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

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Survivin prevents the polycomb repressor complex 2 from methylating histone 3 lysine 27

Jensen, Maja; Chandrasekaran, Venkataragavan; García-Bonete, María-José; Li, Shuxiang; Anindya, Atsarina Larasati; Andersson, Karin; Erlandsson, Malin C.; Oparina, Nina Y.; Burmann, Björn M.; Brath, Ulrika; Panchenko, Anna R.; Bokarewa I., Maria; Katona, Gergely
iScience.
Jul 2023
10.1016/j.isci.2023.106976

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.

Machine learning-driven multifunctional peptide engineering for sustained ocular drug delivery

Hsueh, Henry T.; Chou, Renee Ti; Rai, Usha; Liyanage, Wathsala; Kim, Yoo Chun; Appell, Matthew B.; Pejavar, Jahnavi; Leo, Kirby T.; Davison, Charlotte; Kolodziejski, Patricia; Mozzer, Ann; Kwon, HyeYoung; Sista, Maanasa; Anders, Nicole M.; Hemingway, Avelina; Rompicharla, Sri Vishnu Kiran; Edwards, Malia; Pitha, Ian; Hanes, Justin; Cummings, Michael P.; Ensign, Laura M.
Nat Commun.
May 2023
10.1038/s41467-023-38056-w
Abstract Sustained drug delivery strategies have many potential benefits for treating a range of diseases, particularly chronic diseases that require treatment for years. For many chronic ocular diseases, patient adherence to eye drop dosing regimens and the need for frequent intraocular injections are significant barriers to effective disease management. Here, we utilize peptide engineering to impart melanin binding properties to peptide-drug conjugates to act as a sustained-release depot in the eye. We develop a super learning-based methodology to engineer multifunctional peptides that efficiently enter cells, bind to melanin, and have low cytotoxicity. When the lead multifunctional peptide (HR97) is conjugated to brimonidine, an intraocular pressure lowering drug that is prescribed for three times per day topical dosing, intraocular pressure reduction is observed for up to 18 days after a single intracameral injection in rabbits. Further, the cumulative intraocular pressure lowering effect increases ~17-fold compared to free brimonidine injection. Engineered multifunctional peptide-drug conjugates are a promising approach for providing sustained therapeutic delivery in the eye and beyond.

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.

IgE and IgG4 Epitopes of Dermatophagoides and Blomia Allergens before and after Sublingual Immunotherapy

Figo, Daniele Danella; Cordeiro Macedo, Priscilla Rios; Gadermaier, Gabriele; Remuzgo, Cesar; Castro, Fábio Fernandes Morato; Kalil, Jorge; Galvão, Clovis Eduardo Santos; Santos, Keity Souza
IJMS.
Feb 2023
10.3390/ijms24044173
Sublingual immunotherapy (SLIT) is used worldwide to treat house dust mites (HDM) allergy. Epitope specific immunotherapy with peptide vaccines is used far less, but it is of great interest in the treatment of allergic reactions, as it precludes the drawbacks of allergen extracts. The ideal peptide candidates would bind to IgG, blocking IgE-binding. To better elucidate IgE and IgG4 epitope profiles during SLIT, sequences of main allergens, Der p 1, 2, 5, 7, 10, 23 and Blo t 5, 6, 12, 13, were included in a 15-mer peptide microarray and tested against pooled sera from 10 patients pre- and post-1-year SLIT. All allergens were recognized to some extent by at least one antibody isotype and peptide diversity was higher post-1-year SLIT for both antibodies. IgE recognition diversity varied among allergens and timepoints without a clear tendency. Der p 10, a minor allergen in temperate regions, was the molecule with more IgE-peptides and might be a major allergen in populations highly exposed to helminths and cockroaches, such as Brazil. SLIT-induced IgG4 epitopes were directed against several, but not all, IgE-binding regions. We selected a set of peptides that recognized only IgG4 or were able to induce increased ratios of IgG4:IgE after one year of treatment and might be potential targets for vaccines.

