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

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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.

Severity-dependent IgG epitope profiling in COVID-19 reveals differential recognition of pathogen-derived antigens

Do Nascimento, Lais Alves; Machado, NicolleRakanidis; Borges, João Vitor Da Silva; Fagundes, Beatriz Oliveira; Bergamasco, Isabella Siuffi; Sgnotto, Fabio Da Ressureição; Bachi, André Luis Lacerda; Sato, Maria Notomi; Victor, Jefferson Russo
Front. Immunol..
Sep 2025
10.3389/fimmu.2025.1668223
Background The contribution of antibody-mediated responses to COVID – 19 outcomes remains unclear, particularly regarding cross-reactivity with unrelated pathogens. While co-infections are known to influence disease progression, the broader landscape of IgG reactivity during SARS-CoV-2 infection has not been systematically explored. Methods We employed a high-density peptide microarray containing 4,344 linear epitopes from 37 viruses, 27 bacteria, 17 parasites, and 8 fungi to characterize serum IgG repertoires from individuals with moderate (n = 39) or severe (n = 40) COVID – 19. Controls included pre-pandemic healthy donors and a pooled intravenous immunoglobulin (IVIg) formulation. Data analysis included intensity ranking, epitope mapping, and comparative analysis of mean signal intensities for each epitope between the COVID-Mod and COVID-Sev groups. Results COVID – 19 patients showed widespread IgG reactivity against diverse pathogens, with patterns differing by disease severity. Severe cases displayed broader and more intense reactivity, notably against hepatitis C virus (HCV), SARS-CoV-1, influenza A, Mycobacterium tuberculosis, and Plasmodium falciparum. Moderate cases showed preferential recognition of epitopes from HTLV-I, Neisseria meningitidis, and Trypanosoma cruzi. These findings suggest that SARS-CoV-2 infection modulates pre-existing humoral memory, possibly through epitope spreading or immune reprogramming. Conclusions SARS-CoV-2 infection reshapes the IgG epitope repertoire in a severity-dependent manner, extending to antigens from unrelated pathogens. This phenomenon may reflect underlying immune dysregulation or idiotype-driven interactions. Comprehensive profiling of pathogen-related IgG responses may reveal potential biomarkers of disease severity. This phenomenon may inform future investigations aimed at improving personalized management strategies for co-infected or immunocompromised patients.

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.

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.

HCV immunodominant peptide mapping reveals unique HLA-A*02-restricted signatures: insights for CD8+ T-cell-based vaccines and immunotherapies

Cardoso Corrêa-Dias, Laura; Lopes-Ribeiro, Ágata; Marques-Ferreira, Geovane; Gomes-de-Pontes, Letícia; Pereira-Santos, Thaiza Aline; De Sousa Reis, Erik Vinicius; Silva Moraes, Thaís De Fátima; Assis Martins-Filho, Olindo; Figueiredo Barbosa-Stancioli, Edel; Guimarães Da Fonseca, Flávio; Coelho-dos-Reis, Jordana Grazziela
Immunogenetics.
Jan 2025
10.1007/s00251-025-01370-2
Several barriers for the development of an HCV vaccine still exist, including the genetic diversity of the virus, and the shortage of assessable models for in vitro and in vivo assays. Therefore, in this study, HCV epitope mapping was performed for 59 polyprotein sequences from 7 HCV genotypes. Around 2,880 peptides were considered epitopes for CD8+ T cells. The peptide induction of cytokines from Th1 and/or Th2 axes of the cellular immune response was assessed, indicating a tendency for Th2 axis. In vitro evaluation was performed using peptide microarray and a recombinant HLA-A*02:01 molecule. A total of 615 peptides of high reactivity to HLA-A*02:01 were identified, with predominance of leucine and tryptophan residues, highlighting their importance for TCR-epitope binding and CD8+ T activation. Finally, HCV-derived peptide patterns restricted to HLA-A2*02:01 observed in this study provide important information for the development of a multi-epitope-based pan-genotypic vaccine against the virus.

