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

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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.

Targeting FLT3 by new-generation antibody-drug-conjugate in combination with kinase inhibitors for treatment of AML

Roas, Maike; Vick, Binje; Kasper, Marc-André; Able, Marina; Polzer, Harald; Gerlach, Marcus; Kremmer, Elisabeth; Hecker, Judith S.; Schmitt, Saskia; Stengl, Andreas; Waller, Verena; Hohmann, Natascha; Festini, Moreno; Ludwig, Alexander Edmund; Rohrbacher, Lisa; Herold, Tobias; Subklewe, Marion; Götze, Katharina S.; Hackenberger, Christian P.R.; Schumacher, Dominik; Helma-Smets, Jonas; Jeremias, Irmela; Leonhardt, Heinrich; Spiekermann, Karsten
Aug 2022
10.1182/blood.2021015246
Fms like tyrosine kinase 3 (FLT3) is often overexpressed or constitutively activated by internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations in acute myeloid leukemia (AML). Despite the use of receptor tyrosine kinase inhibitors (TKI) in FLT3-ITD positive AML, the prognosis of patients is still poor and further improvement of therapy is required. Targeting FLT3 independent of mutations by antibody‑drug‑conjugates (ADCs) is a promising strategy for AML therapy. Here, we report the development and preclinical characterization of a novel FLT3‑targeting ADC, 20D9-ADC, which was generated by applying the innovative P5 conjugation technology. In vitro, 20D9‑ADC mediated potent cytotoxicity to Ba/F3 cells expressing transgenic FLT3 or FLT3-ITD, to AML cell lines and to FLT3-ITD positive patient derived xenograft AML cells. In vivo, 20D9‑ADC treatment led to a significant tumor reduction and even durable complete remission in AML xenograft models. Further, 20D9‑ADC demonstrated no severe hematotoxicity in in vitro colony formation assays using concentrations that were cytotoxic in AML cell line treatment. The combination of 20D9-ADC with the TKI midostaurin showed strong synergy in vitro and in vivo, leading to reduction of aggressive AML cells below the detection limit. Our data indicate that targeting FLT3 with an advanced new-generation ADC is a promising and potent antileukemic strategy, especially when combined with FLT3-TKI in FLT3‑ITD positive AML.

Development of broadly neutralizing antibodies targeting the cytomegalovirus subdominant antigen gH

Parsons, Andrea J.; Ophir, Sabrina I.; Duty, J. Andrew; Kraus, Thomas A.; Stein, Kathryn R.; Moran, Thomas M.; Tortorella, Domenico
Commun Biol.
Apr 2022
10.1038/s42003-022-03294-z
Human cytomegalovirus (HCMV) is a β-herpesvirus that increases morbidity and mortality in immunocompromised individuals including transplant recipients and newborns. New anti-HCMV therapies are an urgent medical need for diverse patient populations. HCMV infection of a broad range of host tissues is dependent on the gH/gL/gO trimer and gH/gL/UL28/UL130/UL131A pentamer complexes on the viral envelope. We sought to develop safe and effective therapeutics against HCMV by generating broadly-neutralizing, human monoclonal antibodies (mAbs) from VelocImmune® mice immunized with gH/gL cDNA. Following high-throughput binding and neutralization screening assays, 11 neutralizing antibodies were identified with unique CDR3 regions and a high-affinity (KD 1.4-65 nM) to the pentamer complex. The antibodies bound to distinct regions within Domains 1 and 2 of gH and effectively neutralized diverse clinical strains in physiologically relevant cell types including epithelial cells, trophoblasts, and monocytes. Importantly, combined adminstration of mAbs with ganciclovir, an FDA approved antiviral, greatly limited virus dissemination. Our work identifies several anti-gH/gL mAbs and sheds light on gH neutralizing epitopes that can guide future vaccine strategies.

