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

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Severity-dependent IgG epitope profiling in COVID-19 reveals differential recognition of pathogen-derived antigens

Do Nascimento, Lais Alves; Machado, NicolleRakanidis; Borges, João Vitor Da Silva; Fagundes, Beatriz Oliveira; Bergamasco, Isabella Siuffi; Sgnotto, Fabio Da Ressureição; Bachi, André Luis Lacerda; Sato, Maria Notomi; Victor, Jefferson Russo
Front. Immunol..
Sep 2025
10.3389/fimmu.2025.1668223
Background The contribution of antibody-mediated responses to COVID – 19 outcomes remains unclear, particularly regarding cross-reactivity with unrelated pathogens. While co-infections are known to influence disease progression, the broader landscape of IgG reactivity during SARS-CoV-2 infection has not been systematically explored. Methods We employed a high-density peptide microarray containing 4,344 linear epitopes from 37 viruses, 27 bacteria, 17 parasites, and 8 fungi to characterize serum IgG repertoires from individuals with moderate (n = 39) or severe (n = 40) COVID – 19. Controls included pre-pandemic healthy donors and a pooled intravenous immunoglobulin (IVIg) formulation. Data analysis included intensity ranking, epitope mapping, and comparative analysis of mean signal intensities for each epitope between the COVID-Mod and COVID-Sev groups. Results COVID – 19 patients showed widespread IgG reactivity against diverse pathogens, with patterns differing by disease severity. Severe cases displayed broader and more intense reactivity, notably against hepatitis C virus (HCV), SARS-CoV-1, influenza A, Mycobacterium tuberculosis, and Plasmodium falciparum. Moderate cases showed preferential recognition of epitopes from HTLV-I, Neisseria meningitidis, and Trypanosoma cruzi. These findings suggest that SARS-CoV-2 infection modulates pre-existing humoral memory, possibly through epitope spreading or immune reprogramming. Conclusions SARS-CoV-2 infection reshapes the IgG epitope repertoire in a severity-dependent manner, extending to antigens from unrelated pathogens. This phenomenon may reflect underlying immune dysregulation or idiotype-driven interactions. Comprehensive profiling of pathogen-related IgG responses may reveal potential biomarkers of disease severity. This phenomenon may inform future investigations aimed at improving personalized management strategies for co-infected or immunocompromised patients.

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.

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.

A tumor-binding antibody with cross-reactivity to viral antigens

Campa, Michael J.; Gottlin, Elizabeth B.; Wiehe, Kevin; Patz, Edward F.
Cancer Immunol Immunother.
Feb 2025
10.1007/s00262-025-03975-8
**Background** We previously identified in non-small cell lung cancer (NSCLC) patients an autoantibody to complement factor H (CFH) that is associated with non-metastatic disease and longer time to progression in patients with stage I disease. A recombinant human antibody, GT103, was cloned from single B cells isolated from patients with the autoantibody. GT103 inhibits tumor growth and establishes an antitumor microenvironment. The anti-CFH autoantibody and GT103 recognize the epitope PIDNGDIT within the SCR19 domain of CFH. Here, we asked if this autoantibody could have originally arisen as a humoral response to a similar epitope in a viral protein from a prior infection. **Methods** Homologous viral peptides with high sequence identity to the core PIDNGDIT epitope sequence were identified and synthesized. NSCLC patient plasma containing anti-CFH autoantibodies were assayed by ELISA against these peptides. GT103 was assayed on a 4345-peptide pathogen microarray. **Results** Epitopes similar to the GT103 epitope are present in several viruses, including human metapneumovirus-1 (HMPV-1) that contains a sequence within attachment glycoprotein G that differs by one amino acid. Anti-CFH autoantibodies in NSCLC patient plasma weakly bound to an HMPV-1 peptide containing the epitope. GT103 cross-reacted with multiple viral epitopes on a peptide microarray, with the top hits being peptides in the human endogenous retrovirus-K polymerase (HERV-K pol) protein and measles hemagglutinin glycoprotein. GT103 bound the viral HMPV-1, HERV-K pol, and measles epitope peptides but with lower affinity compared to the GT103 epitope peptide. **Conclusion** These findings suggest that memory B cells against a viral target could have affinity matured to produce an antibody that recognizes a similar epitope on tumor cells and exhibits antitumor properties.

