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

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Coupling Far-Western Blotting with Peptide Microarrays Reveals Novel E-Cadherin Spore-Surface Ligands in Clostridioides difficile

Lopez-Garcia, Osiris K.; Pizarro-Guajardo, Marjorie; Kocurek, Klaudia I.; Rezenom, Yohannes H.; Paredes-Sabja, Daniel
J Proteome Res.
Jun 2026
10.1021/acs.jproteome.5c01166
Clostridioides difficile is an anaerobic spore-forming bacterium and the leading cause of nosocomial diarrhea. A major clinical challenge is C. difficile infection recurrence, affecting 20–30% of patients, mainly driven by spore persistence. The mechanisms underlying spore persistence in the gut remain poorly understood. Recently, our group showed that E-cadherin acts as a spore receptor mediating adherence to intestinal epithelial cells (PMID: 36448839), but the identity of the E-cadherin-binding proteins remains unknown. Here, far-Western blotting coupled with MS/MS-identified E-cadherin-binding spore ligands. The spore surface proteins CotE (CDIF27147_01458) and CdeM (CDIF27147_01682), along with two uncharacterized proteins, CDIF27147_03838 and CDIF27147_02282, interact with E-cadherin. Peptide microarray analysis mapped discrete E-cadherin-binding regions within these proteins, corresponding to 9–20 residue motifs, predicted to be surface-exposed by AlphaFold. However, competitive E-cadherin pull-down assays using synthetic peptides of these motifs did not reduce E-cadherin binding to C. difficile spores. Comparative genomics showed that these ligands and motifs are conserved across all five classical C. difficile clades (C1–C5). Similar levels of E-cadherin binding were observed in spores from all five classical clades. Collectively, this work identifies CDIF27147_03838 and CDIF27147_02282 as novel E-cadherin ligands and suggests additional roles for CotE and CdeM, expanding insights into spore-host interactions.

Epitope mapping of humoral immunogenicity of orvacabtagene autoleucel shows an IgM response with minimal impact on CAR T cellular kinetics

Liu, Xianghong; Hu, Hongxiang; Dai, Yanshan; Pazos, Michael; Gokemeijer, Jochem; Ogasawara, Ken; Stoevesandt, Oda; Stadler, Volker; Mora, Johanna; Jawa, Vibha
Mol Ther Adv.
May 2026
10.1016/j.omta.2026.201763
Orvacabtagene autoleucel (orva-cel) is a fully human B cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T cell therapy evaluated in a phase 1/2 study in patients with relapsed or refractory multiple myeloma (RRMM). To assess treatment-related immunogenicity, anti-CAR therapeutic domain-specific antibodies (ATAs) were monitored in 157 treated patients. The ATAs were detected in 44.6% of patients over the course of study, with titers and incidence increasing over time. The goal of this study was to further characterize the observed immune response. The ATA status did not affect CAR T cell expansion or patient survival outcomes, though reduced persistence was observed in ATA-positive patients. Comprehensive immune profiling—including isotype analysis and B cell epitope mapping—identified five immunodominant consensus peptide sequences within the CAR domain. These epitopes were targeted by both Immunoglobulin G (IgG) and Immunoglobulin M (IgM) isotypes, with a persistent IgM response detected in most ATA-positive individuals. Despite the presence of ATAs, no adverse impact on cellular expansion was observed, potentially due to lymphodepletion and baseline immune suppression characteristic of B cell malignancies. These data suggest that the limited functional T- and B-cell capacity in RRMM may attenuate the clinical consequences of ATA development. The in vitro immunogenicity risk assessment and epitope mapping identified immunogenic hotspots within the CAR structure, which could have led to the high incidence of immune response observed in the patients. However, the analysis from this study points to a weak clinically non-relevant nature of the response that could be attributed to the patient’s immune status and diseased state.

