Characterisation of the Pathophysiological and Immunological Responses to Snake Venom Proteins: Avenues to Designing and Testing Novel Therapies

Home , Chinese cobra, Egyptian cobra, Snouted cobra, Venombin A

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

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.

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 throughout 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.

I would like also to thank Paul Rawley for all venom extractions and Mark Wilkinson for technical advice and assistance.

Thank you to my sponsor Ministry of Higher Education in Saudi Arabia for funding and supporting me during my PhD.

I am indebted to all my dear friends and my family for their undying support during this academic journey for the achievement of a doctorate. Their constant love and prayers have been so helpful in emboldening me to finish the present doctoral work.

I am particularly grateful to my wife, Nessrin, who has always encouraged me and sacrificed so much for me to be able to attain my doctoral degree.

Declaration:

Entitled Characterisation of the pathophysiological and immunological responses to snake venom proteins: avenues to designing and testing novel therapies, the present PhD work has been initiated and undertaken by myself in collaboration with my primary and second supervisors, Professor Robert Harrison and Professor Nicholas Casewell, respectively, who supervised my research and offered assistance with the experiments. Apart from a few exceptions, I carried out the work by myself.

Chapter 2: Ethical approval was obtained for the collection of the eyes and I conducted every collection, optimisation and experiment on my own. The histopathological analysis of the dissected cornea was conducted at the University of Liverpool, while the veterinary pathologist Dr Gail Leaming undertook revision of the slide analysis that I conducted. Ahmed Kzali and Shaun Pennington assisted me in carrying out the measurements of opacity and fluorescent intensity.

Chapter 3: Ethical approval was obtained for the collection and preparation of the skin sample, for which I worked together with Genoskin, France. I formulated, performed and validated the experiment design. Dr Gail Leaming supervised the histopathological examination of every injected skin sample, which was undertaken at the University of Liverpool.

Chapter 4: Professor Robert Harrison undertook the process of blood sampling, while I undertook slide and serum separation and storage. Furthermore, I supervised an MSc student in the processing of the acute phase proteins and inflammatory markers. I undertook formulation and validation of every in vitro assay and biochemical assay. Revision of the work performed by the MSc student (Ms Chloe Evans) was conducted.

Chapter 5: The work of this chapter is part of a major MRC-funded project to generate a new ‘antivenomics’ approach to develop a single an antivenom effective against envenoming by sub-Saharan African snake species. To this end, I benefitted from the expertise of Professor Juan Calvate from Spain and Professor Jose M. Gutierrez from Costa Rica in performing antivenomics and investigating the capacity of the experimental antivenoms to counteract venom effects. I undertook the optimisation and performance of IgG purification and every immunological assay (ELISA endpoint titration, ELISA avidity and immunoblotting).

Chapter 2-5 (Results chapters) are in manuscript form and yet to be published. Since the work is meant for publication, the introductory part to every chapter outlines the main

v themes, which are inevitably repetitive, but iterations have been minimised as much as possible. A methods section, and appropriate reference list is included in each chapter.

Abbreviations:

3FTxs Three Finger Toxins APP Acute Phase Protein APR Acute Phase Response CAM Cell Adhesion Molecules CBC Complete Blood Count CRP C-reactive protein COX Cyclooxygenase Cl Confidence Limits CTL C-type lectins DAMP Damage Associated Molecular Patterns DALYs The disability- adjusted life year ELISA Enzyme-Linked Immunosorbent Assay EDTA Ethylenediaminetetraacetic acid ESR An erythrocyte sedimentation rate

ED50 Effective dose prevents 50% death GM-CSF Granulocyte-Macrophage Colony-Stimulating Factor Hb haemoglobin Hp Haptoglobin IFN Interferon IL Interleukin IgG Immunoglobulin G IgM Immunoglobulin M kDa Kilodalton

LD50 Median Lethal Dose (50%) MCP-1 Monocyte Chemoattractant Protein 1 MIP Macrophage Inflammatory Protein MMP Matrix metalloproteinase NTD Neglected Tropical Disease NO Nitric Oxide

PLA2 Phospholipase A2 PAGE Polyacrylamide gel electrophoresis PBS Phosphate-Buffered Saline RBC Red Blood Cells SVMPs Snake Venom Metalloproteases

vii

SDS sodium dodecyl sulfate SVSPs Snake venom Proteases TBST tris-buffered saline Tween WB Western Blots WBC White Blood Cell WHO World Health Organization

viii

List of Figures

Figure 1.1 Estimation of snakebites mortality in 138 countries and the annual estimated number of deaths. Figure 1.2 Examples of Viperidae snakes of medically importance snakes in Sub- Saharan Africa

Figure 1.3 Examples of Elapidae snakes of medically importance snakes in Sub- Saharan Africa

Figure 1.4 Structure and classification of SVMP classes

Figure 1.5 Overview of the clinical symptoms presented by victims of snakebite envenoming Figure 2.1 Image of the exit orifice obtained by scanning electron micrographs of (A) non- spitting cobra N. kaouthia and (B) spitting cobra N. pallida.

Figure 2.2 Experimental design of ex vivo bovine eye model to study the ocular envenomation after topical administration of lyophilised/ fresh venom from spitting and non-spitting cobra.

Figure 2.3 Corneas treated with the positive and negative control Figure 2.4 Corneas treated with different doses of Naja nigricollis venom.

Figure 2.5 Corneas treated with different doses of Naja haje venom

Figure 2.6 Corneas treated with different doses of Naja nivea venom

Figure 2.7 Characterisation of venom proteins penetrating the cornea after topical application of fresh venom. Figure 2.8 The intervention studies using water / milk following the topical application of fresh venom

Figure 3.1 Identification and characterisation of venom proteins penetrating the skin

Figure 3.2 The metalloprotease activities of venoms in the collected culture medium Figure 3.3 The serine protease activities of venoms in the collected culture medium.

Figure 3.4 The macroscopic observation of the injected biopsies (pre and post the subcutaneous injection).

Figure 3.4 Viability and structural integrity assessment following the subcutaneous injection of snake venoms Figure 3.5 Assessment of apoptotic cells following the subcutaneous injection of snake venoms.

Figure 3.6 DNA fragmentation assessment following the subcutaneous injection of snake venoms.

Figure 3.7 Dynamic changes in pro-inflammatory cytokine following subcutaneous injection by Viperidae venom (E. ocellatus and B. arietans) and Elapid venoms (N. nigricollis and N. haje).

Figure 3.8 Dynamic changes in pro-inflammatory chemokines following subcutaneous injection by Viperidae venom (E. ocellatus and B. arietans) and Elapids venoms (N. nigricollis and N. haje).

Figure 3.9 Dynamic changes in growth factors following subcutaneous injection by Viperidae venom (E. ocellatus and B. arietans) and Elapid venoms (N. nigricollis and N. haje). Figure 4.1 The Experimental outline of the in vivo murine acute phase model to study the acute envenoming by Acute Phase Reactants (APR) and acute inflammatory mediators. Figure 4.2 Abnormal red blood cell morphology and haemolysis induced by snake venoms.

Figure 4.3 B. arietans, N. nigricollis, and N. melanoleuca venoms cause increased WBC counts and elevated neutrophils

Figure 4.4 Elapids activate platelet aggregation. Figure 4.5 Venoms elicit different acute phase and inflammatory responses in the systemic circulation of mice

Figure 4.6 Serum biochemistry parameters suggest acute hepatic and renal damage following N. nigricollis envenoming

Figure S 4.1 Examples of platelet aggregation (x 100 objective) were observed in envenomed mice.

Figure 5.1 Immune reactivity of sheep sera post 12 months immunisation to snake venoms Figure 5.2 ELISA End-Point Titration (EPT); Titration curves for poly-specific antibodies against antigen of IgGs purified from immunisation bleeds at six months

Figure 5.3 ELISA End-Point Titration (EPT): Titration curves for poly-specific antibodies against antigen of IgGs purified from immunisation bleeds at twelve months

Figure 5.4 Figure 5.4: Chaotropic ELISA (Avidity): Titration curves for IgGs purified from immunisation bleeds at six months Figure 5.5 Chaotropic ELISA (Avidity): Titration curves for IgGs purified from immunisation bleeds at and twelve months

Figure 5.6 Percentage reduction of IgG titre (twelve months immunisation) after addition of 4M ammonium thiocyanate.

Figure 5.7 Western blotting analysis

Figure 5.8 Comparison of the antivenomics analysis of the immunoreactivity of experimental antivenoms (purified IgGs from immunisation bleeds at six months and twelve months). Figure 6.1 Summaries of the most significant findings in the current PhD, focusing on the pathologies induced by snake venom proteins

List of Tables Table 1.1 Syndromic management of snakebites in sub-Saharan Africa Table 1.2 Antivenom reactions among individuals bitten by snakes Table 2.1 Details of snake venom and venom doses used to study the corneal damage induced by lyophilised and fresh venom from spitting and non-spitting cobras. Table 2.2 Observations of the pathology of corneas treated with fresh venom from Asian and African spitting and non-spitting cobras Table 3.1 Details of snake venoms species and doses used in the study

Table 4.1 LD50 doses of African snake venoms in mice was used for blood and sera analyses Table 4.2. Grading of platelet aggregation in mice injected with medically important snake venoms Table S 4.1 Prior murine in vivo studies of the inflammatory response following dosing with snake venom. Table 5.1 Details of the venoms and adjuvants used for the generation of the experimental antivenoms after six months and twelve months by ovine immunisation

Table 5.2 Estimation of LD50s of the venoms used in this study.

Table 5.3 Values for the Median Effective Dose (ED50)

Chapter 1

General Introduction

1.1 Introduction:

1.1 Snakebite is a neglected tropical disease;

Throughout the world, snakebite envenoming is a severe problem of such large proportions that national and regional health authorities need to accord it greater priority than they have done so far. Young people working in agriculture and living in deprived rural communities in developing nations of Africa, Asia, Latin America and Oceania are at greatest risk of being bitten by venomous snakes (Gutiérrez 2012b; Gutiérrez et al. 2017b; Gutiérrez et al. 2010). This group of people are the primary income earners and food producers in their communities, which is why snakebite envenoming is known as a ‘disease of poverty’ (Harrison et al. 2009). Snakebites estimated (Figure 1.1) at global level of almost 5.4 million, with more than 2.7 million envenoming cases and about 138,000 deaths and about 400,000 disabled people/year. About 32,000 deaths annually at the level of the entire sub-Saharan Africa (Gutiérrez et al. 2017b; WHO 2019). The majority of victims in these rural, remote communities take hours, sometimes days to access healthcare facilities. There are many reasons contributing to this delay, including people’s preference for traditional healing over hospital care, insufficient health centres, high costs of hospital treatment and the fact that many Africans distrust health centres and the equipment used to treat snakebites. Furthermore, despite being the sole treatment of incontrovertible efficiency, antivenom is not widely available, particularly in sub-Saharan Africa. This has prompted endeavours over the past ten years to make raise awareness of the burden of disease and paucity of antivenom supply – all in an effort to stimulate action making antivenom more accessible and to reduce health complications and mortality associated with envenomation (Brown 2012; Habib 2013). The burden of disease posed by snakebite was highlighted in a paper demonstrating that the Disability Adjusted Life Years (DALYs) lost due to mortality and morbidity in 16 Western African countries was 0.32 million DALYs. This exceeds not only the approximated burden of Buruli ulcer, Echinococcosis, Leprosy, Trachoma, Yaws and Yellow Fever at global level, but also the burden of African Trypanosomiasis, Leishmaniasis and Onchocerciasis in those same sixteen West African countries (Habib et al. 2015).

Figure 1.1: Estimation of snakebites mortality in 138 countries and the annual estimated number of deaths. Adopted from (Gutiérrez et al. 2017b; Harrison et al. 2009) with some modification. Highest snakebites mortality (Darker colour).

1.2 Types and classes of snakes

Snakes are believed to have developed either from aquatic lizards (e.g. mosasaurs) or from burrowing lizards, with the second hypothesis having gained greater support thanks to molecular findings that have been recently produced (Vidal & Hedges 2004). At global level, the number of snake species exceeds 3,500, belonging to the order Squamata and the suborder Serpentes. Apart from Antarctica, New Zealand and Madagascar snakes live in a wide range of environments, including the Himalayan Mountains and the Pacific coral reefs. Some snakes are exclusively aquatic, others are terrestrial or live underground in cavities created by them or other animals, and others are arboreal. As regards reproduction, the majority of snakes are oviparous, although there are also certain species that are viviparous. Some species of oviparous snakes (e.g. python and king cobra) even keep watch over their eggs. All snake species feed solely on other animals, and numerous species can swallow prey that greatly exceeds their own size and bulk. Venomous snakes are also known as advanced snakes and all of them are members of the superfamily Colubroidea or Caenophidia. Atractaspididae, Elapidae, Viperidae and Colubridae are the four families of Colubroidea. The first three of these, as well as the Colubridae subfamily of sea snakes, Hydrophidae, comprise all the venomous snakes of medical importance. Furthermore, of the four families, the one with the highest number of species (approximately 1,700) is Colubridae (Lawson et al. 2005; Panfoli et al. 2010). Snakes in this family used to be considered non-dangerous to humans, it

3 has been ascertained that more than 40 species can in fact envenom humans and even cause death (Ismail & Memish 2003; LIMPUS 2013; Vidal 2002).

There are about 351 snake species that make up the Elapidae family and they are found in all continents apart from Europe and Antarctica. The Elapinae (e.g. coral snakes, cobras, mambas and kraits) and Hydrophiinae (e.g. sea snakes) are the two subfamilies of venomous Elapidae. All the Elapidae have fangs at the front of the mouth and the majority are medically important because of the potent neurotoxic venom they produce to immobilise their prey. There are about 311 species of Viperidae that can be found throughout North and South America, Africa, Europe and Asia [examples of medically importance snakes in Sub-Sharan Africa in figure 1.2 and 1.3]. The Viperinae (pit-less vipers such as Bitis, Cerastes, Echis and Vipera genera) and Crotalinae (pit-vipers such as Agkistrodon, Bothrops and Crotalus genera) are the two major subfamilies of Viperidae. The Viperidae possess hinged fangs at the front of the mouth and, like the Elapinae, are associated with considerable human mortality owing to their potent haemotoxic effects of their venom (O'Shea 2008).

Figure 1.2: Examples of Viperidae snakes of medically importance snakes in Sub-Saharan

Africa (WHO, 2019)

Figure 1.3: Examples of Elapidae snakes of medically importance snakes in Sub-Saharan Africa (WHO, 2019).

1.3 The development of envenoming mechanisms

Elapidae, Viperidae and Atractaspididae produce and store venom in a large gland situated behind the orbit and wrapped in a fibrous sheath covered by muscles. Apart from these shared characteristics, however, the venom glands of these three families differ structurally (Jackson 2003). During biting, the venom is delivered to the tubular fangs via the venom ducts as a result of the compression to the venom gland applied by a compressor glandulae muscle presented by Elapidae, Viperidae and Atractaspididae. It is possible to view the occurrence of this muscle as a synapomorphy of true venomous snakes. However, this muscle is supplied with blood and innervated in each of the three families with a different external adductor muscle attached to the temporal skull plate (Jackson 2003, 2007).

1.4 Snake venom

Snake venom consists of different toxins designed to rapidly kill and immobilise prey or for defence against aggressors (Casewell et al. 2013). These toxins typically have more than one action. Furthermore, venom composition varies not only between venomous snakes of the same species but also within the same snake. Various enzymatic and non-enzymatic proteins account for the greatest proportions of snake venom toxins. Many different enzymatic toxins are present in just one sample of snake venom, but they are categorised into a handful of protein groups (McCue 2005). Apart from the enzymes L-amino acid oxidase, phospholipases

A2 and phosphodiesterase, which are present in most snake venoms (Kang et al. 2011), the other enzymes are associated exclusively with specific snake taxonomic groups. For instance, the proteolytic enzymes endopeptidases, arginine ester hydrolases, thrombin-like enzymes, kininogenases and pro-coagulant enzymes are typically present in the venom of Viperidae and low proportion in the venom of Elapidae (< 5%) (Lomonte & Calvete 2017).

The following section demonstrates that snake venoms contain a complex array of species- specific enzymatic and non-enzymatic proteins that bind numerous different physiological targets to cause a variety of pathologies, including neurotoxicity, disruption of the cardiovascular system/haemostasis and specific effects upon different vital organs, including heart, kidneys and muscles.

1.4.1 Snake venom metalloproteinases (SVMPs); The ability of snakes to adapt to various environments depends significantly on snake venom metalloproteinases (SVMPs) that consist of zinc-based enzymes of variable molecular mass. SVMPs represent the chief component in the venom of the majority of vipers (Calvete et al.

2009; Gutiérrez & Rucavado 2000; Sousa et al. 2013) and are capable of disruptive interaction with various proteins regulating haemostasis or tissues involved in key physiological activities in both prey and aggressors (Bernardoni et al. 2014; Moura-da-Silva, Butera & Tanjoni 2007). SVMPs are a multi-isoform family of great complexity owing to the considerable variability exhibited by their domain structure, tertiary confirmation and function. SVMPs can be categorised into four groups (Figure 1.4), namely, P-I, P-II, P-III and P-IV (Fox & Serrano 2008). With molecular masses of 60-100 kDa, the PI class SVMPs comprising solely immature propeptide and metalloproteinase domains and have the most basic structure. Proteolytic processing of the propeptide occurs in the enzymes that have achieved maturity. The highly conserved metalloproteinase domain with a canonical zinc- binding motif is present in every SVMP (Herrera et al. 2015). Meanwhile, PII class SVMPs display a canonical disintegrin domain occurring in the immature protein, which is generally subjected to post-translational and proteolytic processing by the proteinase domain. It must be highlighted that, given their isolation from crude venom, apparent stability is exhibited by the proteolytic products, the proteinase domain and the disintegrin domain (Fox & Serrano 2008). PII SVMPs are divided into five subtypes: the PIIa class with proteolytic cleavage from the metalloproteinase domains of the disintegrin domain, which is typically distinguished by the occurrence of an RGD motif, resulting in a ‘free’ disintegrin; the PIIb class with proteolytic processing of the disintegrin domain, which nevertheless stays integral to the enzyme structure; the PIIc class, which is a dimeric version of the PIIb class; and the PIId class and the PIIe class, which represent precursor enzyme forms yielding disintegrins with a content of homodimeric and heterodimeric RGD. The group P-III SVMPs comprise a structure consisting of sequential proteinase, disintegrin-like elements and cysteine-rich domains. The 68 kDa haemorrhagic toxin-a (HT), in the venom of C. atrox, is one example of this class of SVMPs (Markland & Swenson 2013). SVMPs primarily cause bleeding by damaging the integrity of capillaries and interfering with the blood coagulation system, which leads to coagulation plasma factors being consumed (Moura-da-Silva, Butera & Tanjoni 2007). Various targets are incorporated in the action mechanisms of different SVMPs, including coagulation Factor X activation (Siigur et al. 2001), Factor II activation (Modesto et al. 2005), fibrino(gen)olytic activity, as well as capillary binding and disruption (Baldo et al. 2010; Escalante et al. 2011). The venom pool of one species may contain SVMPs in interaction with specific haemostatic targets, causing disruption at the level of the entire haemostatic system and resulting in prey overpowering generally by shock (Kamiguti et al. 1994).

Figure 1.4: Structure and classification of SVMP classes (Fox & Serrano 2008). P=signal peptide, Pro= prodomain and S= spacer domain.

1.4.2 Snake venom phospholipase A2 (PLA2); An esterolytic enzyme occurring in multi-molecular forms and present both at intracellular and extracellular level, phospholipase A2 (PLA2) undertakes the hydrolysis of fatty acids at the Sn-2 position of glycerophospholipids. It also has action on a range of natural substrates, including phosphotidyl choline, phosphotidyl ethanolamine, phosphotidyl serine, phosphotidyl inositol, plasmogen and platelet aggregation factor. Secretions produced by the pancreas of mammals and snake venoms are the two most abundant sources of PLA2

(Kini 2005a). Furthermore, unlike the majority of proteins, the PLA2 enzyme is typically of small size and displays stability, most likely because of the many (6-7) intramolecular disulphide bridges that are present. Moreover, PLA2 can survive at extremes of temperature and pH. The molecular weight of PLA2 comprises 119-143 amino acid residues and usually varies between 12 and 16 kDa (Scott & Sigler 1994). PLA2 has come to be considered an extensive superfamily of different enzymes that are crucially involved in a range of biological activities. For example, in mammals, PLA2 is involved in the maintenance of cellular phospholipid pool, membrane repair, fertilisation, proliferation, inflammation and membrane homeostasis (Kini 1997). By contrast, PLA2 found in snake venom underpins neurotoxic, myotoxic, cardiotoxic and cytotoxic effects, suppresses or activates platelet

9 aggregation, and is involved in haemolysis, anticoagulation, convulsion, hypotension, and oedema formation (Gutiérrez & Ownby 2003; Kini & Evans 1989; Petan et al. 2005). According to the sequence of amino acids, 3D structure and disulphide bond patterns, there are two types of snake venom PLA2s, namely, group I and group II PLA2s. Present in the pancreas of mammals and the venom of Elapidae and Colubridae (Harris & Scott-Davey

2013), group I PLA2s usually display a length of 115-120 amino acid residues with seven disulphide bridges. PLA2s in the venom of Elapidae and in mammalian pancreas are respectively distinguished by the so-called elapid loop, which links the catalytic α-helix and the β-wing, and the pancreatic loop, which is an extra extension of five amino acids.

Meanwhile, group II PLA2s are found in the venom of Viperidae and generally have a length of 120-125 amino acid residues with seven disulphide bridges. Unlike group I, this group has an extra C-terminal extension instead of the elapid or pancreatic loop. According to the

th amino acid residue at the 49 position, several subtypes of group II PLA2s are recognised.

D49 enzymes are group II PLA2s containing an aspartic acid residue. As noted by Scott et al.

(1990), the majority of group II PLA2s conserve aspartic acid as it is essential for catalysis. Meanwhile, K49 (Maraganore et al. 1984), S49 (Polgar et al. 1996), N49 (Inn-Ho et al. 2004) and R49 (Chijiwa et al. 2006) enzymes are those respectively characterised by the replacement of the amino acid residue at the 49th position with lysine, serine, asparagine or arginine. Hydrolytic activity is poor or absent in enzymes with replacement of the aspartic acid residue, as this hinders cofactor Ca2+ from attaching to the Ca2+ binding loop (Maraganore & Heinrikson 1986). The envenomation capability of Elapidae is crucially dependent on neurotoxic PLA2s because they paralyse and therefore kill the victim.

1.4.3 Hyaluronidase: An enzyme that breaks down hyaluronan, hyaluronidase represents an endoglycosidase believed to be a ubiquitous protein in snake venoms known as the “spreading factor”. Consisting of groups of glycosidases, hyaluronidase has recently attracted a great deal of interest owing to its hyaluronan (HA) substrate. Tu and Hendon (1983) reported that the local haemorrhagic effect of a toxin from the venom of Trimeresurus flaoviridis was supported by hyaluronidase, reflecting its spreading property. In the process of enzyme-facilitated spreading that occurs during snake envenomation, a crucial event is believed to be the breakdown of hyaluronan in the extracellular matrix of local tissues. Several studies have described how hyaluronan breaks down in vitro (Kudo & Tu 2001; Pukrittayakamee et al. 1988), while the degeneration of extracellular matrix in tissue samples from humans and other organisms has been discussed by Girish et al. (2004).

This property is considered to aid other deadly toxins to spread rapidly. Therefore, ample attention has been paid to the ability of hyaluronidase to spread venom haemorrhagic toxins and this enzyme is thought to play an essential role in the activation of local tissue deterioration due to extracellular matrix breakdown. Hyaluronidase displays synergistic interaction with other venom toxins and this interaction has been identified as the source of its toxicity. This enzyme has been isolated and characterised from the venom of various snake species, including Agkistrodon acutus (Xu et al. 1982), Vipera russelii (Pukrittayakamee et al. 1988) and Agkistrodon contortrix contortrix (Kudo & Tu 2001). Furthermore, hyaluronidase isoenzymes were found by Girish et al. (2004) to be present in the venom of Naja naja (Girish et al. 2004). Whilst the amino acid sequences of the most pathogenic venom proteins show considerable species-specific variation, sequences encoding snake venom hyaluronidases are very conserved (Harrison et al. 2007).

1.4.4 Snake Venom Serine Proteases (SVSPs) A non-homogeneous class of single-chain proteins (Evans, 1984), snake venom serine proteases have molecular masses in the range of (15-67) kDa. A number of additional enzymes represent multi-subunit proteins (Fry 1999; Zaganelli et al. 1996). Based on their structure, the SVSPs are considered to be either serine proteases or metalloproteases. Apart from Hydrophidae, the venoms of all snakes contain proteases. Proteolytic enzymes are thought to be particularly abundant in the venom of Viperidae and proteases are the source of most toxic effects of the venom of pit-vipers such as rattlesnakes, water moccasins and puff adders. The proteolytic enzymes are in fact hydrolases that have a digestive action since they are responsible for the degradation of proteins. Endopeptidases, peptidases, arginine ester hydrolases, kininogenases, procoagulants and anticoagulants are among the major proteolytic enzymes. Pharmacological effects have been attributed to some of these. For instance, local capillary damage and tissue necrosis are triggered by proteinase and arginine ester hydrolases (Gutiérrez & Rucavado 2000; Gutiérrez et al. 2005). Furthermore, coagulant and haemorrhagic effects have also been exhibited by proteases (Lu, Clemetson & Clemetson 2005; Markland 1998). The pain and acute low blood pressure associated with the discharge of vasoactive peptides are triggered by enzymes that release kinin (kininogenase) (Felicori et al. 2003; Matsui, Fujimura & Titani 2000; White 2005). Procoagulant and anticoagulant mechanisms are employed by several venom proteases to break down fibrinogen and influence blood coagulation (Andrews, Gardiner & Berndt 2004; Markland 1998). Plasma coagulation is triggered by the proteases present in the venom of

Trimeresurus flavovirides, Bitis gabanica, Cerastes vipera, Trimeresurus stejnegeri and Agkistrodon caliginosus (Hiremath et al. 2014). Meanwhile, plasma coagulation is extended by proteases present in the venom of Naja nigricollis, Bothrops castelnaudi, Echis carinatus and Vipera lebetina (Babaie, Salmanizadeh & Zolfagharian 2013).

1.4.5 L-amino acid oxidases (LAAO):

The flavoenzymes known as L-amino acid oxidases are present not only in the venom of Viperidae, Crotalidae and Elapidae, but also in organisms such as bacteria, fungi, algae, fish and snails (Costa et al. 2014). The concentration in which LAAOs occur in snake venom differs from one species to another and contributes to the level of venom toxicity. LAAOs manifest catalytic specificity for long chain hydrophobic and aromatic amino acids and can function at varying pH and temperature. Considerable variability is also exhibited by their structures, molecular masses and isoelectric points. They can alter platelet function, leading to local effects on plasma clotting abnormalities and other effects. Furthermore, as reported by Izidoro et al. (2014), LAAOs can cause different cell lines to die as well as exhibiting effects against microbes and parasites. Ande et al. (2008) even suggested that LAAOs may protect against natural, parasitic and bacterial agents.

1.4.6 C-type lectins (CTL): Of the different haemorrhagic elements in snake venom, C-type lectin-like proteins are particularly significant. These proteins derive from the folding in classic C-type lectins (e.g. mannose-binding proteins and selectins), which represent a form of carbohydrate-binding proteins (Chou 1996). The C-type refers to the requirement of calcium ions for binding to the carbohydrate substrate. Various roles are fulfilled by proteins incorporating C-type lectin domains, such as adhesion between cells, anti-pathogenic immune response and cell death. Calcium dependence is displayed only by some binding interactions between C-type lectins and protein ligands, including the C-type lectins of coagulation factors IX/X, Von Willebrand factor (VWF) binding proteins and natural killer cell receptors. According to the manner in which the different protein domains are ordered in every protein, the C-type lectins were initially divided into seven subgroups (I-VII) before seven other groups (VIII-XIV) were added in 2002. Three additional subgroups (XV-XVII) were introduced more recently (Drickamer 1999; Drickamer & Taylor 2002; Zelensky & Gready 2005).

Interaction occurs between several components of venom, especially the venom of Elapidae and Viperidae, and proteins of the coagulation cascade and fibrinolytic pathway. As previously mentioned, coagulation can either be promoted or inhibited by toxins in snake venom. Indeed, multiple such inhibitors or promoters or coagulation are present in the venom of numerous snake species. The venom proteins that causes coagulation are distinguished as pro-coagulant factors, anticoagulant factors and fibrinolysis (Kini 2005b).

1.4.6.1 Factor V activator:

A multifunctional glycoprotein that is essentially involved in both promotion and suppression of coagulation is factor V activator. Factor V is activated by thrombin through cleavage at Arg709, Arg1018 and Arg1545, resulting in the formation of factor Va, which is a heterodimer comprising a 105 kDa heavy chain and 72 or 74 kDa light chain doublet. In the activation of prothrombin catalysed by factor Xa, factor Va serves as cofactor and promotes a thousand- fold increase in thrombin production. The venoms of Bothrops atrox, Doboia russelli, Vipera lebetina, Vipera ursine, Naja naja oxiana and Naja nigricollis nigricollis have all been reported to contain multiple factor V activators (Thorelli 1999).

1.4.6.2 Factor X activator:

The venoms of both Viperidae and Elapidae contain factor X activators, which are metalloproteinases or serine proteases (Tans & Rosing 2001). Ample research has been dedicated to human blood coagulation factor X (RVV-X), powerful activators of which have been found in the venom of Russell’s viper (Markland 1998). The venom of Bothrops atrox (Markland 1998). and that of a number of other species (e.g. Ophiophagus Hannah and Bungarus fasciatus) (Lee et al. 1995; Zhang, Xiong & Bon 1995)has also revealed factor X activators. RVV-X has been classified as a metalloproteinase, unlike the factor X activators from the venom of the Elapidae Ophiophagus hannah and Bungarus faciatus, which are serine proteinases (Tans & Rosing 2002).

1.4.6.3 Prothrombin activators:

Prothrombin or factor II is classified as a single-chain 72 kDa glycoprotein (Kini 2005b). Prothrombin activators are present in the venom of numerous snake species, transforming prothrombin into either meizothrombin or thrombin (Sachetto & Mackman 2019). There are four types of prothrombin activators, depending on their morphology, functionality and cofactor specifications. Group A prothrombin activators represent metalloproteinases that drive prothrombin activation in the absence of cofactors, like phospholipids (PLs) or cofactor

Va. Group B prothrombin activators are dependent upon Ca2+ and comprise two, non- covalently linked subunits, namely, a metalloproteinase and a disulphide-linked dimmer similar to C-type lectin. The Group C prothrombin activators are serine proteases in the venom of Australasian Elapidae, whose bioactivity is dependent upon Ca2+, PLs or factor Va. Thus, Factor VII is activated by oscutarin from the venom of Oxyuranus scutellatus as well. Group D prothrombin activators are also serine proteases that exhibit a strong dependence on Ca2+, PL with negative charge and factor Va (Kini 2005b).

1.4.6.4 Thrombin-like enzymes:

A range of activities are attributed to thrombin and some snake venom enzymes are categorised as ‘thrombin-like’ because they are capable of clotting fibrinogen (Hutton & Warrell 1993; Marsh 1994; Ouyang, Teng & Huang 1992). Thus, venombin A (from Calloselasma rhodostoma), venombin B (from Agkistrodon contortrix contortrix) and venombin AB (from Gaboon viper, Bitis gabonica) are the three types of thrombin-like enzymes (Markland 1998). Additionally, regarding their capability to convert fibrinogen, these enzymes are species-specific to some extent. Meanwhile, although serine protease inhibitors suppress thrombin-like enzymes, thrombin inhibitors (e.g. anti-thrombin III and hirudin) have no effect on them. Hence, thrombin-like enzymes present clinical potential as defibrinogenating agents because the fibrin they produce can be eliminated from circulation without difficulty (Kini & Koh 2016).

1.5 Snakebite envenoming pathology

Venom injection in the skin or muscle of an animal or human is called snake envenomation and it is associated with three pathologies. Two of them are ‘hypotensive’ and ‘paralytic’ strategies and are both designed to restrict the mobility of the prey/victim. In the third strategy, snake envenomation triggers internal tissue breakdown before the prey is even seized. Snake envenomation pathophysiology consists of a sequence of complex events mediated by the integrated effect of toxic and non-toxic elements (Aird 2002; Warrell 1996). It acts both at local and systemic levels (Figure 1.5).

1.5.1 Local manifestations:

A bite by a venomous snake is usually indicated by two puncture wounds, differing from the bite of a non-venomous snake, which takes the form of several, small puncture wounds with an arc-like distribution. The bite is typically instantly followed by burning, bursting or throbbing pain that disseminates upwards from the bite site. Soon after, draining lymph

14 nodes begin to feel painful. However, the amount of pain and progression of pathology is dictated by different drivers, including the snake species, and amount of venom injected and the anatomical site of envenoming. For instance, no significant pain is associated with the bite of kraits and sea snakes, while the local response caused by the bite of vipers is of greater intensity compared to the bite of other snakes, with swelling occurring within 15 minutes and reaching extreme proportion on 2-3 days, lasting for 1-3 weeks (Mehta & Sashindran 2002). The diffusion of the swelling from the bite site happens fast (minutes) and may affect not only the entire limb but the adjacent trunk as well. The development of regional lymphadenopathy is possible. Furthermore, ischaemia occurs if the envenoming site is a tight fascial segment, such as the pulp space of the digit or the anterior tibial segment. Envenomation is unlikely to have occurred if a bite by a viper does not cause swelling within two hours. Moreover, a couple of days after a viper bite, the tissue may appear bruised, develop blisters and start to necrotise. The bites of Asian pit vipers and certain rattlesnakes also cause severe necrosis. On the other hand, swelling is localised and tender and blisters form as a result of bites by Asian cobras, whereas no local response is generally triggered by krait bites. Venom ophthalmia may occur in the case of individuals that have been hit in the face by the venom of spitting elapids. Secondary infection develops as a result of the bacterial flora present in the snakes’ mouth (Warrell 1996).

1.5.2 Systemic manifestations:

The pathophysiological alterations triggered by the venom of specific snake species determine the systemic features of snakebite envenoming. The first signs of elapid envenoming emerge within five minutes or with a delay of up to ten hours (Mehta & Sashindran 2002), while the signs of viper envenoming occur usually within 20 minutes of the bite. By contrast, the myotoxic symptoms of sea snake venom, and some of the Australasian elapids, occur within two hours of the bite (Paul, Chawla & Jatana 1993). The type and concentration of toxins, their bioactivity and how fast they spread all determine the severity of systemic effects. Three classes of snakes were initially distinguished according to the major venom components, namely, neurotoxic (e.g. cobras and kraits), haemorrhagic (e.g. vipers) and myotoxic (e.g. sea snakes). This classification has been recently dismissed because a combination of manifestations can be generated by the venom of different species (Moura-da-Silva, Butera & Tanjoni 2007; Paul, Chawla & Jatana 1993).

Figure 1.5: Overview of the clinical symptoms presented by victims of snakebite envenoming (Gold & Barish 1992; Wangoda, Watmon & Kisige 2004; Warrell 2010b).

1.5.2.1 Neurotoxicity.

The two main groups of neurotoxins are distinguished by their interaction with the neuromuscular junction. The β-neurotoxins or presynaptic neurotoxins cause anatomic disruption of the the presynaptic site, while the α-neurotoxins or postsynaptic neurotoxins bind to molecules releases from the postsynaptic site (Ranawaka, Lalloo & de Silva 2013; Rossetto et al. 2006).

Presynaptic neurotoxicity: Two stages are usually involved in the presynaptic action. In the first stage, the toxin binds to receptors in the neuromuscular junction presynaptic site, and in the second stage, the neurotransmitter release is inhibited by physical destruction of the presynaptic membrane. In recent times, ample research has been dedicated to presynaptic neurotoxins, all of which are structurally PLA2s (Montecucco & Rossetto 2000; Schiavo, Matteoli & Montecucco 2000; Westerlund et al. 1992). Meanwhile, venoms contain monomeric as well as multimeric β-neurotoxins. The single-chain toxins that have been extensively investigated include Notexin from the venom of Notechis scutatus, Caudoxin from Bitis caudalis and Ammodytoxin from Vipera ammodytes ammodytes, while the multimeric neurotoxins that have been the focus of numerous studies are Taipoxin, Textilotoxin from Pseudonaja textiles, Mojave toxin, Crotoxin and β-bungarotoxin (Wilson et al. 1996). Furthermore, as explained by Hodgson and Wickramaratan (2002), non-

16 depolarising inhibition of neuromuscular transmission is triggered by postsynaptic neurotoxins through their specific binding to the nicotinic acetylcholine receptor (nAchR) at the motor end plate.

Postsynaptic neurotoxicity: display competitive suppression of acetylcholine receptors, thus targeting the neuromuscular junction at extracellular level; however, administration of antivenom counteracts this effect. Group II PLA2s are predominant in the venom of Viperidae and some of them, like crotoxin and notexin, exhibit myotoxicity and are capable of triggering myonecrosis or system myotoxicity (Gopalakrishnakone et al. 1984; Mebs & Ownby 1990).

Pro-inflammatory effects are manifested by other group II PLA2s; for instance, penetration of inflammatory cells is facilitated by PLA2 from the venom of Naja naja atra (Zhang & Gopalakrishnakone 1999), whereas macrophage phagocytosis is promoted by Asp49 and

Lys49 PLA2s contained in the venom of Bothrops asper (Zuliani et al. 2005).

1.5.2.2 Haemotoxicity

Snakebite envenomation, especially by Viperidae, often results in haemotoxicity. Haemor- rhage at local or systemic level taking the form of either external bleeding (e.g. from gums) or internal bleeding (e.g. intracranially) is an effect of venom-induced disruption of haemostasis (Slagboom et al. 2017). Another pervasive and complicated effect of envenomation by Viperidae and some Elapidae and Colubridae (Mackessy & Modahl 2019) is snakebite-induced coagulopathy, otherwise called venom-induced consumption coagulopathy (VICC). By increasing susceptibility to heavy bleeding, VICC is associated with a high mortality risk. The common assumption is that administration of antivenom is the only solution that can help counteract the triggered pathophysiological effects of high complexity (Isbister et al. 2013; Maduwage & Isbister 2014). As explained by Isbister et al. (2009), the venom of some snake species contains procoagulant toxins that digest the clotting factors, causing the blood clotting cascade to activate at excessively high levels and thus leading to the onset of VICC. The procoagulant toxins are so named because they promote the quick development of clots in vitro, despite the fact that they cause serious factor consumption and heighten haemorrhage probability in vivo. Known under various terms at different times, such as procoagulant coagulopathy, defibrination syndrome, and disseminated intravascular coagulation (DIC), VICC is understood to represent post-envenomation fibrinogen depletion (Maduwage & Isbister 2014; Warrell 2010a). TLEs or fibrinogenases cleave the fibrinogen α-chain or β-chain, thus leading to the occurrence of fibrinogen consumption without fibrin development owing to fibrinopeptide A or B (Maduwage & Isbister 2014). The venom of certain species of snakes,

17 such as the Echis spp., Elapidae found in Australia, and Russell’s viper, contains a number of factor deficiencies, like activators of factor X or of prothrombin, and they exert their effect higher up the clotting cascade (Slagboom et al. 2017)

1.5.2.3 Cardiotoxicity

The cardiotoxins in snake venom are very simple proteins of 5.5-7 kDa molecular mass and four disulphide cross-linkages (Munawar et al. 2014). They cause cardiac, skeletal and smooth muscles to contract and lose excitability by depolarising them. Furthermore, cardiotoxins participate in the fusion, haemolysis, and cytotoxicity of membranes, the apoptosis of particular tumour cells and suppression of the activity of protein kinase C (Gasanov, Dagda & Rael 2014). Barrington et al., (1986) reported that the cardiotoxicity of a basic PLA2 isolated from the venom of N. nigricollis manifested in the form of a rise in the concentration of intracellular calcium ions. Moreover, other studies described a number of polypeptides and PLA2 enzymes that also exhibit cardiotoxicity (Huang & Mackessy 2004; Torres et al. 2003; Xiao et al. 2017).

1.5.2.3.1 Haemolytic effects

Venom factors acting directly or via a complex mechanism comprising multiple steps can disrupt erythrocytes, a process called haemolysis. Snake venoms causing haemolysis can be grouped by their mechanism of action. Thus, all venom PLA2s have been reported capable of inducing indirect haemolysis (Williams et al. 2018), by the direct lytic venoms trigger the hydrolysis of phospholipids in various domains of the red blood cells to cause haemolysis. In contrast, the venom of Agkistrodon halys blomhoffi, Trimeresurus flavoviridis and Naja naja contain PLA2s capable of direct haemolysis (Díaz et al. 2001; Wilson et al. 1997; Yamamoto et al. 2001). The mechanism of action of these enzymes likely involves membrane disruption with effect on the external as well as internal phospholipid bilayer leaflets

1.5.2.3.2 Hypotensive effects

Various strategies are used by many snake venoms to rapidly lower blood pressure, resulting in circulatory shock that immobilises the prey and eventually causes it to die. The release of autocoids with pharmacological activity, such as histamine, 5-hydroxy tryptamine and leukotrienes cause an abrupt decrease in blood pressure (Andrião-Escarso et al. 2002; Joseph et al. 2011; Lumsden et al. 2004). Hypotensive peptides of 5-13 amino acids that are N- terminally blocked with pyroglutamic acid are present in the venom of numerous pit vipers. Since they can augment the hypotensive action of bradykinin, these peptides are usually

18 called bradykinin-potentiating peptides (BPPs) (Péterfi et al. 2019). Huang and Lee (1984) and Andriao-Escarso et al. (2002) have attributed the low blood pressure to the acidic PLA2s in the venom of Bothrops jararacussu (BthA-I-PLA2) and Vipera russelii.

1.5.2.3.3 Convulsive effects

Paralysis of the respiratory system and additional pre-agonal effects trigger asphyxia that in turn drives convulsive symptoms that frequently precedes death after envenoming by some cobras. Because of the high potassium ion influx, the supplies of acetylcholine are exhausted by snake venom components. This symptom is also caused by the aforementioned prevention of neurotransmitter release following the actions of presynaptic neurotoxins (Silva, Hodgson & Isbister 2017). The venoms of N. naja (Bhat & Gowda 1989), Vipera russelii (Jayanthi & Gowda 1990; Kasturi & Gowda 1989) and E. carinatus (Kamiguti et al. 1994) have all been reported to trigger convulsions.

1.6 Clinical treatment of snakebite envenoming

1.6.1 First aid

Gaining immediate access to medical care is the most important step in managing snakebite envenoming (Warrell 2010b). Widely used measures such as application of a tourniquet or drawing out the venom by cleaning or cutting into the wound are not advised because they actually lack efficiency (Blaylock 2005). Another measure of doubtful efficiency is the Sutherland pressure immobilisation method in which compression bandage is applied to the bitten limb along a splinted support to slow down the spread of the venom. This method may be helpful in cases where venom spread relies on the lymphatic system. By contrast, experiments showed that venom transport was reduced by direct pressure pad application. However, the clinical efficiency of this approach is uncertain. What is more, the use of the methods outlined above by practitioners and first-aiders may do more harm than good due to a lack of proper education. For example, a tourniquet effect may be engendered by the excessively tight application of bandaging (Canale, Isbister & Currie 2009) and, apart from bites known to have been caused by species with neurotoxic venom (e.g. mambas), tourniquets should not be used as a first-aid measure. Furthermore, even when they are used, they should not be kept in place for more than an hour and a half, because they can generate ischaemic effects that aggravate tissue damage caused by cytotoxic envenomation, which occurs in 90% of bites in Africa (Blaylock 2005; Dreyer & Dreyer 2013). Therefore, tourniquets can lead to a long hospital stay and unfavourable outcomes.

1.6.2 Antivenom and syndromic management of snakebite:

Snakebite management is made difficult primarily by the inadequate availability of antivenom throughout the world and especially in rural areas. Nevertheless, because a range of clinical manifestations can be engendered by the different snake venom components, the management of these manifestations can be successful, regardless of the availability of antivenom. Indeed, in most cases where there is uncertainty about the species that caused the bite, symptom management is considered the best approach (Table 1.1).

Snakebite envenoming can only be effectively treated with antivenom, but the use of this medication is associated with significant risks and often it is the adverse reactions to antivenom rather than the actual envenomation that is the cause of patient death. Thus, managing snakebite envenomation is a difficult task, even for medical practitioners with ample experience, because most cases occur in rural areas without access to emergency transportation, and therefore the envenomation effects are advanced by the time the victims arrive at a medical centre. Another reason making snakebite management challenging is people’s preference for traditional rather than modern treatments and reliance on traditional healers. Furthermore, the first-aid measures taken frequently do more harm than good, as people and healthcare practitioners in rural areas are inadequately educated. However, Dreyer and Dreyer (2013) refuted this argument, noting that envenomation by snakes with neurotoxic venom can cause respiratory failure and hypoxic death if treated late, while lack of treatment of envenomation by snakes with hemotoxic venom can result in fatal coagulopathy.

The unavailability of antivenom and modern medical equipment aggravates the challenging task of snakebite management even more. Snakebite envenoming is particularly prevalent in rural regions of developing countries with an inadequate healthcare infrastructure and poor resources. Practitioners frequently have no antivenom at their disposal, so they are forced to come up with other methods of treating cases where envenomation has progressed significantly. Under such circumstances, the implementation of a systematic approach to the management of snakebite clinical manifestations is essential and could be efficient even in the absence of necessary resources (Blaylock 2005; Kasturiratne et al. 2008).

Antivenom was first produced in 1894, when Mr Albert Calmette injected animals with small amounts of snake venom and then gradually with higher amounts, thus immunising them against venom and saving them. Currently, antivenom of animal origin is produced in 46 laboratories at global level, although recent alterations to this number cannot be ruled out.

Most laboratories are present in Asia, the Americas and Europe, with a couple in Africa and only one in Australia (Gutiérrez 2019). Regarding production scale and antivenom properties, the existing laboratories differ considerably. Three types of producers can be distinguished, namely, national, regional and global producers, which respectively manufacture antivenom for use in their own countries, in their own countries and adjacent ones, and in countries throughout the world (Gutiérrez 2012a). Another classification system of the laboratories is public laboratories, which number 31, and private laboratories, which number 15. A greater antivenom amount is generally manufactured by private than by public laboratories. Nevertheless, although some public laboratories produce just enough to satisfy the demand in their particular countries, a few manufacture substantial amounts as well.

The available quality and amount of antivenom are less than ideal throughout the world. According to statistics, less than 5% to 20% of the antivenom required for treatment of snakebite envenomation cases is available in certain countries with low and middle income (Brown 2012; Chippaux & Habib 2015). To put it differently, the amount of antivenom available in Africa is only enough to treat about 80,000 patients (Brown 2012), whereas envenomation on this continent has an incidence of around 435,000 per year (Chippaux & Habib 2015; Gutiérrez et al. 2017a). The real proportion of successful treatments is even lower because certain antivenoms vary in their efficacy or are not marketed adequately. Similarly, the amount of antivenom available worldwide is sufficient to treat approximately 600,000 victims, but it is doubtful how effective the existing antivenoms are. Thus, it means that less than a third of the about 1.8 million people bitten by venomous snakes every year receive antivenom treatment, and over 100,000 victims would be saved if antivenom availability were higher (Brown 2012; Gutiérrez et al. 2017a). The situation is further compounded by manufacture suspension by some producers and suboptimal regulation of novel antivenom quality, reducing the availability of antivenom of proven reliability. Both state and non-state bodies are partially responsible for this situation because they have failed to provide the funds needed to ensure proper monitoring and promote a healthy and competitive antivenom market.

The development of an efficient and sustainable solution to the problem is significantly hindered by the fact that antivenom is highly expensive for the majority of envenomation victims. Indeed, disadvantaged individuals living in deprived areas will always struggle to afford antivenom treatment. In India, efficient antivenom treatment costs R1000-5000 on average (Vaiyapuri et al. 2013), while in Africa, antivenom treatment must cost less than £3 to be affordable (Theakston & Warrell 2000). According to Brown (2012), in sub-Saharan

Africa, a single antivenom vial costs $18-135, but the total cost can reach $60-640 if multiple vials are necessary for successful treatment. This effectively makes antivenom treatment inaccessible to the majority of victims. The situation is worsened by the fact that not all antivenoms are equally effective and of good general quality. Furthermore, some people may be attracted by the low cost of one antivenom vial, although the total cost can be astronomical if more than 20 vials are needed (Habib & Brown 2018).

