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

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Peptide Epitopes of NC16A BP180 in the Diagnostics of Bullous Pemphigoid

Lytton, Simon D.; Wagger, Christine; Meyersburg, Damian; Mussnig, Birgit; Lang, Roland; Maglie, Roberto; Anzengruber, Florian; Antiga, Emiliano; Hall, Russell P.; Bauer, Johann W.
JID Innovations.
Nov 2025
10.1016/j.xjidi.2025.100415

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.

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.

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

Linear epitope mapping in the E and NS1 proteins of dengue and Zika viruses: Prospection of peptides for vaccines and diagnostics

Aquino, Victor Hugo; Fumagalli, Marcilio J.; Silva, Angélica; De Moura Negrini, Bento Vidal; Rojas, Alejandra; Guillen, Yvalena; Bernal, Cynthia; Figueiredo, Luiz Tadeu Moraes
PLoS ONE.
Oct 2023
10.1371/journal.pone.0292451
The arrival of the Zika virus (ZIKV) in dengue virus (DENV)-endemic areas has posed challenges for both differential diagnosis and vaccine development. Peptides have shown promise in addressing these issues. The aim of this study was to identify the linear epitope profile recognized by serum samples from dengue and Zika patients in the E and NS1 proteins of DENV and ZIKV. This cross-sectional study included individuals of all ages with laboratory-confirmed DENV and ZIKV infections, who were selected through convenience sampling. The serum samples from dengue and Zika patients detected epitopes evenly distributed across the viral proteins in a peptide microarray platform. However, several epitopes were located within “epitope hotspots”, characterized by clusters of peptides recognized in more than 30% of the sub-arrays analyzed using individual or pooled serum samples. The serum samples from dengue and Zika patients showed a high level of cross-reactivity with peptides in the DENV and ZIKV proteins. Analysis using an additional peptide microarray platform, which contained peptides selected based on the results of the initial screening, revealed that two DENV and one ZIKV peptide, highly specific to their related viruses, were located within the epitope hotspots; however, they presented low detection rates (32.5, 35.0, and 28.6%, respectively). In addition, two DENV peptides detected at similarly high rates by both dengue and Zika patients were also found within the epitope hotspots. These hotspots contain several immunodominant epitopes that are recognized by a larger number of individuals when compared to 15-amino acid (aa) sequence peptides. Thus, epitope hotspots may have greater potential to serve as antigens in diagnostic tests and vaccine development than peptides composed of only 15 amino acids.

Novel anti-CD30/CD3 bispecific antibodies activate human T cells and mediate potent anti-tumor activity

Faber, Mary L.; Oldham, Robyn A. A.; Thakur, Archana; Rademacher, Mary Jo; Kubicka, Ewa; Dlugi, Theresa A.; Gifford, Steven A.; McKillop, William M.; Schloemer, Nathan J.; Lum, Lawrence G.; Medin, Jeffrey A.
Front. Immunol..
Aug 2023
10.3389/fimmu.2023.1225610
CD30 is expressed on Hodgkin lymphomas (HL), many non-Hodgkin lymphomas (NHLs), and non-lymphoid malignancies in children and adults. Tumor expression, combined with restricted expression in healthy tissues, identifies CD30 as a promising immunotherapy target. An anti-CD30 antibody-drug conjugate (ADC) has been approved by the FDA for HL. While anti-CD30 ADCs and chimeric antigen receptors (CARs) have shown promise, their shortcomings and toxicities suggest that alternative treatments are needed. We developed novel anti-CD30 x anti-CD3 bispecific antibodies (biAbs) to coat activated patient T cells (ATCs) ex vivo prior to autologous re-infusions. Our goal is to harness the dual specificity of the biAb, the power of cellular therapy, and the safety of non-genetically modified autologous T cell infusions. We present a comprehensive characterization of the CD30 binding and tumor cell killing properties of these biAbs. Five unique murine monoclonal antibodies (mAbs) were generated against the extracellular domain of human CD30. Resultant anti-CD30 mAbs were purified and screened for binding specificity, affinity, and epitope recognition. Two lead mAb candidates with unique sequences and CD30 binding clusters that differ from the ADC in clinical use were identified. These mAbs were chemically conjugated with OKT3 (an anti-CD3 mAb). ATCs were armed and evaluated in vitro for binding, cytokine production, and cytotoxicity against tumor lines and then in vivo for tumor cell killing. Our lead mAb was subcloned to make a Master Cell Bank (MCB) and screened for binding against a library of human cell surface proteins. Only huCD30 was bound. These studies support a clinical trial in development employing ex vivo -loading of autologous T cells with this novel biAb.

