Into the Preclinical Assessment of Snake Venom Toxicity and Antivenom Efficacy
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INCORPORATING THE 3RS (REFINEMENT, REPLACEMENT AND REDUCTION OF ANIMALS IN RESEARCH) INTO THE PRECLINICAL ASSESSMENT OF SNAKE VENOM TOXICITY AND ANTIVENOM EFFICACY Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor of Philosophy By Fiona Margaret Simpson Bolton March 2017 Noah said to the creatures ‘Go forth and multiply’ ‘We can’t’ said the snakes ‘We’re adders.’ ABSTRACT Antivenom is the only effective treatment for snakebite and comprise immunoglobulins obtained from venom-immunised horses or sheep. Globally, more than 45 manufacturers make over 120 snake antivenoms; it is a regulatory requirement that the venom-neutralising efficacy of all antivenoms are assessed preclinically. The World Health Organisation (WHO) recommended preclinical tests of efficacy are the median lethal venom dose (LD50) and median effective antivenom dose (ED50) assays performed in mice. They result in substantial pain and suffering to the mice with death/survival as their metric. With NC3R-funding, we sought to apply the ‘Refine, Reduce and Replace’ principles of animal experimentation to these murine assays. Pain is a near-universal symptom of snake envenoming, and one of our objectives was to identify an effective analgesic that could be utilised without invalidating the assay results. The Mouse Grimace Scale and Activity scores were used to measure pain. We examined the effects of two opioid analgesics, buprenorphine and morphine, in a range of venom LD50 and ED50 assays. Both were effective at reducing pain scores, but death rates were higher in those which had received buprenorphine, hence morphine is preferable. We demonstrate that each venom exhibits a distinct set of lesions, the severity of which appears time and dose dependent, and that the observed murine pathological lesions show significant similarities to those reported in envenomed human victims. Applying the 3R principles, we have used pathological observations, in combination with ante mortem observations, to establish more humane end-points, consequently reducing the duration of LD50 and ED50 assays from 24 to 6 hours. In addition, we have implemented a ‘dose- staging’ element into experimental design in which one dose is given and the next dose(s) selected based on the results of the previous dose, reducing total mice required. To reduce the numbers of assays, and therefore mice, we have shown an excellent correlation between in vitro binding assays, cytotoxicity neutralisation assays and in vivo ED50 using antivenoms derived from the same pool of donor animals. Comparison of the results of in vitro binding assays between 35 different venom/antivenom combinations showed a poor correlation overall, but the correlation improved when each of five venoms were considered separately. The possibility of replacing the in vivo LD50 and ED50 tests, using a cell-based neutralisation assay was investigated using two cell lines from diverse tissues of origin, namely VERO epithelial-type cells and neural SH SY5Y. All venoms studied produced a cytopathic effect in both cell lines, with the VERO cells being more sensitive to viper venoms and SH SY5Y cells to the effects of elapid venoms, when both cell lines were grown in co-culture. However, variability of results made optimisation of a neutralisation assay inadequate for use as an alternative to in vivo tests. Acknowledgements I would like to thank my supervisor, Rob Harrison for his help, encouragement and endless patience. I am also indebted to Nick Casewell and Professor John Landon for their guidance and invaluable advice. Special thanks go to my friends and colleagues, past and present, at LSTM – Richard and his family, Rachel, Camilla, Maimonah, Paul Rowley (snake technician extraordinaire), Gareth, Stuart; and at MicroPharm – Brenda, Ibrahim, Caryl, the three Chris’s, Dewi, Matt, Sarah, Rossen, Hayley, Elise, Megan, Ruth, Ian, Cheyenne. My husband, David has patiently proof read my thesis and supported me through the difficult times; my family and friends have patiently listened to me pontificating about snakes, venom, snakebites and their treatment. Special thanks to my prosthetist and the team at the Artificial Limb Centre for keeping me mobile. Thanks to Professor Brian Faragher for help with the statistics, to Matt Leach, Newcastle University for explaining how to assess pain in mice and to Lynn McLaughlin and the staff of the BSU, Liverpool University for assistance with the in vivo work. Without the help of all these people, and many more, this thesis would never have come to fruition. I would like to acknowledge the financial support in the form of a PhD studentship from NC3Rs - the National Centre for Refinement, Replacement and Reduction of animals in Research; MicroPharm Ltd and the MRC. LIST OF TABLES Table 1.2-1: Major viper venom enzymes, proteins peptides and toxic components showing their biological action and induced pathological lesions (Mackessy, 2010). ........................ 