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.

Investigation of Immunoreactivity Profiles and Epitope Landscape in Divergent COVID-19 Trajectories and SARS-CoV-2 Variants

Bihani, Surbhi; Ray, Arka; Borishetty, Dhanush; Tuckley, Chaitanya; Salkar, Akanksha; Acharjee, Arup; Shrivastav, Prithviraj; Shrivastav, Om; Shastri, Jayanthi; Agrawal, Sachee; Duttagupta, Siddhartha; Srivastava, Sanjeeva
J. Proteome Res..
Jan 2025
10.1021/acs.jproteome.4c00791
This study aimed to elucidate the complexity of the humoral immune response in COVID-19 patients with varying disease trajectories using a SARS-CoV-2 whole proteome peptide microarray chip. The microarray, containing 5347 peptides spanning the entire SARS-CoV-2 proteome and key variants of concern, was used to analyze IgG responses in 10 severe-to-recovered, 9 nonsevere-to-severe cases, and 10 control case (5 pre-pandemic and 5 SARS-CoV-2-negative) plasma samples. We identified 1151 IgG-reactive peptides corresponding to 647 epitopes, with 207 peptides being cross-reactive across 124 epitopes. Nonstructural protein 3 (nsp3) exhibited the highest number of total and unique epitopes, followed by the spike protein. nsp12 had the most number of cross-reactive epitopes. Peptides from the spike protein and nsps 2, 3, 5, and 13 were notably associated with recovery. Additionally, specific mutations in SARS-CoV-2 variants were found to alter peptide immunoreactivity, with some mutations (e.g., G142D, L452R, and N501Y) enhancing and others (e.g., R190S and E484 K) reducing immune recognition. These findings have critical implications for the development of diagnostics, vaccines, and therapeutics. Understanding the distribution of epitopes and the impact of viral mutations on antigenicity provides insights into immune evasion mechanisms, informing strategies for controlling COVID-19 and future coronavirus outbreaks.

High-throughput identification of immunoreactive peptides and corresponding proteins from Anaplasma platys and Ehrlichia canis using peptide microarray chips

Llanes, Alejandro; Madesh, Swetha; Brangulis, Kalvis; Rajeev, Sreekumari
Front Cell Infect Microbiol.
Jan 2025
10.3389/fcimb.2025.1671309
INTRODUCTION: Anaplasma platys and Ehrlichia canis are rickettsial pathogens infecting dogs, with a worldwide distribution. Both species are obligate intracellular pathogens and colonize bone marrow-derived cells, with coinfections frequently reported in dogs. Although E. canis immunodominant proteins have been thoroughly characterized, very few high-throughput studies have been conducted to identify immunogenic proteins from Anaplasma spp. In this study, we used a methodology based on peptide microarray chips to identify immunoreactive peptides, either shared or species-specific, in the complete theoretical proteomes of both pathogens. METHODS: B-cell epitopes were predicted in the corresponding proteins from both species and ranked for synthesis on the peptide microarrays. These microarrays were screened with serum samples from antibody-positive dogs, as well as negative control sera from unexposed dogs. Additionally, we assessed the feasibility of integrating evidence gathered at the level of individual peptides to identify potentially immunogenic proteins contributing to the patterns of immunoreactivity observed on microarrays. RESULTS: Screening of peptide microarrays resulted in complex antibody reactivity patterns against thousands of peptides. After discarding peptides with cross-reactivity to negative control sera, we identified over 1,200 immunoreactive peptides, including ~80 peptides shared between the two species with almost identical sequences. Despite screening linear peptides, we were able to identify proteins previously reported as immunodominant in E. canis, some of which contain predominantly conformational epitopes. DISCUSSION: Our results suggest that a high-throughput strategy based on peptide microarrays is an effective approach for the rapid identification of immunoreactive peptides and the underlying immunogenic proteins. This study provides a foundation for developing novel diagnostic tools and vaccine candidates against A. platys and E. canis, including potential combined or multivalent formulations targeting both pathogens.

