{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,13]],"date-time":"2026-04-13T03:13:26Z","timestamp":1776050006099,"version":"3.50.1"},"reference-count":84,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2023,11,28]],"date-time":"2023-11-28T00:00:00Z","timestamp":1701129600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000781","name":"Horizon 2020 FET OPEN","doi-asserted-by":"publisher","award":["899670"],"award-info":[{"award-number":["899670"]}],"id":[{"id":"10.13039\/501100000781","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebites and threefold more victims suffer life-altering disabilities. The current treatment of snakebite relies solely on antivenom\u2014polyclonal antibodies isolated from the plasma of hyperimmunised animals\u2014which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through the use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein; it has 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology and mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability.<\/jats:p>","DOI":"10.3390\/toxins15120673","type":"journal-article","created":{"date-parts":[[2023,11,28]],"date-time":"2023-11-28T07:41:32Z","timestamp":1701157292000},"page":"673","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["ADDovenom: Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9273-9296","authenticated-orcid":false,"given":"Stefanie K.","family":"Menzies","sequence":"first","affiliation":[{"name":"Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK"},{"name":"Centre for Drugs & Diagnostics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1050-335X","authenticated-orcid":false,"given":"Raquel","family":"Arinto-Garcia","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Technol\u00f3gica, Apartado 12, 2781-901 Oeiras, Portugal"},{"name":"ITQB NOVA, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1453-3185","authenticated-orcid":false,"given":"Fernanda Gobbi","family":"Amorim","sequence":"additional","affiliation":[{"name":"Mass Spectrometry Laboratory, MolSys Research Unit, All\u00e9e du six Aout 11, Quartier Agora, Li\u00e8ge Universit\u00e9, 4000 Li\u00e8ge, Belgium"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0288-4706","authenticated-orcid":false,"given":"Iara Aim\u00ea","family":"Cardoso","sequence":"additional","affiliation":[{"name":"Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK"}]},{"given":"Camille","family":"Abada","sequence":"additional","affiliation":[{"name":"Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-3272-8233","authenticated-orcid":false,"given":"Thomas","family":"Crasset","sequence":"additional","affiliation":[{"name":"Mass Spectrometry Laboratory, MolSys Research Unit, All\u00e9e du six Aout 11, Quartier Agora, Li\u00e8ge Universit\u00e9, 4000 Li\u00e8ge, Belgium"}]},{"given":"Fabien","family":"Durbesson","sequence":"additional","affiliation":[{"name":"Architecture et Fonction des Macromol\u00e9cules Biologiques, CNRS, Aix-Marseille Universit\u00e9, 13009 Marseille, France"}]},{"given":"Rebecca J.","family":"Edge","sequence":"additional","affiliation":[{"name":"Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK"}]},{"given":"Priscila","family":"El-Kazzi","sequence":"additional","affiliation":[{"name":"Architecture et Fonction des Macromol\u00e9cules Biologiques, CNRS, Aix-Marseille Universit\u00e9, 13009 Marseille, France"}]},{"given":"Sophie","family":"Hall","sequence":"additional","affiliation":[{"name":"School of Biochemistry, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK"},{"name":"Max Planck Bristol Centre for Minimal Biology, Cantock\u2019s Close, Bristol BS8 1TS, UK"}]},{"given":"Damien","family":"Redureau","sequence":"additional","affiliation":[{"name":"Mass Spectrometry Laboratory, MolSys Research Unit, All\u00e9e du six Aout 11, Quartier Agora, Li\u00e8ge Universit\u00e9, 4000 Li\u00e8ge, Belgium"}]},{"given":"Richard","family":"Stenner","sequence":"additional","affiliation":[{"name":"School of Biochemistry, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK"},{"name":"Max Planck Bristol Centre for Minimal Biology, Cantock\u2019s Close, Bristol BS8 1TS, UK"}]},{"given":"Johara","family":"Boldrini-Fran\u00e7a","sequence":"additional","affiliation":[{"name":"School of Biochemistry, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK"},{"name":"Max Planck Bristol Centre for Minimal Biology, Cantock\u2019s Close, Bristol BS8 