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

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Combinatorial Synthesis of Macromolecular Arrays by Microchannel Cantilever Spotting (µCS)

Atwater, Jordyn; Mattes, Daniela S.; Streit, Bettina; von Bojničić-Kninski, Clemens; Loeffler, Felix F.; Breitling, Frank; Fuchs, Harald; Hirtz, Michael
Adv. Mater..
Aug 2018
10.1002/adma.201801632
Surface-bound microarrays of multiple oligo- and macromolecules (e.g., peptides, DNA) offer versatile options in biomedical applications like drug screening, DNA analysis, or medical diagnostics. Combinatorial syntheses of these molecules in situ can save significant resources in regard to processing time and material use. Furthermore, high feature densities are needed to enable high-throughput and low sample volumes as generally regarded in combinatorial chemistry. Here, a scanning-probe-lithography-based approach for the combinatorial in situ synthesis of macromolecules is presented in microarray format. Feature sizes below 40 µm allow for the creation of high-density arrays with feature densities of 62 500 features per cm2. To demonstrate feasibility of this approach for biomedical applications, a multiplexed array of functional protein tags (HA- and FLAG-tag) is synthesized, and selective binding of respective epitope recognizing antibodies is shown. This approach uses only small amounts of base chemicals for synthesis and can be further parallelized, therefore, opening up a route to flexible, highly dense, and cost-effective microarrays.

Universal detection of foot and mouth disease virus based on the conserved VP0 protein

Loureiro, Silvia; Porta, Claudine; Maity, Hemanta K.; Perez, Eva; Bagno, Flavia F.; Kotecha, Abhay; Fry, Elizabeth; Ren, Jingshan; Stuart, David I.; Hoenemann, Holger; Serrano, Amaya; van den Born, Erwin; Charleston, Bryan; Jones, Ian M.
Wellcome Open Res.
Jul 2018
10.12688/wellcomeopenres.14655.1
Background : Foot and mouth disease virus (FMDV), a member of the picornaviridae that causes vesicular disease in ungulates, has seven serotypes and a large number of strains, making universal detection challenging. The mature virion is made up of 4 structural proteins, virus protein (VP) 1 – VP4, VP1-VP3 of which form the outer surface of the particle and VP4 largely contained within. Prior to mature virion formation VP2 and VP4 occur together as VP0, a structural component of the pre-capsid which, as a result of containing the internal VP4 sequence, is relatively conserved among all strains and serotypes. Detection of VP0 might therefore represent a universal virus marker. Methods : FMDV virus protein 0 (VP0) was expressed in bacteria as a SUMO fusion protein and the SUMO carrier removed by site specific proteolysis. Rabbit polyvalent sera were generated to the isolated VP0 protein and their reactivity characterised by a number of immunoassays and by epitope mapping on peptide arrays. Results : The specific VP0 serum recognised a variety of FMDV serotypes, as virus and as virus-like-particles, by a variety of assay formats. Epitope mapping showed the predominant epitopes to occur within the unstructured but highly conserved region of the sequence shared among many serotypes. When immunogold stained VLPs were assessed by TEM analysis they revealed exposure of epitopes on the surface of some particles, consistent with particle breathing hitherto reported for some other picornaviruses but not for FMDV. Conclusion : A polyvalent serum based on the VP0 protein of FMDV represents a broadly reactive reagent capable of detection of many if not all FMDV isolates. The suggestion of particle breathing obtained with this serum suggests a reconsideration of the FMDV entry mechanism.

Reductionist Approach in Peptide-Based Nanotechnology

Gazit, Ehud
Annu. Rev. Biochem..
Jun 2018
10.1146/annurev-biochem-062917-012541
The formation of ordered nanostructures by molecular self-assembly of proteins and peptides represents one of the principal directions in nanotechnology. Indeed, polyamides provide superior features as materials with diverse physical properties. A reductionist approach allowed the identification of extremely short peptide sequences, as short as dipeptides, which could form well-ordered amyloid-like β-sheet-rich assemblies comparable to supramolecular structures made of much larger proteins. Some of the peptide assemblies show remarkable mechanical, optical, and electrical characteristics. Another direction of reductionism utilized a natural noncoded amino acid, α-aminoisobutryic acid, to form short superhelical assemblies. The use of this exceptional helix inducer motif allowed the fabrication of single heptad repeats used in various biointerfaces, including their use as surfactants and DNA-binding agents. Two additional directions of the reductionist approach include the use of peptide nucleic acids (PNAs) and coassembly techniques. The diversified accomplishments of the reductionist approach, as well as the exciting future advances it bears, are discussed.