Investigation of Immunoreactivity Profiles and Epitope Landscape in Divergent COVID-19 Trajectories and SARS-CoV-2 Variants

Bihani, Surbhi; Ray, Arka; Borishetty, Dhanush; Tuckley, Chaitanya; Salkar, Akanksha; Acharjee, Arup; Shrivastav, Prithviraj; Shrivastav, Om; Shastri, Jayanthi; Agrawal, Sachee; Duttagupta, Siddhartha; Srivastava, Sanjeeva
J. Proteome Res..
Jan 2025
10.1021/acs.jproteome.4c00791
This study aimed to elucidate the complexity of the humoral immune response in COVID-19 patients with varying disease trajectories using a SARS-CoV-2 whole proteome peptide microarray chip. The microarray, containing 5347 peptides spanning the entire SARS-CoV-2 proteome and key variants of concern, was used to analyze IgG responses in 10 severe-to-recovered, 9 nonsevere-to-severe cases, and 10 control case (5 pre-pandemic and 5 SARS-CoV-2-negative) plasma samples. We identified 1151 IgG-reactive peptides corresponding to 647 epitopes, with 207 peptides being cross-reactive across 124 epitopes. Nonstructural protein 3 (nsp3) exhibited the highest number of total and unique epitopes, followed by the spike protein. nsp12 had the most number of cross-reactive epitopes. Peptides from the spike protein and nsps 2, 3, 5, and 13 were notably associated with recovery. Additionally, specific mutations in SARS-CoV-2 variants were found to alter peptide immunoreactivity, with some mutations (e.g., G142D, L452R, and N501Y) enhancing and others (e.g., R190S and E484 K) reducing immune recognition. These findings have critical implications for the development of diagnostics, vaccines, and therapeutics. Understanding the distribution of epitopes and the impact of viral mutations on antigenicity provides insights into immune evasion mechanisms, informing strategies for controlling COVID-19 and future coronavirus outbreaks.

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.

Discovery of putative breast cancer antigens using an integrative platform of genomics-driven immunoproteomics

Qendro, Veneta; Lundgren, Deborah H.; Palczewski, Samuel; Hegde, Poornima; Stevenson, Christina; Perpetua, Laurie; Latifi, Ardian; Merriman, Jesse; Bugos, Grace; Han, David K.
Proteomics.
Aug 2018
10.1002/pmic.201600318
Recent advances in cancer immuno-therapeutics such as checkpoint inhibitors, chimeric antigen-receptor T cells, and tumor infiltrating T cells (TIL) are now significantly impacting cancer patients in a positive manner. Although very promising, reports indicate no more than 25% of cases result in complete remission. One of the limitations of these treatments is the identity of putative cancer antigens in each patient, as it is technically challenging to identify cancer antigens in a rapid fashion. Thus, identification of cancer antigens followed by targeted treatment will increase the efficacy of cancer immunotherapies. To achieve this goal, a combined technologies platform of deep genomic sequencing and personalized immune assessment was devised, termed Genomics Driven Immunoproteomics (GDI). Using this technological platform, we report the discovery of 149 tumor antigens from human breast cancer patients. Significant number of these putative cancer antigens arise from single nucleotide variants (SNVs), as well as insertions and deletions that results into frame-shift mutations. We propose a general model of anti-cancer immunity and suggest that the GDI platform may help identify patient-specific tumor antigens in a timely fashion for precision immunotherapies.

Circulating GRP78 antibodies from ovarian cancer patients: a promising tool for cancer cell targeting drug delivery system?

Van Hoesen, Kylie; Meynier, Sonia; Ribaux, Pascale; Petignat, Patrick; Delie, Florence; Cohen, Marie
Oncotarget.
Dec 2017
10.18632/oncotarget.22412
Glucose-regulated protein 78 (GRP78) is a chaperone protein that has a high frequency in tumor cells. Normally it is found in the endoplasmic reticulum to assist in protein folding, but under cellular stress, GRP78 influences proliferative signaling pathways at the cell surface. The increased expression elicits autoantibody production, providing a biomarker of ovarian cancer, as well as other types of cancer. This study aims to determine the epitope recognition of GRP78 autoantibodies isolated from serum of ovarian cancer patients and use the identified antibodies to design new drug delivery systems to specifically target cancer cells. We first confirmed that the membrane GRP78 levels are increased in ovarian cancer cells and positively correlate with proliferation. However, the level of circulating GRP78 autoantibodies did not correlate with membrane GRP78 expression in ovarian cancer cells and was lower, although not significantly, compared to control patients. We then determined the epitope recognition of GRP78 autoantibodies and showed that treatment with paclitaxel-loaded nanoparticles coated with anti-GRP78 antibodies significantly decreased tumor development in chick embryo culture of ovarian cancer cell tumors compared to paclitaxel treatment alone. This evidence suggests that nanoparticle drug delivery systems coupled with antibodies against GRP78 has potential as a powerful therapy against ovarian cancer.

