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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.

A Quantum Vaccinomics Approach for the Design and Production of MSP4 Chimeric Antigen for the Control of Anaplasma phagocytophilum Infections

de la Fuente, José; Moraga-Fernández, Alberto; Alberdi, Pilar; Díaz-Sánchez, Sandra; García-Álvarez, Olga; Fernández-Melgar, Rubén; Contreras, Marinela
Vaccines.
Nov 2022
10.3390/vaccines10121995
Anaplasma phagocytophilum Major surface protein 4 (MSP4) plays a role during infection and multiplication in host neutrophils and tick vector cells. Recently, vaccination trials with the A. phagocytophilum antigen MSP4 in sheep showed only partial protection against pathogen infection. However, in rabbits immunized with MSP4, this recombinant antigen was protective. Differences between rabbit and sheep antibody responses are probably associated with the recognition of non-protective epitopes by IgG of immunized lambs. To address this question, we applied quantum vaccinomics to identify and characterize MSP4 protective epitopes by a microarray epitope mapping using sera from vaccinated rabbits and sheep. The identified candidate protective epitopes or immunological quantum were used for the design and production of a chimeric protective antigen. Inhibition assays of A. phagocytophilum infection in human HL60 and Ixodes scapularis tick ISE6 cells evidenced protection by IgG from sheep and rabbits immunized with the chimeric antigen. These results supported that the design of new chimeric candidate protective antigens using quantum vaccinomics to improve the protective capacity of antigens in multiple hosts.

In silico and in vitro arboviral MHC class I-restricted-epitope signatures reveal immunodominance and poor overlapping patterns

Lopes-Ribeiro, Ágata; Araujo, Franklin Pereira; Oliveira, Patrícia de Melo; Teixeira, Lorena de Almeida; Ferreira, Geovane Marques; Lourenço, Alice Aparecida; Dias, Laura Cardoso Corrêa; Teixeira, Caio Wilker; Retes, Henrique Morais; Lopes, Élisson Nogueira; Versiani, Alice Freitas; Barbosa-Stancioli, Edel Figueiredo; da Fonseca, Flávio Guimarães; Martins-Filho, Olindo Assis; Tsuji, Moriya; Peruhype-Magalhães, Vanessa; Coelho-dos-Reis, Jordana Grazziela Alves
Front. Immunol..
Nov 2022
10.3389/fimmu.2022.1035515
Introduction The present work sought to identify MHC-I-restricted peptide signatures for arbovirus using in silico and in vitro peptide microarray tools. Methods First, an in-silico analysis of immunogenic epitopes restricted to four of the most prevalent human MHC class-I was performed by identification of MHC affinity score. For that, more than 10,000 peptide sequences from 5 Arbovirus and 8 different viral serotypes, namely Zika (ZIKV), Dengue (DENV serotypes 1-4), Chikungunya (CHIKV), Mayaro (MAYV) and Oropouche (OROV) viruses, in addition to YFV were analyzed. Haplotype HLA-A*02.01 was the dominant human MHC for all arboviruses. Over one thousand HLA-A2 immunogenic peptides were employed to build a comprehensive identity matrix. Intending to assess HLAA*02:01 reactivity of peptides in vitro, a peptide microarray was designed and generated using a dimeric protein containing HLA-A*02:01. Results The comprehensive identity matrix allowed the identification of only three overlapping peptides between two or more flavivirus sequences, suggesting poor overlapping of virus-specific immunogenic peptides amongst arborviruses. Global analysis of the fluorescence intensity for peptide-HLA-A*02:01 binding indicated a dose-dependent effect in the array. Considering all assessed arboviruses, the number of DENV-derived peptides with HLA-A*02:01 reactivity was the highest. Furthermore, a lower number of YFV-17DD overlapping peptides presented reactivity when compared to non-overlapping peptides. In addition, the assessment of HLA-A*02:01-reactive peptides across virus polyproteins highlighted non-structural proteins as “hot-spots”. Data analysis supported these findings showing the presence of major hydrophobic sites in the final segment of non-structural protein 1 throughout 2a (Ns2a) and in nonstructural proteins 2b (Ns2b), 4a (Ns4a) and 4b (Ns4b). Discussion To our knowledge, these results provide the most comprehensive and detailed snapshot of the immunodominant peptide signature for arbovirus with MHC-class I restriction, which may bring insight into the design of future virus-specific vaccines to arboviruses and for vaccination protocols in highly endemic areas.

