Venoms and Toxins 2019 28Th – 29Th August 2019 the Edward Boyle Auditorium, St Hilda’S College Oxford OX4 1DY, United Kingdom

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6th International Toxinology Meeting Venoms and Toxins 2019 28th – 29th August 2019 The Edward Boyle Auditorium, St Hilda’s College Oxford OX4 1DY, United Kingdom

Email: [email protected] Web: http://lpmhealthcare.com/venoms-and-toxins-2019 Twitter: @LPMHealthcare | @VenomsOxford | Hashtag: #VenOx19

TABLE OF CONTENTS 4: General Information 5: Insurance and Liability 5: Disclaimer 5: Local Map 6: Podium Agenda 9: Podium Abstracts 28: Poster Abstracts 43: Sponsors and Supporters

Website: http://jvenomres.co.uk LinkedIn: https://www.linkedin.com/groups/3553905 Twitter: @VenomsOxford | hashtag #JVenRes

Publish free until 15 th December 2019 We are happy to waive the basic open access fee for Venoms and Toxins 2019 participants of the manuscript submitted until 15 th December 2019 , as long as the manuscripts are prepared according to the published guidelines. Editor-in-Chief: Dr Edward G Rowan PhD Strathclyde Institute of Pharmacy and Biomedical Sciences University of Strathclyde GLASGOW G4 0NR United Kingdom Email: [email protected] >EDITORIAL BOARD< About the Journal: The Journal (ISSN: 2044-0324) will publish high-quality peer-reviewed articles addressing most aspects of therapeutic, molecular, biochemical and evolutionary research on venoms, and envenomation . The Journal aims at providing an efficient route to fast-track publication, within 10-12 weeks of manuscript submission if accepted. Furthermore, manuscripts will be made available online in their final form generally within 2-3 weeks of final acceptance . The Journal will be primarily published online but on sufficient demand for printed version journal hard copies may also be provided to individuals and institutions. Manuscripts in the following categories will be considered for publication: reviews , mini-reviews , research articles, research reports , research letters , clinical reports , case studies, new methods and technologies , meeting reports and commercial, patent and product news and editorials .

The Journal is currently indexed in: • PubMed Central • UKPMC • PMC Canada •The Directory of Open Access Journals • Google Scholar • OPEN J-Gate • Chemical Abstracts Service (CAS) • Academic Search Complete (EBSCO)

For further information, please visit http://JVenomRes.co.uk or email [email protected].

We gratefully acknowledge support of our sponsors and supporters.

Event Dates: 28th - 29 th August 2019 Event Website: http://lpmhealthcare.com/venoms-and-toxins-2019 Venue: St Hilda’s College , Cowley Place, Oxford, OX4 1DY, England, UK Tel: +44 (0) 1865 276884, Website: http://www.st-hildas.ox.ac.uk Registration Desk: Located outside the meeting hall Name Badges: The College requests all delegates to wear name badges while on the premises to avoid any confusion. Refreshments/Lunch: JdP Foyer Mobile Phones: As a courtesy to speakers and participants, please switch off your mobile phone during oral presentations. Speaker Presentations: We will not be distributing speaker presentations. Therefore, if you interested in presentation slides of any speakers, please get in touch with them directly. Internet access: Please use edurom if you can. Otherwise, WiFi Code and instructions for internet access via your laptop/mobile device can be obtained at the time of registration. Health and Safety: Please do not leave your belongings unattended or in passageways and familiarise yourself with emergency exits. Smoking: In addition to any local venue regulations, UK no-smoking regulations apply on the College premises.

© Copyright Information : This handbook is for use by the Venoms and Toxins 2019 Oxford (28 th -29 th August 2019) participants only. Textual and graphical contents of this handbook are copyright of presenters, sponsors, instructors and/or LibPubMedia Ltd, unless explicitly stated otherwise. No part of this handbook may be reproduced, distributed or transmitted in any form or by any means, electronic or mechanical, including but not limited to, photocopy, recording, or any other information storage or retrieval system, without the prior written permission of the legal copyright owners.

INSURANCE AND LIABILITY Participants are strongly advised to be comprehensively insured at all times to cover all costs and consequences of medical treatment, repatriation, personal/public liability, personal injury/accident, damage/theft/loss of personal belongings, legal expenses, recovery of conference fees, alternative accommodation and flights booked, or needed, in the event of cancellation. The event organisers and hosts are not responsible for personal accidents, any travel costs or the loss of private property, and will not be liable for any claims. Event participants shall be responsible for compensating any loss, should they cause any damage to the host’s property or the venue. DISCLAIMER The information specified in oral and poster presentations, written abstracts, biographies and exhibitions come from diverse sources and it is not in the capacity of event organisers to validate it, and is provided on an ‘as-is’ basis. Responsibility for the literary and scientific content of the abstracts and the presentations, both oral and poster, remains with the authors and the presenters. Therefore, the event organisers accept no responsibility for literary or scientific correctness of this information, and shall accept no liability of any kind, should any of the information be incorrect. The event organisers and hosts make no representation or warranty of gain of business or profits as a result of use of services or information provided in connection with the even and shall not be liable for any direct or indirect damages, loss of business, employment, profits or anticipated savings resulting from the use of the services or information provided in connection with the event, in any country or court of law. Furthermore, the materials contained in the event handbook are provided on the understanding that speakers or presenters have the right to their presentation in this manner. Therefore, event organisers and hosts shall not be liable for infringement of third party rights by an event presenter, participant, sponsor, supporter or exhibitor.

LOCAL MAP

PODIUM AGENDA (subject to change)

DAY 0: Tuesday 27 th August | Venue TBC

1830: Meet up at St Hilda’s Lodge 1900-2200: Pre-conference social meet-up, Angel and Greyhound , St Clement’s Street DAY 1: Wednesday 28 th August | The Edward Boyle Auditorium, St Hilda’s College

0745: Registration, welcome coffee and networking 0820: Welcome and Housekeeping Session I: Omics in Toxinology – Chair Eddie Rowan

0830: Professor Juan Calvete – keynote , Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain Phylovenomics of daboia russelii across the indian subcontinent. Bioactivities and comparative in vivo neutralization and in vitro 3G antivenomics of antivenoms against venoms from India, Bangladesh and Sri Lanka 0900: Dr Timothy Jenkins , University of Cambridge, Cambridge, United Kingdom The unlocked potential of venom gland transcriptomics, droplet microfluidics, and alternative protein scaffolds 0920: Miss Lucka Bibic , University of East Anglia, Norwich Research Park, Norwich, United Kingdom Discovery of a small molecule toxin with inhibitory activity at human P2X4 ion channels 0935: Dr Cassandra Modahl , National University of Singapore, Singapore Rear-fanged snake venom novelty: Adaptive toxicity and metalloproteinase activity 0955: Professor José María Gutiérrez – Hamish Ogston Foundation Lecture , Univ de Costa Rica, San José, Costa Rica Unraveling the mechanisms of action of hemorrhagic toxins of viperid snake venoms 1035: Refreshment Break, poster and networking Session 2: Venoms and toxins: evolution, effects and functions – Chair Dietrich Mebs

1105: Professor Denise Tambourgi , Butantan Institute, São Paulo, SP, Brazil Complement system inhibition as means for controlling inflammation associated to envenomation 1125: Dr Sebastien Dutertre , Université Montpellier – Centre Nationale de la Recherche Scientifique, Montpellier, France Evolutionary considerations on separate predatory and defensive venoms 1145: Dr Timothy Jackson , University of Melbourne, Melbourne, Australia Venom in context: evolution, ecology and snakebite 1205: Miss Chloe Evans , Liverpool School of Tropical Medicine, Liverpool, United Kingdom Medically important African snakes cause variable acute phase and inflammatory responses following envenoming (12+3) 1220: Life-time achievement award – Professor Alan Harvey 1230: Group Photo and Lunch Session 3: Drugs from Toxins – Chair Juan Calvete

1330: Professor Alan Harvey – keynote , Strathclyde University, Glasgow, United Kingdom Drugs from toxins? Some dos and don’ts? 1400: Professor Glenn King , The University of Queensland, St Lucia, Australia Venom peptide inhibitors of acid-sensing ion channel 1a for treating ischemic injuries of the heart and brain 1420: Dr Elaine Fitches , Durham University, Durham, United Kingdom Demonstrating the potential of a novel spider venom based biopesticide for target-specific control of the small hive beetle, a serious pest of the European honey bee

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 7 1440: Dr Keith Miller , Sheffield Hallam University, Sheffield, United Kingdom Improving the Therapeutic Index of Venom Derived Antimicrobial Peptides 1500: Professor Jan Tytgat , KU Leuven, Leuven, Belgium Beyond hemostasis: a potassium channel blocker snake venom serine protease with potential antitumor activity 1520: Mr Ernesto Pinheiro-Junior , University of São Paulo, SP, Brazil PEGylating toxins: a new trend in toxinology? A successful example of a PEGylated snake venom serine protease 1535: Mr Steve Peigneur , Toxicology and Pharmacology, KU Leuven, Leuven, Belgium The insecticidal wasp toxin γ–pompilidotoxin is a linear peptide modulating voltage-gated sodium channel 1550: Dr Aneesh Karatt-Vellatt , IONTAS Ltd, Iconic Park, London Road, Pampisford, Cambridge, UK KnotBodies: Fusing venom derived mini-proteins to antibody CDR loops to treat ion channel mediated pathologies 1610: Refreshment Break, poster and networking Session 4: Inspiration, Innovation and Investment in Snake-bite Research – Chair David Warrell

1630: Dr Bernadette Abela-Ridder and Dr David Williams, World Health Organization, Switzerland 1700: Professor Mike Turner, The Wellcome Trust, United Kingdom 1720: Professor José María Gutiérrez and Professor Juan Calvete, Global Snakebite Initiative 1730: Dr Cathy Roth, Department for International Development, United Kingdom 1750: Dr Michael Vaughan, Hamish Ogston Foundation, United Kingdom 1805: Dr Benjamin Waldmann, Health Action International, Netherlands 1820: Dr Tamar Ghosh, Royal Society of Tropical Medicine and Hygiene, United Kingdom 1830: End of Day 1 1915: Networking Dinner (by prior booking or invitation only)

DAY 2: Thursday 29 th August | The Edward Boyle Auditorium, St Hilda’s College

Session 5: Newly Recognised Venomous Taxa – Chair Alan Harvey

0830: Professor Dietrich Mebs – keynote , Goethe-University of Frankfurt, Frankfurt, Germany What is cheaper: synthesis or acquisition of toxins from exogenous sources? 0900: Dr Ronald Jenner , Natural History Museum, London, United Kingdom There is no such thing as ‘centipede venom’ – comparative proteotranscriptomic analyses uncover deep divergence of venom composition in Chilopoda 0920: Dr Christopher Lynch , University of Oxford, Oxford, United Kingdom A knottin scaffold directs CXC-chemokine binding specificity of tick evasins 0940: Mr James Dobson , University of Queensland, St. Lucia, Australia Anguimorph lizard venoms disrupt the clotting ability of human fibrinogen via destructive cleavage 0955: Mr Jonas Krämer , University of Cologne, Cologne, Germany Venom collection and analysis in the pseudoscorpion Chelifer cancroides 1010: Mr Bjoern Marcus von Reumont , LOEWE Centre for Translational Biodiversity Genomics, Frankfurt, Germany Comprehensive insights into the venom system and the genomic processes of toxin evolution in robber flies (Asilidae, Diptera) 1025: Refreshment Break, poster and networking 1050: Professor Paul Long , King’s College London, London, United Kingdom Is the cnidarian parasite of caviar, ‘Polypodium hydriforme ’, a venomous animal?

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 8 1110: Dr Gary Bucciarelli , University of California, Los Angeles, USA Assessing rangewide variation of toxin defenses in a poisonous amphibian Session 6: Bacterial Toxins – Chair Jan Tytgat

1130: Professor Oliver Dolly , Dublin City University, Glasnevin, Dublin, Ireland A chimeric botulinum neurotoxin unveiled that TNF α–induced surface trafficking of TRP/V1 and /A1 channels in sensory neurons is dependent on SNAP-25 1150: Dr John Barr , Centers for Disease Control and Prevention, Atlanta, GA, USA Comprehensive characterization of anthrax toxins during the course of inhalation infection in non-human primate models 1210: Dr Suzanne Kalb , Centers for Disease Control and Prevention, Atlanta, GA, USA Mass Spectrometric Detection and Characterization of Botulinum Neurotoxins 1230: Dr Celia Carlini , Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil Proteus mirabilis urease: unsuspected non-enzymatic biological properties relevant to pathogenesis 1250: Lunch and networking Session 7: Monoclonal Antivenoms – Chair José María Gutiérrez

1340: Dr Andreas Laustsen , Technical University of Denmark, Kongens Lyngby, Denmark Discovery strategies for recombinant antivenoms based on broadly-neutralizing recyclable human monoclonal antibodies 1400: Dr Paddy Waters , University of Oxford, Oxford, United Kingdom A snake in the test 1420: Miss Charlotte Dawson , Liverpool School of Tropical Medicine, Liverpool, United Kingdom Cross-Species and Geographic Potential of Epitope Strings Identified for Generation of an Africa-specific Snake Venom- induced Necrosis Serotherapy 1435: Mrs Nessrin Alomran, Liverpool School of Tropical Medicine, Liverpool, United Kingdom The immunological cross-reactivity and in vitro neutralising efficacy of novel antivenoms against haemotoxic snake venoms Session 8: Clinical Toxinology – Chair David Williams

1450: Dr Naira Ayvazyan , Orbeli Institute of Physiology of the National Academy of Sciences, Yerevan, Armenia Macrovipera lebetina obtusa: Venomics, Bioactivity and Preclinical Assessment of new ovine antivenom (Armenia) 1510: Refreshment Break, poster and networking 1530: Professor Abdulsalami Nasidi , University of Africa, Toru-orua, Bayelsa State, Nigeria EchiTAb antivenom – A global solution to a local problem 1550: Dr Aniruddha Ghose , Chittagong Medical College & Hospital, Chittagong, Bangladesh Coagulopathy in Green Pit Viper bite in Bangladesh: an unrecognized and unaddressed issue 1610: Dr Andrew Watt , Australian Venom Research Unit, University of Melbourne, Australia PNG Snakebite Partnership: Antivenom distribution and snakebite incidence across Papua New Guinea 1630: Professor David Warrell , Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom Myanmar Snake-bite Project: an Australian DFAT-GPFD programme 1650: Dates of the next meeting 1700: Discussion and close

Phylovenomics of daboia russelii across the indian subcontinent. Bioactivities and comparative in vivo neutralization and in vitro 3G antivenomics of antivenoms against venoms from India, Bangladesh and Sri Lanka

Davinia Pla 1, Libia Sanz 1, Sarai Quesada-Bernat 1, Mauren Villalta 2, Joshua Baal 3, Mohammad Abdul Wahed Chowdhury 4, Guillermo León 2, José M. Gutiérrez 2, Ulrich Kuch 5, Juan J. Calvete 1

1Evolutionary and Translational Venomics Laboratory, Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain 2Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica 3Technische Universität Kaiserslautern, Erwin-Schrödinger-Straße 52, Kaiserslautern, Germany 4Department of Zoology, University of Chittagong, Chittagong, Bangladesh 5Institute of Occupational Medicine, Social Medicine and Environmental Medicine, Goethe University, Frankfurt, Germany

Russell's viper ( Daboia russelii ) is, together with Naja naja , Bungarus caeruleus and Echis carinatus , a member of the medically important ‘Big Four’ species responsible for causing a large number of morbidity and mortality cases across the Indian subcontinent. Despite the wide distribution of Russell's viper and the well-documented ubiquity of the phenomenon of geographic variability of intraspecific snake venom composition, Indian polyvalent antivenoms against the “Big Four” venoms are raised against venoms sourced mainly from Chennai in the southeastern Indian state of Tamil Nadu. Biochemical and venomics investigations have consistently revealed notable compositional, functional, and immunological differences among geographic variants of Russell's viper venoms across the Indian subcontinent. However, these studies, carried out by different laboratories using different protocols and involving venoms from a single geographical region, make the comparison of the different venoms difficult. To bridge this gap, we have conducted bioactivities and proteomic analyses of D. russelii venoms from the three corners of the Indian subcontinent, Pakistan, Bangladesh, and Tamil Nandu (India) and Sri Lanka, along with comparative in vivo neutralization and in vitro third-generation antivenomics of antivenoms used in India, Bangladesh and Sri Lanka. These analyses let us to propose two alternative routes of radiation for Russell's viper in the Indian subcontinent. Both radiations, towards the northeast of India and Bangladesh and towards south India and Sri Lanka, have a common origin in Pakistan, and provide a phylovenomics ground for rationalizing the geographic variability in venom composition and their distinct immunoreactivity against available antivenoms. The unlocked potential of venom gland transcriptomics, droplet microfluidics, and alternative protein scaffolds

Thomas Fryer 1 and Timothy P Jenkins 2,3

1Department of Biochemistry, University of Cambridge, Cambridge CB3 0ES, UK 2Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, UK 3Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark

Snakebite envenoming has for a long time been an overlooked problem, despite the hundreds of thousands of deaths and disabilities caused by this disease each year. Fortunately, technological advances, alongside an increase in public funding, are now presenting a new horizon for snakebite therapeutics. Hence, researchers are exploring novel approaches towards treating snakebite envenomations, such as the deployment of monoclonal immunoglobulin G antibodies (IgG). However, whilst there have been promising results in the development of IgG based recombinant antivenoms, non-antibody protein scaffolds provide an intriguing alternative or addition to the development of next-generation antivenoms. Indeed, such scaffolds are likely to overcome some of the limitations of IgGs, while retaining several of their benefits. Particularly, designed ankyrin repeat proteins

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 10 (DARPins) and nanobodies carry significant promise due to their impressive safety profile in humans (established through rigorous clinical testing), high thermostability (potentially making cold chain distribution to remote locations redundant), low cost of manufacture, and high engineerability. Furthermore, recent developments in phage display and droplet microfluidics have the potential to enable ultra-high-throughput discovery and development of antitoxins, while venom gland transcriptomics could hold the key to global key-toxin identification. If we harness the combined potential of these scaffolds and of high-throughput and cutting edge- technologies, we could provide a novel, cutting edge, and high-tech solution for global envenomings that is still affordable to those most in need. Discovery of a small molecule toxin with inhibitory activity at human P2X4 ion channels