Antivenom efficiency exhibits a significant degree of variability and is mainly determined by the existing levels of particular antibodies targeting a specific venom. One or two ampoules must be administered for the most effective antivenoms to successfully counteract envenomation (Abubakar et al. 2010b), whereas more than 20 ampoules must be administered for antivenoms with poor efficiency but greater affordability to be effective (Das, Sankar & Dev 2015). Another issue is the commercialisation of fake or inefficient antivenoms in areas with snake species whose venom is not targeted by the supplied antivenom (Warrell 2008). By robbing health systems of their already scarce financial resources and increasing suspicion about what is otherwise reliable medical treatment, such fake and inefficient antivenoms can have widespread negative consequences. Given the above considerations, the formulation of envenomation treatments of extensive efficiency is considerably thwarted by the lack of homogeneity in venom components.

All venomous snake species have a complex composition of venom toxins. This complexity is increased in cases of attempts to counteract the venom of more than one species, especially as toxins with multiple functions do not have the same immunogenicity. Thus, antivenom is usually effective only against species with venom employed as immunogen and occasionally against species of near affiliation whose venom contains enough similar toxins to activate the produced antibodies (Casewell et al. 2010; Segura et al. 2010; Williams et al. 2011a). It is important to comprehend how the venom of species of medical significance is constituted as venom composition variability is interspecific, intraspecific and ontogenetic (Casewell et al. 2014; Chippaux, Williams & White 1991; Gibbs et al. 2013). Such comprehension is helpful in estimating how effective an antivenom is likely to be against the venom of multiple species and, implicitly, the geographical range over which it can be used. Therefore, antivenom paraspecificity has been explored based on a combination of examinations of venom toxicity and pathology and venom proteomics, antivenomics and/or immunological analyses (Calvete et al. 2009; Madrigal et al. 2012; Pla et al. 2013).

Besides the quantity in which antivenom is manufactured and the area over which it is made

22 available, discrepancies exist with regard to certain technical features of antivenom development and manufacture as well. For immunisation purposes, horses are employed by the majority of producers, but sheep (WHO 2010b) and donkeys (Gutiérrez et al. 2007) are respectively used by two producers. Based on the immunogen used in the manufacturing process, there are two types of antivenom, namely, monovalent antivenom, which is obtained through animal immunisation with the venom from just one specie, and polyvalent antivenom, which is obtained through immunisation with the venom of multiple species of medical significance from a certain geographical area. The latter promotes the generation of antibodies targeting multiple toxins in the venom of various species and prevents problems associated with administration of the incorrect antivenom owing to the absence of diagnostic protocols, thus overcoming the restricted paraspecific cross-reactivity of monovalent antivenoms (Abubakar et al. 2010a; O'leary & Isbister 2009). On the downside, polyvalent antivenoms present a higher risk of side-effects and may be more expensive and thus less accessible to poor people, as they must be administered in greater dosage to be efficient (Hoogenboom 2005; O'leary & Isbister 2009). F(ab’)2 fragments constitute an active substance found in most antivenoms and are obtained by subjecting hyperimmune plasma to pepsin digestion and salting out processes to fractionate it. Meanwhile, entire IgG molecules make up the composition of other antivenoms and are produced by precipitating non-immunoglobulin plasma proteins with caprylic acid; papain digestion of ovine plasma immunoglobulins is another method for manufacturing Fab antivenoms (WHO 2010b). Additional differences among antivenoms may be related to the employed excipients and preservatives as well as to the final form they take (e.g. liquid, freeze-dried), which raises issues regarding how the antivenoms are stored and transported (e.g. a cold chain system is necessary for antivenoms in liquid form).

Antivenom typically causes adverse reactions in over 10% of patients, and these reactions occur either immediately after administration or after a couple of days (Table 1.2). Although dosage is the main reason for the occurrence of antivenom reactions, there are some cases of sensitisation (IgE-mediated Type I hypersensitivity) to late serum sickness type reactions. Use of animal serum is what causes hypersensitivity and the risk of this is especially high among individuals who were previously exposed to animal serum. Several researchers recommended avoiding sensitivity tests prior to antivenom administration, because they lack accuracy and delay the treatment of individuals in critical condition (Dreyer & Dreyer 2013; Warrell 2010a).

Further research on polyvalent antivenoms is required to enhance the efficacy of these antivenoms by refining specificity and the ability to counteract venom effects, reducing costs and increasing safety compared to traditional antivenoms. Nevertheless, conversion of such research into concrete envenomation therapy requires better knowledge of the toxins targeted by the antivenoms. In particular, investigation of major pathogenic toxins with multiple functions from the venom of various species of medical significance is essential to develop, assess and improve novel envenomation treatments.

Table 1.1: syndromic management of snakebites in sub-Saharan Africa (Blaylock 2005; Dreyer & Dreyer 2013; WHO 2010a).

Syndrome Description Species bites Treatment Localised swelling Localised swelling is Saw-scaled or carpet Monospecific Echis accompanied by painful and spreads fast antivenom should non-blood clotting and is occasionally vipers of the Echis be used for bites accompanied by blister species sustained south of formation and necrosis, the Sahara and with blood that does Desert horned-vipers north of the not coagulate (based on (Cerastes cerastes). Equator, while 20WBCT detection) and polyspecific bleeding from gums, Certain types of puff antivenom should nose, and adders (Bitis arietans) be used in all other gastrointestinal/urino- African regions. genital tracts or bleeding at the bite site that does not stop. Progressive paralysis Local swelling is not Cobra and mamba Polyspecific due to neurotoxic significant but is species with neurotoxic antivenom should effect accompanied by venom be used or else paralysis that typically anticholinesterase progresses downwards. therapy may be a viable option as well. Breathing should be monitored closely, with intubation and support ventilation if/when required. Only moderate It is usually limited to Night adders of the Antivenom is not swelling half of the bitten limb, Causus species. required as with minor or lack of symptomatic systemic Burrowing asps of the therapy is enough. manifestations. Atractaspis species.

Certain dwarf, bush and desert vipers Local swelling that is An infrequent Boomslangs Monospecific moderate and lack occurrence, it involves (Dispholidus typus). antivenom and of blood coagulation minor local swelling supportive with blood that does Vine snakes of the treatment should not coagulate (based on Thelotornis species be used for 20WBCT detection) and (rare) boomslang and frequently systemic vine snake bites, bleeding that occurs respectively. spontaneously.

Local swelling of Local swelling is Dwarf adders, such as Antivenom is moderate to moderate to severe and Péringuey’s desert or unavailable, only significant degree is accompanied by sidewinding adder (Bitis symptomatic with neurotoxic neurotoxic effects. peringueyi). therapy can be effect Despite being a rare used. clinical occurrence, it is Desert mountain adders particularly associated (B. xeropaga) with the bite caused by the Berg adder (Bitis atropos).

Table 1.2: Antivenom reactions among individuals bitten by snakes (Morais & Massaldi 2009; Warrell 2010a).

Reaction (s) Clinical presentation

Early anaphylaxis A series of symptoms within 10-180 minutes after antivenom administration, including itching, especially on the scalp, urticaria, dry cough, fever, nausea, vomiting, abdominal colic, diarrhoea and tachycardia.

Anaphylaxis, including low blood pressure, bronchospasm and angio- oedema, that may prove fatal is also developed by some patients.

Antivenom reactions are more probably caused by complement activation by IgG aggregates or residual Fc fragments or else by direct stimulation of mast cells or basophils by antivenom protein.

Pyrogenic Developing 1-2 hours’ post-administration, these reactions manifest (endotoxin) as shaking chills (rigors), fever, vasodilatation, and hypotension. reactions Children may experience intense febrile convulsions.

Late reactions of the Developing 1-12 days’ post-administration, these reactions trigger serum sickness type fever, nausea, vomiting, diarrhoea, itching, recurrent urticaria, arthralgia, myalgia, lymphadenopathy, periarticular swellings, mononeuritis multiplex, proteinuria with immune complex nephritis, and, sometimes, encephalopathy.

1.7 Rationale:

The above literature review provides a detailed summary of the global distribution of venomous snakes, the bioactivity of the toxins within their venoms and the different pathologies they cause and culminates in a review of antivenom-treatment of snake envenoming. The latter identified that there exists an urgent need for research to develop new therapies with substantially improved:

• Antivenom efficacy against multiple snakes within any given region:

The efficacy of an antivenom is restricted to envenoming by the snake(s) whose venom(s) was used in production. Antivenoms should be produced with minimal immunising venoms but be capable of counteracting the maximum number of clinically relevant toxins. The efficacy of antivenoms manufactured by immunisation with multiple venom mixtures is affected by the significant within- and between-species variability in venom composition. Novel antivenoms can be introduced in a geographical area only after thorough assessment, such as through the mouse lethality test and other assays according to venom toxicology (Warrell et al. 2013), of their efficiency against the venom of the majority of clinically significant species in that area. For example, geographical intraspecific discrepancies in venom composition signify that antivenoms manufactured in India may have poorer efficiency against certain Nepalese species. This may be the reason why Indian polyvalent antivenoms have been poorly effective or ineffective against the venom of snake species in Sri Lanka (Shrestha et al. 2017).

• Antivenom efficiency against envenomation-induced necrosis: Envenomation- induced local tissue damage is primarily caused by hydrolases like metalloproteinases, PLA2s and potentially hyaluronidases. Cytotoxicity manifests as bite area pain and gradual swelling, followed by blistering and bruising, occasionally accompanied by hypovolaemic shock and other systemic effects (WHO 2010a). Local tissue damage frequently aggravates into necrosis, which, because antivenom is ineffective against local venom effects, necessitates debridement or amputation (Slagboom et al. 2017).

The severity of local tissue damage increases rapidly, a significant issue because the majority of rural snakebite victims take several hours to access health facilities (Habib & Warrell 2013; Harrison & Gutiérrez 2016) due to distance from homes to the nearest roads, lack of local ambulances, transport cost. Furthermore, efficient clinical management is rarely available in deprived areas. Toxin neutralisation by IgG and its fragments is another issue (Gutiérrez,

Theakston & Warrell 2006). Development of fast-acting, thermostable, affordable and polyspecific intervention to manage envenomation-induced tissue necrosis within field settings is also an important objective (Harrison & Gutiérrez 2016).

• Antivenom safety: Since they contain non-human immunoglobin, antivenoms can have side-effects and therefore proper production and quality control are essential in preventing inclusion of impurities, contaminants, immunoglobulin aggregates or deterioration products as well as excessively high levels of proteins, thus ensuring antivenom safety; additionally, negative reactions may also be induced by some preservatives in antivenoms (Chippaux et al. 2015). Early antivenom administration is vital for venom counteraction (Agarwal et al. 2005). The venom of Elapidae has poor antigenicity and fast absorption and therefore requires higher antivenom dosage. Irrespective of age, victims are administered an initial 100-200 ml equine antivenom dosage, triggering an immediate response, but also hypersensitivity reactions due to the equine serum. Higher dosage and intravenous injection increase antivenom response time (Mehta & Sashindran 2002). In up to 80% of cases, antivenom causes anaphylaxis, pyrogenic reactions, or serum sickness (Alirol et al. 2010; de Silva, Ryan & de Silva 2016), which is why health professionals in remote areas of India are reticent to manage envenomation. Furthermore, health professionals are mistrusted because of antivenom unaffordability and inefficiency, determining victims to prefer going to traditional healers, although the latter’s interventions are seldom safe and efficient (Harrison & Gutiérrez 2016). Side-effects can be attenuated by better compliance with good manufacturing practices.

• Antivenom affordability: Despite existing for over a century, immunotherapy has not advanced significantly due to funding unavailability, diminishing markets and lack of initiatives from public health entities. Snakebite envenomation is most efficiently treated with antivenom, but this is often unavailable to at-risk individuals in deprived rural areas of low- and middle-income countries (Williams et al. 2011b), even though the situation could be improved with only moderate investment. In Africa, antivenom scarcity has fostered the production of fake and inefficient products (Theakston & Warrell 2000; Theakston, Warrell & Griffiths 2003; Warrell 2008), which often escape detection by underfunded national regulatory agencies. Inexpensive but ineffective products are unfortunately preferred over costly but effective ones (Bregani, Maraffi & Van Tien 2011), resulting in severe antivenom scarcity and suboptimal treatment outcomes. A profitable black market has thus emerged, with prices for stolen antivenoms reaching US$3200 for one vial (Warrell 2008).

Improvement of envenomation treatment requires novel ‘omic' technologies and equal attention must be paid to making sure that antivenom products are safe, effective and affordable. For instance, a new treatment will be pointless if it is too costly for the rural inhabitants of low-income Asian and African countries who are most in need of it, despite having greater efficiency and safety than existing treatments. In the following part, an overview of the latest endeavours to enhance envenomation treatment with omic technologies is provided.

• Improvement of antivenom dose efficiency through venom-affinity chromatography: antivenom IgGs can be made fully venom specific and efficiency can be increased five-fold by purifying IgGs.

• Improvement of antivenom dose efficiency based on monoclonal antibodies: in theory, the therapeutic potential of all IgGs can be ensured by producing monoclonal antibodies with venom toxin specificity. The development of monoclonal antibodies capable of counteracting bleeding triggered by venom has been achieved.

• Improvement of antivenom dose efficiency based on recombinant immunoglobulins: recombinant antivenom is characterised by full IgG specificity to venom toxins and its production involves isolation of B cells from animals immunised to venom, followed by sub-cloning of genes with expression of IgGs of particular specificity into bacteria, and generation of recombinant IgG at a certain concentration.

• Development of antivenom with efficiency suitable for a particular area and snake species: so-called ‘venomics’ methods have been devised owing to notable innovations in proteomic technology, facilitating detection of venom protein components. Furthermore, ‘antivenomics' enables the development of mixtures for venom immunisation that comply with the clinical requirements in a particular area.

• Development of antivenom with toxin specificity and with efficiency in broad areas and against multiple snake species through transcriptome-based creation of artificial immunogens: an obvious and reasonable strategy for enhancing dosage efficiency and, implicitly, safety and affordability, is antivenom with toxin specificity. However, this requires differentiation between pathogenic and non-pathogenic venom toxins.

• Improvement of IgG purity by altering processes of antivenom production: this is a strategy that is not only cost-effective but can also eliminate certain contaminating viruses and enhance the IgG yield.

• Possible improvement of antivenom efficiency based on the singular structure of camelid IgG: standard light chains are absent from more than half of their immunoglobulin.

• Management of local pathological effects of venom based on peptide inhibitors of matrix metalloproteinases

• Improvement of antivenom production cost-effectiveness: this can be achieved by replacing horses with animals that are cheaper to keep; for instance, most antivenoms, including EchiTabG, are now manufactured based on ovine immunisation.

• Improvement of antivenom cost-effectiveness: various novel methods, such as antivenomics, could be used to make antivenom more cost-effective by implementing scale economies in its production.

Measurement of the degree to which novel and traditional treatments eliminate venom from the circulatory system provides an impartial evaluation of how effective those treatments are, as well as facilitating the approximation of the extent of circulating treatments at a particular moment following intervention commencement. Assays endorsed by the WHO and by both pharmaceutical companies and national authorities must be conducted for testing all treatments prior to their application to human envenomation cases. These assays have two goals, namely, assessment of how effective treatments are in counteracting the effects of the venoms they have been designed to target and assessment of how safe the treatments are. Examples of such assays are lethality assays and lethality elimination assays, which are conducted on experimental models of different animals. Furthermore, antivenom resolution of pathologies caused by venom (e.g. haemorrhage, coagulopathy, defibrinogenation, local necrosis) can also be evaluated through various in vitro and in vivo tests. However, research is presently focused on the creation of other testing options as in vivo tests are harmful to experimental animals. Moreover, the pharmacokinetics of envenomation and treatment have been addressed in a large number of animal-based investigations, but these are not relevant to human envenomation due to the preference for

31 intravenous injection of venom, whereas intradermal or intramuscular injection of venom is the norm in the case of human victims.

1.8 Project Aims:

The aim of this PhD project was therefore to develop new models enabling the research community to investigate mechanisms of envenoming to guide their strategies and test the efficacies of their candidate therapies.

1. ex vivo bovine eye model seeks (i) to measure the opacity of bovine corneas exposed to snake venom from spitting and non-spitting cobra, (ii) to assess damage to corneal tissue using sodium fluorescein imaging, and to (iii) determine the anatomical depth of damage to the cornea using histopathology, and (iv) to identification and characterisation of venom proteins penetrating the cornea and (v) seek for the ideal management strategies following the ocular envenomation.

2. ex vivo human skin model, to characterize the pathogenesis of dermonecrosis induced by the venom of Viperidae and Elapidae. In addition, the toxins responsible for this effect were identified, and the potential role of inflammatory mediators’ responses in tissue damage. Finally, the efficacy antivenoms constituted to neutralize venom-induced dermonecrosis.

3. To expand our knowledge of the systemic inflammatory and acute phase responses to African snake venoms. By assessing, in a mouse model, whether inflammatory and acute phase responses following snake envenomation is associated with snake venoms causing distinct pathologies.

4. A comparative serological and immunological analyses of ovine IgG post short and long immunisation protocol, to improve the immunogenicity, cross- reactivities and the neutralisation efficacy.

1.9 References:

Abubakar, I.S., Abubakar, S.B., Habib, A.G., Nasidi, A., Durfa, N., Yusuf, P.O., Larnyang, S., Garnvwa, J., Sokomba, E. & Salako, L. (2010a) 'Randomised controlled double-blind non-inferiority trial of two antivenoms for saw-scaled or carpet viper (Echis ocellatus) envenoming in Nigeria', PLoS Negl Trop Dis, vol. 4, no. 7, p. e767. Abubakar, S., Abubakar, I., Habib, A., Nasidi, A., Durfa, N., Yusuf, P., Larnyang, S., Garnvwa, J., Sokomba, E. & Salako, L. (2010b) 'Pre-clinical and preliminary dose-finding and safety studies to identify candidate antivenoms for treatment of envenoming by saw-scaled or carpet vipers (Echis ocellatus) in northern Nigeria', Toxicon, vol. 55, no. 4, pp. 719-723. Agarwal, R., Aggarwal, A.N., Gupta, D., Behera, D. & Jindal, S.K. (2005) 'Low dose of snake antivenom is as effective as high dose in patients with severe neurotoxic snake envenoming', Emergency Medicine Journal, vol. 22, no. 6, pp. 397-399. Aird, S.D. (2002) 'Ophidian envenomation strategies and the role of purines', Toxicon, vol. 40, no. 4, pp. 335-393. Alirol, E., Sharma, S.K., Bawaskar, H.S., Kuch, U. & Chappuis, F. (2010) 'Snake bite in South Asia: a review', PLoS neglected tropical diseases, vol. 4, no. 1, p. e603. Andrews, R.K., Gardiner, E.E. & Berndt, M.C. (2004) 'Snake venom toxins affecting platelet function', Platelets and Megakaryocytes: Volume 2: Perspectives and Techniques, pp. 335-347. Andrião-Escarso, S.l.H., Soares, A.M., Fontes, M.R.M., Fuly, A.L., Corrêa, F.M.A., Rosa, J.C., Greene, L.J. & Giglio, J.R. (2002) 'Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A2 from Bothrops jararacussu snake venom', Biochemical pharmacology, vol. 64, no. 4, pp. 723-732. Babaie, M., Salmanizadeh, H. & Zolfagharian, H. (2013) 'Blood Coagulation Induced by Iranian Saw-Scaled Viper (Echis Carinatus) Venom: Identification, Purification and Characterization of a Prothrombin Activator', Iranian journal of basic medical sciences, vol. 16, no. 11, p. 1145. Baldo, C., Jamora, C., Yamanouye, N., Zorn, T.M. & Moura-da-Silva, A.M. (2010) 'Mechanisms of vascular damage by hemorrhagic snake venom metalloproteinases: tissue distribution and in situ hydrolysis', PLoS Negl Trop Dis, vol. 4, no. 6, p. e727. Bernardoni, J.L., Sousa, L.F., Wermelinger, L.S., Lopes, A.S., Prezoto, B.C., Serrano, S.M., Zingali, R.B. & Moura-da-Silva, A.M. (2014) 'Functional variability of snake venom

metalloproteinases: adaptive advantages in targeting different prey and implications for human envenomation', PloS one, vol. 9, no. 10, p. e109651. Bhat, M.K. & Gowda, T.V. (1989) 'Purification and characterization of a myotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom', Toxicon, vol. 27, no. 8, pp. 861-873. Blaylock, R. (2005) 'The identification and syndromic management of snakebite in South Africa', South African Family Practice, vol. 47, no. 9, pp. 48-53. Bregani, E.R., Maraffi, T. & Van Tien, T. (2011) 'Snake bites in Moyen Chari district, Chad: a five-year experience', Tropical doctor, vol. 41, no. 2, pp. 123-126. Brown, N.I. (2012) 'Consequences of neglect: analysis of the sub-Saharan African snake antivenom market and the global context', PLoS Negl Trop Dis, vol. 6, no. 6, p. e1670. Calvete, J.J., Sanz, L., Angulo, Y., Lomonte, B. & Gutiérrez, J.M. (2009) 'Venoms, venomics, antivenomics', FEBS letters, vol. 583, no. 11, pp. 1736-1743. Canale, E., Isbister, G.K. & Currie, B.J. (2009) 'Investigating pressure bandaging for snakebite in a simulated setting: bandage type, training and the effect of transport', Emergency Medicine Australasia, vol. 21, no. 3, pp. 184-190. Casewell, N.R., Cook, D.A., Wagstaff, S.C., Nasidi, A., Durfa, N., Wüster, W. & Harrison, R.A. (2010) 'Pre-clinical assays predict pan-African Echis viper efficacy for a species- specific antivenom', PLoS neglected tropical diseases, vol. 4, no. 10, p. e851. Casewell, N.R., Wagstaff, S.C., Wüster, W., Cook, D.A.N., Bolton, F.M.S., King, S.I., Pla, D., Sanz, L., Calvete, J.J. & Harrison, R.A. (2014) 'Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms', Proceedings of the National Academy of Sciences, vol. 111, no. 25, pp. 9205-9210. Casewell, N.R., Wüster, W., Vonk, F.J., Harrison, R.A. & Fry, B.G. (2013) 'Complex cocktails: the evolutionary novelty of venoms', Trends in ecology & evolution, vol. 28, no. 4, pp. 219-229. Chijiwa, T., Tokunaga, E., Ikeda, R., Terada, K., Ogawa, T., Oda-Ueda, N., Hattori, S., Nozaki, M. & Ohno, M. (2006) 'Discovery of novel [Arg49] phospholipase A2 isozymes from Protobothrops elegans venom and regional evolution of Crotalinae snake venom phospholipase A2 isozymes in the southwestern islands of Japan and Taiwan', Toxicon, vol. 48, no. 6, pp. 672-682. Chippaux, J.-P. & Habib, A.G. (2015) Antivenom shortage is not circumstantial but structural, Oxford University Press.

Chippaux, J.-P., Massougbodji, A., Diouf, A., Baldé, C.M. & Boyer, L.V. (2015) 'Snake bites and antivenom shortage in Africa', The Lancet, vol. 386, no. 10010, pp. 2252-2253. Chippaux, J.-P., Williams, V. & White, J. (1991) 'Snake venom variability: methods of study, results and interpretation', Toxicon, vol. 29, no. 11, pp. 1279-1303. Chou, K.-C. (1996) 'Knowledge-based model building of the tertiary structures for lectin domains of the selectin family', Journal of protein chemistry, vol. 15, no. 2, pp. 161- 168. Costa, T.R., Burin, S.M., Menaldo, D.L., de Castro, F.A. & Sampaio, S.V. (2014) 'Snake venom L-amino acid oxidases: an overview on their antitumor effects', Journal of Venomous Animals and Toxins including Tropical Diseases, vol. 20, no. 1, p. 23. Das, R.R., Sankar, J. & Dev, N. (2015) 'High-dose versus low-dose antivenom in the treatment of poisonous snake bites: a systematic review', Indian journal of critical care medicine: peer-reviewed, official publication of Indian Society of Critical Care Medicine, vol. 19, no. 6, p. 340. de Silva, H.A., Ryan, N.M. & de Silva, H.J. (2016) 'Adverse reactions to snake antivenom, and their prevention and treatment', British journal of clinical pharmacology, vol. 81, no. 3, pp. 446-452. Dreyer, S. & Dreyer, J. (2013) 'Snake Bite: A review of Current Literature', East and Central African Journal of Surgery, vol. 18, no. 3, pp. 45-52. Drickamer, K. (1999) 'C-type lectin-like domains', Current opinion in structural biology, vol. 9, no. 5, pp. 585-590. Drickamer, K. & Taylor, M.E. (2002) 'Glycan arrays for functional glycomics', Genome biology, vol. 3, no. 12, p. reviews1034. 1031. Díaz, C., León, G., Rucavado, A., Rojas, N., Schroit, A.J. & Gutiérrez, J.M. (2001) 'Modulation of the susceptibility of human erythrocytes to snake venom myotoxic phospholipases A2: role of negatively charged phospholipids as potential membrane binding sites', Archives of biochemistry and biophysics, vol. 391, no. 1, pp. 56-64. Escalante, T., Rucavado, A., Fox, J.W. & Gutiérrez, J.M. (2011) 'Key events in microvascular damage induced by snake venom hemorrhagic metalloproteinases', Journal of Proteomics, vol. 74, no. 9, pp. 1781-1794. Felicori, L.F., Souza, C.T., Velarde, D.T., Magalhaes, A., Almeida, A.P., Figueiredo, S., Richardson, M., Diniz, C.R. & Sanchez, E.F. (2003) 'Kallikrein-like proteinase from bushmaster snake venom', Protein expression and purification, vol. 30, no. 1, pp. 32- 42.

Fox, J.W. & Serrano, S.M. (2008) 'Insights into and speculations about snake venom metalloproteinase (SVMP) synthesis, folding and disulfide bond formation and their contribution to venom complexity', The FEBS journal, vol. 275, no. 12, pp. 3016-3030. Fry, B.G. (1999) 'Structure–function properties of venom components from Australian elapids', Toxicon, vol. 37, no. 1, pp. 11-32. Gasanov, S.E., Dagda, R.K. & Rael, E.D. (2014) 'Snake venom cytotoxins, phospholipase A2s, and Zn2+-dependent metalloproteinases: mechanisms of action and pharmacological relevance', Journal of clinical toxicology, vol. 4, no. 1, p. 1000181. Gibbs, H.L., Sanz, L., Sovic, M.G. & Calvete, J.J. (2013) 'Phylogeny-based comparative analysis of venom proteome variation in a clade of rattlesnakes (Sistrurus sp.)', PloS one, vol. 8, no. 6, p. e67220. Girish, K., Shashidharamurthy, R., Nagaraju, S., Gowda, T.V. & Kemparaju, K. (2004) 'Isolation and characterization of hyaluronidase a “spreading factor” from Indian cobra (Naja naja) venom', Biochimie, vol. 86, no. 3, pp. 193-202. Gold, B.S. & Barish, R. (1992) 'Venomous snakebites. Current concepts in diagnosis, treatment, and management', Emergency medicine clinics of North America, vol. 10, no. 2, pp. 249-267. Gopalakrishnakone, P., Dempster, D.W., Hawgood, B.J. & Elder, H.Y. (1984) 'Cellular and mitochondrial changes induced in the structure of murine skeletal muscle by crotoxin, a neurotoxic phospholipase A2 complex', Toxicon, vol. 22, no. 1, pp. 85-98. Gutiérrez, J. (2019) 'Global availability of antivenoms: The relevance of public manufacturing laboratories', Toxins, vol. 11, no. 1, p. 5. Gutiérrez, J., Solano, G., Pla, D., Herrera, M., Segura, Á., Vargas, M., Villalta, M., Sánchez, A., Sanz, L. & Lomonte, B. (2017a) 'Preclinical evaluation of the efficacy of antivenoms for snakebite envenoming: state-of-the-art and challenges ahead', Toxins, vol. 9, no. 5, p. 163. Gutiérrez, J.M. (2012a) 'Improving antivenom availability and accessibility: science, technology, and beyond', Toxicon, vol. 60, no. 4, pp. 676-687. Gutiérrez, J.M. (2012b) 'Snakebite envenoming: a public health perspective', Public health- methodology, environmental and systems issues. InTech, pp. 131-162. Gutiérrez, J.M., Calvete, J.J., Habib, A.G., Harrison, R.A., Williams, D.J. & Warrell, D.A. (2017b) 'Snakebite envenoming', Nature reviews. Disease primers, vol. 3, p. 17063.

Gutiérrez, J.M., Higashi, H.G., Wen, F.H. & Burnouf, T. (2007) 'Strengthening antivenom production in Central and South American public laboratories: report of a workshop', Toxicon, vol. 49, no. 1, pp. 30-35. Gutiérrez, J.M. & Rucavado, A. (2000) 'Snake venom metalloproteinases: their role in the pathogenesis of local tissue damage', Biochimie, vol. 82, no. 9, pp. 841-850. Gutiérrez, J.M., Rucavado, A., Escalante, T. & Díaz, C. (2005) 'Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage', Toxicon, vol. 45, no. 8, pp. 997-1011. Gutiérrez, J.M., Theakston, R.D.G. & Warrell, D.A. (2006) 'Confronting the neglected problem of snake bite envenoming: the need for a global partnership', PLoS medicine, vol. 3, no. 6, p. e150. Gutiérrez, J.M., Williams, D., Fan, H.W. & Warrell, D.A. (2010) 'Snakebite envenoming from a global perspective: Towards an integrated approach', Toxicon, vol. 56, no. 7, pp. 1223-1235. Gutiérrez, J.M.a. & Ownby, C.L. (2003) 'Skeletal muscle degeneration induced by venom phospholipases A 2: insights into the mechanisms of local and systemic myotoxicity', Toxicon, vol. 42, no. 8, pp. 915-931. Habib, A.G. (2013) 'Public health aspects of snakebite care in West Africa: perspectives from Nigeria', Journal of Venomous Animals and Toxins including Tropical Diseases, vol. 19, no. 1, p. 27. Habib, A.G. & Brown, N.I. (2018) 'The snakebite problem and antivenom crisis from a health- economic perspective', Toxicon, vol. 150, pp. 115-123. Habib, A.G., Kuznik, A., Hamza, M., Abdullahi, M.I., Chedi, B.A., Chippaux, J.-P. & Warrell, D.A. (2015) 'Snakebite is Under Appreciated: Appraisal of Burden from West Africa', Plos Neglected Tropical Diseases, vol. 9, no. 9, pp. e0004088-e0004088. Habib, A.G. & Warrell, D.A. (2013) 'Antivenom therapy of carpet viper (Echis ocellatus) envenoming: effectiveness and strategies for delivery in West Africa', Toxicon, vol. 69, pp. 82-89. Harris, J.B. & Scott-Davey, T. (2013) 'Secreted phospholipases A2 of snake venoms: effects on the peripheral neuromuscular system with comments on the role of phospholipases A2 in disorders of the CNS and their uses in industry', Toxins, vol. 5, no. 12, pp. 2533-2571.

Harrison, R. & Gutiérrez, J. (2016) 'Priority actions and progress to substantially and sustainably reduce the mortality, morbidity and socioeconomic burden of tropical snakebite', Toxins, vol. 8, no. 12, p. 351. Harrison, R.A., Hargreaves, A., Wagstaff, S.C., Faragher, B. & Lalloo, D.G. (2009) 'Snake envenoming: a disease of poverty', PLoS neglected tropical diseases, vol. 3, no. 12, p. e569. Harrison, R.A., Ibison, F., Wilbraham, D. & Wagstaff, S.C. (2007) 'Identification of cDNAs encoding viper venom hyaluronidases: cross-generic sequence conservation of full- length and unusually short variant transcripts', Gene, vol. 392, no. 1-2, pp. 22-33. Herrera, C., Escalante, T., Voisin, M.-B., Rucavado, A., Morazán, D., Macêdo, J.K.A., Calvete, J.J., Sanz, L., Nourshargh, S. & Gutiérrez, J.M. (2015) 'Tissue localization and extracellular matrix degradation by PI, PII and PIII snake venom metalloproteinases: clues on the mechanisms of venom-induced hemorrhage', PLoS neglected tropical diseases, vol. 9, no. 4, p. e0003731. Hiremath, V., Yariswamy, M., Nanjaraj Urs, A., Joshi, V., Suvilesh, K., Ramakrishnan, C., Nataraju, A. & Vishwanath, B. (2014) 'Differential action of Indian BIG FOUR snake venom toxins on blood coagulation', Toxin Reviews, vol. 33, no. 1-2, pp. 23-32. Hoogenboom, H.R. (2005) 'Selecting and screening recombinant antibody libraries', Nature biotechnology, vol. 23, no. 9, p. 1105. Huang, P. & Mackessy, S.P. (2004) 'Biochemical characterization of phospholipase A2 (trimorphin) from the venom of the Sonoran Lyre Snake Trimorphodon biscutatus lambda (family Colubridae)', Toxicon, vol. 44, no. 1, pp. 27-36. Hutton, R. & Warrell, D. (1993) 'Action of snake venom components on the haemostatic system', Blood reviews, vol. 7, no. 3, pp. 176-189. Inn-Ho, T., Ying-Ming, W., Yi-Hsuan, C., Tein-Shun, T. & Ming-Chung, T.U. (2004) 'Venom phospholipases A2 of bamboo viper (Trimeresurus stejnegeri): molecular characterization, geographic variations and evidence of multiple ancestries', Biochemical Journal, vol. 377, no. 1, pp. 215-223. Isbister, G.K., Buckley, N.A., Page, C.B., Scorgie, F.E., Lincz, L.F., Seldon, M., Brown, S.G.A. & Investigators, A.S.P. (2013) 'A randomized controlled trial of fresh frozen plasma for treating venom-induced consumption coagulopathy in cases of A ustralian snakebite (ASP-18)', Journal of Thrombosis and Haemostasis, vol. 11, no. 7, pp. 1310-1318.

Ismail, M. & Memish, Z. (2003) 'Venomous snakes of Saudi Arabia and the Middle East: a keynote for travellers', International journal of antimicrobial agents, vol. 21, no. 2, pp. 164-169. Jackson, K. (2003) 'The evolution of venom-delivery systems in snakes', Zoological Journal of the Linnean Society, vol. 137, no. 3, pp. 337-354. Jackson, K. (2007) 'The evolution of venom-conducting fangs: insights from developmental biology', Toxicon, vol. 49, no. 7, pp. 975-981. Jayanthi, G. & Gowda, T.V. (1990) 'Synergistic interaction of a protease and protease inhibitors from Russell's viper (Vipera russelli) venom', Toxicon, vol. 28, no. 1, pp. 65- 74. Joseph, B., Raj, S.J., Edwin, B.T. & Sankarganesh, P. (2011) 'Pliarmacognostic and Biochemical Properties of Certain Biomarkers in Snake Venom'. Kamiguti, A., Slupsky, J., Zuzel, M. & Hay, C. (1994) 'Properties of fibrinogen cleaved by Jararhagin, a metalloproteinase from the venom of Bothrops jararaca', Thrombosis and haemostasis, vol. 72, no. 2, pp. 244-249. Kang, T.S., Georgieva, D., Genov, N., Murakami, M.T., Sinha, M., Kumar, R.P., Kaur, P., Kumar, S., Dey, S. & Sharma, S. (2011) 'Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis', The FEBS journal, vol. 278, no. 23, pp. 4544-4576. Kasturi, S. & Gowda, T.V. (1989) 'Purification and characterization of a major phospholipase A2 from Russell's viper (Vipera russelli) venom', Toxicon, vol. 27, no. 2, pp. 229-237. Kasturiratne, A., Wickremasinghe, A.R., de Silva, N., Gunawardena, N.K., Pathmeswaran, A., Premaratna, R., Savioli, L., Lalloo, D.G. & de Silva, H.J. (2008) 'The global burden of snakebite: a literature analysis and modelling based on regional estimates of envenoming and deaths', PLoS medicine, vol. 5, no. 11, p. e218. Kini, R. (1997) 'Phospholipase A2: a complex multifunctional protein puzzle', Venom phospholipase A2 enzymes: structure, function and mechanism, pp. 1-28. Kini, R. & Koh, C. (2016) 'Metalloproteases affecting blood coagulation, fibrinolysis and platelet aggregation from snake venoms: definition and nomenclature of interaction sites', Toxins, vol. 8, no. 10, p. 284. Kini, R.M. (2005a) 'Structure–function relationships and mechanism of anticoagulant phospholipase A 2 enzymes from snake venoms', Toxicon, vol. 45, no. 8, pp. 1147- 1161.

Kini, R.M. (2005b) 'The intriguing world of prothrombin activators from snake venom', Toxicon, vol. 45, no. 8, pp. 1133-1145. Kini, R.M. & Evans, H.J. (1989) 'A model to explain the pharmacological effects of snake venom phospholipases A2', Toxicon, vol. 27, no. 6, pp. 613-635. Kudo, K. & Tu, A.T. (2001) 'Characterization of hyaluronidase isolated from Agkistrodon contortrix contortrix (Southern Copperhead) venom', Archives of biochemistry and biophysics, vol. 386, no. 2, pp. 154-162. Lawson, R., Slowinski, J.B., Crother, B.I. & Burbrink, F.T. (2005) 'Phylogeny of the Colubroidea (Serpentes): new evidence from mitochondrial and nuclear genes', Molecular phylogenetics and evolution, vol. 37, no. 2, pp. 581-601. Lee, W.-H., Zhang, Y., Wang, W.-Y., Xiong, Y.-L. & Gao, R. (1995) 'Isolation and properties of a blood coagulation factor X activator from the venom of king cobra (Ophiophagus hannah)', Toxicon, vol. 33, no. 10, pp. 1263-1276. LIMPUS, C.J. (2013) 'THE VENOM APPARATUS AND VENOM YIELDS OF SUB-TROPICAL QUEENSLAND', Toxins: Animal, Plant and Microbial, p. 39. Lomonte, B. & Calvete, J.J. (2017) 'Strategies in ‘snake venomics’ aiming at an integrative view of compositional, functional, and immunological characteristics of venoms', Journal of Venomous Animals and Toxins including Tropical Diseases, vol. 23, no. 1, p. 26. Lu, Q., Clemetson, J. & Clemetson, K. (2005) 'Snake venoms and hemostasis', Journal of Thrombosis and Haemostasis, vol. 3, no. 8, pp. 1791-1799. Lumsden, N.G., Fry, B.G., Ventura, S., Kini, R.M. & Hodgson, W.C. (2004) 'The in vitro and in vivo pharmacological activity of Boiga dendrophila (mangrove catsnake) venom', Autonomic and Autacoid Pharmacology, vol. 24, no. 4, pp. 107-113. Mackessy, S.P. & Modahl, C.M. (2019) 'Venoms of Rear-fanged Snakes: New Proteins and Novel Activities', Frontiers in Ecology and Evolution, vol. 7, p. 279. Madrigal, M., Sanz, L., Flores-Díaz, M., Sasa, M., Núñez, V., Alape-Girón, A. & Calvete, J.J. (2012) 'Snake venomics across genus Lachesis. Ontogenetic changes in the venom composition of Lachesis stenophrys and comparative proteomics of the venoms of adult Lachesis melanocephala and Lachesis acrochorda', Journal of proteomics, vol. 77, pp. 280-297. Maduwage, K. & Isbister, G.K. (2014) 'Current treatment for venom-induced consumption coagulopathy resulting from snakebite', PLoS neglected tropical diseases, vol. 8, no. 10, p. e3220.

Maraganore, J.M. & Heinrikson, R.L. (1986) 'The lysine-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus. Relation of structure and function to other phospholipases A2', Journal of Biological Chemistry, vol. 261, no. 11, pp. 4797-4804. Maraganore, J.M., Merutka, G., Cho, W., Welches, W., Kezdy, F.J. & Heinrikson, R.L. (1984) 'A new class of phospholipases A2 with lysine in place of aspartate 49. Functional consequences for calcium and substrate binding', Journal of Biological Chemistry, vol. 259, no. 22, pp. 13839-13843. Markland, F.S. (1998) 'Snake venoms and the hemostatic system', Toxicon, vol. 36, no. 12, pp. 1749-1800. Markland, F.S. & Swenson, S. (2013) 'Snake venom metalloproteinases', Toxicon, vol. 62, pp. 3-18. Marsh, N. (1994) 'Snake venoms affecting the haemostatic mechanism--a consideration of their mechanisms, practical applications and biological significance', Blood coagulation & fibrinolysis: an international journal in haemostasis and thrombosis, vol. 5, no. 3, pp. 399-410. Matsui, T., Fujimura, Y. & Titani, K. (2000) 'Snake venom proteases affecting hemostasis and thrombosis', Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, vol. 1477, no. 1, pp. 146-156. McCue, M.D. (2005) 'Enzyme activities and biological functions of snake venoms', Applied Herpetology, vol. 2, no. 2, pp. 109-123. Mebs, D. & Ownby, C.L. (1990) 'Myotoxic components of snake venoms: their biochemical and biological activities', Pharmacology & therapeutics, vol. 48, no. 2, pp. 223-236. Mehta, S. & Sashindran, V. (2002) 'Clinical features and management of snake bite', Medical Journal, Armed Forces India, vol. 58, no. 3, p. 247. Modesto, A., Junqueira-de-Azevedo, I.L.M., Neves-Ferreira, A.G.C., Fritzen, M., Oliva, M.L.V., Ho, P.L., Perales, J. & Chudzinski-Tavassi, A.M. (2005) 'Insularinase A, a prothrombin activator from Bothrops insularis venom, is a metalloprotease derived from a gene encoding protease and disintegrin domains', Biological chemistry, vol. 386, no. 6, pp. 589-600. Montecucco, C. & Rossetto, O. (2000) 'How do presynaptic PLA 2 neurotoxins block nerve terminals?', Trends in biochemical sciences, vol. 25, no. 6, pp. 266-270. Morais, V.M. & Massaldi, H. (2009) 'Snake antivenoms: adverse reactions and production technology', Journal of Venomous Animals and Toxins including Tropical Diseases, vol. 15, no. 1, pp. 2-18.

Moura-da-Silva, A.M., Butera, D. & Tanjoni, I. (2007) 'Importance of snake venom metalloproteinases in cell biology: effects on platelets, inflammatory and endothelial cells', Current pharmaceutical design, vol. 13, no. 28, pp. 2893-2905. Munawar, A., Trusch, M., Georgieva, D., Hildebrand, D., Kwiatkowski, M., Behnken, H., Harder, S., Arni, R., Spencer, P. & Schlüter, H. (2014) 'Elapid snake venom analyses show the specificity of the peptide composition at the level of genera Naja and Notechis', Toxins, vol. 6, no. 3, pp. 850-868. O'leary, M.A. & Isbister, G.K. (2009) 'Commercial monovalent antivenoms in Australia are polyvalent', Toxicon, vol. 54, no. 2, pp. 192-195. O'Shea, M. (2008) Venomous snakes of the world, New Holland Publishers. Ouyang, C., Teng, C.-M. & Huang, T.-F. (1992) 'Characterization of snake venom components acting on blood coagulation and platelet function', Toxicon, vol. 30, no. 9, pp. 945- 966. Panfoli, I., Calzia, D., Ravera, S. & Morelli, A. (2010) 'Inhibition of hemorragic snake venom components: old and new approaches', Toxins, vol. 2, no. 4, pp. 417-427. Paul, V., Chawla, D. & Jatana, V. (1993) 'Animal and insect bites', Medical emergencies in children, vol. 2. Petan, T., Krizaj, I., Gelb, M.H. & Pungercar, J. (2005) 'Ammodytoxins, potent presynaptic neurotoxins, are also highly efficient phospholipase A2 enzymes', Biochemistry, vol. 44, no. 37, pp. 12535-12545. Pla, D., Sanz, L., Molina-Sánchez, P., Zorita, V., Madrigal, M., Flores-Díaz, M., Alape-Girón, A., Núñez, V., Andrés, V. & Gutiérrez, J.M. (2013) 'Snake venomics of Lachesis muta rhombeata and genus-wide antivenomics assessment of the paraspecific immunoreactivity of two antivenoms evidence the high compositional and immunological conservation across Lachesis', Journal of proteomics, vol. 89, pp. 112- 123. Polgar, J., Magnenat, E.M., Peitsch, M.C., Wells, T.N.C. & Clemetson, K.J. (1996) 'Asp-49 is not an absolute prerequisite for the enzymic activity of low-Mr phospholipases A2: purification, characterization and computer modelling of an enzymically active Ser- 49 phospholipase A2, ecarpholin S, from the venom of Echis carinatus sochureki (saw-scaled viper)', Biochemical Journal, vol. 319, no. 3, pp. 961-968. Pukrittayakamee, S., Warrell, D.A., Desakorn, V., McMichael, A.J., White, N.J. & Bunnag, D. (1988) 'The hyaluronidase activities of some Southeast Asian snake venoms', Toxicon, vol. 26, no. 7, pp. 629-637.

Péterfi, O., Boda, F., Szabó, Z., Ferencz, E. & Bába, L. (2019) 'Hypotensive Snake Venom Components—A Mini-Review', Molecules, vol. 24, no. 15, p. 2778. Ranawaka, U.K., Lalloo, D.G. & de Silva, H.J. (2013) 'Neurotoxicity in snakebite--the limits of our knowledge', Plos Neglected Tropical Diseases, vol. 7, no. 10, pp. e2302-e2302. Rossetto, O., Morbiato, L., Caccin, P., Rigoni, M. & Montecucco, C. (2006) 'Presynaptic enzymatic neurotoxins', Journal of neurochemistry, vol. 97, no. 6, pp. 1534-1545. Sachetto, A.T.A. & Mackman, N. (2019) 'Modulation of the mammalian coagulation system by venoms and other proteins from snakes, arthropods, nematodes and insects', Thrombosis research. Schiavo, G., Matteoli, M. & Montecucco, C. (2000) 'Neurotoxins affecting neuroexocytosis', Physiological reviews, vol. 80, no. 2, pp. 717-766. Scott, D.L. & Sigler, P.B. (1994) 'Structure and catalytic mechanism of secretory phospholipases A 2', Advances in protein chemistry, vol. 45, pp. 53-88. Segura, Á., Villalta, M., Herrera, M., León, G., Harrison, R., Durfa, N., Nasidi, A., Calvete, J.J., Theakston, R.D.G. & Warrell, D.A. (2010) 'Preclinical assessment of the efficacy of a new antivenom (EchiTAb-Plus-ICP®) for the treatment of viper envenoming in sub- Saharan Africa', Toxicon, vol. 55, no. 2-3, pp. 369-374. Shrestha, B.R., Pandey, D.P., Acharya, K.P., Thapa-Magar, C., Mohamed, F. & Isbister, G.K. (2017) 'Effective, polyvalent, affordable antivenom needed to treat snakebite in Nepal', Bulletin of the World Health Organization, vol. 95, no. 10, p. 718. Siigur, E., Tõnismägi, K., Trummal, K., Samel, M., Vija, H., Subbi, J. & Siigur, J. (2001) 'Factor X activator from Vipera lebetina snake venom, molecular characterization and substrate specificity', Biochimica et Biophysica Acta (BBA)-General Subjects, vol. 1568, no. 1, pp. 90-98. Silva, A., Hodgson, W. & Isbister, G. (2017) 'Antivenom for neuromuscular paralysis resulting from snake envenoming', Toxins, vol. 9, no. 4, p. 143. Slagboom, J., Kool, J., Harrison, R.A. & Casewell, N.R. (2017) 'Haemotoxic snake venoms: their functional activity, impact on snakebite victims and pharmaceutical promise', British journal of haematology, vol. 177, no. 6, pp. 947-959. Sousa, L.F., Nicolau, C.A., Peixoto, P.S., Bernardoni, J.L., Oliveira, S.S., Portes-Junior, J.A., Mourão, R.H.V., Lima-dos-Santos, I., Sano-Martins, I.S. & Chalkidis, H.M. (2013) 'Comparison of phylogeny, venom composition and neutralization by antivenom in diverse species of bothrops complex', PLoS Negl Trop Dis, vol. 7, no. 9, p. e2442.