A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein

Tan, Joshua; Sack, Brandon K; Oyen, David; Zenklusen, Isabelle; Piccoli, Luca; Barbieri, Sonia; Foglierini, Mathilde; Fregni, Chiara Silacci; Marcandalli, Jessica; Jongo, Said; Abdulla, Salim; Perez, Laurent; Corradin, Giampietro; Varani, Luca; Sallusto, Federica; Sim, Betty Kim Lee; Hoffman, Stephen L; Kappe, Stefan H I; Daubenberger, Claudia; Wilson, Ian A; Lanzavecchia, Antonio
Nat Med.
Mar 2018
10.1038/nm.4513
Immunization with attenuated Plasmodium falciparum sporozoites (PfSPZs) has been shown to be protective against malaria, but the features of the antibody response induced by this treatment remain unclear. To investigate this response in detail, we isolated IgM and IgG monoclonal antibodies from Tanzanian volunteers who were immunized with repeated injection of Sanaria PfSPZ Vaccine and who were found to be protected from controlled human malaria infection with infectious homologous PfSPZs. All isolated IgG monoclonal antibodies bound to P. falciparum circumsporozoite protein (PfCSP) and recognized distinct epitopes in its N terminus, NANP-repeat region, and C terminus. Strikingly, the most effective antibodies, as determined in a humanized mouse model, bound not only to the repeat region, but also to a minimal peptide at the PfCSP N-terminal junction that is not in the RTS,S vaccine. These dual-specific antibodies were isolated from different donors and were encoded by VH3-30 or VH3-33 alleles that encode tryptophan or arginine at position 52. Using structural and mutational data, we describe the elements required for germline recognition and affinity maturation. Our study provides potent neutralizing antibodies and relevant information for lineage-targeted vaccine design and immunization strategies.

A Trifunctional Linker for Purified 3D Assembled Peptide Structure Arrays

Mattes, Daniela S.; Rentschler, Simone; Foertsch, Tobias C.; Münch, Stephan W.; Loeffler, Felix F.; Nesterov-Mueller, Alexander; Bräse, Stefan; Breitling, Frank
Small Methods.
Feb 2018
10.1002/smtd.201700205
Microarrays are an important tool in modern research that allow the rapid screening of many different interactions simultaneously. Peptide arrays, which bear different peptides arranged in separate spots, permit high-throughput screening to investigate linear and cyclic binding sites. To study conformational or discontinuous binding sites, protein arrays are the major choice. However, the tremendous costs for the generation of high-density protein arrays of high purity restrict progress in protein research. Therefore, peptide-based arrays, which can mimic assembled peptide structures, have an enormous potential. Here, a method is presented to create such structures in the array format as an alternative to protein arrays. A trifunctional linker is developed with an azide, a protected alkyne, and a carboxyl group, which can react with two or three different peptides. Due to the spatial proximity, the peptides interact and can form an assembled peptide structure. As a proof of concept, assembled peptide structures are demonstrated on beads and on a polymer surface and the approach can be validated via matrix-assisted laser desorption/ionization spectrometry. Furthermore, a multistep transfer of peptide arrays is shown, generating purified assembled peptide structure arrays in high density.

Development and characterization of a human monoclonal antibody targeting the N-terminal region of hepatitis C virus envelope glycoprotein E1

Mesalam, Ahmed Atef; Desombere, Isabelle; Farhoudi, Ali; Van Houtte, Freya; Verhoye, Lieven; Ball, Jonathan; Dubuisson, Jean; Foung, Steven K.H.; Patel, Arvind H.; Persson, Mats A.A.; Leroux-Roels, Geert; Meuleman, Philip
Virology.
Jan 2018
10.1016/j.virol.2017.10.019
Monoclonal antibodies (mAbs) targeting the hepatitis C virus (HCV) envelope have been raised mainly against envelope protein 2 (E2), while the antigenic epitopes of envelope protein 1 (E1) are not fully identified. Here we describe the detailed characterization of a human mAb, designated A6, generated from an HCV genotype 1b infected patient. ELISA results showed reactivity of mAb A6 to full-length HCV E1E2 of genotypes 1a, 1b and 2a. Epitope mapping identified a region spanning amino acids 230–239 within the N-terminal region of E1 as critical for binding. Antibody binding to this epitope was not conformation dependent. Neutralization assays showed that mAb A6 lacks neutralizing capacity and does not interfere with the activity of known neutralizing antibodies. In summary, mAb A6 is an important tool to study the structure and function of E1 within the viral envelope, a crucial step in the development of an effective prophylactic HCV vaccine.

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.

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.

Identification of two conserved B-cell epitopes in the gp90 of reticuloendothelial virus using peptide microarray

Khairy, Wiaam O.A.; Qian, Kun; Shao, Hongxia; Ye, Jianqiang; Qin, Aijian
Veterinary Microbiology.
Nov 2017
10.1016/j.vetmic.2017.10.009
Since the gp90 protein of Reticuloendotheliosis virus (REV) plays vital roles in virus neutralization, so detailed analysis of REV-gp90 epitopes is important for the development of epitope based marker vaccines and diagnostic tools for REV infections. In this study, two monoclonal antibodies (mAbs) namely 6C12 and 09980 were used to map the epitopes in REVgp90 using peptide microarray and ELISA. Peptide microarray revealed that mAbs 6C12 and 09980 recognized 216YHPLA220 and 230DPQTSDILEA239 motifs, respectively. This result was confirmed by ELISA using synthetic peptides. Moreover, homology analysis indicated that mAbs defined epitopes are highly conserved among REV strains used in this study. The mAbs and their epitopes identified in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines for control of REV infections.