Funneling modulatory peptide design with generative models: Discovery and characterization of disruptors of calcineurin protein-protein interactions

Tubiana, Jérôme; Adriana-Lifshits, Lucia; Nissan, Michael; Gabay, Matan; Sher, Inbal; Sova, Marina; Wolfson, Haim J.; Gal, Maayan
PLoS Comput Biol.
Feb 2023
10.1371/journal.pcbi.1010874
Design of peptide binders is an attractive strategy for targeting “undruggable” protein-protein interfaces. Current design protocols rely on the extraction of an initial sequence from one known protein interactor of the target protein, followed by in-silico or in-vitro mutagenesis-based optimization of its binding affinity. Wet lab protocols can explore only a minor portion of the vast sequence space and cannot efficiently screen for other desirable properties such as high specificity and low toxicity, while in-silico design requires intensive computational resources and often relies on simplified binding models. Yet, for a multivalent protein target, dozens to hundreds of natural protein partners already exist in the cellular environment. Here, we describe a peptide design protocol that harnesses this diversity via a machine learning generative model. After identifying putative natural binding fragments by literature and homology search, a compositional Restricted Boltzmann Machine is trained and sampled to yield hundreds of diverse candidate peptides. The latter are further filtered via flexible molecular docking and an in-vitro microchip-based binding assay. We validate and test our protocol on calcineurin, a calcium-dependent protein phosphatase involved in various cellular pathways in health and disease. In a single screening round, we identified multiple 16-length peptides with up to six mutations from their closest natural sequence that successfully interfere with the binding of calcineurin to its substrates. In summary, integrating protein interaction and sequence databases, generative modeling, molecular docking and interaction assays enables the discovery of novel protein-protein interaction modulators.

Probing peptide sequences on their ability to generate affinity sites in molecularly imprinted polymers

Piletska, Elena V; Guerreiro, Antonio; Mersiyanova, Margarita; Cowen, Todd; Canfarotta, Francesco; Piletsky, Stanislav S.; Karim, Kal; Piletsky, Sergey A.
Langmuir.
Dec 2019
10.1021/acs.langmuir.9b03410
An array of 4000 defined and addressable tripeptides on a polymer-coated glass slide is used to synthesize molecularly imprinted polymer (MIP) nanoparticles. This work is undertaken to systematically probe the impact of the peptide sequence on the ability to generate affinity MIPs. The polymer affinity is assessed by measuring the fluorescence of bound MIP nanoparticles at each peptide spot on the surface after washing the array to remove any low-affinity polymer. The generic composition commonly used in the preparation of MIPs against proteins seems to be equally suitable for imprinting hydrophobic and hydrophilic tripeptides. The amino acids frequently contributing to the formation of high-affinity MIPs include T, F, D, N, Y, W, and P. The amino acids that rarely contribute to the formation of high-affinity interactions with MIPs are G, V, A, L, I, and M. These observations are confirmed by computational modeling. The basic technique proposed here may be applicable in optimizing polymer compositions for the production of high-affinity MIPs or, more specifically, for the selection of appropriate amino acid sequences when peptide epitopes are used instead of whole protein imprinting.

Diagnostic Profiling of the Human Public IgM Repertoire With Scalable Mimotope Libraries

Pashov, Anastas; Shivarov, Velizar; Hadzhieva, Maya; Kostov, Victor; Ferdinandov, Dilyan; Heintz, Karen-Marie; Pashova, Shina; Todorova, Milena; Vassilev, Tchavdar; Kieber-Emmons, Thomas; Meza-Zepeda, Leonardo A.; Hovig, Eivind
Front. Immunol..
Dec 2019
10.3389/fimmu.2019.02796
Specific antibody reactivities are routinely used as biomarkers, but the antibody repertoire reactivity (igome) profiles are still neglected. Here, we propose rationally designed peptide arrays as efficient probes for these system level biomarkers. Most IgM antibodies are characterized by few somatic mutations, polyspecificity, and physiological autoreactivity with housekeeping function. Previously, probing this repertoire with a set of immunodominant self-proteins provided a coarse analysis of the respective repertoire profiles. In contrast, here, we describe the generation of a peptide mimotope library that reflects the common IgM repertoire of 10,000 healthy donors. In addition, an appropriately sized subset of this quasi-complete mimotope library was further designed as a potential diagnostic tool. A 7-mer random peptide phage display library was panned on pooled human IgM. Next-generation sequencing of the selected phage yielded 224,087 sequences, which clustered in 790 sequence clusters. A set of 594 mimotopes, representative of the most significant sequence clusters, was shown to probe symmetrically the space of IgM reactivities in patients’ sera. This set of mimotopes can be easily scaled including a greater proportion of the mimotope library. The trade-off between the array size and the resolution can be explored while preserving the symmetric sampling of the mimotope sequence and reactivity spaces. BLAST search of the non-redundant protein database with the mimotopes sequences yielded significantly more immunoglobulin J region hits than random peptides, indicating a considerable idiotypic connectivity of the targeted igome. The proof of principle predictors for random diagnoses was represented by profiles of mimotopes. The number of potential reactivity profiles that can be extracted from this library is estimated at more than 1070. Thus, a quasi-complete IgM mimotope library and a scalable representative subset thereof are found to address very efficiently the dynamic diversity of the human public IgM repertoire, providing informationally dense and structurally interpretable IgM reactivity profiles.