Lucka Bibic 1, Volker Herzig 2, Glenn F. King 2, Mark Searcey 1, Leanne Stokes 1

1School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, UK 2Institute for Molecular Bioscience, University of Queensland, Australia

Today, one in five adults experience chronic pain and this figure increases for those > 65 years old. However, frustration is mounting over the inadequate treatment for chronic neuropathic pain since its symptoms are difficult to treat and often resistant to opioids. Processing of pain signals relies on the activities of ion channels with some of the P2X receptors being important players. Animal venoms can be used to gain insights into potential new modulators for ion channels. Our aim is to investigate whether crude venoms contain modulators of key P2X receptors including P2X4, P2X3 and P2X7. Using high-throughput fluorescent screening assays (poster) we tested crude venoms and performed fractionation on those with either inhibitory or potentiating effects on human P2X4. Fractionation of crude spider venoms was performed using RP-HPLC and molecular identification of the inhibitory fractions (compounds 1 and 2) was performed by MALDI-TOF, LC-MS and MS-MS. This analysis indicates the inhibitory toxins to be two structurally uncharacterized small molecules, found in a number of spider species. Additionally, with compound 1 we found no modulating effect on rat P2X4, hP2X7, hP2X3, hNMDA 1A/2A receptors. The inhibitory activity on hP2X4 was confirmed on mouse P2X4, using both stable HEK-293 and a mouse microglial BV2 cell lines. Spider venoms typically contain small molecular components in abundance. However, none of the tested small molecules in the venoms such as nucleotides or polyamines showed an effect on hP2X4 responses. In addition to small molecules, our screening assay also showed some potential inhibitory peptides that might play a role in blocking the human P2X4 receptor. Further characterisation and validation is required to understand whether these novel compounds could be useful as analgesics. This work is supported by BBSRC NRP Doctoral Training Program and the Royal Society. Rear-fanged snake venom novelty: Adaptive toxicity and metalloproteinase activity

Cassandra M. Modahl 1,2 and Stephen P. Mackessy 2

1Department of Biological Sciences, National University of Singapore, Science Drive 4, Singapore 117543 2School of Biological Sciences, University of Northern Colorado, 501 20th St., CB 92, Greeley, CO 80639-0017 USA

Venom research has focused on front-fanged venomous snakes with fewer than three percent of rear-fanged snake venom proteomes characterized. These venoms have been neglected due to difficulties obtaining venom for characterization and a lack of toxin sequences in databases for proteomic identifications. We characterized venom gland transcriptomes, venom proteomes, and toxin biological activities, using both enzymatic and toxicity assays, for two Old World and two New World rear-fanged snake species. Venoms were dominated by either three-finger toxins (3FTxs) or metalloproteinases, paralleling the venom dichotomy of front-fanged venomous snakes. Rear-fanged snakes Boiga irregularis and Spilotes sulphureus had venoms dominated by 3FTxs, and shared similar heterodimeric 3FTxs with lizard specific toxicity. In addition, S. sulphureus venom contained a monomeric 3FTx with mammal specific toxicity, allowing this non-constricting snake to capture mammalian prey, whereas B. irregularis uses constriction. Metalloproteinase dominated venoms from Ahaetulla

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 11 prasina and Borikenophis portoricensis rapidly degraded the alpha subunit of fibrinogen, and within five minutes A. prasina venom also degraded beta subunits. Beta subunit degradation rate for A. prasina was even faster than observed for the front-fanged rattlesnake Crotalus viridis viridis. Other common snake venom enzymes, including serine proteases, L-amino acid oxidases, acetylcholinesterases, phosphodiesterases, and phospholipase A 2s, were absent or present at negligible levels in the four rear-fanged snake venoms. The majority of bites from rear-fanged venomous snakes do not produce systemic effects in humans, however these venoms provide insight into toxin binding selectivity and mechanisms of action, as well as predator-prey toxin evolution. Unraveling the mechanisms of action of hemorrhagic toxins of viperid snake venoms

José María Gutiérrez

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica

Local and systemic hemorrhages are common clinical manifestations in envenomings by viperid snakes. Zinc- dependent metalloproteinases (SVMPs) are the main hemorrhagic toxins in snake venoms. From a structural standpoint, SVMPs are classified within classes P-I, P-II and P-III, depending on their domain composition. In general, SVMPs of the P-III class are more hemorrhagic than those of the P-I class, suggesting relevant roles for the disintegrin-like and the cysteine-rich domains, probably related to the presence of exosites responsible for their binding to key targets in the microvasculature. The variation in hemorrhagic potency within the class of P-I SVMPs has been associated with variations in the flexibility of a loop located close to the active site. The key event in the onset of microvascular damage induced by SVMPs is the cleavage of substrates of the basement membrane (BM) that provide mechanical support to endothelial cells in capillaries. Experimental evidence strongly suggests that hydrolysis of type IV collagen at the BM is the key step in the onset of hemorrhage, owing to the structural role of this collagen. However, cleavage of other proteins associated with the BM and with endothelial cell-cell junctions may be also involved. Proteomic analyses of exudates collected from envenomed tissues have identified hitherto unknown substrates of SVMPs in the extracellular matrix, which may have implications for the pathogenesis of hemorrhage. Once the mechanical stability of the BM is affected, the biophysical hemodynamic forces operating in the circulation cause the distension and eventual disruption of the capillary wall. Studies on hemorrhage in muscle tissue, skin and lungs reveal similarities and differences in the pathogenesis of local and systemic hemorrhage. In addition, the action of viperid venom components on hemostasis, i.e. on blood clotting and platelets, further contributes to bleeding through complex synergistic patterns. Low molecular mass metalloproteinase inhibitors are being explored as potential therapeutic tools that would complement antivenoms in the treatment of snake venom-induced hemorrhage. Complement system inhibition as means for controlling inflammation associated to envenomation

Denise V. Tambourgi , Lygia Samartin Gonçalves Luchini, Giselle Pidde, Carla C. Squaiella Baptistão

Immunochemistry Laboratory, Butantan Institute, São Paulo, SP, Brazil

The complement (C) system, one of the first lines of defence of the innate immunity, is a major mechanism by which the body recognizes foreign substances and pathogens. In nature a wide range of animal venoms and animal secretions are found to interact with C-system, which activation can contribute to both the spread of the venom and the inflammatory response. An in-depth characterization of the action of venoms on the activation of the C-system as well as the use of C-inhibitors in experimental models of envenomation may shed light on potentially specific therapies targeting this system. We previously showed that Bothrops venoms are able to activate C-system and induce the generation of anaphylatoxins, potent pro-inflammatory mediators, whose mechanisms include the direct cleavage of C-components by snake venom metalloproteinases and serine proteinases present in the venoms. A metalloproteinase able to activate the three C-pathways and generate

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 12 active anaphylatoxins, named C-SVMP, was purified from the venom of Bothrops pirajai . C-SVMP was able to activate the C-system in the ex-vivo whole blood model, generating C3a/C3a desArg, C5a/C5a desArg and the Terminal Complement Complex (TCC). This protein was able to promote an increase in the expression of CD11b, CD14, C3aR, C5aR1, TLR2 and TLR4 markers in leukocytes. Inhibition of component C3 by compstatin, a C- inhibitor, significantly reduced the production of anaphylatoxins and the TCC in blood plasma treated with the toxin, as well as the expression CD11b, C3aR and C5aR on leukocytes. C-SVMP was able to induce increased production of cytokines and chemokines in the human whole blood model. The addition of compstatin to the reactions caused a significant reduction in the production of these mediators. The data obtained with the use of compstatin indicate that Complement inhibition may significantly control the inflammatory process initiated by Bothrops snake venom toxins. Financial support: CETICS/FAPESP # 2013/07467-1. Evolutionary considerations on separate predatory and defensive venoms

Sebastien Dutertre

Institut des Biomolécules Max Mousseron, Département des Acides aminés, Peptides et Protéines, Université Montpellier - Centre Nationale de la Recherche Scientifique, Place Eugène Bataillon, 34095 Montpellier CEDEX 5, France

It is generally accepted that prey capture efficiency is the major driver of venom evolution, yet most venomous animals use these complex mixtures of toxic components not only to facilitate prey capture but also to defend against predators. For instance, venomous snakes exist in ecosystems as both predators and prey, and in a typical arm race fashion, the venomous snake predator is expected to evolve more potent toxins to rapidly subdue its prey, as the latter keeps fighting back the debilitating effects of the venom (e.g. by modifying its toxin-binding receptors to prevent the interaction with the toxins, as in rattlesnake vs ground squirrel). However, in a similar way, a non-venomous predator can also adapt its physiology to avoid the deleterious effects of the defensive injections from a venomous snake prey (e.g. mongoose vs cobra). This suggests that the defensive use of venom maybe an underestimated evolutionary force capable of driving equal or even greater adaptations to the species compared to predatory use. Whereas it is largely assumed (but not often demonstrated!) that the same combination of toxins is injected regardless of the intended purpose (predation and defence), presumably exploiting conserved target pharmacology across prey and predators, recent research on carnivorous marine snails of the genus Conus strongly question this assumption. Indeed, in a new paradigm, cone snails were shown to rapidly and reversibly switch between two types of venoms in response to predatory or defensive stimulus, providing an unprecedented opportunity to challenge the role of predation and defence in venom evolution and specialization. Venom in context: evolution, ecology and snakebite

Timothy N. W. Jackson

Australian Venom Research Unit, Department of Pharmacology & Therapeutics, University of Melbourne, Australia

Snake venom is a functional trait designed (by natural selection) to facilitate the subjugation or deterrence of one animal (the target organism) by another (the snake). Venom is thus an intrinsically “ecological” trait – there can be no “venom” without interaction between the venomous and the envenomed. Unfortunately, that interaction, all too frequently in parts of the tropical world, involves a venomous snake and a human. This ecological interaction, in which the human is perceived as a potential predator by a defensive snake, can have tragic consequences. In order to understand venom, we must situate it in its evolutionary context. In order to significantly reduce the global impact of snakebite envenoming, we must (at least instrumentally) understand both venom and the circumstances in which snakebite occurs. In this talk, I will highlight recent research on the importance of context for the evolution of venom, from the deep-evolutionary history of venom genes and their “recruitment” into venom systems, to the origins of the functional diversity that contributes to making the design of broad-spectrum antivenoms an ongoing challenge. In addition, I will describe snakebite in ecological

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 13 terms and argue that this approach to understanding the “multifactorial” crisis of snakebite envenoming may deliver important insights applicable to its mitigation. Medically important African snakes cause variable acute phase and inflammatory responses following envenoming

Chloe Evans , Jaffer Alsolaiss, Nicholas R Casewell, Robert A Harrison

Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK

The local and systemic effects of snakebite envenoming can result in severe pathology, leading to long term morbidity and mortality. The severity of envenoming pathology is likely amplified by acute phase and inflammatory responses. During these responses, various inflammatory mediators are synthesized, stimulating hepatic cells to generate acute phase proteins and causing physiological and biochemical changes. However, the acute mammalian response to different snake venoms responsible for causing diverse pathologies are yet to be explored. We collected blood and sera from mice six hours post envenomation with ten medically important snake species of sub-Saharan Africa ( Bitis , Echis , Naja , and Dendroaspis ). Venoms from the African cobras Naja nigricollis and Naja melanoleuca caused significant elevations in the levels of circulating inflammatory markers, including haptoglobin, P-selectin, and in the case of N. melanoleuca envenoming, Serum Amyloid A. N. nigricollis venom also increased systemic production of interleukin 6 (IL-6) by 100-fold. We generated white blood cell counts from thin blood films, revealing lymphopenia with neutrophilic leukocytosis in mice envenomed by both cobra venoms, and the red blood cell morphology in all cases of envenoming was abnormal and indicative of membrane abnormalities and mechanical damage. An in vitro assay confirmed direct haemolysis in the presence of N. nigricollis venom, and biochemical analyses of sera revealed elevated concentrations of bilirubin, evidencing red blood cell breakdown. In combination, our results demonstrate that acute inflammation is a systemic pathology of snake envenomation that is not restricted to previously studied viper venoms. These findings advocate for further clinical research on this topic to better understand whether the acute phase and inflammatory responses stimulated following envenoming can be abrogated by existing therapeutics to the benefit of snakebite victims. Drugs from toxins? Some dos and don’ts

Alan L Harvey

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0NR, UK

Toxins and venom components have provided successful leads to new medicines. The main successes are well- known: curare-like muscle relaxants, ACE inhibitors, integrin antagonists, pain-relieving channel blockers and, of course, botulinum toxins. However, successful toxin-based drug discoveries are few in number despite continued interest in this area by the toxinology community. Why should this be? And what might help to improve the transition from toxin research to the development of new medicines? Perhaps the biggest obstacle is the most common starting point: most toxin research is conducted in academic laboratories and the focus of the research is on the toxin; most successful drug discovery efforts focus on an unmet therapeutic need. Many toxin-centred drug discovery projects are, in effect, searches for potential therapeutic applications of newly discovered or otherwise interesting toxins. It is better to seek to marry unique bioactivity properties of a toxin with poorly treated pathology: a good example is how the extremely long-lived blockade of acetylcholine release from motor nerve endings by botulinum toxin is uniquely successful in dampening the overactive nerve activity in various conditions involving uncontrolled muscular spasms. More generally, there can be dangers in assuming that the high specificity shown by many toxins will sidestep the possibility of side-effects when used in humans. Toxin researchers aiming at drug development must always test thoroughly the selectivity, specificity and potential for side-effects of their lead compound: although the toxin may have exquisite affinity for a single

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 14 molecular target, the target may not be nicely localised to the tissue affected by the disease. Another difficulty facing academic-based drug seekers is that the range of skills needed for a successful drug discovery and development project is vast. In most cases, collaborators and partners are needed to provide the required resources and expertise. Venom peptide inhibitors of acid-sensing ion channel 1a for treating ischemic injuries of the heart and brain

Glenn F King

Institute for Molecular Bioscience, The University of Queensland, St Lucia QLD 4072, Australia

Abstract excluded on author’s request Demonstrating the potential of a novel spider venom based biopesticide for target- specific control of the small hive beetle, a serious pest of the European honey bee

Elaine C. Fitches 1,2 , Hannah M. Bradish 1, Min Cao 1, Rebecca Mackinson 1, J.A. Gatehouse 1, Michelle Powell 2

1School of Biosciences, Durham University, South Road, Durham DH1 3LE, UK 2Fera Science Ltd., Sand Hutton, York YO41 1LZ, UK

Spider venoms contain cysteine-rich peptides that interact with ion channels in the nervous system (NS) of prey causing paralysis. High potency, stability and specificity of action towards invertebrates makes such peptides ideal candidates for development as insecticides. However, whilst these peptides may be lethal when injected, efficacy is typically low when delivered orally to insects due to failure to access their target sites of action in the NS. We have developed a novel method that enables the oral delivery of insecticidal peptides to their site of action via fusion to a “carrier” protein that directs transport of an attached toxin across the insect gut to the circulatory system. Constructs encoding a selected toxin linked to snowdrop lectin (GNA) are used to produce recombinant fusion proteins by microbial fermentation. Fusion proteins comprised of toxins derived from venom of the cellar spider ( Segestria florentina; Sfl1) ; the red scorpion ( Buthus tamulus ; ButaIT) and the funnel-web spider ( Hadroncyhe versuta ; ω-ACTX-Hv1a) have shown oral activity towards a range of insect pests.The parasitic small hive beetle ( Aethina tumida ) is a serious pest of the European honey bee ( Apis mellifera ), the most important global crop pollinator. This invasive beetle feeds on pollen, honey and bee brood causing honey to ferment, combs to be destroyed and often structural collapse and abandonment of hives. Establishment of A. tumida in North America and Australia has resulted in significant economic damage to the apiculture industry and further threat is evidenced by its detection across a wide range of countries, including Canada, Egypt, Mexico, Sudan, Hawaii, Philippines, Portugal and Italy. The inherent toxicity of chemical control measures to bees makes effective treatment problematic. Here we report potential for the “in hive” use of a fusion protein comprised of the ω-ACTX-Hv1a toxin linked to GNA that is toxic to this invasive beetle pest but harmless to honey bees. Improving the Therapeutic Index of Venom Derived Antimicrobial Peptides.