Characterization of antibodies against the replication protein (Rep) encoded by bovine meat and milk factors (BMMFs)

Frehtman, Veronika; Shukla, Gunjan; Gentz, Michael; Müller, Marcus; Duduyemi, Oladimeji Paul; Grewe, Imke; Ernst, Claudia; Tessmer, Claudia; Didier, Andrea; Hofmann, Ilse; Bund, Timo; Leuchs, Barbara
Appl Microbiol Biotechnol.
Apr 2026
10.1007/s00253-026-13809-x
Bovine Meat and Milk Factors (BMMFs) are DNA elements with similarity to bacterial plasmids, are frequently identified in bovine meat and milk and were proposed to contribute to cancer development. All known BMMFs encode a conserved replication protein (Rep), allowing for histologic BMMF detection in clinical specimens based on Rep-directed mouse monoclonal antibodies (mAbs), which, however, have only been partially characterized so far. Here, 20 anti-BMMF Rep antibodies were assessed for biophysical properties, reactivity, specificity and binding sensitivity to five distinct BMMF Reps and other prokaryotic/eukaryotic target antigens using an enzyme-linked immunosorbent assay (ELISA)-based anti-BMMF Rep antibody binding assay. We demonstrated sensitive and specific antibody reaction with their respective Rep targets, according to the antibody immunization. Consensus antibodies raised against defined peptides of conserved Rep amino acid stretches interacted with most of the Rep antigens. Antibodies produced based on immunization with the Rep encoded on the BMMF isolate H1MSB.1, including rabbit and human chimeric variants, reacted only with the cognate H1MSB.1 Rep, with only two outliers targeting additional Reps. Completely new antibodies raised against the Rep of another isolate (C1HB.4) specifically detected the cognate C1HB.4 Rep antigen – not interacting with other Reps. New antibodies generated by triple Rep immunization (H1MSB.2/C1MI.3M.1/C1MI.9M.1 Rep) reacted to either all three or two immunization antigens without interacting with any other Reps. None of the antibodies cross-reacted against Reps of bacteria occurring during milk production or lysates of mammalian hosts. Competitive inhibition confirmed antigen-specificity across the antibody panel, which additionally did not show aberrancies concerning purity or antibody size for the majority of the tested Abs. These findings authenticate a highly specific panel of anti-BMMF Rep antibodies, which can serve as tools for BMMF detection in cancer and chronic diseases.**Key Points** • Anti-BMMF Rep antibodies are important to judge BMMFs’ role as cancer risk factors. • Selective binding of anti-BMMF Rep antibodies to BMMF Rep antigens. • No cross-reactivity of anti-BMMF Rep antibodies with bacterial and mammalian outgroup specimens.

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.

Identification of a conformational epitope on the E antigen implicated in anti-E alloimmunization

Matsuura, Hideaki; Yamada, Ayuna; Doi, Hiroki; Fujii, Sumie; Miura, Yasuo
Blood Adv.
Mar 2026
10.1182/bloodadvances.2025018046

Selective Targeting of Tip Endothelial Cells as a Therapeutic Strategy for Tumor Angiogenesis

Kim, Byoungmo; Lee, Ha Kyeong; Azam, Zulfikar; Choi, Jeong Uk; Wahab, Riajul; Lee, Na Kyeong; Ko, Yoon Gun; Choi, So‐Young; Lee, Se‐Ra; Shim, Wan Seob; Kim, Taeeung; Kim, In‐San; Alam, Farzana; Kim, Sang Yoon; Kim, Seong Who; Byun, Youngro; Al‐Hilal, Taslim A
Advanced Science.
Mar 2026
10.1002/advs.202512975
ABSTRACT Tip endothelial cells (TipEC), the leading edge of angiogenic sprouts, are essential for pathological neo‐vascularization but remain difficult to target due to the lack of specific druggable markers. Here, we identify Doppel as a selective and druggable regulator of endothelial tip cell function. Doppel expression enhances TipEC selection, directional migration, and regulates tip‐stalk cell dynamics by spatially controlling VEGFR2/Dll4/Src pathway. Genetic ablation of PRND (Doppel) reduces tip cell formation without affecting the stalk cells (StalkECs) number in tumors, indicating its selective role in TipECs. Importantly, depletion of TipECs using the first‐in‐class monoclonal antibodies against a highly conserved WQF‐motif of Doppel robustly decreased the growth of tumors by selectively downregulating VEGFR2+ TipECs but not StalkECs. These findings position Doppel as a tumor TipEC‐specific, druggable target that may offer a new avenue to enhance and refine anti‐angiogenic therapies in cancer treatment.