Immunity to Influenza is dependent on MHC II polymorphism: study with 2 HLA transgenic strains

Luckey, David; Weaver, Eric A.; Osborne, Douglas G.; Billadeau, Daniel D.; Taneja, Veena
Sci Rep.
Dec 2019
10.1038/s41598-019-55503-1
Major histocompatibility complex II (MHC II) molecules are involved in antigen presentation and the development of a functional adaptive immune response. Evolutionary selection for MHC molecules that effectively clear infectious agents provides an advantage to humans. However, certain class II molecules are associated with autoimmune diseases. In this study we infected autoimmune-susceptible DRB1*0401.AEo and non-susceptible *0402.AEo mice with H1N1 influenza and determined clearance and protective immunity to H3N2 virus. *0401 mice generated a robust TLR-triggered immune response and cleared H1N1 influenza virus infection. After vaccination and challenge with H1N1, *0401 mice, when challenged with H3N2, generated cross-protective immunity to heterosubtypic H3N2 influenza strain whereas *0402 mice cleared the H1N1 infection but did not generate cross-protective immunity against the H3N2 influenza strain. The intracellular trafficking route of MHCII revealed that *0401 molecules traffic through the late endosome/lysosomes while *0402 molecules traffic into early endosomes. This suggested that trafficking of MHCII could affect the functional output of the innate immune response and clearance of viral infections. Also, DRB1*0401 mice live longer than HLA-DRB1*0402 mice. The study provides a potential hypothesis for evolutionary selection of *0401 molecule, even though it is associated with autoreactivity, which may be dependent on the availability of peptide repertoire of self-antigens.

Analysis of humoral immune responses in chikungunya virus (CHIKV) infected patients and individuals vaccinated with a candidate CHIKV vaccine

Henss, Lisa; Yue, Constanze; von Rhein, Christine; Tschismarov, Roland; Lewis-Ximenez, Lia Laura; Dölle, Albert; Baylis, Sally A; Schnierle, Barbara S
Dec 2019
10.1093/infdis/jiz658
Abstract Background Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe flu like symptoms. The acute symptoms disappear after one week, but chronic arthralgia can persist for years. Here, humoral immune responses in CHIKV-infected patients and vaccinees were analyzed. Methods Alphavirus neutralization activity was analyzed with pseudotyped lentiviral vectors and antibody epitope mapping was performed with a peptide array. Results Greatest CHIKV neutralization activity was observed 60-92 days after onset of symptoms. The amount of CHIKV-specific antibodies, their binding avidity and cross-reactivity with other alphaviruses increased over time. CHIKV and o’nyong-nyong virus (ONNV) were both neutralized to a similar extent. Linear antibody binding epitopes were mainly found in E2 domain B and the acid-sensitive regions (ASRs). In addition, serum samples from healthy volunteers vaccinated with a measles-vectored chikungunya vaccine candidate, MV-CHIK, were analyzed. Neutralization activity in the samples from the vaccine cohort was 2–6-fold lower than in samples from CHIKV-infected patients. In contrast to infection, vaccination only induced cross-neutralization with ONNV and the E2 ASR1 was the major antibody target. Conclusion These data could assist vaccine design and enable the identification of correlates of protection necessary for vaccine efficacy.