K Miller, K.M. Rawson & P.N. Strong

Biomolecular Sciences Research Centre, Sheffield Hallam University, Sheffield, S1 1WB UK

Antimicrobial peptides (AMPs) are naturally occurring compounds which possess a rapid killing mechanism and low resistance potential. AMPs are typically cationic and interact preferentially with the weakly anionic bacterial membrane. The binding mechanisms are multi-site and are more complex than a simple cation-anion binding reaction and other domains, such as charged and hydrophobic regions, have been associated with antimicrobial activity. One of the major factors limiting further development of AMPs is off target toxicity. We have previously characterised AMPs derived from the venom of the scorpion Scorpio maurus palmatus. This study seeks to

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 15 better understand the relationship between structure, function and bacterial selectivity by modifying the sequence of these peptides. It has previously been shown that increasing the charge of an AMP can increase antimicrobial activity. A systematic amino acid substitution approach has been taken to create mutant peptides with increased charge. As hydrophobicity has also been implicated in AMP function, amino acid substitutions have been performed to create more hydrophobic mutants. The structure-function relationship of our AMPs has been investigated by performing N-terminal, mid-chain and C-terminal amino acid substitutions and determining the effect this has on their antimicrobial and cytotoxic activity. Increased charge at the N, mid and C-terminus of the peptide resulted in increased antimicrobial activity. Increased hydrophobicity at the N- terminus resulted in reduced haemolysis and cytotoxicity. Reduced antimicrobial, haemolytic and cytotoxic activity was observed by increased hydrophobicity at the mid-chain. Functional improvements have been made to modified peptides when compared with native AMPs, which has produced peptides with enhanced therapeutic indices. Beyond hemostasis: a potassium channel blocker snake venom serine protease with potential antitumor activity

Johara Boldrini-França 1,2 , Ernesto Lopes Pinheiro-Junior 1,2 , Steve Peigneur 2, Manuela Berto Pucca 1, Felipe Augusto Cerni 1, Rafael Junqueira Borges 3, Tássia Rafaella Costa 1, Sante Emmanuel Imai Carone 1, Marcos Roberto de Mattos Fontes 3, Suely Vilela Sampaio 1, Eliane Candiani Arantes 1 and Jan Tytgat 2

1School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/nº, Ribeirão Preto, SP Brazil 2Toxicology and Pharmacology, KU Leuven, O&N2 Herestraat 49 – PO Box 922, 3000 Leuven, Belgium 3Institute of Biosciences, São Paulo State University (UNESP), Rua Prof. Dr. Antonio Celso Wagner Zanin, Botucatu, SP, Brazil

Snake venom serine proteases (SVSPs) are complex and multifunctional enzymes that act primarily on hemostasis. In this work, we report the hitherto unknown inhibitory effect of a SVSP isolated from the venom of Crotalus durissus collilineatus , named collinein-1, on the voltage-gated and cancer-relevant h EAG 1 potassium channel (Kv10.1). Of all potassium channel types tested, it was found that collinein-1 selectively inhibits h EAG 1 and that the inhibition occurs independently from its enzymatic activity. Corroboratively, it was found that collinein-1 reduces the cell viability of human breast cancer MCF7 (high expression of h EAG ), but neither affects cells from liver carcinoma HepG2 (low expression of hEAG ) nor from non-tumorigenic epithelial breast cell line (also low expression of h EAG ). In order to obtain both functional and structural confirmation of this unexpected finding, where an unusually large ligand acts as an inhibitor of an ion channel, a recombinant and catalytically inactive mutant of collinein-1 (His43Arg) was made and found to preserve its capability of inhibiting h EAG 1. A molecular docking model is also proposed. Financial support: CAPES, CNPq, FAPESP and FWO-Vlaanderen. PEGylating toxins: a new trend in toxinology? A successful example of a PEGylated snake venom serine protease

Ernesto L. Pinheiro-Junior 1,5 , Johara Boldrini-França 2, Tássia R. Costa 3, Agnes A. S. Takeda 4, Suely V. Sampaio¹, Marcos Roberto M. Fontes 4, Steve Peigneur 5, Jan Tytgat 5, Eliane C. Arantes¹

1School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil 2Graduate Program in Ecosystem Ecology, University of Vila Velha, Brazil 3Institute of Biotechnology, Federal University of Uberlandia, UFU, Uberlandia, MG, Brazil 4Institute of Biosciences – São Paulo State University, Brazil 5Toxicology and Pharmacology, KU Leuven, Belgium

PEGylation has been used for more than 20 years as a strategy to decrease immunogenicity and improve pharmacokinetic properties of biopharmaceuticals. However, it remains poorly employed in toxinology, even though it may be a promising strategy to empower molecule candidates in therapeutics. rCollinein-1 is a recombinant snake venom serine protease (SVSP) derived from Crotalus durissus collilineatus venom, with

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 16 effects on fibrinogen consumption and inhibitory activity on hEAG channels. This work compared the functional, structural and immunogenic properties of the non-PEGylated (rCollinein-1) and the PEGylated form (PEG- rCollinein-1) of this SVSP. PEG-rCollinein-1 shares similar kinetic parameters with rCollinein-1, presenting comparable K m, k cat and K m/k cat values. PEGylation also maintained its capability of degrading fibrinogen, but strongly reduced its blocking activity on hEAG channels. Both PEGylated and non-PEGylated enzymes showed to be non-toxic to peripheral blood mononuclear cells (PBMC), even in high concentrations, with cell viability higher than 85%. Structurally, circular dichroism (CD) analysis revealed the maintenance of protein conformation after PEGylation, sharing similar content of secondary elements with the non-PEGylated form. Regarding their immunogenicity, in silico analysis indicated four putative amino acid epitopes, located in the surface of the protein structure, near to possible sites of PEGylation. Interestingly, immune response on mice showed PEG- rCollinein-1 was devoid of immunogenicity, even after four sensibilizations, whereas the non-PEGylated enzyme showed low immunogenicity. Consequently, besides reducing the immunogenicity of this SVSP, PEGylation also directed its activity towards hemostasis control, broadening its possibilities to be employed as a defibrinogenant agent in conditions related to imbalances in this system, such as stroke, thrombosis, and pulmonary embolism. The insecticidal wasp toxin γ–pompilidotoxin is a linear peptide modulating voltage- gated sodium channels

Steve Peigneur 1, Andrey V. Tsarev 2, Alexander S. Paramonov 2, Christina I. Schroeder 3 Maria P. Ikonomopoulou 4, Hidetoshi Inagaki 5, David J. Craik 3, Katsuhiro Konno 6, Zakhar O. Shenkarev 2 & Jan Tytgat 1*

1Toxicology and Pharmacology, Katholieke Universiteit (KU) Leuven, Campus Gasthuisberg, Leuven, Belgium 2M.M. Shemyakin & Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, the Russian Academy of Sciences, Moscow, Russia 3Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia 4Madrid Institute for Advanced Studies (IMDEA) in Food, CEI UAM+CSIC, Madrid, E28049, Spain 5Biomedical Research Institute, National Institute of Advanced Industrial Science 6Technology (AIST), Tsukuba, Ibaraki, Japan/Institute of Natural Medicine, University of Toyama, Toyama, Toyama, Japan

The fascinating observation that solitary wasps, such as spider wasps (Pompilidae), paralyze their prey instead of killing it, suggests their venoms to be interesting reservoirs of components acting on the nervous system. Hence, their venom promises to contain neurotoxins acting on ion channels and receptors. Pompilidotoxins (PMTx) are small (13-15 residue), non-disulfide bridge peptides isolated from solitary wasp venom. PMTxs interact with voltage-gated sodium (Nav) channels. γ-PMTX, isolated from Cyphononyx fulvognathus venom, is a novel member of the PMTx family. Electrophysiological characterization indicated that γ-PMTX acts by slowing down channel inactivation. γ-PMTX was screened against a panel of 7 mammalian Nav (Nav1.1-Nav1.6 and Nav1.8), 1 insect Nav from Blattella germanica (BgNav1) and 1 arachnid channel from Varroa destructor (VdNav1). γ-PMTX displays an interesting specificity for insect Nav channels over mammalian isoforms. Moreover, γ-PMTX induces cytolysis and histamine release from mast cells at higher concentrations. The structure of γ-PMTX was determined by CD and NMR spectroscopies and indicated that the peptide adopts an α-helical, amphipathic conformation in the presence of POPC/POPG lipid vesicles and micelles composed of DPC, in contrast to a disordered state in an aqueous solution. The senolytic and antitumoral properties of γ-PMTX was evaluated in different cell assays. An Ala-scan was performed indicating the relevance of a Phe residue in the middle of its sequence, determinant for the peptide’s biological activity. Cyclized analogs of γ-PMTX were also designed and investigated for their activity on Nav channels. The obtained results indicate that the structural and pharmacological properties of γ-PMTX render this peptide as a potential lead compound for further development of novel insecticides targeting Nav channels. To the best of our knowledge, γ-PMTX is the shortest, Cys-free peptide modulating Nav channel inactivation gating.

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 17 KnotBodies: Fusing venom derived mini-proteins to antibody CDR loops to treat ion channel mediated pathologies

Aneesh Karatt-Vellatt , Damian C Bell, Sachin B Surade, Tim Luetkens, Edward W Masters, John McCafferty

IONTAS Ltd, Iconic Park, London Road, Pampisford, Cambridge CB22 3EG, UK

Venom derived mini-proteins (e.g. knottins) have potential as therapeutic agents to modulate ion channels involved in cancer, autoimmunity and pain but can suffer from manufacturing difficulties, short half-lives and a lack of specificity. We have developed a novel molecular format wherein a peripheral CDR loop of an antibody has been replaced by a knottin. In this format (termed KnotBody TM ), the antibody gains additional diversity within a scaffold pre-disposed to the blockade of ion channels and the knottin enjoys the extended half-life and the additional specificity conferred by the antibody molecule. A proof-of-concept fusion protein of one structural domain within another was initially achieved by inserting a trypsin inhibiting knottin (EETI-II) flanked by diverse repertoire of short linker sequences into the CDR2 position of naïve antibody light chain sequences. Functional KnotBody TM molecules then were selected from this library using phage display technology on the basis of retained trypsin binding, with the correct folding of both domains confirmed using X-ray crystallography. To further demonstrate the benefits of this novel format, the modular nature of the KnotBody TM binding surface was exploited to: (i) improve existing knottin binding by introducing additional V H contacts; (ii) create a bispecific molecule by introducing a V H chain that binds to a different target; (iii) generate ion channel modulating KnotBody TM molecules by substituting the proof-of-concept knottin (EETI-II, a trypsin inhibitor) with Kv1.3, ASIC1a and Nav1.7 blocking toxins. What is cheaper: synthesis or acquisition of toxins from exogenous sources?

Dietrich Mebs

Institute of Legal Medicine, Goethe-University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt, Germany

In the animal kingdom, acquiring toxicity is achieved using intrinsic synthesis of toxins either by gene expression or by complex metabolic pathways, but also through the uptake, accumulation and storing toxic compounds produced by other organisms, such as microbes, plants or other animals. When economic aspects are concerned, it is difficult to evaluate which way of acquiring toxicity is cheaper or most efficient. Resistance needs to be developed when sequestering toxic chemicals, such as changes at the molecular level of receptors or ion- channels. This may eventually outweigh the benefits of saving metabolic energy. On the other hand, adaptation and resistance to toxins enables their entering into marine as well as terrestrial food webs. As the result, the role they may play in ecosystems is not always clear. Moreover, conclusive answers to questions do not exist, like what forces drive the evolution of toxins, are selection pressures shaping optimal structures and/or are genetic mechanisms involved promoting the adaptation to a specific target. Fitness to survive does not depend on the possession of the best toxin and there exist examples that toxins have lost their original significance or function. However, both ways to acquire toxicity, synthesis or sequestration, proved to be equally successful during evolution, leading to the huge diversity of toxic compounds. There is no such thing as ‘centipede venom’ – comparative proteotranscriptomic analyses uncover deep divergence of venom composition in Chilopoda

Ronald A. Jenner 1, Eivind AB Undheim 2, 3, 4

1Department of Life Sciences, Natural History Museum, Cromwell Road, London SW7 5BD, UK 2Centre for Ecology and Evolutionary Synthesis, Department of Bioscience, University of Oslo, Blindern, Oslo, Norway 3Centre for Advanced Imaging, University of Queensland, St Lucia, QLD 4072, Australia 4Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD 4072, Australia

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 18 Centipedes (Arthropoda: Chilopoda) are one of the oldest groups of venomous terrestrial predators. Since their origin in the Silurian centipedes have diverged into five extant lineages that are classified as orders. Yet, our understanding of centipede venom composition is based almost entirely on just one of these orders, Scolopendromorpha, which includes the most familiar species, including large tropical ones. Proteotranscriptomic venom profiles are available for just six scolopendromorph species, as well as one species from the order Scutigeromorpha (house centipedes). In this study we conducted the first comparative proteotranscriptomic analyses of centipede venom composition including representatives of all five orders. This allowed us to trace the macroevolutionary dynamics of centipede venom composition, as well as to reconstruct the nature of ancestral venoms in several parts of the centipede tree. Our results show that there is no such thing as a typical centipede venom. Compositional evolution of venoms is concentrated within the five orders. Venoms remained relatively simple cocktails during the early evolutionary history of centipedes, after which venom complexity increased in parallel in each of the orders via numerous recruitments of protein and peptide families. However, the loss of gene families streamlined venoms as well, and Strigamia maritima , the only centipede with a published genome, has lost from its venom all toxin families that we inferred to be present in the ancestral centipede venom. A knottin scaffold directs CXC-chemokine binding specificity of tick evasins

Christopher Lynch 1, Maud Deruaz 2, Angela W. Lee 1, Graham Davies 1, Kamayani Singh 1, Yara Alenazi 1, James R.O. Eaton 1,3 , Akane Kawamura 1,3 , Jeffrey Shaw 2, Amanda Proudfoot 2, João M. Dias 2 & Shoumo Bhattacharya 1

1RDM Division of Cardiovascular Medicine, University of Oxford, Oxford, United Kingdom 2Serono Pharmaceutical Research Institute, Geneva, Switzerland 3Department of Chemistry, University of Oxford, Oxford, United Kingdom

Tick evasins bind either CC or CXC-chemokines by a poorly understood promiscuous or “one-to-many” mechanism to neutralize inflammation. Here we identify 27 CXC-binding evasins homologous to EVA3 and define two functional classes. The first, which includes EVA3, exclusively binds ELR+ CXC-chemokines, whereas the second binds both ELR+ and ELR- CXC-chemokines, including in several cases CXCL10, but, surprisingly, not CXCL8. The X-ray crystal structure of EVA3 reveals a single anti-parallel β-sheet with six conserved cysteine residues that form a disulfide-bonded knottin scaffold creating a contiguous solvent-accessible surface. Swapping and deletion analysis identifies distinct knottin scaffold segments responsible for different CXC- chemokine binding activities, indicating that differential ligand positioning, at least in part, plays a role in promiscuous binding. The solvent-accessible surfaces of these scaffold segments have distinctive shape and charge, which we suggest drives chemokine binding specificity. These studies provide insight into how CXC- binding tick evasins achieve class specificity but allow promiscuous binding. Anguimorph lizard venoms disrupt the clotting ability of human fibrinogen via destructive cleavage

James S. Dobson 1, Christina N. Zdenek 1, Chris Hay 1, Aude Violette 2, Rudy Fourmy 2, Chip Cochran 3, Bryan G. Fry 1

1Venom Evolution Lab, School of Biological Sciences, University of Queensland, St. Lucia, QLD 4072, Australia 2Alpha Biotoxine, Montroeul-au-bois B-7911, Belgium 3Department of Earth and Biological Sciences, Loma Linda University, Loma Linda, CA 92350, USA.

Anguimorph lizard venom is a controversial topic, partly due to the lack of demonstrated functional activity. Despite numerous reports of bite victims having adverse effects beyond the mechanical damage of the bite, little research has been conducted on the toxins responsible or the pathways in which they act. Persistent bleeding is commonly reported after bites from helodermatid and varanid lizards. Previous studies have shown that one mechanism in which the anticoagulants could act is via the non-specific cleaving of fibrinogen chains. However, the ability of the venoms to prevent clot formation has not been demonstrated. Using a

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 19 Thromboelastograph (TEG 5000), clot strength was measured after incubating human fibrinogen with helodermatid and varanid lizard venoms. Clot strengths were found to be highly variable across the 27 species tested, with the weakest clots produced by incubation with varanid lizard venoms from the Odatria and Euprepriosaurus clades. Some of the most fibrinogenolytically active venoms belonged to V. prasinus and V. scalaris , which are arboreal species and therefore prey escape potential is a strong evolutionary selection pressure. The results are also consistent with reports of profusive bleeding from bites from these species as well as from other notably active species commonly kept in captivity, such as V. giganteus . Our results provide evidence in favour of the predatory role of Anguimorpha venom, thus shedding light on the evolution of venom in reptiles and revealing potential new sources of bioactive molecules useful as lead compounds in drug design and development. Venom collection and analysis in the pseudoscorpion Chelifer cancroides

Jonas Krämer 1, Hans Pohl 2, Reinhard Predel 1

1Institute for Zoology, University of Cologne, Cologne, Germany 2Institut für Zoologie und Evolutionsforschung, Friedrich-Schiller-Universität Jena, Jena, Germany

Pseudoscorpions are tiny arachnids adapted to crevice habitats. Some pseudoscorpions possess pedipalps equipped with a unique venom apparatus for paralyzing prey. The venom of these animals is poorly known. We performed a proteomic analysis of the pseudoscorpion venom, which identified the first genuine venom compounds of these animals. To this end, we have developed a non-invasive approach for extracting minute amounts of venom, enabling the collection of pure venom samples from pseudoscorpions with minimal contamination and high reproducibility. To our knowledge, pseudoscorpions are the smallest venomous animals that have ever been milked individually. Venom release needed to be triggered by a combined mechanical and electrical stimulation, indicating the presence of a mechanism that prevents the wasteful release of venom. High-resolution scanning electron micrographs of the venom delivery system confirmed the presence of a modified seta ( lamina defensor ) at the base of all venom teeth, which might be involved in the venom release. Extracted venom was used for top down proteomics, which, in combination with transcriptomic data, enabled the identification of venom compounds that show sequence similarity to Megicin , an antimicrobial peptide known from the venom of the scorpion Mesobuthus gibbosus . Comprehensive insights into the venom system and the genomic processes of toxin evolution in robber flies (Asilidae, Diptera)

Bjoern Marcus von Reumont 1,2,3 , Stephan Holger Drukewitz 3

1LOEWE Centre for Translational Biodiversity Genomics (LOEWE-TBG), Senckenberganlage 25, 60325 Frankfurt, Germany 2Justus Liebig University, Institute for Insect Biotechnology, Heinrich Buff Ring 58, 35394, Gießen, Germany 3Fraunhofer Institute for Molecular Biology and Applied Ecology, Project group Bioressources, Animal Venomics, Germany

Venoms and the toxins they contain represent molecular adaptations that have evolved on numerous occasions throughout the animal kingdom. However, the processes that shape venom protein evolution are poorly understood because of the scarcity of whole genome data available for comparative analyses of venomous species. Here, we present a broad comparative toxicogenomic analysis on several species to gain insight into the venom system of robber flies (Diptera, Asilidae). Additionally we sequenced a new genome of the hymenopteran hunting asilid Dasypogon diadema to understand the genomic mechanisms of venom evolution in these neglected predatory dipterans. We then applied a comparative genomics approach, based on one additional asilid genome, ten high-quality dipteran genomes, and two lepidopteran genomes as outgroup to reveal the mechanisms and evolutionary origin of the identified venom proteins in robber flies. From 30 identified predominant venom proteins in the non-asilid genomes, 15 highly expressed venom proteins appear to be unique to robber flies. Our results reveal that the venom of D. diadema likely evolves in a multimodal fashion

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 20 comprising 1) neofunctionalization after gene duplication, 2) expression-dependent co-option of proteins and 3) asilid lineage-specific orphan genes with enigmatic origin. The occurrence of orphan genes is currently discussed in evolutionary genomics, but has not yet extensively been tested in the context of toxin evolution. Our results display an unexpected dynamic venom evolution in asilid insects, which contrasts the findings of the only other insect toxicogenomic evolutionary analysis, in parasitoid wasps (Hymenoptera), where toxin evolution is dominated by single gene co-option. These findings underpin the significance of further genomic studies to cover more neglected lineages of venomous taxa and to understand the importance of orphan genes as possible drivers for venom evolution. Is the cnidarian parasite of caviar, ‘ Polypodium hydriforme ’, a venomous animal?

