Characterisation of the Pathophysiological and Immunological Responses to Snake Venom Proteins: Avenues to Designing and Testing Novel Therapies
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CHARACTERISATION OF THE PATHOPHYSIOLOGICAL AND IMMUNOLOGICAL RESPONSES TO SNAKE VENOM PROTEINS: AVENUES TO DESIGNING AND TESTING NOVEL THERAPIES Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor of Philosophy By Jaffer Alsolaiss 23rd September 2019 Table of Contents ABSTRACT i DEDICATION iv ACKNOWLEGMENTS v DECLARATION vi ABBREVIATIONS viii LIST OF FIGURES x LIST OF TABLES xii Chapter 1: General Introduction 1.1 Snakebite is a neglected tropical disease 2 1.2 Types and classes of snakes 3 1.3 The development of envenoming mechanisms 7 1.4 Snake venom 7 1.4.1 Snake venom metalloproteinases (SVMPs) 7 1.4.2 Snake venom phospholipase A2 (PLA2) 9 1.4.3 Hyaluronidase 10 1.4.4 Snake Venom Serine Proteases (SVSPs) 11 1.4.5 L-amino acid oxidases (LAAO) 12 1.4.6 C-type lectins (CTL) 12 1.5 Snakebite envenoming pathology 14 1.5.1 Local manifestations 14 1.5.2 Systemic manifestations 15 1.5.2.1 Neurotoxicity 16 1.5.2.2 Haemotoxicity 17 1.5.2.3 Cardiotoxicity 18 1.6 Clinical treatment of snakebite envenoming 19 1.6.1 First aid 19 1.6.2 Antivenom and syndromic management of snakebite 20 1.7 Rationale 28 1.8 Project Aims 32 1.9 References 33 Chapter 2: A new bovine eye model of venom-induced ophthalmia reveals cellular damage is averted by rapid irrigation with water but not milk 2.1 Introduction 48 2.2 Materials and methods 51 2.3 Results 58 2.3.1 Assessment of corneas treated with negative and positive 58 controls 2.3.2 Assessment of corneas exposed to different concentrations 60 of N. nigricollis venom 2.3.3 Assessment of cornea exposed to different concentrations of 63 N. haje venom 2.3.4 Assessment of corneas exposed to different concentrations 65 of N. nivea venom 2.3.5 The effects of fresh venom from spitting and non-spitting 67 cobra on the cornea 2.3.6 Identification of the venom proteins penetrating the cornea 68 2.3.7 Prevention of damage to cornea caused by venom 70 2.4 Discussion 72 2.5 Conclusions 74 2.6 References 76 Chapter 3: A new ex vivo human skin model of venom-induced dermonecrosis 3.1 Introduction 80 3.2 Material and methods 83 3.3 Results 87 3.3.1 Which venom proteins penetrate human skin tissue? 87 3.3.2 Detection of SVMP and SVSP activities detected in culture 89 media of venom-injected biopsies 3.3.3 What is the anatomical consequence of injecting venom into 92 this tissue? 3.3.4 To what extent is cell apoptosis associated with this skin 95 pathology? 3.3.5 Does injection of skin with venoms from vipers, and spitting 98 and non-spitting cobras stimulate distinct expression of pro- inflammatory cytokines, chemokines and growth factors? 3.4 Discussion 104 3.5 Conclusions 111 3.6 References 113 Chapter 4: Defining acute phase and inflammatory responses to better understand the pathology of envenoming by medically important African snakes 4.1 Introduction 123 4.2 Material and methods 127 4.3 Results 133 4.3.1 Haematological analyses of envenomed mice 133 4.3.1.1 RBC abnormalities suggest venom-induced haemolysis in 133 mice 4.3.1.2 N. nigricollis, N. melanoleuca, and B. arietans venoms cause 136 neutrophilia in mice 4.3.1.3 Mouse thrombocytosis following envenoming with six elapid 138 venoms 4.3.2 Acute phase and acute inflammatory responses in 141 envenomed mice 4.3.2.1 P-selectin involvement in acute inflammatory response 141 activation in envenomed mice 4.3.2.2 Envenomation-related immunoglobulin response. 141 4.3.2.3 APR stimulation by Elapidae venoms 142 4.3.2.4 Naja nigricollis venom causes rapid release of systemic 142 inflammatory mediators 4.3.3 Serum biochemistry following snake envenoming 144 4.4 Discussion 146 4.5 Conclusions 150 4.6 References 151 4.7 Supplementary information 159 Chapter 5: Comparative value of different in vitro assays to predict the in vivo preclinical efficacy of new experimental antivenoms designed to improve treatment of envenoming by the most medically-important African snakes. 5.1 Introduction 164 5.2 Material and methods 167 5.3 Results 174 5.3.1 Time course ELISA of sheep sera 174 5.3.2 IgG titres determined by end-point ELISA 176 5.3.3 IgG avidity determined by chaotropic ELISA 180 5.3.4 IgG specificity revealed by Western blotting 185 5.3.5 Antivenomics profiling of venom protein and antivenom 188 antibody interactions 5.3.6 The venom neutralising efficacy of experimental antivenoms 193 5.4 Discussion 196 5.5 Conclusions 201 5.6 References 202 5.7 Supplementary information 207 Chapter 6: Concluding discussion ex vivo ocular envenomation model 215 ex vivo human dermonecrosis model 216 in vivo systemic envenomation model 217 Low molecular weight proteins 218 Neurotoxicity 219 Research priorities 220 References 222 Abstract: Farmers living in deprived rural regions of south and southeast