Tans, G. & Rosing, J. (2001) 'Snake venom activators of factor X: an overview', Pathophysiology of Haemostasis and Thrombosis, vol. 31, no. 3-6, pp. 225-233. Tans, G. & Rosing, J. (2002) 'Snake venom activators of factor X: an overview', Pathophysiology of Haemostasis and Thrombosis, vol. 31, no. 3-6, pp. 225-233. Theakston, R.D.G. & Warrell, D.A. (2000) 'Crisis in snake antivenom supply for Africa', The Lancet, vol. 356, no. 9247, p. 2104. Theakston, R.D.G., Warrell, D.A. & Griffiths, E. (2003) 'Report of a WHO workshop on the standardization and control of antivenoms', Toxicon, vol. 41, no. 5, pp. 541-557. Thorelli, E. (1999) 'Mechanisms that regulate the anticoagulant function of coagulation factor V', Scandinavian Journal of Clinical and Laboratory Investigation, vol. 59, no. 229, pp. 19-26. Torres, A.M., Wong, H.Y., Desai, M., Moochhala, S., Kuchel, P.W. & Kini, R.M. (2003) 'Identification of a novel family of proteins in snake venoms purification and structural characterization of nawaprin from naja nigricollis snake venom', Journal of Biological Chemistry, vol. 278, no. 41, pp. 40097-40104. Vaiyapuri, S., Vaiyapuri, R., Ashokan, R., Ramasamy, K., Nattamaisundar, K., Jeyaraj, A., Chandran, V., Gajjeraman, P., Baksh, M.F. & Gibbins, J.M. (2013) 'Snakebite and its socio-economic impact on the rural population of Tamil Nadu, India', PloS one, vol. 8, no. 11, p. e80090. Vidal, N. (2002) 'Colubroid systematics: evidence for an early appearance of the venom apparatus followed by extensive evolutionary tinkering', Journal of Toxicology: Toxin Reviews, vol. 21, no. 1-2, pp. 21-41. Vidal, N. & Hedges, S.B. (2004) 'Molecular evidence for a terrestrial origin of snakes', Proceedings of the Royal Society of London B: Biological Sciences, vol. 271, no. Suppl 4, pp. S226-S229. Wangoda, R., Watmon, B. & Kisige, M. (2004) 'Snakebite management: experiences from Gulu Regional Hospital Uganda', East and Central African Journal of Surgery, vol. 9, no. 1. Warrell, D. (1996) Injuries, envenoming, poisoning and allergic reactions caused by animals. Weatherall, DJ, Ledingham, JGG, Warrell, DA, eds, Oxford Textbook of Clinical Medicine. Warrell, D.A. (2008) 'Unscrupulous marketing of snake bite antivenoms in Africa and Papua New Guinea: choosing the right product—‘what's in a name?’', Transactions of the Royal Society of Tropical Medicine and Hygiene, vol. 102, no. 5, pp. 397-399.

Warrell, D.A. (2010a) 'Guidelines for the management of snake-bites', Guidelines for the management of snake-bites. Warrell, D.A. (2010b) 'Snake bite', The Lancet, vol. 375, no. 9708, pp. 77-88. Warrell, D.A., Gutiérrez, J.M., Calvete, J.J. & Williams, D. (2013) 'New approaches & technologies of venomics to meet the challenge of human envenoming by snakebites in India', The Indian journal of medical research, vol. 138, no. 1, p. 38. Westerlund, B., Nordlund, P., Uhlin, U., Eaker, D. & Eklund, H. (1992) 'The three-dimensional structure of notexin, a presynaptic neurotoxic phospholipase A2 at 2.0 Å resolution', FEBS letters, vol. 301, no. 2, pp. 159-164. White, J. (2005) 'Snake venoms and coagulopathy', Toxicon, vol. 45, no. 8, pp. 951-967. WHO (2010a) 'Guidelines for the prevention and clinical management of snakebite in Africa'. WHO (2010b) 'WHO guidelines for the production, control and regulation of snake antivenom immunoglobulins', Geneva: WHO, vol. 134. WHO (2019) Neglected tropical diseases [Online], World Health Organization, Available from: http://www.who.int/neglected_diseases/diseases/en/ (Accessed: 19th September). Williams, D.J., Gutiérrez, J.-M., Calvete, J.J., Wüster, W., Ratanabanangkoon, K., Paiva, O., Brown, N.I., Casewell, N.R., Harrison, R.A. & Rowley, P.D. (2011a) 'Ending the drought: new strategies for improving the flow of affordable, effective antivenoms in Asia and Africa', Journal of proteomics, vol. 74, no. 9, pp. 1735-1767. Williams, H., Hayter, P., Ravishankar, D., Baines, A., Layfield, H., Croucher, L., Wark, C., Bicknell, A., Trim, S. & Vaiyapuri, S. (2018) 'Impact of Naja nigricollis Venom on the Production of Methaemoglobin', Toxins, vol. 10, no. 12, p. 539. Williams, S.S., Wijesinghe, C.A., Jayamanne, S.F., Buckley, N.A., Dawson, A.H., Lalloo, D.G. & de Silva, H.J. (2011b) 'Delayed psychological morbidity associated with snakebite envenoming', Plos Neglected Tropical Diseases, vol. 5, no. 8, pp. e1255-e1255. Wilson, H.A., Huang, W., Waldrip, J.B., Judd, A.M., Vernon, L.P. & Bell, J.D. (1997) 'Mechanisms by which thionin induces susceptibility of S49 cell membranes to extracellular phospholipase A2', Biochimica et Biophysica Acta (BBA)-Lipids and Lipid Metabolism, vol. 1349, no. 2, pp. 142-156. Wilson, J.M., Kalasinsky, K.S., Levey, A.I., Bergeron, C., Reiber, G., Anthony, R.M., Schmunk, G.A., Shannak, K., Haycock, J.W. & Kish, S.J. (1996) 'Striatal dopamine nerve terminal markers in human, chronic methamphetamine users', Nature medicine, vol. 2, no. 6, pp. 699-703.

Xiao, H., Pan, H., Liao, K., Yang, M. & Huang, C. (2017) 'Snake venom PLA2, a promising target for broad-spectrum antivenom drug development', BioMed research international, vol. 2017. Xu, X., Wang, X., Xi, X., Liu, J., Huang, J. & Lu, Z. (1982) 'Purification and partial characterization of hyaluronidase from five pace snake (Agkistrodon acutus) venom', Toxicon, vol. 20, no. 6, pp. 973-981. Yamamoto, C., Tsuru, D., Oda-Ueda, N., Ohno, M., Hattori, S. & Kim, S.T. (2001) 'Trimeresurus flavoviridis (habu snake) venom induces human erythrocyte lysis through enzymatic lipolysis, complement activation and decreased membrane expression of CD55 and CD59', Pharmacology & toxicology, vol. 89, no. 4, pp. 188-194. Zaganelli, G.L., Zaganelli, M.G., Magalhães, A., Diniz, C.R. & De Lima, M.E. (1996) 'Purification and characterization of a fibrinogen-clotting enzyme from the venom of jararacuçu (Bothrops jararacussu)', Toxicon, vol. 34, no. 7, pp. 807-819. Zelensky, A.N. & Gready, J.E. (2005) 'The C-type lectin-like domain superfamily', Febs Journal, vol. 272, no. 24, pp. 6179-6217. Zhang, C. & Gopalakrishnakone, P. (1999) 'Histopathological studies of the acute inflammation in synovial tissue of rat knee joint following intra-articular injection of PLA2 from Chinese Cobra (Naja naja atra) venom', Toxicon, vol. 37, no. 5, pp. 783- 799. Zhang, Y., Xiong, Y.-L. & Bon, C. (1995) 'An activator of blood coagulation factor X from the venom of Bungarus fasciatus', Toxicon, vol. 33, no. 10, pp. 1277-1288. Zuliani, J.P., Gutiérrez, J.M., e Silva, L.L.C., Sampaio, S.C., Lomonte, B. & Teixeira, C.d.F.P. (2005) 'Activation of cellular functions in macrophages by venom secretory Asp-49 and Lys-49 phospholipases A2', Toxicon, vol. 46, no. 5, pp. 523-532.

Chapter 2

Title: A new bovine eye model of venom-induced ophthalmia reveals cellular damage is averted by rapid irrigation with water but not milk

Jaffer Alsolaiss1, Gail leaming2, Ahmed Kazaili3, Shaun Pennington4, Paul Rowley1, Mark Wilkinson 1, Nicholas R Casewell1, Robert A Harrison1

Affiliations:

1Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, Liverpool, UK.

2Institute of infection and Global Health, University of Liverpool, Liverpool, L3 5RF, UK

3Department of Mechanical, Materials and Aerospace Engineering, School of Engineering, University of Liverpool, L69 3GH, UK

2.1 Introduction

Snakebite envenoming (SBE) is a major cause of morbidity, mortality and economic hardship in many communities in the developing world. It adversely affects local and national productivity, with disability ratings that surpass many other endemic tropical diseases (Habib & Brown 2018). Snakebite envenoming is a potentially life-threatening condition that results from the snake injecting a mixture of different venom toxins into tissues (WHO 2019), that also includes ophthalmia that can occur when some snakes (mainly the spitting cobras) spit venom into the eyes of victims.

The spitting cobras lie within the Elapidae and are believed to have undergone three separate evolutionary processes to attain their “spitting” trait (Wuester & Broadley 2007), namely, twice in the “true” cobras of the genus Naja and the third time in their closest- related species, Hemachatus haemachatus, the sole species of the genus Hemachatus. Venom spitting is considered an evolutionary adaptation serving defensive purposes only – spitting cobras do not spit at their prey, only aggressors. The venom spitting trait is exhibited by every species of the subgenus Afronaja and the majority of species of the subgenus Naja, apart from the Asian N. kaouthia, N. naja and N. oxiana species (Wüster & Thorpe 1992). The spitting mechanism is made possible by anatomic changes to the fang architecture such that the orifice in spitting cobras is uniquely forward facing and smaller than that of other venomous snakes (Figure 2.1). This adaptation ensures that the venom coursing down the duct is under unusually high pressure and thus ejected from the modified fang over a distance ranging from 2 to 5 metres (Chu et al. 2010). African spitting cobras of medical significance include N. nigricollis, N. mossambica, N. pallida, N. katiensis, N. ashei, N. nubiae, N. nigricincta and H. haemachatus and Asian species include N. sputatrix, N. philippinensis, N. sumatrana, N. mandalayensis, N. siamensis, N. samarensis and and possibly N. kaouthia. N. nigricollis is the species thought to be responsible for most incidents of venom ophthalmia (Chu et al. 2010).

Figure 2.1: Image of the exit orifice obtained by scanning electron micrographs of (A) non- spitting cobra N. kaouthia and (B) spitting cobra N. pallida (Young et al. 2004)

The immediate clinical manifestations of venom entering the eye are identical for Asian and African spitting cobras. The victim feels an agonising burning sensation and the eye goes red and begins to weep. Complications are more likely to be serious if irrigation is not effected quickly. For example, the venom of Naja siamensis can result in mild bleparospasm and bilateral periorbital oedema, moderate conjunctival chemosis, and causes discrete vertical nebulous patches on the corneal epithelium (Ang, Sanjay & Sangtam 2014; Rita et al. 2011). Clinical reports of cases N. nigricollis ophthalmia describe an instant sharp stinging pain and conjunctival hyperaemia. After a few hours, the pain and irritation lessen and, after two or more days, blepharitis with little discharge may develop (Chu et al. 2010). Instant and severe pain, although apparently of lesser intensity compared to Naja nigricollis and Naja siamensis, also accompanies ocular envenoming by the African species N. pallida and H. haemachatus and the Asian Naja species (Chu et al. 2010; Fung et al. 2009; Rita et al. 2011). Furthermore, the eye is mildly irritated when coming into contact with the venom of other species, such as Rhabdophis tigrinus formosanus and the rattlesnake, Crotalus atrox.

A few studies have explored ocular envenomation in animal models. Koch and Sachs employed dog and rabbit eyes to investigate the effects of venom spat by the African cobras N. nigricollis and H. haemachatus. Other studies used rabbit eyes to examine corneal opacification and subconjunctival haemorrhage by the venom of N. nigricollis, H.

49 haemachatus and N. melanoleuca in both crude and fractionated venoms (fractions VIII) (Ismail & Ellison, 1986; Ismail et al., 1993). The former study examined temporary corneal oedema, extensive chemosis and pupillary due to N. nigricollis venom on albino and pigmented rabbits. Cham et al. (2006) also employed a rabbit model to study whether corneal injury caused by the venom of N. sumatrana could be treated with topical heparin, antivenom, tetracycline and dexamethasone.

The selection of bovine cornea for this study is based on a comprehensive study by Hayashi et al. (2002) comparing the observation on corneas in mammals and amphibians which revealed that the bovine cornea has the closest morphology to the human cornea in terms of the thickness and measurements of different cornea layers (Bowman’s membrane, diameter of collagen, thickness of Descemet’s membrane). Venom testing and toxicological screening are relevant procedures to comprehend how pathology is caused by venom and thus enabling novel approaches for treating snakebites or ocular envenoming (ophthalmia). This ex vivo bovine eye model also bypasses the frequent ethical concerns of the aforementioned studies, because the eyes were sourced from local abattoirs where the cows are used for meat production.

Here, this ex vivo model was used to assess the precise corneal pathology caused by venoms from both spitting and non-spitting cobras, as well as an additional test substance. The metrics used to gauge pathogenicity were (i) reduced light transmission (opacity), (ii) greater penetration of sodium fluorescein dye (permeability), (iii) penetration of corneal layers by venom proteins and (iv) histopathological definition of the temporal progress of ocular damage.

The comparative ocular-pathology analyses utilised lyophilised venoms, freshly extracted venoms of the most medically important African and Asian spitting and non-spitting cobras and culminated with an assessment of the optimal ‘first aid strategy’ to reduce onset of venom ophthalmia after ocular envenoming.

2.2 Materials and methods

2.2.1 Eye collection and preparation;

The killing of cattle intended for human consumption or other commercial purposes is usually carried out in slaughterhouses. Bovine eyes for research purposes are derived solely from healthy cattle deemed appropriate for human consumption. In this study they were sourced from Blacklidge Brothers Abattoir, Wigan, UK. Excision of the eyes was undertaken immediately after death, exsanguination and decapitation of the cows. Animal heads were rinsed with non-detergent solutions to avoid eye irritations. The eyes were immediately preserved and transported in cold (4-6 oC) Hanks’ Balanced Salt Solution (HBSS) or phosphate buffered saline (PBS) and corneas used immediately after delivery to the laboratory (less than 3-4 hours after dissection). For the experimental outline see Figure 2.2.

Figure 2.2 Experimental design of ex vivo bovine eye model to study the ocular envenomation after topical administration of lyophilised/ fresh venom from spitting and non-spitting cobra. (A) using whole eye and (B) using dissected cornea.

2.2.2 Selection criteria and preparation of eyes and corneas

The eyes were subjected to a detailed examination for any defects, scratches or neovascularisation. Every treatment group was made up of at least two eyes (i.e. one eye exposed to venom from spitting or non-spitting snakes, or the positive control and one eye as a negative control). Corneas without defects were positioned on a holder and washed thoroughly with 10 ml PBS. To ensure as normal physiological conditions as possible, the next step was equilibration at 32±3 °C for a minimum of ten minutes (the in vivo corneal surface has a temperature of about 32°C). It is advised to maintain this temperature by employing a water bath, with necessary measures, such as holder pre-warming, being taken to keep the temperature stable.

2.2.3 Preparation of controls and snake venoms

The Centre for Snakebite Research & Interventions at the Liverpool School of Tropical Medicine supplied the lyophilised and fresh venoms (spitting and non-spiting cobra) (Table 2.1). Lyophilised venom was resuspended in PBS at the different doses required (2.5 μg and 10 μg), while fresh venom was extracted on the day of the experiment and applied at the quantity described (Table 2.1).

Table 2.1: Details of snake venom and venom doses used to study the corneal damage induced by lyophilised and fresh venom from spitting and non-spitting cobras.

Spitting/ Lyophilised Fresh Fresh Fresh Snake venom Origin non- venom venom venom with venom spitting Dose/ 100 with dissected Dose/ 10 mechanism μl whole cornea μl eye (penetration 2.5 10 experiment) μg μg Naja Nigeria Spitting ✓ ✓ ✓ ✓ 1000 μg nigricollis Naja haje Uganda Non-spiting ✓ ✓ ✓ - Naja nivea South Non- ✓ ✓ ✓ ✓ 1000 μg Africa spitting Naja Tanzania Spitting ✓ 1000 μg mossambica Naja pallida Tanzania Spitting ✓ 1000 μg Naja nubiae Eastern Spitting ✓ 1000 μg Africa Hemachatus Southern Spitting ✓ 1000 μg haemachatus Africa Naja Thailand Spitting ✓ 1000 μg siamensis Naja naja India Non- ✓ 1000 μg spitting

2.2.3 Exposure of corneas to snake venoms, and to negative and positive controls

Negative control (PBS), positive control (NaOH) or a concentration of lyophilised or fresh snake venom was applied topically to the cornea for one-minute, followed by 100 μl of 2% sodium fluorescein applied for one minute, after which the corneas were thoroughly washed with 10 ml PBS (it is essential that the epithelial surface is completely coated by the test substance, and then completely eliminated by thorough rinsing). Accordingly, a silicon O ring was positioned in the middle of the cornea and a micropipette was used to apply 100 μl (or any other amount sufficient to cover the O ring fully) of the control or venom straight to the epithelial surface of the cornea. For using fresh venoms, a quantity of 10 μl was used to replicate as closely as possible the amount of venom usually discharged through spitting.

Because it lacks blood and lymphatic vessels, the cornea is classified as an immune-privileged tissue. Despite the lack of blood vessels, however, neovascularisation, scarring and corneal blindness can be induced by a range of conditions (Gandhi & Jain 2015). When a substance is applied topically, it usually causes damage to the epithelium as well as stromal oedema.

Following Kuckelkorn et al. (2002) method an investigation into the mechanism of action of the main acidic and alkaline agents associated with severe damage to the eye, with emphasis on chemical type, volume, concentration, pH and exposure length. The assessment was carried out ‘top-down’, i.e. from the superior corneal layer (epithelium) to the stroma and endothelium, due to the topical application of the test substance. The manner of corneal layer penetration by the test substance determines how deep corneal damage can become.

To assess the extent of corneal damage, we measured the opacity of bovine corneas exposed to varying concentrations of the positive and negative controls and snake venoms (N. nigricollis, N. haje and N. nivea at concentrations of 2.5 μg, 10 μg of lyophilised venom or freshly-extracted venom). The treated corneas were exposed to 2% sodium fluorescein and stained with haematoxylin and eosin to describe the histopathological damage to the corneal epithelium. The inclusion of the controls (NaOH and PBS) was intended to make it easier to interpret the results, with the negative control serving as the baseline for comparison of histopathological modifications.

2.2.4 Evaluation of epithelial damage via confocal laser scanning fluorescence microscopy

A Zeiss LSM880 confocal microscope was employed for fluorescence imaging. PBS and NaOH- treated eyes were used for optimisation of 488 nm laser power (3.6%), gain (260), and virtual filters (493 – 624). The characteristics of the 15 images of 12-bit size obtained over a 760.78 μm section (with a 54.34 μm interval), centring on the peak emission plane, were 10× magnification, 0.6× digital zoom, and 1,024 × 1,024 resolution. The fluorescent intensities for all images were then measured using ImageJ software (Java-based image processing program). These setting measurements were applied for all selected areas (the area integrated intensity and mean grey value were selected), both for areas with fluorescence and areas without fluorescence (as background). The intensities for all selected regions were measured and the corrected total cell florescence (CTCF) was calculated [CTCF= Integrated Density- (Area of selected cell x Mean fluorescence of background reading)], and the reading then plotted using GraphPad. The P-value was calculated and compared with a cornea treated with the negative control (PBS).

2.2.5 Measuring the cornea transparency

To determine the light transmission in the visible region the method described previously by Doutch, Quantock and Meek (2007) was used with some modifications, the cornea was stored in PBS after the topical application of lyophilised or fresh venom and then mounted

54 and clamped in specially constructed half cells, that possess flat, polished glass apertures. Each half chamber was filled with PBS with a refractive index similar to that of the cornea so as to minimise reflections. Both cells allow appropriate, positive hydrostatic pressure to be applied to the preparation. After application of the 2% sodium fluorescein, an opacimeter with a wavelength in the visible light range (370-800 mm) was employed to measure opacity. The beam size was adjusted to 1 x 1 mm using customised apertures fitted to the sample cell and reference beam. Measurements were taken at optical wavelengths in the 400-800 nm region. The baseline was the sample cell filled with PBS only. More light transmission indicated a healthy cornea and less light transmission indicated increased opacity of the cornea. The readings were reported in terms of percentage of opacity, using GraphPad.

2.2.6 Evaluation of histopathology

The bovine corneas were fixed in 10 % neutral buffered formalin solution and embedded in paraffin. Tissue sections were stained with haematoxylin and eosin (H&E), and the corneal epithelium keratocyte, condition of the endothelium and the extent of disorganisation of the stromal collagen all being examined.

2.2.7 Identification of toxins from spitting and non-spitting fresh venoms penetrating the treated corneas

Freshly collected corneas were examined and dissected before being placed in the top of a Franz diffusion cell. The corneas were sealed into the donor compartment and the receptor cell filled with 5 ml sterile PBS (Gibco, pH 7.4) and placed in a water bath at 32 oC for 10 minutes before the venom was applied. 1 ml of PBS was collected (pre-sample), and the silicon O ring placed at the middle of the cornea before 10 μl of freshly collected venom was applied. The supernatant was collected after five minutes, ten minutes and then every ten minutes until the end of the experiment (60 minutes after initial exposure).

2.2.7.1 SDS-PAGE and Immunoblotting:

20 μl of the collected supernatant was diluted 2:1 with reduced SDS-PAGE loading buffer and boiled for five minutes at 100 oC. 8 μg of this reduced sample was then loaded to 15% hand cast gel prepared following the manufacturer’s instructions. The gel was run for 55 minutes at 200 V and then stained for two hours on a rocker at room temperature (RT) and then de- stained for one hour. For immunoblotting, the samples were prepared as described previously and the proteins in the gel electrophoretically transferred onto 0.45 um nitrocellose membrane (13A, 24A, for seven minutes), the membrane stained with Ponceau

S (confirming protein transfer) and washed with Tris Buffered Saline Tween (TBST). The membrane was incubated overnight in 5% skimmed milk to ‘block’ all proteins with weakly bound whey proteins. The membrane was next washed for 15 minutes in TBST (with three changes). SAIMR polyvalent antivenom (X02646, EXP Sep 2012) was used as the primary antibody diluted 1:2000 in blocking buffer (5% skimmed milk in TBST) for two hours at RT. Finally, the blots were washed with TBST as in the previous washing step, before being incubated at RT with secondary antibody (horseradish peroxidase- conjugated donkey anti- sheep IgG; Sigma, UK) diluted 1:1800. The blots were then washed as before and visualised by the addition of DAB substrate for 25 seconds (50 mg 3', 3'-diaminobenzidine, 100 ml PBS, 0.024% hydrogen peroxide; Sigma Aldrich, UK).

2.2.7.2 In-gel digestion Gel slices from SDS-PAGE were washed (2 x 30 min) with 50% acetonitrile, 0.2M ammonium bicarbonate pH 8.9 and then dried in a rotary evaporator. The slices were re-swollen in RHB [2 M urea, 0.2 M ammonium bicarbonate pH 7.8] containing 0.1 μg trypsin and incubated at 37ºC overnight. Excess RHB was then removed to a second microfuge tube and peptides were extracted from the gel slices with 60% acetonitrile, 0.1% TFA. The total peptide extract was then concentrated to 10 μl in a rotary evaporator and then desalted using C18 ZipTips according to the manufacturer’s instructions. 2.2.7.3 LC-MS/MS The desalted digests were delivered into a Triple TOF 6600 mass spectrometer (Sciex) by automated in-line reversed phase liquid chromatography (LC), using an Eksigent NanoLC 415 System equipped with trap (nanoACQUITY Symmetry C18 trap column, 100 Å, 5 µm, 180 µm x 20 mm, Waters) and analytical column (nanoACQUITY BEH C18 column, 300 Å, 1.7 µm, 75 µm X 250 mm, Waters). A NanoSpray III source was fitted with a 10 μm inner diameter PicoTip emitter (New Objective). Samples were loaded in 0.1% formic acid onto the trap which was then washed with 2% ACN/0.1% formic acid for 10mins at 2 µL/min before switching in-line with the analytical column. A gradient of 2-50 % (v/v) ACN, 0.1 % (v/v) formic acid over 90 min was applied to the column at a flow rate of 300 nL/min. Spectra were acquired automatically in positive ion mode using information-dependent acquisition powered by Analyst TF 1.7 software (Sciex), using mass ranges of 400-1500 atomic mass units (amu) in MS and 100-1600 amu in MS/MS. Up to 25 MS/MS spectra were acquired per cycle (approx 10 Hz) using a threshold of 300 counts per sec, with dynamic exclusion for 12 secs and rolling collision energy.

2.2.8 Intervention strategy using water or milk following ocular envenoming;

To assess the optimal irrigation of eyes subjected to venom, we considered that most rural victims would likely use either water or milk. The whole bovine eye was subjected to fresh venom from N. nigricollis applied topically to all eyes (16 eyes/ group), and then subjected to 10 ml of water/ milk

Because these solutions might not be available immediately, we exposed the eyes to venom for different durations before rinsing with either water or milk, as follows: 1 minute, 5 minutes, 10 minutes and every 10 minutes thereafter to a maximum of 60 minutes.

In summary, this study seeks to answer the following questions:

• Do venoms from spitting cobras exert greater ocular pathology than venoms from non- spitting cobras? • What is the time scale and anatomical manifestation of venom-induced ocular pathology? • Does freshly-extracted venom exert greater ocular pathology than lyophilised venom (which is most often used in venom research)? • Do the multiple layers of the cornea prevent ingress of venom into the vascular circulation? If not- are all venom proteins capable of cornea-penetration? • What is the most effective first aid irrigation – water or milk? And is the rapidity of irrigation important?

2.3. Results

2.3.1 Assessment of corneas treated with negative and positive controls

Evaluation of the epithelium was conducted by staining it transiently with 2% sodium fluorescein, following application of PBS to the cornea for a duration of one minute. With PBS, the epithelium did not exhibit any damage or degeneration in either of the two corneas. The result was corroborated by measurement of opacity using a light transmittance method, which is indicative of the extent to which light penetrates the cornea and thus, inversely, of damage (Figure 2.3.1-A). NaOH (1M) was used as a potent positive control, instantly causing the colour of the cornea to change to a white pigment. The measurement of opacity indicated high opacity (59%). Furthermore, there was indication of severe corneal damage following treatment with NaOH, as confirmed by H&E staining of the cornea section. There was multifocal erosion of the squamous layer with multifocal vacuolation of the cells, and multifocal thickening of the epithelium with loss of eosinophilia. Polygonal cell cytoplasm exhibited increased eosinophilia and mild cytoplasmic vacuolation and nuclei were vesicular. In addition, oedema, keratocyte pyknosis and vacuolisation were the main manifestations of stromal damage, accompanying disruption of the regular structured array of extracellular collagen matrix fibres, which also has a direct correlation with corneal opacity (see Figure 2.3-2A).

Figure 2.3: Corneas treated with the positive and negative control. The whole eye was used to study the corneal damage caused by PBS and NaOH. (1 - Aa) The opacity measurement following treatment with PBS: with no visual sign of opacity; (Ab) following treatment with NaOH the opacity appeared as a white pigment at the central part of the cornea. (1- B) Measurement of fluorescent intensities: (Ba) Sodium fluorescein did not permeate the cornea, indicating no damage. (Bb) Sodium fluorescein stained the cornea and permeation indicated extensive damage of the epithelium. (2) Histological results (2A) With PBS the epithelium did not exhibit any damage or degeneration. (2B) With NaOH multifocal erosion of the squamous layer with multifocal vacuolation of the cells was evident, alongside multifocal thickening of the epithelium with loss of eosinophilia. Polygonal cell cytoplasm exhibited increased eosinophilia with mild cytoplasmic vacuolation, and nuclei are vesicular. Significant P-value (***P ≤ 0.001).

2.3.2 Assessment of corneas exposed to different concentrations of N. nigricollis venom

To observe how venom caused corneal opacification, N. nigricollis venom was applied to the bovine corneas at different doses (2.5 μg, 10 μg and fresh) and at different durations of exposure (60 seconds and 30 minutes). The fluorescence intensities of the corneas after treatment showed a significant increase in corneas treated with fresh extracted venom after 30 minutes - about 10-fold more than the cornea treated with NaOH. Opacity measurements revealed a significant increase for all treated corneas in comparison with corneas treated with PBS.

The histopathological examination of stained sections revealed (Figure 2.4-2) that, at low doses of venom (2.5 μg concentration), the corneal epithelium exhibited mild degeneration, with top-down assessment of the dissected cornea identifying mild fragmentation/sloughing of the squamous epithelium and mild swelling of the squamous epithelium with multifocal mild vacuolation. Tissue morphology remained more or less the same at both time points. The mild degeneration of the epithelium was validated by the sodium fluorescein staining and opacity measurement. The opacity was slightly lower (60 %) after 30 minutes with this dose.

Meanwhile, at 10 μg dose, the corneal epithelium exhibited mild to moderate alterations, with thinning and swelling of the squamous epithelium and moderate swelling of polygonal and squamous epithelium. There was also more widespread mild cytoplasmic vacuolation (perinuclear). The same doses of the venom with a longer cornea exposure revealed moderate cell swelling; more frequent foci than with corneas treated for one minute and cytoplasmic vacuolation seen in all cell layers of anterior epithelium. Nonetheless, structural integrity was maintained, and the effects did not differ greatly between the two exposure times. The stroma was mildly swollen and mild stromal collagen matrix vacuolisation was observed as well. When the corneas were stained with sodium fluorescein, it was observed that there was a significant increase in the aggregation of dead and apoptotic cells between the two-time points.

After direct application of 10 μl of fresh venom (a natural venom dose), the corneas sustained significant damage and, by comparison to PBS-treated cornea, manifest by substantial multifocal erosion and swelling of the squamous epithelium and fewer swollen polygonal cells. Focally extensive ulceration and stroma thickening were more significant. By contrast,

60 the endothelium was not damaged. Consequently, the fluorescent dye could better permeate the epithelium. Furthermore, owing to coagulation and condensation, higher opacity was associated with this dose, which was confirmed by the increasing corneal cloudiness and loss of lustre at the half-hour time point.

Figure 2.4: Corneas treated with different doses of Naja nigricollis venom. The whole eye was used to study the corneal damage caused by lyophilised and fresh venom. (1A) Opacity measurement following the topical application of the N.nigricollis venom, P-value significantly increased with all venom doses and exposure durations; (1B) Fluorescence intensities using 2% sodium fluorescein for corneas treated with different doses of N. nigricollis venom, lyophilised and fresh. More significant increase of the fluorescence intensity after treatment with NaOH and fresh venom in comparison with cornea treated with PBS. (2) Histopathological section (5 μm) of (A) 2.5 μg concentration N. nigricollis venom for 1 minute and (B) for 30 minutes. (C) 10 μg of venom for one minute and (D) 30 minutes treatment with fresh venom. (A and B) show mild fragmentation and swelling of the squamous epithelium with a mild vacuolation with low dose of venom (2.5 ug); (C) shows mild thinning and swelling of the squamous epithelium and more widespread mild cytoplasmic vacuolation with the increase in the venom dose to 10 μg; (D) shows extensive loss of anterior epithelium, splitting of the polygonal epithelium and separation between the Bowman’s membrane and anterior epithelium with 10 μl of fresh venom. Significant P-value (****P ≤ 0.0001)

2.3.3 Assessment of cornea exposed to different concentrations of N. haje venom

N. haje is a non-spitting cobra and its venom was used here as a negative control since, in nature, it is not associated with ocular envenoming. The opacity induced by different venom doses were compared with the negative control (PBS). Fluorescence measurement revealed a slight increase of the intensities with corneas treated with 10 μg after both 1 minute and 30 minutes of topical application, and with fresh venom after 30 minutes. This suggests mild damage of the corneal epithelium since the high fluorescein staining was indicative of cell apoptosis.

The effects of N. haje venom were then assessed in the H& E section (Figure 2.5) and this revealed that with 2.5 μg of N. haje venom the corneal section morphology was identical to that observed with N. nigricollis venom; corneal epithelium being only mild damaged, with mild multifocal thinning of the anterior epithelium, swelling of the squamous epithelium and mild erosion of the most superficial squamous epithelium. The damage was progressive with a longer treatment course (30 minutes), with full thickness loss of the anterior epithelium, although the endothelium was unaffected. On the other hand, at 10 μg concentration for 1 minute, the N. haje venom penetrated the middle layer of polygonal cells and showed a moderate cell swelling and cytoplasmic vacuolation in all cell layers of the anterior epithelium and splitting between the anterior epithelium and Bowman’s membrane. Full thickness loss of the anterior epithelium was observed. After 30 minutes treatment, swelling of the anterior epithelium with scattered cytoplasmic vacuolation was noted. The fresh venom caused extensive variation in the thickness in the anterior epithelium, vesicle formation in the squamous epithelium with some swelling in adjacent polygonal cells. Moreover, by comparison to N. nigricollis venom, aggregation of dead and apoptotic cells, alongside reduced fluorescein staining, was revealed by cornea treatment with sodium fluorescein. The corneas treated with the three different concentrations exhibited a progressive increase in opacity, which was confirmed by alterations to cells, although cloudiness was not visible macroscopically.

Figure 2.5: Corneas treated with different doses of Naja haje venom. The whole eye was used to study the corneal damage caused by lyophilised and fresh venom. (1A) Opacity measurement following the topical application of the N. haje venom, P-value significantly ≤ 0.0001) increased with all venom doses.; (B) Fluorescence intensities using 2% sodium fluorescein for corneas treated with different doses of N. haje lyophilised and fresh venom for two exposure durations (1 minute and 30 minutes). Increase in the fluorescence intensity after treatment with NaOH, treatment with 10 μg (30 minutes) and fresh venom (30 minutes) in comparison with corneas treated with PBS. (2) Histopathological section (5 μm) of (A) 2.5 μg of resuspended N. haje venom for 1 minute showing mild multifocal thinning of the anterior epithelium with swelling of the squamous epithelium; (B) 2.5 μm for 30 minutes, showing mild multifocal thinning (erosion) of the anterior epithelium, two foci of full thickness loss of anterior epithelium. (C) 1 minute treatment with 10 μg of venom showing moderate cell swelling; more frequent foci, cytoplasmic vacuolation in all cell layers of anterior epithelium, focal full thickness loss of anterior epithelium (D) 30 minutes treatment with fresh venom showing increase in the corneal damage with extensive variation in thickness in the anterior epithelium ;multifocal vesicle formation in the squamous epithelium and some swelling in adjacent polygonal cells. Significant P-value (*P ≤ 0.05, and ****P ≤ 0.0001)

2.3.4 Assessment of corneas exposed to different concentrations of N. nivea venom

Despite its powerful neurotoxicity, N. nivea venom, like that of N. haje, is not known to cause eye damage. In the present study, this venom was used both in diluted form at 2.5 and 10 μg doses, and as a freshly extracted fresh venom, and was compared with the venom of the spitting N. nigricollis and the non-spitting N. nivea. Treatment of the corneas with 2% sodium fluorescein revealed no significant increase in fluorescence intensity with any of the corneas compared to corneas treated with PBS. There was slight increase (28%) in the opacity of the cornea treated with low venom doses (2.5 μg) and significant increased (50-70%) with all other venom preparations (10 μg and fresh venom) (Figure 2.6).

The corneal morphology was altered by only moderate swelling/ballooning degeneration of the squamous epithelium and the superficial polygonal epithelium. The corneas treated for 30 minutes showed a cellular swelling (intracellular oedema), cytoplasmic vacuolation and nuclear pyknosis indicating rapid necrotic cell degeneration. Higher venom concentrations led to some multifocal epithelial degeneration, slight stromal alterations and full thickness loss of the anterior epithelium (ulceration) adjacent to the altered anterior epithelium. Full thickness swelling/intracytoplasmic oedema was noted after 30 minutes after initial exposure. It was noted that the fresh venoms were highly viscous, and the corneal surface was coated in a sticky substance after topical application, which necessitated repeated thorough washing to be removed. The aggregation of dead cells in the treated area was validated by staining with sodium fluorescein. Overall however, the damage was superficial, and despite the fact that cells were lost, the epithelium was maintained with only very mild swelling of the superficial squamous epithelium. What is more, although the matrix collagen expanded to some extent, the endothelium was unaffected.

Figure 2.6: Corneas treated with different doses of Naja nivea venom. The whole eye was used to study the corneal damage caused by lyophilised and fresh venom. (1A) Opacity measurement following the topical application of the N. nivea venom. The opacity significantly increased (P-value <0.0001) with all venom doses. (1B) Fluorescence intensities using 2% sodium fluorescein for corneas treated with different doses of N. nivea, lyophilised and fresh venom, for two exposure durations (1 minute and 30 minutes). Although there was a significant increase of the fluorescence intensity after treatment with NaOH in comparison with corneas treated with PBS, no significant increase was observed after topical treatment with lyophilised and fresh venom. (2) Histopathological section (5 μm) of (A) 2.5 μg of resuspended N nivea venom for 1 minute showing extensive moderate swelling/ballooning degeneration of the squamous epithelium and also the superficial polygonal epithelium and mild vesicle formation within the squamous epithelium; (B) 2.5 μg for 30 minutes showing mild thinning (erosion) of the anterior epithelium and moderate cellular swelling, cytoplasmic vacuolation and nuclear pyknosis were observed; (C) 1-minute treatment with 10 μg of venom showing cytoplasmic vacuolation in all cell layers of anterior epithelium and full thickness loss of anterior epithelium (ulceration), adjacent to altered anterior epithelium; (D) 30 minutes treatment with with 10 μg of the N. nivea venom showing swelling/intracytoplasmic oedema. . Significant P-value (****P ≤ 0.0001)

2.3.5 The effects of fresh venom from spitting and non-spitting cobra on the cornea

Optimising experiments were conducted to establish the best venom concentration and exposure/incubation duration. To Investigate the effect of fresh venom of African and Asian spitting and non-spitting cobras on corneas, a comparative analysis of visually observable opacity and alterations in morphology induced by one-hour corneal exposure to fresh venom was undertaken.

According to macroscopic observations, application of venom from both spitting and non- spitting cobras caused the occurrence of opacification. The extent of corneal layer opacification and damage varied with each venom applied: Asian or African spitting or non- spitting cobras (Table 2.2). Thus, the venom of the spitting cobras, N. pallida and H. haemachatus, gave rise to a similar sharp line in the centre of the cornea and the white pigment did not diffuse beyond the area where the venom was applied. The corneal epithelium was mildly to moderately damaged, as indicated by corneal morphology, by both snake venoms.

Table 2.2; Observations of the pathology of corneas treated with fresh venom from Asian and African spitting and non-spitting cobras (scoring; Severe [+++], Moderate [++], Mild [+], and no effect [-]).

Epithelium Bowman's Stroma thickness species Superficial layer Middle layer Deep later Epithelium thickness Ulceration Vaculation membrane µm (Mean ±SD) (3-4 layers) (polygonal) (basel cell) µm (Mean ±SD) PBS - - - - - 141.6 (± 9.39) - 895 (± 44.39) NaoH +++ +++ + - +++ 175.3 (± 39.1) + 978 (± 39.1) N.nigricollis +++ +++ +++ +++ +++ 128.6 (± 28.05) +++ 901 (± 84.72) N. mossambica + + + - ++ 94.66 (± 29.48) - 1482 (± 130.47) N. pallida +++ +++ +++ - - 158 (± 25.45) +++ 1385 (± 253.5) N. nubia + + + - - 101 (± 16.30) ++ 1288 (± 84.44) N. siamensis + + + - + 182 (± 21.96) - 1260 (± 49.81) hemachatus haemachatus + + + - - 149 (± 44.95) - 1220.3 (± 105.7) N.naja + + + - + 180 (± 14.165) - 1546 (± 196.0) N. nivea + ++ + +++ - 205.6 (± 7.71) - 1062.3 (± 35.74)

The corneas exposed to the venom of the African spitting cobras, N. nigricollis and N. pallida, exhibited significant loosening of cells and damage to the superficial, middle and deep corneal layers. More specifically, the squamous epithelium exhibited marked ulceration, vacuolation and splitting due to exposure to fresh venom of N. nigricollis, whereas N. pallida venom caused significant separation in the anterior epithelium. Table 2.2 summarises the most significant findings observed following the topical application of fresh venom from

67 spitting and non-spitting cobra. Interestingly, the thickness of corneas treated with N. nigricollis showed reduced epithelial thickness and a normal stroma thickness, while other spitting and non-spitting venoms induced a significant increase in the thickness of both epithelium and stroma as a result of swelling and oedema.

2.3.6 Identification of the venom proteins penetrating the cornea

To detect whether venom proteins are capable of penetrating the multiple layers of the cornea (within one hour of the topical application of fresh venom), supernatants from prior experiments were collected and analysed. Freshly collected cobra venom was diluted in PBS and the protein concentration measured using Nanodrop spectrometry (A280). Based on this measurement, every 10 μl of topically applied venom contains 1,000 ug of proteins. Based upon SDS-PAGE analysis (Figure 2.6), supernatants from eyes subjected to only N. nigricollis, N. siamensis and N. naja venoms contained venom proteins that had penetrated through the corneal layers five minutes after application. None of the other venoms had proteins with such capability.

Two approaches were used to confirm the identified proteins. The first involved the use of western blots decorated with SAIMR polyvalent antivenom (manufactured with venoms from Bitis, Naja and Dendroaspis specuis) (Figure 2.7). Identification of two proteins was possible five minutes after topical application of N. nigricollis: band (1) and band (2), respectively classified as SVMP molecular weight (~55kDa) and Nerve Growth Factor (NGF) molecular weight (~25kDa). Furthermore, although no visible proteins were identified by SDS-PAGE in the supernatant derived from application of Asian N. siamensis venom, immunoblotting revealed clear detection of one protein with ~25kDa molecular weight, which was the same as the protein identified in the supernatant from N. nigricollis venom (NGF). Notably, extremely strong bands of two proteins were detected when analysing the supernatant from non-spitting N. naja venom but no equivalence could be established between these and the venom protein profile. In addition, extremely strong bands were identified by immunoblotting with smearing with the other time point (10-20 minutes). It was concluded that the venom proteins and corneal enzymes interacted with each other since both identified proteins had molecular weight that differed from the venom profile.

The second approach involved the use the venom proteomic analysis using in-gel digestion and LC-MS/MS which identified that the two bands were PLA2 and 3FTx, respectively with analysing the supernatant from non-spitting N. naja venom. Analysing the two detected

68 bands from N. nigricollis venoms revealed, respectively classified as SVMP (band 1) and NGF (band 2).

Figure 2.7: Characterisation of venom proteins penetrating the cornea after topical application of fresh venom. Dissected corneas were placed on a Franz diffusion cell and sealed on the top with a donor chamber. The receptor chamber was filled with 5 ml of PBS and incubated at 32 °C for 5 minutes before the venom was applied. 10 μl of fresh venom was applied to the central part of the cornea and the all supernatant collected and replaced with fresh PBS (Detailed in the method section. The collection of the supernatant was after 5 minutes for the first collection and every 10 minutes for the rest of the collection period up to 60 minutes). (1) N. nigricollis African spitting cobra; (2) N. siamensis Asian spitting cobra and (3) N. naja Asian non-spitting cobra. (A) the dissected cornea after the treatment showing the spread of the visible white pigment after 60 minutes. (B) SDS-PAGE of all the supernatants with venom protein profiles and pre-sample. (C) western blots of all supernatants and the venom to visualise the venom proteins. SAIMR polyvalent antivenom was used as the primary antibody. The red circle indicates venom proteins that had penetrated the cornea within 5 minutes of the topical application.

2.3.7 Prevention of damage to cornea caused by venom

Establishing the ideal strategy for managing eye exposure to fresh venom was the focus of this study. As explained in the method section, 10 ml water or milk were used to rinse several eyes for different lengths of time. The damage to the cornea was assessed based on macroscopic observation and alterations in morphology (Figure 2.8). The topically applied venom caused minor loss of lustre in every cornea. Corneal layers were only mildly damaged when rinsing with water was performed within 1-5 minutes of venom exposure. By contrast, damage was more significant and widespread when rinsing with water was performed later. On the other hand, the cornea was more likely to be damaged when it was rinsed with milk than with water, even if the procedure was performed early. One possible explanation for this may be that the milk and venom proteins interact in a way that prevents the milk proteins from inhibiting the damage-inducing toxins.

Figure 2.8: The intervention studies using water / milk following the topical application of fresh venom. 10 μl of venom was applied followed by rinsing the cornea with 10 ml of water/ milk. The corneas were then visualised with H&E staining (1) cornea rinsed with water after (A) 1 minutes, (B) 5 minutes and (C) 10 minutes. Delay in rinsing the cornea with water was associated with increased tissue damage. For example, (1A) rinsing the cornea with water 1 minute after the fresh venom was applied was associated with multifocal mild to moderate cytoplasmic vacuolation in the mid to deep anterior epithelium and mild multifocal linear separation of the squamous epithelium. (1C) Rinsing 10 minutes after application of venom was associated with mild vacuolation and separation of the basal epithelium while the mid to deep anterior epithelium exhibits multifocal mild to moderate cytoplasmic vacuolation. The nuclei in the squamous epithelium are plump/rounded. Following irrigation with milk, meanwhile, the corneas exhibited moderate damage. For example (2C), moderate multifocal cytoplasmic vacuolation and mild to moderate erosion of squamous epithelium.

2.4 Discussion

Deliberate spitting of venom for defence purposes is a defining characteristic of Elapidae. The Elapidae of medical importance are the African N. nigricollis, N. mossambica, N. pallida and N. nubiae, and the Asian N. siamensis, N. philippinensis, N. sumatrana and N. mandalayensis. The present study investigates the specific anatomical effects of spitting and non-spitting cobra venom on the eye in this ex vivo bovine eye model. This enabled determination of the most effective first aid strategies to reduce damage to the cornea. Systemic envenoming of patients with ocular envenoming has never been recorded. This suggests that the cornea acts as a barrier to prevent systemic envenoming.

Three-finger toxins (3FTxs) constitute around 70% of the venom of Naja species, while phospholipases A2 and class III metalloproteases constitute the remaining 30% (Cardoso et al. 2019; Chong et al. 2019; Theakston & Reid 1983). Elapidae venom is rich in a class of single-chain, basic, short polypeptides of high hydrophobicity known as cytotoxins (cardiotoxins) (Munawar et al. 2018). Evidence exists that interaction occurs between Lys and Arg, the basic cytotoxin residues, and cell membrane phospholipids, contributing to potentially serious membrane damage and necrotic cell apoptosis by promoting the development of pores on cell membranes (Gasanov, Dagda & Rael 2014; Konshina et al. 2011). Furthermore, probably owing to the ability of cytotoxins to bind to unknown membrane proteins or acidic phospholipids, there is temporary binding of the cytotoxin-PLA2 complex to cell membranes, thus enabling phospholipid hydrolysis by PLA2. This leads to the secreted fatty acid tails encircling the cytotoxin areas of hydrophilicity and these molecular interactions then serve to free cytotoxin from the enzyme-toxin complex (Gasanov et al. 1991).

Covering the external ocular mucosal surfaces, the tear film is highly effective in protecting the eye surface against microbes of potential pathogenicity (Dartt & Willcox 2013). There is also interaction between this film and a protective glycocalyx that the cornea generates from membrane-spanning mucins present in the corneal cell apical membrane (Hodges & Dartt 2013; Shirai & Saika 2015). The produced mucins mediate the removal of allergens, cell debris and pathogens from mucosal surfaces by capturing them. As Li et al. (2018) explained, Membrane-Associated Mucins (MAMs) play a role in adhesion prevention, lubrication, water retention, pathogen barrier and inflammation prevention. The ex vivo bovine model employed in this study clearly indicated that, in the context of topical application of spitting and non-spitting cobra venom, the venom proteins interacted with the external mucosal

72 surface of the cornea, as reflected in the diminished corneal lustre and the lack of tear film and corneal epithelium following corneal exposure to both high and low venom dosage (Sridhar 2018). An alternative explanation is that the venom proteins undergo hydrolysis and cause the cornea to opacify, making the corneal epithelium more permeable and promoting apoptosis and squamous metaplasia.

The results obtained by the present study suggest that the venom of spitting and non-spitting cobras have the same effects on the cornea. Use of a higher venom dosage or fresh venom led to a degree of damage in every cornea, as revealed by corneal morphology. Two-phased development of the damage induced by the venom of N. nigricollis, with vesicular and necrotising effects induced by venom toxins, followed by the action of the discharged pro- inflammatory prostaglandins and leukotrienes. Consistent with such findings, this study observed that lyophilised venom caused mild to moderate corneal damage. While, fresh venoms caused severe damage and marked opacification of the corneas. An additional observation of this study is that mild to moderate corneal damage and marked augmentation of corneal opacification were caused by the venom of the African non-spitting N. nivea and N. haje, as well as the Asian N. naja. Based on this, it was deduced not only that the venom proteins interfaced with the surface of the cornea, but also that the venom could penetrate the corneal layers, thus having further deleterious effects. It must be noted that the venom of Asian Elapidae has not been reported to result in the complications associated with the venom of African Elapidae, such as ophthalmia, corneal ulceration, scarring and opacification (Chu et al. 2010). The present study confirmed that the venom of the Asian spitting cobra N. siamensis and non-spitting cobra N. naja did not lead to ulceration or scarring but did induce opacification and mild splitting of the squamous epithelium as well as cytoplasmic vacuolation.