Antigen discovery by bioinformatics analysis and peptide microarray for the diagnosis of cystic echinococcosis

Batisti Biffignandi, Gherard; Vola, Ambra; Sassera, Davide; Najafi-Fard, Saeid; Gomez Morales, Maria Angeles; Brunetti, Enrico; Teggi, Antonella; Goletti, Delia; Petrone, Linda; Tamarozzi, Francesca
PLoS Negl Trop Dis.
Apr 2023
10.1371/journal.pntd.0011210
Background Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato, is a neglected zoonosis. Its diagnosis relies on imaging, supported by serology, while only imaging is useful for staging and follow-up. Since diagnostic tools and expertise are not widely available, new accurate and easily implementable assays for the diagnosis and follow-up of CE are highly needed. Methodology/Principal Findings We aimed to identify new E . granulosus antigens through a bioinformatics selection applied to the parasite genome, followed by peptide microarray screening and validation in ELISA, using independent panels of sera from patients with hepatic CE and clinically relevant controls. From 950 proteins selected in silico , 2,379 peptides were evaluated by microarray for IgG reactivity and eight candidates selected for validation. Reactivity to one peptide was significantly higher in the CE group (p = 0.044), but had suboptimal diagnostic accuracy. Conclusions/Significance Here we performed bioinformatics analysis and peptide microarray for antigen discovery, useful for the diagnosis of CE. Eight candidates were selected and validated. Reactivity to one peptide associated to CE but had suboptimal diagnostic accuracy. Importantly, the database developed in this study may be used to identify other antigenic candidates for CE diagnosis and follow-up.

Immunoglobulin profiling with large high-density peptide microarrays as screening method to detect candidate proteins for future biomarker detection in dogs with steroid-responsive meningitis-arteritis

Nessler, Jasmin Nicole; Tipold, Andrea
PLoS ONE.
Apr 2023
10.1371/journal.pone.0284010
Steroid responsive meningitis arteritis (SRMA) is an aberrant Th2-mediated systemic inflammatory disease in dogs. The etiopathogenesis still remains unclear as no triggering pathogen or autoantigen could be found so far. Hypothesis . Large high-density peptide microarrays are a suitable screening method to detect possible autoantigens which might be involved in the pathogenesis of SRMA. Methods . The IgA and IgG profile of pooled serum samples of 5 dogs with SRMA and 5 dogs with neck pain due to intervertebral disc herniation (IVDH) without ataxia or paresis were compared via commercially available high-density peptide microarrays (Discovery Microarray) containing 29,240 random linear peptides. Canine distemper virus nucleoprotein (CDVN) served as positive control as all dogs were vaccinated. Common motifs were compared to amino acid sequences of known proteins via databank search. One suitable protein was manually selected for further analysis with a smaller customized high-density peptide microarray. Results . Pooled serum of dogs with SRMA and IVDH showed different IgA and IgG responses on Discovery Microarray. Only top IgG responses of dogs with SRMA showed a common motif not related to the control protein CDVN. This common motif is part of the interleukin 1 receptor antagonist protein (IL1Ra). On IL1Ra, dogs with SRMA displayed IgA binding to an additional epitope, which dogs with IVDH did not show. Discussion . IL1Ra is an anti-inflammatory acute phase protein. Different immunoglobulin binding patterns on IL1Ra could be involved in the pathogenesis of SRMA and IL1Ra might be developed as future biomarker for SRMA.