Construction of a Large Size Human Immunoglobulin Heavy Chain Variable (VH) Domain Library, Isolation and Characterization of Novel Human Antibody VH Domains Targeting PD-L1 and CD22

Sun, Zehua; Li, Wei; Mellors, John W.; Orentas, Rimas; Dimitrov, Dimiter S.
Front Immunol.
Apr 2022
10.3389/fimmu.2022.869825
Phage display is a well-established technology for in vitro selection of monoclonal antibodies (mAb), and more than 12 antibodies isolated from phage displayed libraries of different formats have been approved for therapy. We have constructed a large size (10^11) human antibody VH domain library based on thermo-stable, aggregation-resistant scaffolds. This diversity was obtained by grafting naturally occurring CDR2s and CDR3s from healthy donors with optimized primers into the VH library. This phage-displayed library was used for bio-panning against various antigens. So far, panels of binders have been isolated against different viral and tumor targets, including the SARS-CoV-2 RBD, HIV-1 ENV protein, mesothelin and FLT3. In the present study, we discuss domain library construction, characterize novel VH binders against human CD22 and PD-L1, and define our design process for antibody domain drug conjugation (DDC) as tumoricidal reagents. Our study provides examples for the potential applications of antibody domains derived from library screens in therapeutics and provides key information for large size human antibody domain library construction.

Inhibition of lung microbiota-derived proapoptotic peptides ameliorates acute exacerbation of pulmonary fibrosis

D’Alessandro-Gabazza, Corina N.; Yasuma, Taro; Kobayashi, Tetsu; Toda, Masaaki; Abdel-Hamid, Ahmed M.; Fujimoto, Hajime; Hataji, Osamu; Nakahara, Hiroki; Takeshita, Atsuro; Nishihama, Kota; Okano, Tomohito; Saiki, Haruko; Okano, Yuko; Tomaru, Atsushi; Fridman D’Alessandro, Valeria; Shiraishi, Miyako; Mizoguchi, Akira; Ono, Ryoichi; Ohtsuka, Junpei; Fukumura, Masayuki; Nosaka, Tetsuya; Mi, Xuenan; Shukla, Diwakar; Kataoka, Kensuke; Kondoh, Yasuhiro; Hirose, Masaki; Arai, Toru; Inoue, Yoshikazu; Yano, Yutaka; Mackie, Roderick I.; Cann, Isaac; Gabazza, Esteban C.
Nat Commun.
Mar 2022
10.1038/s41467-022-29064-3
Idiopathic pulmonary fibrosis is an incurable disease of unknown etiology. Acute exacerbation of idiopathic pulmonary fibrosis is associated with high mortality. Excessive apoptosis of lung epithelial cells occurs in pulmonary fibrosis acute exacerbation. We recently identified corisin, a proapoptotic peptide that triggers acute exacerbation of pulmonary fibrosis. Here, we provide insights into the mechanism underlying the processing and release of corisin. Furthermore, we demonstrate that an anticorisin monoclonal antibody ameliorates lung fibrosis by significantly inhibiting acute exacerbation in the human transforming growth factorβ1 model and acute lung injury in the bleomycin model. By investigating the impact of the anticorisin monoclonal antibody in a general model of acute lung injury, we further unravel the potential of corisin to impact such diseases. These results underscore the role of corisin in the pathogenesis of acute exacerbation of pulmonary fibrosis and acute lung injury and provide a novel approach to treating this incurable disease.

In vitro evolution of myc-tag antibodies: in-depth specificity and affinity analysis of Myc1-9E10 and Hyper-Myc