A. Hartigan 1, L. Doonan 2, A. Jaimes-Becerra 3, A. C. Marques 3, B. Okamura 1 and P. F. Long 2,3

1Natural History Museum, London, UK 2King’s College London, UK 3University of São Paulo, Brazil

Polypodium hydriforme is a unique cnidarian adapted to parasitism of developing oocytes of acipenseriform fish (sturgeons and paddlefishes). Phylogenomic and morphological evidence place P. hydriforme as a monotypic sister clade to the diverse Myxozoa, which are also important parasites of fish but additionally use invertebrate hosts in their complex life cycles. Polypodium ’s one-host life cycle involves a parasitic larval stage in the oocytes and a free-living stolon of connected tentaculate individuals that emerges from spawned eggs. Subsequent fragmentation releases single individual polyps that take up a freshwater benthic life. Free-living P. hydriforme are approximately the same size as the model freshwater cnidarian Hydra. Both Hydra and free-living P. hydriforme can ‘walk’ on their tentacles. However, four tentacles of P. hydriforme are apparently specialised for this form of locomotion while eight longer sensory tentacles are used for prey capture and in defence . The tentacles of Hydra are all morphological similar. Unlike Hydra , the cnidome of P. hydriforme possesses no specialised penetrant nematocysts. Instead, Polypodium uses unusual holotrichous isorhizas that act as penetrants because their discharged tubule is covered by two helical rows of tiny spines - a peculiar feature of P. hydriforme that has never been described in nematocysts of other cnidarians. There are no published studies that describe the toxin content of Polypodium nematocysts. Herein we present genomics, transcriptomics and proteomics approaches to identify toxin and toxin-like proteins from free-living P. hydriforme polyps. Discovering and confirming such toxins allows comparison with the well-studied venom of Hydra which lacks a parasitic stage. Additional results will be presented to determine if inclusion of myxozoans alters this perspective. Whether toxins in endocnidozoans ( Polypodium + Myxozoa) are deployed differently than in free-living relatives will also be discussed. Assessing rangewide variation of toxin defenses in a poisonous amphibian

Gary M. Bucciarelli 1, David B. Green 2, Brad H. Shaffer 1, Lee B. Kats 2

1Department of Ecology and Evolutionary Biology, University of California, Los Angeles, Los Angeles, CA, USA 2Natural Science Division, Pepperdine University, Malibu, CA 90263, USA

Comparisons of range-wide phenotypic variation can reveal the evolutionary and ecological forces that have produced distinct trait differences between populations. When interacting species have well-matched phenotypes they may become tightly linked in an antagonistic relationship that leads to coevolution, if the traits are heritable and there is reciprocal selection. Variation of a neurotoxin chemical defense (tetrodotoxin, TTX) in newts (genus Taricha) and resistance in predatory snakes (genus Thamnophis) has remained an exemplar of coevolution and an escalating arms-race. Under this coevolutionary model, traits evolved in lock-step, leading to greater and greater chemical defenses and resistance in populations. Given this model, (i) newt TTX defenses should be greatest in populations where snake predators have the greatest resistance, (ii) variation of TTX should be minimal between populations in close geographic proximity, and (iii) TTX should be a stable trait within populations and individuals

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 21 that does not fluctuate. In this study, we characterize the spatial and temporal patterns of newt TTX from nearly 50 populations across California, assess the magnitude of variation across space and time, and ultimately determine the degree to which TTX within populations matches known predator resistance. Ultimately, our results suggest that a coevolutionary model does not explain the evolution of TTX in newts. A chimeric botulinum neurotoxin unveiled that TNF α–induced surface trafficking of TRP/V1 and /A1 channels in sensory neurons is dependent on SNAP-25

J Oliver Dolly

International Centre for Neurotherapeutics, Dublin City University, Ireland

Transient receptor potential (TRP) vallinoid 1 (TRPV1) and ankyrin 1 (TRPA1) are two transducing channels expressed on peripheral sensory nerves involved in pain sensation. Upregulation of their expression, stimulated by inflammatory cytokines and growth factors in animal pain models, correlate with the induction of nociceptive hyper-sensitivity. Herein, we demonstrate by immuno-cytochemical labelling that TNF α augments the surface content of these channels on cultured dorsal root ganglion (DRG) neurons from early postnatal rats and this, in turn, enhances the electrophysiological and functional responses of the latter to their specific agonists. A molecular basis underlying this TNF α-dependent enhancement was unveiled by pre-treating DRGs with a recently-published chimeric protein, consisting of the protease light chain (LC) of botulinum neurotoxin (BoNT) serotype E fused to full-length BoNT/A (LC/E-BoNT/A). This cleaves synaptosomal-associated protein of Mr 25k (SNAP-25) in vitro and was reported previously to exhibit anti-nociceptive activity in a rat model of neuropathic pain. Low pM concentrations of this chimera were found to prevent the TNF α-stimulated delivery of TRPV1/A1 to the neuronal plasmalemma and, accordingly, decreased their incremental functional activities relative to those of control cells, an effect accompanied by SNAP-25 cleavage. Advantageously, LC/E-BoNT/A did not reduce the basal surface contents of the two channels or their pharmacological responses. Thus, use of multiple complementary methodologies provides evidence that LC/E-BoNT/A abolishes the TNF α-dependent augmented, but not the resting, surface trafficking of TRPV1/A1. As TNF α is known to induce nociceptive hyper- sensitivity in vivo, our observed inhibition by LC/E-BoNT/A of its action in vitro could contribute to its potential alleviation of pain.

Comprehensive characterization of anthrax toxins during the course of inhalation infection in non-human primate models John R. Barr 1, Anne E. Boyer 1, Adrian R. Woolfitt 1, Maribel Gallegos Candela 1, Renato C. Lins 2, Maria I. Solano 1, James L. Pirkle 1

1Centers for Disease Control and Prevention, Atlanta, Georgia, USA 2Battelle Atlanta Analytical Services, Atlanta, Georgia, USA

Inhalation anthrax has been described as a triphasic clinical staging system that includes the early prodromal phase (high cure rate), an intermediate progressive stage (cure possible), and the late-fulminant stage (low cure rate). The toxins of B. anthracis are virulence factors that promote disease progression by disabling host immune defenses. These toxins are composed of three proteins, protective antigen (PA), edema factor (EF), and lethal factor (LF). PA combines with EF and LF to form the binary toxins edema toxin (ETx) and lethal toxin (LTx). We have developed sensitive mass spectrometry-based methods that quantify all the toxins including total LF (free LF+LTx), LTx, total EF (free EF+ETx), ETx and PA. We have applied these methods to non-human primate studies and shown that anthrax toxemia follows a triphasic progression with an initial rise (phase-1), a plateau/decline (phase-2), and a final rise (phase-3). The NHP study protocol was approved by Battelle’s and CDC’s Institutional Animal Care and Use Committees and adhered to the NIH guidelines for the ethical and humane care and use of laboratory animals. All aspects of the studies were designed to minimize stress in the animals. LF was the earliest toxin detected followed by EF and both toxins were detected well before symptom onset and before bacteremia.

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 22 Toxins levels at symptom onset were highest for PA (87.4±0.69-ng/mL) and LF (19.5±0.68-ng/mL). Toxin ratios changed throughout the course of infection. The LF/EF ratios were high in early infection (118±0.236) but converged to 9.8±0.283 in late infection. The LTx/LF and ETx/EF levels were low in early infections but were almost all in the complex form (LTx and ETx) at the time of death. Collectively, the results defined all toxin levels at all stages, elucidated critical toxemic indices with the potential for predicting treatment failures, and provided guidance for development of point-of-care diagnostics and advanced therapeutics. Mass Spectrometric Detection and Characterization of Botulinum Neurotoxins

Suzanne R. Kalb and John R. Barr

Centers for Disease Control and Prevention, Atlanta, GA 30341

Botulism is a life-threatening disease caused by intoxication with botulinum neurotoxins (BoNTs), protein toxins produced by C. botulinum which paralyze by cleaving proteins in the SNARE complex that are needed for nerve impulse transmission. Rapid clinical diagnosis of botulism is important in a public health emergency, and characterization of the BoNT causing botulism assists with epidemiological investigations. Our laboratory has developed, validated, and currently uses a mass spectrometric botulism diagnostic. Additionally, we have developed mass spectrometric techniques to characterize toxin subtypes/variants and to discover novel BoNTs and their enzymatic activity, independent of the presence of genetic information. We have used these techniques to characterize both a novel BoNT and to discover the enzymatic activity of a novel protein similar to BoNT. The enzymatic activity of a novel BoNT can be characterized by measuring the cleavage of native SNARE proteins by BoNT. We developed mass spectrometric methods to analyse intact and cleaved SNARE proteins and then applied these methods to identify the enzymatic activity of a novel protein from S. enterococcus whose genetic sequence was similar to that of the bont gene, yet the function of the protein was unknown. Additionally, we developed mass spectrometric methods to determine the amino acid sequence of a novel BoNT in absence of the gene sequence. The methods rely upon tryptic digestion of the novel BoNT and comparison of the mass spectral fragmentation patterns to patterns from well-characterized BoNTs. In combination, these mass spectrometry techniques allow for detection of BoNT in a clinical or other sample, allow for characterization of the toxin itself, and allow for characterization of the enzymatic activity of the toxin, all in the absence of genetic material. Proteus mirabilis urease: unsuspected non-enzymatic biological properties relevant to pathogenesis

Valquiria Broll 1, Fernanda C. Lopes 1, Anne H. S. Martinelli 1, Natalia R. Moyetta 2, Leonardo L. Fruttero 2, Matheus V. C. Grahl 2, Augusto F. Uberti 2, Diogo R. Demartini 1, Rodrigo Ligabue-Braun 1, Celia R. Carlini 2

1Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil 2Brain Institute and School of Medicine, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil

Infection by the uropathogen Proteus mirabilis leads to kidney and bladder stones and incrustation of catheters due to the enzymatic activity of its urease (PMU). The hydrolysis of urea catalyzed by PMU, an enzyme composed of subunits PmUre α, PmUre β and PmUre γ, in an ( αβγ )3 organization, releases ammonia and alkalinizes the medium, leading to precipitation of urine’s salts and stone formation. During the last two decades, ureases have been characterized as multifunctional toxins, which besides catalyzing the hydrolysis of urea, may contributed non-enzymatically to the development of diseases caused by urease-producing microorganisms. Particularly, neurotoxicity, exocytosis-inducing and pro-inflammatory effects were described for plant (ex: Canavalia ensiformis ) ureases as well as for the urease of the gastric pathogen Helicobacter pylori . Here, we evaluated non-enzymatic properties of a recombinant PMU, and of its separate subunits. The holourease displayed fungitoxicity, lethal activity in insects, and promoted activation and aggregation of insect hemocytes as well as of human platelets. The late effect was previously shown to closely correlate to the pro-

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 23 inflammatory properties of C. ensiformis’ and H. pylori ’s ureases. Like jaburetox, a recombinant polypeptide derived from a C. ensiformis urease, PmUre β was toxic against yeasts and insects, and also aggregated platelets. PmUre α (containing the catalytic site) and PmUre α were weakly or not active in the bioassays, hence PmUre β carries most of the non-enzymatic activities of PMU. Bioinformatics analyses revealed gene/segment duplication of PmUre β, and homologous sequences were found in plant ureases (jaburetox) as well as in the PmUre α subunit, uncovering the evolutionary divergence among ureases. Our data indicate that P. mirabilis urease may contribute in other unsuspected ways to the pathogenesis of urinary infection besides promoting stone formation. FAPERGS/CNPq/CAPES. Discovery strategies for recombinant antivenoms based on broadly-neutralizing recyclable human monoclonal antibodies

Shirin Ahmadi 1,2 , Manuela B. Pucca 1,3 , Felipe A. Cerni 1,4 , Jonas A. Jürgensen 1, Cecilie Knudsen 1, Urska Pus 5, Line Ledsgaard 1, Christoffer V. Sørensen 1, Sofie Føns 1, Rahel Janke 1, Julius Knerr 1, Andreas Bertelsen 1, Rasmus I. Dehli 1, Andrea Martos Esteban 6, Aneesh Keratt-Vellatt 6, John McCafferty 6, Bruno Lomonte 7, Eliane Candiani Arantes 4, Andreas H. Laustsen 1

1Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads 224, Lyngby, Denmark 2Department of Biotechnology and Biosafety, Eskisehir Osmangazi University, Meselik Yerleskesi 26480, Eskisehir, Turkey 3Medical School, Federal University of Roraima, Capitão Ene Garcez 2413, 69310-000, Boa Vista, Brazil 4Dept of Physics & Chemistry, School of Pharma Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil 5The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet, Lyngby, Denmark 6IONTAS Ltd., Iconix Park, London Road, Pampisford, Cambridgeshire CB22 3EG, United Kingdom 7Instituto Clodomiro Picado, Facultad de Microbiologi ́a, Universidad de Costa Rica, San Jose ,́ Costa Rica

Recombinant antivenoms based on mixtures of human monoclonal antibodies have the potential to become the next generation of envenoming therapies, as they can be designed to have improved therapeutic properties, such as better safety profiles, enhanced efficacy, and improved manufacturability compared to existing animal plasma-derived polyclonal antivenoms. Significant scientific developments within this area have been reported in the later years, including the development of the first experimental antivenom based on a mixture of fully human monoclonal immunoglobulin G antibodies and strategies for broadening the neutralization capacity of toxin-neutralizing antibodies. Nevertheless, many technical challenges need to be resolved before it is feasible to manufacture recombinant antivenoms at larger scale and evaluate them in the clinical setting. These challenges include how to discover and rationally engineer broadly-neutralizing antibodies that target multitudes of toxins, and how to design antibody mixtures that can be administered at very low dose while retaining therapeutic efficacy. Here, new strategies for high-throughput discovery of broadly-neutralizing and recyclable antibodies are presented with examples of recently discovered antibodies against snake, spider, scorpion, and bee toxins from our lab. A snake in the test

Patrick Waters 1, Thashi Chang 2, Hannah Fox 1, Neelika Malavige 3, Ariaranee Gnanathasan 2, Sarosh Irani 1

1Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, Oxford, UK 2Department of Clinical Medicine, Faculty of Medicine, University of Colombo, Sri Lanka 3Faculty of Medical Sciences, University of Sri Jayawardenapura, Sri Lanka

Researchers from Oxford developed a radioimmunoassay (RIA) to identify individuals with antibodies to the shaker family of voltage-gated potassium channels (VGKC). Although it eventually turned out that the antibody targets were other proteins complexed in the detergent extract with the VGKC, the assay demonstrated the possibility of antibodies causing disease in the ‘immunopriviledged’ brain. This finding has led to the evolution of the field of neuroimmunology and the subsequent discovery of more than 10 antibody-mediated, treatable,

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 24 central nervous system diseases over the last two decades. In this RIA the substrate is made up of homogenised rabbit brain extract which is incubated with I 125 -α-dendrotoxin from the Eastern green mamba ( Dendroaspis angusticeps ). This α-dendrotoxin labels some of the shaker family VGKCs. While running this test we identified indivduals who were seropositive but did not have any disease. Further investigation showed that individuals had antibodies that were specific for the radiolabelled dendrotoxin rather than the VGKC itself. We have no evidence that these individuals were ever bitten by snakes but their profession is suggestive: snake-handlers from east Africa. In our laboratory we routinely examine the function of target specific antibodies from different CNS diseases by cloning the variable portion of their BCRs and expressing full length antibodies in vitro. We will do the same with venom specific antibodies and have developed a simple test to detect serum antibodies to - dendrotoxin. Cross-Species and Geographic Potential of Epitope Strings Identified for Generation of an Africa-specific Snake Venom-induced Necrosis Serotherapy

Charlotte A Dawson, Stuart Ainsworth, Mark Wilkinson, Robert Harrison

Center for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK

Every year 1.8-2.7 million people suffer snakebite envenoming, of these, an estimated 137’000 die and 400’000 are left disfigured or disabled. Fundamental to alleviating this burden is improving antivenom efficacy. Linear B-cells epitopes are short peptides displayed by antigen presenting cells to elicit an immune response. Multiple toxin epitopes can be identified and arranged as ‘beads on a string’, increasing the probability of a broadly-neutralising immune response. Prior studies have shown epitope-string immunogens are able to generate antibodies capable of neutralising haemotoxic venom proteins. I intend to adopt this approach to create snakebite necrosis specific serotherapies. Snakebite necrosis causes significant physical, mental, and economic distress. Whilst there is a debate over the efficacy of antivenom for necrosis treatment, a targeted therapeutic does not exist to prevent lifelong disability and disfigurement. A necrosis therapeutic requires a focus on the aetiological venom proteins; snake venom metalloproteinases, phospholipase A 2s, hyaluronidases, and cytotoxic three finger toxins. Through bioinformatic interrogation of sequence data from three medically important African snakes ( Echis ocellatus, Bitis arietans, and Naja nigricollis ) 6 epitope strings were generated for delivery via a hepatitis B core virus like particle (VLP) carrier. To refine immunisation experiments, I investigated the ability of antivenom used in Africa to recognise the selected epitopes. To understand the geographical utility of those immunogens I also assessed their recognition by antivenom used in Asia and South America. Determining the cross-genera and cross-continent potential of the epitope-string immunogens will provide valuable information on the possibility of a global anti- necrotic. Identifying where cross-specificity is minimal will strengthen the approach, potentially avoiding epitope identification across all necrotic species and their toxin sequences. The immunological cross-reactivity and in vitro neutralising efficacy of novel antivenoms against haemotoxic snake venoms

