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

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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
Abstract 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.

Systematic analysis of the RGS2 degron reveals characteristics of substrate recognition by the F-box protein FBXO44

McNabb, Harrison J.; Cho, Eugene; Pitman, Mary; Rushton, Phillip S.; Mobley, David; Sjögren, Benita
Journal of Biological Chemistry.
Nov 2025
10.1016/j.jbc.2025.110757
Regulator of G protein signaling 2 (RGS2) negatively modulates signaling downstream of G protein–coupled receptors by accelerating GTP hydrolysis at Gα subunits of heterotrimeric G proteins. Decreased RGS2 levels are implicated in numerous diseases, including cardiovascular disease and asthma. Thus, identifying selective means of enhancing RGS2 protein levels would be a viable therapeutic strategy. RGS2 is rapidly degraded through the ubiquitin–proteasomal pathway, and we previously identified F-box only protein 44 (FBXO44) as the substrate recognition component of the E3 ligase responsible for facilitating RGS2 degradation. As such, the RGS2–FBXO44 interaction is a potential target for pharmacological intervention. Detailed information on the FBXO44 recognition site (degron) in RGS2 will aid in structure-based small-molecule inhibitor design, as well as in identifying additional FBXO44 targets, which would help predict possible side effects of targeting this interaction. Thus, the goal of this study was to dissect the molecular properties for FBXO44 binding of the RGS2 degron. We used a peptide array utilizing systematic residue substitution, combined with AlphaFold modeling and molecular dynamics simulations, to identify several amino acid changes that altered binding both positively and negatively. Finally, we experimentally confirmed our results in cells through coimmunoprecipitation and proteasomal inhibition, using full-length RGS2. Altogether, these results provide structural insights into RGS2–FBXO44 binding, which will aid in structure-guided drug discovery efforts. It also provides a framework for building a consensus recognition motif for FBXO44, which could aid in identifying more substrates for this understudied F-box protein.

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.

Anti-allodynic effect of intrathecal antibodies against macrophage-inducible C-type lectin in spinal nerve ligation model in rat

Kang, Dong Ho; Kim, Woong Mo; Bae, Hong Beom; Yang, Jihoon; Choi, Jeong Il
Heliyon.
Nov 2024
10.1016/j.heliyon.2024.e40694
*Introduction* Macrophage-inducible C-type lectin (Mincle) has emerged as a potential contributor to neuropathic pain induction and neuroinflammatory responses within the spinal cord. Moreover, evidence suggests a close association between toll-like receptor (TLR) and Mincle expression in myeloid cells. This study evaluated the effectiveness of Mincle antibodies in neuropathic pain and identified the epitope of these antibodies. In addition, the mode of interaction between Mincle and TLR inhibition was explored using isobolographic analysis. *Methods* Three different Mincle antibodies and a specific TLR4 inhibitor (TAK-242) were intrathecally administered, and mechanical allodynia was evaluated using the von Frey test in a rat model of spinal nerve ligation (SNL). Isobolographic analysis was conducted on the effect of combination of TAK-242 and Mincle Ab. Microarray analysis examined the specific region of Mincle targeted by the antibodies. *Results* All Mincle antibodies and TAK-242 significantly alleviated mechanical allodynia in a dose-dependent manner. However, the maximal possible effects (MPE) produced by the antibodies ranged widely from 37.1% to 91.8%, comparable to that of TAK-242 (88.7%). The combination of TAK-242 and the antibody with the highest MPE resulted in an additive interaction for their anti-allodynic effects. Epitope mapping revealed that each antibody targeted the extracellular domain, with epitope lengths ranging from 5 to 15 amino acids. *Conclusions* The current study demonstrates the anti-allodynic effect of Mincle antibodies and additive interaction with TLR4 inhibition in spinal nerve ligation model, suggesting the potential of blocking of Mincle signaling with its antibodies as a novel treatment strategy for neuropathic pain.

Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

Chu, Xiaojie; Shin, Seungmin; Baek, Du-San; Zhang, Liyong; Conard, Alex; Shi, Megan; Kim, Ye-Jin; Adams, Cynthia; Hines, Maggie; Liu, Xianglei; Chen, Chuan; Sun, Zehua; Jelev, Dontcho V.; Mellors, John W.; Dimitrov, Dimiter S.; Li, Wei
mAbs.
Aug 2024
10.1080/19420862.2024.2387240
Prostate stem cell antigen (PSCA) is expressed in all stages of prostate cancer, including in advanced androgen-independent tumors and bone metastasis. PSCA may associate with prostate carcinogenesis and lineage plasticity in prostate cancer. PSCA is also a promising theranostic marker for a variety of other solid tumors, including pancreatic adenocarcinoma and renal cell carcinoma. Here, we identified a novel fully human PSCA antibody using phage display methodology. The structure-based affinity maturation yielded a high-affinity binder, F12, which is highly specific and does not bind to 6,000 human membrane proteins based on a membrane proteome array assay. F12 targets PSCA amino acids 63–69 as tested by the peptide scanning microarray, and it cross-reacts with the murine PSCA. IgG1 F12 efficiently internalizes into PSCA-expressing tumor cells. The antimitotic reagent monomethyl auristatin E (MMAE)-conjugated IgG1 F12 (ADC, F12-MMAE) exhibits dose-dependent efficacy and specificity in a human prostate cancer PC-3-PSCA xenograft NSG mouse model. This is a first reported ADC based on a fully human PSCA antibody and MMAE that is characterized in a xenograft murine model, which warrants further optimizations and investigations in additional preclinical tumor models, including prostate and other solid tumors.