According to earlier research, eye pathology caused by the venom of African spitting Naja species in animal models was the outcome of venom cardiotoxins (Ismail et al. 1993). This is corroborated by the present study, which found that the cornea was damaged, and tissues were opacified and destroyed possibly owing to the action of PLA2 and 3FTx. As regards snake venom metalloproteinases (SVMPs), they trigger haemorrhage but not fast toxicity to endothelial cells (Fox & Gutiérrez 2018; Gutiérrez et al. 2016). The cornea was observed to be permeated by SVMPs and NGF five minutes after fresh venom of N. nigricollis was topically applied, implying that SVMPs and NGF triggered neither pathological effects nor corneal damage. This strongly suggested a future investigation of pathological effects of

73 those proteins following penetrating the cornea layers. Drug administration in therapeutic doses at the intended site is made more difficult by such eye tissue barriers (Gaudana et al. 2010). It is worth noting that corneal penetration was achieved by venom proteins within five minutes of application of non-spitting N. naja venom. Mass spectrometry validated that

PLA2 and 3FTx caused damage to the cornea. This is consistent with other research that has reported the rapid penetration of membrane by cytotoxins, with subsequent activation of various biological effects within cells (Gasanov, Dagda & Rael 2014).

Among the recommendations recently issued by the WHO for dealing with ophthalmia triggered by venom in Africa and South-East Asia (WHO 2010, 2016) are immediate administration of local anaesthetic in the form of eyedrops to open the eye and make it easier to undertake the subsequent procedure of rinsing the eye with water. In the present study, both tap water and fresh milk were used to rinse eyes exposed to fresh venom of N. nigricollis. It was found that eye rinsing with water within the first 5-10 minutes after exposure attenuated the effects of the venom on the cornea, ensuring that the damage did not become aggravated and thus making it more straightforward to manage. Conversely, late rinsing heightened damage severity. By contrast, corneal damage was not reduced by rinsing with milk. This result corroborates Delafontaine et al. (2018), who reported that damage was alleviated by rinsing with tap water.

Damage and oedema persisted following exposure to venom, particularly in the case of the venom of African Naja. Although the findings supported the WHO guidelines regarding eye rinsing, they also emphasise the need to manage with greater specificity by an urgent irrigating the eyes and other mucous membrane following the incidence with copious quantities of water or any liquid (Warrell 2010). Furthermore, although the WHO suggests topical treatment in the event of corneal erosion, no particular treatment for venom-induced eye damage is available because antivenom can cause irritation if applied topically so it is contraindicated (Chu et al. 2010). Nevertheless, Cham et al. (2006) found that eye pathology in rabbit models was enhanced by topical application of diluted antivenom and heparin, but not of tetracycline.

2.5 Conclusions

Investigation of both specific management strategies and the mechanisms of venom ophthalmia is facilitated by the use of an ex vivo empirical model, which also averts animal suffering and costly long-term follow-up. This model can reproduce corneal opacification,

74 the keratitis that precedes the development of ophthalmia induced by the venom of African Naja. The stromal structure, oedema, and cellular damage to the epithelium and endothelium can be evaluated based on measurement of the fluorescence intensity of the corneal epithelium and histological combined analysis. In the future, research should use lengthier follow-up to investigate corneal re-epithelisation and recovery after venom- induced keratitis. The results of this study confirm that the venom of both spitting and non- spitting cobras causes severe damage to the cornea if untreated; however, despite not being completely avoidable, damage can be attenuated by immediate rinsing with water, validating recently issued recommendations. Moreover, the results enhance knowledge of the process of venom keratitis resulting in ophthalmia and can serve as the basis for the development of treatment strategies of greater specificity and efficacy.

2.6 References:

Ang, L.J., Sanjay, S. & Sangtam, T. (2014) 'Ophthalmia due to spitting cobra venom in an urban setting-A report of three cases', Middle East African journal of ophthalmology, vol. 21, no. 3, p. 259. Cardoso, F.C., Ferraz, C.R., Arrahman, A., Xie, C., Casewell, N.R., Lewis, R.J. & Kool, J. (2019) 'Multifunctional toxins in snake venoms and therapeutic implications: from pain to hemorrhage and necrosis', Frontiers in Ecology and Evolution, vol. 7, p. 218. Chong, H.P., Tan, K.Y., Tan, N.H. & Tan, C.H. (2019) 'Exploring the diversity and novelty of toxin genes in Naja sumatrana, the Equatorial spitting cobra from Malaysia through de novo venom-gland transcriptomics', Toxins, vol. 11, no. 2, p. 104. Chu, E.R., Weinstein, S.A., White, J. & Warrell, D.A. (2010) 'Venom ophthalmia caused by venoms of spitting elapid and other snakes: report of ten cases with review of epidemiology, clinical features, pathophysiology and management', Toxicon, vol. 56, no. 3, pp. 259-272. Dartt, D.A. & Willcox, M.D.P. (2013) 'Complexity of the tear film: importance in homeostasis and dysfunction during disease', Experimental eye research, vol. 117, p. 1. Fox, J. & Gutiérrez, J.M. (2018) Snake venom metalloproteinases, MDPI. Fung, H.T., Choy, C.H., Lau, K.H., Lam, T.S.K. & Kam, C.W. (2009) 'Ophthalmic injuries from a spitting Chinese cobra', Hong Kong Journal of Emergency Medicine, vol. 16, no. 1, pp. 26-28. Gandhi, S. & Jain, S. (2015) The anatomy and physiology of cornea, in, Keratoprostheses and Artificial Corneas, Springer, pp. 19-25. Gasanov, S.E., Dagda, R.K. & Rael, E.D. (2014) 'Snake venom cytotoxins, phospholipase A2s, and Zn2+-dependent metalloproteinases: mechanisms of action and pharmacological relevance', Journal of clinical toxicology, vol. 4, no. 1, p. 1000181. Gasanov, S.E., Kolusheva, S.O., Salakhutdinov, B.A., Beknazarov, U.M. & Aripov, T.F. (1991) The mechanism of cobra venom phospholipase A2 and cytotoxin synergistic action on phospholipid membrane structure, pp. 44-46. Gaudana, R., Ananthula, H.K., Parenky, A. & Mitra, A.K. (2010) 'Ocular drug delivery', The AAPS journal, vol. 12, no. 3, pp. 348-360. Gutiérrez, J., Escalante, T., Rucavado, A., Herrera, C. & Fox, J. (2016) 'A comprehensive view of the structural and functional alterations of extracellular matrix by snake venom

metalloproteinases (SVMPs): novel perspectives on the pathophysiology of envenoming', Toxins, vol. 8, no. 10, p. 304. Habib, A.G. & Brown, N.I. (2018) 'The snakebite problem and antivenom crisis from a health-economic perspective', Toxicon, vol. 150, pp. 115-123. Hodges, R.R. & Dartt, D.A. (2013) 'Tear film mucins: front line defenders of the ocular surface; comparison with airway and gastrointestinal tract mucins', Experimental eye research, vol. 117, pp. 62-78. Ismail, M., Al-Bekairi, A.M., El-Bedaiwy, A.M. & Abd-El Salam, M.A. (1993) 'The ocular effects of spitting cobras: II. Evidence that cardiotoxins are responsible for the corneal opacification syndrome', Journal of Toxicology: Clinical Toxicology, vol. 31, no. 1, pp. 45-62. Konshina, A.G., Boldyrev, I.A., Utkin, Y.N., Omel'kov, A.V. & Efremov, R.G. (2011) 'Snake cytotoxins bind to membranes via interactions with phosphatidylserine head groups of lipids', PloS one, vol. 6, no. 4, p. e19064. Li, X., Kang, B., Woo, I.H., Eom, Y., Lee, H.K., Kim, H.M. & Song, J.S. (2018) 'Effects of topical mucolytic agents on the tears and ocular surface: a plausible animal model of mucin-deficient dry eye', Investigative ophthalmology & visual science, vol. 59, no. 7, pp. 3104-3114. Munawar, A., Ali, S., Akrem, A. & Betzel, C. (2018) 'Snake venom peptides: Tools of biodiscovery', Toxins, vol. 10, no. 11, p. 474. Rita, P., Animesh, D.K., Aninda, M., Benoy, G.K., Sandip, H. & Datta, K. (2011) 'Snake bite, snake venom, anti-venom and herbal antidote. A review', Int J Res Ayurveda pharm, vol. 2, no. 4, pp. 1060-1067. Shirai, K. & Saika, S. (2015) Ocular surface mucins and local inflammation—studies in genetically modified mouse lines, vol. 15, BioMed Central, p. 154. Sridhar, M.S. (2018) 'Anatomy of cornea and ocular surface', Indian journal of ophthalmology, vol. 66, no. 2, p. 190. Theakston, R.D.G. & Reid, H.A. (1983) 'Development of simple standard assay procedures for the characterization of snake venoms', Bulletin of the world health organization, vol. 61, no. 6, p. 949. Warrell, D.A. (2010) 'Snake bite', The Lancet, vol. 375, no. 9708, pp. 77-88. WHO (2010) 'Guidelines for the prevention and clinical management of snakebite in Africa'. WHO (2016) 'Guidelines for the Management of Snake Bites WHO. Regional Office for South-East Asia', World Health Organization.

WHO (2019) Neglected tropical diseases [Online], World Health Organization, Available from: http://www.who.int/neglected_diseases/diseases/en/ (Accessed: 19th September). Wuester, W. & Broadley, D.G. (2007) 'Get an eyeful of this: a new species of giant spitting cobra from eastern and north-eastern Africa (Squamata: Serpentes: Elapidae: Naja)', Zootaxa, vol. 1532, no. 1, pp. 51-68. Wüster, W. & Thorpe, R.S. (1992) 'Dentitional phenomena in cobras revisited: spitting and fang structure in the Asiatic species of Naja (Serpentes: Elapidae)', Herpetologica, pp. 424-434. Young, B.A., Dunlap, K., Koenig, K. & Singer, M. (2004) 'The buccal buckle: the functional morphology of venom spitting in cobras', Journal of Experimental Biology, vol. 207, no. 20, pp. 3483-3494.

Chapter 3

Title: A new ex vivo human skin model of venom-induced dermonecrosis

Jaffer Alsolaiss1, Gail Leaming2, Paul Rowley1, Nicholas R Casewell1, Robert A Harrison1

Affiliations:

1Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, Liverpool, UK.

2Institute of infection and Global Health, University of Liverpool, Liverpool, L3 5RF, UK

3.1 Introduction

Annually, up to 138,000 individuals die and over 400,000 individuals suffer life-altering disabilities as a result of the neglected tropical disease of snakebite envenoming. This disease is especially prevalent among deprived inhabitants of rural tropical regions, keeping them in a state of poverty (WHO 2019). Snake venom comprises a complex mixture of proteins with varying bioactive effects, which in human victims causes hemotoxic, neurotoxic and cytotoxic and other effects – depending upon the snake species responsible. The aetiological toxins include PLA2s, SVMPs, SVSPs and 3FTxs, acting on their own or in synergy. For instance, specific members of the PLA2 and 3FTx protein groups can cause neurotoxic effects leading to respiratory paralysis. Similarly, specific venom PLA2s, SVMPs, SVSPs, CTLs are responsible for haemorrhage and acute renal damage following envenoming by some but not all snakes (Gutiérrez et al. 2017; Gutiérrez & Rucavado 2000a; Gutiérrez et al. 2005; Harris & Scott- Davey 2013; Rivel et al. 2016). The venom of certain Elapidae and Viperidae trigger muscle damage at the bite site (Isbister 2010; Sitprija 2006) due to myotoxic non-catalytic PLA2 proteins. Viper and elapid venom PLA2s, 3FTxs and SVMPs can result in local tissue damage (e.g. swelling, blisters, oedema, necrosis), which frequently necessitates tissue-debridement or limb-amputation (Gasanov, Dagda & Rael 2014; Isbister et al. 2013; Mehta & Sashindran 2002).

Symptoms of local envenoming can include instant radiating pain around the bite site, tender swelling that expands quickly and accompanied by hot inflammatory erythema within two hours of the envenomation, lymph vessel inflammation manifesting as red lines on the skin (lymphangitis), protracted haemorrhage from the puncture wounds caused by the fangs, blistering (bullae), bruising (ecchymosis), tenderness and an increase in the size of local lymph nodes, superficial necrosis of the soft tissue and muscles, and secondary infection such as cellulitis or abscess (Warrell 1999; Warrell 2004; Who 2010). Skip lesions and ‘soft’ tissue necrosis often accompany envenoming by spitting cobras and some Asian elapids.

Severe pain can be engendered by certain PLA2s, SVSPs and SVMPs through the activation of ion channels like transient receptor potential vanilloid type 1 (TRPV1) and acid-sensing ion channel (ASIC) to modulate pain pathways (Bohlen et al. 2011; Zhang et al. 2017) and/or through the activation of inflammatory mediators to cause pain sensitisation (Ferraz et al. 2015; Mamede et al. 2016; Menaldo et al. 2013). Pain is triggered by certain mediators as well. By releasing ATP and activating purinergic receptors, sensory neurons associated with pain are excited by PLA2 homologues of catalytic inactivity (Rucavado et al. 2016; Zhang et

80 al. 2017). Numerous studies have shown that both humans and experimental models experience pain or hyperalgesia due to the inflammation triggered by the venom of Elapidae and Viperidae (Bucaretchi et al. 2016; Hifumi et al. 2015; Kleggetveit, Skulberg & Jørum 2016; Mamede et al. 2016).

Eicosanoids, nitric oxide (NO), bradykinin, complement anaphylatoxins, histamine and cytokines are synthesised and released, resident macrophages and additional types of cells are activated, and leukocytes are mobilised in the context of the large-scale local inflammatory process triggered by envenomation of tissue (Teixeira et al. 2009; Teixeira et al. 2003). vessels become more permeable as a result of such an inflammatory process and the exudate that develops contains plasma proteins alongside intracellular and extracellular protein fragments, chemokines, cytokines and molecular patterns related to damage, which probably aggravate the inflammation and intensify tissue damage.

The inflammatory reaction triggered by envenoming is underpinned by SVMPs, leading to the development of oedema, mobilisation of neutrophils, triggering of the complement system, and release of cytokines such as interleukins, IL-1β, IL-6, and IL-10 and tumour necrosis factor α (TNF-α) (Bernardes et al. 2015; Clissa et al. 2001; Farsky et al. 2000; Zychar et al. 2010). The PLA2s in snake venom, meanwhile, have been reported by many studies to promote inflammatory effects under various circumstances and the changes in physiology and immunity that they trigger are based on a series of major mechanisms that have been discovered through research (Teixeira et al. 2005).

Both non-neurogenic and neurogenic (substance-P dependent) elements are associated with

PLA2-triggered inflammation (Câmara et al. 2003; Zhang et al. 2017). The hydrolysis of membrane lipids producing powerful lipid mediators that promote inflammation is the main source of the non-neurogenic element (Costa, Camargo & Antunes 2017). PLA2s also trigger other non-neurogenic and neurogenic inflammations, but the complexity of their underlying mechanisms has so far precluded comprehensive understanding. Furthermore, PLA2s play a role in activating mast cells and the subsequent release of histamine and inflammatory mediators like platelet-activating factor, serine proteases, leukotrienes, and prostaglandins, which enhance their nociceptive action (Chiu, Chen & Teng 1989; Wang & Teng 1990; Wei et al. 2009). Oedema, skin rash, intestinal irritability and bronchoconstriction are among the local and systemic effects that can be induced by the released mediators, according to the location of the bite site (Camargo et al. 2005; Câmara et al. 2003; Ferreira et al. 2009; Landucci et al. 2000).

Viperidae envenomation often results in blistering and ulceration. Using experimental mouse models, some researchers have documented the division of the dermis and epidermis and the ulceration reported clinically, showing how these are caused by haemorrhagic SVMPs, especially the ones belonging to the P-I class (Freitas-de-Sousa et al. 2017). Blistering probably develops due to protein hydrolysis at the interface between the dermis and epidermis, as suggested by the dermis-epidermis division alongside the occurrence of basement membrane protein fragments in exudates gathered after skin sectioning in mouse models administered P-I SVMP (Escalante et al. 2009). Furthermore, lasting sequelae accompanied by contractures or extensive fibrosis are a frequent outcome of the cutaneous necrosis caused by the venom of a number of cobras of the genus Naja, including the African spitting cobras Naja nigricollis and N. mossambica (Warrell 2017). The precise role of toxins and inflammatory responses in mediating the acute and longer-term local tissue pathologies remains uncertain. Further research is needed to shed more light on the development of the clinically significant local necrosis (Gutiérrez et al. 2018).

Our knowledge therefore of the pathology of local envenoming by African vipers and spitting cobras primarily derives from clinical observation studies, with very little input from animal models to better define the mechanism resulting in this pathology. Conversely, our knowledge on the mechanisms of local envenoming emanates primarily from subjecting murine models with venoms from Latin American snakes. In this study, we developed a new ex vivo human skin model that occupies the important research space between clinical observations and animal models. To address knowledge gaps on the local tissue-destructive effects of African vipers and spitting cobras, we utilised this new model of local envenoming to answer the following research questions:

• Which venom proteins penetrate human skin tissue?

• What is the anatomical consequence of injecting venom into this tissue?

• To what extent is cell apoptosis associated with this skin pathology?

• Does injection of skin with venoms from vipers, and spitting and non-spitting cobras stimulate distinct expression of pro-inflammatory cytokines, chemokines and growth factors?

3.2 Material and methods:

3.2.1 Skin collection procedure:

Genoskin collected anonymised human skin samples from donors who had undergone an abdominoplasty procedure and who had given their written informed consent. Donors did not have any record of allergies or dermatological disorders and had not use glucocorticoid or steroid treatment. Full ethical approval for the study protocol was obtained from the French ethical research committee (Comite de Protection des Personnes) and authorisation was given by the French Ministry of Research. All studies were conducted according to the Declaration of Helsinki protocols. The biopsies were prepared in Genoskin France and delivered to the Liverpool School of Tropical Medicine (LSTM) the next day. The skin experiments were initiated that day.

3.2.2 Snake venom and the injection doses;

The Centre for Snakebite Research & Interventions at the Liverpool School of Tropical Medicine supplied the lyophilised venoms (Viperidae and Elapidae) from medically importance snakes (details in table 3.1). The lyophilised venom was resuspended in PBS at the different doses required for the injection.

Table 3.1: Details of snake venoms species and doses used in the study;

Snake species Family Common name Origin Venom

(MND50) dose Echis ocellatus Viperidae West African carpet viper Nigeria 120 µg Bitis arietans Viperidae African puff adder Nigeria 195 µg Naja nigricollis Elapidae Black-necked spitting cobra Nigeria 165 µg Naja haje Elapidae The Egyptian cobra/ African Uganda 156 µg (Control venom) banded cobra

MND50: refers to the minimum amount of venom (in µg) that shows a necrotic lesion (5 mm diameter) 72 hours after being injected intradermally in groups of mice.

3.2.3 Culture and treatments for Hyposkin models:

Hyposkin models (n=28) 15 mm diameter with 10 mm thick adipose tissue were produced from a female donor and cultured under cell culture conditions (37 °C, 5% CO2 and max humidity) for six hours, 12 hours, 24 hours, 36 hours, 48 hours and biopsies at seven days with 2 ml standard NativeSkin medium renewed every day. Three venoms and one venom

83 control (details and doses in Table 1) were tested, with the selected doses being based on the Minimum Necrotising Dose (MND50 performed in mouse model). 20 µl venom was injected subcutaneously (at day 1) with a Hamilton LT 250 μl syringe and 27G needle. Two Hyposkin models were left untreated (Negative control). Two fresh biopsies (referred to as the Uncultured control) from day 0 were fixed in 10% buffered formalin for 48 hours and processed for paraffin wax embedding (for later comparison).

3.2.4 Sampling and histological analysis:

The culture medium (2 ml) was collected daily and stored at -80 °C. At each collection time point, the biopsies were split into two parts. One half was fixed in 10% buffered formalin for 48 hours at room temperature (RT) and processed for paraffin wax embedding and histopathological analysis. The second half was stored in RNAlater solution for gene expression analysis (the method and the results not included in the study). Culture medium were collected, and histological analysis was performed for all samples as follows:

3.2.5 Haematoxylin and Eosin staining

Performed on 5 μm thick cross sections with a transmitted light image being acquired with a Leica DMi1 microscope with 40x lens.

3.2.6 Anti-active CASEPASE-3 fluorescence immunostaining:

Performed on 5 μm thickness skin cross sections incubated in blocking buffer (Goat serum) for 30 minutes at 37 °C. Then rinsed with PBS for five minutes (three times) before being incubated overnight with primary antibody (Rabbit polyclonal; Abcam ab2302) at a dilution of 1:100 with gentle agitation. Next day, the section was washed as in the previous step and incubated with HRP-conjugated secondary antibody for one hour at RT. Following the incubation and rinsing, mounting medium (containing DAPI) was applied and the sample was examined under the microscope.

3.2.7 TUNEL Assay for Detection of DNA Fragmentation and cellular Damage:

The assay was performed using (ApopTag® Fluorescein In Situ Apoptosis Detection Kit, Ref. S7110, Millipore). Briefly, 5 μm thick skin cross sections were washed with a range of buffers (i) Xylene for five minutes (three changes), (ii) Absolute ethanol for five minutes (two changes); (iii) 95% ethanol (once); (iv) 70% ethanol for three minutes (once); (v) and a final washing with PBS for five minutes (one change). A freshly diluted digesting enzyme (proteinase K; 20 μg/ml) was applied to the pre-treated tissues for 15 minutes at RT in a

Coplin jar. Following incubation, the tissues were washed with PBS for two minutes (two changes), excess liquid was gently tapped off and equilibration buffer immediately applied directly to the tissues and incubated for at least ten seconds at RT. The excess liquid was gently tapped off and working strength TdT enzyme was immediately pipetted onto the sections and incubated for one hour at 37 °C. The sections were then transferred to a Coplin jar containing a working strength stop/wash buffer with gentle agitation for 15 seconds and incubated at RT for ten minutes. After the incubation, the sections were washed with PBS for one minute (three changes), and warmed working strength Anti-Digoxigenin conjugate was applied to the slide and then incubated for 30 minutes at RT in a humidified chamber while avoiding exposure to the light. This was then washed with PBS for two minutes at RT (four changes), followed by applying a mounting medium containing (0.5-1.0 ug/ml) DAPI and microscopic examination using Leica DM5000, processed with ImageJ software.

3.2.8 Quantification of human cytokines by multiplex bead array

The Human Cytokine Magnetic 30-Plex Panel (based on xMAP™ technology) was used for quantifying human cytokines, chemokines and growth factors (EGF, Eotaxin, FGF basic, G- CSF, GM-CSF, HGF, IFN-α, IFN-γ, IL-1RA, IL-1β, IL-2, IL-2r, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40/p70), IL-13, IL-15, IL-17, IP-10, MCP-1, MIG, MIP-1α, MIP-1β, RANTES, TNF-α, and VEGF) of the collected culture medium. The standards and samples were prepared and reconstituted following the kit manual. Briefly, antibody beads were added to each well and incubated on the magnet for 60 seconds before the liquid was decanted and washed twice with 200 μl of washing buffer. 50 μl of incubation buffer was added followed by 100 μl of diluted standard and blank (PBS). 50 μl of assay diluent was added before addition of 50 μl of neat samples. The plate was covered to shield it from light and kept on the rocker overnight at 4 °C. The following day, the plate washed as before and 100 μl of biotinylated detector antibody was added and incubated for one hour at RT, followed by washing three times and the addition of 150 μl of wash buffer. Luminex® 100/200 technology (Mag-Plex®- Avidin Microspheres) was used for measuring bead fluorescence readings. xPonent Luminex software was used for the analysis of the results and the mean and standard error was calculated for each group.

3.2.9 SDS-PAGE and western blot profiles of daily collected culture medium:

20 μl of the daily-collected culture medium were diluted 2:1 in reduced buffered, heated for five minutes at 100 °C and 8 µg of the reduced sample was loaded into 15% SDS-PAGE gel hand cast gels prepared following the manufacturer’s instructions. The gel was run for 55

85 minutes at 200V, stained for two hours on the rocker at RT and then de-stained for one hour. For western blots, the samples were prepared as described previously and the proteins electrophoretically transferred from the gel to 0.45 μm nitrocellose membrane (13A, 24A, for seven minutes). The membrane was stained using Ponceau S stain to confirm effective protein transfer and then washed with TBST. The membrane was incubated overnight in 5% non-fat skimmed milk. Next day, the membrane was washed for 15 minutes (three changes). SAIMR polyvalent antivenom (used against culture medium of Bitis arietans, Naja nigricollis and Naja haje) and SAIMR Echis antivenom (used against Echis ocellatus) were used as primary antibodies diluted 1:2000 in blocking buffer (5% skimmed milk in TBST) for two hours at RT. This was followed by washing with TBST as in the previous washing step, before the blots were incubated at RT with diluted 1:1800 secondary antibody (horseradish peroxidase- conjugated donkey anti-sheep IgG; Sigma, UK). The blots then washed as before and visualised by addition of DAB substrate for 25 seconds (50 mg 3', 3'-diaminobenzidine, 100 ml PBS, 0.024% hydrogen peroxide; Sigma Aldrich, UK).

3.2.10 Quantification of the activities of snake venom metalloproteins and of serine proteases in collected culture medium;

The SVMP activity of the venoms and of the daily-collected culture medium was measured using a quenched fluorogenic substrate (ES010, R&D Biosystems). Briefly, 10 µl of the substrate (supplied as a 6.2 mM stock) was used per 5 ml reaction buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5). Reactions consisted of 10 µl of venom (1 µg/reaction) in PBS, or 10 µl of undiluted culture medium sample and 90 µl of substrate. The venom and the samples were incubated at 37 °C for 30 minutes and then pipetted in triplicate onto 384-well plates (Greiner). A multichannel was used to dispense the substrate. The plate was run on an Omega FluoSTAR (BMG Labtech) instrument at an excitation wavelength of 320 nm and emission wavelength of 405 nm at 25 °C for one hour. The means and average of duplicate or triplicate measurements with standard error (SE) were plotted using GraphPad Prism7.

To determine the serine protease activity in the daily-collected culture medium, we used a chromogenic kinetic assay and the specific substrate S-2288 (Cambridge Biosciences). 5 µl samples were plated onto 384-well plates and then overlaid with Tris buffer (100 mM Tris, 100 mM NaCl, pH 8.5), and 6 mM of S-2288. Changes in absorbance were measured at 405 nm for ~30 mins. Negative control readings were taken from PBS. The means and average of triplicate measurements with standard error (SE) were plotted using GraphPad Prism7.

3.3 Results

The following experiments were performed after week-long ‘assay-standardising’ experiments were conducted by the PhD student with Genoskin colleagues in France. The following sections describe the results to the original research questions.

3.3.1 Which venom proteins penetrate human skin tissue?

The skin biopsies were injected intradermally with venom and the daily-collected culture medium examined for presence of venom proteins. Comparison of the protein profiles of each sample was undertaken against the venom protein profiles, untreated homogenies skin biopsy, and untreated control culture medium.

In the initial 24 hours, there was extensive damage, as shown by the profiles of the treated Echis ocellatus biopsy (Figure 3.1), with natural tissue loss to the medium being a probable reason for the high molecular weight protein, similar to the untreated control. SDS-PAGE analysis of the culture medium identified two proteins with 25kDa molecular weight and proteins with 10kDa molecular weight were observed, with release up to the fifth day. The venom proteins in the samples were detected with the antivenom SAIMR Echis monovalent. Moreover, in the initial 24 hours, weak bands of proteins with 25kDa molecular weight were identified, but no additional specific proteins were identified with the culture medium material.

Analysis of the culture medium of the biopsies injected with B. arietans venom identified further tissue damage in the initial two days. Interrogating immunoblots of the culture medium with SAIMR polyvalent revealed continuous release of venoms from the tissue into the culture medium for the duration of the 7-day experiment. The culture medium of the biopsy injected with N. nigricollis venom exhibited additional tissue deterioration which intensified until the third day. The use of the SAIMR polyvalent antivenom did not allow identification of any specific venom proteins. Skin tissue damage was not detected in the biopsy injected with N. haje venom, which was employed as a negative venom control at a dosage of 156 µg. Tissue deterioration was not observed after the initial two days. A protein with low molecular weight of around 6-7 kDa and a protein with high molecular weight of around 25kDa, respectively indicative of 3FTx and NGF, were identified by the SAIMR polyvalent antivenom.

Figure 3.1; Identification and characterisation of venom proteins penetrating the skin: Daily collected samples subjected to SDS-PAGE to identify the presence of venom toxins in withthe hicollectegh moleculard samples. weight (2:1) of of around samples 25kDa, treated respectively with a reduce indicative buffer of and PLA run2 and in aNGF, handmade were identifiedcast. (A) byprotein the SAIMR profiles polyvalent of individual antivenom. samples compared with homogenies skin (s), snake venom (v) and untreated samples (U). (B) immunoblotting of all collected samples against (1 B) SAIMR Echis monospecific antivenom, (2B-4B) against SAIMR polyvalent.

3.3.2 Detection of SVMP and SVSP activities detected in culture media of venom-injected biopsies

The evaluation of the SVMPs (Figure 3.2) and SVSPs (Figure 3.3) activities was performed in all collected culture medium. The in vitro kinetic fluorescent assay was performed to measure the SVMP activity in the culture medium, followed by comparison of the outcomes against PBS and untreated biopsy (negative control) and the venom of B. arietans (as positive control). According to the results, only samples gathered in the first 24 hours and on the third day after injection with E. ocellatus venom exhibited significant SVMP activities. By contrast, the culture medium gathered from biopsies injected with the venom of B. arietans, N. nigricollis and N. haje did not identify any activity. Chromogenic assay was performed to measure the SVSPs activity of every sample, with B. arietans venom and PBS, respectively, serving as positive and negative controls. From the third to the seventh day a slight increase in SVSP activity was exhibited by the culture medium gathered from samples injected with E. ocellatus venom, whereas, during the same time frame, a significant increase was displayed in the SVSP activity in the culture medium gathered from samples injected with B. arietans venom. By contrast, injection with Elapidae venom was not associated with any increase in SVSP activity.

Figure 3.2: The metalloprotease activities of venoms in the collected culture medium; All collected culture medium were subjected to a chromogenic kinetic assay. The samples collected after injection with venoms (A) Echis ocellatus; (B) Bitis arietans; (C) N. nigricollis and

(D) N. haje. In each case one biopsy was injected with Echis ocellatus and another was injected with N. nigricollis, then followed after one hour by an antivenom interventions. The culture medium collected 48 hours after the injection was subjected to a quenched fluorogenic substrate and measured at an excitation wavelength of 320 nm and an emission wavelength of 405 nm at 25 °C for 1 h. The results were compared with the results from culture medium collected from day 0. Significant values [****(<0.0001), *** (0.0001), ** (0.0045), * (0.0206)]. NGA (Nigeria), UGA (Uganda).

Figure 3.3: The serine protease activities of venoms in the collected culture medium; All collected culture medium were subjected to a chromogenic kinetic assay. The samples collected after injection with venoms (A) Echis ocellatus; (B) Bitis arietans; (C) N. nigricollis and (D) N. haje. In each case one biopsy was injected with Echis ocellatus and another injected with N. nigricollis then followed after one hour by antivenom interventions. The culture medium collected 48 hours after the injection was subjected to chromogenic assays and measured at 405 nm for ~30 minutes. The results were compared with the results from culture medium collected from day 0. Significant values [****(<0.0001), *** (0.0001), ** (0.0045), * (0.0206)]. NGA (Nigeria), UGA

(Uganda).

3.3.3 What is the anatomical consequence of injecting venom into this tissue?

The experimental model presented in this study reproduces in explants of human skin the characteristic effects described in clinical cases of envenoming by Viperidae and Elapidae venoms. The clinical descriptions recount oedema, blistering and necrosis of the skin and subcutaneous connective tissue. Blistering forms are observed after seven days, followed by prominent necrosis of the dermis and loss of the epidermis. These effects are then followed by the sloughing of the epidermis, thus generating an ulcer, which is then filled with a hyaline fibrinoid material.

The control skin samples (both the untreated controls and the negative venom controls (treated with N. haje venom)) had a normal macroscopic appearance. A progressive series of pathological events was observed in skin injected with E.ocellatus, B.arietans and N. nigricollis venom. Macroscopically, the ex vivo model showed evidence of mild damage in the epidermis and/or superficial dermis at seven days, which was most pronounced with B. arietans venom. The necrotic area was characterised by a faint brownish lesion surrounded by a whitish area.

Figure 3.4: The macroscopic observation of the injected biopsies (pre and post the subcutaneous injection). 20 μl of venom was injected and the macroscopic changes on the injected tissues were observed daily. Day 0 refers to before the subcutaneous injection and Day 7 to seven days after the injection. (A) untreated biopsy, and biopsies injected with venoms (B) E. ocellatus (120 μg); (C) B. arietans (195 μg); (D) N. nigricollis (165 μg) and (E) N. haje (156 μg). The macroscopic observations revealed that after seven days in culture, the untreated control and the biopsy treated with N. haje venom did not display any changes. All the other biopsies exhibited a slight darkness following the injection with venoms (E. ocellatus, B. arietans and N. nigricollis).

Histopathological analysis of control biopsies at day 7 (both for untreated and N. haje venom- treated samples) revealed an unaltered epidermis and dermis, including skin appendages (Fig. 3.4 A and E). All biopsies of venom-injected skin collected at day 7 showed evident pathological alterations. Loss of skin viability and structure was observed with all three venoms (E. ocellatus, B. arietans and N. nigricollis). The epidermis structure was preserved in a similar condition as seen in the non-injected control, although a few pyknotic nuclei were observed in epidermal crests (pyknotic nuclei could mean apoptotic or necrotic cells). These pyknotic nuclei were present in the deep dermis in both snake venom control, and all three snake venom experiments. There was separation of the epidermis from the dermis and blistering forms in biopsies injected with the three venoms (E. ocellatus, B. arietans and N. nigricollis). The dermis and epidermis exhibit pyknotic cells in biopsies injected with B. arietans and the biopsies injected with B. arietans and N. nigricollis exhibited ballooning degeneration of the epidermal cells. No morphological changes were observed with N. haje and untreated biopsy.

Figure 3.4: Viability and structural integrity assessment following the subcutaneous injection of snake venoms. The H&E section of tissues obtained from day 7 after the injection. (A) Untreated control section; tissues treated with venoms (B) E. ocellatus (120 μg); (C) B. arietans (195 μg); (D) N. nigricollis (165 μg) and (E) N. haje (156 μg). The examination revealed an important loss of skin viability and structure with three venoms (E. ocellatus, B. arietans and N. nigricollis) whereas no changes were detected in the tissues of the untreated control and the tissue treated with N. haje venom.

3.3.4 To what extent is cell apoptosis associated with this skin pathology?

The cytoplasm of cells whose nuclear morphology was indicative of apoptosis was selectively stained, indicating the presence of antibodies specific for active caspases-3. The cytoplasm of morphologically healthy-looking cells were also stained (Bressenot et al. 2009), however, thus suggesting that these antibodies recognised activated protein at the early stage of apoptosis. Active caspase-3 was occasionally detected in nuclei, thus indicating a translocation of the protein to the nucleus. Strikingly, our study showed evidence of apoptosis in all the biopsies subcutaneously injected with snake venoms, except the non- injected control (Figure 3.5). It may be that this is due to the trauma of injection.

DNA fragmentation usually results from either apoptosis or necrosis and, in apoptosis, is associated with ultrastructural changes in cellular morphology. The extent of such fragmentation can be studied using the TUNEL assay (Figure 3.6). When TUNEL staining was applied to our samples apoptotic cells were in all envenomed biopsies. The TUNEL staining results thus confirmed the presence of apoptotic cells and revealed the presence of necrotic cells, in particular the model injected with venoms only. Cell mortality was observed in all injected biopsies, albeit with different levels of TUNEL positive cells. Both cobra venoms (N. nigricollis and N. haje) showed less cell damage confirmed by TUNEL staining. While B. arietans venoms showed the presence of more necrotic and apoptotic cells in the collected sample

Figure 3.5: Assessment of apoptotic cells following the subcutaneous injection of snake venoms. Anti-active caspase-3 fluorescence immunostaining was performed on 5 μm thick skin collected at day 7 following the injection. (A) Untreated control section; tissues treated with venoms (B) E. ocellatus (120 μg); (C) B. arietans (195 μg); (D) N. nigricollis (165 μg) and (E) N. haje (156 μg). The examination revealed a few apoptotic cells (white arrow) in all injected conditions except untreated control. Similar levels of apoptosis were detected in all sections following the venom injection.

Figure 3.6: DNA fragmentation assessment following the subcutaneous injection of snake venoms. TUNEL assay was performed on 5 μm thick skin collected at day 7 after the injection using ApopTag® Fluorescein In Situ Apoptosis Detection Kit. (A) Untreated control section; tissues treated with venoms (B) E. ocellatus (120 μg); (C) B. arietans (195 μg); (D) N. nigricollis (165 μg) and (E) N. haje (156 μg). TUNEL staining confirmed apoptotic cells (White arrow) presence and revealed the presence of necrotic cells (Yellow arrow) in biopsies injected with venom. Untreated biopsies did not present any dead cells.

3.3.5 Does injection of skin with venoms from vipers, and spitting and non-spitting cobras stimulate distinct expression of pro-inflammatory cytokines, chemokines and growth factors? The cytokines, chemokines and growth factors arising from the local tissue damage caused by the venom were measured in order to examine in more detail the impact of venom on the employed human skin model. Glycoproteins are generated by inflammatory and epithelial cells, while cytokines are responsible for immune response regulation and underpin communication between cells, as well as being employed by inflammatory cells (e.g. neutrophils, macrophages, lymphocytes) for coordination of the whole inflammatory reaction process (Juhn et al. 2008). Local tissue damage pathogenesis relies most extensively on inflammatory cytokines, since these compounds affect most skin layer cells and influence the production of other inflammatory compounds and enzymes through intracellular pathways of signal transduction. There are 19 inflammatory cytokines with the major ones being G-CSF, GM-CSF, IL-1β, IL-1RA and IL-12. Biopsies injected with the venom of E. ocellatus and B. arietans revealed a rapid increase in IL-1B in the first two days, which then declined somewhat by the fourth day, while biopsies injected with the venom of N. nigricollis and N. haje revealed an incremental increase in IL-1B in the first day, before reverting to normal by the third day.

The pleiotropic effects of IL1 are modulated by the anti-inflammatory IL-1RA through competitive inhibition of its adherence to receptors on the cell surface (Arend & Guthridge 2000). According to the results obtained, the same activities were associated with a marked increase in both IL-1RA and IL-1β. Furthermore, the levels of granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte-colony stimulating factor 3 (CSF-3), both peaked early in the inflammatory cascade before rapidly declining so that the inflammation could not be clinically proven. Injection with viper venoms (E. ocellatus and B. arietans) led to an incremental rise in GM-CSF and CSF-3 in the first two days and then a rapid increase on the fifth day, whereas injection with elapid venoms (N. nigricollis and N. haje) led to an increase on the first and fifth days. Moreover, the immune response was significantly modulated by interleukin 12 (IL-12) (Sun et al. 2015). Compared to the normal control, all injected biopsies had marked elevation of IL-12, whereas some cytokines (e.g. IL- 4) did not exhibit a notable increase by contrast to the normal control (Figure 3.7).

Figure 3.7. Dynamic changes in pro-inflammatory cytokines following subcutaneous injection by Viperidae venom (E. ocellatus and B. arietans) and Elapid venoms (N. nigricollis and N. haje). The Human Cytokine Magnetic 30-Plex Panel for the Luminex™ platform was performed to quantify the cytokines’ responses in the culture medium collected from the injected skin between Days 0 and 5. Six cytokines analytes simultaneously showed responses; (A) IL1-β, (B) IL-1RA, (C) IL-12, (D) G-CSF/CSF-3, (E) IL-4 and (F) GM-CSF. The sample collected from the untreated skin tissue was used to compare with the pro-inflammatory cytokines’ response following the subcutaneous injection. (Av= antivenom).

Mobilisation of leukocytes at the infection or damage site is the main reason why cells generate pro-inflammatory chemokines. When cells release chemokines, however, immune cells can invade and damage vital tissues and organs (Turner et al. 2014). On stimulation by IL-1 or TNF, endothelial cells, fibroblasts and epithelial cells express monocyte chemotactic protein-1 (MCP-1) (Deshmane et al. 2009). Biopsies injected with E. ocellatus venom exhibited elevated levels of MCP-1 in the initial two days, before reverting to normal. Meanwhile, biopsies injected with B. arietans venom displayed an increase in MCP-1 at 12 and 36 hours, whilst biopsies injected with N. nigricollis venom displayed maximum levels at three days, followed by a gradual decline to normal by the fifth day. Lastly, biopsies injected with N. haje venom showed an increase in MCP-1 on the first day. Moreover, injection with E. ocellatus, B. arietans and N. haje venom determined peak levels of the chemokines’ (Figure 3.8) macrophage inflammatory protein MIP1-ALPHA, RANTES at two days, followed by gradual reversal to normal by the fifth day. In biopsies injected with N. nigricollis venom, these chemokines increased at three days and reverted to normal after a further two days.

100

Figure 3.8. Dynamic changes in pro-inflammatory chemokines following subcutaneous injection by Viperidae venom (E. ocellatus and B. arietans) and Elapids venoms (N. nigricollis and N. haje). The Human Cytokine Magnetic 30-Plex Panel for the Luminex™ platform was performed in order to quantify the chemokines’ responses in the culture medium collected from the injected skin on Days 0 to 5. Four analytes simultaneously showed responses; (A) MCP-1, (B) RANTES, (C) MIP1-ALPHA, and (D) MIG. The sample collected from untreated skin tissue was used to compare with the pro-inflammatory chemokines’ response following the subcutaneous injection. (Av= antivenom).

101

In order to proliferate and differentiate, myogenic cells rely on a number of growth factors and cytokines produced by myogenic and inflammatory cells and those secreted from storage sites within the extracellular matrix (ECM) constituents of muscle tissue (Gutiérrez et al. 2018). These include hepatocyte growth factor (HGF) and fibroblast growth factor (FGF) (Karalaki et al. 2009). Biopsies injected with E. ocellatus and B. arietans venom, exhibited an increase in FGF after 12 hours and at two time points (one day and three days). Meanwhile, biopsies injected with N. nigricollis and N. haje venom, respectively, exhibited a minor FGF increase after 12-36 hours and no modification at all. It is notable that injection with B. arietans venom caused a significant increase in HGF (Figure 3.8). The development of blood vessels and vascular permeability are critically modulated by vascular endothelial growth factor (VEGF-A) and the related protein family (Shibuya 2011). Injection with E. ocellatus, B. arietans and N. nigricollis led to progressive increase in VEGF-A after two days, which peaked on the fourth day and reverted to normal on the fifth day. By contrast, injection with N. haje venom did not trigger a marked increase. The levels of proinflammatory cytokines, chemokines and growth factors reduced to normal levels after 48 hours of systemic and local injection of the antivenoms (both SAMIR polyvalent and SAIMR Echis a monovalent) in comparison with the samples collected from the skin injected with the venoms only.

102

Figure 3.9. Dynamic changes in growth factors following subcutaneous injection by Viperidae venom (E. ocellatus and B. arietans) and Elapid venoms (N. nigricollis and N. haje). The Human Cytokine Magnetic 30-Plex Panel for the Luminex™ platform was performed to quantify the growth factor responses in the culture medium collected from the injected skin on Days 0 to 5. three analytes simultaneously showed responses; (A) HGF, (B) FGF-Basic and (C) VEGF-A. The sample collected from untreated skin tissue was used to compare with the growth factors’ responses following the subcutaneous injection. (Av= antivenom).

103

3.4 Discussion

Strengths and weaknesses of the new ex vivo human skin model of envenoming

As stated earlier, the new ex vivo human skin model occupies the research space between clinical observations and animal models. An obvious weakness of this model is the inability to mimic intravenous interventions, which, in the field of snakebite therapy, places clear limits on the utility of the model. The need for experimentation within a day of biopsy excision, and the costs of the human skin biopsies, will inevitably restrict experimentation to well-funded groups close to surgical facilities. There were concerns that the lack of vascularisation of the ex vivo biopsies might exert physiological conditions restricting the duration of experiments and the cells/systems that can be validly examined. The distinctions between control and venom-injected biopsies in terms of skin morphology (histopathology analysis), cell death (caspase and TUNEL analysis) and skin-cell expression of a wide variety of cytokines, chemokines and growth factors (Multiplex luminex) identified that the biopsies were physiologically viable and responsive to tissue injury for at least six days after excision. The ability to capture these datasets from human skin tissue therefore represents a significant ‘clinical-relevancy’ advance over the murine models of local envenoming. The ability to interrogate, for up to five to six days, physiological responses of biopsies subjected to injurious substances that would be ethically forbidden in patients illustrates the significant clinical research utility of this model. Nevertheless, we have been careful to factor the lack of vascularisation into our interpretations of the results gained using this model.

Human skin responses to venoms of the most medically-important snakes of sub-Saharan Africa

The pathophysiological response to skin injury is a complicated process whereby cells and the extracellular matrix (ECM) are exposed to toxic substances and inflammatory mechanisms, which, while designed to limit injury, can inflict additional tissue damage. Standard empirical instruments are inappropriate for gaining insight into these complex interactions. As our knowledge of the tissue pathology following African snake envenoming is limited to clinical observations, we used this new ex vivo human skin model to identify (i) the most pathogenic snake venoms and toxin groups, (ii) the temporal pathophysiological response to this venom injury and (iii) the efficacy of antivenom to neutralise skin pathology by adding antivenom into the venom-injected biopsy culture media – in an attempt to mimic intravenous antivenom treatment.

104

The African snake venoms and toxin groups most pathogenic to ex vivo human skin

The Group II PLA2, SVMP, SVSP, CTL, disintegrin toxin groups dominate the composition of

Viperidae venoms (Gutiérrez et al. 2017). By contrast, Group I PLA2s and 3FTxs are the major components of Elapidae venoms, alongside reduced concentrations of other types of proteins (Calvete 2011). Gutiérrez et al. (2016) reported that the death of endothelial cells in culture was triggered by several SVMPs. Consistent with this, the present work observed that SVMPs and SVSPs were discharged by the venom of Viperidae into the culture medium. Functional biochemical assays confirmed that several toxic-enzymatic constituents were present in the culture medium. As noted by Kang et al. (2011), the occurrence of proteinases in venom can have a range of clinical effects during envenomation, including inflammatory events, tissue injury and abnormal coagulation and haemorrhage. Envenomation with E. ocellatus venom led to the release of abundant SVMPs in the skin, beginning in the initial 24 hours and between the third and seventh days, implying that tissue and endothelial damage was induced by SVMP release. The haemorrhagic SVMPs in the venom of Viperidae initiate tissue ischaemia due to the extensive extravasation accompanying the induced capillary vessel damage, and therefore they are involved in the development of myonecrosis (Gutiérrez & Rucavado 2000b).

SVSPs resemble thrombin in their fibrinogenolytic functional activities and many are capable of breaking down fibrinogen into fibrinopeptides through proteolytic cleavage, which is why they are also known as thrombin-like enzymes (TLEs) (Phillips et al. 2010). SVSP functional activity was observed in the culture medium derived from skin biopsy exposed to the venom of B. arietans, with release from the third until the seventh day post-administration. It was thus concluded that SVSPs played a role in dermal permeation by the venom, leading to systemic envenomation.

Empirical models have reported significant inflammation caused by PLA2 in venom (Teixeira et al. 2003). This protein has been implicated in various inflammation events, including oedema, infiltration of inflammatory cells and activation of mast cells (Wei et al. 2009). This could elucidate oedema formation in the present work. On the other hand, metalloproteases are known to have a role in both local and systemic effects of venom and may also induce oedema, as well as bleeding and muscle necrosis (Fernandes et al. 2007). Several authors have suggested that the defensive mechanisms of the body may be significantly reliant on oedema and inflammatory cell infiltration (Teixeira et al. 2003; Zuliani et al. 2005). In this work, the culture medium collected from N. nigricollis venom were subjected to

105 immunoblotting, but PLA2 was not found to occur in the culture medium, implying its participation in the local tissue damage induced by the venom of N. nigricollis. Lysophospholipids and unsaturated fatty acids are produced through the hydrolysis of phospholipids with unsaturated fatty acid tails at the sn-position by PLA2 (Kang et al. 2011). In turn, the hydrolysis products can induce extensive cellular pathological effects by altering the physical properties of cell membranes and activating downstream signal transduction pathways (Gasanov, Dagda & Rael 2014).

The temporal pathophysiological response of human skin to injury by venoms of African snakes

Envenomation-related inflammation mechanisms can be distinguished based both on the pathologies formed after subcutaneous injection of the venom of Viperidae and Elapidae, but also through measurement of pro-inflammatory markers like cytokines, chemokines and growth factors. Such mechanisms can help to achieve a better understanding of how venom exerts its effects, leading to more effective management of envenomation cases (Boda et al. 2018; Gutierrez et al. 2007; Moura-da-Silva, Butera & Tanjoni 2007). The chain of immune responses triggered by trauma, tissue injury or infection halts the damage, restricts external effects, inhibits microorganisms and induces repair mechanisms (Harris & Gelfand 1995). This section of the discussion explores the contribution of this study in each of these areas.