Ara h 7 isoforms share many linear epitopes: Are 3D epitopes crucial to elucidate divergent abilities?

Ehlers, Anna M.; Klinge, Marco; Suer, Waltraud; Weimann, Yvonne; Knulst, André C.; Besa, Frithjof; Le, Thuy‐My; Otten, Henny G.
Clin Exp Allergy.
Nov 2019
10.1111/cea.13496
Background The peanut allergens Ara h 2, h 6, and h 7 are potent allergens and can trigger severe reactions. Ara h 7 consists of three isoforms differing in their ability to induce basophil degranulation, whereas the ability of Ara h 7.0201 is comparable to Ara h 2 and 6 as shown in previous literature. Objective To identify linear epitopes of Ara h 7.0101, Ara h 7.0201 and Ara h 7.0301 recognized by IgE and IgG4 from patients sensitized to Ara h 7 and to investigate their potential to elucidate divergent abilities of the Ara h 7 isoforms in inducing basophil activation. Methods Linear epitopes recognized by IgE and IgG4 were mapped by peptide microarray analysis containing 15-mer peptides of Ara h 2.0201, 6, 7.0101, 7.0201 and 7.0301 and 39 peanut allergic patients sensitized to Ara h 7 (discovery). For validation, 20-mer peptides containing the minimal epitope and surrounding amino acids were incubated with 25 sensitized patients and 10 controls (validation). Results Three out of 14 linear epitopes were unique for each isoform (Ara h 7.0101: aa 97-109; Ara h 7.0201: aa 122-133; Ara h 7.0301: aa 65-74) but scarcely recognized by IgE. The main linear IgE epitope (aa 51-57) located in the long flexible loop of all Ara h 7 isoforms was bound by antibodies from 31% of the patients (discovery and validation cohort). Regarding IgG4, 55% of the patients recognized an epitope present on all isoforms (aa 55-65), whereas epitope aa 129-137, only present on Ara h 7.0101/0.0301, was recognized by 38% of the patients. Recognition was highly individual, although 20% of the patients recognized any linear epitope neither by IgE nor by IgG4 despite a low mean z-score of ≥ 1.7. Remarkably, only 50% of the patients recognized one or more epitopes by IgE. Conclusion & Clinical Relevance Ara h 7 isoforms share many linear epitopes being easily accessible for antibody binding. Unique epitopes, essential to elucidate divergent potencies, were scarcely recognized, suggesting a crucial involvement of conformational epitopes.

A Low‐Cost Laser‐Based Nano‐3D Polymer Printer for Rapid Surface Patterning and Chemical Synthesis of Peptide and Glycan Microarrays

