13 research outputs found
Peptide microarray fabrication by laser-based in situ synthesis and utilization for infectious disease research
Due to the circulation of infectious diseases and the emergence of new pathogens, fundamental research, as well as the development of vaccines are of utmost importance. Peptide microarrays (PMAs) can facilitate the investigation of an immune response to an antigen by detecting linear B cell epitopes. They consist of a miniaturized spot pattern containing different peptide sequences. These reproduce all potential linear epitopes of a protein, usually as a map of overlapping peptides. Therefore, PMAs allow for high-throughput screening in a fast manner. To show their versatility, PMAs were applied for the detection of linear B cell epitopes elicited by infectious pathogens or a vaccine-delivered antigen. First, PMAs of the Ebola virus spike glycoprotein were used to analyze the development of antibodies, recognizing linear peptide epitopes, in vaccine recipients and an Ebola virus disease survivor. Second, PMAs covering the SARS-CoV-2 coronavirus proteome were used to identify epitopes. The antibodies elicited by patients with COVID-19 disease were studied during the course of the disease. Today, commercially available PMAs do either lack peptide sequence flexibility and/or a high peptide density. Thus, the price per analyzed sample is high and therefore, reducing the usage of PMAs. Hence, the combinatorial laser-induced forward transfer (cLIFT) technology was developed for the fabrication of high-density PMAs. Thereby, a polymer and an amino acid are transferred via laser irradiation from a donor to an acceptor in a spot pattern. Together with intermittent chemical processing, this laser based technique can be used to in situ synthesize microarrays. With the implementation of an automated synthesizer and optimal synthesis parameters, it was possible to produce up to 20-residue peptides with controlled spot size. Finally, a full combinatorial synthesis of overlapping 15-mer peptides containing the Ebola virus proteome with 4444 and 10 000 spots per cm2 was performed. The antibody binding was compared to a commercial peptide microarray containing the same peptides of the spike glycoprotein. The results revealed an excellent quality up to a density of 4444 spots per cm2. Moreover, the flexibility of this method allows the exchange of building blocks and thus, enables the synthesis of other molecules.Angesichts der Verbreitung von Infektionskrankheiten und dem Auftreten neuer Krankheitserreger sind Grundlagenforschung und die Entwicklung von Impfstoffen von gröĂter Bedeutung. Peptid Microarrays (PMAs) können die Untersuchung einer Immunantwort auf ein Antigen durch den Nachweis linearer B-Zell-Epitope erleichtern. Sie bestehen aus einem miniaturisierten Spotmuster, welches verschiedene Peptidsequenzen enthĂ€lt. Diese bilden alle potentiellen linearen Epitope eines Proteins ab, in der Regel als ĂŒberlappende Peptide. Daher ermöglichen PMAs schnelle Hochdurchsatz-Untersuchungen. Um ihre Vielseitigkeit zu zeigen, wurden PMAs fĂŒr den Nachweis linearer B-Zelle-Epitope eingesetzt, die durch infektiöse Erreger oder durch ein Impfstoff-verabreichtes Antigen ausgelöst wurden. Zuerst wurden PMAs des Ebolavirus Spike-Glykoproteins verwendet, um die Entwicklung von Antikörpern, welche lineare Peptidepitope erkennen, in Geimpften und einem Ăberlebenden der Ebolavirus Erkrankung zu analysieren. Zweitens wurden PMAs, die das Proteom des Coronavirus SARS-CoV 2 umfassen, zur Identifizierung von Epitopen eingesetzt. Somit konnten die gebildeten Antikörper von Patienten mit der COVID-19-Erkrankung im Verlauf der Erkrankung untersucht werden. Heutzutage, mangelt es kommerziell erhĂ€ltlichen PMAs entweder an PeptidsequenzflexibilitĂ€t und/oder an hoher Peptiddichte. Dadurch ist der Preis pro analysierter Probe hoch und schrĂ€nkt somit die Anwendung von PMAs ein. Daher wurde der kombinatorische Laser-induzierte VorwĂ€rtstransfer (cLIFT) zur Herstellung von PMAs mit hoher Dichte entwickelt. Dabei werden Spots, die ein Polymer und eine AminosĂ€ure enthalten, von einem Donator auf einen Akzeptor ĂŒbertragen, um ein Spotmuster zu erzeugen. Zusammen mit intermittierenden chemischen Schritten kann diese Laser-basierte Technik zur in situ Synthese von Microarrays verwendet werden. Mit der EinfĂŒhrung einer automatisierten Synthesemaschine und optimaler Syntheseparameter war es möglich Peptide mit bis zu 20 AminosĂ€uren und kontrollierter SpotgröĂe herzustellen. SchlieĂlich wurde eine vollstĂ€ndig kombinatorische Synthese von ĂŒberlappenden 15-mer Peptiden, die das Proteom des Ebolavirus umfassen, mit 4444 und 10 000 Spots pro cm2 durchgefĂŒhrt. Die Antikörperbindung wurde mit einem kommerziellen Peptid Microarray verglichen, der die gleichen Peptide des Spike-Glykoproteins enthĂ€lt. Die Ergebnisse zeigten eine hervorragende QualitĂ€t bis zu einer Dichte von 4444 Spots pro cm2. DarĂŒber hinaus ermöglicht die FlexibilitĂ€t dieser Methode den Austausch von Bausteinen und damit die Synthese anderer MolekĂŒle
Rapid response to pandemic threats: immunogenic epitope detection of pandemic pathogens for diagnostics and vaccine development using peptide microarrays
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-, responsible for yet more than 800âŻ000 deaths globally. Outbreaks caused by viruses, such as SARS-CoV-, HIV, Ebola, influenza, and Zika, have increased over the past decade, underlining the 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 for a multiplexed, high-throughput antibody analysis. This enabled for example the identification of discriminant/diagnostic epitopes in Zika or influenza and mapping epitope evolution in natural infections versus vaccinations. In this review, we highlight synthesis platforms that facilitate fast and 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
Development of Neutralizing and Non-neutralizing Antibodies Targeting Known and Novel Epitopes of TcdB of Clostridioides difficile.