1TS, UK"}]},{"given":"Huan","family":"Sun","sequence":"additional","affiliation":[{"name":"School of Biochemistry, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK"},{"name":"Max Planck Bristol Centre for Minimal Biology, Cantock\u2019s Close, Bristol BS8 1TS, UK"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0414-4899","authenticated-orcid":false,"given":"Ant\u00f3nio","family":"Rold\u00e3o","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Technol\u00f3gica, Apartado 12, 2781-901 Oeiras, Portugal"},{"name":"ITQB NOVA, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1445-3556","authenticated-orcid":false,"given":"Paula M.","family":"Alves","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Technol\u00f3gica, Apartado 12, 2781-901 Oeiras, Portugal"},{"name":"ITQB NOVA, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Robert A.","family":"Harrison","sequence":"additional","affiliation":[{"name":"Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK"},{"name":"Centre for Drugs & Diagnostics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9667-0196","authenticated-orcid":false,"given":"Renaud","family":"Vincentelli","sequence":"additional","affiliation":[{"name":"Architecture et Fonction des Macromol\u00e9cules Biologiques, CNRS, Aix-Marseille Universit\u00e9, 13009 Marseille, France"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7518-9045","authenticated-orcid":false,"given":"Imre","family":"Berger","sequence":"additional","affiliation":[{"name":"School of Biochemistry, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK"},{"name":"Max Planck Bristol Centre for Minimal Biology, Cantock\u2019s Close, Bristol BS8 1TS, UK"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8153-9590","authenticated-orcid":false,"given":"Lo\u00efc","family":"Quinton","sequence":"additional","affiliation":[{"name":"Mass Spectrometry Laboratory, MolSys Research Unit, All\u00e9e du six Aout 11, Quartier Agora, Li\u00e8ge Universit\u00e9, 4000 Li\u00e8ge, Belgium"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8035-4719","authenticated-orcid":false,"given":"Nicholas R.","family":"Casewell","sequence":"additional","affiliation":[{"name":"Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK"},{"name":"Centre for Drugs & Diagnostics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1516-9760","authenticated-orcid":false,"given":"Christiane","family":"Schaffitzel","sequence":"additional","affiliation":[{"name":"School of Biochemistry, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK"}]}],"member":"1968","published-online":{"date-parts":[[2023,11,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"17063","DOI":"10.1038\/nrdp.2017.63","article-title":"Snakebite envenoming","volume":"3","author":"Calvete","year":"2017","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Harrison, R.A., Hargreaves, A., Wagstaff, S.C., Faragher, B., and Lalloo, D.G. (2009). Snake Envenoming: A Disease of Poverty. PLoS Negl. Trop. Dis., 3.","DOI":"10.1371\/journal.pntd.0000569"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/j.toxicon.2018.03.004","article-title":"Pros and cons of different therapeutic antibody formats for recombinant antivenom development","volume":"146","author":"Laustsen","year":"2018","journal-title":"Toxicon"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1279","DOI":"10.1016\/0041-0101(91)90116-9","article-title":"Snake venom variability: Methods of study, results and interpretation","volume":"29","author":"Chippaux","year":"1991","journal-title":"Toxicon"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"835","DOI":"10.1093\/trstmh\/try134","article-title":"The time is now: A call for action to translate recent momentum on tackling tropical snakebite into sustained benefit for victims","volume":"113","author":"Harrison","year":"2019","journal-title":"Trans. R. Soc. Trop. Med. Hyg."},{"key":"ref_6","unstructured":"World Health Organization (2023, November 03). Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins. Available online: https:\/\/cdn.who.int\/media\/docs\/default-source\/biologicals\/blood-products\/document-migration\/antivenomglrevwho_trs_1004_web_annex_5.pdf?sfvrsn=ef4b2aa5_3&download=true."