Binding epitope for recognition of human TRPM4 channel by monoclonal antibody M4M

Wei, Shunhui; Behn, Julian; Poore, Charlene Priscilla; Low, See Wee; Nilius, Bernd; Fan, Hao; Liao, Ping
Sci Rep.
Nov 2022
10.1038/s41598-022-22077-4
Abstract Mouse monoclonal antibody M4M was recently designed to block human TRPM4 channel. The polypeptide for generating M4M is composed of peptide A1 between the transmembrane segment 5 (S5) and the pore, and a second peptide A2 between the pore and the transmembrane segment 6 (S6). Using peptide microarray, a 4-amino acid sequence EPGF within the A2 was identified to be the binding epitope for M4M. Substitution of EPGF with other amino acids greatly reduced binding affinity. Structural analysis of human TRPM4 structure indicates that EPGF is located externally to the channel pore. A1 is close to the EPGF binding epitope in space, albeit separated by a 37-amino acid peptide. Electrophysiological study reveals that M4M could block human TRPM4, but with no effect on rodent TRPM4 which shares a different amino acid sequence ERGS for the binding motif. Our results demonstrate that M4M is a specific inhibitor for human TRPM4.

Antibody Response to HML ‐2 May Be Protective in Amyotrophic Lateral Sclerosis

Garcia‐Montojo, Marta; Simula, Elena Rita; Fathi, Saeed; McMahan, Cynthia; Ghosal, Anubrata; Berry, James D.; Cudkowicz, Merit; Elkahloun, Abdel; Johnson, Kory; Norato, Gina; Jensen, Peter; James, Tony; Sechi, Leonardo A.; Nath, Avindra
Annals of Neurology.
Nov 2022
10.1002/ana.26466

Peptide microarray analysis of in-silico predicted B-cell epitopes in SARS-CoV-2 sero-positive healthcare workers in Bulawayo, Zimbabwe

Vengesai, Arthur; Naicker, Thajasvarie; Midzi, Herald; Kasambala, Maritha; Muleya, Victor; Chipako, Isaac; Choto, Emilia; Moyo, Praise; Mduluza, Takafira
Acta Tropica.
Nov 2022
10.1016/j.actatropica.2022.106781

Vivaxin genes encode highly immunogenic, non-variant antigens on the Trypanosoma vivax cell-surface