Russo, Giulio; Unkauf, Tobias; Meier, Doris; Wenzel, Esther Veronika; Langreder, Nora; Schneider, Kai-Thomas; Wiesner, Rebecca; Bischoff, Ralf; Stadler, Volker; Dübel, Stefan
Mar 2022
10.1515/hsz-2021-0405
One of the most widely used epitope tags is the myc-tag, recognized by the anti-c-Myc hybridoma antibody Myc1-9E10. Combining error-prone PCR, DNA shuffling and phage display, we generated an anti-c-Myc antibody variant (Hyper-Myc) with monovalent affinity improved to 18 nM and thermal stability increased by 37%. Quantification of capillary immunoblots and by flow cytometry demonstrated improved antigen detection by Hyper-Myc. Further, three different species variants of this antibody were generated to allow the use of either anti-human, anti-mouse or anti-rabbit Fc secondary antibodies for detection. We characterized the specificity of both antibodies in depth: individual amino acid exchange mapping demonstrated that the recognized epitope was not changed by the in vitro evolution process. A laser printed array of 29,127 different epitopes representing all human linear B-cell epitopes of the Immune Epitope Database allowing to chart unwanted reactivities with mimotopes showed these to be very low for both antibodies and not increased for Hyper-Myc despite its improved affinity. The very low background reactivity of Hyper-Myc was confirmed by staining of myc-tag transgenic zebrafish whole mounts. Hyper-Myc retains the very high specificity of Myc1-9E10 while allowing myc-tag detection at lower concentrations and with either anti-mouse, anti-rabbit or anti human secondary antibodies.

SARS-CoV-2 epitope mapping on microarrays highlights strong immune-response to N protein region

Musicò, Angelo; Frigerio, Roberto; Mussida, Alessandro; Barzon, Luisa; Sinigaglia, Alessandro; Riccetti, Silvia; Gobbi, Federico; Piubelli, Chiara; Bergamaschi, Greta; Chiari, Marcella; Gori, Alessandro; Cretich, Marina
Nov 2020
10.1101/2020.11.09.374082

Abstract

A workflow for SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 Spike (S), Nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155-171) providing 92% sensitivity and 100% specificity of IgG detection in Covid-19 samples thus being a promising candidate for rapid implementation in serological tests.

Generation of Chicken IgY against SARS-COV-2 Spike Protein and Epitope Mapping

Lu, Yan; Wang, Yajun; Zhang, Zhen; Huang, Jingliang; Yao, Meicun; Huang, Guobin; Ge, Yuanyuan; Zhang, Peichun; Huang, Huaxin; Wang, Yong; Li, Huiliang; Wang, Wen
Oct 2020
This new decade has started with a global pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), precipitating a worldwide health crisis and economic downturn. Scientists and clinicians have been racing against time to find therapies for COVID-19. Repurposing approved drugs, developing vaccines and employing passive immunization are three major therapeutic approaches to fighting COVID-19. Chicken immunoglobulin Y (IgY) has the potential to be used as neutralizing antibody against respiratory infections, and its advantages include high avidity, low risk of adverse immune responses, and easy local delivery by intranasal administration. In this study, we raised antibody against the spike (S) protein of SARS-CoV-2 in chickens and extracted IgY (called IgY-S) from egg yolk. IgY-S exhibited high immunoreactivity against SARS-CoV-2 S, and by epitope mapping, we found five linear epitopes of IgY-S in SARS-CoV-2 S, two of which are cross-reactive with SARS-CoV S. Notably, epitope SIIAYTMSL, one of the identified epitopes, partially overlaps the S1/S2 cleavage region in SARS-CoV-2 S and is located on the surface of S trimer in 3D structure, close to the S1/S2 cleavage site. Thus, antibody binding at this location could physically block the access of proteolytic enzymes to S1/S2 cleavage site and thereby impede S1/S2 proteolytic cleavage, which is crucial to subsequent virus-cell membrane fusion and viral cell entry. Therefore, the feasibility of using IgY-S or epitope SIIAYTMS-specific IgY as neutralizing antibody for preventing or treating SARS-CoV-2 infection is worth exploring.

Detection of specific IgE against linear epitopes from Gal d 1 has additional value in diagnosing hen’s egg allergy in adults