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1016\/j.toxicon.2009.10.024","article-title":"Pre-clinical and preliminary dose-finding and safety studies to identify candidate antivenoms for treatment of envenoming by saw-scaled or carpet vipers (Echis ocellatus) in northern Nigeria","volume":"55","author":"Abubakar","year":"2010","journal-title":"Toxicon"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1016\/j.biologicals.2012.11.001","article-title":"Assessment of snake antivenom purity by comparing physicochemical and immunochemical methods","volume":"41","author":"Segura","year":"2013","journal-title":"Biologicals"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1016\/0041-0101(94)90107-4","article-title":"A new antivenom to treat eastern coral snake (Micrurus fulvius fulvius) envenoming","volume":"32","author":"Rawat","year":"1994","journal-title":"Toxicon"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"887","DOI":"10.4269\/ajtmh.14-0333","article-title":"Antivenomic Characterization of Two Antivenoms Against the Venom of the Taipan, Oxyuranus scutellatus, from Papua New Guinea and Australia","volume":"91","author":"Herrera","year":"2014","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Harrison, R.A., and Guti\u00e9rrez, J.M. (2016). Priority Actions and Progress to Substantially and Sustainably Reduce the Mortality, Morbidity and Socioeconomic Burden of Tropical Snakebite. Toxins, 8.","DOI":"10.3390\/toxins8120351"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1598","DOI":"10.3389\/fimmu.2019.01598","article-title":"History of Envenoming Therapy and Current Perspectives","volume":"10","author":"Pucca","year":"2019","journal-title":"Front. Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Guti\u00e9rrez, J.M., Albulescu, L.-O., Clare, R.H., Casewell, N.R., Abd El-Aziz, T.M., Escalante, T., and Rucavado, A. (2021). The Search for Natural and Synthetic Inhibitors That Would Complement Antivenoms as Therapeutics for Snakebite Envenoming. Toxins, 13.","DOI":"10.3390\/toxins13070451"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Jenkins, T.P., Fryer, T., Dehli, R.I., J\u00fcrgensen, J.A., Fuglsang-Madsen, A., F\u00f8ns, S., and Laustsen, A.H. (2019). Toxin Neutralization Using Alternative Binding Proteins. Toxins, 11.","DOI":"10.3390\/toxins11010053"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Lewin, M.R., Carter, R.W., Matteo, I.A., Samuel, S.P., Rao, S., Fry, B.G., and Bickler, P.E. (2022). Varespladib in the Treatment of Snakebite Envenoming: Development History and Preclinical Evidence Supporting Advancement to Clinical Trials in Patients Bitten by Venomous Snakes. Toxins, 14.","DOI":"10.3390\/toxins14110783"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"90","DOI":"10.12688\/wellcomeopenres.17682.1","article-title":"TRUE-1: Trial of Repurposed Unithiol for snakebite Envenoming phase 1 (safety, tolerability, pharmacokinetics and pharmacodynamics in healthy Kenyan adults)","volume":"7","author":"Abouyannis","year":"2022","journal-title":"Wellcome Open Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"eaaw2853","DOI":"10.1126\/sciadv.aaw2853","article-title":"Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display","volume":"5","author":"Vragniau","year":"2019","journal-title":"Sci. Adv."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Besson, S., Vragniau, C., Vassal-Stermann, E., Dagher, M.C., and Fender, P. (2020). The Adenovirus Dodecahedron: Beyond the Platonic Story. Viruses, 12.","DOI":"10.3390\/v12070718"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1913","DOI":"10.1016\/j.ymthe.2022.02.011","article-title":"Elicitation of potent SARS-CoV-2 neutralizing antibody responses through immunization with a versatile adenovirus-inspired multimerization platform","volume":"30","author":"Chevillard","year":"2022","journal-title":"Mol. Ther."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Luo, C., Yan, Q., Huang, J., Liu, J., Li, Y., Wu, K., Li, B., Zhao, M., Fan, S., and Ding, H. (2022). Using Self-Assembling ADDomer Platform to Display B and T Epitopes of Type O Foot-and-Mouth Disease Virus. Viruses, 14.","DOI":"10.3390\/v14081810"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Buzas, D., Bunzel, H.A., Staufer, O., Milodowski, E.J., Edmunds, G.L., Bufton, J.C., Mateo, B.V.V., Yadav, S.K.N., Gupta, K., and Fletcher, C. (2023). In vitro generated antibodies guide thermostable ADDomer nanoparticle design for nasal vaccination and passive immunization against SARS-CoV-2. Antib. Ther., tbad024.","DOI":"10.1093\/abt\/tbad024"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Zochowska, M., Paca, A., Schoehn, G., Andrieu, J.