Morphology: The reported clinical manifestations of the venom of Viperidae and Elapidae, including oedema, blistering and dermal and subdermal necrosis (WHO 2010), were among the alterations in skin structure and morphology that were observed in the present work after subcutaneous venom injection. Dermal oedema formed quickly, followed by blisters after one week and then significant dermal necrosis and epidermis loss (Gutiérrez et al. 2005). Subsequently, there was shedding of the epidermis and the greater part of the dermis, leading to the formation of an ulcer and permeation of the damaged site by a hyaline fibrinoid material. It can thus be deduced that; besides the direct cytotoxicity of the venom, the development of dermonecrosis may be promoted by ischaemia accompanying thrombosis and vascular damage. Necrosis followed by epidermis and dermis shedding is a more probable reason than venom metalloproteinase or endogenous MMP activity as to why a significant proportion of collagen is lost from the dermis. This is consistent with clinical evidence (Warrell 2017; WHO 2010). The dermonecrosis caused by the venom of N. nigricollis was investigated by Rivel et al. (2016), who reported that initial dermis oedema degenerated into blistering, skin appendage loss and a decrease in cellularity. Dermis

106 necrosis became obvious by 24 hours, with loss of epidermis portions and permeation of the damaged sites by a fibrinoid hyaline material (Rivel et al. 2016). In the present work, subcutaneous injection of the human skin model did not produce any notable effects in the initial two days, highlighting that envenomation had distinct effects on human and animal and that more directly above.

Cytokine responses: Envenomation causes local tissue damage by triggering the cellular immune response whereby tissue macrophages and blood monocytes activate a reaction chain. Cytokines such as IL-1 (α or β) and TNF-α are among the key mediators released by activated macrophages (Akdis et al. 2011; Dinarello 2011). Cytokines participate in regulating every inflammatory response stage, hence they are crucial for interaction between cells (Arend & Gabay 2004). Cytokine involvement in local pathologies triggered by the venom of Viperidae and Elapidae was assessed in the current work based on the empirical model.

In the context of inflammation, the immune response and tissue repair unfold with the involvement of anti- and pro-inflammatory cytokines and other proteins released by leukocytes. In this work, the occurrence of venom-triggered local pathologies was signalled by increases in the pro-inflammatory cytokines IL-1β, IL-1RA, IL_12, G-CSF/CSF-3 and GM- CSF. The pro-inflammatory cytokine IL-1β not only regulates prostaglandin synthesis and chemokine release, but also activates lymphocytes, stimulates macrophages and promotes leukocyte attachment to endothelial cells (Rucavado et al. 2002). In this work, injection of the venom of E. ocellatus and B. arietans led to IL-1β elevation two to three days afterwards, whereas the venom of N. nigricollis and N. haje increased IL-1β only slightly after the initial day, before a decrease on the next day. Hyperalgesia, expansion of peripheral oedema and allodynia can all be triggered by IL-1β release (Fink 2005; Kulmatycki & Jamali 2001). Escocard et al. (2006) reported that the proteolytic activity of metalloproteinases in Bothrops atrox venom enabled cleavage of IL-1β, IL-6 and TNF-α. In the current work, IL-1β was 20 times higher compared to untreated biopsy due to venom SVMPs.

IL-1RA likely inhibits additional IL-1 activity and participates in inflammatory response cessation as IL-1RA elevation occurs after IL-1 elevation in the inflammatory response. In a study employing a mouse model, Polin et al. (2011) found that inflammation in different conditions was diminished by IL-1ra administration. Furthermore, the involvement of anti- inflammatory reactions in the regulation of inflammation is implied by the correlation of IL- 1RA increase and IL-1β increase.

107

Infection by different intracellular pathogens triggers IL-12 production by mature dendritic cells (DCs) (Brzoza, Rockel & Hiltbold 2004; Hunter 2005). IL-12 is considered a key mediator between the innate and adaptive immunity because it strongly affects activation of T cells, especially production of interferon gamma (IFN-γ), which is critically involved in intracellular pathogen eradication (Henry et al. 2008). In the present work, all biopsies injected with venom showed an increase in IL-12 until the fifth day post-injection. Moreover, with the venom of E. ocellatus and B. arietans, the effects of SVMP and SVSP on IL-12 maintained IL- 12 elevation on the fifth day after injection. Similarly, Delafontaine et al. (2017) indicated that IL-12 was targeted by SVMPs and SVSPs from Bothrops venom.

Neutrophil progenitors proliferate, differentiate and survive based on the activity of the primary cytokine granulocyte colony-stimulating factor (G-CSF). Mammalian mature neutrophils also rely on G-CSF for improved trafficking and immunological activities. Monocytes and macrophages, fibroblasts, endothelial cells and bone marrow stromal cells all generate G-CSF in humans (Katakura et al. 2019). Naito et al. (2009) reported that G-CSF therapy boosted repair and activation of established anabolic signalling pathways (e.g. Akt) in the skeletal muscle of mice injected with myonecrosis-inducing venom. This implies that muscle repair was promoted by elevated G-CSF, as attested by heightened levels of myogenic satellite cells. Pitzer et al. (2008) also noted that exogenous G-CSF therapy enhanced muscle repair and survival rates in a murine model of muscular dystrophy, while it also promoted motor function and increased muscle fibre size by 55% in a murine model of amyotrophic lateral sclerosis. In the present work, G-CSF increased two days after administration of N. nigricollis venom. Additionally, G-CSF exhibited heightened levels on day three following E. ocellatus venom injection and a minor increase after injection with B. arietans and N. haje venom.

Cells including macrophages, mast cells, T cells, fibroblasts and endothelial cells generate GM-CSF in reaction to the activation of an immune response and the release of pro- inflammatory cytokines. As well as occurring in the majority of tissues, GM-CSF also occurs in the extracellular matrix and as an integral membrane protein (Shi et al. 2006). Pathologies like wounds, tumour stimulation or inflammatory skin conditions trigger keratinocytes to release GM-CSF into the skin. Infiltrating cells (e.g. monocytes, macrophages, neutrophils, eosinophils, mast cells) and resident skin cells are among the cells targeted by GM-CSF (Mann et al. 2001). Currently, research has not been able to specify how GM-CSF acts, but the cytokine has been employed in a clinical setting as a drug regimen to diminish drug treatment-induced apoptosis of hematopoietic cells in the context of HIV (Hewitt et al. 1993),

108 while Kaplan et al. (1992) conducted an in vitro investigation showing that GM-CSF induced keratinocytes to proliferate and helped skin wounds to heal faster in cases of lepromatous leprosy. Furthermore, there is evidence that cytokine co-delivery improves cellular or humoral responses (Kim et al. 1997). In this work, GM-CSF increased within one to two days of administration of the venom of N. nigricollis and N. haje; GM-CSF secretion might be promoted by the high proportion of 3FTxs occurring in the venom of these two cobra species (Tasoulis & Isbister 2017). Based on the above observations, it is concluded that post- envenomation wound healing is signalled and aided by high GM-CSF levels.

Chemokines: Chemoattractants like chemokines are as important as adhesion molecules for the targeting processes of extravasation and tissue homing (Moser & Willimann 2004). Chemokines afford selective mediation of area-specific mobilisation of neutrophils, macrophages and lymphocytes owing to their property of target-cell specificity.

Preferential attraction of neutrophils and, potentially, lymphocytes is exhibited by C-X-C chemokines like IL-8, growth-related oncogene α (GROα), and epithelial-derived neutrophil attractant-78 amino acids (ENA-78) with the glutamin acid-leucine-arginine motif (Rudack et al. 2003). Conversely, preferential attraction of lymphocytes is exhibited by C-X-C chemokines without the glutamin acid-leucine-arginine motif, such as monokine induced by interferon-γ (MIG) and interferon-γ-inducible protein-10 (IP-10). Constituting the second chemokine subfamily, C-X-C chemokines encompass monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α and β (MIP-1α and β), and the regulated-on activation normal T cells expressed and secreted (RANTES). Regarding specificity for macrophages, lymphocytes and non-neutrophil granulocytes, a slight overlap is exhibited by this chemokine family (Morimoto et al. 2004; Widney et al. 2000).

In this work, N. nigricollis venom heightened the levels of the c-c chemokines MCP-1, MIP1- ALPHA and RANTES and the C-X-C chemokine MIG one day after being injected subcutaneously. The highest levels of MCP-1 and MIP1-ALPHA were recorded at two days, followed by reversal to normal on day three. B. arietans venom increased MIG at one day following injection before a gradual reversal to normal by day five. One reason for this is that different chemokine sets provide dynamic control of the movement and aggregation of key leukocytes in damaged areas, including neutrophils, monocytes/macrophages and lymphocytes, with fast upregulation immediately following wound formation. Employing a human blood model, Pitzer et al. (2008) observed that MIG and MCP-1 were generated in higher amounts due to the action of SVMPs from Bothrops pirajai venom. Empirical work

109 based on incisional adult skin wounds revealed that robust MCP-1 expression, beginning from the second day, was indicative of macrophage migration (Pitzer et al. 2008). Engelhardt et al. (1998) reported a correlation between migration of lymphocytes and expression of MCP-1, MIG and IP-10 after day four. In human skin wounds, MCP-1 seems to function mainly to chemoattract and activate monocytes and macrophages, which subsequently initiate production of cytokines conducive to growth under stimulation by other signals. DiPietro et al. (1995) used a human model to show that, unlike skin wound healing in mice, the inflammatory network underpinning wound repair in humans depended greatly on basal keratinocytes within the neighbouring epidermis along with MCP-1 production. In a different study employing a mouse model, MCP-1 expression only started to increase after day 16 of

C. adamanteus venom injection, due to the action of basic PLA2 in that venom (Samy et al. 2014). Such observations imply that human skin displays a high level of sensitivity and is prone to inflammation after envenomation.

Growth factors: Cells activate, differentiate and proliferate under stimulation by growth factors released by proteolytic processing of ECM constituents during inflammatory events, including insulin growth factor (IGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF), (Arroyo & Iruela-Arispe 2010). In terms of angiogenesis, constituents that both promote (e.g. vascular endothelial growth factor, VEGF) and inhibit (e.g. endostatin, endorepelin) this process can be released due to SVMP activity. Various mediators may therefore be activated when SVMPs hydrolyse the ECM, resulting in more significant tissue changes, disruption of the interplay between cells and matrix, stimulation and suppression of cell proliferation, and repair activity in the context of complicated tissue interaction (Gutiérrez et al. 2016). The present work observed that HGF, FGF and VEGF increased one day after injection of the venom of E. ocellatus and B. arietans. Such mediators signal the activity of SVMPs due to their targeting of the ECM. Meanwhile, VEGF increased only four days following injection of N. nigricollis venom, whilst N. haje venom did not elicit any modifications.

The efficacy of antivenom to neutralise local skin pathology induced by African snake venoms

The efficacy of antivenom in clinically counteracting dermonecrosis caused by N. nigricollis venom is unclear, with some researchers maintaining that such efficacy is usually poor (Warrell & Ormerod 1976), owing to suboptimal composition of antibodies targeting

110 cytotoxins or PLA2s in the antivenom, or incompatibility between venom toxicokinetics and antivenom antibody pharmacokinetics, with local tissue damage occurring before antibodies reach the site (Rivel et al. 2016). The present work employed the non-specific antivenom SAIMR polyvalent and a specific antivenom to counteract N. nigricollis and E. ocellatus venom, respectively. The antivenoms were subcutaneously and systemically injected 60 minutes after the venom was injected. Notably, there was no difference in local inflammatory response between the biopsies taken two days after administration of antivenom administration and those taken injection only with the venom.

Real envenomation settings were mimicked through intravenous antivenom injection straight after intradermal venom injection. The challenges posed by delayed antivenom treatment, as is frequently the case in sub-Saharan Africa, were highlighted by the fact that dermonecrosis was solely partially counteracted by a high dosage of IgG antivenom delivered straight after envenomation (Chippaux 2009). Given the difficulty of using antivenom to counteract local dermonecrosis caused by the venom of Elapidae and Viperidae, antivenom delivery immediately post-envenomation and the identification of new viable suppressors should be prioritised in a public health setting. The results obtained indicate that dermonecrosis caused by the venom of Elapidae and Viperidae could be counteracted by early antivenom administration, since IgG and F(ab’)2 antivenoms are available in Africa (Scheske, Ruitenberg & Bissumbhar 2015).

3.5 Conclusions Acute tissue damage triggered by snake results in inflammation which aggravates that damage through fibrillar collagen hydrolysis by MMPs and other endogenous proteinases stemming from resident tissue cells or invading leukocytes, leading to extensive ECM breakdown. Physiologically active protein fragments derived from ECM hydrolysis are involved in tissue changes and processes of inflammation and repair. Furthermore, cell behaviour may be influenced by tissue biochemical alterations caused by PLA2 or/ and SVMP- induced modifications in ECM stiffness. Under these circumstances, the identification of new

SVMP or PLA2 inhibitors and inflammation modulators demands comprehension of how envenomation-induced ECM breakdown occurs. Local tissue damage may be mitigated, thus minimising the sequelae suffered by victims of Viperidae envenomation, through an integrated approach that consists of fast delivery of SVMP and PLA2 inhibitors alongside administration of inflammation inhibitors and antivenom. Moreover, tissue repair and regeneration can be enhanced by acquiring greater knowledge and comprehension of ECM

111 damage caused by snakebite envenomation, thus diminishing the rate of morbidity that is associated with this neglected tropical disease.

112

3.6 References: Akdis, M., Burgler, S., Crameri, R., Eiwegger, T., Fujita, H., Gomez, E., Klunker, S., Meyer, N., O’Mahony, L. & Palomares, O. (2011) 'Interleukins, from 1 to 37, and interferon-γ: receptors, functions, and roles in diseases', Journal of allergy and clinical immunology, vol. 127, no. 3, pp. 701-721. Arend, W.P. & Gabay, C. (2004) 'Cytokines in the rheumatic diseases', Rheumatic diseases clinics of North America, vol. 30, no. 1, pp. 41-67. Arend, W.P. & Guthridge, C.J. (2000) 'Biological role of interleukin 1 receptor antagonist isoforms', Annals of the rheumatic diseases, vol. 59, no. suppl 1, pp. i60-i64. Arroyo, A.G. & Iruela-Arispe, M.L. (2010) 'Extracellular matrix, inflammation, and the angiogenic response', Cardiovascular research, vol. 86, no. 2, pp. 226-235. Bernardes, C.P., Menaldo, D.L., Mamede, C.C.N., Zoccal, K.F., Cintra, A.C.O., Faccioli, L.H., Stanziola, L., de Oliveira, F. & Sampaio, S.V. (2015) 'Evaluation of the local inflammatory events induced by BpirMP, a metalloproteinase from Bothrops pirajai venom', Molecular immunology, vol. 68, no. 2, pp. 456-464. Boda, F., Banfai, K., Garai, K., Curticapean, A., Berta, L., Sipos, E. & Kvell, K. (2018) 'Effect of Vipera ammodytes ammodytes Snake Venom on the Human Cytokine Network', Toxins, vol. 10, no. 7, p. 259. Bohlen, C.J., Chesler, A.T., Sharif-Naeini, R., Medzihradszky, K.F., Zhou, S., King, D., Sánchez, E.E., Burlingame, A.L., Basbaum, A.I. & Julius, D. (2011) 'A heteromeric Texas coral snake toxin targets acid-sensing ion channels to produce pain', Nature, vol. 479, no. 7373, p. 410. Bressenot, A., Marchal, S., Bezdetnaya, L., Garrier, J., Guillemin, F. & Plénat, F. (2009) 'Assessment of apoptosis by immunohistochemistry to active caspase-3, active caspase-7, or cleaved PARP in monolayer cells and spheroid and subcutaneous xenografts of human carcinoma', Journal of Histochemistry & Cytochemistry, vol. 57, no. 4, pp. 289-300. Brzoza, K.L., Rockel, A.B. & Hiltbold, E.M. (2004) 'Cytoplasmic entry of Listeria monocytogenes enhances dendritic cell maturation and T cell differentiation and function', The Journal of Immunology, vol. 173, no. 4, pp. 2641-2651. Bucaretchi, F., De Capitani, E.M., Vieira, R.J., Rodrigues, C.K., Zannin, M., Da Silva Jr, N.J., Casais-e-Silva, L.L. & Hyslop, S. (2016) 'Coral snake bites (Micrurus spp.) in Brazil: a review of literature reports', Clinical Toxicology, vol. 54, no. 3, pp. 222-234.

113

Calvete, J.J. (2011) 'Proteomic tools against the neglected pathology of snake bite envenoming', Expert review of proteomics, vol. 8, no. 6, pp. 739-758. Camargo, E.A., Esquisatto, L.C.M., Esquisatto, M.A., Ribela, M.T.C.P., Cintra, A.C., Giglio, J.R., Antunes, E. & Landucci, E.C.T. (2005) 'Characterization of the acute pancreatitis induced by secretory phospholipases A2 in rats', Toxicon, vol. 46, no. 8, pp. 921-926. Chippaux, J.P. (2009) 'Snakebite in Africa: current situation and urgent needs', Handbook of venoms and toxins of reptiles, pp. 453-473. Chiu, H.-F., Chen, J. & Teng, C.-M. (1989) 'Edema formation and degranulation of mast cells by a basic phospholipase A2 purified from Trimeresurus mucrosquamatus snake venom', Toxicon, vol. 27, no. 1, pp. 115-125. Clissa, P.B., Laing, G.D., Theakston, R.D.G., Mota, I., Taylor, M.J. & Moura-da-Silva, A.M. (2001) 'The effect of jararhagin, a metalloproteinase from Bothrops jararaca venom, on pro-inflammatory cytokines released by murine peritoneal adherent cells', Toxicon, vol. 39, no. 10, pp. 1567-1573. Costa, S.K.P., Camargo, E.A. & Antunes, E. (2017) 'Inflammatory Action of Secretory Phospholipases A 2 from Snake Venoms', Toxins and Drug Discovery, pp. 35-52. Câmara, P.R.S., Esquisatto, L.C.M., Camargo, E.A., Ribela, M.T.C.P., Toyama, M.H., Marangoni, S., De Nucci, G. & Antunes, E. (2003) 'Inflammatory oedema induced by phospholipases A2 isolated from Crotalus durissus sp. in the rat dorsal skin: a role for mast cells and sensory C-fibers', Toxicon, vol. 41, no. 7, pp. 823-829. Deshmane, S.L., Kremlev, S., Amini, S. & Sawaya, B.E. (2009) 'Monocyte chemoattractant protein-1 (MCP-1): an overview', Journal of interferon & cytokine research, vol. 29, no. 6, pp. 313-326. Dinarello, C.A. (2011) 'Interleukin-1 in the pathogenesis and treatment of inflammatory diseases', Blood, vol. 117, no. 14, pp. 3720-3732. Escalante, T., Rucavado, A., Pinto, A.F.M., Terra, R.M.S., Gutiérrez, J.M. & Fox, J.W. (2009) 'Wound exudate as a proteomic window to reveal different mechanisms of tissue damage by snake venom toxins', Journal of proteome research, vol. 8, no. 11, pp. 5120-5131. Farsky, S.H.P., Gonçalves, L.R.C., Gutiérrez, J.M., Correa, A.P., Rucavado, A., Gasque, P. & Tambourgi, D.V. (2000) 'Bothrops asper snake venom and its metalloproteinase BaP–1 activate the complement system. Role in leucocyte recruitment', Mediators of inflammation, vol. 9, no. 5, pp. 213-221.

114

Fernandes, C.M., de Fatima Pereira Teixeira, C., Leite, A., Gutiérrez, J.M. & Rocha, F.A.C. (2007) 'The snake venom metalloproteinase BaP1 induces joint hypernociception through TNF-α and PGE2-dependent mechanisms', British journal of pharmacology, vol. 151, no. 8, pp. 1254-1261. Ferraz, C.R., Calixto-Campos, C., Manchope, M.F., Casagrande, R., Clissa, P.B., Baldo, C. & Verri Jr, W.A. (2015) 'Jararhagin-induced mechanical hyperalgesia depends on TNF- α, IL-1β and NFκB in mice', Toxicon, vol. 103, pp. 119-128. Ferreira, T., Camargo, E.A., Ribela, M.T.C.P., Damico, D.C., Marangoni, S., Antunes, E., De Nucci, G. & Landucci, E.C.T. (2009) 'Inflammatory oedema induced by Lachesis muta muta (Surucucu) venom and LmTX-I in the rat paw and dorsal skin', Toxicon, vol. 53, no. 1, pp. 69-77. Fink, W.A. (2005) 'The pathophysiology of acute pain', Emergency Medicine Clinics, vol. 23, no. 2, pp. 277-284. Freitas-de-Sousa, L., Colombini, M., Lopes-Ferreira, M., Serrano, S. & Moura-da-Silva, A. (2017) 'Insights into the mechanisms involved in strong hemorrhage and dermonecrosis induced by atroxlysin-Ia, a PI-class snake venom metalloproteinase', Toxins, vol. 9, no. 8, p. 239. Gasanov, S.E., Dagda, R.K. & Rael, E.D. (2014) 'Snake venom cytotoxins, phospholipase A2s, and Zn2+-dependent metalloproteinases: mechanisms of action and pharmacological relevance', Journal of clinical toxicology, vol. 4, no. 1, p. 1000181. Gutierrez, J.M., Lomonte, B., León, G., Rucavado, A., Chaves, F. & Angulo, Y. (2007) 'Trends in snakebite envenomation therapy: scientific, technological and public health considerations', Current Pharmaceutical Design, vol. 13, no. 28, pp. 2935-2950. Gutiérrez, J., Escalante, T., Rucavado, A., Herrera, C. & Fox, J. (2016) 'A comprehensive view of the structural and functional alterations of extracellular matrix by snake venom metalloproteinases (SVMPs): novel perspectives on the pathophysiology of envenoming', Toxins, vol. 8, no. 10, p. 304. Gutiérrez, J.M., Calvete, J.J., Habib, A.G., Harrison, R.A., Williams, D.J. & Warrell, D.A. (2017) 'Snakebite envenoming', Nature reviews. Disease primers, vol. 3, p. 17063. Gutiérrez, J.M. & Rucavado, A. (2000a) 'Snake venom metalloproteinases: their role in the pathogenesis of local tissue damage', Biochimie, vol. 82, no. 9, pp. 841-850. Gutiérrez, J.M. & Rucavado, A. (2000b) 'Snake venom metalloproteinases: their role in the pathogenesis of local tissue damage', Biochimie, vol. 82, no. 9-10, pp. 841-850.

115

Gutiérrez, J.M., Rucavado, A., Escalante, T. & Díaz, C. (2005) 'Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage', Toxicon, vol. 45, no. 8, pp. 997-1011. Gutiérrez, J.M., Rucavado, A., Escalante, T., Herrera, C., Fernández, J., Lomonte, B. & Fox, J.W. (2018) 'Unresolved issues in the understanding of the pathogenesis of local tissue damage induced by snake venoms', Toxicon, vol. 148, pp. 123-131. Harris, B.H. & Gelfand, J.A. (1995) The immune response to trauma, vol. 4, Philadelphia, PA: WB Saunders Co., c1992-, pp. 77-82. Harris, J.B. & Scott-Davey, T. (2013) 'Secreted phospholipases A2 of snake venoms: effects on the peripheral neuromuscular system with comments on the role of phospholipases A2 in disorders of the CNS and their uses in industry', Toxins, vol. 5, no. 12, pp. 2533-2571. Henry, C.J., Ornelles, D.A., Mitchell, L.M., Brzoza-Lewis, K.L. & Hiltbold, E.M. (2008) 'IL-12 produced by dendritic cells augments CD8+ T cell activation through the production of the chemokines CCL1 and CCL17', The journal of immunology, vol. 181, no. 12, pp. 8576-8584. Hewitt, R.G., Morse, G.D., Lawrence, W.D., Maliszewski, M.L., Santora, J., Bartos, L., Bonnem, E. & Poiesz, B. (1993) 'Pharmacokinetics and pharmacodynamics of granulocyte-macrophage colony-stimulating factor and zidovudine in patients with AIDS and severe AIDS-related complex', Antimicrobial agents and chemotherapy, vol. 37, no. 3, pp. 512-522. Hifumi, T., Sakai, A., Kondo, Y., Yamamoto, A., Morine, N., Ato, M., Shibayama, K., Umezawa, K., Kiriu, N. & Kato, H. (2015) 'Venomous snake bites: clinical diagnosis and treatment', Journal of intensive care, vol. 3, no. 1, p. 16. Hunter, C.A. (2005) 'New IL-12-family members: IL-23 and IL-27, cytokines with divergent functions', Nature Reviews Immunology, vol. 5, no. 7, p. 521. Isbister, G.K. (2010) 'Antivenom efficacy or effectiveness: the Australian experience', Toxicology, vol. 268, no. 3, pp. 148-154. Isbister, G.K., Brown, S.G.A., Page, C.B., McCoubrie, D.L., Greene, S.L. & Buckley, N.A. (2013) 'Snakebite in Australia: a practical approach to diagnosis and treatment', Medical journal of Australia, vol. 199, no. 11, pp. 763-768. Juhn, S.K., Jung, M.-K., Hoffman, M.D., Drew, B.R., Preciado, D.A., Sausen, N.J., Jung, T.T.K., Kim, B.H., Park, S.-Y. & Lin, J. (2008) 'The role of inflammatory mediators in the

116

pathogenesis of otitis media and sequelae', Clinical and Experimental Otorhinolaryngology, vol. 1, no. 3, p. 117. Kang, T.S., Georgieva, D., Genov, N., Murakami, M.T., Sinha, M., Kumar, R.P., Kaur, P., Kumar, S., Dey, S. & Sharma, S. (2011) 'Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis', The FEBS journal, vol. 278, no. 23, pp. 4544-4576. Karalaki, M., Fili, S., Philippou, A. & Koutsilieris, M. (2009) 'Muscle regeneration: cellular and molecular events', In vivo, vol. 23, no. 5, pp. 779-796. Katakura, F., Nishiya, K., Wentzel, A.S., Hino, E., Miyamae, J., Okano, M., Wiegertjes, G.F. & Moritomo, T. (2019) 'Paralogs of common carp granulocyte colony-stimulating factor (G-CSF) have different functions regarding development, trafficking and activation of neutrophils', Frontiers in immunology, vol. 10, p. 255. Kim, J.J., Ayyavoo, V., Bagarazzi, M.L., Chattergoon, M.A., Dang, K., Wang, B., Boyer, J.D. & Weiner, D.B. (1997) 'In vivo engineering of a cellular immune response by coadministration of IL-12 expression vector with a DNA immunogen', The Journal of Immunology, vol. 158, no. 2, pp. 816-826. Kleggetveit, I.P., Skulberg, P.K. & Jørum, E. (2016) 'Complex regional pain syndrome following viper-bite', Scandinavian journal of pain, vol. 10, no. 1, pp. 15-18. Kulmatycki, K.M. & Jamali, F. (2001) 'Therapeutic relevance of altered cytokine expression', Cytokine, vol. 14, no. 1, pp. 1-10. Landucci, E.C.T., de Castro, R.C., Toyama, M., Giglio, J.R., Marangoni, S., De Nucci, G. & Antunes, E. (2000) 'Inflammatory oedema induced by the Lys-49 phospholipase A2 homologue piratoxin-i in the rat and rabbit: effect of polyanions and p- bromophenacyl bromide', Biochemical Pharmacology, vol. 59, no. 10, pp. 1289- 1294. Mamede, C.C.N., de Sousa, B.B., da Cunha Pereira, D.F., Matias, M.S., de Queiroz, M.R., de Morais, N.C.G., Vieira, S.A.P.B., Stanziola, L. & de Oliveira, F. (2016) 'Comparative analysis of local effects caused by Bothrops alternatus and Bothrops moojeni snake venoms: enzymatic contributions and inflammatory modulations', Toxicon, vol. 117, pp. 37-45. Mann, A., Breuhahn, K., Schirmacher, P., Wilhelmi, A., Beyer, C., Rosenau, A., Özbek, S., Rose-John, S. & Blessing, M. (2001) 'Up-and down-regulation of granulocyte/macrophage-colony stimulating factor activity in murine skin increase

117

susceptibility to skin carcinogenesis by independent mechanisms', Cancer research, vol. 61, no. 5, pp. 2311-2319. Mehta, S. & Sashindran, V. (2002) 'Clinical features and management of snake bite', Medical Journal, Armed Forces India, vol. 58, no. 3, p. 247. Menaldo, D.L., Bernardes, C.P., Pereira, J.C., Silveira, D.S.C., Mamede, C.C.N., Stanziola, L., de Oliveira, F., Pereira-Crott, L.S., Faccioli, L.H. & Sampaio, S.V. (2013) 'Effects of two serine proteases from Bothrops pirajai snake venom on the complement system and the inflammatory response', International immunopharmacology, vol. 15, no. 4, pp. 764-771. Morimoto, J., Yoneyama, H., Shimada, A., Shigihara, T., Yamada, S., Oikawa, Y., Matsushima, K., Saruta, T. & Narumi, S. (2004) 'CXC chemokine ligand 10 neutralization suppresses the occurrence of diabetes in nonobese diabetic mice through enhanced β cell proliferation without affecting insulitis', The Journal of Immunology, vol. 173, no. 11, pp. 7017-7024. Moser, B. & Willimann, K. (2004) 'Chemokines: role in inflammation and immune surveillance', Annals of the rheumatic diseases, vol. 63, no. suppl 2, pp. ii84-ii89. Moura-da-Silva, A.M., Butera, D. & Tanjoni, I. (2007) 'Importance of snake venom metalloproteinases in cell biology: effects on platelets, inflammatory and endothelial cells', Current pharmaceutical design, vol. 13, no. 28, pp. 2893-2905. Phillips, D.J., Swenson, S.D., Francis, S., Markland, J. & Mackessy, S.P. (2010) 'Thrombin-like snake venom serine proteinases', Handbook of venoms and toxins of reptiles, vol. 139, p. 154. Pitzer, C., Krüger, C., Plaas, C., Kirsch, F., Dittgen, T., Müller, R., Laage, R., Kastner, S., Suess, S. & Spoelgen, R. (2008) 'Granulocyte-colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis', Brain, vol. 131, no. 12, pp. 3335-3347. Rivel, M., Solano, D., Herrera, M., Vargas, M., Villalta, M., Segura, Á., Arias, A.S., León, G. & Gutiérrez, J.M. (2016) 'Pathogenesis of dermonecrosis induced by venom of the spitting cobra, Naja nigricollis: An experimental study in mice', Toxicon, vol. 119, pp. 171-179. Rucavado, A., Escalante, T., Teixeira, C.F.P., Fernandes, C.M., Diaz, C. & Gutierrez, J.M. (2002) Increments in cytokines and matrix metalloproteinases in skeletal muscle after injection of tissue-damaging toxins from the venom of the snake Bothrops asper, vol. 11, pp. 121-128.

118

Rucavado, A., Nicolau, C., Escalante, T., Kim, J., Herrera, C., Gutiérrez, J. & Fox, J. (2016) 'Viperid envenomation wound exudate contributes to increased vascular permeability via a DAMPs/TLR-4 mediated pathway', Toxins, vol. 8, no. 12, p. 349. Rudack, C., Maune, S., Eble, J. & Schroeder, J.-M. (2003) 'The primary role in biologic activity of the neutrophil chemokines IL-8 and GRO-α in cultured nasal epithelial cells', Journal of interferon & cytokine research, vol. 23, no. 2, pp. 113-123. Samy, R.P., Kandasamy, M., Gopalakrishnakone, P., Stiles, B.G., Rowan, E.G., Becker, D., Shanmugam, M.K., Sethi, G. & Chow, V.T.K. (2014) 'Wound healing activity and mechanisms of action of an antibacterial protein from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus)', PLoS One, vol. 9, no. 2, p. e80199. Scheske, L., Ruitenberg, J. & Bissumbhar, B. (2015) 'Needs and availability of snake antivenoms: relevance and application of international guidelines', International journal of health policy and management, vol. 4, no. 7, p. 447. Shi, Y., Liu, C.H., Roberts, A.I., Das, J., Xu, G., Ren, G., Zhang, Y., Zhang, L., Yuan, Z.R. & Tan, H.S.W. (2006) 'Granulocyte-macrophage colony-stimulating factor (GM-CSF) and T- cell responses: what we do and don't know', Cell research, vol. 16, no. 2, p. 126. Shibuya, M. (2011) 'Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) signaling in angiogenesis: a crucial target for anti-and pro-angiogenic therapies', Genes & cancer, vol. 2, no. 12, pp. 1097-1105. Sitprija, V. (2006) 'Snakebite nephropathy', Nephrology, vol. 11, no. 5, pp. 442-448. Sun, L., He, C., Nair, L., Yeung, J. & Egwuagu, C.E. (2015) 'Interleukin 12 (IL-12) family cytokines: Role in immune pathogenesis and treatment of CNS autoimmune disease', Cytokine, vol. 75, no. 2, pp. 249-255. Tasoulis, T. & Isbister, G. (2017) 'A review and database of snake venom proteomes', Toxins, vol. 9, no. 9, p. 290. Teixeira, C., Cury, Y., Moreira, V., Picolo, G. & Chaves, F. (2009) Inflammation induced by Bothrops asper venom, p. 988 Teixeira, C.d.F.P., Fernandes, C.M., Zuliani, J.P. & Zamuner, S.F. (2005) 'Inflammatory effects of snake venom metalloproteinases', Memórias do Instituto Oswaldo Cruz, vol. 100, pp. 181-184. Teixeira, C.F.P., Landucci, E.C.T., Antunes, E., Chacur, M. & Cury, Y. (2003) 'Inflammatory effects of snake venom myotoxic phospholipases A2', Toxicon, vol. 42, no. 8, pp. 947-962.

119

Turner, M.D., Nedjai, B., Hurst, T. & Pennington, D.J. (2014) 'Cytokines and chemokines: at the crossroads of cell signalling and inflammatory disease', Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, vol. 1843, no. 11, pp. 2563-2582. Wang, J.P. & Teng, C.M. (1990) 'Rat paw oedema and mast cell degranulation caused by two phospholipase A2 enzymes isolated from Trimeresurus mucrosquamatus venom', Journal of pharmacy and pharmacology, vol. 42, no. 12, pp. 846-850. Warrell, D.A. (1999) The clinical management of snake bites in the Southeast Asian region, Southeast Asian Journal of Tropical Medicine and Public Helath. Warrell, D.A. (2004) 'Snakebites in Central and South America: Epidemiology, clinical features, and clinical management. Bothrops lancelatus', The Venomous Reptiles of the Western Hemisphere. Cornell University, pp. 743-744. Warrell, D.A. (2017) Clinical toxicology of snakebite in Africa and the Middle East/Arabian Peninsula, in, Handbook of clinical toxicology of animal venoms and poisons, CRC Press, pp. 433-492. Warrell, D.A. & Ormerod, L.D. (1976) 'Snake venom ophthalmia and blindness caused by the spitting cobra (Naja nigricollis) in Nigeria', The American journal of tropical medicine and hygiene, vol. 25, no. 3, pp. 525-529. Wei, J.-F., Wei, X.-L., Mo, Y.-Z. & He, S.-H. (2009) 'Induction of mast cell accumulation, histamine release and skin edema by N49 phospholipase A 2', BMC immunology, vol. 10, no. 1, p. 21. WHO (2010) 'Guidelines for the prevention and clinical management of snakebite in Africa'. WHO (2019) Neglected tropical diseases [Online], World Health Organization, Available from: http://www.who.int/neglected_diseases/diseases/en/ (Accessed: 19th September). Who, A. (2010) 'Guidelines for the prevention and clinical management of snake bite in Africa', http://www. afro. who. int/index. php? option= com_docman&task= doc_download&gid= 5529. Widney, D.P., Xia, Y.-R., Lusis, A.J. & Smith, J.B. (2000) 'The murine chemokine CXCL11 (IFN- inducible T cell α chemoattractant) is an IFN-γ-and lipopolysaccharide-inducible glucocorticoid-attenuated response gene expressed in lung and other tissues during endotoxemia', The Journal of Immunology, vol. 164, no. 12, pp. 6322-6331. Zhang, C., Medzihradszky, K.F., Sánchez, E.E., Basbaum, A.I. & Julius, D. (2017) 'Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP

120

release', Proceedings of the National Academy of Sciences, vol. 114, no. 12, pp. E2524-E2532. Zuliani, J.P., Gutiérrez, J.M., e Silva, L.L.C., Sampaio, S.C., Lomonte, B. & Teixeira, C.d.F.P. (2005) 'Activation of cellular functions in macrophages by venom secretory Asp-49 and Lys-49 phospholipases A2', Toxicon, vol. 46, no. 5, pp. 523-532. Zychar, B.C., Dale, C.S., Demarchi, D.S. & Gonçalves, L.R.C. (2010) 'Contribution of metalloproteases, serine proteases and phospholipases A2 to the inflammatory reaction induced by Bothrops jararaca crude venom in mice', Toxicon, vol. 55, no. 2-3, pp. 227-234.

121

Chapter 4

Title: Defining acute phase and inflammatory responses to better understand the pathology of envenoming by medically important African snakes

Authors: Jaffer Alsolaiss1, Chloe A Evans1, Nicholas R Casewell1, Robert A Harrison1

Affiliations:

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

122

4.1 Introduction

Snakebite is a neglected tropical disease (NTD) that affects over 5 million people each year (WHO 2019). Of those bites, around 138,000 people suffer from mortality, and over 400,000 are disabled, disfigured or suffer from psychological disturbances (Chippaux 1998; Gutiérrez et al. 2017; Gutiérrez et al. 2010; WHO 2019). The burden of snakebite is greatest in the rural tropical regions of South and Southeast Asia, Latin America and sub-Saharan Africa (Kasturiratne et al. 2008). In Africa, over 32,000 deaths are thought to occur each year, and it suffers the greatest case fatality rate of any continent (Gutiérrez et al. 2017). In part, this is due to the restricted availability of snakebite therapies known as antivenom. Although these medicines save thousands of lives every year, they are costly to produce, are not effectively distributed and are typically not subsidised by national Ministries of Health (Habib et al. 2015). In addition, antivenom has variable efficacy against bites by different snake species and cause high rates of adverse reactions (de Silva, Ryan & de Silva 2016). In cases where treatment is available, victims and their families are burdened by the substantial cost of treatment and often suffer an ensuing loss of income due to disability (Harrison et al. 2009). Hence, in deprived rural tropical areas, poverty is both a cause and consequence of snakebite.

Medically important venomous snakes can be broadly grouped into two major families: Elapidae and Viperidae (Tasoulis & Isbister 2017). The Echis (saw-scaled or carpet vipers) and Bitis (puff adders) species of the Viperidae family are the most important cause of snakebite in the most impoverished regions of Africa (Habib & Warrell 2013). The clinical symptoms of viper envenomation include local oedema, bleeding and necrosis, and if the venom disseminates into the systemic circulation, spontaneous bleeding, coagulopathy and cardiovascular shock can occur (Gutiérrez et al. 2017). Dendroaspis (mambas) and Naja (spitting and non-spitting cobras) are medically important snake species from the Elapidae family. Venoms from this snake family are typically associated with a neurotoxic syndrome, typified by ptosis proceeding to respiratory paralysis. However, the spitting cobras (some snakes of the genus Naja) induce severe local cytotoxic effects, resulting in extensive swelling and local tissue damage. Pathogenesis in each of these groups is caused by the combined action of different venom components, most notably those from four major toxin families: the snake venom metalloproteinases (SVMPs), snake venom serine proteases (SVSPs), phospholipase A2 (PLA2), and three-finger toxins (3FTxs) (Tasoulis & Isbister 2017). Venom variation is inherent among snake species, and major differences occur between snake

123 families (e.g. viperid venoms are abundant in SVMP, SVSP and PLA2 vs the 3FTx and PLA2 toxins abundant in elapid venoms) (Casewell et al. 2014a; Tasoulis & Isbister 2017).

One of the first observed effects of snake envenoming is local inflammation at the bite site, manifesting as heat, pain, redness and/or swelling within two hours (Warrell 2005). This response is a fundamental part of the body’s defence system, intended to generically protect against injury and stimulate tissue repair, whilst hindering the systemic spread of foreign bodies. During this protective physiological process, myriads of inflammatory mediators are released at the inflammation site, including alarmins, histamines, chemokines and cytokines (e.g. interleukin 1 [IL-1], interleukin 6 [IL-6], and tumour necrosis factor alpha [TNF-α]) (Arango Duque & Descoteaux 2014). These mediators cause increased vascular permeability, vasodilation and chemotaxis, which ultimately facilitates the diffusion of plasma and leucocytes to the interstitial tissue (Teixeira et al. 2003). In addition, a non-specific systemic reaction termed the acute phase response (APR) is also initiated in responses to injury, which results in major alterations in the concentration of liver-generated plasma proteins (Gruys et al. 2005). Two types of plasma proteins encompass this response: positive acute-phase proteins, which are elevated during APR (e.g. C-reactive protein [CRP], serum amyloid A (SAA), fibrinogen and α-globulins), and negative acute-phase proteins, which are reduced (e.g. albumin) (Cray, Zaias & Altman 2009). Quantification of individual acute-phase proteins can provide relative insight into the magnitude of the triggering event, although the most pertinent acute-phase proteins differ amongst species (Cray, Zaias & Altman 2009). In addition, various physiological and biochemical modifications accompany the APR, including increased levels of circulating leucocytes, activation of complement and blood coagulation, and biosynthesis of adreno-corticotropic hormones (Baumann & Gauldie 1994; Stone et al. 2013).

Human inflammatory responses to snakebite envenomation have been comprehensively examined in relatively few studies (Avila-Aguero et al. 2001; Barraviera et al. 1995b; Stone et al. 2013), and only Barraviera et al. (1995) investigated standard acute phase responses. Stone et al. (2013) found envenoming by Daboia russelii (Russell’s viper; n=113) to be associated with elevations in anaphylatoxins and pro- and anti-inflammatory cytokines [IL-6 and interleukin-10 (IL-10)], whilst Avila-Agüero et al. (2001) identified elevations in IL-6, TNF- α, and IL-8 in just nine of eighteen victims of Bothrops (pit viper) envenoming in their study. Barraviera et al. (1995) assessed several parameters in victims of Bothrops (n=16) and Crotalus (n=15) envenoming and detected elevated levels of IL-6 and IL-8, but no significant change in IL-1β in either type of envenoming. The authors also reported marked leucocytosis

124 with neutrophilia and lymphopenia and significant changes in Acute Phase proteins (APPs), although only four patients were assessed for changes in C-reactive protein (CRP) – a key biomarker of the APR and inflammation in humans – and no other APPs were investigated (Sproston & Ashworth 2018).

Due to the frequency of envenomation, slightly more research effort relates to responses in veterinary cases of snakebite, including some examples from snake species of medical importance to Africa (de sousa-e-Silva et al. 2003; Langhorn et al. 2014; Lobetti & Joubert 2004; Nagel et al. 2014; Nogueira & Sakate 2006). Utilising CRP as a marker of systemic inflammation, Laghorm et al. (2014) investigated three groups of dogs envenomed by Vipera berus (European Viper; n=24), Bitis arietans (African puff adder; n=5) and Naja annulifera (snouted cobra; n=9), and found that the median CRP value increased significantly from admission to 12 hours post-admission amongst all groups, corresponding to a time- dependent systemic inflammatory response in 83%, 100%, and 86% of dogs in each respective group. Nagel et al. (2014) also investigated the haemostatic and inflammatory responses in dogs envenomed by the African puff adder (n=9) and snouted cobra (n=9) at admission and 24 hours later, and the study showed systemic fibrinogen and CRP concentrations were significantly elevated in both groups.

Supplementing these relatively sparse clinical studies is data from in vivo studies using animal models of envenoming (summarised in Supplementary Table S 4.1). For example, two studies previously used mouse models of Bothrops atrox (common lancehead) envenoming to elucidate the mechanism by which venom-induced local pathologies associated with inflammation are caused (Rucavado et al. 2016). Rucavado et al. (2016) performed proteomic analysis of wound exudate following B. atrox injection into mice, and found an abundance of cytokines, chemokines and damage associated molecular patterns (DAMPs). DAMPs are endogenous molecules, derived from the proteolysis of proteins by venoms toxins and host proteinases, and they generate most of their activity through interaction with toll-like receptors (TLRs). When a TLR4 antagonist was used as a pre-treatment in mice, reduced vascular permeability was observed. Further to this, Moreira et al. (2016) injected wild-type and TLR2 gene knockout mice with B. atrox venom, and found the knockout mice had higher levels of IL-6 and monocyte chemoattractant protein-1 (CCL2), as well as an accumulation of polymorphonuclear cells. Together, these findings indicate that venom proteins induce and modulate inflammation at the wound site through TLR4 and TLR2 signalling respectively, and this signalling pathway offers some level of regulation. However, to our knowledge, the systemic responses to envenoming in this model remain unknown,

125 and the majority of these experimental small animal studies (Supplementary Table 1) relate to envenoming by snakes of just two genera (Crotalus and Bothrops).

Consequently, our study utilised a murine model of envenoming to characterise the acute inflammatory and acute phase responses associated with medically-important African snake venoms that cause distinct pathologies. In addition, we supplemented these experiments with human in vitro whole blood assays to assess whether our murine findings could be replicated in model’s representative of human envenoming. Here we show that N. nigricollis and N. melanoleuca venoms drive a strong APR in mice, and we present evidence of the complex diversity of pathologies that can manifest following envenoming, including haemostatic changes, haemolysis, and damage to vital organs. This data provides evidence that inflammation is a systemic pathology of snake envenomation that is not restricted to cytotoxic snake venoms, and a further understanding of these processes could facilitate future effective treatment of snakebite.

126

4.2 Materials and methods

4.2.1 Snake venoms

Lyophilised venoms from medically-important African snake species were donated by Biological E. Limited, India. The venoms were from: (i) four vipers: Bitis gabonica (Gaboon viper), B. arietans (puff adder), Echis ocellatus (West African saw-scaled viper) and E. leucogaster (white-bellied carpet viper), and, (ii) six elapids: Naja haje (Egyptian cobra), N. melanoleuca (forest cobra), N. nigricollis (black-necked spitting cobra), Dendroaspis polylepis (black mamba), D. viridis (Western green mamba) and D. jamesoni (Jameson’s mamba). Lyophilised venoms were stored long-term at 4°C, before reconstitution in phosphate- buffered saline (PBS, pH 7.2) ready for use, with stocks stored short-term at -20°C.

* Note: N. melanoleuca snake has been split up recently into four species by Wüster et al. 2018. The venom used in this study is now from Naja subfulva.

4.2.2 Animals and research design

Murine in vivo experiments were undertaken with approvals from The Animal Welfare and Ethical Review Boards of the Liverpool School of Tropical Medicine and the University of Liverpool, and the UK Home Office, under the Animals (Scientific Procedures) Act 1986. In keeping with assay recommendations by WHO (2016), first the median lethal dose (LD50) for each venom was determined by intravenously (tail vein) injecting different venom doses in 100 μl volumes into groups of five male CD-1 mice (20-22 g; Charles River, UK). In addition, a control group (n=2) received 100 μl of PBS instead of venom. Experimental animals were monitored for the duration of the experiment for signs of severe envenoming and were euthanised if external signs of haemorrhage, seizure, pulmonary distress or paralysis were observed. Survival was documented six hours post-envenomation (end of experiment). Following euthanasia, either due to the effects of envenoming or at the end of the experiment, blood samples were collected via cardiac puncture and stored in sterile conical vials with no anticoagulant. Immediately after blood collection, a thin blood film (n = 50) was processed for every mouse that survived until the end of the experiment, while the remainder of the blood sample (n = 50) was centrifuged for 10 minutes (9,600 x g at 4°C) and sera collected and stored in aliquots at -20°C. For serum and blood film analysis, we selected the LD50 (See Table 4.1).

127

Figure 4.1. The Experimental outline of the in vivo murine acute phase model to study the acute envenoming by Acute Phase Reactants (APR) and acute inflammatory mediators. (details of the experiment see 4.2.2).

Table 4.1: LD50 doses of African snake venoms in mice was used for blood and sera analyses

Venom LD50 doses (µg/mouse) (95 CI) Bitis gabonica 18.4 (17.4- 19.53) Bitis arietans 17.7 (17.24- 18.29) Echis leucogaster 56 (31.1- 100.5) Echis ocellatus 58 (33.01- 101.94) Naja haje 6.96 (5.36- 9.07) Naja melanoleuca 14.5 (13.57- 15.51) Naja nigricollis 27.5 (25.23- 29.97) Dendroaspis polylepis 11.5 (10.524- 12.576) Dendroaspis viridis 13.2 (11.79- 14.82) Dendroaspis jamesoni 54 (46.99- 61.81)

128

4.2.3 Blood film preparation and haematological analysis

After initial collection, blood films were left to air-dry overnight, fixed for 60-seconds in methyl alcohol and stained with 10% Giemsa. A Leica ICC50W microscope was used to morphologically profile the resulting films, including manual counting of leucocytes, erythrocytes and platelets in a cell monolayer, with documentation of cell irregularities, such as erythrocyte agglutination or platelet aggregation. As we were unable to generate total platelet counts as platelets were often aggregated for accurate estimation, we qualitatively assessed the levels of platelet clumping using the following grading system: i) no aggregation, <1 small colony/field; ii) mild aggregation, 2-5 colonies/field and/or small colonies only; iii) moderate aggregation, 2-8 colonies/field and/or small and medium colonies; iv) marked aggregation, >5 colonies/field and/or medium and large colonies present. Small, medium and large colonies were defined as 2-10 platelets, 11-20 platelets and >20 platelets, respectively.