Clinical outcomes-dependent IgG epitope profiling in HTLV-1 reveals differential recognition of pathogen-derived antigens

Cilento, Natali Espasiani; Borges, João Vitor Da Silva; Machado, Nicolle Rakanidis; Do Nascimento, Lais Alves; Moreira, Anna Luisa Baratelli; Passos, Lhays Ozório; Santamarina, Aline Boveto; Casseb, Jorge; Sanabani, Sabri Saeed; Victor, Jefferson Russo
Front. Immunol..
Feb 2026
10.3389/fimmu.2026.1755133
Human T-lymphotropic virus type 1 (HTLV-1) infection presents a wide clinical spectrum ranging from lifelong asymptomatic carriage to severe inflammatory neurodegeneration (HAM/TSP) or adult T-cell leukemia/lymphoma (ATLL). Although IgG responses contribute to viral control and immunopathology, the extent to which HTLV-1 clinical outcomes shape pathogen-derived IgG repertoires remains unclear. In this study, we applied a high-density infectious-disease epitope microarray containing 4,345 linear epitopes from viral, bacterial, parasitic, and fungal pathogens to profile IgG responses in healthy controls (HCs), asymptomatic carriers (ACs), HAM/TSP patients, and ATLL patients. Signal intensities were quantified in arbitrary units, and recognized epitopes were evaluated using similarity clustering (80% identity threshold) to assess repertoire structure. HTLV-1–infected individuals exhibited extensive remodeling of humoral immunity, with marked differences in the breadth and intensity of IgG recognition across clinical groups. HAM/TSP patients displayed broad and high-magnitude responses consistent with chronic inflammation and heightened Th1 activation, whereas ATLL patients recognized the largest number of epitopes but with distinct patterns indicative of altered B-cell regulation. Enhanced IgG responses to Mycobacterium tuberculosis, Strongyloides stercoralis, Toxoplasma gondii, and Plasmodium species were consistent with known co-infection susceptibilities in HTLV-1. Epitope similarity analysis revealed hundreds of low-redundancy clusters across all groups, arguing against simple linear cross-reactivity and suggesting phenotype-specific reshaping of B-cell selection and idiotypic networks. These findings demonstrate that HTLV-1 infection produces distinct, clinically dependent IgG epitope signatures across multiple pathogen classes, with potential relevance for understanding HTLV-1 pathogenesis and informing future studies integrating epitope mapping with B-cell repertoire analysis.

Syndecan-1-targeted therapeutic antibody impairs macropinocytosis and elicits antitumor immunity in pancreatic cancer

Yang, Zecheng; Theardy, Madelaine S.; Chen, Shuaitong; Wei, Yongkun; Takeda, Mitsunobu; Zeng, Yue; Wang, Xiaofei; Yao, Jun; Li, Jennifer; Thirasastr, Prapassorn; Park, Jangho; Zheng, Yangxi; Vien, Long T.; Wani, Khalida M.; Wang, Huamin; Gao, Sisi; Heffernan, Tim; Kwong, Lawrence; Wistuba, Ignacio I.; Bover, Laura; Draetta, Giulio F.; Ying, Haoqiang; Yao, Wantong
Cell Reports Medicine.
Feb 2026
10.1016/j.xcrm.2026.102613
Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies, with a 5-year survival rate of just 13%. While the development and early clinical use of small molecules targeting oncogenic KRAS mutations, key drivers of PDAC, have shown promise, resistance to these targeted therapies remains a significant challenge. We recently identified Syndecan-1 (SDC1), a highly expressed heparan sulfate proteoglycan, as a critical KRAS effector protein that promotes nutrient salvage and tumor growth. Here, we report the development of a human-specific monoclonal antibody (anti-SDC1 mAb) that inhibits PDAC cell proliferation in vitro and suppresses PDAC tumor growth in vivo. Mechanistically, the anti-SDC1 mAb blocks macropinocytosis and induces antibody-dependent cellular cytotoxicity (ADCC). In vivo, anti-SDC1 mAb synergizes with standard chemotherapy, KRAS∗ inhibitors, and immunotherapies, resulting in tumor regression and near-complete response. These findings highlight the anti-SDC1 mAb as a promising therapeutic strategy for PDAC and potentially other KRAS∗ and SDC1-driven tumors.