-P., Chroboczek, J., Dublet, B., and Szolajska, E. (2009). Adenovirus Dodecahedron, as a Drug Delivery Vector. Jagetia GC, editor. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0005569"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Buzas, D., Sun, H., Toelzer, C., Yadav, S.K.N., Borucu, U., Gautam, G., Gupta, K., Bufton, J., Capin, J., and Sessions, R.B. (2023). Engineering the ADDobody protein scaffold for generation of high-avidity ADDomer super-binders. BioRxiv.","DOI":"10.1101\/2023.09.09.556966"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Guti\u00e9rrez, J.M., Theakston, R.D.G., and Warrell, D.A. (2006). Confronting the Neglected Problem of Snake Bite Envenoming: The Need for a Global Partnership. PLoS Med., 3.","DOI":"10.1371\/journal.pmed.0030150"},{"key":"ref_25","first-page":"33","article-title":"Poisoning by bites of the saw-scaled or carpet viper (Echis carinatus) in Nigeria","volume":"46","author":"Warrell","year":"1977","journal-title":"Q. J. Med."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Dingwoke, E.J., Adamude, F.A., Mohamed, G., Klein, A., Salihu, A., Abubakar, M.S., and Sallau, A.B. (2021). Venom proteomic analysis of medically important Nigerian viper Echis ocellatus and Bitis arietans snake species. Biochem. Biophys. Rep., 28.","DOI":"10.1016\/j.bbrep.2021.101164"},{"key":"ref_27","first-page":"689","article-title":"Snake toxins from mamba venoms: Unique tools for the physiologist","volume":"58","author":"Rowan","year":"2011","journal-title":"Acta Chim. Slov."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1016\/j.jprot.2016.02.003","article-title":"Toxicovenomics and antivenom profiling of the Eastern green mamba snake (Dendroaspis angusticeps)","volume":"136","author":"Lauridsen","year":"2016","journal-title":"J. Proteom."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1016\/j.jprot.2015.02.002","article-title":"Unveiling the nature of black mamba (Dendroaspis polylepis) venom through venomics and antivenom immunoprofiling: Identification of key toxin targets for antivenom development","volume":"119","author":"Laustsen","year":"2015","journal-title":"J. Proteom."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3216","DOI":"10.1021\/pr070142t","article-title":"New Method for Characterizing Highly Disulfide-Bridged Peptides in Complex Mixtures: Application to Toxin Identification from Crude Venoms","volume":"6","author":"Quinton","year":"2007","journal-title":"J. Proteome Res."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Dunbar, J.P., Fort, A., Redureau, D., Sulpice, R., Dugon, M.M., and Quinton, L. (2020). Venomics Approach Reveals a High Proportion of Lactrodectus-like Toxins in the Venom of the Noble False Widow Spider Steatoda nobilis. Toxins, 12.","DOI":"10.3390\/toxins12060402"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1736","DOI":"10.1016\/j.febslet.2009.03.029","article-title":"Venoms, venomics, antivenomics","volume":"583","author":"Calvete","year":"2009","journal-title":"FEBS Lett."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Wilson, D., and Daly, N.L. (2018). Venomics: A Mini-Review. High Throughput, 7.","DOI":"10.3390\/ht7030019"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1038\/s41570-022-00393-7","article-title":"The chemistry of snake venom and its medicinal potential","volume":"6","author":"Oliveira","year":"2022","journal-title":"Nat. Rev. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"9205","DOI":"10.1073\/pnas.1405484111","article-title":"Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms","volume":"111","author":"Casewell","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2501","DOI":"10.1021\/acs.jproteome.9b00044","article-title":"Multi-Enzymatic Limited Digestion: The Next-Generation Sequencing for Proteomics?","volume":"18","author":"Morsa","year":"2019","journal-title":"J. Proteome Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2337","DOI":"10.1002\/rcm.1196","article-title":"PEAKS: Powerful software for peptide de novo sequencing by tandem mass spectrometry","volume":"17","author":"Ma","year":"2003","journal-title":"Rapid Commun. Mass Spectrom."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/j.jprot.2017.08.016","article-title":"The medical threat of mamba envenoming in sub-Saharan Africa revealed by genus-wide analysis of venom composition, toxicity and antivenomics profiling of available antivenoms","volume":"172","author":"Ainsworth","year":"2018","journal-title":"J. Proteom."