Nessrin O Alomran , Laura-Oana Albulescu, Jaffer A Alsolaiss, Paul D Rowley, Robert A Harrison and Nicholas R Casewell

Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Pembroke Place Liverpool, UK

Snakebite is a neglected tropical disease that causes high rates of mortality (~138,000 deaths annually worldwide) and disability. Snake venoms are complex mixtures of peptides/proteins that vary from one species to another and cause a variety of pathologies in snakebite victims. Venom-induced consumption coagulopathy (VICC), which results in the depletion of fibrinogen and exacerbates haemorrhage, is one of the most potentially lethal pathologies encountered after snakebite. Antivenoms are polyclonal antibodies derived from animal sera that are used to treat snakebite victims. Despite saving thousands of lives, these therapies have many limitations

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 25 associated with them, including limited cross-species efficacy, which restricts their utility to particular geographical regions. As a proof of concept, here I explored the feasibility of generating pathology-specific, rather than region-specific, antivenoms by using two sets of venom immunogens to raise ovine polyclonal antibodies against haemotoxic snake venoms. The immunogens consisted of 7 and 12 different haemotoxic snake venoms of medical importance and/or broad geographical diversity. Using a variety of immunological and in vitro functional assays I quantified the binding and neutralising capabilities of the resulting experimental antivenoms to each of the venoms used in the immunising mixtures. My findings revealed that both antivenoms exhibited desirable immunological and neutralising characteristics against the majority of venoms used as immunogens. Interestingly, the inclusion of additional venoms to the immunogen mixture was found to only provide minor increases in immunological binding and in vitro neutralisation, suggesting that a core venom pool of diverse immunogens may be sufficient to stimulate broad neutralisation of particular venom activities. These findings suggest that pathology-specific antivenoms show promise as global, modular, treatments for the world’s snakebite victims. Macrovipera lebetina obtusa : Venomics, Bioactivity and Preclinical Assessment of new ovine antivenom (Armenia)

Naira Ayvazyan

Orbeli Institute of Physiology of the National Academy of Sciences, Yerevan, Armenia

Snakebite envenoming is an important medical problem in Armenia, with an estimated 300 hospital admissions annually. Despite the magnitude of this problem, there is no specific antivenom for Armenia, where antivenom manufactured in Uzbekistan is being used without proven preclinical and clinical confirmation of efficacy and safety. As a polyspecific product of immunization with the venom of snakes, which are not presented in the herpetofauna of Armenia, this antivenom has been shown to induce a high incidence of adverse reactions. Moreover, there is no antivenom specific for 3 other species of very endemic vipers responsible for the remarkable number of cases in our country. This situation prompted the development of monospecific antivenom for Armenia against the Levantine viper’s snakebites with the further goal to develop also polyvalent antivenom for the rest of viperid species of Armenia. This study describes in detail the procedure for antivenom production, but also the reason and criteria for selection of animals (sheep) intended to be used for Fab antivenom production. Existing protocols for antivenom treatment of snake envenomations are generally not well optimized due partly to inadequate knowledge of the toxicokinetics of venoms in patients with different blood types. Our results suggest that both the severity of envenomation and the antivenom efficacy for human could be defined also by the blood group of the patient. EchiTAb antivenom – A global solution to a local problem

Abdulsalami Nasidi ,1,2 Nandul Durfa, 1 Isa S Abubakar 1, Saidu B Abubakar 1, John Garnvwa, 1 Peter O Yusuf 1, José- María Gutiérrez 1, Robert Harrison 1, Abdulrazaq G Habib 1, David RG Theakston 1, David A. Warrell 1 and John Landon 1

1EchiTAb Study Group Nigeria/UK 2University of Africa, Toru-orua, Bayelsa State, Nigeria

Nigeria and a dozen other countries of the West African savanna, most snake-bite mortality and morbidity is attributable to carpet vipers ( Echis ocellatus complex). To address this problem, the Nigerian Government funded a collaboration with Liverpool School of Tropical Medicine and University of Oxford to encourage development of new antivenoms for the region, raised against venoms of Nigerian snakes. Eventually, two whole IgG antivenoms were produced: monospecific Echis ocellatus/romani (“EchiTAb G” by MicroPharm UK) and polyspecific Echis ocellatus/romani-Bitis arietans-Naja nigricollis (“EchiTAb Plus ICP by Instituto Clodomiro Picado Costa Rica). By 2010, intensive pre-clinical and clinical testing had confirmed that both were highly effective in correcting Echis - induced coagulopathy. Subsequent use in Nigeria has reduced case-fatality to less than 1%, while pre-clinical

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 26 “antivenomics” testing has suggested wider para-specific neutralization and geographical relevance. As a key element of WHO’s “Strategy for prevention and control of snakebite envenoming” (2019), selected antivenoms will be stockpiled for distribution to hospitals most in need. In future, the necessary technology must be transferred to African countries to allow local manufacture of these essential medicines. Coagulopathy in Green Pit Viper bite in Bangladesh: an unrecognized and unaddressed issue

Aniruddha Ghose 1, 2 , Tazrian Khurshid 1, Abdullah A Sayeed 1, 2 , Muhammed S Alam 1, Abu S M Zahed 1, Shoman Sarkar 1, Mohammed H Rahman 1, Kajal K Das 1, Asok K Dutta 1, 2 , Mohammad A Faiz 2

1Chittagong Medical College & Hospital, Chittagong, Bangladesh 2Venom Research Centre, Chittagong Medical College, Bangladesh

Green pit viper bite is the commonest cause of venomous snakebite in Bangladesh. Though mortality is rare but morbidity and potential threat to victims’ health due to coagulopathy and local tissue swelling is substantial. We present a series of 112 cases of Green pit viper bite admitted to a tertiary hospital in Southeast part of Bangladesh over 1 year period. Having informed written consent, patients’ epidemiological profile, presentation and examination findings were recorded in predesigned case record forms. Extent of local swelling was determined by the differences in circumferences of the bitten limb and non-bitten limbs, measured in cm at the same distance from the nearby anatomical points. Bedside 20 min WBCT was done in every patient on admission and 12 hourly to detect and follow coagulopathy. Any sort of spontaneous bleeding was searched for and recorded. Coagulopathy and its reversal were confirmed by lab coagulation profile – Activated partial thromboplastin time (APTT), prothrombin time( PT) and platelet count. Median age of the victims was 32 years (SD+19.5 yrs) ranging from 6 – 75 years, maximum (31.3%) belonged to 21 – 30yrs age group. Most of them were from the rural areas. Male to female ratio was 4:1. Farmers were the commonest (N23; 10.5%) victims. Most happened to pedestrians while walking on village roads (N 24; 21.4%). Most common body parts were foot 41% (N46), fingers 27.6% (N31) and hands 13.4% (N15). 80.4% (N 90) of the victims attended Chittagong Medical College Hospital within 5 hrs. 97.3% (N 109) applied tourniquets and maximum had at least 2. Swelling of the bitten part was the commonest local clinical manifestation and was present in 91% (N 102) cases. Blisters (14.3% (N 16) and necrosis (6.3% (N 7) were also observed. Spontaneous bleeding in case of 4.5% (N 5) from bite site was observed. Local swelling gradually increased and became maximum in Day 3 and Day 4 followed by gradual declining circumference of the swelling. In this study 43% (N48) patients developed coagulopathy. Only 6.25% (N 3) had so during admission and remaining 36.75% (N 45) victims developed coagulopathy over time ranging from 7-72 hours after hospital admission, detected on follow up 20 min WBCT that was done 12 hourly. Only one patient developed echymosis in the left calf muscle. Only 1% patient had systemic bleeding. Gradually blood started to become coagulable and 8 patients recovered within eight days. Forty patients took discharge on their own without improvement in coagulation status and others got gradual reversal of coagulopathy. Forty four (39.28%) patients developed thrombocytopenia. Remaining 57.14%(N 64) patient who did not develop coagulopathy were observed and followed up by doing 20 minute WBCT 12 hourly for 48 hours. All these patients were managed conservatively with prophylactic antibiotics. Patients with coagulopathy had a significantly higher hospital stay and the duration was even more for those who developed spontaneous bleeding. The study provides a clinical picture of Green pit viper bite patients demonstrating it as a cause of significant morbidity having a potential for mortality. The coagulation abnormality by Green Pit venom is an under evaluated and under addressed issue. This study provides an overview of the extent of this problem and emphasizes the need for further evaluation of this problem. Currently there is no antivenom for Green Pit Viper venom in Bangladesh. The patients are being treated conservatively (in other word leaving their body on their own to deal with the venom). This may not result in a favourable outcome in all patients. We need to have antivenom against Green Pit Viper venom.

Andrew D Watt 1, Geno Roalakona 2, Ben Bande 2, Emmanuel Singi 2, Owen Paiva 2, Timothy NW Jackson 1, David Williams 1,2

1Australian Venom Res Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Melbourne, Australia 2Charles Campbell Toxinology Centre, University of Papua New Guinea, Port Moresby, Papua New Guinea

Papua New Guinea (PNG) has one of the highest region-specific snakebite rates in the world. The World Health Organization data attributes >1000 deaths across PNG to snakebite every year and in some parts of the country case fatality rates can be three times higher than those from diseases such as malaria or tuberculosis. Australian- made effective antivenoms have been available for more than 70 years, but their high cost (up to ~US$1,500 vial) has limited access to these essential medicines, resulting in chronic shortages and supply failures. Launched in March 2018, the PNG Snakebite Partnership is a three-year antivenom distribution and epidemiological project involving the National Department of Health (PNG), the Australian Government, Seqirus Pty Ltd, and the Australian Venom Research Unit (Department of Pharmacology & Therapeutics, University of Melbourne). 600 vials of antivenoms against snake and stonefish venoms (donated by Seqirus) will be distributed to health centres across PNG each year and vital incidence and clinical data collected on each suspected snakebite patient. Here we present an overview of the initial 12months of the projects. Myanmar Snake-bite Project: an Australian DFAT-GPFD programme

David A Warrell 1, Chen Au Peh 2, Julian White 3, Afzal Mahmood 4

1Nuffield Department of Clinical Medicine, University of Oxford, UK 2Department of Renal Medicine, Royal Adelaide Hospital and University of Adelaide, Australia 3Toxinology Department, Women’s and Children’s Hospital Adelaide, Australia 4School of Public Health, University of Adelaide, Australia

In 2015, the Australian Department of Foreign Affairs and Trade (DFAT) - Government Partnerships for Development (GPFD) awarded to the University of Adelaide funding (Aus$4 million=US$2.7 million) for “Improving the health outcomes for snakebite patients in Myanmar” in collaboration with the Ministries of Industry and Health, Union of Myanmar. The project ran from 2015-2018 with the specific aims of 1- improving the quantity and quality of antivenom production, 2- increasing the availability of antivenom to health centres especially in rural regions, and 3- optimising the management of snakebite patients at the community level. With the help of experts from Australia, all aspects of antivenom production and distribution were tackled, including improving captive snake husbandry and venom collection, horse health and longevity, and refinement and lyophilisation of hyperimmune plasma. Antivenom production by the national manufacturer, Burma Pharmaceutical Industry (BPI), has increased, obviating the need to import less specific antivenoms from India and Thailand. The entire production is now lyophilised, allowing for greater variation in storage temperatures in rural areas. WHO/GMP level quality control has been introduced. Model electronic medical record systems and guidelines have been introduced while training of Health Assistants and Township Medical Officers has been successfully piloted in Mandalay Division. Community-based studies of snake-bite incidence and mortality and attitudes to allopathic medical treatment, suggested that as many as 1,000 deaths from snake-bite each year may occur across the seven high incidence regions of Mandalay, Sagaing, Bago, Magway, Yangon, Nay Pyi Taw and Ayeryarwady. Early antivenom treatment reduced the risk of developing acute kidney injury and the duration of coagulopathy. Hospital-based collections of dead snakes brought by the people they had bitten or spat at confirm the predominant medical importance of Eastern Russell’s viper ( Daboia siamensis ), but revealed the Mandalay spitting cobra ( Naja mandalayensis ) as the commonest cause of elapid envenoming in Upper Myanmar.

Hemachatus haemachatus snake cross-reactivity with other vipers and elapids

Rae Ahamed

University of Reading, School of Pharmacy, Whiteknights PO Box 217, Reading Berkshire, RG6 6AH

In Sub-Saharan Africa, approximately 1 million snakebites occur annually with up to fifty thousand permanent disabilities and mortalities. With three hundred and twenty species in sixty one genera are recognised as Elapidae, causing up to Forty percent of snake bites in Sub-Saharan countries. Elapid’s venom is mostly neurotoxic, however exceptions occur where some snake species may display both cytotoxicity and neurotoxicity. One example is the Hemachatus haemachatus, a southern african spitting cobra who’s snake venom composition includes phosphodiesterase, PLA2, prothrombin activators, three finger toxin and neurotoxin. Accordingly, treatment difficulties are as a result of poor antivenom availability, permitting only 2% of victims to receive antivenom and resulting in roughly 40-80% morbidities. Further difficulties in treatment arise in identification of the responsible snake (unless snake is bought in) but most importantly lack of a universal antivenom. To create a universal antivenom, the compositional differences of different snake venoms are to be identified in order to target a common key player. However, the cross-reactivity between viper and elapid snakes is yet to be assessed. Here, we report the cross-reactivity between Hemachatus haemachatus, other elapids and viper venoms by purifying antibodies against Hemachatus haemachatus, followed by using direct and indirect enzyme linked immunosorbent assay. Hemachatus haemachatus snake venom cross reacts with vipers but more so with elapids. This study, therefore hypothesises that Hemachatus haemachatus has similar venom composition to other vipers and elapids, an observation that may become significant in terms of current universal antivenom development. The linear epitope recognition landscape of commercial antivenoms to snake venoms of sub-Saharan Africa

Stuart Ainsworth 1, Carina Skaarup 2, Kamille Elvstrøm Krause 2, Mikael Engmark 2,4 , Timothy Jenkins 4, Charlotte Dawson 1, Nicholas Casewell 1, Robert Harrison 1, Bruno Lomonte 3, Julián Fernández 3, José María Gutiérrez 3, Andreas H. Laustsen 4 and Ole Lund 2

1Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK 2Department of Bio and Health Informatics, Technical University of Denmark, Kongens Lyngby, Denmark 3Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica 4Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark

Antivenoms are polyclonal mixtures of purified immunoglobulin G (IgG) antibodies or fragments thereof from venom-immunised animals. Currently, there are multiple antivenom therapeutics available for treating envenomation in sub-Saharan Africa. Although each product is manufactured in a quite similar manner, the venoms used for immunisation vary substantially between products, in terms of numbers of venoms used and where the species from which the venoms were obtained originate from. It is not known if the use of different venom immunising mixtures results in substantially different epitope recognition profiles for antivenoms, especially in terms of para-specificity, which may or may not reflect an antivenom’s pre-clinical or clinical efficacy. Here, we present a comprehensive analysis of the ability of antibodies in eight commercial antivenoms to recognise linear epitopes of 286 toxins from 31 medically important snake species of sub-Saharan Africa.

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 29 Unravelling the diversity of fish venoms – Inter and instraspecific differences in Atlantic and Mediterranean scorpionfish (Scorpaena )

Frederico Almada 1, Marta Araújo 1, Francisca Rodrigues 1, Sara M. Francisco 1, Américo G. Duarte 2, Sérgio Moreno 3, Cristina S. Lima 1, Luisa Maia 4, José Moura 4, Juan Calvete 5

1MARE—Marine and Environmental Sciences Centre, ISPA Instituto Universitário, Rua Jardim do Tabaco 34, Lisboa, Portugal 2Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, Oeiras, Portugal 3BIOECOMAC, Universidad de La Laguna, Avenida Astrofísico Francisco Sánchez, Canary Islands, Spain 4LAQV, FCT NOVA, 2829-516 Caparica, Portugal 5Evolutionary and Translational Venomics Laboratory, CSIC, Jaume Roig 11, 46010 Valencia, Spain

Research in blue biotechnology is thriving with the study of a vast array of novel molecules obtained from marine organisms. Surprisingly, thousands of species of venomous fish have been largely overlooked as a source of potential bioactive compounds. Furthermore, many species are morphologically similar and present cryptic behaviour which frequently results in broad taxonomic identifications in field surveys and unknown phylogenies. However, knowledge of their evolutionary relationships and the diversity of their venoms would allow effective predictions on characteristics that have not yet been studied. The genus Scorpaena , with 62 species distributed worldwide, is an excellent model group to study the evolution of venom in fish. Currently, its phylogeny has only been partially addressed and, excluding one single species, nothing is known about the composition or properties of their venoms. SDS-PAGE analysis of crude venom extracts from five north-eastern Atlantic and Mediterranean species revealed a remarkable diversity in venom composition. The potential interspecific venom diversity is further emphasized by the differences between populations of S. maderensis from the archipelagos of the Azores and Madeira compared to the Canary Islands. Differences in venom composition do not seem to follow phylogenetic or phylogeographic topologies inferred from neutral DNA markers. Fish venoms are part of a defensive mechanism against predators and their venom systems probably evolved multiple times. Further studies are needed to evaluate if these defensive cocktails resulted from different selective pressures and/or to divergence throughout their evolutionary history. Unravelling common/exclusive venom components and the relationships between these taxa will allow us to prioritize subsequent research in fish toxinology and pave the way to future applications across multiple disciplinary fields. Discovery of cross-reactive and pH-sensing monoclonal antibodies against neurotoxins from the notorious Tityus serrulatus scorpion

Felipe A Cerni 1,2 , Manuela B Pucca 2,3 , Eliane C Arantes 1, Andreas H Laustsen 2

1Dept of Physics and Chemistry, School of Pharma Sciences, Ribeirão Preto, University of São Paulo, São Paulo, Brazil 2Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Kongens Lyngby, Denmark 3Medical School, Federal University of Roraima, Av. Cap. Ene Garcês 2413, 69310-000, Boa Vista, Roraima, Brazil