Efficacy of an Adenoviral Vectored Multivalent Centralized Influenza Vaccine

Lingel, Amy; Bullard, Brianna L.; Weaver, Eric A.
Sci Rep.
Nov 2017
10.1038/s41598-017-14891-y
Mice were immunized with Adenovirus expressing the H1-con, H2-con, H3-con and H5-con HA consensus genes in combination (multivalent) and compared to mice immunized with the traditional 2010–2011 FluZone and FluMist seasonal vaccines. Immunized mice were challenged with 10–100 MLD50 of H1N1, H3N1, H3N2 and H5N1 influenza viruses. The traditional vaccines induced robust levels of HA inhibition (HI) titers, but failed to protect against five different heterologous lethal influenza challenges. Conversely, the multivalent consensus vaccine (1 × 1010 virus particles (vp)/mouse) induced protective HI titers of ≥40 against 8 of 10 influenza viruses that represent a wide degree of divergence within the HA subtypes and protected 100% of mice from 8 of 9 lethal heterologous influenza virus challenges. The vaccine protection was dose dependent, in general, and a dose as low as 5 × 107 vp/mouse still provided 100% survival against 7 of 9 lethal heterologous influenza challenges. These data indicate that very low doses of Adenovirus-vectored consensus vaccines induce superior levels of immunity against a wide divergence of influenza subtypes as compared to traditional vaccines. These doses are scalable and translatable to humans and may provide the foundation for complete and long-lasting anti-influenza immunity.

Identification of a Tetanus Toxin Specific Epitope in Single Amino Acid Resolution

Palermo, Andrea; Weber, Laura K.; Rentschler, Simone; Isse, Awale; Sedlmayr, Martyna; Herbster, Karin; List, Volker; Hubbuch, Jürgen; Löffler, Felix F.; Nesterov-Müller, Alexander; Breitling, Frank
Biotechnol. J..
Oct 2017
10.1002/biot.201700197
Vaccinations are among the most potent tools to fight infectious diseases. However, cross-reactions are an ongoing problem and there is an urgent need to fully understand the mechanisms of the immune response. For the development of a methodological workflow, the linear epitopes in the immune response to the tetanus toxin is investigated in sera of 19 vaccinated Europeans applying epitope mapping with peptide arrays. The most prominent epitope, appearing in nine different sera (923IHLVNNESSEVIVHK937), is investigated in a substitution analysis to identify the amino acids that are crucial for the binding of the corresponding antibody species − the antibody fingerprint. The antibody fingerprints of different individuals are compared and found to be strongly conserved (929ExxEVIVxK937), which is astonishing considering the randomness of their development. Additionally, the corresponding antibody species is isolated from one serum with batch chromatography using the amino acid sequence of the identified epitope and the tetanus specificity of the isolated antibody is verified by ELISA. Studying antibody fingerprints with peptide arrays should be transferable to any kind of humoral immune response toward protein antigens. Furthermore, antibody fingerprints have shown to be highly disease-specific and, therefore, can be employed as reliable biomarkers enabling the study of cross-reacting antigens.

Antibody fingerprints in lyme disease deciphered with high density peptide arrays

Weber, Laura K.; Isse, Awale; Rentschler, Simone; Kneusel, Richard E.; Palermo, Andrea; Hubbuch, Jürgen; Nesterov-Mueller, Alexander; Breitling, Frank; Loeffler, Felix F.
Eng. Life Sci..
Oct 2017
10.1002/elsc.201700062
Lyme disease is the most common tick-borne infectious disease in Europe and North America. Previous studies discovered the immunogenic role of a surface-exposed lipoprotein (VlsE) of Borreliella burgdorferi. We employed high density peptide arrays to investigate the antibody response to the VlsE protein in VlsE-positive patients by mapping the protein as overlapping peptides and subsequent in-depth epitope substitution analyses. These investigations led to the identification of antibody fingerprints represented by a number of key residues that are indispensable for the binding of the respective antibody. This approach allows us to compare the antibody specificities of different patients to the resolution of single amino acids. Our study revealed that the sera of VlsE-positive patients recognize different epitopes on the protein. Remarkably, in those cases where the same epitope is targeted, the antibody fingerprint is almost identical. Furthermore, we could correlate two fingerprints with human autoantigens and an Epstein-Barr virus epitope; yet, the link to autoimmune disorders seems unlikely and must be investigated in further studies. The other three fingerprints are much more specific for B. burgdorferi. Since antibody fingerprints of longer sequences have proven to be highly disease specific, our findings suggest that the fingerprints could function as diagnostic markers that can reduce false positive test results.

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