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1016\/j.jprot.2009.01.008","article-title":"Snake venomics and antivenomics: Proteomic tools in the design and control of antivenoms for the treatment of snakebite envenoming","volume":"72","author":"Lomonte","year":"2009","journal-title":"J. Proteom."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1287","DOI":"10.1038\/82407","article-title":"Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display","volume":"18","author":"Hanes","year":"2000","journal-title":"Nat. Biotechnol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1007\/978-1-4939-8648-4_13","article-title":"Rapid Selection of High-Affinity Antibody scFv Fragments Using Ribosome Display","volume":"1827","author":"Dreier","year":"2018","journal-title":"Methods Mol. Biol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1707","DOI":"10.1038\/s41596-020-0304-x","article-title":"Generation of synthetic nanobodies against delicate proteins","volume":"15","author":"Zimmermann","year":"2020","journal-title":"Nat. Protoc."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"575","DOI":"10.1038\/nbt962","article-title":"High-affinity binders selected from designed ankyrin repeat protein libraries","volume":"22","author":"Binz","year":"2004","journal-title":"Nat. Biotechnol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"18870","DOI":"10.1074\/jbc.M309169200","article-title":"Directed in Vitro Evolution and Crystallographic Analysis of a Peptide-binding Single Chain Antibody Fragment (scFv) with Low Picomolar Affinity","volume":"279","author":"Zahnd","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Rivera-de-Torre, E., Rimbault, C., Jenkins, T.P., S\u00f8rensen, C.V., Damsbo, A., Saez, N.J., Duhoo, Y., Hackney, C.M., Ellgaard, L., and Laustsen, A.H. (2022). Strategies for Heterologous Expression, Synthesis, and Purification of Animal Venom Toxins. Front. Bioeng. Biotechnol., 9.","DOI":"10.3389\/fbioe.2021.811905"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/j.toxicon.2014.06.009","article-title":"Production in Escherichia coli, folding, purification and characterization of notexin with wild type sequence and with N-terminal and catalytic site mutations","volume":"88","author":"Simonato","year":"2014","journal-title":"Toxicon"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"890","DOI":"10.1002\/psc.1020","article-title":"Mutations on N-terminal region of Taiwan cobra phospholipase A2 result in structurally distorted effects","volume":"14","author":"Chiou","year":"2008","journal-title":"J. Pept. Sci."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1186\/s12934-016-0617-1","article-title":"High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery","volume":"16","author":"Turchetto","year":"2017","journal-title":"Microb. Cell Fact."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1186\/s12934-016-0618-0","article-title":"Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli","volume":"16","author":"Sequeira","year":"2017","journal-title":"Microb. Cell Fact."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1007\/978-1-4939-2272-7_12","article-title":"Site-specific biotinylation of purified proteins using BirA","volume":"1266","author":"Fairhead","year":"2015","journal-title":"Methods Mol. Biol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/1475-2859-10-1","article-title":"Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E. coli","volume":"10","author":"Nguyen","year":"2011","journal-title":"Microb. Cell Fact."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1021","DOI":"10.1038\/nmeth983","article-title":"Protein complex expression by using multigene baculoviral vectors","volume":"3","author":"Fitzgerald","year":"2006","journal-title":"Nat. Methods"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1016\/0378-1119(91)90171-7","article-title":"Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide","volume":"98","author":"Tessier","year":"1991","journal-title":"Gene"},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Wagstaff, S.C., Laing, G.D., Theakston, R.D.G., Papaspyridis, C., and Harrison, R.A. (2006). Bioinformatics and Multiepitope DNA Immunization to Design Rational Snake Antivenom. PLoS Med., 3.","DOI":"10.1371\/journal.pmed.0030184"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"617429","DOI":"10.3389\/fimmu.2020.617429","article-title":"In Vitro Tests for Assessing the Neutralizing Ability of Snake Antivenoms: Toward the 3Rs Principles","volume":"11","author":"Vargas","year":"2021","journal-title":"Front. Immunol."