Diagnostic Profiling of the Human Public IgM Repertoire With Scalable Mimotope Libraries

Pashov, Anastas; Shivarov, Velizar; Hadzhieva, Maya; Kostov, Victor; Ferdinandov, Dilyan; Heintz, Karen-Marie; Pashova, Shina; Todorova, Milena; Vassilev, Tchavdar; Kieber-Emmons, Thomas; Meza-Zepeda, Leonardo A.; Hovig, Eivind
Front. Immunol..
Dec 2019
10.3389/fimmu.2019.02796
Specific antibody reactivities are routinely used as biomarkers, but the antibody repertoire reactivity (igome) profiles are still neglected. Here, we propose rationally designed peptide arrays as efficient probes for these system level biomarkers. Most IgM antibodies are characterized by few somatic mutations, polyspecificity, and physiological autoreactivity with housekeeping function. Previously, probing this repertoire with a set of immunodominant self-proteins provided a coarse analysis of the respective repertoire profiles. In contrast, here, we describe the generation of a peptide mimotope library that reflects the common IgM repertoire of 10,000 healthy donors. In addition, an appropriately sized subset of this quasi-complete mimotope library was further designed as a potential diagnostic tool. A 7-mer random peptide phage display library was panned on pooled human IgM. Next-generation sequencing of the selected phage yielded 224,087 sequences, which clustered in 790 sequence clusters. A set of 594 mimotopes, representative of the most significant sequence clusters, was shown to probe symmetrically the space of IgM reactivities in patients’ sera. This set of mimotopes can be easily scaled including a greater proportion of the mimotope library. The trade-off between the array size and the resolution can be explored while preserving the symmetric sampling of the mimotope sequence and reactivity spaces. BLAST search of the non-redundant protein database with the mimotopes sequences yielded significantly more immunoglobulin J region hits than random peptides, indicating a considerable idiotypic connectivity of the targeted igome. The proof of principle predictors for random diagnoses was represented by profiles of mimotopes. The number of potential reactivity profiles that can be extracted from this library is estimated at more than 1070. Thus, a quasi-complete IgM mimotope library and a scalable representative subset thereof are found to address very efficiently the dynamic diversity of the human public IgM repertoire, providing informationally dense and structurally interpretable IgM reactivity profiles.

Predicting HBsAg clearance in genotype A chronic hepatitis B using HBsAg epitope profiling: A biomarker for functional cure

Walsh, Renae; Hammond, Rachel; Yuen, Lilly; Deerain, Joshua; O’Donnell, Tanya; Leary, Thomas; Cloherty, Gavin; Gaggar, Anuj; Kitrinos, Kathryn; Subramanian, Mani; Wong, Darren; Locarnini, Stephen
Liver Int.
Nov 2019
10.1111/liv.14207
Background and Aim Functional cure is the major goal of chronic hepatitis B (CHB) therapy though few biomarkers predict this outcome. HBsAg epitope occupancy can be influenced by therapeutic and immune pressure. The aim of this study was to map the HBsAg epitope profiles during long-term nucleos(t)ide analogue therapy in patients with genotype A CHB, in the context of HBsAg loss (SL)/seroconversion. Methods We evaluated 25 genotype A CHB patients in the GS-US-174-0103 trial of HBeAg-positive CHB patients treated with tenofovir or adefovir for 4 years, 14 who achieved SL whilst 11 had no change. We epitope mapped the major domains of HBsAg to identify those patients with HBsAg clearance profile (CP) (loss of binding at both loops 1 and 2 epitopes of the ‘a’ determinant) vs non-clearance profile (no change in epitope recognition, or loss of epitope binding at one loop only), correlating this to on-treatment HBsAg responses. Complexed anti-HBs was also measured. Results Analysis of the HBsAg epitope profiles of the 25 patients at baseline identified no predictive correlation with SL. In contrast, analysis at week 48 and end of study (week 192) or prior to SL identified significant predictive associations between development of HBsAg CPs and outcome of functional cure. The detection of a CP also correlated with the development of an alanine aminotransferase flare and detection of anti-HBs complexed with HBsAg. Conclusion The detection of HBsAg CPs by epitope mapping represents a novel viral biomarker, reflecting an emerging anti-HBs selection pressure prior to functional cure.

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