Ehlers, Anna M.; Otten, Henny G.; Wierzba, Eva; Flügge, Ulrike; Le, Thuy-My; Knulst, André C.; Suer, Waltraud
Sep 2020
10.1111/cea.13730
Background Although hen’s egg allergy is more prevalent in children, up to 0.6% of adults from different European countries suffers from a persistent or newly-onset hen’s egg allergy, making accurate diagnosis in adults necessary. However, sensitisation to hen’s egg extracts, components and linear epitopes are solely studied in children. Methods Hen’s egg allergic (n=16) and tolerant (n=20) adults were selected by sensitisation towards recombinant components rGal d 1 and/or 3. Sensitisation profiles towards egg white and yolk extract and the native components Gal d 1, 2, 3 and 4 were respectively evaluated with the ImmunoCAP or the EUROLINE system. Characterisation of linear epitopes was performed with a peptide microarray containing 15mer peptides representing the entire sequence of mature Gal d 1 and 3. Results Overall, sIgE titres against hen’s egg extracts and single components overlapped largely between allergic and tolerant adults. Although the median sIgE/sIgG4 ratio to Gal d 1 was increased in allergic adults, the range was comparable between both groups. Clinically relevant sensitisation to Gal d 1 was confirmed by sIgE-binding to the linear epitopes aa30-41, aa39-50 or aa84-95 in 6/13 allergic adults, mainly suffering from objective symptoms. In comparison, these epitopes were recognized by 1/15 tolerant patient. Only a few linear epitopes were detected for Gal d 3, suggesting a greater importance of conformational epitopes for the recognition of Gal d 3. Conclusion and Clinical Relevance Specific IgE-binding to linear epitopes of Gal d 1 is highly specific in identifying hen’s egg allergic adults with objective symptoms.

Rapid response to pandemic threats: immunogenic epitope detection of pandemic pathogens for diagnostics and vaccine development using peptide microarrays

Heiss, Kirsten; Heidepriem, Jasmin; Fischer, Nico; Weber, Laura K; Dahlke, Christine; Jaenisch, Thomas; Loeffler, Felix F
J. Proteome Res..
Sep 2020
10.1021/acs.jproteome.0c00484
Emergence and re-emergence of pathogens bearing the risk of becoming a pandemic threat are on the rise. Increased travel and trade, growing population density, changes in urbanization, and climate have a critical impact on infectious disease spread. Currently, the world is confronted with the emergence of a novel coronavirus SARS-CoV-2, responsible for yet more than 500 000 deaths globally. Outbreaks caused by viruses such as SARS-CoV-2, HIV, Ebola, influenza, and Zika have increased over the last decade, underlining the urgent need for a rapid development of diagnostics and vaccines. Hence, the rational identification of biomarkers for diagnostic measures on the one hand, and antigenic targets for vaccine development on the other, are of utmost importance. Peptide microarrays can display large numbers of putative target proteins translated into overlapping linear (and cyclic) peptides. Using these highly diverse libraries, covering tens of thousands of peptides, allow for the in-depth analysis of antibody signatures in a multiplexed, high-throughput fashion. In this review, we highlight synthesis platforms that facilitate fast and highly flexible generation of high-density peptide microarrays. We further outline the multifaceted applications of these peptide array platforms for the development of serological tests and vaccines, to quickly encounter pandemic threats.

Expression of different L1 isoforms of Mastomys natalensis papillomavirus as mechanism to circumvent adaptive immunity

Fu, Yingying; Cao, Rui; Schäfer, Miriam; Stephan, Sonja; Braspenning-Wesch, Ilona; Schmitt, Laura; Bischoff, Ralf; Müller, Martin; Schäfer, Kai; Vinzón, Sabrina E; Rösl, Frank; Hasche, Daniel
Aug 2020
10.7554/eLife.57626
Although many high-risk mucosal and cutaneous human papillomaviruses (HPVs) theoretically have the potential to synthesize L1 isoforms differing in length, previous seroepidemiological studies only focused on the short L1 variants, co-assembling with L2 to infectious virions. Using the multimammate mouse Mastomys coucha as preclinical model, this is the first study demonstrating seroconversion against different L1 isoforms during the natural course of papillomavirus infection. Intriguingly, positivity with the cutaneous MnPV was accompanied by a strong seroresponse against a longer L1 isoform, but to our surprise, the raised antibodies were non-neutralizing. Only after a delay of around 4 months, protecting antibodies against the short L1 appeared, enabling the virus to successfully establish an infection. This argues for a novel humoral immune escape mechanism that may also have important implications on the interpretation of epidemiological data in terms of seropositivity and protection of PV infections in general.

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