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"6094","DOI":"10.1038\/s41467-020-19981-6","article-title":"A therapeutic combination of two small molecule toxin inhibitors provides broad preclinical efficacy against viper snakebite","volume":"11","author":"Albulescu","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_57","doi-asserted-by":"crossref","unstructured":"Gopalakrishnakone, P., and Calvete, J.J. (2016). Venom Genomics and Proteomics, Springer.","DOI":"10.1007\/978-94-007-6416-3"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1042\/BST20200110","article-title":"Cytotoxicity of snake venom enzymatic toxins: Phospholipase A2 and l-amino acid oxidase","volume":"48","author":"Hiu","year":"2020","journal-title":"Biochem. Soc. Trans."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1016\/j.toxicon.2013.05.017","article-title":"Cytotoxic activities of [Ser49]phospholipase A2 from the venom of the saw-scaled vipers Echis ocellatus, Echis pyramidum leakeyi, Echis carinatus sochureki, and Echis coloratus","volume":"71","author":"Conlon","year":"2013","journal-title":"Toxicon"},{"key":"ref_60","doi-asserted-by":"crossref","unstructured":"Still, K.B.M., Nandlal, R.S.S., Slagboom, J., Somsen, G.W., Casewell, N.R., and Kool, J. (2017). Multipurpose HTS Coagulation Analysis: Assay Development and Assessment of Coagulopathic Snake Venoms. Toxins, 9.","DOI":"10.3390\/toxins9120382"},{"key":"ref_61","doi-asserted-by":"crossref","unstructured":"Navred, K., Martin, M., Ekdahl, L., Zetterberg, E., Andersson, N.G., Strandberg, K., and Norstrom, E. (2019). A simplified flow cytometric method for detection of inherited platelet disorders\u2014A comparison to the gold standard light transmission aggregometry. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0211130"},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"106982","DOI":"10.1016\/j.toxicon.2022.106982","article-title":"How snake venom disintegrins affect platelet aggregation and cancer proliferation","volume":"221","author":"Kolvekar","year":"2023","journal-title":"Toxicon"},{"key":"ref_63","doi-asserted-by":"crossref","unstructured":"Patel, R.N., Clare, R.H., Ledsgaard, L., Nys, M., Kool, J., Laustsen, A.H., Ulens, C., and Casewell, N.R. (2023). An in vitro assay to investigate venom neurotoxin activity on muscle-type nicotinic acetylcholine receptor activation and for the discovery of toxin-inhibitory molecules. Biochem. Pharmacol., 216.","DOI":"10.1016\/j.bcp.2023.115758"},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.toxicon.2018.04.022","article-title":"Neurotoxicity fingerprinting of venoms using on-line microfluidic AChBP profiling","volume":"148","author":"Slagboom","year":"2018","journal-title":"Toxicon"},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"24279","DOI":"10.1074\/jbc.272.39.24279","article-title":"Only Snake Curaremimetic Toxins with a Fifth Disulfide Bond Have High Affinity for the Neuronal \u03b17 Nicotinic Receptor","volume":"272","author":"Servent","year":"1997","journal-title":"J. Biol. Chem."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/S0041-0101(00)00162-8","article-title":"Twenty years of dendrotoxins","volume":"39","author":"Harvey","year":"2001","journal-title":"Toxicon"},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"87","DOI":"10.2174\/1875397301206010087","article-title":"A Comparison of the Performance and Application Differences between Manual and Automated Patch-Clamp Techniques","volume":"6","author":"Yajuan","year":"2012","journal-title":"Curr. Chem. Genom."},{"key":"ref_68","first-page":"949","article-title":"Development of simple standard assay procedures for the characterization of snake venom","volume":"61","author":"Theakston","year":"1983","journal-title":"Bull. World Health Organ."},{"key":"ref_69","doi-asserted-by":"crossref","unstructured":"Knudsen, C., Casewell, N.R., Lomonte, B., Guti\u00e9rrez, J.M., Vaiyapuri, S., and Laustsen, A.H. (2020). Novel Snakebite Therapeutics Must Be Tested in Appropriate Rescue Models to Robustly Assess Their Preclinical Efficacy. Toxins, 12.","DOI":"10.3390\/toxins12090528"},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s00253-003-1223-9","article-title":"Insect cell culture for industrial production of recombinant proteins","volume":"62","author":"Ikonomou","year":"2003","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"10061","DOI":"10.1021\/acs.iecr.8b00985","article-title":"The Coming Age of Insect Cells for Manufacturing and Development of Protein Therapeutics","volume":"57","author":"Yee","year":"2018","journal-title":"Ind. Eng. Chem. Res."