Clostridioides difficile is the causative bacterium in 15-20% of all antibiotic associated diarrheas. The symptoms associated with C. difficile infection (CDI) are primarily induced by the two large exotoxins TcdA and TcdB. Both toxins enter target cells by receptor-mediated endocytosis. Although different toxin receptors have been identified, it is no valid therapeutic option to prevent receptor endocytosis. Therapeutics, such as neutralizing antibodies, directly targeting both toxins are in development. Interestingly, only the anti-TcdB antibody bezlotoxumab but not the anti-TcdA antibody actoxumab prevented recurrence of CDI in clinical trials. In this work, 31 human antibody fragments against TcdB were selected by antibody phage display from the human naive antibody gene libraries HAL9/10. These antibody fragments were further characterized by in vitro neutralization assays. The epitopes of the neutralizing and non-neutralizing antibody fragments were analyzed by domain mapping, TcdB fragment phage display, and peptide arrays, to identify neutralizing and non-neutralizing epitopes. A new neutralizing epitope within the glucosyltransferase domain of TcdB was identified, providing new insights into the relevance of different toxin regions in respect of neutralization and toxicity
Automated LaserâTransfer Synthesis of HighâDensity Microarrays for Infectious Disease Screening
Laser-induced forward transfer (LIFT) is a rapid laser-patterning technique for high-throughput combinatorial synthesis directly on glass slides. A lack of automation and precision limits LIFT applications to simple proof-of-concept syntheses of fewer than 100 compounds. Here, an automated synthesis instrument is reported that combines laser transfer and robotics for parallel synthesis in a microarray format with up to 10â000 individual reactions cmâ2. An optimized pipeline for amide bond formation is the basis for preparing complex peptide microarrays with thousands of different sequences in high yield with high reproducibility. The resulting peptide arrays are of higher quality than commercial peptide arrays. More than 4800 15-residue peptides resembling the entire Ebola virus proteome on a microarray are synthesized to study the antibody response of an Ebola virus infection survivor. Known and unknown epitopes that serve now as a basis for Ebola diagnostic development are identified. The versatility and precision of the synthesizer is demonstrated by in situ synthesis of fluorescent molecules via Schiff base reaction and multi-step patterning of precisely definable amounts of fluorophores. This automated laser transfer synthesis approach opens new avenues for high-throughput chemical synthesis and biological screening
Automated Laser-Transfer Synthesis of High-Density Microarrays for Infectious Disease Screening
Laser-induced forward transfer (LIFT) is a rapid laser-patterning technique for high-throughput combinatorial synthesis directly on glass slides. A lack of automation and precision limits LIFT applications to simple proof-of-concept syntheses of fewer than 100 compounds. Here, an automated synthesis instrument is reported that combines laser transfer and robotics for parallel synthesis in a microarray format with up to 10â000 individual reactions cmâ2. An optimized pipeline for amide bond formation is the basis for preparing complex peptide microarrays with thousands of different sequences in high yield with high reproducibility. The resulting peptide arrays are of higher quality than commercial peptide arrays. More than 4800 15-residue peptides resembling the entire Ebola virus proteome on a microarray are synthesized to study the antibody response of an Ebola virus infection survivor. Known and unknown epitopes that serve now as a basis for Ebola diagnostic development are identified. The versatility and precision of the synthesizer is demonstrated by in situ synthesis of fluorescent molecules via Schiff base reaction and multi-step patterning of precisely definable amounts of fluorophores. This automated laser transfer synthesis approach opens new avenues for high-throughput chemical synthesis and biological screening
Epitopes of Naturally Acquired and Vaccine-Induced Anti-Ebola Virus Glycoprotein Antibodies in Single Amino Acid Resolution