Romero-Ramirez, Alessandra; Casas-Sánchez, Aitor; Autheman, Delphine; Duffy, Craig W.; Brandt, Cordelia; Clare, Simon; Harcourt, Katherine; André, Marcos Rogério; de Almeida Castilho Neto, Kayo José Garcia; Teixeira, Marta M. G.; Machado, Rosangela Zacharias; Coombes, Janine; Flynn, Robin J.; Wright, Gavin J.; Jackson, Andrew P.
PLoS Negl Trop Dis.
Sep 2022
10.1371/journal.pntd.0010791
Trypanosoma vivax is a unicellular hemoparasite, and a principal cause of animal African trypanosomiasis (AAT), a vector-borne and potentially fatal livestock disease across sub-Saharan Africa. Previously, we identified diverse T. vivax-specific genes that were predicted to encode cell surface proteins. Here, we examine the immune responses of naturally and experimentally infected hosts to these unique parasite antigens, to identify immunogens that could become vaccine candidates. Immunoprofiling of host serum shows that one particular family (Fam34) elicits a consistent IgG antibody response. This gene family, which we now call Vivaxin, encodes at least 124 transmembrane glycoproteins that display quite distinct expression profiles and patterns of genetic variation. We focused on one gene (viv-β8) that encodes one particularly immunogenic vivaxin protein and which is highly expressed during infections but displays minimal polymorphism across the parasite population. Vaccination of mice with VIVβ8 adjuvanted with Quil-A elicits a strong, balanced immune response and delays parasite proliferation in some animals but, ultimately, it does not prevent disease. Although VIVβ8 is localized across the cell body and flagellar membrane, live immunostaining indicates that VIVβ8 is largely inaccessible to antibody in vivo. However, our phylogenetic analysis shows that vivaxin includes other antigens shown recently to induce immunity against T. vivax. Thus, the introduction of vivaxin represents an important advance in our understanding of the T. vivax cell surface. Besides being a source of proven and promising vaccine antigens, the gene family is clearly an important component of the parasite glycocalyx, with potential to influence host-parasite interactions.

Anti-neuronal antibodies against brainstem antigens are associated with COVID-19

Lucchese, Guglielmo; Vogelgesang, Antje; Boesl, Fabian; Raafat, Dina; Holtfreter, Silva; Bröker, Barbara M.; Stufano, Angela; Fleischmann, Robert; Prüss, Harald; Franke, Christiana; Flöel, Agnes
eBioMedicine.
Sep 2022
10.1016/j.ebiom.2022.104211
Background Understanding how SARS-CoV-2 affects respiratory centres in the brainstem may help to preclude assisted ventilation for patients in intensive care setting. Viral invasion appears unlikely, although autoimmunity has been implicated, the responsible antigens remain unknown. We previously predicted the involvement of three epitopes within distinct brainstem proteins: disabled homolog 1 (DAB1), apoptosis-inducing-factor-1 (AIFM1), and surfeit-locus-protein-1 (SURF1). Methods Here, we used microarrays to screen serum from COVID-19 patients admitted to intensive care and compared those with controls who experienced mild course of the disease. Findings The results confirm the occurrence of IgG and IgM antibodies against the hypothesised epitopes in COVID-19 patients. Importantly, while IgM levels were similar in both groups, IgG levels were significantly elevated in severely ill patients compared to controls, suggesting a pathogenic role of IgG. Interpretation The newly discovered anti-neuronal antibodies might be promising markers of severe disease and the targeted peptide epitopes might be used for targeted immunomodulation. Further work is needed to determine whether these antibodies may play a role in long-COVID.

Identification of Equine Arteritis Virus Immunodominant Epitopes Using a Peptide Microarray

Mayers, Jo; Westcott, David; Steinbach, Falko
Viruses.
Aug 2022
10.3390/v14091880
Using the commercially available PEPperCHIP® microarray platform, a peptide microarray was developed to identify immunodominant epitopes for the detection of antibodies against Equine arteritis virus (EAV). For this purpose, the whole EAV Bucyrus sequence was used to design a total of 1250 peptides that were synthesized and spotted onto a microarray slide. A panel of 28 serum samples representing a selection of EAV strains was tested using the microarray. Of the 1250 peptides, 97 peptides (7.76%) showed reactivity with the EAV-positive samples. No single peptide was detected by all the positive serum samples. Seven peptides repeatedly showed reactivity above the cut-off and were considered to have diagnostic potential. Five of these peptides were within the immunodominant GP5 protein and two were within the replicase polyprotein regions NSP2 and NSP10, located in ORF1. The diagnostic sensitivity of the seven peptides selected was low, ranging from 5% to 55%; however, the combined diagnostic sensitivity and specificity of the seven peptides was 90% and 100%, respectively. This data demonstrate that multiple peptide sequences would be required to design a comprehensive serological test to cover the diversity of the EAV strains and the individual immune responses of horses.

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