In Brazil, scorpion envenomings are considered an important public health problem. The yellow scorpion, Tityus serrulatus is considered the most dangerous species in the country, annually causing more than 120,000 envenomings. T. serrulatus is highly tolerant to food deprivation (surviving up to 400 days without food) and is able to produce offspring throughout the year by parthenogenesis. The major components from T. serrulatus venom are ion channel-modulating neurotoxins. These induce a neuroexcitatory syndrome, resulting in the severe envenoming and mortality. Among T. serrulatus neurotoxins, Ts1, Ts2, and Ts5 are considered key toxins

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 30 for envenoming pathology due to their ability to inhibit sodium channels. Currently, the administration of antivenom derived from the plasma of hyper-immunized horses is the only specific treatment for envenomings caused by scorpions. These antivenoms comprise polyclonal horse antibodies that due to their heterologous nature may cause anaphylaxis and serum sickness. Using phage display technology, this study presents recent results on the discovery of human monoclonal antibodies presenting cross-reactivity against sodium-channel T. serrulatus neurotoxins. Moreover, using a new method to discover and engineer therapeutic antibodies with conditional binding properties, cross-reactive monoclonal antibodies that also display pH-sensing capabilities were discovered in this study with the aim at generating antibodies that can be recycled by the FcRn (neonatal Fc receptor) in the early endosomes. This FcRn-recycling mechanism is the fundamental reason for the long serum half-life of IgG antibodies, and exploiting this mechanism may allow for a dramatic decrease in dosing of therapeutic antibodies. This study thus reports the first discovery of cross-reactive and pH-sensing antibodies against scorpion neurotoxins, and the hope is that this new therapeutic concept may help pave the way for next-generation scorpion antivenoms in Brazil. Fibrinogenolytic activity of Causus snake venoms

Francisco C. P. Coimbra , Bryan G. Fry

Venom Evolution Laboratory, School of Biological Sciences, University of Queensland, St. Lucia QLD 4072, Australia

Night Adders (Genus Causus) are responsible for a large proportion of envenomings in sub-Saharan Africa. Although not usually life-threatening, clinical reports indicate that Causus envenomings impair coagulation via anticoagulant mechanisms; however, these venoms have been poorly characterised. The aim of this study was to determine proteomic composition and characterise the fibrinogenolytic activity of venom from night adder species C. lichtensteinii and C. rhombeatus . SDS-PAGE and tandem-mass spectrometry sequencing were performed to identify toxin components. Fibrinogenolysis was qualified in time-dependent incubation assays using ten venom concentrations (1.525 to 800 µg/ml), visualized via 1D SDS-PAGE, and quantified using ImageJ software. To determine functional effect of fibrinogenolysis, venom was incubated with fibrinogen and thrombin added post-incubation in a Claussian method. Time until clot formation was then measured using a Stago STA-R max coagulation analyzer. To assess clot strength, venom was incubated with fibrinogen, thrombin added post- incubation, and resulting clot measured via a thromboelastograph (TEG). Venoms presented similar qualitative toxin composition and functional fibrinogenolytic activity. Both venoms produced similar progressive proteolysis of A α and B β chains, leaving γ chain unaffected. At concentration 0.4 mg/ml and above clotting impairment was absolute. Clot strength measured in maximum amplitude of clotting curve (MA – mm) was reduced by roughly half of control strength (i.e. from 10.9 +/-0.30 mm to 4.27 +/-0.85 mm in C. lichtensteinii and 5.00 +/-0.36 mm in C. rhombeatus ) at 0.2 mg/ml venom concentration. Although further work is required to understand the full functional activity of these venoms, the characterization of fibrinogenolytic activity in snake venom can lead to the mapping of various fibrinogenolytic loci of exploitable interest – as venoms and fractions can have unique cleavage sites. Ornithogalum plants, an unrecognized risk to animals and possible humans

Marieke A Dijkman , Irma de Vries

Dutch Poisons Information Center (DPIC), University Medical Center Utrecht, Utrecht University, The Netherlands

Several Ornithogalum species are held as ornamental plants and flowers. Some species are considered extremely toxic due to suspected involvement of cardiac glycosides. We present a retrospective overview of consultations to the DPIC concerning Ornithogalum plant exposures. From 2007-2018, 25 cases involving 28 patients were recorded; 14

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 31 animals (12 dogs/2 cats) and 14 humans. In 56% the Ornithogalum species was known: O. arabicum (1 dog), O. dubium (1 human), O. saundersiae (4 dogs, 1 human) and O. thyrsoides (7 humans). Predominantly young children (n=10, 0-4 yr) and dogs (n=7, 0-1 yr) were involved. Humans and cats were mainly exposed to above ground parts while the bulbs were more attractive to dogs. Mean delay between exposure and consultation by veterinarians was 12 hours (range 4-48) at which time most animals were symptomatic (12/14); (severe) gastrointestinal signs (10/12), lethargy (5/12), visual dysfunction including blindness (3/12) and an irregular heartbeat (1/12). Ophthalmologic examination in two dogs showed severe retinal degeneration. At least 3 dogs died. In contrast, human consultations were often made within 1 hour after exposure (median: 45 min., range 0.17-24 hr) and most patients were asymptomatic (11/14). One patient developed nausea after eating the stem of an Ornithogalum plant, which was mistaken for basil. Two young children developed drowsiness and weakness, hours after sucking and chewing on Ornithogalum plant material. Poisonings with Ornithogalum plants in humans and animals are rare but do occur. Considering the severity of the veterinary cases, further studies are necessary to confirm the suggested involvement of cardiac glycoside-like toxins and their effects on the retina. Also, a more thorough follow-up, including ophthalmologic examinations, in humans seems necessary. Toxin-Specific Phospholipase A 2 Antibodies in the Detection and Neutralisation of a Range of Snake Venoms

Alice Filipe , Harry F. Williams, Sakthivel Vaiyapuri

School of Pharmacy, Whiteknights Campus, University of Reading, Reading, Berkshire, RG6 6AH, UK

Snakebite envenomation is a neglected tropical disease which causes over 100,000 deaths a year. Different compositions of proteins/toxins present in each species’ venom causes difficulty in treatment. Currently, if the snake remains unidentified, a polyvalent antivenom treatment, which has remained unchanged for 125 years, is the only treatment available. This treatment has a low specificity causing a less effective treatment. Resultantly, more antivenom must be administered, increasing the risk of an anaphylactic reaction. The impact of creating a more specific treatment for different snake venoms or a quick form of species identification, would increase patient survival and decrease damage caused by snakebites. Here, we use toxin-specific phospholipase A 2 (PLA 2) antibodies from a viper snake called Agkistrodon piscivorous leucostoma (APL) to determine the extent of neutralisation and detection on a range of snake venoms. Preliminary results show significant cross reactivity with our toxin specific antibody derived from a viper with other Viperidae species as well as the spitting cobras. As expected, following experiments showed a higher cross reactivity with vipers compared to elapids, possibly signifying structurally similar PLA 2s to APL. Subsequent assays however showed that there are antibodies raised against serine protease present contaminating our PLA 2 antibodies. This could have caused some cross- reactivity and therefore further refinements are required to produce toxin-specific PLA 2 antibodies. This study provides novel insights into the similarities of toxin-specific PLA 2 with other viper and elapid venoms as well as providing the initial steps towards a more toxin specific treatment approach. Discovery of human antibody fragments against α-latrotoxin from the Mediterranean black widow spider

Sofie Føns , Line Ledsgaard & Andreas H. Laustsen

Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark

Widow spiders are among the few spider species worldwide that can cause serious envenoming in humans. The mainstay treatment of spider bite envenoming is antivenom consisting of antibodies derived from the plasma of hyperimmunised animals. While these antivenoms save lives, some products have suboptimal safety, efficacy,

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 32 affordability, and availability. To address these disadvantages, the development of recombinant antivenoms based on human monoclonal antibodies targeting key spider toxins may be a promising solution. The aim of this project is to harness phage display technology to discover human monoclonal antibodies that target α- latrotoxin from the Mediterranean black widow spider, Latrodectus tredecimguttatus. Though the venom of the black widow spider consists of a multitude of toxins, α-latrotoxin appears to be the sole toxin responsible for the clinical effects of envenoming observed in humans. In this project, a phage display library consisting of 1.6 x 10 10 human antibody fragments was successfully employed to accumulate phages displaying human single-chain variable fragments (scFvs) that bind α-latrotoxin. The genes encoding the scFvs were subcloned into Escherichia coli BL21 (DE3) cells and expressed. The binding characteristics of these scFvs was then evaluated through ELISA experiments. Based on ELISA and DNA sequencing six unique monoclonal scFvs were identified. The six clones were expressed and ranked based on their affinity to α-latrotoxin through expression-normalized ELISA experiments. Next, the scFvs will be tested for neutralizing ability in vitro . Eventually, the most promising scFvs will be converted into human monoclonal IgG antibodies that can be expressed in mammalian cell cultures and tested for neutralizing ability in vivo. A novel method to determine prey-specific nicotinic acetylcholine receptor orthosteric binding of elapid venoms

Richard J. Harris 1, Christina N. Zdenek 1, Sanjaya Kuruppu 2, Bryan G. Fry 1

1Venom Evolution Lab, School of Biological Sciences, The University of Queensland, St. Lucia, Australia 2Department of Pharmacology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia

The α-neurotoxin family shown to target nicotinic acetylcholine receptors (nAChRs) belong to a molecular group known as three-finger toxins (3FTxs). These toxins are ubiquitous throughout the elapid snake venoms, more recently having been isolated from some colubrids and other advanced snakes. The plesiotypic function of 3FTxs is neuromuscular targeting of the orthosteric site on α1 nAChR subunits and show a greater potency toward diapsids (reptiles and birds) than synapsids (mammals), indicating prey-specific targeting. Yet despite this, very few studies have investigated the prey-specific nAChR binding of elapid venoms. Current analytical methods in determining venom binding to nAChRs are in vitro skeletal muscle preparations (chick biventer cervicis nerve- muscle preparation or mouse/rat phrenic nerve hemidiaphragm), oocyte patch-clamp systems and Fluorescence Imaging Plate Reader (FLIPR). Although these pose many experimental and mechanical drawbacks, the two main issues are being taxonomically limited and difficulties differentiating orthosteric and allosteric binding of venoms to nAChRs. Here, we demonstrate a novel analyte-ligand binding analysis assay, which combines Bio- layer interfermetry (BLI) accuracy with precisely designed taxon-specific mimotopes, to measure the postsynaptic binding of elapid neurotoxins to the orthosteric site of α1-nAChR subunits. Understanding how these venoms selectively target nAChRs can give insight into venom evolution and snake ecology. How snake venoms evolve to target prey-specific nAChR receptors might be key in understanding evolutionary facets such as antagonistic coevolution, prey-driven toxin evolution and neurotoxin resistance. Design, Synthesis and Characterisation of Potent Conotoxin Peptidomimetics Incorporating a Triazole Disulfide Bond Mimic

A. Knuhtsen, 1 C. Whitmore, 2 F. S. McWhinnie, 1 L. McDougall, 1 B. O. Smith, 3 C. M. Timperley, 2 A. C. Green, 2 K. I. Kinnear 2 and A. G. Jamieson 1

1School of Chemistry, University of Glasgow, Glasgow, UK. 2Chemical, Biological and Radiological Division, Defence Science and Technology Laboratory, Salisbury, UK 3Institute of Molecular, Cell & Systems Biology, University of Glasgow, Glasgow, UK

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 33 The potency and selectivity of conotoxin peptides for neuropathic receptors has made them attractive lead compounds in the development of new therapeutics. However, these disulfide bond rich peptides are limited as lead drug compounds due to their unfavourable physicochemical properties. We have used a peptidomimetic triazole disulfide bridge surrogate to replace each disulfide bridge in turn within α-conotoxin GI. This produced a mimetic with an order of magnitude increase in blood plasma stability, whilst retaining full biological activity. Our studies were performed in human CN21 cells expressing human AChRs, making them applicable towards drug development in humans. Bespoke force field descriptions of the triazole mimetic were developed that allowed us to determine the solution structure by NMR spectroscopy, we discovered significant similarities in conformation between the mimetic and the peptide bioactive toxin. Harnessing monoclonal antibodies for the development of a next generation antivenom against Naja nigricollis envenoming

Jonas A. Jürgensen , Rahel Janke, Rasmus I. Dehli, Andrea M. Esteban, Sofie Føns, Line L. Jensen, Andreas H. Laustsen

Department of Biotechnology and Biomedicine, Technical University of Denmark

The Black-necked spitting cobra ( Naja nigricollis ) is one of the most infamous snake species found in the African continent, being classified as a category 1 snake of highest medical importance by the WHO. Its venom consists of highly potent mixture of cytotoxins, leaving many of its victims crippled for life. Currently, the African continent is affected by an antivenom shortage. Furthermore, due to the heterologous origin and low specificity of these antivenoms, they have a propensity to cause severe adverse reactions, including serum sickness and anaphylaxis, which could lead to death of the patient. Here we explore recombinant antibodies as a therapeutic alternative. We are developing an antivenom based on recombinant human monoclonal antibodies of the immunoglobulin G (IgG) format, predicted to be safer, cost-competitive, and more efficacious compared to the existing treatment. Through the utilization of phage display technology, it has proven possible to discover and express novel human single-chain variable antibody fragments (scFv) against the five most medically relevant venom toxins from Naja nigricollis (African black-necked spitting cobra), four being cytotoxins and one being a

Phospholipase A 2. Out of 486 monoclonal scFvs analyzed, 133 were considered good binders. Of these, 94 were sequenced, resulting in the identification of 30 unique scFvs. The binding properties of these scFvs will be evaluated, and the most promising leads will be converted into the IgG format and assessed for their ability to neutralize N. nigricollis toxins in vivo . It is our hope that the presented work can pave the way for snakebite therapeutics entering the 21 st century. Snake venom proteinase activity profiling through screening of peptide substrates

Konstantinos Kalogeropoulos 1, Andreas Frederik Treschow 1, Ulrich auf dem Keller 1, Teresa Escalante 2, Alexandra Rucavado 2, José María Gutiérrez 2, Andreas Hougaard Laustsen 1, Christopher T. Workman 1

1Department of Biotechnology and Biomedicine, Technical University of Denmark, 2800 Lyngby, Denmark 2Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José 11501-2060, Costa Rica

Metalloproteinases and serine proteinases are among the most abundant enzymes in many snake venoms, particularly among viperids. These proteinases are responsible for some of the clinical manifestations classically seen in viperid snakebites, including hemorrhage, necrosis, and coagulopathies. The study investigated the activities of said proteinases using a rich peptide library of biologically relevant proteins, as a method of screening for the target substrates of the venom proteinases of five viperid (Echis carinatus, Bothrops asper, Daboia russelii, Bitis arietans, Bitis gabonica) and one elapid (Naja nigricollis) species of high medical

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 34 importance. The venoms were each tested against 360 peptide substrates, yielding 2160 enzymatic activity profiles. A nonlinear regression model that accurately described the observed activities was constructed based on the experimental data, allowing for cleavage rate comparison across species. In this study, previously unknown protein targets of snake venom proteinases were identified, potentially implicating novel human and animal proteins that may be involved in the pathophysiology of snake envenomings. These new findings may contribute to our understanding of the clinical manifestations and underlying biochemical mechanisms of snakebite envenoming by the investigated snake species. Discovery and evaluation of immunoglobulin G antibodies for rapid stratification of snakebites from Bothrops , Crotalus , and Lachesis species in Brazil

Cecilie Knudsen 1,2 , Søren H. Dam 1, Aleksander M. Haack 1, Rasmus U. W. Friis 1, Jonas A. Jürgensen 1, Jan K. Andersen 2, Andreas H. Laustsen 1

1Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark 2BioPorto Diagnostics A/S, Tuborg Havnevej 15, DK-2900 Hellerup, Denmark Approximately 600 species of venomous snakes roam the Earth. Out of these, around 200 species are considered to be of medical importance because of their contribution to the Neglected Tropical Disease (NTD) of snakebite envenoming. While it is broadly agreed upon that snakebite envenoming causes pain, death, and suffering, the extent of the mortality and morbidity, which can be attributed to the disease, is debated due to a lack of epidemiological data. Here, we present a study on discovery and evaluation of monoclonal murine immunoglobulin G (IgG) antibodies with the purpose of employing these antibodies in the development of an assay for rapid stratification of snake venoms in envenomed patients in Brazil. Hybridoma technology was used to generate 117 antibodies against whole venoms from Bothrops atrox , Crotalus durissus terrificus , and Lachesis muta muta . The specificities of the IgGs were tested in ELISAs against whole venoms from these same species, and 41 IgGs were found to be specific for one snake venom out of the three. These 41 IgGs were tested again against a panel of 21 different venoms from Bothrops, Crotalus, and Lachesis species. Out of the 41 antibodies, one was found to be specific for Crotalus species, four were specific for Lachesis species, and three were specific for Bothrops species. The next step of the study is using further ELISAs to determine which antibodies can form sandwich pairs for use in a lateral flow assay. Such an assay might be able to detect and identify toxins in the blood of victims before clinical manifestations become apparent to the treating physician, thereby potentially allowing physicians to start treatment sooner. It is also possible that the device can be used by epidemiologists to support mapping of snakebite incidence. Increased knowledge of incidence could help raise awareness, improve resource management during antivenom procurement, and it might guide the design of novel antivenoms. Bangladesh’s Venom Research Centre: towards a national production of snake venoms and antivenom testing capacity

Aniruddha Ghose 1, 2, M Farid Ahsan 2, 3 , M A Wahed Chowdhury 2, 3 , M Mizanur Rahman 2, Mohammed Noman 2, Ibrahim K A Haidar 2, Dilruba A Rumi 2, M Borhan Biswas Romon 2, Abdullah A Sayeed 1, 2 , Sujat Paul 1, 2 , Anupam Barua 1, 2 , M A Sattar 1, 2 , Forhad U H Chowdhury 2, 4 , M Robed Amin 2, 5 , M Ridwanur Rahman 2, 4 , Selim M Jahangir 1, 2, Asok K Dutta 1, 2 , M Abul Faiz 2, 4, 5 , Ulrich Kuch 2, 6

