (mAbs) against Tc24-C4 were generated. Here, we report new findings made with mAb Tc24-C4/884 that detects Tc24-WT and Tc24-C4, as well as native Tc24 in T. cruzi on ELISA, western blots, and different imaging techniques. Surprisingly, detection of Tc24 by Tc24-C/884 in fixed T. cruzi trypomastigotes required permeabilization of the parasite, revealing that Tc24 is not exposed on the surface of T. cruzi, making a direct role of antibodies in the induced protection after Tc24-C4 immunization less likely. We further observed that after immunostaining T. cruzi–infected cells with mAb Tc24-C4/884, the expression of Tc24 decreases significantly when T. cruzi trypomastigotes enter host cells and transform into amastigotes. However, Tc24 is then upregulated in association with parasite flagellar growth linked to re-transformation into the trypomastigote form, prior to host cellular escape. These observations are discussed in the context of potential mechanisms of vaccine immunity.

Serum Peptide Immunoglobulin G Autoantibody Response in Patients with Different Central Nervous System Inflammatory Demyelinating Disorders

Lee, Hye Lim; Park, Jin-Woo; Seok, Jin Myoung; Jeon, Mi Young; Kim, Hojin; Lim, Young-Min; Shin, Ha Young; Kang, Sa-Yoon; Kwon, Oh-Hyun; Lee, Sang-Soo; Seok, Hung Youl; Min, Ju-Hong; Lee, Sung-Hyun; Kim, Byung-Jo; Kim, Byoung Joon
Diagnostics.
Jul 2021
10.3390/diagnostics11081339
Previous efforts to discover new surrogate markers for the central nervous system (CNS) inflammatory demyelinating disorders have shown inconsistent results; moreover, supporting evidence is scarce. The present study investigated the IgG autoantibody responses to various viral and autoantibodies-related peptides proposed to be related to CNS inflammatory demyelinating disorders using the peptide microarray method. We customized a peptide microarray containing more than 2440 immobilized peptides representing human and viral autoantigens. Using this, we tested the sera of patients with neuromyelitis optica spectrum disorders (NMOSD seropositive, n = 6; NMOSD seronegative, n = 5), multiple sclerosis (MS, n = 5), and myelin-oligodendrocyte glycoprotein antibody-associated disease (MOGAD, n = 6), as well as healthy controls (HC, n = 5) and compared various peptide immunoglobulin G (IgG) responses between the groups. Among the statistically significant peptides based on the pairwise comparisons of IgG responses in each disease group to HC, cytomegalovirus (CMV)-related peptides were most clearly distinguishable among the study groups. In particular, the most significant differences in IgG response were observed for HC vs. MS and HC vs. seronegative NMOSD (p = 0.064). Relatively higher IgG responses to CMV-related peptides were observed in patients with MS and NMOSD based on analysis of the customized peptide microarray.

Landscape and selection of vaccine epitopes in SARS-CoV-2

Smith, Christof C.; Olsen, Kelly S.; Gentry, Kaylee M.; Sambade, Maria; Beck, Wolfgang; Garness, Jason; Entwistle, Sarah; Willis, Caryn; Vensko, Steven; Woods, Allison; Fini, Misha; Carpenter, Brandon; Routh, Eric; Kodysh, Julia; O’Donnell, Timothy; Haber, Carsten; Heiss, Kirsten; Stadler, Volker; Garrison, Erik; Sandor, Adam M.; Ting, Jenny P. Y.; Weiss, Jared; Krajewski, Krzysztof; Grant, Oliver C.; Woods, Robert J.; Heise, Mark; Vincent, Benjamin G.; Rubinsteyn, Alex
Genome Medicine.
Jun 2021
10.1186/s13073-021-00910-1
Early in the pandemic, we designed a SARS-CoV-2 peptide vaccine containing epitope regions optimized for concurrent B cell, CD4+ T cell, and CD8+ T cell stimulation. The rationale for this design was to drive both humoral and cellular immunity with high specificity while avoiding undesired effects such as antibody-dependent enhancement (ADE).

SARS-CoV-2 spike protein stabilized in the closed state induces potent neutralizing responses.

Carnell, George W.; Ciazynska, Katarzyna A.; Wells, David A.; Xiong, Xiaoli; Aguinam, Ernest T.; McLaughlin, Stephen H.; Mallery, Donna; Ebrahimi, Soraya; Ceron-Gutierrez, Lourdes; Asbach, Benedikt; Einhauser, Sebastian; Wagner, Ralf; James, Leo C.; Doffinger, Rainer; Heeney, Jonathan L.; Briggs, John A. G.
May 2021
10.1128/JVI.00203-21
The majority of SARS-CoV-2 vaccines in use or advanced development are based on the viral spike protein (S) as their immunogen. S is present on virions as pre-fusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described against both open and closed conformations. The long-term success of vaccination strategies depends upon inducing antibodies that provide long-lasting broad immunity against evolving SARS-CoV-2 strains. Here we have assessed the results of immunization in a mouse model using an S protein trimer stabilized in the closed state to prevent full exposure of the receptor binding site and therefore interaction with receptor. We compared this with other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induced a T cell response and long-lived, strongly neutralizing antibody responses against 2019 SARS-CoV-2 and variants of concern B.1.248 and B.1.351. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralizing responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, immune responses than open spikes, and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. Together with their improved stability and storage properties we suggest that closed spikes may be a valuable component of refined, next-generation vaccines. Importance Vaccines in use against SARS-CoV-2 induce immune responses against the spike protein. There is intense interest in whether the antibody response induced by vaccines will be robust against new variants, as well as in next-generation vaccines for use in previously infected or immunized individuals. We assessed the use as an immunogen of a spike protein engineered to be conformationally stabilized in the closed state where the receptor binding site is occluded. Despite occlusion of the receptor binding site, the spike induces potently neutralizing sera against multiple SARS-CoV-2 variants. Antibodies are raised against a different pattern of epitopes to those induced by other spike constructs, preferring conformational epitopes present in the closed conformation. Closed spikes, or mRNA vaccines based on their sequence, can be a valuable component of next generation vaccines.

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