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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.

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.

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.

Monoclonal antibodies to HLA-E bind epitopes carried by unfolded β 2 m-free heavy chains: Molecular immunology

Tremante, Elisa; Lo Monaco, Elisa; Ingegnere, Tiziano; Sampaoli, Camilla; Fraioli, Rocco; Giacomini, Patrizio
Eur. J. Immunol..
Aug 2015
10.1002/eji.201545446
Since HLA-E heavy chains accumulate free of their light β2-microglobulin (β2m) subunit, raising mAbs to folded HLA-E heterodimers has been difficult, and mAb characterization has been controversial. Herein, mAb W6/32 and 5 HLA-E-restricted mAbs (MEM-E/02, MEM-E/07, MEM-E/08, DT9, and 3D12) were tested on denatured, acid-treated, and natively folded (both β2m-associated and β2m-free) HLA-E molecules. Four distinct conformations were detected, including unusual, partially folded (and yet β2m-free) heavy chains reactive with mAb DT9. In contrast with previous studies, epitope mapping and substitution scan on thousands of overlapping peptides printed on microchips revealed that mAbs MEM-E/02, MEM-E/07, and MEM-E/08 bind three distinct α1 and α2 domain epitopes. All three epitopes are linear since they span just 4–6 residues and are “hidden” in folded HLA-E heterodimers. They contain at least one HLA-E-specific residue that cannot be replaced by single substitutions with polymorphic HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G residues. Finally, also the MEM-E/02 and 3D12 epitopes are spatially distinct. In summary, HLA-E-specific residues are dominantly immunogenic, but only when heavy chains are locally unfolded. Consequently, the available mAbs fail to selectively bind conformed HLA-E heterodimers, and HLA-E expression may have been inaccurately assessed in some previous oncology, reproductive immunology, virology, and transplantation studies.

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