},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1016\/j.pep.2016.02.014","article-title":"Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system","volume":"122","author":"Maghodia","year":"2016","journal-title":"Protein Expr. Purif."},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"3248","DOI":"10.1002\/bit.27494","article-title":"A new nodavirus-negative Trichoplusia ni cell line for baculovirus-mediated protein production","volume":"117","author":"Maghodia","year":"2020","journal-title":"Biotechnol. Bioeng."},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"174","DOI":"10.1016\/j.nbt.2017.07.010","article-title":"Production of virus-like particles for vaccines","volume":"39","author":"Fuenmayor","year":"2017","journal-title":"New Biotechnol."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"1149","DOI":"10.1586\/erv.10.115","article-title":"Virus-like particles in vaccine development","volume":"9","author":"Mellado","year":"2010","journal-title":"Expert Rev. Vaccines"},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1016\/j.copbio.2018.04.003","article-title":"How scalable and suitable are single-use bioreactors?","volume":"53","author":"Junne","year":"2018","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"116272","DOI":"10.1016\/j.ces.2020.116272","article-title":"Continuous downstream bioprocessing for intensified manufacture of biopharmaceuticals and antibodies","volume":"231","author":"Gerstweiler","year":"2021","journal-title":"Chem. Eng. Sci."},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"2122","DOI":"10.1002\/bit.28118","article-title":"An integrated and continuous downstream process for microbial virus-like particle vaccine biomanufacture","volume":"119","author":"Gerstweiler","year":"2022","journal-title":"Biotech. Bioeng."},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"834","DOI":"10.1016\/j.vaccine.2013.12.029","article-title":"How much does it cost to get a dose of vaccine to the service delivery location? Empirical evidence from Vietnam\u2019s Expanded Program on Immunization","volume":"32","author":"Mvundura","year":"2014","journal-title":"Vaccine"},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"2697","DOI":"10.1016\/j.vaccine.2015.03.084","article-title":"Estimating the costs of the vaccine supply chain and service delivery for selected districts in Kenya and Tanzania","volume":"33","author":"Mvundura","year":"2015","journal-title":"Vaccine"},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"811","DOI":"10.1080\/14760584.2022.2053678","article-title":"Thermostable vaccines: An innovative concept in vaccine development","volume":"21","author":"Kumar","year":"2022","journal-title":"Expert Rev. Vaccines"},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"1357","DOI":"10.1111\/pbi.13060","article-title":"Cold chain and virus-free oral polio booster vaccine made in lettuce chloroplasts confers protection against all three poliovirus serotypes","volume":"17","author":"Daniell","year":"2019","journal-title":"Plant Biotechnol. J."},{"key":"ref_83","doi-asserted-by":"crossref","unstructured":"Guo, M., Li, J., Teng, Z., Ren, M., Dong, H., Zhang, Y., Ru, J., Du, P., Sun, S., and Guo, H. (2021). Four Simple Biomimetic Mineralization Methods to Improve the Thermostability and Immunogenicity of Virus-like Particles as a Vaccine against Foot-and-Mouth Disease. Vaccines, 9.","DOI":"10.3390\/vaccines9080891"},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"231","DOI":"10.4149\/av_2017_301","article-title":"Viral vaccine stabilizers: Status and trends","volume":"61","author":"Cardoso","year":"2017","journal-title":"Acta Virol."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/15\/12\/673\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T21:32:14Z","timestamp":1760131934000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/15\/12\/673"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,11,28]]},"references-count":84,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2023,12]]}},"alternative-id":["toxins15120673"],"URL":"https:\/\/doi.org\/10.3390\/toxins15120673","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,11,28]]}}}