4.2.4 Quantification of C-reactive protein and cytokines by multiplex bead array

The CRP Mouse ProcartaPlex™ Simplex Kit was used for serum CRP analysis, while the Th1/Th2 Cytokine 11-Plex Mouse ProcartaPlex™ Panel (both ThermoFisher Scientific, Vienna, Austria) was employed for serum cytokine analysis. All samples and standards were run in duplicate and for both approaches we used the manufacturer’s instructions with minor modifications, as follows: (i) Universal Assay Buffer (1x) was used for three-fold dilutions of the sera samples for the CRP simplex immunoassay, but the samples were not diluted for the cytokine multiplex immunoassay, (ii) every incubation step involved shaking the 96-well plate at 220 rpm, and (iii) after beads, serum, standards and PBS were added, the mixture was left to incubate overnight. Luminex® 100/200 technology (Mag-Plex®-Avidin Microspheres) was used for measuring bead fluorescence, and if the count did not exceed 100 or was not within the identifiable range, the data was excluded from downstream analysis. ProcartaPlex Analyst software was used for the analysis of results and the mean and standard deviation for each group was calculated.

4.2.5 Quantification of serum amyloid A, P-selectin and murine IgM by ELISA

The PHASE™ SAA Murine Assay Kit (Tridelta Development Limited, Ireland), P-selectin Mouse ELISA Kit (ThermoFisher Scientific, Vienna, Austria) and Mouse IgM ELISA Ready-SET-Go! ™ Kit (Invitrogen™ eBioscience™, ThermoFisher Scientific, Vienna, Austria) were used in accordance with manufacturer’s guidelines to quantify serum amyloid A, P-selectin and

129 murine IgM. Sera dilutions were performed in the supplied buffers for SAA, P-selectin and IgM at 1:200, 1:500 and 1:20, respectively. All samples were analysed in duplicate. Resulting absorbance measurements were taken at 450 nm (SAA, P-selectin) and 570 nm (murine IgM) using the LT-4500 microplate absorbance reader (BMG LabTech). The mean values and standard deviation for each group was recorded.

4.2.6 Quantification of haptoglobin by colorimetric assay

We used the colorimetric PHASE™ Haptoglobin Assay (Tridelta Development Limited, Ireland) to quantify serum haptoglobin. The supplied stock calibrator and diluent were used for manual preparation of haptoglobin standards. Samples were not diluted before assessment and each sample was run in duplicate. After the samples were incubated with chromogen for five minutes, absorbance was read at 620 nm with an LT-4500 automatic microplate absorbance reader. For each group, the mean and standard deviation was calculated.

4.2.7 Standard biochemical analysis of serum samples

To quantify markers related to liver function, cardiac damage and renal function, we used an automated clinical chemistry analyser (Roche cobas c 701). All enzymatic and colorimetric reagent kits, calibrators and controls were supplied from Roche Diagnostics UK Ltd. The mouse sera were diluted 1:20 before duplicate analysis alongside non-envenomed controls. Recording of the following analytes were performed using colorimetric and enzymatic methods: (i) liver function tests; alanine aminotransferase (ALT), total protein (TP), albumin and total bilirubin (TB); (ii) cardiac enzymes; creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST); (iii) renal function tests; creatinine (CRT).

4.2.8 Collection of human blood for in vitro assays

Blood samples were collected from healthy human volunteers in to tubes containing different anticoagulants (acid citrate dextrose adenine [ACD-A] and ethylenediaminetetraacetic acid [K3-EDTA]). Blood samples were obtained according to ethically approved protocols (LSTM research tissue bank, REC ref. 11/H1002/9), and all volunteers confirmed they had not taken any anticoagulant treatments for at least three months prior to blood collection.

130

4.2.8.1 Quantifying venom haemolytic activity with human erythrocytes

Fresh human whole blood from healthy volunteers was collected and used to determine venom toxicity to erythrocytes. The red blood cells (RBCs) were collected in K3-EDTA and 100 μl of RBCs were first washed three times with 3 ml of PBS. Next, 100 μl of washed RBCs were resuspended with 3 ml PBS and added (100 μl/well) to 96-well microtiter plates (Nunc). Next, the RBCs in each well were overlaid with the LD50 dose of each venom in 100 μl PBS in duplicate. The plates were incubated for 3 hours at 37°C and then centrifuged for five minutes at 1000 x g. Following this, 100 μl of culture medium from each sample was transferred to new 96-well microtiter plate (Nunc) and an LT-4500 automatic microplate absorbance reader was used to generate haemolysis readings at 405 nm.

4.2.8.2 Quantifying venom-induced platelet aggregation with human platelets

To separate platelets, plasma and other blood components from human blood, we used a standard platelet-rich plasma (PRP) separation technique (Dhurat and Sukesh, 2014), whereby ACD-A blood samples were subjected to an 11-minute centrifugation step at 150 x g. Following collection of the culture medium, platelets were manually counted to confirm normal levels (350 x 109 platelets per microliter of blood) (Daly 2011). For the platelet aggregation assay, 96-well microtiter plates (Nunc) were loaded with 72 μl/well platelet-rich plasma and incubated with 8 μl of the LD50 dose of each venom in duplicate. Next, we used a FLUOstar Omega microplate reader for kinetic measurement of the absorbance of the wells at 405 nm (40 mins at 37oC). Samples containing venom only and platelets only served as negative controls, while 72 μl of platelet was incubated with 8 μl of 50 µg/ml human placenta collagen (Sigma-Aldrich®, United Kingdom) served as positive control. Absorbance was plotted against time and platelet aggregation was measured by calculating the mean AUC of mean readings.

4.2.8.3 Quantification of venom-induced release of human haemoglobin via colorimetric assay

Cyanmethemoglobin techniques were employed for haemoglobin (Hb) measurements. First, a standard curve was prepared by diluting different concentrations of commercial Cyanmethemoglobin Standard Solution (180 mg/ml, 120 mg/ml, 60 mg/ml and blank) of appropriate haemoglobin in Drabkin’s Solution (used for quantitative, colorimetric determination of haemoglobin). The mixture was then incubated at room temperature for 5 minutes and before absorbance was read at 540 nm using an LT-4500 automatic microplate

131 reader (BMG LabTech). The resulting absorbances were plotted to produce a standard curve (absorbance vs cyanmethemoglobin concentration (mg/ml). For venom experiments, human blood from healthy volunteers was collected in 4 ml ethylenediaminetetraacetic acid [K3- EDTA] tubes. Subsequently, the collected RBCs in EDTA were washed (three times in PBS). Washed RBCs were then resuspended in PBS (1:1), and 100 μl aliquots were incubated with

100 μl of the LD50 dose (Table 4.1) of each venom for 60 minutes at 37°C. High (21 g/dl) and low (6.0 g/dl) controls of Hb (Sigma-Aldrich®, United Kingdom) and washed RBCs with no venom were also used. All samples were mixed before 10 μl of each sample was added to 625 μl Drabkin’s solution (Sigma-Aldrich®, United Kingdom) in duplicate and incubated for 20 mins at room temperature. The resulting absorbances were measured at 540 nm using an LT-4500 automatic microplate reader (BMG LabTech). The formula from the standard calibration curve was used to quantitate the Hb levels in each sample.

4.2.9 Statistical analyses

For all analyses, the significance of the differences of mean values between experimental groups was assessed by analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test to compare means between experimental groups (mice injected with venom) and controls (mice injected with PBS). A P value < 0.05 was considered significant for all statistical tests. All analyses were conducted using GraphPad Prism 7.0.

132

4.3 Results

4.3.1 Haematological analyses of envenomed mice

4.3.1.1 RBC abnormalities suggest venom-induced haemolysis in mice

Groups of mice were injected with LD50 dose of ten medically important snake venoms and after 6 hours, blood samples were taken from each mouse and used to generate blood films. We microscopically analysed blood films (100x objective) of PBS-injected mice (normal control group) to generate a standard for RBC morphology (Figure 4.2A). Comparison of these with envenomed murine samples revealed prominent changes in the cytological features of RBCs, indicating various pathophysiological mechanisms. For example, B. arietans (Figure 4.2B) envenoming caused echinocyte (spiculated RBC) formation which is likely due to phospholipase-induced membrane abnormalities (Walton et al. 1997), and D. jamesoni (Figure 4.2C) venom caused dacrocyte (tear-drop RBC) formation, which are typical of mechanical trauma. The most pronounced pathology however, was hyperchromic microspherocytosis (RBC whose diameter is less than normal, but thickness is increased), which was observed in 8 out of the 10 species investigated (both Bitis species, E. ocellatus, all three Naja species, and D. viridis and D. jamesoni), though this was most prominent in N. nigricollis (Figure 4.2D) and N. melanoleuca envenomed mice. These findings are indicative of red blood cell destruction, which can occur within the vascular system (intravascular haemolysis) or within the spleen or liver (extravascular haemolysis). Ghost cells (post- haemolytic cell-like structure) were also observed in all cases exhibiting spherocytosis, suggesting a loss of haemoglobin in to the extracellular space (Hoffman 1992).

To relate these findings to direct venom activity, we incubated human red blood cells with the same venom doses determined from our murine in vivo study (Table 4.1) and used separate in vitro assays to quantify: (i) the haemolytic activity of each of the venoms on RBCs, and (ii) the Hb content of venom-treated RBCs. Our findings demonstrated that all ten snake venoms caused a decrease in Hb compared to the control (RBCs incubated with PBS) (Figure 4.2E). While these reductions are relatively small (e.g. 86.9-97.7 % of the control), eight of the ten venoms (B. arietans, E. ocellatus, N. haje, N. melanoleuca, N. nigricollis, D. polylepis, D. viridis, D. jamesoni) were found to be statistically significant (P < 0.05; one-way ANOVA, Dunnett’s multiple comparisons test). For the haemolysis assay, PBS and Triton™ X-100 were used to generate negative and positive controls respectively, representing 0% and 100% haemolysis (Figure 4.2F). N. nigricollis venom caused marked haemolytic activity (86.8 %) which was significantly greater than the negative control (P < 0.05; one-way ANOVA,

133

Dunnett’s multiple comparisons test). All other venoms exhibited a low level of direct haemolytic activity at these doses (1.7-5.3 %), none of which were significantly elevated compared with the negative control.

134

Figure 4.2 Abnormal red blood cell morphology and haemolysis induced by snake venoms. Mice were injected with venom or PBS (normal), and after 6 hours, thin blood smears were made, and RBC morphology was observed at x 100 objective. Representative blood smears are shown. (A) non-envenomed control. (B) B. arietans envenoming caused RBC membrane abnormalities, illustrated by echinocytes (E) and helmet cells (H), and cell fragmentation [schistocytes (Sch)]. Ghost cells (G) are present, indicating loss of hemoglobin due to hemolysis; (C) D. jamesoni venom caused codocyte (C), stomatocyte (St), and dacrocyte (D) formation; (D) N. nigricollis venom shows hyperchromic microspherocytosis. Human RBCs were incubated with the same LD50 dose of venom and used in vitro absorbance assays to: (E) determine haemoglobin levels (cyanmethemoglobin technique), which were slightly decreased in most cases (B. arietans, E. ocellatus, N. haje, N. melanoleuca, N. nigricollis, D. polylepis, D. viridis, D. jamesoni) compared to the negative control (RBCs incubated with PBS); and (F) determine the percentage of direct haemolysis, using PBS (0% activity) and Triton X-100 (100% activity) as negative and positive control respectively. N. nigricollis venom caused significant haemolysis (86.8 %) compared to the PBS control, whilst all other venoms caused low level of haemolytic activity (1.7-5.3 %). Key: Venoms; BG (B. gabonica), BA (B. arietans), EL (E. leucogaster), EO (E. ocellatus), NH (N. haje), NM (N. melanoleuca), NN (N. nigricollis), DP (D. polylepis), DV (D. viridis), DJ (D. jamesoni). Parameters from venom-incubation groups (n = 3) were compared to those from the negative control (PBS) by Dunnett’s multiple comparison test. Significant values are indicated by *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. Bars represent means of triplicate measurements, and error bars represent.

135

4.3.1.2 N. nigricollis, N. melanoleuca, and B. arietans venoms cause neutrophilia in mice

White blood cells (WBCs) are major components of the inflammatory process and as such, estimation of the total WBC count and differentiation of cell types is typically used as an early indicator of the APR (Chmielewski & Strzelec 2018). We analysed blood films at 40x objective for total WBC counts, and then at 100x objective to generate an absolute WBC count describing the differential WBCs (Figure 4.3). The control group had a mean total count of 12,400/μl with a distribution of differential cell types within the reference range for mice: 72–76% lymphocytes, 11–12% neutrophils, 7–9% monocytes, 6–7% basophils, and <1% eosinophils (assessed online https://phenome.jax.org/strains/12). We found that WBC counts were greatly reduced (P < 0.05; one-way ANOVA, Dunnett’s multiple comparisons test) in mice envenomed with N. haje, D. polylepis, D. viridis, D. jamesoni and B. gabonica venoms, with N. haje venom resulting in the lowest mean count (3,480/μl) (Figure 4.3A). With the exception of D. viridis, for which there was a high level of inter-group variation observed, all of the aforementioned venoms also caused a significant reduction in the number of lymphocytes (Figure 4.3B; P < 0.05) compared to the control and, with the addition of E. leucogaster venom, all of these venoms also caused decreased basophil counts (Figure 4.2C; P < 0.05). Contrasting with these findings, the total WBC counts for B. arietans, N. melanoleuca and N. nigricollis envenomed animals were elevated (Figure 4.3A), and their venom caused significant increases in neutrophil counts which were 3-5 times higher than the control (Figure 4.3D; P < 0.05). In the case of N. nigricollis, absolute monocyte counts were also significantly elevated (Figure 2E; P < 0.05). Furthermore, the eosinophil levels were highly elevated only in mice injected with N. nigricollis and D. viridis compared to the control group (Figure 4.3F).

136

Figure 4.3. B. arietans, N. nigricollis, and N. melanoleuca venoms cause increased WBC counts and elevated neutrophils. Groups of mice were envenomed with ten medically important snake venoms and after 6 hours blood samples were used to make blood smears, which were stained with Giemsa and observed at x 40 objective to complete the (A) total white blood cell (WBC) count, and x 100 objective to complete the absolute counts of (B) neutrophils, (C) lymphocytes, (D) monocytes, (E) basophils, and (F) eosinophils (number of cells per µl blood). B. gabonica, N. haje, D. polylepis, D. viridis, and D. jamesoni caused significant reductions in the total levels of circulating leucocytes, which for all, except D. viridis, also caused a significant reduction in the number of lymphocytes. Basophil levels were also decreased in all of these groups. Meanwhile, B. arietans, N. melanoleuca, and N. nigricollis caused increased total WBC counts, including significant elevations in neutrophils in all cases and elevated monocytes in mice injected with N. nigricollis venom. Eosinophils were low in all cases. Venoms: BG (B. gabonica), BA (B. arietans), EL (E. leucogaster), EO (E. ocellatus), NH (N. haje), NM (N. melanoleuca), NN (N. nigricollis), DP (D. polylepis), DV (D. viridis), DJ (D. jamesoni). Parameters from envenomed groups were compared to those from the control group (Normal: injected with PBS) by Dunnett’s multiple comparison test. Significant values are indicated by *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. Bars represent means of duplicate counts, and error bars represent.

137

4.3.1.3 Mouse thrombocytosis following envenoming with six elapid venoms

Platelets are active cells in inflammation and haemostasis, and are known to exert their effects on lymphocytes, neutrophils and monocytes (Ferrer-Acosta et al. 2014). Consequently, we examined the effect of venoms on platelet aggregation via the analysis of blood films sourced from envenomed mice. As murine platelets have a propensity to clump (O'Connell et al. 2015), we were unable to accurately estimate total platelet counts, as the normal samples studied here had artifactually low counts due to a high level of platelet clumping. Instead, we qualitatively assessed the level of platelet clumping, with the results displayed in Table 4.2. The normal had marked platelet clumping, as described by either 2 to 3 small colonies (5-8 platelets/colony). For B. gabonica. N. melanoleuca, N. nigricollis, and D. jamesoni envenomed mice (3+ colonies per field, and/or medium (11-20 platelets/colony), and thus we cannot determine whether envenoming affected platelet aggregation. Contrastingly, B. arietans and B. gabonica venom appeared to potently inhibit platelet aggregation, as there were less than one small colonies per field. E. leucogaster and N. haje venoms also caused inhibition, as only mild platelet aggregation was observed, whilst E. ocellatus, D. polylepis, and D. viridis venom caused a small reduction in aggregation compared with the control (Supplementary Figure S 4.1 examples of platelet aggregation observed in the blood film).

138

Table 4.2. Grading of platelet aggregation in mice injected with medically important snake venoms. Platelet aggregation was recorded according to relative size and distribution of platelet colonies per field of view (x 100 objective): i) no aggregation, <1 small colony/field; ii) mild aggregation, 2-5 colonies/field and/or small colonies only; iii) moderate aggregation, 2-8 colonies/fi eld and/or small and medium colonies; iv) marked aggregation, >5 colonies/field and/or medium and/or large colonies present. Small, medium and large colonies were defined as 2-10 platelets, 11-20 platelets and >20 platelets, respectively.

Platelet aggregation Snake species Comments No Mild Moderate Marked Mild platelet aggregation Normal + (2 colonies/field, small colonies) Bitis gabonica - No platelet aggregation Bitis arietans - No platelet aggregation Mild platelet aggregation Echis - + (2 colonies/field, small- leucogaster medium colonies) Mild/ No platelet Echis aggregation (<1 + ocellatus colonies/field, small colonies) one small (2-8 Naja haje + platelets/colony) colonies per field Marked platelet Naja aggregation (3-4 +++ melanoleuca colonies/field, small, and medium colonies) Moderate platelet Naja aggregation (>2 ++ nigricollis colonies/field, small and medium colonies) Mild platelet aggregation Dendroaspis (2-3 small colonies/field, ++ polylepis medium and large colonies) Mild platelet aggregation Dendroaspis ++ (3-4 colonies/field, small viridis colonies) Marked platelet Dendroaspis aggregation (3-4 +++ jamesoni colonies/field, small, and medium colonies)

139

To confirm this, we employed an in vitro assay (Figure 4.4) to study venom-induced platelet aggregation or inhibition in human plasma. Compared to the control group, all venoms caused a degree of platelet aggregation (5-25%) greater than negative control (PBS). There was a significantly increased of platelet aggregation caused by N. nigricollis venom 10% greater than positive control. Some Viperidae venoms (B. gabonica, B. arietans, and E. leucogaster) shows a low level of aggregation (> 10%) compared to E. ocellatus venom that induced a significant level of aggregation. All elapids in (Naja and Dendroaspis species) caused the greatest levels of platelet aggregation (15-24%) compared to the negative control.

Figure 4.4 Elapids activate platelet aggregation. To measure levels of platelet aggregation induced by directly by venoms, a platelet aggregation in vitro assay was performed, to measure levels of platelet aggregation induced by directly by venoms, using PBS as a negative control and collagen as a positive control. All results were compared to collagen, as the negative control was standardised at 0% activity. All venoms caused greater levels of platelet aggregation than the negative control, and N. nigricollis venom caused platelets to aggregate significan tly more than the positive control. With the exception of E. ocellatus, venoms form species of the Elapidae family caused the greatest levels of platelet aggregation. Venoms: BG (B. gabonica), BA (B. arietans), EL (E. leucogaster), EO (E. ocellatus), NH (N. haje), NM (N. melanoleuca), NN (N. nigricollis), DP (D. polylepis), DV (D. viridis), DJ (D. jamesoni). Parameters were compared by Dunnett’s multiple comparison test. Significant values are indicated by *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. Mean ± SD of replicate counts shown.

140

4.3.2 Acute phase and acute inflammatory responses in envenomed mice

4.3.2.1 P-selectin involvement in acute inflammatory response activation in envenomed mice

Following an inflammatory stimulus, several molecules cause platelets and endothelial cells to become activated, leading to the surface expression of the transmembrane protein P- selectin. P-selectin is then shed into the systemic circulation through cleavage, giving rise to a soluble form (Lorant et al. 1993). Consequently, we used a commercial ELISA assay to quantify soluble P-selectin in the sera of envenomed mice, as a proxy for the level of endothelial cell and platelet activation (Figure 4.5A). Mice injected with PBS had soluble P- selectin concentrations of 544 ± 84 ng/ml (mean ± SD), whereas those envenomed with N. melanoleuca and N. nigricollis venom exhibited around a two-fold increase (1197 ± 68 ng/ml and 907 ± 78 ng/ml, respectively; P < 0.01 vs control). Conversely, envenoming by B. arietans resulted in significant decreases in P-selectin levels (147 ± 79 ng/ml; P < 0.01 vs control), while none of the remaining venoms exhibited significant differences in P-selectin levels when compared with the PBS control.

4.3.2.2 Envenomation-related immunoglobulin response.

Pathogens and toxins are detected by natural IgM antibodies. When an antigen-antibody complex forms, the classical complement pathway can become activated, heightening the antibody response (Baumgarth, Tung & Herzenberg 2005; Ochsenbein et al. 1999). Consequently, we next used a specific ELISA to quantify the levels of systemic IgM in the sera of the envenomed mice (Figure 4.5B). We found that treatment with B. gabonica venom led to marked increases (46 %) in circulating IgM (P < 0.05), while significant reductions in IgM with B. arietans (66 %), E. leucogaster (60 %), D. viridis (60 %), and D. jamesoni (66 %) venoms caused (all P < 0.05). No significant differences were observed when comparing IgM levels caused by the other snake venoms in comparison with the control samples.

4.3.2.3 APR stimulation by Elapidae venoms

The APR is an innate, non-specific, systemic reaction to local or systemic disturbances. When stimulated, there are detectable and quantifiable changes in acute phase reactants in comparison to baseline: namely, positive acute phase proteins will increase and negative acute phase proteins will decrease (Quinton et al., 2009). In this study we quantified levels of haptoglobin, SAA and CRP in mice envenomed with the 10 African snake venoms and compared these with non-envenomed controls. We observed five-fold increases in

141 haptoglobin levels with those mice dosed with N. melanoleuca and N. nigricollis venoms (P < 0.0001 vs control), while all other venoms caused haptoglobin levels to decrease (Figure 4.5C). While these reductions were modest, they were significant in the case of B. gabonica, B. arietans, E. leucogaster, D. polylepis, and D. jamesoni. Interestingly, N. melanoleuca venom caused SAA to increase (P < 0.0001), but none of the other venoms, including that of N. nigricollis, resulted in significant changes compared with the control (Figure 4.4D). We observed no significant changes in the levels of CRP across the different envenomed mice groups, which may the result of CRP not being a major acute phase protein in mice (Pathak & Agrawal 2019), and although albumin appeared to decrease slightly in most groups treated with snake venom, no significant reductions were identified when compared to the control.

4.3.2.4 Naja nigricollis venom causes rapid release of systemic inflammatory mediators

Cytokines are a diverse array of signalling peptides released by cells, and they are key regulators of the inflammatory and acute phase response. Once released, cytokines trigger many biological processes including cell activation, proliferation, growth, differentiation, migration and apoptosis (Faqi 2016). We investigated the levels of circulating cytokines in the different groups of mice dosed with African snake venoms via multiplex analysis of the resulting sera (Figure 4.5E). The control group (PBS only) exhibited untraceable levels of most of the cytokines investigated, although low levels of IFN-γ (2.08 pg/ml) and interleukin (IL)- 1β (0.15 pg/ml) were detected. Noticeably, experimental animals dosed with N. nigricollis venom exhibited increased levels of several pro-inflammatory cytokines (IL-5, IL-6, IL-13, IL- 18, GM-CSF, TNF-α). However, of these, significant elevations were only detected for IL-6 (P<0.0001 vs control), where a dramatic increase was detected (119.57 ± 4.6 pg/ml vs 0 pg/ml). Elevations of IL-6 were also detected in mice envenomed with E. leucogaster, E. ocellatus, N. haje, N. melanoleuca, and D. polylepis venoms, although these increases were modest (not significant vs control), and still represented low circulating levels. No statistically significant differences in TNF-α, IL-1β, IL-13, IL-5, GM-CSF, IL-12p70, IL-2, Il-4, IL-18 and IFN- γ levels were caused by any of the different snake venoms. With exception, IFN-γ significantly elevated with D. polylepis venom and IL-18 was significantly elevated in mouse envenomed with N. nigricollis venom.

142

Figure 4.5 Venoms elicit different acute phase and inflammatory responses in the systemic circulation of mice. Sera was obtained from mice six hours post-injection of different snake venoms and APPs were quantified. (A) P-selectin was significantly elevated in mice dosed with N. melanoleuca and N. nigricollis venoms, and significantly reduced in those dosed with Bitis arietans venom. (B) B. gabonica appears to increase circulating IgM compared to the normal, whilst B. arietans, E. leucogaster, D. viridis, and D. jamesoni caused significant reductions in the levels of circulating IgM. (C) Hp was significantly elevated in mice inoculated with Naja melanoleuca and Naja nigricollis venom, as quantified by a colorimetric assay, whilst (D) SAA was found to be elevated in mice injected with N. melanoleuca venom only, as quantified by ELISA). (E) Sera from mice were assayed for 11 cytokines using a multiplex Th1/Th2 immunoassay kit. Cytokines in the normal mouse sera (control) were low or undetected in all cases, and all other groups were compared to this control. Darker red indicates high elevation, pink indicates moderate elevation and white indicates minor or no change (see colour legend). Venoms: BG (B. gabonica), BA (B. arietans), EL (E. leucogaster), EO (E. ocellatus), NH (N. haje), NM (N. melanoleuca), NN (N. nigricollis), DP (D. polylepis), DV (D. viridis), DJ (D. jamesoni). Values found to be significant compared to the normal control (PBS only) are indicated by *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and 4.3.3*** Serum*P ≤ 0.0001. biochemistry following snake envenoming

143

Blood biochemistry is one of the most fundamental diagnostic indicators of muscle breakdown and organ failure. Consequently, we assessed a range of biochemical parameters to gain insight into the acute pathologies induced by this diverse groups of ten African snake venoms (Figure 4.6). The first marker measured was bilirubin, which accumulates in the systemic circulation as a breakdown product of RBCs during haemolysis and/or a functional failure of the liver to assist in its excretion (Kalakonda & John 2018). We found that all venoms caused a significant elevation of total bilirubin, with the exception N. haje and N. melanoleuca (Figure 4.6A). Quantification of the total protein (Figure 4.6B) and albumin levels (Figure 4.6C) in the sera of the envenomed mice revealed no changes from the control, with the exception of animals dosed with B. arietans venom, which caused significant reductions in both levels, suggesting acute liver and renal problems. Similarly, only one venom resulted in significant changes to AST (Figure 4.6D) and ALT (Figure 4.6E). The venom of N. nigricollis venom induced notable increases to both markers, indicating hepatocellular damage, and the combination of significant elevated (2-10 folds) levels of AST, along with LDH (Figure 4.6F) and CK (Figure 4.6G) caused by this venom is suggestive of cardiac damage. Finally, N. melanoleuca venom appeared to affect renal function, as creatine levels were moderately raised (indication as poor clearance of creatinine by the kidneys) (Figure 4.6H) while significant increases in LDH (as indication of haemolysis), in a comparable manner to N. nigricollis, were observed (Figure 4.6F). Aside from bilirubin levels, and a couple of other exceptions, the venoms of B. gabonica, E. leucogaster, E. ocellatus, N. haje and the three Dendroaspis spp. had little effect on the serum biochemistry of the envenomed mice under the experimental conditions used here (Figure 4.6).

144

Figure 4.6 Serum biochemistry parameters suggest acute hepatic and renal damage following N. nigricollis envenoming. Mice were injected with ten medically important snake venoms and after six hours, blood and sera samples were obtained. Several biochemical markers were then quantified using an automated clinical chemistry analyser and compared to sera obtained from the normal group (mice injected with PBS). We found that (A) total bilirubin was elevated in all cases of viper envenoming and also by N. nigricollis and Dendroaspis spp., and (B) total protein was significantly decreased following dosing with B. arietans venom. (C) B. arietans venom also caused albumin to reduce compared to the normal control. N. nigricollis venom caused significant elevations in AST (D) and ALT (E) levels. (F) Both N. melanoleuca and N. nigricollis caused significant elevations in the sera levels of LDH, while (G) N. nigricollis venom caused significant alterations to CK levels (elevated more than nine times). (H) Creatine levels were mostly comparable to the normal, with the exception that both Echis species caused significant reductions. Venoms: BG (B. gabonica), BA (B. arietans), EL (E. leucogaster), EO (E. ocellatus), NH (N. haje), NM (N. melanoleuca), NN (N. nigricollis), DP (D. polylepis), DV (D. viridis), DJ (D. jamesoni). Values found to be significant compared to the normal (PBS inoculation only) are indicated by *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. 145

4.4 Discussion

Snake venoms are extremely diverse in their composition, both interspecifically and intraspecifically (Casewell et al. 2014b). It is this variation in venom composition that mediates the diverse array of clinical symptoms presented by snakebite victims (Gutiérrez et al. 2017). Both in vitro and in vivo research indicates that inflammatory processes may exacerbate the local and systemic pathologies caused by elapid and viperid envenomation. Here, we report that snake venoms prompt very diverse responses in mice following acute envenoming, and that the venom of two African cobra venoms in particular – N. nigricollis and N. melanoleuca – cause marked inflammatory and non-specific acute phase responses. We also report venom-induced haemolytic activity in several cases of envenoming. The variable findings presented herein emphasise the importance of understanding the acute responses that manifest following envenoming, and further research in this area may facilitate new diagnostic and treatment approaches, which in turn may lead to better clinical outcomes for affected individuals.

Despite the clinical relevance of cobra (Naja spp.) envenoming in Africa, there have been few studies on the pathogenesis of inflammation-associated local tissue damage caused by such species (Iddon, Theakston & Ownby 1987; Rivel et al. 2016) and there is a paucity of knowledge on the systemic response to these venoms. IL-6, TNF-α and IL-1β are known to be the primary mediators of the APR (Heinrich 1990), and we found that serum IL-6 was dramatically elevated in mice injected with N. nigricollis venom. However, serum levels of TNF-α and IL-1β remained close to baseline. Elevated serum IL-6 has been reported in human snakebite victims, and although this correlated with non-specific clinical symptoms (nausea, vomiting, headache, abdominal pain, diarrhoea) in one case (Stone et al. 2013), there does not appear to be any significant clinical association between elevated serum cytokine concentration and severity of envenoming (Avila-Aguero et al. 2001). Additionally, anti- cytokine antibodies (anti-TNF-α, anti-IL-1β, anti-IL-6) failed to reduce lethality, haemorrhage, necrosis and oedema following Bothrops venom injection. This said, there is a lack of literature regarding these responses in cobra envenoming.

Once cytokines become systemically distributed, they can act on the bone marrow to modify leucocyte production, and IL-6 has been shown to be important in leucocyte recruitment and neutrophil migration during the inflammatory response (Rosales 2018). We found that envenoming by both N. nigricollis and N. melanoleuca caused significant changes to the leukocyte profile in circulation, with a significantly higher ratio of neutrophils to lymphocytes

146 compared to the control within only six hours of envenomation. This response is typical of acute stress syndrome and inflammation, and while this has previously been reported in human snakebite victims following viper envenoming (Barraviera et al. 1995a; Santoro et al. 2008), it is unreported for elapid envenoming. The number of leukocytes and their subtypes has been recognised as a marker of inflammation in disease (Fest et al. 2018) and the neutrophil:lymphocyte ratio has been reported as a risk factor for the severity of outcome of snakebite (Aktar et al. 2016; Elbey et al. 2017). Aktar and Tekin, (2017) investigated several parameters in 142 children with snakebite, and those that were grouped as ‘severe’ had significantly higher leucocytes and neutrophils than the other groups. The clinical symptoms of these children were distinct in that they suffered extensive swelling extending to the trunk, necrosis or compartment syndrome, as well as clinical signs of systemic effects (systemic bleeding, hypotension, disseminated intravascular coagulation (DIC) or renal failure, cerebral haemorrhage, or multi-system failure).

Another fundamental part of the acute phase response occurs when inflammatory mediators stimulate hepatocytes to alter the production of certain proteins. Haptoglobin is an acute phase protein in humans and mice, and although its key role is to bind free haemoglobin following haemolysis to prevent oxidative damage (Williams, 2018), haptoglobin will also increase rapidly in response to infection and/or injury, exhibiting a range of immunomodulatory properties (Eckersall et al. 2001; Shapiro & Black 1992). In support of our previous findings, we identified physiologically relevant elevations in haptoglobin in mice injected with both N. melanoleuca and N. nigricollis venoms. SAA is also a major acute phase protein, although it is produced in a time-dependent manner and typically reaches its peak after 24-72 hours. We found SAA to be elevated in mice envenomed with N. melanoleuca venom, although the reasons for a lack of increase observed in mice envenomed with N. nigricollis venom remain unclear. Contrastingly, haptoglobin levels in mice envenomed with the viper venoms from B. gabonica and B. arietans envenomed mice, were significantly lower than the normal control, suggesting a predominantly haemolytic response. Low levels of haptoglobin has been used as a marker of coagulopathy in snakebite victims previously (Kim et al. 2008), and both B. gabonica and B. arietans venoms have been shown to cause consumption coagulopathy (Maduwage & Isbister 2014). Interestingly, in B. gabonica envenoming, we found IgM to be elevated and serum albumin to be decreased, which has been shown to be characteristic of endothelium disturbances induced by haemotoxic snake venoms, allowing fluid to leak into the interstitial space and contributing to oedema.

147

Biochemical analysis of serum also indicated that mice envenomed with N. nigricollis and N. melanoleuca venoms showed unique pathological characteristics in comparison with the other venoms tested. Both groups had significantly elevated LDH, which is indicative of intravascular haemolysis (Barcellini & Fattizzo 2015), and marked spherocytosis was apparent in the blood film analysis. Spherocytes are damaged cells that are highly susceptible to destruction. Typically, they can be found in all haemolytic anaemias to some degree – a medically relevant consequence of snakebite – and occur following complement and/or immunoglobulin fixation (immune-mediated haemolytic anaemia [IMHA]) or due to direct damage (McCullough 2003). Secondary IMHA has been reported in dogs following elapid envenomings with tiger snake (Notechis scutatus) based on similar diagnostic parameters to those included in this study (e.g. evidence of haemolysis (ghost cells), marked spherocytosis, and raised AST, ALT and total bilirubin) (Ong et al. 2015). Our findings here suggest that IMHA is the direct result of the action of venom toxins. Supporting this hypothesis is evidence from Tambourgi et al. (1994), who performed in vitro haemolysis assays using sheep RBCs, and found that both N. nigricollis and N. melanoleuca venoms caused lysis of cells. On the other hands, with the in vitro direct haemolysis assays, N. nigricollis show a significant haemolysis. While, N. melanoleuca wasn’t and that maybe due to the dose dependent effects. This activity may be the result of the high content of cytotoxic 3FTx and PLA2 in these venoms

(Lauridsen et al. 2017), particularly since such PLA2 toxins have previously been shown to cause haemolysis in vivo (Arce-Bejarano, Lomonte & Gutiérrez 2014; Fernández et al. 2018).

Biochemical analysis of serum from mice injected with N. nigricollis venom also showed dramatically elevated CK levels, with a ten-fold increase compared with control exceeding the five-fold threshold used as a clinical indicator in humans for the presence of rhabdomyolysis (Cleary 2016). These findings were also supported by elevations in LDH in the same experimental animals, as LDH is a marker of muscle breakdown (Torres et al. 2015). Rhabdomyolysis is a major pathology that can occur following bites by a variety of elapid and viperid snakes (e.g. Australian red-bellied black snake (Pseudechis porphyriacus) and can lead to the accumulation of muscle breakdown products that cause damage to the kidneys (Lim, Singh & Isbister 2016). Vikrant et al. (2017) investigated acute injury in 121 snakebite victims in India and found intravascular haemolysis and rhabdomyolysis to be the most important risk factors for developing acute kidney injury. Additionally, those who died (9.1 %) had elevated WBC counts, elevated bilirubin, and low levels of albumin compared to the surviving group – all of which were identified in the N. nigricollis envenomed mice used in this study. Furthermore, elevations in ALT, AST and total bilirubin in the N. nigricollis envenomed mice

148 are also indicative of acute hepatocellular damage; a finding which is in line with the findings of James et al. (2013) who used rat models to investigate pathology caused by N. nigricollis venom. Liver injury is to be said one of the most common and serious symptoms of cobra snake envenomation (N. haje). Several mechanisms have been proposed to explain how such damage develops, dependent on venom type (Gutiérrez et al. 2017).

Thrombosis and haemostasis are vital cellular processes, and different snake venoms disrupt these processes in a variety of ways. Thrombocytopenia, which is a reduction in the platelet count, is a common clinical observation in snakebite victims and is thought to be caused by venom-induced hyper-aggregation of platelets, particularly in those bitten by vipers (Cheng et al. 2017; Lavonas et al. 2002; Odeleye et al. 2004; Santoro et al. 2008). However, a wide variety of snake venom toxins have been demonstrated to interact with platelets, and act via various mechanisms to either inhibit or promote their aggregation (Slagboom et al. 2017). We found that elapid venoms caused the greatest level of platelet aggregation, whilst viperid venoms appeared to minimise platelet aggregation (B. arietans and E. leucogaster).

Platelets are the major source of soluble P-selectin (Tscharre et al. 2019), which is shed into the circulation to dampen the inflammatory potential of the cell. N. melanoleuca and N. nigricollis envenomation caused elevated levels of P-selectin in the systemic circulation, suggesting high levels of platelet activation and endothelial dysfunction. P-selectin has rarely been documented in snakebite models, apart from a comprehensive physiological investigation conducted by Sun et al. (2016). The authors reported that P-selectin is physiologically involved in the neutralisation of venom-induced coagulopathy following Crotalus atrox envenoming. Both cobra venoms discussed in this study have potent anticoagulant effects (Bittenbinder et al. 2019) and have been shown here to stimulate inflammatory responses, suggesting that high levels of circulating P-selectin could perhaps stimulate some protective responses against systemic coagulopathy (Dole et al. 2005). Interestingly, we found that B. arietans envenomation significantly reduced P-selectin levels compared to the control and, as such, we propose that the venom somehow interferes with the function and/or activation of platelets, preventing shedding. These findings also correlate with our blood film analysis, which revealed that B. arietans caused markedly decreased levels of platelet clumping compared with the controls. Indeed, it is well- established that B. arietans and E.ocellatus venom contains several anti-platelet peptides, including arietin (from B.arietans) and Echistatin (from E.ocellatus), which suppresses platelet aggregation by interacting with fibrinogen receptors on platelet membranes (Huang et al. 1991; Smith et al., 2002) and platelet aggregation has also previously been shown to

149 be strongly suppressed by whole B. arietans venom in vitro (Strydom et al. 2016). Given the importance of platelet receptors for platelet function, we suggest that anti-platelet toxins in B. arietans venom are interacting and/or overlapping with the binding sites of those receptors, thus preventing both their activation and their downstream functions (de Queiroz et al. 2017).

4.5 Conclusion

The present study used a murine model to detect inflammatory and acute phase markers in the systemic circulation in response to envenoming by ten snake venoms of medical importance in Africa. We also assessed the biochemical profile of envenomed mice and completed haematological analyses of blood to further elucidate the clinicopathological mechanisms of snakebite envenoming. We found that N. melanoleuca and N. nigricollis venoms – whose venoms are typically associated with neurotoxic and cytotoxic clinical pathologies respectively – exhibit a similar systemic response that can readily be detected only 6 hours post-envenomation. We also identified major systemic pathologies associated with diverse envenomings, including variable. In addition, we find that SAA and haptoglobin show promise for exploring further for their potential clinical value as biomarkers to assess acute responses in the early stages of envenoming, particularly since these tests would be substantially faster and cheaper than measuring the standard biomarker of CRP, and since evidence from other studies suggests they may be more effective markers for assessing acute responses in other clinical scenarios (Christensen et al. 2014; Watson, Round & Hamilton 2012). Despite highlighting novel insights into the acute phase and inflammatory responses occurring immediately after African snake envenoming, much remains unknown about the mechanisms involved in the induction and development of the immediate systemic response to the venoms under study, in particular the specific venom toxins that initiate the reaction and their respective mechanisms of action. The variable findings reported here strongly advocate for further research effort on this topic to aid the future treatment and clinical management of African snakebite victims.

150

4.6 References:

Aktar, F., Aktar, S., Yolbas, I. & Tekin, R. (2016) 'Evaluation of risk factors and follow-up criteria for severity of snakebite in children', Iranian journal of pediatrics, vol. 26, no. 4. Arango Duque, G. & Descoteaux, A. (2014) 'Macrophage cytokines: involvement in immunity and infectious diseases', Frontiers in immunology, vol. 5, p. 491. Arce-Bejarano, R., Lomonte, B. & Gutiérrez, J.M. (2014) 'Intravascular hemolysis induced by the venom of the Eastern coral snake, Micrurus fulvius, in a mouse model: Identification of directly hemolytic phospholipases A2', Toxicon, vol. 90, pp. 26-35. Avila-Aguero, M., Paris, M.M., Hu, S., Peterson, P.K., Gutierrez, J.M., Lomonte, B., Faingezicht, I. & The Snakebite Study, G. (2001) Systemic cytokine response in children bitten by snakes in Costa Rica, p. 425 Barcellini, W. & Fattizzo, B. (2015) 'Clinical applications of hemolytic markers in the differential diagnosis and management of hemolytic anemia', Disease markers, vol. 2015. Barraviera, B., Lomonte, B., Tarkowski, A., Hanson, L. & Meira, D.A. (1995a) 'Acute-phase reactions, including cytokines, in patients bitten by Bothrops and Crotalus snakes in Brazil', Journal of Venomous Animals and Toxins, vol. 1, no. 1, pp. 11-22. Barraviera, B., Lomonte, B., Tarkowski, A., Hanson, L.Å. & Meira, D.A. (1995b) 'Acute-phase reactions, including cytokines, in patients bitten by Bothrops and Crotalus snakes in Brazil', Toxicon, vol. 33, p. 283. Barros, S.F., Friedlanskaia, I., Petricevich, V.L. & Kipnis, T.L. (1998) 'Local inflammation, lethality and cytokine release in mice injected with Bothrops atrox venom', Mediators Of Inflammation, vol. 7, no. 5, pp. 339-346. Baumann, H. & Gauldie, J. (1994) The acute phase response, Great Britain, p. 74. Baumgarth, N., Tung, J.W. & Herzenberg, L.A. (2005) 'Inherent specificities in natural antibodies: a key to immune defense against pathogen invasion', Springer Seminars In Immunopathology, vol. 26, no. 4, pp. 347-362. Bittenbinder, M.A., Dobson, J.S., Zdenek, C.N., op den Brouw, B., Naude, A., Vonk, F.J. & Fry, B.G. (2019) 'Differential destructive (non-clotting) fibrinogenolytic activity in Afro-Asian elapid snake venoms and the links to defensive hooding behavior', Toxicology in Vitro. Casewell, N.R., Wagstaff, S.C., Wüster, W., Cook, D.A.N., Bolton, F.M.S., King, S.I., Pla, D., Sanz, L., Calvete, J.J. & Harrison, R.A. (2014a) 'Medically important differences in

151

snake venom composition are dictated by distinct postgenomic mechanisms', Proceedings Of The National Academy Of Sciences Of The United States Of America, vol. 111, no. 25, pp. 9205-9210. Cheng, C.-L., Mao, Y.-C., Liu, P.-Y., Chiang, L.-C., Liao, S.-C. & Yang, C.-C. (2017) 'Deinagkistrodon acutus envenomation: a report of three cases', Journal of venomous animals and toxins including tropical diseases, vol. 23, no. 1, p. 20. Chippaux, J.P. (1998) Snake-bites: appraisal of the global situation, p. 515 Chmielewski, P.P. & Strzelec, B. (2018) 'Elevated leukocyte count as a harbinger of systemic inflammation, disease progression, and poor prognosis: a review', Folia morphologica, vol. 77, no. 2, pp. 171-178. Christensen, M.B., Langhorn, R., Goddard, A., Andreasen, E.B., Moldal, E., Tvarijonaviciute, A., Kirpensteijn, J., Jakobsen, S., Persson, F. & Kjelgaard-Hansen, M. (2014) Comparison of serum amyloid A and C-reactive protein as diagnostic markers of systemic inflammation in dogs, vol. 55, pp. 161-168. Cleary, M.A. (2016) 'Creatine kinase, exertional rhabdomyolysis, and exercise-associated hyponatremia in ultra-endurance athletes: A critically appraised paper'. Cray, C., Zaias, J. & Altman, N.H. (2009) 'Acute phase response in animals: a review', Comparative medicine, vol. 59, no. 6, pp. 517-526. de Queiroz, M.R., de Sousa, B.B., da Cunha Pereira, D.F., Mamede, C.C.N., Matias, M.S., de Morais, N.C.G., de Oliveira Costa, J. & de Oliveira, F. (2017) 'The role of platelets in hemostasis and the effects of snake venom toxins on platelet function', Toxicon, vol. 133, pp. 33-47. de Silva, H.A., Ryan, N.M. & de Silva, H.J. (2016) 'Adverse reactions to snake antivenom, and their prevention and treatment', British journal of clinical pharmacology, vol. 81, no. 3, pp. 446-452. de sousa-e-Silva, M.C., Tomy, S.C., Tavares, F.L., Navajas, L., Larsson, M.H., Lucas, S.R., Kogika, M.M. & Sano-Martins, I.S. (2003) 'Hematological, hemostatic and clinical chemistry disturbances induced by Crotalus durissus terrificus snake venom in dogs', Human & experimental toxicology, vol. 22, no. 9, pp. 491-500. Dole, V.S., Bergmeier, W., Mitchell, H.A., Eichenberger, S.C. & Wagner, D.D. (2005) 'Activated platelets induce Weibel-Palade-body secretion and leukocyte rolling in vivo: role of P-selectin', Blood, vol. 106, no. 7, pp. 2334-2339. Eckersall, P.D., Gow, J.W., McComb, C., Bradley, B., Rodgers, J., Murray, M. & Kennedy, P.G.E. (2001) 'Cytokines and the acute phase response in post-treatment reactive

152

encephalopathy of Trypanosoma brucei brucei infected mice', Parasitology international, vol. 50, no. 1, pp. 15-26. Elbey, B., Baykal, B., Yazgan, Ü.C. & Zengin, Y. (2017) 'The prognostic value of the neutrophil/lymphocyte ratio in patients with snake bites for clinical outcomes and complications', Saudi journal of biological sciences, vol. 24, no. 2, pp. 362-366. Faqi, A.S. (2016) A comprehensive guide to toxicology in nonclinical drug development, Academic Press. Fernández, M.L., Quartino, P.Y., Arce-Bejarano, R., Fernández, J., Camacho, L.F., Gutiérrez, J.M., Kuemmel, D., Fidelio, G. & Lomonte, B. (2018) 'Intravascular hemolysis induced by phospholipases a2 from the venom of the eastern coral snake, micrurus fulvius: Functional profiles of hemolytic and non-hemolytic isoforms', Toxicology letters, vol. 286, pp. 39-47. Ferrer-Acosta, Y., González, M., Fernández, M. & Valance, W.A. (2014) 'Emerging roles for platelets in inflammation and disease', Journal of infectious disease and therapy, vol. 2, no. 4. Fest, J., Ruiter, R., Ikram, M.A., Voortman, T., van Eijck, C.H.J. & Stricker, B.H. (2018) 'Reference values for white blood-cell-based inflammatory markers in the Rotterdam Study: a population-based prospective cohort study', Scientific reports, vol. 8, no. 1, p. 10566. Gruys, E., Toussaint, M.J.M., Niewold, T.A. & Koopmans, S.J. (2005) 'Acute phase reaction and acute phase proteins', Journal of Zhejiang University. Science. B, vol. 6, no. 11, p. 1045. Gutiérrez, J.M., Calvete, J.J., Habib, A.G., Harrison, R.A., Williams, D.J. & Warrell, D.A. (2017) 'Snakebite envenoming', Nature reviews. Disease primers, vol. 3, p. 17063. Gutiérrez, J.M., Williams, D., Fan, H.W. & Warrell, D.A. (2010) 'Snakebite envenoming from a global perspective: Towards an integrated approach', Toxicon, vol. 56, no. 7, pp. 1223-1235. Habib, A.G., Kuznik, A., Hamza, M., Abdullahi, M.I., Chedi, B.A., Chippaux, J.-P. & Warrell, D.A. (2015) 'Snakebite is under appreciated: appraisal of burden from West Africa', PLoS neglected tropical diseases, vol. 9, no. 9, p. e0004088. Habib, A.G. & Warrell, D.A. (2013) 'Antivenom therapy of carpet viper (Echis ocellatus) envenoming: effectiveness and strategies for delivery in West Africa', Toxicon, vol. 69, pp. 82-89.