Immunoanalytical Approach for Detecting and Identifying Ancestral Peptide Biomarkers in Early Earth Analogue Environments

Severino, Rita; Moreno-Paz, Mercedes; Puente-Sánchez, Fernando; Sánchez-García, Laura; Risso, Valeria A.; Sanchez-Ruiz, Jose M.; Cabrol, Nathalie; Parro, Victor
Anal. Chem..
Mar 2023
10.1021/acs.analchem.2c05386

Evaluation of tumor antigen-specific antibody responses in patients with metastatic triple negative breast cancer treated with cyclophosphamide and pembrolizumab

Routh, Eric D; Woodcock, Mark G; Beckabir, Wolfgang; Vensko, Steven P; Serody, Jonathan S; Vincent, Benjamin G
J Immunother Cancer.
Mar 2023
10.1136/jitc-2022-005848
The role of B cells in antitumor immunity is becoming increasingly appreciated, as B cell populations have been associated with response to immune checkpoint blockade (ICB) in patients with breast cancer and murine models of breast cancer. Deeper understanding of antibody responses to tumor antigens is needed to clarify the function of B cells in determining response to immunotherapy. We evaluated tumor antigen-specific antibody responses in patients with metastatic triple negative breast cancer treated with pembrolizumab following low-dose cyclophosphamide therapy using computational linear epitope prediction and custom peptide microarrays. We found that a minority of predicted linear epitopes were associated with antibody signal, and signal was associated with both neoepitopes and self-peptides. No association was observed between signal presence and subcellular localization or RNA expression of parent proteins. Patient-specific patterns of antibody signal boostability were observed that were independent of clinical response. Intriguingly, measures of cumulative antibody signal intensity relative to immunotherapy treatment showed that the one complete responder in the trial had the greatest increase in total antibody signal, which supports a potential association between ICB-dependent antibody boosting and clinical response. The antibody boost in the complete responder was largely driven by increased levels of IgG specific to a sequence of N-terminal residues in native Epidermal Growth Factor Receptor Pathway Substrate 8 (EPS8) protein, a known oncogene in several cancer types including breast cancer. Structural protein prediction showed that the targeted epitope of EPS8 was in a region of the protein with mixed linear/helical structure, and that this region was solvent-exposed and not predicted to bind to interacting macromolecules. This study highlights the potential importance of the humoral immune response targeting neoepitopes as well as self epitopes in shaping clinical response to immunotherapy.

Identification of Zika Virus NS1-Derived Peptides with Potential Applications in Serological Tests

Prudencio, Carlos Roberto; Gomes da Costa, Vivaldo; Rocha, Leticia Barboza; Costa, Hernan Hermes Monteiro; Orts, Diego José Belato; da Silva Santos, Felipe Rocha; Rahal, Paula; Lino, Nikolas Alexander Borsato; da Conceição, Pâmela Jóyce Previdelli; Bittar, Cintia; Machado, Rafael Rahal Guaragna; Durigon, Edison Luiz; Araujo, João Pessoa; Polatto, Juliana Moutinho; da Silva, Miriam Aparecida; de Oliveira, Joyce Araújo; Mitsunari, Thais; Pereira, Lennon Ramos; Andreata-Santos, Robert; de Souza Ferreira, Luís Carlos; Luz, Daniela; Piazza, Roxane Maria Fontes
Viruses.
Feb 2023
10.3390/v15030654
Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.

Exploring the Immunodominant Epitopes of SARS-CoV-2 Nucleocapsid Protein as Exposure Biomarker

Vashisht, Kapil; Goyal, Bharti; Pasupureddy, Rahul; Na, Byoung-Kuk; Shin, Ho-Joon; Sahu, Dibakar; De, Sajal; Chakraborti, Soumyananda; Pandey, Kailash C
Feb 2023
10.7759/cureus.34827

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