1Chattogram Medical College, Chattogram, Bangladesh 2Venom Research Centre, Chattogram Medical College, Bangladesh 3Department of Zoology, University of Chittagong, Chattogram, Bangladesh

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 35 4Bangladesh Association for Advancement of Tropical Medicine, Dhaka, Bangladesh 5Toxicology Society of Bangladesh, Dhaka, Bangladesh 6Institute of Occupational Medicine, Health Sciences Centre, Goethe University, Frankfurt am Main, Germany

Snakebite is a neglected health problem in Bangladesh with more than 600,000 bites and more than 6,000 deaths annually. The currently used antivenom is produced against the venoms of four species of snake of India but does not cover the medically most important snakes of Bangladesh. Following WHO recommendations, the Government of Bangladesh has taken an initiative to produce specific antivenom for the country under its non- communicable disease control programme. Towards this goal, a Venom Research Centre (VRC) was established in Chattogram Medical College which is known internationally for its snakebite clinic and related research. The primary objective of VRC is to establish a modern, scientifically managed facility for the sustainable long-term keeping and breeding of venomous snakes in order to collect quality venoms for pre-procurement efficacy testing of commercial antivenoms, and for production of improved, regionally appropriate, safe and effective antivenoms. VRC also provides an excellent opportunity for collaborative research on venomics, antivenomics and herpetological topics. So far, a floor space of 400 m 2 has been converted into a serpentarium, a laboratory, a quarantine area and a mouse breeding area. At present VRC hosts more than 60 snakes of different species including cobras, kraits and vipers of different ages collected from different georeferenced locations of Bangladesh to look for and ensure representation of species and venom variability. A population of captive-born cobras and pit vipers is being reared. The operation and maintenance of this centre and procedures follow WHO guidelines and advice from our collaborators in Costa Rica, Germany, Spain, Thailand as well as Chittagong Veterinary and Animal Sciences University and University of Rajshahi, with a vision of achieving GMP certification for snake venom production in the future. As an additional task VRC also maintains a snakebite registry in Chattogram Medical College Hospital recording the details of more than 1,000 snakebite patients per year. We are also engaged in the training of healthcare professionals and creating public awareness about snakebite, and open to collaboration proposals. Isolation, purification, characterization and an insight of in silico analysis of hyaluronidase from Naja species (Nigricollis, Haje and Katiensis) snake venom

Binta G Kurfi 1, Jamila Abdullahi 2, Ibrahim Aminu 3, Nasiru Abdullahi 4

1Department of Biochemistry, Faculty of basic medical Sciences, Bayero University, Kano, Nigeria

Elapid venom is highly valuable and possesses medically and pharmacologically important peptides, Naja species venom retains elapid neurotoxins in combination with cytotoxins and cardiotoxins, venom. In this report, hyaluronidase (Hyase) was purified and characterized from the venom of three species of Naja ( Nigricollis, haje and katiensis) involved in accidents with humans in the sub-Saharan Africa particularly Nigeria, and an insight of Insilco analysis of the enzyme. Purification of the enzyme was done using three steps; which includes protamine sulphate precipitation, gel filtration on sephadex G-75column. Active fraction were applied to ion –exchange chromatography on DEAE cellulose which was eluted using convex Nacl gradient. Hyaluronidase was purified and presented a specific activity of 39.129tru/mg, 20.732tru/mg and 17.110TRU/Mg and (crude venom 14.7635tru/mg, 7.5108tru/mg, and 8.4836tru/mg for N. nigircollis, N. Kateinsis and N.haje respectively. Hyase displayed molecular weight of 44kDa, 30kDa and 28kDa for N.nigricollis, N.haje and N.kateinsis respectively. The optimum pH were 4-8, for the species respectively, the optimum temperature ranging from 37OC- 42 OC. The insilico analysis predicted similar amino acid found at the catalytic site of human hyaluronidase (Hyal4) the conserve domain region also shows that the enzyme belongs to glyco hydrolase 56 super family and the phylogenetic tree also shows that they have similar evolution. The physicochemical properties of this Hyaluronidase are comparable to other hyaluronidase characterized specifically with spider hyaluronidase and

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 36 other snakes from same and different genus insight on insilico analysis revealed that hyaluronidases from snakes are similar to hyase from humans. Discovery of cross-reactive human monoclonal antibodies against short neurotoxins from the black mamba and the forest cobra

Line Ledsgaard 1, Andrea Martos-Esteban 2, Shirin Ahmadi 1,3 , Aneesh Karatt-Vellatt 2, John McCafferty 2, Andreas H. Laustsen 1

1Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Kongens Lyngby, Denmark 2IONTAS Ltd., Iconix Park, London Road, Pampisford, Cambridgeshire CB22 3EG, United Kingdom 3Department of Biotechnology and Biosafety, Eskisehir Osmangazi University, Meselik Yerleskesi, Eskisehir, Turkey

Snakebite envenoming is one of the world’s most neglected tropical diseases and constitutes a serious global health challenge in tropical regions of the world. Each year, snakebite envenoming has a death toll of 125,000 people and causes around 400,000 amputations and other sequalae. The only effective therapy specific for envenoming is antivenom, which is derived from the plasma of animals immunized with snake venom. However, due to its heterologous nature, antivenom can be associated with severe adverse reactions. The black mamba (Dendroaspis polylepis ) and the forest cobra ( Naja melanoleuca ) are among the most venomous and feared African snakes and with good reason: Both are on the World Health Organization’s list of Category 1 snakes, which includes those snakes that present the largest threat to humankind. The main toxicity of their venoms is derived from α-neurotoxins. The most important of these α-neurotoxins is a short neurotoxin, which is present in the venom of both snakes (called short neurotoxin 1). Here, we report the most recent results of our ongoing work aiming at discovering human monoclonal immunoglobulin G (IgG) antibodies with cross-neutralizing effects against short neurotoxins from D. polylepsis and N. melanoleuca . Using phage display selection, we discovered a range of human scFv antibodies from the IONTAS phage display library, and their binding to short neurotoxins was assessed using different ELISA approaches. In future experiments, the scFv-encoding genes will be sequenced and all unique clones will be subjected to thorough testing of cross-reactivity and binding strength in different ELISA- based assays. Once the top 5-10 clones have been selected, their affinity will be determined using Surface Plasmon Resonance, and they will be converted to fully human IgGs and expressed in mammalian cell cultures. Finally, their ability to neutralize short neurotoxins in vivo will be determined in rodent models. Examining the insecticidal potential of spitting cobra venom to address the global health threat posed by insecticide resistance

Rebecca Lees, Hatim Altumari, Mark Wilkinson, Gareth Lycett, Robert Harrison

Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK

Mosquito-transmitted parasitic and viral diseases collectively cause approximately a million deaths annually. Vector control strategies involve the deployment of adulticide and larvicide formulations and the distribution of bed-nets impregnated with insecticide. The rapid spread of insecticide-resistance is therefore a global health threat. Invertebrate venoms have previously been investigated for molecules with biocidal activity against human and agricultural disease vectors with success. Like invertebrate venoms, snake venoms additionally contain a range of enzymes, peptide toxins, polyamines, and small molecules. However, to date, the rich source of material present in snake venoms has been under-explored for identification of leads that could potentially control disease vectors. Having screened a range of venoms from several species of snake originating from various regions of the world, African spitting cobra venoms have shown to have marked toxicity to Aedes aegypti larvae and adult mosquito. The assessment of the activity of both heat-treated and dialysed whole venom against mosquitos suggests the active component

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 37 is protein in nature. The successful fractionation of the venom from an African spitter, Naja mossambica , into distinct toxin groups has allowed further investigation into the active components. Those components showing promise in insecticide-susceptible adult Adese aegypti have been tested in insecticide-resistant strains of both Adese and Anopheles and have proven to cause mortality. It is the hope that validation of the mode of action of those toxins found to be adulticidal will be possible in future, facilitating the development of new insecticides to combat the spread of vector-borne tropical disease. Comparison of five protocols for the whole IgG snake antivenom purification in terms of stability, purity and immunoglobulin subclass composition

Sanja Mateljak Luka čevi ć, Tihana Kurtovi ć, Marija Brgles and Beata Halassy

University of Zagreb, Centre for Research and Knowledge Transfer in Biotechnology, Rockefellerova, Zagreb, Croatia

The whole IgG antivenoms are obtained from the hyperimmune animal plasma by various refinement strategies. Besides therapeutic action, the “refined” preparations still commonly cause clinical side effects attributable to contaminating proteins and /or aggregates. There has been a hypothesis in the literature, that some purification procedures (precipitation of IgGs, low pH-mediated elution of IgGs in chromatography), which induce transient conformational changes of IgG molecules, make their structure less stable and more prone to aggregation, in contrast to others that leave them in solution throughout (caprylic acid precipitation of non-IgG plasma proteins). Our goal was to verify this hypothesis by comparing IgGs isolated by five different commonly employed protocols from the same plasma in terms of stability, purity and immunoglobulin subclass composition. The fractionation methods were: ammonium sulfate precipitation (ASP), anion (AEC) and cation (CEC) exchange chromatography, affinity chromatography (AC) and caprylic acid precipitation (CAP). The highest purity was achieved by CAP and AC, while the highest aggregates content was generated in AC, CEC and ASP, as shown by SDS-PAGE and SEC- HPLC. Some protocols influenced IgG subclass composition, with AC generating the highest loss of IgG(T), as shown by ELISA. Less pure IgG fractions (from ASP, AEC and CEC) were additionally purified by CAP step prior stability study. Pure IgGs had different melting temperatures ( Tm ) in thermal shift assay, which might be the consequence of diverse subclass composition. However, one month storage of IgGs at 37 °C did not influence either Tm or aggregate content of analysed preparations. Our so far results indicate that different procedures gain IgGs of variable purity and subclass composition which might affect both safety and effectiveness. Still, conformational changes during purification procedures might not be the trigger for increased aggregation. Pro-inflammatory properties of B. lanceolatus venom: analysis in the human whole blood model

Joel José Megale Gabrili 1, Felipe França 1, Laurence Mathieu 2, Joël Blomet 2, Denise V. Tambourgi 1

1Immunochemistry Laboratory, Butantan Institute, São Paulo, Brazil 2Prevor Laboratory, Valmondois, France

Bothrops lanceolatus , commonly named Martinique lancehead (“Fer-de-lance”), is a native species confined to the Caribbean island of Martinique. Its venom causes systemic thrombotic syndrome but also local inflammation involving extensive oedema, pain, and haemorrhage. Systemic thrombotic syndrome may lead to fatal pulmonary embolism and myocardial and cerebral infarction. B. lanceolatus venom, in vitro , is able to activate the complement system in normal human serum and induce the generation of anaphylatoxins, whose

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 38 mechanisms include the direct cleavage of complement components by snake venom metalloproteinases and serine proteinases present in the venoms. Considering the inflammatory nature of B. lanceolatus venom and its complement-activation property, in the present work, we investigated the inflammatory effects of this venom in a human whole blood model. The anticoagulant used in this model is lepirudin, a recombinant form of hirudin derived from leeches that direct acts on thrombin and does not affect complement system activity. B. lanceolatus venom was able to activate the complement system in the whole blood model, just after one hour after treatment, generating C3a/C3a desArg, C4a/C4a desArg, C5a/C5a desArg. This venom was also able to induce an increased production of Tumor Necrosis factor a (TNF ), the chemokines CXCL8/IL-8, CCL2/MCP-1 and CCL5/RANTES, as well as of Leukotriene B4 (LTB4), Prostaglandin 2 (PGE2) and Thromboxane B2 (TxB2) in the human whole blood model. Data presented here suggests that B. lanceolatus venom can be directly recognized by pattern of recognition receptors in human whole blood, triggering several inflammatory effects, including complement activation, production of cytokine, chemokines and eicosanoids. Thus, this model seems to be suitable for further studies aiming to test new pharmacological interventions for the treatment of B. lanceolatus snake envenomation. Snakebite Education Resources

Bethany S Moos

Health Education England, Southern House, Otterbourne, Winchester SO21 2RU, UK

Introduction: Snakebite remains a public health concern of significant standing in Myanmar. Many patients still use inappropriate first aid methods or manipulations, which can lead to a delay in presentation and be associated with detrimental health outcomes. Patient education about the importance of prevention and appropriate first aid measures are essential if we are to improve outcomes in keeping with the World Health Organization target of halving death and disability from snakebite by 2030. Existing resources were reviewed and areas for improvement highlighted. Methods: There were three snakebite education resources available; feedback on the resources was sought from 25 members of healthcare staff at Yangon General Hospital and stakeholders were consulted, where strong themes emerged. The cartoon character ‘Tut Pi’ received strong positive feedback and thus was used as the foundation for the new patient education materials. Approval and copyright was obtained from ‘Tut Pi Publishing’. The ‘Venomous Snakes of Myanmar’ poster has been updated with new pictures and text. Continuous feedback from staff was gained throughout the development process. Results: Eight new posters have been created. The content and pictures have been approved by Dr Yi Yi Khine (Program Manager of the Snakebite Control Project) and submitted to the Health Literacy Promotions Unit of the Ministry of Health and Sport for field-testing, with a view to using across the country. Conclusion: These new snakebite education materials will contribute to increasing public awareness of snakebite prevention and appropriate first aid measures, in addition to increased staff awareness of local snakes of medical importance. I am very grateful to Professor David Warrell for the photographs of venomous snakes and for reviewing the resources. Exposures to hogweed ( Heracleum spp.) reported to the DPIC, including systemic effects after ingestion of 'giant hogweed soup'

Henneke N Mulder-Spijkerboer 1, Anne A Kan 1, Moniek CM Koomen 2, Dylan W de Lange 1

1Dutch Poisons Information Center, University Medical Center Utrecht, Utrecht University, The Netherlands 2Independent general practitioner

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 39 In this study, we retrospectively describe all human exposures to hogweed ( Heracleum spp.) reported to the Dutch National Poisons Information Center (DPIC) from 2014 to 2018. Additionally, we report the development of systemic effects in an adult after ingestion of giant hogweed ( Heracleum mantegazzianum ). Results: The DPIC was consulted about 106 patient with exposure to hogweed. The most common exposures were dermal and/or eye contact in adults while gardening (43%), and dermal contact and/or ingestion of small amounts in children while playing outside (42%). Skin contact in combination with exposure to sunlight led to severe skin effects, due to the phototoxic effects of psoralens. Ingestion mostly led to irritation of the mouth and/or gastrointestinal tract. However, the DPIC was consulted about one case with unexpected systemic effects after consumption of giant hogweed as a meal. A 75 year-old man prepared soup from 150-200 g cooked leaves of a young plant, which he consumed at lunchtime. Initially, he displayed no symptoms. On day 2 he spend part of the day naked in his garden, as it was a warm, sunny day. That evening he developed skin effects: his entire body was red and itchy, with the hands, forearms, and face particularly affected. On the morning of day 3 there was a fluid-filled swelling under the right eye. He visited his general practitioner, who consulted the DPIC. The patient was advised to avoid further exposure to sunlight, and was given desloratadine (an antihistamine). The following days, the symptoms increased: the left eye and the hands became swollen, and the skin of the face and hands felt tight. More than a week after exposure the symptoms improved. On day 10 his face and hands were still red, but there was no more swelling. Conclusion: In addition to local effects of exposure to giant hogweed, one should also be aware of the possibility of systemic effects after ingestion of this plant in combination with sun exposure. Mass spectrometry analysis of a new aggregation platelet inhibitor from Crotalus durissus collilineatus snake venom

Isadora S Oliveira 1,2 , Manuela B Pucca 3, Marco A Sartim 4, Dominique Baiwir 2, Gabriel Mazzucchelli 2, Suely V Sampaio 5, Eliane C Arantes 1, Loïc Quinton 2

1Department of Physics and Chemistry, School of Pharma Sciences of Ribeirão Preto, University of São Paulo, SP, Brazil 2Mass Spectrometry Laboratory, MolSys Research Unit, Liège Université, Liège, Belgium 3Medical School, Federal University of Roraima, Boa Vista, RR, Brazil 4Institute of Biological Sciences, Federal University of Amazonas, Manaus, AM, Brazil 5Toxicology and Food Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, SP, Brazil

Animal venoms are rich sources of biologically active molecules with potential therapeutical properties. Anti- thrombotic drugs based on platelet aggregation inhibitors isolated from snake venoms are currently available for vascular disturbs treatment, such as Aggrastat® and Integrilin®. Among platelet aggregation inhibitors, phosphodiesterases (PDE) show a different mechanism from disintegrins by cleaving phosphodiester bonds of nucleic acids and other components with platelet aggregation activity, as ADP. This study aims to isolate a PDE from Crotalus durissus collilineatus venom, to characterize it through mass spectrometry techniques and to evaluate its inhibitory action into platelet aggregation. PDE was isolated through liquid chromatography (venom recovery 0.7%) and further studied using different mass spectrometric approaches. PDE entire molecular weight was determined by matrix-assisted laser desorption/ionization with time of flight analyzer (MALDI-TOF). To determine PDE amino acid sequence trypsin digestion and Multi Enzymatic Limited Digestion (MELD) were employed, both analyzed by liquid chromatography tandem mass chromatography (LC–MS/MS). Moreover, peptide mass fingerprint and de novo sequence were also performed. Platelet aggregation assay was performed using different concentrations of PDE (0.12, 0.06, 0.03 and 0.015 µg/µL). Our results demonstrated that the PDE was purified and its molecular mass was estimated. Trypsin digestion and MELD analyses resulted in 1,822 and 8,889 peptides, respectively, suggesting that MELD can be considered the best approach to determine the protein sequence. PDE significantly ( p < 0.05) inhibited ADP-induced platelet aggregation in a dose-dependent manner. In conclusion, our results support that the novel PDE from C. d. collilineatus venom is a

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 40 potent inhibitor of platelet aggregation and could be used as a model for designing new PDE-based drugs to treat thrombotic cardiovascular events. Characterization of Angolan snake venoms of medical importance and recognition by an experimental murine serum

Paula R S Oliveira 1, Maria de L Bastos 2, Denise V Tambourgi 3

1Faculty of Medicine, Katyavala Bwila University, Benguela, Angola 2UCIBIO REQUIMTE, Toxicology Laboratory, Faculty of Pharmacy, University of Porto, Porto, Portugal 3Immunochemistry Laboratory, Butantan Institute, São Paulo, Brazil