HIV-1 gp140 epitope recognition is influenced by immunoglobulin DH gene segment sequence

Wang, Yuge; Kapoor, Pratibha; Parks, Robert; Silva-Sanchez, Aaron; Alam, S. Munir; Verkoczy, Laurent; Liao, Hua-Xin; Zhuang, Yingxin; Burrows, Peter; Levinson, Michael; Elgavish, Ada; Cui, Xiangqin; Haynes, Barton F.; Schroeder, Harry
Immunogenetics.
Feb 2026
10.1007/s00251-015-0890-x

Non-Neutralizing Antibody Functions Predict Susceptibility to SARS-CoV-2 Infection after mRNA Booster Vaccination

Levy, Shlomia; Trifkovic, Sanja; Mielke, Dieter; Oppenheimer, Hannah; Goodman, Derrick; Ostrovsky, Daniel; Sanfield-Oakley, Sherry A.; Brackett, Caroline; Friedman, Lilach M; Kerkau, Melissa; Webby, Richard; Tomaras, Georgia; Guido, Ferrari; Nesher, Lior; Hertz, Tomer
Jan 2026
10.2139/ssrn.6019238
Previous studies have shown that neutralizing and binding antibody titers are correlates of protection for symptomatic SARS-CoV-2 infection. We previously reported that individuals with low IgG and IgA baseline immune history (BIH) to SARS-CoV-2 variants were at increased risk of symptomatic infection in study of healthcare workers that received 3 or 4 doses of the Pfizer BNT-1262b2 vaccine. We also found that 1-month post-vaccination with the 4th booster dose, individuals with low-BIH mounted significant rises in binding and neutralizing antibody titers, to levels comparable to those of individuals with high-BIH, demonstrating that their increased risk was not due to inability to respond to vaccination. To further study the underlying factors that are associated with increased risk, we conducted a systems serology study of 40 low-BIH and 40 high-BIH individuals across 7 months of follow-up. We found that individuals with low BIH exhibited a significantly higher risk of symptomatic infection (HR=2.691, p=0.0065) and mounted weaker IgA and antibody-dependent cellular cytotoxicity (ADCC) responses compared to high-BIH individuals, particularly against Omicron and Delta variants. Baseline levels of the chemokine CXCL-11 were elevated in the low-BIH group. We then showed that baseline immune profiles can be used to train a prediction model of infection risk across 7 months of follow-up with 76% accuracy. IgA, ADCC and ADCP baseline features were dominant predictors of susceptibility. Our findings suggest that non-neutralizing antibody functions, especially IgA and ADCC, contribute to protection against symptomatic SARS-CoV-2 infection and that serology-guided stratification can enhance discovery of immune correlates of risk informing future vaccine design and deployment strategies.

Circulating Autoantibodies Targeting TREK-1 in Patients With Short-Coupled Ventricular Fibrillation

