Andersen, Mikael Rørdam

De Masi, Federico

Engmark, Mikael

Gutiérrez, José María

Hansen, Christian Skjødt

Kringelum, Jens Vindahl

Laustsen, Andreas Hougaard

Lomonte, Bruno

Lund, Ole

Patel, Jigar

Sullivan, Eric

Insight into the molecular details of polyclonal antivenom antibody specificity is a prerequisite for accurate prediction of cross-reactivity and can provide a basis for design of novel antivenoms. In this work, a highthroughput approach was applied to characterize linear elements in epitopes in 82 toxins from four African mamba and three neurotoxic cobra snakes obtained from public databases

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Downloaded from orbit.dtu.dk on: Dec 18, 2017High-throughput epitope profiling of snake venom toxinsunveiling the complexity of antigen-antibody interactions of antivenomsEngmark, Mikael; Andersen, Mikael Rørdam; Laustsen, Andreas Hougaard; Patel, Jigar; Sullivan, Eric;De Masi, Federico; Hansen, Christian Skjødt; Kringelum, Jens Vindahl; Lomonte, Bruno; Gutiérrez, JoséMaría; Lund, OlePublication date:2016Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Engmark, M., Andersen, M. R., Laustsen, A. H., Patel, J., Sullivan, E., De Masi, F., ... Lund, O. (2016). High-throughput epitope profiling of snake venom toxins: unveiling the complexity of antigen-antibody interactions ofantivenoms. Poster session presented at The 12th Congress of the Pan-American Section of the InternationalSociety on Toxinology, Miami Beach, United States.High-throughput epitope profiling of snake venom toxins – unveiling the complexity of antigen-antibody interactions of antivenoms IntroductionInsight into the molecular details of polyclonal antivenom antibodyspecificity is a prerequisite for accurate prediction of cross-reactivity andcan provide a basis for design of novel antivenoms1. In this work, a high-throughput approach was applied to characterize linear elements inepitopes in 82 toxins from four African mamba and three neurotoxic cobrasnakes obtained from public databases.Mikael Engmark1,2, Mikael R. Andersen2, Andreas H. Laustsen2,3, Jigar Patel4, Eric Sullivan4, Federico de Masi1, Christian S. Hansen1, Jens V. Kringelum1, Bruno Lomonte5, José María Gutiérrez5, and Ole Lund1ConclusionsCustom-designed high density peptide microarray technology enables parallel automated identification of linear elements of epitopes in snake neurotoxins.Trend: antivenom antibodies recognize and bind to epitopes at the functional sites of toxins.References1. Labhardt, A. M. et al. European Journal of Biochemistry 177, 295–305 (1988) 2. Cheung, J. et al. Journal of Medicinal Chemistry 55, 10282–10286 (2012) 3. Albrand, J. et al. Biochemistry 34, 5923–5937 (1995) 4. Wang, C. A. et al. Biochemistry 53, 3758–3766 (2014)5. Ramos, H. R. et al. PLOS Neglected Tropical Diseases 10, e0004484 (2016) AcknowledgementWe would like to thank Morten Nielsen for scientific discussion and the Novo Nordisk Foundation for financial support(grant number NNF13OC0005613) Affiliations1. Technical University of Denmark, Department of Bio and Health Informatics, Kgs. Lyngby2. Technical University of Denmark, Department of Biotechnology and Biomedicine, Kgs. Lyngby3. University of Copenhagen, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, Denmark4. Roche NimbleGen, Madison, Wisconsin, USA5. Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San JoséFigure 3. Structural presentation of B-cell epitope analysis: (A-B) Short neurotoxin 1 (P01416) from D. polylepis as an example of atype 1 α-neurotoxin. Structure built upon1; (C-D) Fasciculin-2 (P0C1Z0) from D. angusticeps as an example of a fasciculin. TheFasciculin-2 is co-crystallized with the human acetylcholinesterase enzyme. Structure built upon2; (E-F) Toxin FS-2 (P01414) fromD. polylepis as an example of an L-type calcium channel blocker. Structure built upon3; (G-H) α-elapitoxin-Dpp2c (P01397) from D.polylepis as an example of a type 2 α-neurotoxin. Structure built upon4. (A,C,E,G) Residues colored according to alaninesubstitution effect in log2 fold-change, where magenta indicates that a residue is of particular importance for antibody recognition.Residue numbers refer to original sequence and not alignment; (B,D,F,H) Residues colored according to residue score, where darkred refers to resides with high residue score, and blue refers to residues with low residue scores.Figure 1. (A-C) Venn diagrams of peptides classified to bind antivenom antibodies for each pair of experiments conducted with thesame antivenom in two different dilutions; (A) SAIMR Polyvalent Snake Antivenom, (B) VINS African, and (C) VINS Central Africa. (D)Venn diagram of peptides classified as binders for each antivenom. Only peptides identified in both experiments with eachantivenom, corresponding to the overlap in Venn diagram in part A-C, are included.High number of epitopes recognized by SAIMR antivenomKey residues for antivenom toxin recognition Antivenoms antibodies bind to functional sites of toxins Studying linear epitopes using peptide microarraysGeneration of in silico library of 12-mers covering toxin sequencesExpanding the peptide library with alanine-substituted peptides 1) Binding of antivenom 2) fluorescence-labeled anti-horse-IgG Light-directed solid-phase synthesis of custom peptide microarrayBinding data Epitope mappingEpitopesPerspectivesDetermination of linear elements in snake venom toxin epitopes may provide the basis for:• Explaining the molecular basis of antivenoms para-specificity• Guiding next-generation antivenoms based on DNA immunization and immunization with synthetic epitope strings5Figure 2. Examples of B-cell epitope analysis: Type 1 and 2 α-neurotoxin and dendrotoxins recognized by the SAIMR polyvalentantivenom. The filled profiles above each sequence represent the average score of peptides containing a given peptide. The tilebackground represents the average alanine substitution effect. When no 12-mer peptide covering a given residue passed theepitope-threshold, the residue is colored gray. Dark purple indicates that a residue is of particular importance for antibodyrecognition.Loop 1Loop 3Loop 1Loop 2Loop 3Loop 1Loop 2Loop 3Loop 1Loop 2Loop 3Loop 2HGFEDCBAType 1 α-neurotoxinsType 2 α-neurotoxinsDendrotoxins

High-throughput epitope profiling of snake venom toxins:unveiling the complexity of antigen-antibody interactions of antivenoms

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High-throughput epitope profiling of snake venom toxins:unveiling the complexity of antigen-antibody interactions of antivenoms

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