Asia, sub-Saharan Africa and Latin America are the primary group at risk from snakebite envenoming: a priority World Health Organization Neglected Tropical Disease. Depending on the toxins within the venom, and their physiological effects, envenomation can have acute local and systemic manifestations. With a focus upon the most medically-important snakes of sub-Saharan Africa, the present work seeks to address this knowledge gap by developing three new animals’ models of local and systemic envenoming. This enabled identification of the most pathogenic venom toxins and provided preliminary insights into pathophysiological mechanisms. The project culminates in a study to develop ovine antivenoms targeting the neurotoxic, haemorrhagic and coagulopathic systemic effects of envenoming by these snakes. I developed a new ex vivo bovine eye model to interrogate ophthalmic envenoming by spitting cobras. This model identified that corneal damage and opacification were caused by the venom of all cobras, irrespective of their spitting and or non-spitting traits. Notably, venom proteins from the venom of African spitting cobra (Naja nigricollis) and Asian non- spitting cobra (N. naja) were identified as being capable of rapidly (five minutes after topical administration) penetrating through the multi-tissue layers of cornea. I next developed the ex vivo human skin model to examine the relative local tissue injuries exerted by the most medically-important snakes of sub-Saharan Africa. Using this 7-day, non- perfused model of human envenoming allowed me to define the temporal progression of dermal damage for several snake species, identify the aetiological toxins and the extent to which they stimulate proinflammatory cytokines. Greatest dermal damage was affected by SVMPs and serine proteases (SVSPs) of the saw-scaled viper, E. ocellatus and the puff adder, B. arietans. Notable local tissue injury was induced by venoms of both N. nigricollis and Egyptian cobra, N. haje. Seeking to assess envenoming at the next stage in the envenoming process, I next developed the in vivo murine acute phase model. In addition to investigating snake species-specific effects upon the cardiovascular system and other key organs and tissues, I also examined the response to acute envenoming by acute phase reactants, including (Serum Amyloid A (SAA), P-selectin and haptoglobin), as well as acute inflammatory mediators such as cytokines. Severe systemic damage and inflammation were induced by venoms of N. nigricollis and the i forest cobra, N. melanoleuca through the action of acute phase proteins and acute inflammatory mediators. I examined the relative venom-neutralising efficacies of experimental ovine antivenoms prepared by immunisation with different mixtures of neurotoxic venom, adjuvants and duration of immunisation – in comparison to the ovine equivalent of EchiTAB-PLUS-ICP. After a year of immunisation, the experimental anti-neurotoxic antivenoms proved substantially less efficacious and were highly species specific in their efficacy. This study identifies that snake venom-induced neurotoxicity poses the greatest snakebite therapeutic challenge in sub-Saharan Africa – and elsewhere. In summation, this PhD project has contributed new models to improve our understanding of the causative toxins and their mechanisms of action that lead to serious local and systemic effects of snake envenoming, and the role of acute phase reactant and cytokines in this neglected tropical disease. ii Dedication I wish to dedicate this work to the memory of my parents and my sister, who have always had a strong belief in my academic potential. Their belief has supported me throughout my studies, despite their absence. This work is also dedicated to my beloved wife, who is my constant rock through all the difficulties, to my dear siblings, as well as to my darling children, Hadi and Eyad, whom I love enormously. Last but not least, the work is dedicated to my family, my friends, and all the people who love, encourage and support me. iii Acknowledgments: Praise be to Allah, the Most Merciful and Compassionate, Lord of the world, and prayers and peace be upon Mohamed, His servant and messenger. I would like to express my supreme gratitude to Allah, the Ever-Magnificent, the Ever-Thank- ful, for His blessings. His guidance has been essential in bringing this work to fruition. I am greatly thankful to a number of individuals who have provided me assistance through- out this research, namely, my supervisors, Professor Robert Harrison, and Professor Nicholas Caseswell, who have been of enormous help in every stage of the research. I would also like to mention Professor Jose M. Gutierrez, Professor Juan Calvete, Dr Lorenzo Ressel, Dr Gail Leaming, Dr Riaz Aktar, and Genoskin France. I would like to thank all of the CSRI members for all advice and help during my work in the lab or during my writing.