Bayesian Analysis of MicroScale Thermophoresis Data to Quantify Affinity of Protein:Protein Interactions with Human Survivin

Garcia-Bonete, Maria-Jose; Jensen, Maja; Recktenwald, Christian V.; Rocha, Sandra; Stadler, Volker; Bokarewa, Maria; Katona, Gergely
Sci Rep.
Dec 2017
10.1038/s41598-017-17071-0
A biomolecular ensemble exhibits different responses to a temperature gradient depending on its diffusion properties. MicroScale Thermophoresis technique exploits this effect and is becoming a popular technique for analyzing interactions of biomolecules in solution. When comparing affinities of related compounds, the reliability of the determined thermodynamic parameters often comes into question. The thermophoresis binding curves can be assessed by Bayesian inference, which provides a probability distribution for the dissociation constant of the interacting partners. By applying Bayesian machine learning principles, binding curves can be autonomously analyzed without manual intervention and without introducing subjective bias by outlier rejection. We demonstrate the Bayesian inference protocol on the known survivin:borealin interaction and on the putative protein-protein interactions between human survivin and two members of the human Shugoshin-like family (hSgol1 and hSgol2). These interactions were identified in a protein microarray binding assay against survivin and confirmed by MicroScale Thermophoresis.

Development and analytical performance of a new ARCHITECT automated dipeptidyl peptidase-4 immunoassay

Hemken, Philip M.; Jeanblanc, Nicolette M.; Rae, Tracey; Brophy, Susan E.; Datwyler, Maria J.; Xu, Ying; Manetz, T. Scott; Vainshtein, Inna; Liang, Meina; Xiao, Xiaodong; Chowdhury, Partha S.; Chang, Chien-ying; Streicher, Katie; Greenlees, Lydia; Ranade, Koustubh; Davis, Gerard J.
Practical Laboratory Medicine.
Dec 2017
10.1016/j.plabm.2017.10.003
Background Dipeptidyl peptidase-4 (DPP-4) may be a suitable biomarker to identify people with severe asthma who have greater activation of the interleukin-13 (IL-13) pathway and may therefore benefit from IL-13-targeted treatments. We report the analytical performance of an Investigational Use Only immunoassay and provide data on the biological range of DPP-4 concentrations. Methods We assessed assay performance, utilising analyses of precision, linearity and sensitivity; interference from common endogenous assay interferents, and from asthma and anti-diabetic medications, were also assessed. The assay was used to measure the range of serum DPP-4 concentrations in healthy volunteers and subjects with diabetes and severe, uncontrolled asthma. Results The total precision of DPP-4 concentration measurement (determined using percentage coefficient of variation) was ≤5% over 20 days. Dilution analysis yielded linear results from 30 to 1305 ng/mL; the limit of quantitation was 19.2 ng/mL. No notable endogenous or drug interferences were observed at the expected therapeutic concentration. Median DPP-4 concentrations in healthy volunteers and subjects with asthma or Type 1 diabetes were assessed, with concentrations remaining similar in subjects with diabetes and asthma across different demographics. Conclusion These analyses indicate that the ARCHITECT DPP-4 Immunoassay is a reliable and robust method for measuring serum DPP-4 concentration.

A novel neutralizing human monoclonal antibody broadly abrogates hepatitis C virus infection in vitro and in vivo

Desombere, Isabelle; Mesalam, Ahmed Atef; Urbanowicz, Richard A.; Van Houtte, Freya; Verhoye, Lieven; Keck, Zhen-Yong; Farhoudi, Ali; Vercauteren, Koen; Weening, Karin E.; Baumert, Thomas F.; Patel, Arvind H.; Foung, Steven K.H.; Ball, Jonathan; Leroux-Roels, Geert; Meuleman, Philip
Antiviral Research.
Dec 2017
10.1016/j.antiviral.2017.10.015
Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. Conclusion: mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines.

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