Antigen-Heterologous Vaccination Regimen Triggers Alternate Antibody Targeting in SARS-CoV-2-DNA-Vaccinated Mice

Frische, Anders; Krogfelt, Karen Angeliki; Fomsgaard, Anders; Lassaunière, Ria
Vaccines.
Feb 2024
10.3390/vaccines12030218
An in-depth analysis of antibody epitopes following vaccination with different regimens provides important insight for developing future vaccine strategies. B-cell epitopes conserved across virus variants may be ideal targets for vaccine-induced antibodies and therapeutic drugs. However, challenges lie in identifying these key antigenic regions, and directing the immune system to target them. We previously evaluated the immunogenicity of two candidate DNA vaccines encoding the unmodified spike protein of either the SARS-CoV-2 Index strain or the Beta variant of concern (VOC). As a follow-on study, we characterized here the antibody binding profiles of three groups of mice immunized with either the DNA vaccine encoding the SARS-CoV-2 Index strain spike protein only, the Beta VOC spike protein only, or a combination of both as an antigen-heterologous prime-boost regimen. The latter induced an antibody response targeting overlapping regions that were observed for the individual vaccines but with additional high levels of antibody directed against epitopes in the SD2 region and the HR2 region. These heterologous-vaccinated animals displayed improved neutralization breadth. We believe that a broad-focused vaccine regimen increases neutralization breadth, and that the in-depth analysis of B-cell epitope targeting used in this study can be applied in future vaccine research.

Surveillance of SARS-CoV-2 immunogenicity: loss of immunodominant HLA-A*02-restricted epitopes that activate CD8+ T cells

Lopes-Ribeiro, Ágata; Oliveira, Patrícia De Melo; Retes, Henrique Morais; Barbosa-Stancioli, Edel Figueiredo; Da Fonseca, Flávio Guimarães; Tsuji, Moriya; Coelho-dos-Reis, Jordana Grazziela Alves
Front. Immunol..
Nov 2023
10.3389/fimmu.2023.1229712
Introduction and methods In this present work, coronavirus subfamilies and SARS-CoV-2 Variants of Concern (VOCs) were investigated for the presence of MHC-I immunodominant viral peptides using in silico and in vitro tools. Results In our results, HLA-A*02 haplotype showed the highest number of immunodominant epitopes but with the lowest combined prediction score. Furthermore, a decrease in combined prediction score was observed for HLA-A*02-restricted epitopes when the original strain was compared to the VOCs, indicating that the mutations on the VOCs are promoting escape from HLA-A2-mediated antigen presentation, which characterizes a immune evasion process. Additionally, epitope signature analysis revealed major immunogenic peptide loss for structural (S) and non-structural (ORF8) proteins of VOCs in comparison to the Wuhan sequence. Discussion These results may indicate that the antiviral CD8 + T-cell responses generated by original strains could not be sufficient for clearance of variants in either newly or reinfection with SARS-CoV-2. In contrast, N epitopes remain the most conserved and reactive peptides across SARS-CoV-2 VOCs. Overall, our data could contribute to the rational design and development of new vaccinal platforms to induce a broad cellular CD8 + T cell antiviral response, aiming at controlling viral transmission of future SARS-CoV-2 variants.

Anti-Toxin Responses to Natural Enterotoxigenic Escherichia coli (ETEC) Infection in Adults and Children in Bangladesh

Girardi, Petra; Bhuiyan, Taufiqur Rahman; Lundin, Samuel B.; Harutyunyan, Shushan; Neuhauser, Irene; Khanam, Farhana; Nagy, Gábor; Szijártó, Valéria; Henics, Tamás; Nagy, Eszter; Harandi, Ali M.; Qadri, Firdausi
Microorganisms.
Oct 2023
10.3390/microorganisms11102524
A sero-epidemiology study was conducted in Dhaka, Bangladesh between January 2020 and February 2021 to assess the immune responses to ETEC infection in adults and children. (1) Background: Enterotoxigenic Escherichia coli infection is a main cause of diarrheal disease in endemic countries. The characterization of the immune responses evoked by natural infection can guide vaccine development efforts. (2) Methods: A total of 617 adult and 480 pediatric diarrheal patients were screened, and 43 adults and 46 children (below 5 years of age) with an acute ETEC infection completed the study. The plasma samples were analyzed for antibody responses against the ETEC toxins. (3) Results: Heat-stable toxin (ST)-positive ETEC is the main cause of ETEC infection in adults, unlike in children in an endemic setting. We detected very low levels of anti-ST antibodies, and no ST-neutralizing activity. However, infection with ETEC strains expressing the heat-labile toxin (LT) induced systemic antibody responses in less than 25% of subjects. The antibody levels against LTA and LTB, as well as cholera toxin (CT), correlated well. The anti-LT antibodies were shown to have LT- and CT- neutralizing activity. The antibody reactivity against linear LT epitopes did not correlate with toxin-neutralizing activity. (4) Conclusions: Unlike LT, ST is a poor antigen and even adults have low anti-ST antibody levels that do not allow for the detection of toxin-neutralizing activity.

Epitope Mapping of BmpA and BBK32 Borrelia burgdorferi Sensu Stricto Antigens for the Design of Chimeric Proteins with Potential Diagnostic Value

Grąźlewska, Weronika; Holec-Gąsior, Lucyna; Sołowińska, Karolina; Chmielewski, Tomasz; Fiecek, Beata; Contreras, Marinela
ACS Infect. Dis..
Oct 2023
10.1021/acsinfecdis.3c00258

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