Snakebites are a health problem in many countries around the world, including Angola. These accidents are considered a ‘’Neglected Tropical Disease’’, responsible for high morbidity and mortality in the Sub-Saharan Africa. The extent of this health problem in Angola is still unknown. The objectives of this study were to biochemically analyze the venoms from snakes, collected in Angola; to evaluate the immunogenicity of the venoms; to produce experimental antivenom sera. A two-step methodological design was carried out, including the biochemical characterization of venoms from snakes captured in Angola, by analyzing the : i) protein content of the venoms and their electrophoretic profiles; ii ) the glycosylation profile of the venom proteins; iii) the proteolytic, phospholipase and hyaluronidase venoms activities. The second step was related to the evaluation of the immunogenic potential of Angolan snake venoms in a murine animal model and detection of the antigenic components by the experimental murine antivenom. The results allowed to conclude that: 1) the venoms of snakes, involved in accidents, have a remarkable intraspecies biochemical variability, related to the sex of the animals and their regions of origin; 2) venoms of snakes of medical importance in Angola are immunogenic. Viperid venoms ( B. arietans , gabonica ) were more immunogenic than the elapidic venom ( N. nigricollis ) and the experimental murine antivenom recognized a considerable number of venom components. Targeting melittin from Africanized bee venom with pH-sensing human monoclonal antibodies

Manuela B Pucca 1,2 , Felipe A Cerni 1,3 , Eliane C Arantes 3, Andreas H Laustsen 2

1Medical School, Federal University of Roraima, Av. Cap. Ene Garcês 2413, 69310-000, Boa Vista, Roraima, Brazil 2Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Kongens Lyngby, Denmark 3Department of Physics and Chemistry, School of Pharma Sciences of Ribeirão Preto, University of São Paulo, SP, Brazil

Envenomings and deaths caused by bees represent a serious health problem in the Americas. Over 1,000 human deaths due to bee envenoming have been recorded throughout time. The actual number is unknown, but likely to be significantly higher, as some bee sting deaths are erroneously attributed to heart attacks and other causes. Africanized bees ( i.e. killer bees) are the main perpetrators for these attacks due to nesting habits, aggressive nature, and their high propensity to swarm and chase intruders for several miles with the purpose of attacking in high numbers. To many people, severe bee envenoming is not life-threatening, and antivenom has thus not been made available. The main toxic effects of bee envenomings are mainly caused by melittin, which accounts for 50-60% of bee venom. This toxic protein is poorly immunogenic due to its low molecular weight, random structural conformation, and highly lytic activity. Therefore, it is not feasible to develop traditional antivenom against this main venom component using traditional immunization procedures. Unlike conventional animal immunization, phage display technology allows for the generation of fully human antibodies against non- immunogenic targets, making this technology particularly suitable for the development of monoclonal antibodies against melittin. Moreover, by combining phage display and conditional binding and elution steps, we demonstrate that it is possible to select human monoclonal antibodies that bind melittin in a pH-dependent

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 41 manner. The aim is to generate antibodies that can be recycled by the FcRn (neonatal Fc receptor) in the early endosomes, which may enable a recombinant antivenom based on such antibodies to be administered at very low dose. This study presents recent progress in the discovery of pH-sensing anti-melittin human monoclonal antibodies, exploring the strategies employed during phage display panning, subcloning, melittin-binding assays (ELISA), antibody sequencing, and modelling. Structural and functional characterization of Latrodectin II, a low molecular weight component from black widow spider venom

Esperanza Rivera-de-Torre 1, Javier Narbona 1, David Pantoja-Uceda 2, Gustavo Titaux 2, Belén Patiño 3, Pilar Medina 4, José G. Gavilanes 1, Jessica E. Garb 5, María-Ángeles Jiménez 2, Álvaro Martínez-del-Pozo 1

1Dept of Biochemistry and Molecular Biology, Faculty of Chemistry, Complutense University, Madrid, Spain 2Dept of Biological Physical Chemistry, Institute of Physical Chemistry Rocasolano, CSIC, Serrano, Madrid, Spain 3Dept of Microbiology III, Faculty of Biology, Complutense University of Madrid, Madrid, Spain 4Unidad de Protección de Cultivos, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid, Madrid, Spain 5Department of Biological Sciences, University of Massachusetts Lowell, Lowell, MA, USA

Latrodectins (Ltds) are low molecular weight proteins of around 70 amino acids (6-8kDa), with an acidic isoelectric point and a high content of disulphide bridges that have been detected within black widow spider venom ( Latrodectus spp ). They have been isolated in small amounts from the venomous cocktail of black widow species but have not been characterized in deep detail. They usually appear as an omnipresent component in latrotoxin (LTXs) preparations. These LTXs are the most characteristic high molecular weight proteins (110- 140kDa) within Latrodectus spp venom, the toxic activity of which relies in the formation of pores through biological membranes. However, Ltds natural biological function is not yet known. The few available results suggest that they are essential to increase the neurotoxic activity of LTXs by increasing their binding affinity for the membrane. The association between these two groups of proteins (Ltds and LTXs) has even driven some authors to use the concept of what is called the latrotoxin macromolecular complex . In order to contribute to sort out the role of Ltds in black widow spider venom, we have cloned the highly expressed LtdII protein from L. hesperus in a suitable vector for expression in the yeast P. pastoris . The protein was successfully produced and purified to homogeneity through several chromatographic steps. This purified protein did not show neither antimicrobial nor antifungal activities. Its six cysteine residues were forming disulphide bridges, as supported by mass spectrometry data. Circular dichroism characterization was consistent with a highly thermostable and fully folded globular peptide, showing high -helical content. These results were also in full agreement with the analysis of 3D NMR spectra recorded for 15 N , 13 C –labelled protein. Developing high-throughput screening assays for accelerating discovery of P2X receptor modulators from venoms

Lucka Bibic 1, Volker Herzig 2, Carenza Smith 1, Glenn F. King 2, Mark Searcey 1, Leanne Stokes 1

1School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich UK 2Institute for Molecular Bioscience, University of Queensland, Australia

Animal venoms play an important role in drug discovery as they contain a rich source of bioactive molecules evolutionarily fine-tuned to target ion channels and receptors. Evaluation of venom constituents against interesting ion channel and receptor targets requires the use of appropriate screening systems and high throughput platforms. We have established a number of quantitative fluorescent-based high throughput screening assays for assessing activity of venom toxins at purinergic P2X ligand-gated ion channels. Our fluorescent based screens measure agonist-induced calcium responses (P2X3, P2X4) within cells or agonist-

© VENOMS AND TOXINS 2019 | 28 th – 29 th August 2019 | OXFORD | UK Email: [email protected] | http://lpmhealthcare.com/venoms-and-toxins-2019 P a g e | 42 induced dye uptake responses (P2X7) using a Flexstation 3 plate reader. These assays have been robustly tested for reproducibility (Z’ factors > 0.55). A diverse selection of 180 crude venoms from spiders, centipedes, hymenopterans, and cone snails have been screened against human P2X4 in a heterologous expression system (HEK-293 and 1321N21 cells). We next generated a collection of fractionated venoms in a library for hit identification purposes using a combination of chromatographic and mass spectrometric techniques to be able to isolate and purify active venom components. RP-HPLC chromatograms for several crude venoms will be presented, including Nhandu chromatus, Lasiodora klugi, Apis mellifera, Vespula germanica, Vespa velutina nigrithorax, Conus geographus . Example data from testing 48 fractions collected from Nhandu chromatus venom against hP2X4, hP2X3 and hP2X7 will be presented. Our high-throughput assays allow testing of multiple venoms on multiple targets in minimal testing time. These assays are also suitable for the testing of other libraries of natural products to investigate other bioactive molecules that may modulate P2X receptor activity. This work is supported by BBSRC NRP Doctoral Training Program and the Royal Society. Anticancer, antibacterial and insecticidal effect of assassin bug Rhynocoris iracundus (Hemiptera: Reduviidae) venom

Miray Tonk 1*, , Nicolai Rügen 2* , T. Jenkins 3, H. Vogel 4, N. Welsch 4,Andreas Vilcinskas 1,2

1Institute for Insect Biotechnology, Justus Liebig University of Giessen, Winchester Strasse 2, 35392 Giessen, Germany 2Fraunhofer Institute for Molecular Biology and Applied Ecology, Department of Bioresources, Giessen, Germany 3Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark 4Max Planck Institute for Chemical Ecology, Department of Entomology, Jena, Germany *shared first authorship

Animal venoms constitute a rich source of biologically active compounds with high potential for novel structures with therapeutic and biotechnological applications. In this study, we used predatory assassin bugs, Rhynocoris iracundus, which use their venom to capture prey or as a self-defence mechanism. Interestingly, their venom is capable of extra-oral digestion of the prey. Predatory assassin bugs are not medically important, the potential effect of their venom was therefore neglected so far. The aim of the present study is to evaluate the cytotoxic, haemolytic, antibacterial and insecticidal effect of crude venom of R. iracundus . For this purpose, we tested pure and diluted forms of the venom on the following cells: mouse hepatoma Hepa 1-6, neuroblastoma Neuro2a, myoblasts C2C12 and Drosophila melanogaster Schneider 2 cells. We performed antibacterial assay to evaluate potential venom activity on Gram positive and Gram negative bacteria. The venom of R. iracundus showed strong cytotoxic activity on all tested cell lines with relatively low effect against C2C12 (59% of growth inhibition) and strong cytotoxicity to Neuro2a cells (98% growth inhibition). Also, high potent activity on S2 cells (99% of growth inhibition). Furthermore, the venom has antibacterial effect on Escherichia coli D31 . We found that venom has 6% haemolytic effect on pig erythrocytes. The present study aims to identify the therapeutic and agricultural potential of the venom. As part of the characterization of the venom R. iracundus , we are currently investigating which venom components have such strong cytotoxic activity on cells, their targets and mechanisms of action on cancer cells, which may help to unveil novel therapeutic agents in cancer treatment. Additionally, we are performing in vivo assays using meal worm Tenebrio mollitor to find specific compounds for agricultural applications of assassin bug venom components.

The Hamish Ogston Foundation is a charitable foundation driven by a desire to contribute to lasting change, across three pillars: Health, Heritage and Music. Taking advantage of the commercial knowledge of its Founder, Hamish Ogston, and an experienced group of advisors, the Foundation introduces new disciplines and generates wider visions on impactful, fiscally prudent and longer-term giving. The HOF is particularly committed to supporting and raising awareness of some lesser- known and preventable health issues, in particular snakebite. Having recognised the devastating impact snakebite can have, the Foundation aims to alleviate suffering by sponsoring high-quality antivenom research, and has recently committed to support research in three of the Asian countries worst affected by this neglected tropical disease. This will include hospital treatment of snakebite victims and first aid as well as support for studies into the effectiveness and safety of currently available and recently developed antivenoms.

Hamish Ogston CBE is an entrepreneur who co-founded a number of businesses, including Europe’s first retail loyalty reward scheme, an incentive & motivational award programme, a museum operating company, a tour operator & event management company and an international marketing services company. He is an active philanthropist, supporting a range of initiatives related to health, heritage and music in and outside of the UK. Hamish was awarded his CBE in 2011 for services to business and the community in York.

Silanes was born on November 4, 1943 as a medical prescription laboratory. Today is a solid company that has evolved according to the requirements of quality and innovation maintaining a tradition of trust and commitment to its various stakeholders, such as doctors and members of the health sector, employees, customers, partners, researchers, suppliers, authorities, etc., with the motivation of the world every day to provide better solutions to health.

We are an innovative company that set aside 10% of our sales to research and development, patents and generators being agreements academia-industry collaboration. Aware of the challenges imposed on us by an increasingly globalized world, we seek to expand our coverage to other countries by forming strategic alliances with companies in the industry with the launch of a subsidiary in Spain that allows us to generate innovative projects in other continents.

The Instituto Bioclon is a subusidiary company of Silanes and came about as a result of this union 1990. It emerged to take advantage of the opportunities for developing new biotechnologies and for becoming a world leader in the research, development, and production.

Bioclon-Silanes is the creator of third generation antivenoms of worldwide level, whose biotechnological characteristics make them different, that are highly safe, broadly efficacious, and having had no reports of severe secondary effects. They are produced using a technology that is 100% that of Bioclon-Silanes, and protected by patents in several countries. Our antivenoms are currently marketed in Mexico, Central and South America, United States of America, Europe, Africa, and the Middle East through a regionalization program

Bioclon-Silanes is the only Mexican company that has obtained the designation “orphan drug” (the non- existence of a drug or medication for treating a specific disease) by the Food and Drug Administration (FDA) of the United States of America.

VINS Bioproducts Limited entered the arena of biologicals and bio-pharmaceuticals at the turn of the century with a view to produce life-saving drugs for rural areas. Early on, the company focused on finding remedies for diseases that are endemic in India, the tropics and other less developed nations. True to its initial objective, the company commenced work to develop AntiSnake Venom Serum (ASVS), which was launched in early 2000. VINS’ ASVS, is a life-saving product much needed in rural areas where people engaged in agriculture activities are regularly exposed to snake bites.

The product was well accepted and enjoys today, a market share of almost 40% of the Indian ASVS market. Spurred on by the initial success, the company took up the development of ASVS (Polyvalent from different African snake species including for deadly Black Mamba species). Today Vins manufactures several Snake Venom Antiserum country specific to the requirement by the MOH of various countries.

Anti-Rabies Serum (ARS). World Health Organization (W.H.O.) estimates that 75% of deaths caused by rabies world-wide, occur in India, Bangladesh and Pakistan alone. W.H.O. recommends Anti Rabies Serum (ARS) along with anti-rabies vaccine (ARV) to be injected in all Category III and immuno- compromised Category II rabies cases. The other products are Tetanus Antitoxin, and Diphtheria Antitoxin.

MicroPharm Limited is a developer and manufacturer of therapeutic polyclonal antibodies for human and veterinary use. The Company’s core expertise lies in the raising of ovine antisera containing high levels of specific antibodies directed against antigens such as toxic molecules or viruses and the subsequent purification and modification of such antibodies to produce a range of immunotherapeutic products for clinical use. All are designed to treat acute, life-threatening emergencies, have been developed at the request of the medical profession and are required urgently either because no alternative exists or because any alternative is ineffective and/or unsafe. MicroPharm currently produces two antivenoms, ViperaTAb® for the treatment of envenomation by the European adder and EchiTAbG™ for the treatment of the carpet viper, Echis ocellatus . The Company has a number of products in development including: PolyCAb for the treatment of severe C. diff. ; ColchiBIND for the treatment of colchicine poisoning; EBOTAb for the treatment of Ebola Virus Disease and ViperaVet™, for the treatment of dogs envenomed by one of the four medically important species of Vipera (adder) found throughout Western Europe. MicroPharm’s collaborators include Public Health England, University of Oxford, University of Leeds and the University of Edinburgh.

Seqirus , a CSL company, is a leading provider of essential vaccines and pharmaceuticals. We have served Australia’s healthcare needs for over a century and today we are Australia’s only local manufacturing facility for seasonal and pandemic influenza vaccines. As part of our heritage, Seqirus remains the sole provider of a unique range of products made in the national interest for the Australian government, including antivenoms and Q fever vaccine. As the only manufacturer in the world to supply antivenoms specific to a majority of Australia’s most venomous creatures, Seqirus serves a significant public health need. We are committed to reducing the burden of venomous bites and stings through awareness, education and community programmes in the Asia Pacific region. www.seqirus.com.

LATOXAN produces and sells Natural active Ingredients aimed at Pharmaceutics, Animal Health, Cosmetics, Food and Feed Supplements and Life Science Research. Venoms and venom peptides are sourced from our own venomous animals breeding facilities. Plant extracts and small molecules come from an exclusive partnership with a foreign academic Institution specialized in Plant Biochemistry and Pharmacology. Collaboration with several Research Organizations have enabled LATOXAN to expand its catalogue to unique molecules and screening libraries. LATOXAN is also involved in Drug Discovery Research projects on antimicrobials, pain-killing, haematology, cancerology, dermatology. As early as 1982 LATOXAN pioneered in the production of venoms within its facilities based in France and progressively developed a unique know-how for sourcing, farming, breeding and milking over 300 species of snakes, scorpions and batrachians from all over the world. Animals come from breeding or capture missions and are kept several years active and productive. For each production batch LATOXAN is able to provide upon demand certificate of analysis and traceability, certificate of origin, health veterinary certificate and a Venom Production Master File (for pharmaceutical industry).

The Global Snakebite Initiative (http://www.snakebiteinitiative.org) is an internationally-active, non-profit, charitable organisation, registered in Australia, but with world-wide vision, aims and membership. It is led by snake-bite experts who are dedicated to improving access to good quality, robustly tested, safe, effective antivenoms in the world’s poorest communities. It strives to provide a collaborative framework to address the neglected global tragedy of snake-bite envenoming . The Global Snakebite Initiative has been endorsed by the membership of the International Society on Toxinology (IST). Several senior representatives of GSI will be attending the meeting, including Professor José María Gutiérrez, Professor David Warrell, Professor Juan Calvete, Professor Alan Harvey, Professor Robert Harrison, Dr David Williams and Dr Aniruddha Ghose.

OxonLab provides 1-, 2- and 5-day basic biomedical/biosciences laboratory training courses at Oxford. One-day courses include: i) Essential laboratory skills; ii) Essential molecular biology iii) Essential molecular biology & bioinformatics; and iv) Molecular separation methods. Two-day courses include: i) Discovering molecular biology; ii) Discovering molecular biology & bioinformatics; and iii) Discovering microbiology. Five-day courses, providing in-depth training, include: i) Extended molecular biology laboratory skills; and ii) Extended microbiology laboratory skills. The training is provided by experienced laboratory scientists from the University of Oxford, Oxford hospitals and Oxford Brookes University.

In addition, OxonLab also offers one-day workshop in Intellectual property and commercialisation in biosciences, Health & Safety, and Bioethics. These taught workshops provide essential background in important areas, such as intellectual property in biosciences, patent applications, patent searches and regulations, good laboratory practices, biosafety & bioethics, working with GMOs, and COSHH regulations.

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