Eickelmann, Stephan; Tsouka, Alexandra; Heidepriem, Jasmin; Paris, Grigori; Zhang, Junfang; Molinari, Valerio; Mende, Marco; Loeffler, Felix F.
Adv. Mater. Technol..
Nov 2019
10.1002/admt.201900503
A low-cost laser-based printing setup is presented, which allows for the spot-wise patterning of surfaces with defined polymer nanolayers. These nanolayer spots serve as a “solid solvent,” embedding different chemicals, chemical building blocks, materials, or precursors and can be stacked on top of each other. By melting the spot pattern, the polymer-embedded molecules are released for chemical reaction. This enables researchers to quickly pattern a surface with different molecules and materials, mixing them directly on the surface for high-throughput chemical synthesis to generate and screen diverse microarray libraries. In contrast to expensive ink-jet or contact printing, this approach does not require premixing of inks, which enables in situ combinatorial mixing. Easy access and versatility of this patterning approach are shown by generating microarrays of various biomolecules, such as glycans for the first time, to screen interactions of antibodies and lectins. In addition, a layer-by-layer solid-phase synthesis of peptides directly on the microarray is presented. Amino acid–containing nanolayers are repeatedly laser-transferred and reacted with the functionalized acceptor surface in defined patterns. This simple system enables a reproducible array production, down to spot-to-spot distances of 100 µm, and offers a flexible and cheap alternative to expensive spotting robot technology.

Immunization of cats to induce neutralizing antibodies against Fel d 1, the major feline allergen in human subjects

Thoms, Franziska; Jennings, Gary T.; Maudrich, Melanie; Vogel, Monique; Haas, Stefanie; Zeltins, Andris; Hofmann-Lehmann, Regina; Riond, Barbara; Grossmann, Jonas; Hunziker, Peter; Fettelschoss-Gabriel, Antonia; Senti, Gabriela; Kündig, Thomas M.; Bachmann, Martin F.
Journal of Allergy and Clinical Immunology.
Jul 2019
10.1016/j.jaci.2019.01.050
Background Cat allergy in human subjects is usually caused by the major cat allergen Fel d 1 and is found in approximately 10% of the Western population. Currently, there is no efficient and safe therapy for cat allergy available. Allergic patients usually try to avoid cats or treat their allergy symptoms. Objective We developed a new strategy to treat Fel d 1–induced allergy in human subjects by immunizing cats against their own major allergen, Fel d 1. Methods A conjugate vaccine consisting of recombinant Fel d 1 and a virus-like particle derived from the cucumber mosaic virus containing the tetanus toxin–derived universal T-cell epitope tt830-843 (CuMVTT) was used to immunize cats. A first tolerability and immunogenicity study, including a boost injection, was conducted by using the Fel-CuMVTT vaccine alone or in combination with an adjuvant. Results The vaccine was well tolerated and had no overt toxic effect. All cats induced a strong and sustained specific IgG antibody response. The induced anti–Fel d 1 antibodies were of high affinity and exhibited a strong neutralization ability tested both in vitro and in vivo. A reduction in the endogenous allergen level and a reduced allergenicity of tear samples, were observed. Conclusion Vaccination of cats with Fel-CuMVTT induces neutralizing antibodies and might result in reduced symptoms of allergic cat owners. Both human subjects and animals could profit from this treatment because allergic cat owners would reduce their risk of developing chronic diseases, such as asthma, and become more tolerant of their cats, which therefore could stay in the households and not need to be relinquished to animal shelters.

Miniaturized and Automated Synthesis of Biomolecules—Overview and Perspectives

Mattes, Daniela S.; Jung, Nicole; Weber, Laura K.; Bräse, Stefan; Breitling, Frank
Adv. Mater..
Jun 2019
10.1002/adma.201806656
Chemical synthesis is performed by reacting different chemical building blocks with defined stoichiometry, while meeting additional conditions, such as temperature and reaction time. Such a procedure is especially suited for automation and miniaturization. Life sciences lead the way to synthesizing millions of different oligonucleotides in extremely miniaturized reaction sites, e.g., pinpointing active genes in whole genomes, while chemistry advances different types of automation. Recent progress in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging could match miniaturized chemical synthesis with a powerful analytical tool to validate the outcome of many different synthesis pathways beyond applications in the life sciences. Thereby, due to the radical miniaturization of chemical synthesis, thousands of molecules can be synthesized. This in turn should allow ambitious research, e.g., finding novel synthesis routes or directly screening for photocatalysts. Herein, different technologies are discussed that might be involved in this endeavor. A special emphasis is given to the obstacles that need to be tackled when depositing tiny amounts of materials to many different extremely miniaturized reaction sites.

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