The Ebola virus (EBOV) can cause severe infections in humans, leading to a fatal outcome in a high percentage of cases. Neutralizing antibodies against the EBOV surface glycoprotein (GP) can prevent infections, demonstrating a straightforward way for an efficient vaccination strategy. Meanwhile, many different anti-EBOV antibodies have been identified, whereas the exact binding epitopes are often unknown. Here, the analysis of serum samples from an EBOV vaccine trial with the recombinant vesicular stomatitis virus-Zaire ebolavirus (rVSV-ZEBOV) and an Ebola virus disease survivor, using high-density peptide arrays, is presented. In this proof-of-principle study, distinct IgG and IgM antibodies binding to different epitopes of EBOV GP is detected: By mapping the whole GP as overlapping peptide fragments, new epitopes and confirmed epitopes from the literature are found. Furthermore, the highly selective binding epitope of a neutralizing monoclonal anti-EBOV GP antibody could be validated. This shows that peptide arrays can be a valuable tool to study the humoral immune response to vaccines in patients and to support Ebola vaccine development
OnâChip NeoâGlycopeptide Synthesis for Multivalent Glycan Presentation
Single glycanâprotein interactions are often weak, such that glycan binding partners commonly utilize multiple, spatially defined binding sites to enhance binding avidity and specificity. Current array technologies usually neglect defined multivalent display. Laserâbased array synthesis technology allows for flexible and rapid onâsurface synthesis of different peptides. By combining this technique with click chemistry, neoâglycopeptides were produced directly on a functionalized glass slide in the microarray format. Density and spatial distribution of carbohydrates can be tuned, resulting in wellâdefined glycan structures for multivalent display. The two lectins concanavalin A and langerin were probed with different glycans on multivalent scaffolds, revealing strong spacingâ, densityâ, and ligandâdependent binding. In addition, we could also measure the surface dissociation constant. This approach allows for a rapid generation, screening, and optimization of a multitude of multivalent scaffolds for glycan binding
Probing Multivalent Carbohydrate-Protein Interactions With On-Chip Synthesized Glycopeptides Using Different Functionalized Surfaces
Multivalent ligandâprotein interactions are a commonly employed approach by nature in many biological processes. Single glycanâprotein interactions are often weak, but their affinity and specificity can be drastically enhanced by engaging multiple binding sites. Microarray technology allows for quick, parallel screening of such interactions. Yet, current glycan microarray methodologies usually neglect defined multivalent presentation. Our laser-based array technology allows for a flexible, cost-efficient, and rapid in situ chemical synthesis of peptide scaffolds directly on functionalized glass slides. Using copper(I)-catalyzed azideâalkyne cycloaddition, different monomer sugar azides were attached to the scaffolds, resulting in spatially defined multivalent glycopeptides on the solid support. Studying their interaction with several different lectins showed that not only the spatially defined sugar presentation, but also the surface functionalization and wettability, as well as accessibility and flexibility, play an essential role in such interactions. Therefore, different commercially available functionalized glass slides were equipped with a polyethylene glycol (PEG) linker to demonstrate its effect on glycanâlectin interactions. Moreover, different monomer sugar azides with and without an additional PEG-spacer were attached to the peptide scaffold to increase flexibility and thereby improve binding affinity. A variety of fluorescently labeled lectins were probed, indicating that different lectinâglycan pairs require different surface functionalization and spacers for enhanced binding. This approach allows for rapid screening and evaluation of spacing-, density-, ligand and surface-dependent parameters, to find optimal lectin binders.BMBF, 13XP5050A, Erforschung einer neuen Methode fĂŒr die Herstellung von hochdichten MolekĂŒlbibliotheken (cLIFT
On-Chip Neo-Glycopeptide Synthesis for Multivalent Glycan Presentation
Single glycan-protein interactions are often weak, such that glycan binding partners commonly utilize multiple, spatially defined binding sites to enhance binding avidity and specificity. Current array technologies usually neglect defined multivalent display. Laser-based array synthesis technology allows for flexible and rapid on-surface synthesis of different peptides. By combining this technique with click chemistry, neo-glycopeptides were produced directly on a functionalized glass slide in the microarray format. Density and spatial distribution of carbohydrates can be tuned, resulting in well-defined glycan structures for multivalent display. The two lectins concanavalin A and langerin were probed with different glycans on multivalent scaffolds, revealing strong spacing-, density-, and ligand-dependent binding. In addition, we could also measure the surface dissociation constant. This approach allows for a rapid generation, screening, and optimization of a multitude of multivalent scaffolds for glycan binding