153

Harrison, R.A., Hargreaves, A., Wagstaff, S.C., Faragher, B. & Lalloo, D.G. (2009) 'Snake envenoming: a disease of poverty', Plos Neglected Tropical Diseases, vol. 3, no. 12, pp. e569-e569. Hoffman, J.F. (1992) On red blood cells, hemolysis and resealed ghosts, in, The Use of Resealed Erythrocytes as Carriers and Bioreactors, Springer, pp. 1-15. Huang, T.-F., Wang, W.-J., Teng, C.-M. & Ouyang, C. (1991) 'Mechanism of action of the antiplatelet peptide, arietin, from Bitis arietans venom', BBA - General Subjects, vol. 1074, pp. 144-150. Iddon, D., Theakston, R.D.G. & Ownby, C.L. (1987) 'A study of the pathogenesis of local skin necrosis induced by Naja nigricollis (spitting cobra) venom using simple histological staining techniques', Toxicon, vol. 25, no. 6, pp. 665-672. Kalakonda, A. & John, S. (2018) Physiology, Bilirubin, in, StatPearls [Internet], StatPearls Publishing. Kasturiratne, A., Wickremasinghe, A.R., de Silva, N., Gunawardena, N.K., Pathmeswaran, A., Premaratna, R., Savioli, L., Lalloo, D.G. & de Silva, H.J. (2008) 'The global burden of snakebite: a literature analysis and modelling based on regional estimates of envenoming and deaths', PLoS medicine, vol. 5, no. 11, p. e218. Kim, J.S., Yang, J.W., Kim, M.S., Han, S.T., Kim, B.R., Shin, M.S., Lee, J.I., Han, B.G. & Choi, S.O. (2008) 'Coagulopathy in patients who experience snakebite', The Korean journal of internal medicine, vol. 23, no. 2, p. 94. Langhorn, R., Persson, F., Åblad, B., Goddard, A., Schoeman, J.P., Willesen, J.L., Tarnow, I. & Kjelgaard-Hansen, M. (2014) 'Myocardial injury in dogs with snake envenomation and its relation to systemic inflammation', Journal of veterinary emergency and critical care, vol. 24, no. 2, pp. 174-181. Lauridsen, L.P., Laustsen, A.H., Lomonte, B. & Gutiérrez, J.M. (2017) 'Exploring the venom of the forest cobra snake: Toxicovenomics and antivenom profiling of Naja melanoleuca', Journal of proteomics, vol. 150, pp. 98-108. Lavonas, E.J., Tomaszewski, C.A., Ford, M.D., Rouse, A.M. & Kerns Ii, W.P. (2002) 'Severe puff adder (Bitis arietans) envenomation with coagulopathy', Journal of Toxicology: Clinical Toxicology, vol. 40, no. 7, pp. 911-918. Lim, A.Y.L., Singh, P.N. & Isbister, G.K. (2016) 'Severe rhabdomyolysis from red-bellied black snake (Pseudechis porphyriacus) envenoming despite antivenom', Toxicon, vol. 117, pp. 46-48.

154

Lobetti, R.G. & Joubert, K. (2004) 'Retrospective study of snake envenomation in 155 dogs from the Onderstepoort area of South Africa', Journal Of The South African Veterinary Association, vol. 75, no. 4, pp. 169-172. Lomonte, B., Tarkowski, A. & Hanson, L.A. (1993) Host Response to Bothrops asper Snake Venom: Analysis of Edema Formation. Inflammatory Cells, and Cytokine Release in a Mouse Model, PLENUM PUBLISHING CORPORATION, United States, p. 93. Lorant, D.E., Topham, M.K., Whatley, R.E., McEver, R.P., McIntyre, T.M., Prescott, S.M. & Zimmerman, G.A. (1993) 'Inflammatory roles of P-selectin', The Journal Of Clinical Investigation, vol. 92, no. 2, pp. 559-570. Maduwage, K. & Isbister, G.K. (2014) 'Current treatment for venom-induced consumption coagulopathy resulting from snakebite', PLoS neglected tropical diseases, vol. 8, no. 10, p. e3220. McCullough, S. (2003) 'Immune-mediated hemolytic anemia: understanding the nemesis', Veterinary Clinics: Small Animal Practice, vol. 33, no. 6, pp. 1295-1315. Menaldo, D.L., Bernardes, C.P., Zoccal, K.F., Jacob-Ferreira, A.L., Costa, T.R., Del Lama, M.P.F.M., Naal, R.M.Z.G., Frantz, F.G., Faccioli, L.H. & Sampaio, S.V. (2017) 'Immune cells and mediators involved in the inflammatory responses induced by a P-I metalloprotease and a phospholipase A2 from Bothrops atrox venom', Molecular Immunology, vol. 85, pp. 238-247. Moreira, V., Dos-Santos, M.C., Nascimento, N.G., Borges da Silva, H., Fernandes, C.M., D'Império Lima, M.R. & Teixeira, C. (2012) 'Local inflammatory events induced by Bothrops atrox snake venom and the release of distinct classes of inflammatory mediators', Toxicon: Official Journal Of The International Society On Toxinology, vol. 60, no. 1, pp. 12-20. Moreira, V., Teixeira, C., Da Silva, H.B., Lima, M.R.D.I. & Dos-Santos, M.C. (2016) 'The role of TLR2 in the acute inflammatory response induced by Bothrops atrox snake venom', Toxicon, vol. 118, pp. 121-128. Nagel, S.S., Schoeman, J.P., Thompson, P.N., Wiinberg, B. & Goddard, A. (2014) 'Hemostatic analysis of dogs naturally envenomed by the African puffadder (Bitis arietans) and snouted cobra (Naja annulifera)', Journal of Veterinary Emergency and Critical Care, vol. 24, no. 6, pp. 662-671. Nogueira, R.M.B. & Sakate, M. (2006) 'Clinical and hematological alterations in dogs during experimental envenomation with Crotalus durissus terrificus venom and treated

155

with antiophidic serum', Journal of Venomous Animals and Toxins including Tropical Diseases, no. 2, p. 285. O'Connell, K.E., Mikkola, A.M., Stepanek, A.M., Vernet, A., Hall, C.D., Brown, D.E., Sun, C.C., Yildirim, E., Lee, J.T. & Staropoli, J.F. (2015) 'Practical murine hematopathology: A comparative review and implications for research', Comparative Medicine, vol. 65, no. 2, pp. 96-113. Ochsenbein, A.F., Fehr, T., Lutz, C., Suter, M., Brombacher, F., Hengartner, H. & Zinkernagel, R.M. (1999) 'Control of early viral and bacterial distribution and disease by natural antibodies', Science, vol. 286, no. 5447, pp. 2156-2159. Odeleye, A.A., Presley, A.E., Passwater, M.E. & Mintz, P.D. (2004) 'Rattlesnake venom- induced thrombocytopenia', Annals of Clinical & Laboratory Science, vol. 34, no. 4, pp. 467-470. Ong, H.M., Witham, A., Kelers, K. & Boller, M. (2015) 'Presumed secondary immune- mediated haemolytic anaemia following elapid snake envenomation and its treatment in four dogs', Australian veterinary journal, vol. 93, no. 9, pp. 319-326. Pathak, A. & Agrawal, A. (2019) 'Evolution of C-reactive protein', Frontiers in immunology, vol. 10, p. 943. Petricevich, V.L., Teixeira, C.F.P., Tambourgi, D.V. & Gutiérrez, J.M.a. (2000) 'Increments in serum cytokine and nitric oxide levels in mice injected with Bothrops asper and Bothrops jararaca snake venoms', Toxicon, vol. 38, no. 9, pp. 1253-1266. Rivel, M., Solano, D., Herrera, M., Vargas, M., Villalta, M., Segura, Á., Arias, A.S., León, G. & Gutiérrez, J.M. (2016) 'Pathogenesis of dermonecrosis induced by venom of the spitting cobra, Naja nigricollis: An experimental study in mice', Toxicon, vol. 119, pp. 171-179. Rosales, C. (2018) 'Neutrophil: a cell with many roles in inflammation or several cell types?', Frontiers in Physiology, vol. 9, p. 113. Rucavado, A., Escalante, T., Teixeira, C.F.P., Fernandes, C.M., Diaz, C. & Gutierrez, J.M. (2002) Increments in cytokines and matrix metalloproteinases in skeletal muscle after injection of tissue-damaging toxins from the venom of the snake Bothrops asper, vol. 11, pp. 121-128. Rucavado, A., Nicolau, C., Escalante, T., Kim, J., Herrera, C., Gutiérrez, J. & Fox, J. (2016) 'Viperid envenomation wound exudate contributes to increased vascular permeability via a DAMPs/TLR-4 mediated pathway', Toxins, vol. 8, no. 12, p. 349.

156

Santoro, M.L., Sano-Martins, I.S., Fan, H.W., Cardoso, J.L.C., Theakston, R.D.G. & Warrell, D.A. (2008) 'Haematological evaluation of patients bitten by the jararaca, Bothrops jararaca, in Brazil', Toxicon, vol. 51, no. 8, pp. 1440-1448. Shapiro, S.Z. & Black, S.J. (1992) 'Identification of an acute-phase reactant in murine infections with Trypanosoma brucei', Infection and immunity, vol. 60, no. 9, pp. 3921-3924. Slagboom, J., Kool, J., Harrison, R.A. & Casewell, N.R. (2017) 'Haemotoxic snake venoms: their functional activity, impact on snakebite victims and pharmaceutical promise', British journal of haematology, vol. 177, no. 6, pp. 947-959. Sproston, N.R. & Ashworth, J.J. (2018) 'Role of C-Reactive Protein at Sites of Inflammation and Infection', Frontiers In Immunology, vol. 9, pp. 754-754. Stone, S.F., Isbister, G.K., Shahmy, S., Mohamed, F., Abeysinghe, C., Karunathilake, H., Ariaratnam, A., Jacoby-Alner, T.E., Cotterell, C.L. & Brown, S.G.A. (2013) 'Immune response to snake envenoming and treatment with antivenom; complement activation, cytokine production and mast cell degranulation', PLoS neglected tropical diseases, vol. 7, no. 7, p. e2326. Strydom, M.A., Bester, J., Mbotwe, S. & Pretorius, E. (2016) 'The effect of physiological levels of South African puff adder (Bitis arietans) snake venom on blood cells: an in vitro model', Scientific Reports, vol. 6, pp. 35988-35988. Smith, J.B., Theakston, R.D.G., Coelho, A.L.J., Barja-Fidalgo, C., Calvete, J.J. and Marcinkiewicz, C., 2002. Characterization of a monomeric disintegrin, ocellatusin, present in the venom of the Nigerian carpet viper, Echis ocellatus. FEBS letters, 512(1-3), pp.111-115. Tasoulis, T. & Isbister, G. (2017) 'A review and database of snake venom proteomes', Toxins, vol. 9, no. 9, p. 290. Teixeira, C.F.P., Zamunér, S.R., Zuliani, J.P., Fernandes, C.M., Cruz-Hofling, M.A., Fernandes, I., Chaves, F. & Gutiérrez, J.M. (2003) 'Neutrophils do not contribute to local tissue damage, but play a key role in skeletal muscle regeneration, in mice injected with Bothrops asper snake venom', Muscle & Nerve: Official Journal of the American Association of Electrodiagnostic Medicine, vol. 28, no. 4, pp. 449-459. Torres, P.A., Helmstetter, J.A., Kaye, A.M. & Kaye, A.D. (2015) 'Rhabdomyolysis: pathogenesis, diagnosis, and treatment', Ochsner Journal, vol. 15, no. 1, pp. 58-69. Tscharre, M., Vogel, B., Tentzeris, I., Freynhofer, M.K., Rohla, M., Wojta, J., Weiss, T.W., Ay, C., Huber, K. & Farhan, S. (2019) 'Prognostic impact of soluble P-selectin on long-

157

term adverse cardiovascular outcomes in patients undergoing percutaneous coronary intervention', Thrombosis and haemostasis, vol. 119, no. 02, pp. 340-347. Walton, R.M., Brown, D.E., Hamar, D.W., Meador, V.P., Horn, J.W. & Thrall, M.A. (1997) 'Mechanisms of echinocytosis induced by Crotalus atrox venom', Veterinary pathology, vol. 34, no. 5, pp. 442-449. Wanderley, C.W.S., Silva, C.M.S., Wong, D.V.T., Ximenes, R.M., Morelo, D.F.C., Cosker, F., Aragão, K.S., Fernandes, C., Palheta-Júnior, R.C., Havt, A., Brito, G.A.C., Cunha, F.Q., Ribeiro, R.A. & Lima-Júnior, R.C.P. (2014) 'Bothrops jararacussu snake venom- induces a local inflammatory response in a prostanoid- and neutrophil-dependent manner', Toxicon: Official Journal Of The International Society On Toxinology, vol. 90, pp. 134-147. Warrell, D.A. (2005) 'Treatment of bites by adders and exotic venomous snakes', British Medical Journal, vol. 331, no. 7527, pp. 1244-1247. Watson, J., Round, A. & Hamilton, W. (2012) 'Raised inflammatory markers', BMJ: British Medical Journal, vol. 344, no. 7843, p. 43. WHO (2019) Neglected tropical diseases [Online], World Health Organization, Available from: http://www.who.int/neglected_diseases/diseases/en/ (Accessed: 19th September). WÜSTER, W., Chirio, L., Trape, J.F., Ineich, I., Jackson, K., Greenbaum, E., Barron, C., Kusamba, C., Nagy, Z.T., Storey, R. and Hall, C., 2018. Integration of nuclear and mitochondrial gene sequences and morphology reveals unexpected diversity in the forest cobra (Naja melanoleuca) species complex in Central and West Africa (Serpentes: Elapidae). Zootaxa, 4455(1), pp.68-98. Zamuner, S.R., Zuliani, J.P., Fernandes, C.M., Gutiérrez, J.M. & de Fátima Pereira Teixeira, C. (2005) 'Inflammation induced by Bothrops asper venom: release of proinflammatory cytokines and eicosanoids, and role of adhesion molecules in leukocyte infiltration', Toxicon: Official Journal Of The International Society On Toxinology, vol. 46, no. 7, pp. 806-813.

158

4.7 Supplementary Information

Supplementary Table S 4.1. Prior murine in vivo studies of the inflammatory response following dosing with snake venom. Key: IL-1/-6/-8/-10/-12p70: Interleukin-1/-6/-8/-10/-12p70; TNF-α: Tumour Necrosis Factor-α; Interferon-γ: IFN-γ; TXB2 /4; Thromboxane B2/4; NO: Nitric Oxide; LTB4: Leukotriene B4; CCL2: Monocyte Chemotactic Protein-1; COX-2: Cyclooxygenase-2; PGE2/D2: Prostaglandin E2/D2; CysLTs: Cysteinyl Leukotriene Receptors.

Inflammatory Whole Snake Target species mediators (time post- venom/venom Observed pathologies Reference species envenomation in toxin (Mouse strains) hours)

Pro-inflammatory cytokines: IL-6 (1-3), TNF-α (1-6) Leucocyte infiltrate (Zamuner et al. Whole venom Swiss mice Eicosanoids pathway: (neutrophils) 2005) LTB₄ (0.5-1), TXB₂ (0.5- 1)

Myotoxic PLA₂, P-I Pro-inflammatory (Rucavado et al. type Swiss mice cytokines: IL-1 (1-6), Not reported 2002) metalloproteinase IL-6 (1-6)

Leucocyte infiltrate Bothrops Whole venom, (neutrophils, (Lomonte, asper Pro-inflammatory myotoxic PLA₂ Swiss mice mononuclear cells), Tarkowski & cytokines: IL-6 (3-6) isoform reduced platelets, Hanson 1993) oedema

Pro-inflammatory cytokines: IL-1 (2-24), IL-6 (2-6), IFN-γ, TNF-α (Petricevich et Whole venom BALB/c mice (2-18) Not reported al. 2000) Anti-inflammatory cytokines: IL-10 (4-24) Other: NO (2-24)

Pro-inflammatory cytokines: IL-6 (1-4), IL-12p70 (4-8), TNF-α Vascular permeability, leucocyte infiltration Bothrops (1) (Moreira et al. Whole venom Swiss mice (mononuclear and atrox Anti-inflammatory 2012) polymorphonuclear cytokines: IL-10 (8) Chemokines: CCL-2 (1- leucocytes) 8) Eicosanoid pathway:

159

COX-2 (1-4), LTB₄ (1), PGD₂ (1), PGE₂ (1-4), TXB₂ (1)

Pro-inflammatory Leucocyte infiltration cytokines: IL-6 (1-6), (polymorphonuclear IFN-γ (1-12), TNF-α (1- cells), skeletal muscle (Barros et al. Whole venom BALB/c 18) disruption, oedema, 1998) Anti-inflammatory erythema, cytokines: IL-10 (1-18) haemorrhage and Other: NO (1-24) local tissue necrosis

Pro-inflammatory cytokines: IL-1β (4, P-I metalloprotease only), IL-6 (1-2, PLA₂; 2, P-I metalloprotease) Anti-inflammatory Leucocyte infiltration P-I metalloprotease, cytokines: IL-10 (1, (Menaldo et al. C57BL/6 (neutrophils, PLA₂ PLA₂; 1-2, P-I 2017) mononuclear cells) metalloprotease Eicosanoid pathway: CysLTs (1-2, PLA₂ only), LTB₄ (4, PLA₂ only), PGE₂ (4, both toxins)

Pro-inflammatory cytokines: IL-1β (4.5), IL-6 (4.5) Anti-inflammatory Leucocyte infiltration cytokines: IL-10 (4.5) (mononuclear and (Moreira et al. Whole venom C57BL/7 Chemokines: CCL-2 polymorphonuclear 2016) (4.5) leucocytes) Eicosanoid pathway: LTB₄ (4.5), COX-2 (4.5), TXB₂

Pro-inflammatory cytokines: IL-1 (4-24), IL-6 (4-18), TNF-α (2- Bothrops (Petricevich et Whole venom BALB/c mice 18), IFN-γ (2-24) Not reported jararaca al. 2000) Anti-inflammatory cytokines: IL-10 (4-24 hours)

160

Pro-inflammatory Leucocyte infiltration cytokines: IL-1β (4.5), (neutrophils), Bothrops (Wanderley et Whole venom Swiss mice TNF-α (4.5) oedema, jararacaussu al. 2014) Eicosanoid pathway: haemorrhage, and COX-2 (4.5) necrosis

161

Supplementary Figure S 4.1: Examples of platelet aggregation (x 100 objective) were observed in blood film of envenomed mice. Arrow indicated the platelet aggregation colonies compared to normal blood film.

162

Chapter 5

Title: 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

Authors: Jaffer Alsolaiss1, Juan Calvate 2, Jose M. Gutierrez 3, Nicholas R Casewell1, Robert A Harrison1

Affiliations:

1Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, Liverpool, UK.

2Laboratorio de Venomica Estructural y Funcional, Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain 3Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José 11501-2060, Costa Rica

163

5.1 Introduction

Snakebite is one of the world’s most under-researched, high-mortality, high-morbidity neglected tropical diseases (NTD). Global estimates suggest that as many as 2.7 million people are envenomed annually, resulting in around 81,000, or perhaps as many as 138,000, (WHO, 2019) deaths worldwide. The tropics and sub-tropics of South/Southeast Asia, Latin America and sub-Saharan Africa are the areas with the highest frequency of snakebite; here incidence rates can be as high as 11-13 per 100,000 people (Chippaux 2011; Warrell 2010b). In sub-Saharan Africa, perhaps the most neglected region in terms of tackling snakebite, the annual incidence is thought to be more than 314,000 cases per annum, of which 7,300 are thought to be lethal and 6,000 result in amputation (Chippaux 2011). Moreover, snakebite- associated Disability Adjusted Life Years (DALYs) calculated for 16 West African countries have been estimated to be approximately 320,000 (Chippaux 2011; Habib et al. 2015). Hence, in addition to being a major cause of mortality, snakebite envenoming can rapidly and drastically reduce the quality of life of the envenomed victim, placing substantial economic, physically-debilitating and/or psychological burdens on both them and their wider family, thus contributing to and exacerbating the poverty of people in rural areas (Harrison & Gutiérrez 2016). Furthermore, it is often the most impoverished people, particularly those in the economically important age bracket of 10-30 years old, who are the most likely to die or suffer life-changing injuries as a result of snakebite envenomation. The burdens of snakebite envenoming are therefore substantially higher (0.35 million DALYs) than many other neglected tropical diseases (e.g., leishmaniasis, onchocerciasis, trypanosomiasis and schistosomiasis) (Habib et al. 2015).

Antivenom is the first-choice treatment of snakebite envenoming and is included in the World Health Organization’s (WHO) “List of Essential Medicines”, meaning that antivenoms should be readily available in primary healthcare centres in areas with high snakebite prevalence (WHO 2019). Antivenoms are among the least affordable of tropical disease medications, with treatment costing as much as US$400-700 (Harrison & Gutiérrez 2016; Harrison et al. 2009). There is therefore an urgent need for the development of antivenoms that combine safety and effectiveness with affordability if the vulnerable people of the tropics who suffer the greatest risk of snakebite are to be treated in sufficient numbers.

Polyspecific antivenoms contain antibodies generated by immunising donor animals (horses or sheep) with a mixture of venoms from several snake species. Intravenously administered antivenom antibodies bind to and neutralise venom components. Laboratory assessment of

164 the immune response of the venom-immunised animals is essential to identify poor responders and determining the optimal time to harvest antibodies (Gutiérrez et al. 2017c). A variety of in vitro immunological assays can be used to quantify antibody-venom binding and the strength or specificity of that binding (e.g. end-point ELISA, relative avidity ELISA, immunoblotting, small scale affinity purification (Casewell et al. 2010) and antivenomics (Calvete et al. 2016). WHO and pharmacopoeia guidelines (WHO 2010) recommend that in vivo preclinical tests be undertaken to ensure that immune-binding of antivenom immunoglobulins translates into venom-neutralising efficacy.

The gold standard method for validating the preclinical efficiency of antivenom remains the murine effective dose 50 (ED50) assay (Warrell et al. 2013). This method challenges experimental animals (CD1 mice) with antivenom premixed with lethal doses of venom for extended periods of time (7-24 hrs) (e.g. (Bolton 2017; Casewell et al. 2010; Cook et al. 2010). The high costs of these in vivo assays to experimental mice necessitates greater implementation of the 3Rs principles (Replacement, Reduction, Refinement) (Bolton 2017; Gutiérrez et al. 2017a; Gutiérrez et al. 2017b). There is a compelling need for research identifying alternative in vitro tests to assess antivenom efficacy of antivenoms. This aim is exceedingly difficult to attain, however, since venom is a complex mixture of bioactive agents which act on multiple different physiological targets both locally and systemically (Gutiérrez et al. 2017b; Warrell 2010a; Warrell et al. 2013).

In this study, we undertake a comparative assessment of a variety of immunological in vitro assays to determine which best predicts efficacy outcomes of in vivo preclinical testing of antivenom. This study was part of a major CSRI research project to identify the most potent mixture of elapid venoms to generate antivenom with greatest efficacy against envenoming by neurotoxic snakes of sub-Saharan Africa.

The first experiment immunised groups of sheep (2 sheep/group) with a variety of venoms and adjuvants (see Table 5.1) to identify the optimal combination of venoms and adjuvants. Three experimental antivenoms (IgGs) from sheep immunised with different combinations of venoms from the neurotoxic N. melanoleuca, N. nivea and Dendroaspis polylepis snake venoms and Freunds and glycan adjuvants. As a control equivalent to the equine EchiTAb- PLUS-ICP (mentioned previously in this thesis), we also immunised sheep with venoms from the haemotoxic/cytotoxic E. ocellatus, B. arietans and N. nigricollis snakes. Based upon highly encouraging serology results and experimental costs, the PI terminated the experiment at 6 months. In a dramatic contrast to the serology results, the preclinical, in vivo venom-

165 neutralising efficacy results identified that none of the sheep IgGs were effective. This necessitated a repeat experiment, with a slightly revised immunisation protocol (Table 5.1), for a longer duration. I used the 12 months samples for research in this chapter. Sera from these groups of sheep were tested using a panel of in vitro immunological approaches to investigate antibody-venom protein interactions, including (i) end-point ELISA to quantify antibody-venom binding, (ii) avidity ELISA to quantify the strength of these binding interactions, (iii) immunoblotting to visualise the cross-reactivity of antibodies to venom proteins and (iv) second generation antivenomics to determine the components of snake venom that are, or are not, effectively captured by antivenoms. Finally, we compared the results of these experiments with gold-standard preclinical assessments of venom neutralisation, the murine in vivo ED50 assay, to determine whether this ethically challenging protocol can be replaced by in vitro alternatives.

166

5.2 Material and methods

5.2.1 Snakes and venom

The venom samples used in this study were collected from snakes maintained in the Centre for Snakebite Research and Interventions (CSRI) herpetarium at the Liverpool School of Tropical Medicine. The snakes are all medically-important species from sub-Saharan Africa; the vipers E. ocellatus (Nigeria) and Bitis arietans (Nigeria), and the elapids Naja nigricollis (Nigeria), N. melanoleuca (Cameron), N. nivea (South Africa) and Dendroaspis polylepis (Tanzania). Venom samples were collected, lyophilised and stored at 4 °C prior to use.

* Note: N. melanoleuca snake has been split up recently into four species by (Wüster et al. 2018). The venom used in this study is now from Naja subfulva.

5.2.2 Ovine immunisation

We investigated sera from the two immunisation experiments. In the first experiment, in order to generate the experimental antivenoms, we immunised (A) three groups of sheep (n=2) for six months with different immunising mixtures of venom and adjuvants.

(B) four groups of sheep (n=2) immunised for 12 months with a further set of different mixtures of venoms and adjuvants (Table 5.1). In part, this experimental design was applied to take account of the variable immunogenicity of distinct venom toxin families (e.g. high molecular weight snake venom metalloproteinases that are often abundant in viper venoms vs low molecular weight three-finger toxins that typically dominate elapid venoms). One of the generated antivenoms was defined as an anti-haemotoxic/necrotic antivenom and was produced using venoms (from E. ocellatus, B. arietans, N. nigricollis) that cause haemorrhagic, coagulopathic and/or necrotic pathologies in envenomed victims in sub- Saharan Africa. The remaining three antivenoms were generated against snakes responsible for potent neurotoxicity (D. polylepis, N. melanoleuca and N. nivea). One was made using D. polylepis and N. melanoleuca venom, while the remaining three antivenoms used D. polylepis and N. nivea venom but were mixed with different adjuvants for immunisation (Table 5.1).

Sheep were sequentially immunised with 125 μg, 250 μg and 750 μg venom for the first three-month immunisations and 1 mg venom for all subsequent monthly immunisations (until 20 weeks). Each immunisation contained equal ratios of those venoms used for each group, as defined in Table 5.1 (1:1:1 or 1:1). Equal quantities of venom and Freund’s Complete Adjuvant (for primary immunisations) or Freund’s Incomplete Adjuvant (for all subsequent boosting immunisations) were used to prepare the venom immunogens.

167

However, for one of the immunised animals (group C) we emulsified venom with glucan particles and for another (group D) the immunising venoms were mixed with glucan particles and Freund’s Complete/Incomplete Adjuvant (as above Complete for initial, Incomplete for boosters). To induce rapid seroconversion, venom immunisations were administered at the following sites: 0.05 ml/site into five sites for the first and second immunisations; 0.05 ml/site into ten sites for the third immunisation, and 0.05 ml/site into twenty sites for the all remaining immunisations. In all cases, the sites of immunisation were close to major draining lymph nodes in the neck, chest and groin. Blood samples were taken every 30 days to monitor seroconversion. Once antibody titres had reached a plateau (determined by end- point titration ELISA, see details below), one litre of blood was collected, allowed to clot at room temperature (RT), separated via centrifugation, and the sera stored at -20 °C.

Table 5.1: Details of the venoms and adjuvants used for the generation of the experimental antivenoms after six months and twelve months by ovine immunisation;

Sheep Source venom Origin Adjuvant (s) Experimental groups Primary Secondary antivenom Duration of immunisation Group A Dendroaspis polylepis Tanzania Freund’s Freund’s Anti-NmelDpol 6 months (Dpol) Complete and Incomplete Naja melanoleuca Cameroon Freund’s (Nmel) Incomplete Group B Dendroaspis polylepis Tanzania Freund’s Freund’s Anti-NnivDpol.1 6 months (Dpol) Complete and Incomplete Naja nivea (Nniv) South Africa Freund’s Incomplete Group C Dendroaspis polylepis Tanzania glucan/venom anti-NnivDpol.2 6 months (Dpol) South Africa particles NO Naja nivea (Nniv) Group D Dendroaspis polylepis Tanzania glucan/venom Freund’s anti-NnivDpol.3 6 months (Dpol) South Africa particles & Incomplete Naja nivea (Nniv) Freund’s Complete and Freund’s Incomplete Group E Echis ocellatus (Eoc) Nigeria Freund’s Freund’s Anti-EocBarNnig 6 months Bitis arietans (Bar) Nigeria Complete and Incomplete Naja nigricollis (Nnig) Nigeria Freund’s Incomplete

Group F Dendroaspis polylepis Tanzania Freund’s Freund’s Anti-NmelDpol 12 months (Dpol) Complete and Incomplete Naja melanoleuca Cameroon Freund’s (Nmel) Incomplete Group G Dendroaspis polylepis Tanzania Freund’s Freund’s Anti-NnivDpol 12 months (Dpol) Complete and Incomplete Naja nivea (Nniv) South Africa Freund’s Incomplete Group H Dendroaspis polylepis Tanzania Freund’s Freund’s Anti- 12 months (Dpol) Complete and Incomplete NmelNnivDpol Naja melanoleuca Cameroon Freund’s (Nmel) Incomplete Naja nivea (Nniv) South Africa Group I Echis ocellatus (Eoc) Nigeria Freund’s Freund’s Anti-EocBarNnig 12 months Bitis arietans (Bar) Nigeria Complete and Incomplete Naja nigricollis (Nnig) Nigeria Freund’s Incomplete

168

5.2.3 Caprylic acid purification of IgG immunoglobulins

Prior to the purification of IgG antibodies, ovine sera were diluted (1:1) with 0.90% saline. We then used caprylic acid (Sigma, UK), at a final concentration of 6%, to precipitate non-IgG proteins, facilitated by stirring vigorously for 60 minutes (Rojas et al., 1994). The mixture was centrifuged at 13,000 RPM for 60 min at 4 °C, and culture medium containing the immunoglobulin dialysed to remove residual acid (Snakeskin Dialysis Tubing, ThermoFisher Scientific) and three changes of sodium citrate buffer saline (SCS) (pH 6.0) at RT and one at 4 °C overnight. The resulting IgG preparations were lyophilised, reformulated to 50 mg/ml in PBS (pH 7.4), before storage at -20 °C. As a comparator and positive control for our newly- generated antivenoms, we used the SAIMR Polyvalent snake antivenom (batch number X02646; Exp 2012; South African Vaccine Producers, South Africa), which is generated by immunising horses with venoms from snakes of the genera Bitis, Dendroaspis and Naja ( B. arietans, B. gabonica, D. polylepis, D. jamesoni, D. angusticeps, N. nivea, N. melanoleuca, N. annulifera, N. mossambica, and Hemachatus haemachatus). We also standardised this antivenom to the same concentration (50 mg/ml) for use in the immunological assays described below.

5.2.4 In vitro immunological assays

5.2.4.1 Time course ELISA of sheep sera and End-point ELISA of sheep IgGs.

The immune response of sera (1:5000 dilution) from each sheep was assessed by ELISA throughout the experiment. The purified IgG of each sheep at the 6- and 12-month time points was assessed by end-point titration ELISA.

Briefly, ninety-six well ELISA plates (ThermoFisher Scientific) were coated with 100 ng of each venom (1 mg/ml) in coating buffer (100 mM Bicarbonate/ carbonate buffer, pH 9.6) and incubated at 4 °C overnight to allow the venom to bind to the plate (Casewell et al. 2010). The plate was then washed with TBST (0.01 M Tris-HCl, pH 8.5; 0.15 M NaCl; 0.1% Tween 20) three times with three changes (and a five-minute incubation period between each change) to remove the unbound protein. The plate was then blocked with 5% non-fat milk in TBST and incubated at RT for three hours. Subsequently, the blocking buffer was removed by again washing three times with TBST. The plates were next incubated (in triplicate) with each antivenom at an initial dilution of (1:5000) for time course ELISA (a single dilution) or 1∶500 for experimental IgG, followed by 1∶5-fold serial dilutions across the plate, and incubated at 4 °C overnight. The plates were then washed again and incubated for three hours at RT with

169 horseradish peroxidase-conjugated donkey anti-sheep IgG (for all experimental antivenoms) and rabbit anti-horse IgG (for the SAIMR Polyvalent positive control antivenom) (Sigma, UK) at a dilution of 1∶1000. The results were visualised by adding the substrate (0.2% 2,2/-azino- bis (2-ethylbenzthiazoline-6-sulphonic acid) to a citrate buffer (0.5 M, pH 4.0 containing 0.015% hydrogen peroxide (Sigma, UK)) and measured at an optical density (OD) of 405 nm, 15 minutes after the addition of the substrate.

5.2.4.2 Relative avidity ELISA

Relative avidity ELISAs were performed as per the end-point ELISA assays protocols described above (Casewell et al. 2010), but with the addition of ammonium thiocyanate (NH4SCN) after the primary antibodies were washed from the plates. A range of concentrations (0-8 M) of

NH4SCN were added to the plates in triplicate for 15 minutes at RT. The plates were then washed with TBST and the remainder of the protocol undertaken (addition of secondary antibodies, wash and visualisation). As with the end-point ELISA, the plates were measured at an OD of 405 nm 15 minutes after the addition of the substrate.

5.2.4.3 Immunoblotting

For SDS-PAGE, we used 10 µg (1 mg/ml) of each reconstituted snake venom and incubated them at a 1:1 ratio with a reducing protein loading buffer (2xPLOB containing 2, mercaptoethanol) at 100 °C for 12 minutes in order to denature venom proteins. We then ran the samples, alongside 5 µl of protein marker (Promega), on 15% SDS-PAGE gels (hand cast gels) prepared according to the manufacturer’s instructions) at 200 volts for 55 minutes, using a Bio-Rad mini-protean electrophoresis system. The resulting resolved venom proteins were then either stained overnight in Coomassie blue staining (R-250) or transferred to 0.2 µm nitrocellulose membranes using a mini turbo Bio-Rad transfer system as per the manufacturer’s instructions. The resulting immunoblots were blocked overnight with 5% non-fat milk in TBST at 4 °C, and then washed three times every five minutes with TBST. Nitrocellulose membranes were then incubated at RT for three hours with 30 ml of primary antibody (antivenoms) diluted to 1:5,000 in 5% non-fat milk in TBST. The immunoblots were then washed six times in TBST (with a five minute incubation period between each wash), and incubated with 30 ml of horseradish peroxidase-conjugated donkey anti-sheep IgG (for all experimental antivenoms) and rabbit anti-horse IgG (for the SAIMR polyvalent positive control antivenom) (both Sigma, UK) diluted to 1: 2,000 in PBS for three hours at RT. The immunoblots were then washed again (as above) before being developed by DAB substrate

170

(50 mg 3,3’- 222 diaminobenzidine, 100 ml PBS and 0.024% hydrogen peroxide; Sigma) by placing the membrane into the substrate for 30 seconds.

5.2.4.4 Antivenomics

A second generation antivenomics approach (Pla, Gutiérrez & Calvete 2012) was applied in our collaborator Prof Juan Calvete’s laboratory in Valencia in order to examine the immunoreactivity of the ovine IgGs contained in our experimental antivenoms against each of the venoms used for immunisation (Table 5.1). Antivenom IgG affinity columns were prepared in batches of 3 ml. To this end, CNBr-activated Sepharose 4B (Ge Healthcare) matrix was packed in ABT columns and washed with 10x matrix volume of cold 1 mM HCl, followed by 2x matrix volume of 0.2 M NaHCO3, 0.5 M NaCl, pH 8.3 (coupling buffer) to adjust the pH to 7.0-8.0. Purified ovine antivenom IgGs were dialysed against MilliQ® water, lyophilised, and reconstituted in coupling buffer. The concentrations of the stock solutions were determined spectrophotometrically using an extinction coefficient of 1.36 for a 1 mg/mL concentration of IgG at 280 nm using a 1 cm light path length cuvette. One hundred mg of each experimental antivenom IgG was dissolved in 2x matrix volume of coupling buffer and incubated for four hours at RT. The coupling yields, which were estimated by measuring A280 before and after coupling of the antivenoms, were 31.3 (Anti-NmelDpol), 32.6 (Anti- NnivDpol), and 32.2 (Anti-EocBarNnig) mg/ml. After coupling, any remaining active groups were blocked with 6 ml of 0.1 M Tris-HCl, pH 8.5, at RT for four hours. The affinity columns were then alternately washed with three matrix volumes of 0.1 M sodium acetate containing 0.5 M NaCl, pH 4.0-5.0, and three matrix volumes of 0.1 M Tris-HCl, pH 8.5. This procedure was repeated six times.

The affinity columns were next equilibrated with 5 x matrix volumes of 20 mM phosphate buffer, 135 mM NaCl, pH 7.4 (PBS). For the immunoaffinity assays, individual columns containing 9 mg of antivenom IgG coupled to 300 μl matrix were incubated with 200 μg venom dissolved in ½ matrix volume of PBS and incubated for one hour at 25 °C using an orbital shaker. Non-retained fractions were collected with 5 x matrix volume of PBS, and the immunocaptured proteins were eluted with 5 x matrix volume of elution buffer (0.1 M glycine-HCl, pH 2.0) and neutralised with 1 M Tris-HCl, pH 9.0. The non-binding and the immunocaptured venom fractions were dried up to 40 μl. As specificity controls, 300 μl of CNBr-activated Sepharose™ 4B matrix, mock or with 9 mg of immobilised control (naïve) sheep/horse IgGs, were incubated with each one of the venoms and developed in parallel to the immunoaffinity assays.

171

Unbound and bound (immunoretained) fractions were fractionated by reverse-phase HPLC on a Discovery® BIO Wide Pore C18 (15 cm x 2.1 mm, 3 μm particle size, 300 Å pore size) column using an Agilent LC 1100 High Pressure Gradient System equipped with a DAD detector. The column was run at a flow rate of 0.4 ml/min and proteins eluted with a linear gradient of 0.1% TFA in MilliQ® water (solution A) and 0.1% TFA in acetonitrile (solution B), isocratically (5% B for 1 min), followed by 5-25% B for 5 min, 25-45% B for 35 min, and 45- 70% B for 5 min. Protein was detected at 215 nm with a reference wavelength of 400 nm. Proteins were identified by running samples of whole venoms under identical chromatographic conditions that have been previously characterised by venomic analysis (Calvete, 2014). The percentage of non-immunocaptured toxin “i” (% NRtoxin“i”) was calculated as the ratio between the chromatographic areas of the same peak recovered in the non-retained fraction (NRtoxin“i”) and in the injected crude reference venom (Vtoxin“i”), using the equation %NRtoxin“i” = (NRtoxin“i”/Vtoxin“i”) x 100.

5.2.5 In vivo neutralisation studies

The in vivo animal experiments were performed in our collaborator Prof Jose-Maria Gutierrez’s laboratory in Costa Rica using CD-1 male mice (18-20 g body weight). Experimental protocols were approved by the Committee for the Use and Care of Experimental Animals (CICUA) of the University of Costa Rica (CICUA-27-14) as a collaboration between Liverpool school of Tropical Medicine and The University of Costa

Rica. The toxicity of venoms was first assessed using the Median Lethal Dose 50 (LD50) assay (Theakston & Reid 1983). Various amounts of each venom, dissolved in 200 µL of 0.12 M NaCl, 0.04 M phosphates, pH 7.2 (PBS), were injected by the intravenous (i.v.) route (caudal vein) to groups of five mice. Deaths occurring during 24 h were recorded and the LD50 was calculated by Probit analysis (Finney 1952).

For neutralisation experiments, we initially screened the various antivenoms using a single predefined venom/antivenom ratio, which corresponded to 0.2 mg venom/ml antivenom for the neurotoxic venoms (N. nivea, N. melanoleuca and D. polylepis) and antivenoms (Anti- NmelDpol, Anti-NnivDpol.1, Anti-NnivDpol.2 and Anti-NnivDpol.3), and 0.5 mg venom/mL antivenom for the haemorrhagic/cytotoxic venoms (E. ocellatus, B. arietans and N. nigricollis) and corresponding antivenom (Anti-EocBarNnig). Throughout this initial screen, we used a challenge dose that corresponded to 3 x the LD50 dose. The resulting mixtures of venom and antivenom were prepared in 200 µL doses of PBS and incubated at 37 °C for 30 min before i.v. injection into the caudal vein of groups of four mice. Deaths that occurred

172 during 24 h were recorded. Control groups included mice receiving venom incubated with PBS instead of antivenom. In the case of the neurotoxic venoms and antivenoms, an additional set of experiments was performed using a challenge dose of twice the LD50 dose, under otherwise identical conditions. When neutralisation was observed in these initial screening assays, the Median Effective Dose (ED50) of the antivenoms was determined by preparing mixtures of a fixed amount of venom with variable volumes of antivenom, using PBS to attain a final volume of the mixture (200 µl). Resulting mixtures were incubated and injected i.v. as described above, and the ED50 calculated based on survival after 24 h using Probit analysis (Finney 1952).

173

5.3 Results

5.3.1 Time course ELISA of sheep sera

This assay was undertaken to compare the ability of the different venom-immunisation protocols to initiate and sustain seroconversion. The sheep in all the groups after 12 months responded to venom immunisation with a high IgG titre. All the sera samples showed a rapid response to the venom immunisation. We ran the sera with a dilution of 1:5000 for pre samples and sera collected at every second immunisation. The ELISA (Figure 5.1) showed a high titre response from the second immunisation (six weeks after the first immunisation) and continue to reach a plateau. Three venoms (N. melanoleuca, N. nigricollis and B. arietans) showed an unexplained drop in ELISA titre at the tenth immunisation but all showed a rapid recovery at the twelve immunisations. There was clear evidence of cross reactivities of the sera tested against homologues and heterologous venoms with a high titre. There were no differences between any of the animals in response to immunisation (based on results of in vitro immunological assays).

174

Figure 5.1; Immune reactivity of sheep sera post 12 months immunisation to snake venoms. Time course ELISA was employed for the sera (from each sheep in the group) collected for every second immunisation. (A) Dendroaspis polylepis (Dpol), (B) Naja melanoleuca (Nmel), (C) Naja nivea (Nniv), (D) Echis ocellatus (Eoc), (E) Bitis arietans (Bar), (F) Naja nigricollis (Nnig). Each venom was tested against all sera collected from immunised animals. Details of the immunisation protocols are in Table 5.1.

175

5.3.2 IgG titres determined by end-point ELISA

Following the standardisation of each experimental antivenom (anti-neurotoxic) and (anti- haemorrhagic, coagulopathic and/or necrotic) to 50 mg/ml, we quantified venom protein- antibody binding levels for each antivenom via end-point ELISA. Our protocols compared the immune response of the sheep six months after the first immunisation and twelve months post immunisation with those obtained with the following controls: commercial antivenom (SAIMR polyvalent), normal sheep IgG (sourced from non-immunised animals), and PBS. The titre plateau of the twelve-month IgGs was greatly extended in comparison with the six- months IGgs. Although the resulting IgG titres exhibited considerable variation, we observed the greatest binding levels between those venoms used as immunogens, as anticipated (Figure 5.2 and 5.3).

Six months after the immunisation, both experimental anti-neurotoxic antivenoms [Anti- NmelDpol (group A) and Anti-NnivDpol (group B)] exhibited the greatest binding against the homologous neurotoxic venoms from D. polylepis, N. melanoleuca and N. nivea, although cross reactivity against the heterologous elapid venom of N. nigricollis was also observed. Significantly, the venom of E. ocellatus, B. arietans and N. nigricollis was potently recognised by the anti-EocBarNnig (group E) IgG targeting haemorrhagic and/ or coagulopathic venom (Figure 5.2), Thus, the efficiency of anti-venom IgG titres was the same, or even greater, than the clinically effective antivenom employed in sub-Saharan Africa (SAIMR polyvalent). Although the resulting IgG titres exhibited considerable variation, we observed the greatest binding levels between those venoms used as immunogens, as anticipated (Figure 5.2). For example, the experimental antivenoms Anti-NnivDpol.3 (group D) exhibited greatest binding against the homologous venoms from D. polylepis, N. melanoleuca and N. nivea, although cross reactivity against the heterologous elapid venom of N. nigricollis was also observed. However, binding levels obtained with anti-NnivDpol.2 (group C) were low, and the IgG titre declined rapidly even against the venoms used for immunisation. These findings suggested that the sheep immune response directed against these venom proteins was not effectively promoted by the glucan particles used as an adjuvant, or that an immunisation course longer than 20 weeks is required to stimulate high titres of antibodies.

With twelve-months immunisation (the results illustrated in Figure 5.3) the responses continued and were cross-reactive with a higher titre, recording outcomes that were even higher than the SAIMR polyvalent antivenom (positive control). The new group of sheep immunised with a mixture of neurotoxic purified only from twelve months Anti-NmelNivDop

176

(group H) show a good response against N. melanoleuca venoms, similar to Anti-NmelDopl (group F) and Anti-NnivDopl.1 (group G) but with a slightly lower titre against D. polylepis and N. nivea. Notably, the titre of IgG Anti-EocBarNning (group I) increased rapidly against homologous venoms with no remarkable change in respect to heterologous venoms. Interestingly, all four groups immunised for twelve months showed a higher seroconversion in comparison with similar groups that had been immunised with the same immunogens but for a shorter duration (Figure 5.1). These also showed a higher titre and responses than SAIMR polyvalent.

177

Figure 5.2: ELISA End-Point Titration (EPT): Titration curves for poly-specific antibodies against antigen of IgGs purified from immunisation bleeds at six months. Experimental antivenoms (IgGs) (Anti-NmelDpol (group A), Anti-NnivDpol.1 (group B), Anti-NnivDpol.2 (group C), Anti-NivDpol.3 (group D),and Anti-EocBarNnig (group E)), SAIMR polyvalent (positive control) and normal sheep IgG (negative control) were serially diluted by a factor of five and tested by ELISA (EPT) as described in the Experimental Section, against neurotoxic venoms (D. polylepis (A), N. melanoleuca(B) and N. nivea(C)) and hemotoxic / coagulopathic venoms (E. ocellatus (D), B. arietans (E), N. nigricollis (F)).

178

Figure 5.3: ELISA End-Point Titration (EPT): Titration curves for poly-specific antibodies against antigen of IgGs purified from immunisation bleeds at twelve months. Experimental antivenoms (IgGs) (Anti- NmelDpol (group F), Anti-NnivDpol.1 (group G), Anti-NmelNnivDpol (group H) and Anti-EocBarNnig (group I)), SAIMR poly (positive control) and normal sheep IgG (negative control) were serially diluted by a factor of five and tested by ELISA (EPT) as described in the Experimental Section, against neurotoxic venoms (D. polylepis (A), N. melanoleuca(B) and N. nivea(C)) and hemotoxic / coagulopathic venoms (E). ocellatus (D), B. arietans (E), N. nigricollis (F)).

179

5.3.3 IgG avidity determined by chaotropic ELISA

In view of the therapeutic intent of the experimental antivenoms, it is important to determine the binding strength (avidity) of the venom / antivenom interaction. The strength of binding between antivenom IgG and venom proteins can be determined by measuring IgG titres in the presence of a chaotropic agent that disrupts binding. Consequently, the various anti-neurotoxic (Anti-NmelDpol, Anti-NnivDpol) and anti-haemotoxic (Anti-EocBarNnig) standardised antivenoms (1:10,000 dilution of 50 mg/ml IgG) were subjected to chaotropic ELISA experiments with each of the venoms, in the presence of different concentrations of

NH4SCN. The results of greatest immunological relevance were obtained based on comparison between the percentage reduction in OD values of the IgGs in the absence of

NH4SCN and at 4 M NH4SCN (Figures 5.4 & 5.5). Four experimental antivenoms obtained from six months and twelve-months samples were compared with homologous and heterologous venoms. There was a clear improvement in the titration curve between the six months and twelve-months samples after the addition of higher concentrations of ammonium thiocyanate. The addition of 4 M ammonium thiocyanate led to a clear difference in terms of the binding between the venom and the IgG.

