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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.

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.

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

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.

SARS-CoV-2 Rapid Antigen Test Based on a New Anti-Nucleocapsid Protein Monoclonal Antibody: Development and Real-Time Validation

Coelho, Fabiana Fioravante; Da Silva, Miriam Aparecida; Lopes, Thiciany Blener; Polatto, Juliana Moutinho; De Castro, Natália Salazar; Andrade, Luis Adan Flores; Lourenço, Karine Lima; Sato, Hugo Itaru; De Carvalho, Alex Fiorini; Coelho, Helena Perez; Bagno, Flávia Fonseca; Luz, Daniela; Viala, Vincent Louis; Cattony, Pedro Queiroz; Melo, Bruna De Sousa; Moro, Ana Maria; Quintilio, Wagner; Barbosa, Ana Paula; Bomfim, Camila Gasque; Soares, Camila Pereira; Guzzo, Cristiane Rodrigues; Fonseca, Flavio Guimarães; Durigon, Edison Luiz; Gazzinelli, Ricardo Tostes; Ribeiro Teixeira, Santuza M.; Piazza, Roxane Maria Fontes; Fernandes, Ana Paula
Microorganisms.
Sep 2023
10.3390/microorganisms11102422
SARS-CoV-2 diagnostic tests have become an important tool for pandemic control. Among the alternatives for COVID-19 diagnosis, antigen rapid diagnostic tests (Ag-RDT) are very convenient and widely used. However, as SARS-CoV-2 variants may continuously emerge, the replacement of tests and reagents may be required to maintain the sensitivity of Ag-RDTs. Here, we describe the development and validation of an Ag-RDT during an outbreak of the Omicron variant, including the characterization of a new monoclonal antibody (anti-DTC-N 1B3 mAb) that recognizes the Nucleocapsid protein (N). The anti-DTC-N 1B3 mAb recognized the sequence TFPPTEPKKDKKK located at the C-terminus of the N protein of main SARS-CoV-2 variants of concern. Accordingly, the Ag-RDT prototypes using the anti-DTC-N 1B3 mAB detected all the SARS-CoV-2 variants—Wuhan, Alpha, Gamma, Delta, P2 and Omicron. The performance of the best prototype (sensitivity of 95.2% for samples with Ct ≤ 25; specificity of 98.3% and overall accuracy of 85.0%) met the WHO recommendations. Moreover, results from a patients’ follow-up study indicated that, if performed within the first three days after onset of symptoms, the Ag-RDT displayed 100% sensitivity. Thus, the new mAb and the Ag-RDT developed herein may constitute alternative tools for COVID-19 point-of-care diagnosis and epidemiological surveillance.

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.

Multifunctional IgG/IgM antibodies and cellular cytotoxicity are elicited by the full-length MSP1 SumayaVac-1 malaria vaccine

Rosenkranz, Micha; Fürle, Kristin; Hibbert, Julia; Ulmer, Anne; Ali, Arin; Giese, Thomas; Blank, Antje; Haefeli, Walter E.; Böhnlein, Ernst; Lanzer, Michael; Thomson-Luque, Richard
npj Vaccines.
Aug 2023
10.1038/s41541-023-00701-2
Abstract Radical control of malaria likely requires a vaccine that targets both the asymptomatic liver stages and the disease-causing blood stages of the human malaria parasite Plasmodium falciparum . While substantial progress has been made towards liver stage vaccines, the development of a blood stage vaccine is lagging behind. We have recently conducted a first-in-human clinical trial to evaluate the safety and immunogenicity of the recombinant, full-length merozoite surface protein 1 (MSP1 FL ) formulated with GLA-SE as adjuvant. Here, we show that the vaccine, termed SumayaVac-1 , elicited both a humoral and cellular immune response as well as a recall T cell memory. The induced IgG and IgM antibodies were able to stimulate various Fc-mediated effector mechanisms associated with protection against malaria, including phagocytosis, release of reactive oxygen species, production of IFN-γ as well as complement activation and fixation. The multifunctional activity of the humoral immune response remained for at least 6 months after vaccination and was comparable to that of naturally acquired anti-MSP1 antibodies from semi-immune adults from Kenya. We further present evidence of SumayaVac-1 eliciting a recallable cellular cytotoxicity by IFN-γ producing CD8+ T cells. Our study revitalizes MSP1 FL as a relevant blood stage vaccine candidate and warrants further evaluation of SumayaVac-1 in a phase II efficacy trial.