Li, Jin; Janin, Alexandre; Patoughi, Mona; Gaudreault, Nathalie; Kis, Lenke; Moha Ou Maati, Hamid; Bossé, Yohan; Steinberg, Christian
Circulation.
Dec 2024
10.1161/CIRCULATIONAHA.124.070284
*BACKGROUND* Short-coupled ventricular fibrillation (SCVF) is increasingly being recognized as a distinct primary electrical disorder and cause of otherwise unexplained cardiac arrest. However, the pathophysiology of SCVF remains largely elusive. Despite extensive genetic screening, there is no convincing evidence of a robust monogenic disease gene, thus raising the speculations for alternative pathogeneses. The role of autoimmune mechanisms in SCVF has not been investigated so far. The objective of this study was to screen for circulating autoantibodies in patients with SCVF and assess their role in arrhythmogenesis. *METHODS* This is a prospective, single-center, case-control study enrolling cardiac arrest survivors diagnosed with SCVF or idiopathic ventricular fibrillation (IVF) between 2019 and 2023 at the Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval Inherited Arrhythmia Clinic in Canada. Plasma samples were screened for autoantibodies targeting cardiac ion channels using peptide microarray technology. Identified target autoantibodies were then purified from pooled plasma samples for subsequent cellular electrophysiological studies. *RESULTS* Fourteen patients with SCVF (n=4 [29% of patients] female patients; median age, 45 years [interquartile range: 36, 59]; n=14 [100% of patients] non-Hispanic White) and 19 patients with idiopathic ventricular fibrillation (n=8 [42%] female patients; median age, 49 years [38, 57]; n=19 [100%] non-Hispanic White) were enrolled in the study and compared with 38 (n=20 [53%] female subjects; median age, 45 years [29, 66]; n=36 [95%] non-Hispanic White) sex-, age- and ethnicity-matched healthy controls. During the study period, 11 (79%) SCVF probands experienced ventricular fibrillation recurrence after a median of 4.3 months (interquartile range, 0.3–20.7). Autoantibodies targeting cardiac TREK-1 (TWIK [tandem of pore-domains in a weakly inward rectifying potassium channel]–related potassium channel 1 were identified in 7 (50%) patients with SCVF (P=0.049). Patch clamp experiments demonstrated channel-activating properties of anti–TREK-1 autoantibodies that are antagonized by quinidine in both HEK293 cells and human induced pluripotent stem cell–derived cardiomyocytes. *CONCLUSIONS* Patients with SCVF harbor circulating autoantibodies against the cardiac TREK-1 channel. Anti–TREK-1 autoantibodies not only present the first reported biomarker for SCVF, but our functional studies also suggest a direct implication in the arrhythmogenesis of SCVF.

Naturally acquired IgG responses to Plasmodium falciparum do not target the conserved termini of the malaria vaccine candidate Merozoite Surface Protein 2

Zerebinski, Julia; Margerie, Lucille; Han, Nan Sophia; Moll, Maximilian; Ritvos, Matias; Jahnmatz, Peter; Ahlborg, Niklas; Ngasala, Billy; Rooth, Ingegerd; Sjöberg, Ronald; Sundling, Christopher; Yman, Victor; Färnert, Anna; Plaza, David Fernando
Front. Immunol..
Dec 2024
10.3389/fimmu.2024.1501700
Introduction Malaria remains a significant burden, and a fully protective vaccine against Plasmodium falciparum is critical for reducing morbidity and mortality. Antibody responses against the blood-stage antigen Merozoite Surface Protein 2 (MSP2) are associated with protection from P. falciparum malaria, but its extensive polymorphism is a barrier to its development as a vaccine candidate. New tools, such as long-read sequencing and accurate protein structure modelling allow us to study the genetic diversity and immune responses towards antigens from clinical isolates with unprecedented detail. This study sought to better understand naturally acquired MSP2-specific antibody responses. Methods IgG responses against recombinantly expressed full-length, central polymorphic regions, and peptides derived from the conserved termini of MSP2 variants sequenced from patient isolates, were tested in plasma from travelers with recent, acute malaria and from individuals living in an endemic area of Tanzania. Results IgG responses towards full MSP2 and truncated MSP2 antigens were variant specific. IgG antibodies in the plasma of first-time infected or previously exposed travelers did not recognize the conserved termini of expressed MSP2 variants by ELISA, but they bound 13-amino acid long linear epitopes from the termini in a custom-made peptide array. Alphafold3 modelling suggests extensive structural heterogeneity in the conserved termini upon antigen oligomerization. IgG from individuals living in an endemic region, many who were asymptomatically infected, did not recognize the conserved termini by ELISA. Discussion Our results suggest that responses to the variable regions are critical for the development of naturally acquired immunity towards MSP2.

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