8 Table 1.2-2: Major elapid venom enzymes, proteins peptides and toxic components showing their biological action and induced pathological lesions (Mackessy, 2010). ........................ 9 Table 2.2-1: LD50 of Bitis arietans venom........................................................................................ 59 Table 2.3-1: 15% Resolving gel. ....................................................................................................... 62 Table 2.3-2: Stacking gel. ................................................................................................................. 62 Table 2.3-3: Preparation of ammonium thiocyanate ...................................................................... 65 Table 2.4-1: Plate layout ................................................................................................................... 71 Table 2.7-1: Behavioural observations template ............................................................................ 76 Table 2.11-1: Post mortem examination observation and lesion description template .............. 89 Table 3.3-1: Venoms selected showing family, origin, source, batch number (BN) and characteristics .......................................................................................................................... 96 Table 3.3-2: Antivenoms used showing venoms in immunogen, donor, fragment, manufacturer and reason for inclusion .......................................................................................................... 97 Table 3.4-1: ELISA results summary. ............................................................................................ 105 Table 3.4-2: Avidity ELISA Summary............................................................................................. 109 Table 3.4-3: Specific antibody concentrations of a selection of antivenoms determined by SSAC. ...................................................................................................................................... 112 Table 3.4-4: Median cytotoxicity (LC50) of five snake venoms .................................................... 114 Table 3.4-5: Antivenom neutralisation of venom cytotoxicity by a selection of homologous and heterologous antivenoms. ..................................................................................................... 115 Table 3.4-6: Linear correlation of results (R2) between assays - E. ocellatus venom ............... 119 Table 3.4-7: Linear correlation of results (R2) between assays - B. arietans venom................. 119 Table 3.4-8: Linear correlation of results (R2) between assays – V. berus venom .................... 122 Table 3.4-9: Linear correlation of results (R2) between assays - N. nigricollis venom ............. 122 Table 3.4-10: Linear correlation of results (R2) between assays - D. angusticeps venom ....... 122 Table 3.4-11: Linear correlation of results (R2) between assays – pooled venoms .................. 124 Table 3.4-12: Summary of in vitro assay results.. ........................................................................ 125 Table 4.1-1: Assays performed pre- and post-purification on Vipera antiserum samples containing different VSAb concentrations ........................................................................... 132 Table 4.3-1: Antiserum samples selected for study. .................................................................... 134 Table 4.3-2: Dilution of antisera and addition of caprylic acid. αVipH = high VSAb, αVipM = medium VSAb, αVipL = Low VSAb. ...................................................................................... 134 Table 4.3-3: Reformulation of final product .................................................................................. 135 Table 4.4-1: ELISA EC50 titre pre- and post-purification and endpoint titre post- purification. 137 Table 4.4-2: Avidity ELISA – molarity of sodium thiocyanate resulting in 50% disruption of V. berus venom binding (M50) to α-VipH, M and L IgG and ViperaVet antivenom. ................ 139 Table 4.4-3: Summary of VSAbs to V. berus venom measured by SSAC. ................................. 139 Table 4.4-4: Summary of VSAbs to a mix of four Vipera venoms measured by SSAC after purification. ............................................................................................................................. 140 Table 4.4-5: Summary of immunocytotoxicity assays. Neutralisation of VbV cytotoxicity with three different VSAb concentration Vipera