Selection for immune evasion in SARS-CoV-2 revealed by high-resolution epitope mapping and sequence analysis

N’Guessan, Arnaud; Kailasam, Senthilkumar; Mostefai, Fatima; Poujol, Raphaël; Grenier, Jean-Christophe; Ismailova, Nailya; Contini, Paola; De Palma, Raffaele; Haber, Carsten; Stadler, Volker; Bourque, Guillaume; Hussin, Julie G.; Shapiro, B. Jesse; Fritz, Jörg H.; Piccirillo, Ciriaco A.
iScience.
Aug 2023
10.1016/j.isci.2023.107394

AAV-mediated expression of a new conformational anti-aggregated α-synuclein antibody prolongs survival in a genetic model of α-synucleinopathies

Düchs, Matthias; Blazevic, Dragica; Rechtsteiner, Philipp; Kenny, Cynthia; Lamla, Thorsten; Low, Sarah; Savistchenko, Jimmy; Neumann, Manuela; Melki, Ronald; Schönberger, Tanja; Stierstorfer, Birgit; Wyatt, David; Igney, Frederik; Ciossek, Thomas
npj Parkinsons Dis..
Jun 2023
10.1038/s41531-023-00542-9
Abstract Prion-like transmission of pathology in α-synucleinopathies like Parkinson’s disease or multiple system atrophy is increasingly recognized as one potential mechanism to address disease progression. Active and passive immunotherapies targeting insoluble, aggregated α-synuclein are already being actively explored in the clinic with mixed outcomes so far. Here, we report the identification of 306C7B3, a highly selective, aggregate-specific α-synuclein antibody with picomolar affinity devoid of binding to the monomeric, physiologic protein. 306C7B3 binding is Ser129-phosphorylation independent and shows high affinity to several different aggregated α-synuclein polymorphs, increasing the likelihood that it can also bind to the pathological seeds assumed to drive disease progression in patients. In support of this, highly selective binding to pathological aggregates in postmortem brains of MSA patients was demonstrated, with no staining in samples from other human neurodegenerative diseases. To achieve CNS exposure of 306C7B3, an adeno-associated virus (AAV) based approach driving expression of the secreted antibody within the brain of (Thy-1)-[A30P]-hα-synuclein mice was used. Widespread central transduction after intrastriatal inoculation was ensured by using the AAV2HBKO serotype, with transduction being spread to areas far away from the inoculation site. Treatment of (Thy-1)-[A30P]-hα-synuclein mice at the age of 12 months demonstrated significantly increased survival, with 306C7B3 concentration reaching 3.9 nM in the cerebrospinal fluid. These results suggest that AAV-mediated expression of 306C7B3, targeting extracellular, presumably disease-propagating aggregates of α-synuclein, has great potential as a disease-modifying therapy for α-synucleinopathies as it ensures CNS exposure of the antibody, thereby mitigating the selective permeability of the blood-brain barrier.

Differential recognition of influenza A virus H1N1 neuraminidase by DNA vaccine-induced antibodies in pigs and ferrets

Tingstedt, Jeanette Linnea; Stephen, Christine; Risinger, Christian; Blixt, Ola; Gunalan, Vithiagaran; Johansen, Isik Somuncu; Fomsgaard, Anders; Polacek, Charlotta; Lassaunière, Ria
Front. Immunol..
May 2023
10.3389/fimmu.2023.1200718
Neuraminidase (NA) accounts for approximately 10-20% of the total glycoproteins on the surface of influenza viruses. It cleaves sialic acids on glycoproteins, which facilitates virus entry into the airways by cleaving heavily glycosylated mucins in mucus and the release of progeny virus from the surface of infected cells. These functions make NA an attractive vaccine target. To inform rational vaccine design, we define the functionality of influenza DNA vaccine-induced NA-specific antibodies relative to antigenic sites in pigs and ferrets challenged with a vaccine-homologous A/California/7/2009(H1N1)pdm09 strain. Sera collected pre-vaccination, post-vaccination and post-challenge were analyzed for antibody-mediated inhibition of NA activity using a recombinant H7N1 CA09 virus. Antigenic sites were further identified with linear and conformational peptide microarrays spanning the full NA of A/California/04/2009(H1N1)pdm09. Vaccine-induced NA-specific antibodies inhibited the enzymatic function of NA in both animal models. The antibodies target critical sites of NA such as the enzymatic site, second sialic binding site and framework residues, shown here by high-resolution epitope mapping. New possible antigenic sites were identified that potentially block the catalytic activity of NA, including an epitope recognized solely in pigs and ferrets with neuraminidase inhibition, which could be a key antigenic site affecting NA function. These findings show that our influenza DNA vaccine candidate induces NA-specific antibodies that target known critical sites, and new potential antigenic sites of NA, inhibiting the catalytic activity of NA.

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