Among all the six-months experimental antivenoms, the highest cross-snake species IgG- venom binding avidity was demonstrated by the Anti-NmelDpol (group A), Anti-NnivDpol.1 (group B), Anti-NnivDpol.3 (group D) and Anti –EocBarNnig (group E) antivenoms (Figure 5.2). By contrast, the cross-species venom-binding avidity displayed by Anti-NnivDpol.2 (group C) was the lowest. Furthermore, compared to the IgG titre results, the chaotropic ELISA assay indicated that IgG binding avidity and efficiency were more closely associated. Notably, the IgG-binding avidity of various anti-neurotoxic antivenoms (Anti-NmelDpol, Anti-NnivDpol.1, Anti-NnivDpol.3), and that of the anti-haemotoxic antivenom (Anti-EocBarNnig), were higher than that of the SAIMR polyvalent control.

There was a clear improvement in the IgG purified through prolonged immunisation (twelve months), and less reduction of IgG titre. Anti-NmelDpol (group F) and Anti-NnivDpol (group G) showed less reduction in IgG titre against homologous venoms and a significant protection against heterologous venom (E. ocellatus, N. nigricollis and B. arietans). Anti-NmelNnivDpol (group H), meanwhile, showed a strong binding against (D. polylepis, N. melanoleuca and N. nivea) and heterologous venom N. nigricollis, with a reduction in the IgG titre of less than 10%. This IgG showed a lower binding strength against two viper venoms (E. ocellatus and B. arietans), however. The binding of anti-EocBarNnig (group I) against the three haemotoxic

180 coagulopathic venoms (E. ocellatus, B. arietans and N. nigricollis) was improved with twelve months immunisation. The reduction in titre was less than 10 % with this group and less than 20% against the neurotoxic venoms not included in the immunisation protocol.

181

Figure 5.4: Chaotropic ELISA (Avidity): Titration curves for IgGs purified from immunisation bleeds at six months against homologous and heterologous venoms after addition of different concentrations (1,2,3,4,6 and 8 moles) of ammonium thiocyanate (NH4SCN). Experimental antivenoms (IgGs) of (Anti- NmelDpol (group A), Anti-NnivDpol.1(group B), Anti-NnivDpol.2 (group C), Anti-NnivDpol.3 (group D), and Anti-EocBarNnig (group E)), SAIMR poly (as a positive control) and normal sheep IgG (as a negative control) were tested by ELISA assay as described in the Experimental Section, against neurotoxic venoms (D. polylepis, N. melanoleuca and N. nivea)and haemotoxic / coagulopathic venoms (E. ocellatus, B. arietans, N. nigricollis).

182

Figure 5.5: Chaotropic ELISA (Avidity): Titration curves for IgGs purified from immunisation bleeds at and twelve months against homologous and heterologous venoms after addition of different concentrations (1,2,3,4,6 and 8 moles) of ammonium thiocyanate (NH4SCN) with different concentration (1,2,3,4,6 and 8 moles). Experimental antivenoms (IgGs) of (Anti-NmelDpol (group F), Anti-NnivDpol.1( group G), Anti- NmelNnivDpol (Group H) and Anti-EocBarNnig (group I)), SAIMR poly (as a positive control) and normal sheep IgG (as a negative control) were tested by ELISA assay as described in the Experimental Section, against neurotoxic venoms (D. polylepis, N. melanoleuca and N. nivea) and haemotoxic / coagulopathic venoms (E. ocellatus, B. arietans, N. nigricollis).

183

Fig ure 5.6: Percentage reduction of IgG titre (twelve months immunisation) after addition of 4M ammonium thiocyanate. The experimental IgGs (A) Anti-NmelDpol, (B) Anti-NnivDpol.1, (C) Anti- NelNnivDpol and (D) Anti-EocBarNnig were employed against immunised homologous and heterogenous venoms. IgGs purified from immunisation bleeds at six months and twelve months were diluted (1:10,000) and subjected to different concentrations of ammonium thiocyanate (0-8 M). 4 M of the chaotropic agent was used to calculate the percentage of reduction.

184

5.3.4 IgG specificity revealed by Western blotting

Analysis of the protein profiles of resuspended venoms using 15% SDS-PAGE revealed a clear variation in the venom protein profiles between elapid and viperid venoms. The elapid venoms (e.g. D. polylepis, N. melanoleuca, N. nivea and N. nigricollis) predominantly contain phospholipase A2 (PLA2), representative of the three finger toxins family (3FTx), in addition to lower amounts of proteins from other families. In contrast, venoms from Viperidae (E. ocellatus and B. arietans) are predominately composed of various proportions of PLA2, zinc- dependent metalloproteinases (SVMP) and serine proteinases, as well as varying amounts of C-type lectin-like proteins, disintegrins and other types of proteins (Gutiérrez et al. 2018).

A panel of venoms were resolved by SDS-PAGE and then blotted and probed with the ovine IgGs (Our experimental antivenoms). Significant cross reactivities were observed between heterologous and homologous venoms and the experimental antivenoms. As can be observed (Figure 7), the ovine IgGs reacted with a large numbers of venom components (5- 200 KDa). Anti-NmelDpol (group A and group F) successfully recognised its homogenous venoms (D. polylepis and N. melanoleuca) as well as N. nivea and N. nigricollis. Prolonging the immunisation period did not appear to result in any significant changes in the recognition, with the exception of B. arietans venoms, where more proteins appeared in the Western blotting.

Six months following the immunisation, the experimental antivenoms Anti-NmelDpol (group A), Anti-NnivDpol (group B) and Anti-EocBarNnig (group E) recognised well D. polylepis venom proteins. While, N. melanoleuca proteins were strongly recognised by all experimental antivenoms. The majority of our experimental antivenoms bound strongly to N. nivea venoms except Anti-EocBarNning (Group E). It is evident that the antivenom Anti- EocBarNnig generated by immunisation of three venoms (E. ocellatus, N. nigricollis and B. arietans) strongly recognised proteins of E. ocellatus and B. arietans and, less strongly, proteins of N. nigricollis. Both experimental antivenoms, Anti-NnivDpol (group C) and Anti- NiviDpol (group D) poorly recognised homologous and heterologous venoms.

Anti-NnivDpol produced after prolonged immunisation for twelve months (group G), however, did exhibit an improvement, with high intensity and recognition of proteins encompassing most of the venom proteins from the elapids and Viperidae. Interestingly, however, the combination of the three elapids to generate Anti-NmelNinvDpol (group H) showed low binding in comparison with the other two IgG groups. It was noticeable also that Anti-EocBarNnig antivenom strongly reacted with components of homologous venom

185 proteins that had both low and high molecular weight, with no significant changes apparent after prolonged immunisation. B. arietans showed a strong recognition and binding with Anti-EocBarNnig antivenom, to be even higher than SAIMR Polyvalent, which is included (Naja, Bitis and Dendroaspis) in the immunisation protocol of this commercial antivenom. In comparison, to SAIMR Polyvalent, our experimental antivenoms included in the immunisation protocol poly-specific antivenoms which are Naja, Bitis and Dendroaspis. Our experimental antivenom Anti-EocBarNing reacted strongly against Echis, Bitis, while, Anti- NmelDpol (group F) and Anti-NnivDpol (group G) showed a high IgG-venom intensity against homogenous and heterologous venoms compared to SAIMR Polyvalent. No reactivities were observed with the normal sheep IgG.

186

Figure 5.7: Western blotting analysis of neurotoxic venoms (D. polylepis, N. melanoleuca and N. nivea) and haemotoxic / coagulopathic venoms (E. ocellatus, B. arietans, N. nigricollis) against four antivenoms (IgGs) (Anti-NmelDpol, Anti-NnivDpol, Anti-NmelNnivDpol and Anti-EocBarNnig) and compared with SAIMR poly (as a positive control) and normal sheep IgG (as a negative control). The proteins contained in 10 µg of the obtained venoms were separated on 15% acrylamide gels under reducing conditions, and the immunoreactivity of antivenoms was assessed as described in the Experimental Section. Figures (A) Coomassie Brilliant Blue staining of venom profiles: (B) purified IgGs from immunisation bleeds at six months (C) 12 months; (1) Anti-NmelDpol, (2) Anti-NivDpol, (3) Anti-NmelNinvDpol and (4) Anti-EocBarNnig. All IgGs against homologous and heterologous venoms, (D) immunoblots of (1) SAIMR Polyvalent (positive control and (2) normal sheep IgG.

187

5.3.5 Antivenomics profiling of venom protein and antivenom antibody interactions The immunoreactivity of the experimental antivenoms in relation to the employed selection of neurotoxic and haemotoxic coagulopathic venoms was assessed through ELISA, avidity ELISA and western blot analysis. These assays were supplemented with second-generation antivenomics. Reverse-phase HPLC chromatograms of whole venom and immunoaffinity column eluates were quantitatively compared, being both qualitatively and quantitatively informative about the series of toxins carrying epitopes detected by antivenoms, as well as about toxins with suboptimal, incomplete or unsatisfactory immunoreactivity. The comparative antivenomics assessment was focused on determining how effective the four antivenoms of Anti-NmelDpol, Anti-Nnivdpol, Anti-NmelNinivDpol and Anti-EocBarNnig were in terms of immunocapturing separate toxins from three neurotoxic venoms and three haemotoxic coagulopathic venoms of sub-Saharan African snake species. Both homologous and heterologous venoms were employed in the assessment so as to comprehend the efficacy and development of antivenoms more clearly, and to understand better the binding between venom and antivenom. The working premise was that the ability of antivenom to counteract the effect of a venom reflected the level of toxin immunorecognition of that antivenom. Findings revealed that, apart from Anti-EocBarNning, which exhibited partial retention of most homologous venoms (E. ocellatus, B. arietans and N. nigricollis), All experimental antivenoms produced six months after immunisation displayed no immunorecognition with low affinity.

Over a longer term of one-year of immunisation Immunoreactivity was enhanced, however. According to the antivenomics, one-year purification of IgGs led to better binding of 3FTx molecules (peaks 1-12). The Anti-NnivDpol IgGs exhibited partial binding of 3FTx molecules, with minimal or lack of recognition by P-III SVMP (peaks 14-15). Suboptimal immunocapture of the proteins from D. polylepis species was displayed by Anti-NmelDpol and Anti-

EocBarNnig. The main immunodominant proteins (3FTx, PLA2, 3FTx, CRISP and PII-SVMP) from the venom of N. nivea and N. nigricollis were revealed by the chromatographic profiles of entire components. The IgGs of every experimental antivenom demonstrated suboptimal retention or recognition of the N. nivea venom proteins 3FTx and PLA2, whilst they showed partial retention (80-98%) of P-III-SVMP and LAO molecules (peaks 11-13). An immunoaffinity binding yield of 3FTx (peaks 1-3, 9), CRISP (peak 10) and P-III SVMP (peaks

11-13), however, was exhibited by Anti-NnivDpol. None of the antivenoms detected PLA2. Nevertheless, all three antivenoms targeting the neurotoxic venom of N. nigricollis (Anti-

188

NmelDpol, Anti-NnivDpol and Anti-NmelNnivDpol) partially detected 3FTx, CRIPS and SVMP molecules. Furthermore, most proteins had partial binding of Anti-EocBarNning.

The maximum extent of antivenom binding to the PLA2, CTL and SVMP molecules from the venom of E. ocellatus was also investigated via antivenomics, with the non-specific antivenoms Anti-NmelDpol, Anti-NivDpol and Anti-NmelNnivDpol showing inadequate or lack of detection of the main venom proteins. By contrast, partial immunocapture of PLA2 (peak 3) and CTL (peak 5), and strong binding of SVMP molecules were displayed by the specific antivenom Anti-EocBarNning. Disintegrins, SVSP and SVMPs were revealed through reverse-phase HPLC separation of the profiles of the proteins from B. arietans venom. According to the analysis of antivenom immunoreactivity in relation to this venom, none of the experimental antivenoms demonstrated efficient protein detection and immunoprecipitation. Anti-EocBarNning constituted an exception, however, showing strong binding and detection of the main proteins of B. arietans venom, namely, PLA2, SVSP and SVMP, providing evidence of its efficiency in counteracting the actions of highest toxicity.

189

190

191

Figure 5.8: Comparison of the antivenomics analysis of the immunoreactivity of experimental antivenoms (purified IgGs from immunisation bleeds at six months and twelve months) towards neurotoxic venoms (A) D. polylepis, (B) N. melanoleuca, and (C) N. nivea) and haemotoxic coagulopathic/ necrotic venoms (D) E. ocelltus, (E) B.aritens and (F) N. nigricollis). The reverse phase separation of the whole venoms (A1, B1, C1, D1, E1 and F1) and the percentage of immunocaptured (retained) protein fractions are calculated in the table (A2, B2, C2, D2, E2 and F2). The details of these results are included in the supplementary section (Figures S 5.1-S 5.6)

192

5.3.6 The venom neutralising efficacy of experimental antivenoms

In vivo neutralisation of venom We assessed whether the experimental antivenoms were able to prevent the lethal effects of the venom in the WHO-recommended mouse model. We compared the neuralisation activities of purified IgG from six months and twelve-months immunisations with homologue venoms.

When challenged with a dose of 3 x LD50s of D. polylepis venom, the various experimental antivenoms generated from immunisation bleeds at six months using D. polylepis venom as an immunogen (Anti-NmelDpol, Anti-NnivDpolanti) were found to be completely ineffective at neutralising venom lethality at ratios of 0.2 mg venom per ml antivenom. Next, a venom

‘challenge dose’ of only 2 x LD50s of D. polylepis was used. This venom dose resulted in 100% lethality in the control ‘no antivenom’ group. When using this challenge dose of venom, the same antivenoms (Anti-NmelDpol, Anti-NnivDpol) were able to protect against the lethal effects of D. polylepis venom, with the exception of Anti-NnivDpol.2 (Table 5.3). In contrast, the same antivenoms failed to neutralise the lethality of N. melanoleuca venom at the lowest venom/antivenom ratio tested (0.2 mg venom per ml antivenom) when using challenge doses of venom of 3 or 2 x LD50s. We did not test the neutralising capability of the various anti-neurotoxic antivenoms against N. nivea venom, as lethality testing revealed that experimental animals did not succumb to the lethal effects of the venom alone at the highest dose tested (25 µg venom/mouse) (Table 5.2). The neutralisation of the haemotoxic/cytotoxic venoms (Echis ocellatus, Bitis arietans and Naja nigricollis) by the Anti- EocBarNnig (group E) antivenom was assessed by using a ‘challenge dose’ of venom of 3 x

LD50. The experimental antivenom was ineffective at neutralising the lethal activity of these three venoms when tested at ratios of 0.5 mg venom/ml antivenom. Considering commercial monovalent and polyvalent antivenoms typically neutralise more than 3 mg of E. ocellatus venom per ml antivenom (Calvete et al. 2018), lower venom/antivenom ratios were not tested, and the antivenom was considered ineffective for neutralising the lethal effects of these venoms.

The IgGs from immunisation bleeds at twelve months, were assessed in the context of venoms which were neutralised at an ED50 value higher than 0.25 mg venom/ml antivenom. Three venom/antivenom ratios were assessed, i.e. 0.25 mg/ml, 0.5 mg/ml and 0.75 mg/ml

193 for N. melanoleuca and D. polylepis, and 0.75 mg/ml, 1.00 mg/ml and 2.00 mg/ml for N. nivea, using the same challenge dose of 3 LD50s of venom and the same incubation and injection protocol. Anti-NmelDpol (group F), generated with immunisation against N. melanoleuca and D. polylepis) transpired to be ineffective at neutralising N. melanoleuca venom and D. polylepis venom, although it was effective (albeit with an ED50 higher than 0.75 mg /ml) at neutralising N. nivea, which was not included in the immunisation. The antivenom (group G) generated by immunisation of N. nivea and D. polylepis (Anti-NnivDpol.1) was ineffective against these venoms. The mixture of three neurotoxic venoms to generate Anti- NmelNnivDpol (group H) was effective at neutralising homologue venoms used for immunisation (N. melanoleuca, N. nivea and D. polylepis). Here, the ED50 for N. melanoleuca venom was 0.67 mg/ml (95% confidence limits: 0.50 – 0.80), for N. nivea it was > 2.00 mg venom/ml antivenom and for D. polylepis venom 0.75 mg/ml. The neutralisation ability of Anti-EocBarNning (group I) against E. ocellatus, B. arietans and N. nigricollis venoms, meanwhile, showed the highest neutralisation of these homologous venoms at a challenge dose of 3 LD50s (Table 5.3).

Table 5.2. Estimation of LD50s of the venoms used in this study.

Venom (s) LD50 (µg/mouse)

D. polylepis 6.2 (95 CI: 3.1 – 10.2) N. melanoleuca 5.6 (95% CI: 3.1 – 7.0) N. nivea > 25 (no mice died at the highest dose tested (25 µg) E. ocellatus 17.85 (95% CI: 12.46 – 28.53). B. arietans 21.60 (95% CI: 15.96 – 31.60). N. nigricollis 27.81 (95% CI: 22.53 – 41.62).

194

Table 5.3: Values for the Median Effective Dose (ED50)

Venom Immunisation Anti- Anti-NivDpol Anti- Anti- NmelDpol NmelNnivDpol EocBarNnig N. 6 months <0.25 <0.05 ND melanoleuca 12 months <0.25 <0.25 0.67(0.5-0.8) D. polylepis 6 months 0.34 (0.152- 0.673(0.416- ND 0.485) 0.836) ND 12 months <0.25 0.39 (0.24- >0.75 0.48) N. nivea 6 months ND 12 months 2.0 mg >2.0 >2.0 E. ocellatus 6 months <0.5 12 months 1.2(0.75-1.7) B. arietans 6 months ND <0.5 12 months 4.4 (3.2-6.1) N. nigricollis 6 months <0.5 12 months 1.47 (0.16-2.1)

*ED50 of Experimental antivenom [ mg venom/ ml antivenom (95 CI)]. *ND (Not determined).

195

5.4 Discussion The most significant outcome of this study was that antivenom ELISA IgG titres and avidity results must never be used alone as a predictor of antivenom efficacy. This outcome is very important as other groups have published papers with titles such as “Development of an ELISA assay to determine neutralising capacity of horse serum following immunisation with Daboia siamensis venom in Myanmar” (Khaing et al. 2018). These assays are useful immunological tools, particularly when coupled with results of antivenom specificity (e.g. immunoblotting) for several purposes other than efficacy prediction. The paucity of effective and affordable antivenom in many areas has galvanised endeavours to develop antivenom with efficacy against multiple snakes – pan-specific antivenom. The consequent economies of scale improve commercial manufacturing incentives (Gutiérrez et al. 2005; Ratanabanangkoon et al. 2016; Williams et al. 2011). Achieving this snakebite-therapy panacea has proved very problematic.

Selection of immunogens and immunisation protocols Venoms are chosen as immunogens for antivenom manufacture on the basis of their medical significance (WHO 2016a). Venoms of different snake species do not have the same immunogenic profile. Indeed, venom protein composition and hence antigenicity can vary even within one snake species as a consequence of geographic and age distinctions (Alape- Girón et al. 2008; Fry et al. 2003). The immunogenicity of venoms depends on the structural characteristics of every protein family, including molecular mass, tertiary and quaternary structures, glycosylation extent, and additional post-translational changes. Moreover, venom immunogenicity is also determined by the proportion of each protein in the venom. When antivenom is developed, immunoglobulins are usually derived from equine or ovine species, especially horses. Although it was previously thought that the safety of ovine antivenoms exceeded that of equine antivenoms (Landon & Smith 2003), the very limited clinical evidence does not support the belief that one type of antivenom has more side- effects than the other (Abubakar et al. 2010). As a highly glycosylated immunoglobulin, however, equine IgG(T) is considered to have a greater probability of side-effects, particularly serum sickness, because its immunogenicity is higher compared to ovine IgG (Landon & Smith 2003; Sjostrom et al. 1994). Based on the evidence from earlier research, clinical observations and financial considerations, the antivenoms in this study were therefore developed from ovine sources.

196

For the production of an antivenom efficient against neurotoxicity, this work employed the neurotoxic venoms of three clinically significant sub-Saharan African Elapidae (WHO 2016b), namely, Dendroapsis polylepis, Naja melanoleuca and Naja nivea, as immunogens. Meanwhile, for the production of antivenom efficient against haemotoxicity and base dermal necrosis, this work employed the haemotoxic venoms of Echis ocellatus, Bitis arietans and Naja nigricollis. In this study, it was noticed that creating a mixture of the venoms with the gold standard adjuvants, namely, Freund’s Complete and Freund’s Incomplete, resulted in a satisfactory IgG titre being derived from ovine immunisation. On the other hands, using a glucan particle did not show any improvements in the IgG titre after six months immunisation compared to the sheep immunised with the same venoms and different adjuvants. Furthermore, no risks were associated with employing multiple venoms in immunisation. Casewell et al. (2010) reported similar observations, indicating that IgG reached a plateau at 16 weeks following ovine immunisation. Incontrovertible improvement and increased immunogenicity are yielded by immunisation procedures of long duration (6-12 months).

Assessing the immune responses of the experimental antivenoms against homologous and heterologous venoms

Envenomation constitutes an antigen challenge that triggers the adaptive immunity of vertebrates to counteract the effects of the venom by increasing the antibody levels. More specifically, the venom toxins can be counteracted by antibodies attaching to them and preventing the antigen from manifesting its toxicity, or by isolating the antigen in the circulatory system until the toxins are eradicated through lysosomal breakdown (Cheng et al. 2009). When it comes to counteracting the effects of venoms, an important property is antibody cross-reactivity. In relation to polyclonal antivenom, O’Leary et al. (2007) explained cross-reactivity as the capability of whole antivenom to bind more than one antigen. This is a highly sought-after property and can be attained by employing venom from a range of different snake species during the immunisation stage of antivenom development.

In vitro assays such as immunoblotting, immunodiffusion and ELISA can help to determine whether or not a venom and an antivenom have cross-binding. The relevance of such techniques stems from the fact that, if they determine cross-binding to be absent. If in vitro assays indicate the presence of cross-reactivity, however, it will not necessarily mean that cross-neutralisation has taken place, but only that the antivenom antibodies can detect and bind the toxins in the venom (Ledsgaard et al. 2018; León et al. 2018). The reason why cross- neutralisation is not always signalled by cross-reactivity (Kornalik & Taborska 1989; Ownby

197

& Colberg 1990) is that the antibodies produced through immunisation depend on antigenic variations. For instance, in an investigation of the ability of Bothrops jararaca antivenom to counteract the effects of the venoms of nine Bothrops species, Ferreira et al. (1992) found that the actions induced by the PLA2 from the venom of B. alternatus or B. erythromelas were not counteracted by PLA2 from B. jararaca venom. The shortcomings of cross-neutralisation have been researched in the past and have been clearly highlighted for whole venoms of snakes from the families of Viperidae, Crotalidae and Elapidae (Arce et al. 2003; Chinonavanig et al. 1988; Kornalik & Taborska 1989).

In the current study, the immunological assays that were carried out clearly pointed to robust binding and also suggested that the antivenoms displayed immunoreactivity against both homologous and heterologous venoms. This experiment clearly identified that glucan particles are not as effective adjuvants as the conventional Freunds adjuvants. According to the results obtained, a longer duration of immunisation clearly improved avidity and binding between the venom proteins and IgGs. In particular, cross-reactivity against the venom of the spitting N. nigricollis, which was not used in immunisation, was exhibited by three anti- neurotoxic IgGs (Anti-NmelDpol, Anti-NivDpol.1 and Anti-NmelNnivDpol) derived from ovine immunisation with the venom of the non-spitting N. melanoleuca and N. nivea. It can thus be deduced that, owing to the similar proteomic profiles of the venoms of the above species, antivenoms can counteract the effects of the venoms of minor species in the genera, or species who bite less frequently compared to other species in the same taxonomic category (WHO 2016a).

Moreover, in the initial six months, the venom of Elapidae such as D. polylepis exhibited a weaker response, although this became better after one year. One reason for this is that proteins like 3FTx and PLA2 with low molecular weight (6-9 kDa) dominate the composition of Elapidae venom, and they have low immunogenicity, which constitutes a significant issue for the production of antibodies (León et al. 2011). On the other hand, strong antibodies were generated by the venom of Viperidae like E. ocellatus. The composition of the proteins in the venom of Echis species may explain this high immunogenicity. To give an example, the main proteins in the venom of vipers are SVMPs, which contribute significantly to making this venom toxic (Tasoulis & Isbister 2017). Compared to P-I SVMPs, P-III SVMPs with high molecular mass yield a higher antibody response, so the structural characteristics of SVMPs have a major impact on their immunogenicity (Gutiérrez & León 2009). Furthermore, P-III SVMPs undergo glycosylation, whereas P-I SVMPs do not (Fox & Serrano 2009).

198

The findings of the immunological assays therefore showed that the antivenoms demonstrated immunogenicity and cross-reactivity against the venoms of a number of snake species. These findings were contradicted by the second-generation antivenomics, however. Relying on proteomics, the method of antivenomics helps to define the immunological profile and degree of cross-reactivity exhibited by antivenoms to venoms qualitatively and quantitatively in the context of in vivo and in vitro preclinical testing (Calvete et al. 2018; Gutiérrez 2012). According to the antivenomics conducted in the present study, there was weak binding between the venoms of the species of Elapidae (N. melanoleuca, N. nivea and D. polylepis) and anti-neurotoxic antivenoms, whereas partial-to-good binding was exhibited by those antivenoms to the venom of the Viperidae species E. ocellatus. Increasing the duration of immunisation helped to enhance the antigenicity. Furthermore, in comparison to proteins with a high molecular weight, proteins with a low molecular weight displayed a lower immunogenicity. It is challenging to generate antibodies against such proteins because they are small in size and consequently make suboptimal antigens.

Antivenomics confirmed the suboptimal binding and immunogenicity of proteins in the venom of the Elapidae Naja and Dendroaspis with low molecular weight. Moreover, possible reasons for cross-reactivity can also be derived from antivenomics; for instance, antivenomics can indicate whether two particular venoms contain the same subfamily of key toxins, therefore allowing an effective antivenom to be produced using only one of the two venoms (Gutiérrez et al. 2014). The toxic elements present in the venom of mamba species (Dendroaspis) are called dendrotoxins and fasciculins (Harvey 2009; Heyborne & Mackessy 2009) and they have low molecular mass of 6-9 kDa, which means that their immunogenicity is poor, posing an issue for antibody production (León et al. 2011). SVMPs and Anti- EocBarNnig, however, had good binding, according to the antivenomics conducted in this study. Compared to P-I SVMPs, P-III SVMPs with high molecular mass yielded a higher antibody response, so the structural characteristics of SVMPs have a major impact on their immunogenicity. Furthermore, the antigenic attributes of venom antigens depend to a significant extent on their molecular mass. For example, good immunogens include the proteins P-III SVMPs and LAO, which have a molecular mass higher than 70 kDa. On the other hand, apart from elements of Ophiophagus hannah venom, which has low molecular mass

(~25 kDa) but high immunogenicity, the neurotoxins, PLA2, P-I SVMPs and disintegrins in the venom of Elapidae have low molecular mass (5-30 kDa) and therefore lower immunogenicity (Chotwiwatthanakun et al. 2001; Gutiérrez et al. 2009; Ownby & Colberg 1990).

199

Preclinical evaluation of the efficacy of the experimental antivenoms by gold standard in vivo assays

In vivo evaluation of cross-reactivity is based on the gold standard for definition of venom neutralisation, namely, evaluation of whether an antivenom is capable of counteracting the lethal effects of various venoms in mouse models (Ledsgaard et al. 2018). In vivo data should be used to reinforce in vitro cross-reactivity experiments, since precise empirical work of venom neutralisation can only be performed in vivo. Such empirical work is based on the gold standard of assessment of the ability of antivenom to counteract the toxic effects of venom. The procedure implemented to achieve this involves injection of mouse models with venom in various concentrations to calculate the median lethal dose (LD50), followed by determination of the amount of antivenom required to counteract the lethality of a series of median lethal doses. Such experiments yield an ED50, which is indicative of how capable the antivenom is in respect to cross-neutralisation of the lethal effects of venom (Gutiérrez et al.

2017a; Ledsgaard et al. 2018). Neurotoxins and PLA2s with low molecular mass and weak antigenicity are the major components of the venom of Elapidae. Most antivenoms targeting the venom of species in this snake family therefore exhibit ED50 values for lethality neutralisation of 0.5-1.5 mg/ml, with a higher degree of potency attained in some cases (Herrera et al. 2012; Laustsen et al. 2015). On the other hand, SVMPs are the major components in the venom of Viperidae, so antivenoms targeting this venom exhibit potency of at least 2 mg/ml (Segura et al. 2010), with some exceptions (Sánchez et al. 2015).

The antivenoms used in the current experiments to target the venom of Elapidae exhibited suboptimal neutralisation capacity with six-months immunisation, but this capacity improved with twelve-months immunisation, with doses of 0.67-2 mg/ml demonstrating potency. To give an example, homologous venoms of N. melaleuca and D. polylepis were poorly neutralised by Anti-NmelDpol, but N. nivea venom was counteracted by this antivenom when administered in a dose greater than 0.76 mg/ml. N. nivea venom was also efficiently counteracted by the neurotoxic antivenoms Anti-NnivDpol and Anti- NmelNinivDpol at values greater than 2 mg/ml; meanwhile, N. melanoleuca and D. polylepis venoms were counteracted by these two antivenoms at ED50 values greater than 0.67 mg/ml and 0.39 mg/ml, respectively. This is consistent with the findings obtained by Laustsen et al. (2015) regarding neutralisation of D. polylepis venom by VINS African antivenom. Thus, it was concluded that efficient anti-neurotoxic antivenoms could be developed by using a combination of three neurotoxic Elapidae venoms with extended immunisation. Additional research is required to determine how effective the created experimental antivenoms are at

200 counteracting the effects of heterologous venoms, such as those of Naja and Dendroaspis species.

The homologous venoms of E. ocellatus, Bitis arietans and N. nigricollis were weakly or moderately counteracted by Anti-EocBarNnig, with ED50 doses of <1.45 mg/ml, >4 mg/ml and <0.43 mg/ml, respectively. Extension of the immunisation protocol led to better neutralisation, however. These findings are consistent with those obtained by Segura et al. (2010) for antivenom EchiTAB-Plus-ICP, although the present study employed a challenge dose of 3 LD50 and 5 LD50 for tests on ovine samples and standard quality control based on equine samples, respectively. Furthermore, regarding Anti-NmelNnivDpol, the antivenomics and neutralisation assessment were inconsistent, with the latter indicating satisfactory neutralisation capacity. Such findings do not fully agree with the discrepancies among the experimental antivenoms in terms of counteracting lethality, which may be attributed to the fact that antibodies do not have the same affinity to major venom elements, or in other words, antibodies capable of detecting toxins in the affinity column may not extend that capability to neutralising the toxic effects.

5.5 Conclusions:

The results of this study revealed robust immunological responses in vitro, which were enhanced by extension of the immunisation protocol. Antivenomics showed that the four experimental antivenoms demonstrated immunoreactivity with most elements of homologous and heterologous venoms. By contrast, the antivenoms differed significantly in their ability to counteract venom toxicity; thus, Anti-NmelDpol lacked efficiency in counteracting lethal effects, but was capable of effective neutralisation in the context of in vitro assays. Such findings suggest that, to understand antivenom preclinical efficiency better, antivenomics and lethality neutralisation assays should be integrated. Furthermore, the findings demand that WHO and national regulation bodies should implement a minimal appropriate threshold of antivenom neutralisation of venom toxicity in sub-Saharan Africa. In vitro immunological assays are therefore significant for three reasons: assessment of the immunogenicity of each protein in venom composition against particular antivenoms; creation of a hierarchy of protein efficiency according to the significance of the proteins for pathophysiology; and investigation of protein glycosylation, which is a key variable for the development of antibodies that contain particular glycoforms of potential relevance in terms of attaining the targeted efficiency for treatment.

201

5.6 References:

Abubakar, S., Abubakar, I., Habib, A., Nasidi, A., Durfa, N., Yusuf, P., Larnyang, S., Garnvwa, J., Sokomba, E. & Salako, L. (2010) 'Pre-clinical and preliminary dose-finding and safety studies to identify candidate antivenoms for treatment of envenoming by saw-scaled or carpet vipers (Echis ocellatus) in northern Nigeria', Toxicon, vol. 55, no. 4, pp. 719-723. Alape-Girón, A., Sanz, L., Escolano, J., Flores-Diaz, M., Madrigal, M., Sasa, M. & Calvete, J.J. (2008) 'Snake venomics of the lancehead pitviper Bothrops asper: geographic, individual, and ontogenetic variations', Journal of proteome research, vol. 7, no. 8, pp. 3556-3571. Arce, V., Rojas, E., Ownby, C.L., Rojas, G. & Gutiérrez, J.M.a. (2003) 'Preclinical assessment of the ability of polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced in Costa Rica to neutralize the venoms of North American snakes', Toxicon, vol. 41, no. 7, pp. 851-860. Bolton, F. (2017) Incorporating the 3Rs (Refinement, Replacement and Reduction of animals in research) into the preclinical assessment of snake venom toxicity and antivenom efficacy, University of Liverpool. Calvete, J.J., Arias, A.S., Rodríguez, Y., Quesada-Bernat, S., Sánchez, L.V., Chippaux, J.P., Pla, D. & Gutiérrez, J.M. (2016) 'Preclinical evaluation of three polyspecific antivenoms against the venom of Echis ocellatus: Neutralization of toxic activities and antivenomics', Toxicon, vol. 119, pp. 280-288. Calvete, J.J., Rodríguez, Y., Quesada-Bernat, S. & Pla, D. (2018) 'Toxin-resolved antivenomics-guided assessment of the immunorecognition landscape of antivenoms', Toxicon, vol. 148, pp. 107-122. Casewell, N.R., Cook, D.A., Wagstaff, S.C., Nasidi, A., Durfa, N., Wüster, W. & Harrison, R.A. (2010) 'Pre-clinical assays predict pan-African Echis viper efficacy for a species- specific antivenom', PLoS neglected tropical diseases, vol. 4, no. 10, p. e851. Cheng, L.W., Stanker, L.H., Henderson, T.D., Lou, J. & Marks, J.D. (2009) 'Antibody protection against botulinum neurotoxin intoxication in mice', Infection and immunity, vol. 77, no. 10, pp. 4305-4313. Chinonavanig, L., Billings, P.B., Matangkasombut, P. & Ratanabanangkoon, K. (1988) 'Antigenic relationships and relative immunogenicities of venom proteins from six poisonous snakes of Thailand', Toxicon, vol. 26, no. 9, pp. 883-890.

202

Chippaux, J.-P. (2011) 'Estimate of the burden of snakebites in sub-Saharan Africa: a meta- analytic approach', Toxicon, vol. 57, no. 4, pp. 586-599. Chotwiwatthanakun, C., Pratanaphon, R., Akesowan, S., Sriprapat, S. & Ratanabanangkoon, K. (2001) 'Production of potent polyvalent antivenom against three elapid venoms using a low dose, low volume, multi-site immunization protocol', Toxicon, vol. 39, no. 10, pp. 1487-1494. Cook, D.A., Owen, T., Wagstaff, S.C., Kinne, J., Wernery, U. & Harrison, R.A. (2010) 'Analysis of camelid IgG for antivenom development: serological responses of venom- immunised camels to prepare either monospecific or polyspecific antivenoms for West Africa', Toxicon, vol. 56, no. 3, pp. 363-372. Finney, D.J. (1952) Probit analysis: a statistical treatment of the sigmoid response curve, Cambridge university press, Cambridge. Fox, J.W. & Serrano, S.M.T. (2009) 'Timeline of key events in snake venom metalloproteinase research', Journal of proteomics, vol. 72, no. 2, pp. 200-209. Fry, B.G., Winkel, K.D., Wickramaratna, J.C., Hodgson, W.C. & Wüster, W. (2003) 'Effectiveness of snake antivenom: species and regional venom variation and its clinical impact', Journal of Toxicology: Toxin Reviews, vol. 22, no. 1, pp. 23-34. Gutiérrez, J., Solano, G., Pla, D., Herrera, M., Segura, Á., Vargas, M., Villalta, M., Sánchez, A., Sanz, L. & Lomonte, B. (2017a) 'Preclinical evaluation of the efficacy of antivenoms for snakebite envenoming: state-of-the-art and challenges ahead', Toxins, vol. 9, no. 5, p. 163. Gutiérrez, J.M. (2012) 'Improving antivenom availability and accessibility: science, technology, and beyond', Toxicon, vol. 60, no. 4, pp. 676-687. Gutiérrez, J.M., Calvete, J.J., Habib, A.G., Harrison, R.A., Williams, D.J. & Warrell, D.A. (2017b) 'Snakebite envenoming', Nature reviews. Disease primers, vol. 3, p. 17063. Gutiérrez, J.M. & León, G. (2009) 'Snake antivenoms. Technological, clinical and public health issues', Animal Toxins: State of the Art. Perspectives in Health and Biotechnology, pp. 393-421. Gutiérrez, J.M., Lomonte, B., Leon, G., Alape-Giron, A., Flores-Diaz, M., Sanz, L., Angulo, Y. & Calvete, J.J. (2009) 'Snake venomics and antivenomics: proteomic tools in the design and control of antivenoms for the treatment of snakebite envenoming', Journal of proteomics, vol. 72, no. 2, pp. 165-182. Gutiérrez, J.M., Rojas, E., Quesada, L., León, G., Núñez, J., Laing, G.D., Sasa, M., Renjifo, J., Nasidi, A. & Warrell, D.A. (2005) 'Pan-African polyspecific antivenom produced by

203

caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa', Transactions of the Royal Society of Tropical Medicine and Hygiene, vol. 99, no. 6, pp. 468-475. Gutiérrez, J.M., Rucavado, A., Escalante, T., Herrera, C., Fernández, J., Lomonte, B. & Fox, J.W. (2018) 'Unresolved issues in the understanding of the pathogenesis of local tissue damage induced by snake venoms', Toxicon, vol. 148, pp. 123-131. Gutiérrez, J.M., Solano, G., Pla, D., Herrera, M., Segura, Á., Vargas, M., Villalta, M., Sánchez, A., Sanz, L. & Lomonte, B. (2017c) 'Preclinical Evaluation of the Efficacy of Antivenoms for Snakebite Envenoming: State-of-the-Art and Challenges Ahead', Toxins, vol. 9, no. 5, p. 163. Habib, A.G., Kuznik, A., Hamza, M., Abdullahi, M.I., Chedi, B.A., Chippaux, J.-P. & Warrell, D.A. (2015) 'Snakebite is under appreciated: appraisal of burden from West Africa', PLoS neglected tropical diseases, vol. 9, no. 9, p. e0004088. Harrison, R. & Gutiérrez, J. (2016) 'Priority actions and progress to substantially and sustainably reduce the mortality, morbidity and socioeconomic burden of tropical snakebite', Toxins, vol. 8, no. 12, p. 351. Harrison, R.A., Hargreaves, A., Wagstaff, S.C., Faragher, B. & Lalloo, D.G. (2009) 'Snake envenoming: a disease of poverty', Plos Neglected Tropical Diseases, vol. 3, no. 12, pp. e569-e569. Harvey, A.L. (2009) 'Fasciculins: toxins from mamba venoms that inhibit acetylcholinesterase', Handbook of venoms and toxins of reptiles, pp. 317-324. Herrera, M., Fernández, J., Vargas, M., Villalta, M., Segura, Á., León, G., Angulo, Y., Paiva, O., Matainaho, T. & Jensen, S.D. (2012) 'Comparative proteomic analysis of the venom of the taipan snake, Oxyuranus scutellatus, from Papua New Guinea and Australia: Role of neurotoxic and procoagulant effects in venom toxicity', Journal of proteomics, vol. 75, no. 7, pp. 2128-2140. Heyborne, W.H. & Mackessy, S.P. (2009) '16 Cysteine-Rich Secretory Proteins in Reptile Venoms', Handbook of venoms and toxins of reptiles, p. 325. Kornalik, F. & Taborska, E. (1989) 'Cross reactivity of mono-and polyvalent antivenoms with Viperidae and Crotalidae snake venoms', Toxicon, vol. 27, no. 10, pp. 1135-1142. Landon, J. & Smith, D.S. (2003) 'Merits of sheep antisera for antivenom manufacture', Journal of Toxicology: Toxin Reviews, vol. 22, no. 1, pp. 15-22. Laustsen, A.H., Lomonte, B., Lohse, B., Fernández, J. & Gutiérrez, J.M. (2015) 'Unveiling the nature of black mamba (Dendroaspis polylepis) venom through venomics and

204

antivenom immunoprofiling: Identification of key toxin targets for antivenom development', Journal of proteomics, vol. 119, pp. 126-142. Ledsgaard, L., Jenkins, T., Davidsen, K., Krause, K., Martos-Esteban, A., Engmark, M., Rørdam Andersen, M., Lund, O. & Laustsen, A. (2018) 'Antibody cross-reactivity in antivenom research', Toxins, vol. 10, no. 10, p. 393. León, G., Sánchez, L., Hernández, A., Villalta, M., Herrera, M., Segura, A., Estrada, R. & Maria Gutierrez, J. (2011) 'Immune response towards snake venoms', Inflammation & Allergy-Drug Targets (Formerly Current Drug Targets-Inflammation & Allergy), vol. 10, no. 5, pp. 381-398. León, G., Vargas, M., Segura, Á., Herrera, M., Villalta, M., Sánchez, A., Solano, G., Gómez, A., Sánchez, M., Estrada, R. & Gutiérrez, J.M. (2018) 'Current technology for the industrial manufacture of snake antivenoms', Toxicon: Official Journal Of The International Society On Toxinology, vol. 151, pp. 63-73. Ownby, C.L. & Colberg, T.R. (1990) 'Comparison of the immunogenicity and antigenic composition of several venoms of snakes in the family Crotalidae', Toxicon, vol. 28, no. 2, pp. 189-199. Pla, D., Gutiérrez, J.M. & Calvete, J.J. (2012) 'Second generation snake antivenomics: comparing immunoaffinity and immunodepletion protocols', Toxicon, vol. 60, no. 4, pp. 688-699. Ratanabanangkoon, K., Tan, K.Y., Eursakun, S., Tan, C.H., Simsiriwong, P., Pamornsakda, T., Wiriyarat, W., Klinpayom, C. & Tan, N.H. (2016) 'A simple and novel strategy for the production of a pan-specific antiserum against elapid snakes of Asia', PLoS neglected tropical diseases, vol. 10, no. 4, p. e0004565. Segura, Á., Villalta, M., Herrera, M., León, G., Harrison, R., Durfa, N., Nasidi, A., Calvete, J.J., Theakston, R.D.G. & Warrell, D.A. (2010) 'Preclinical assessment of the efficacy of a new antivenom (EchiTAb-Plus-ICP®) for the treatment of viper envenoming in sub- Saharan Africa', Toxicon, vol. 55, no. 2-3, pp. 369-374. Sjostrom, L., Al-Abdulla, I.H., Rawat, S., Smith, D.C. & Landon, J. (1994) 'A comparison of ovine and equine antivenoms', Toxicon, vol. 32, no. 4, pp. 427-433. Sánchez, L.V., Pla, D., Herrera, M., Chippaux, J.P., Calvete, J.J. & Gutiérrez, J.M. (2015) 'Evaluation of the preclinical efficacy of four antivenoms, distributed in sub- Saharan Africa, to neutralize the venom of the carpet viper, Echis ocellatus, from Mali, Cameroon, and Nigeria', Toxicon, vol. 106, pp. 97-107.

205

Tasoulis, T. & Isbister, G. (2017) 'A review and database of snake venom proteomes', Toxins, vol. 9, no. 9, p. 290. Theakston, R.D.G. & Reid, H.A. (1983) 'Development of simple standard assay procedures for the characterization of snake venoms', Bulletin of the world health organization, vol. 61, no. 6, p. 949. Warrell, D.A. (2010a) 'Guidelines for the management of snake-bites', Guidelines for the management of snake-bites. Warrell, D.A. (2010b) 'Snake bite', The Lancet, vol. 375, no. 9708, pp. 77-88. Warrell, D.A., Gutiérrez, J.M., Calvete, J.J. & Williams, D. (2013) 'New approaches & technologies of venomics to meet the challenge of human envenoming by snakebites in India', The Indian journal of medical research, vol. 138, no. 1, p. 38. WHO (2010) 'WHO guidelines for the production, control and regulation of snake antivenom immunoglobulins', Geneva: WHO, vol. 134. WHO (2016a) 'Guidelines for the Management of Snake Bites WHO. Regional Office for South-East Asia', World Health Organization. WHO (2016b) Snake antivenoms [Online], World Health Organization, Available from: http://www.who.int/mediacentre/factsheets/fs337/en/# (Accessed: April 2016). Williams, D.J., Gutiérrez, J.-M., Calvete, J.J., Wüster, W., Ratanabanangkoon, K., Paiva, O., Brown, N.I., Casewell, N.R., Harrison, R.A. & Rowley, P.D. (2011) 'Ending the drought: new strategies for improving the flow of affordable, effective antivenoms in Asia and Africa', Journal of proteomics, vol. 74, no. 9, pp. 1735-1767.

WÜSTER, W., Chirio, L., Trape, J.F., Ineich, I., Jackson, K., Greenbaum, E., Barron, C., Kusamba, C., Nagy, Z.T., Storey, R. and Hall, C., 2018. Integration of nuclear and mitochondrial gene sequences and morphology reveals unexpected diversity in the forest cobra (Naja melanoleuca) species complex in Central and West Africa (Serpentes: Elapidae). Zootaxa, 4455(1), pp.68-98.

206

5.7 Supplementary Figures

D. polylepis D.

ntivenoms purified from six months and 12 months against against months 12 and months six from purified ntivenoms

: Antivenomics analysis of the experimental a experimental the of analysis Antivenomics :

The reverse phase separation of the whole venoms and (B) the percentage of immunocaptured (retained) protein protein (retained) immunocaptured of percentage (B) the and venoms whole the of separation phase The reverse

. (A) (A) .

Figure S 5. FigureS venom fractions.

207

. .

N. melanoleuca N.

al antivenoms purified from six months and 12 months against against months 12 and months six from purified antivenoms al

: Antivenomics analysis of the experiment the of analysis Antivenomics :

The reverse phase separation of the whole venoms and (B) the percentage of immunocaptured (retained) protein fractions protein (retained) immunocaptured of percentage (B) the and venoms whole the of separation phase The reverse

. (A) (A) .

gure S 5. S gure

Fi venom

208

The The

. (A) .

venom

nivea

N. N.

rimental antivenoms purified from six months and 12 months against against months 12 and months six from purified antivenoms rimental

: Antivenomics analysis of the expe the of analysis Antivenomics :

Figure S 5. FigureS fractions. protein (retained) immunocaptured of the percentage (B) and venoms whole the of separation phase reverse

209

. .

nom

E. ocellatus E.

rimental antivenoms purified from six months and 12 months against against months 12 and months six from purified antivenoms rimental

: Antivenomics analysis of the expe the of analysis Antivenomics :

The reverse phase separation of the whole venoms and (B) the percentage of immunocaptured (retained) protein fractions. fractions. protein (retained) immunocaptured of percentage the (B) and venoms whole ofthe separation phase The reverse

Figure S 5. S Figure (A) (A)

210

B. B.

experimental antivenoms purified from six months and 12 months against against months 12 and months six from purified antivenoms experimental

The reverse phase separation of the whole venoms and (B) the percentage of immunocaptured (retained) (retained) ofimmunocaptured percentage the (B) and venoms whole ofthe separation phase reverse The

. (A) (A) .

: Antivenomics analysis of the the of analysis Antivenomics :

ietans venom ietans

ar Figure S 5. FigureS fractions. protein

211

N. N.

he experimental antivenoms purified from six months and 12 months against against months 12 and months six from purified antivenoms experimental he

The reverse phase separation of the whole venoms and (B) the percentage of immunocaptured ofimmunocaptured percentage the (B) and venoms whole the of separation phase reverse The

. (A) (A) .

: Antivenomics analysis of t of analysis Antivenomics :

(retained) protein fractions. fractions. protein (retained) Figure S 5. FigureS venom nigricollis

212

Chapter 6

Concluding discussion

213

The first-choice treatment of snakebite envenoming is antivenom and yet this Neglected Tropical Disease still, annually, causes up to 138,000 deaths and 400,000 disabilities in surviving victims. This disease burden primarily affects the most economically and medically disadvantaged farming communities of Asia, sub-Saharan Africa and Latin America (Harrison et al. 2009; WHO 2019)). While the many barriers victims face to rapidly access effective treatment partly explains this disease burden, the quality of antivenom treatment is also a significant contributor, because:

• Antivenoms are weakly effective (only 10-15% of antivenom immunoglobulins bind venom) multiple vials are needed to effect cure – and each additional vial induces higher levels of adverse effects, and increases treatment costs • Antivenom efficacy is restricted to the snake whose venom was used in manufacture, and most affected regions are home to several different snake species, most manufacturers use multiple snake venoms to produce poly-specific antivenoms. But much higher doses of poly-specific antivenom are required to treat envenoming by any one of these snakes – exacerbating adverse effects and treatment costs • Antivenom is ineffective in treating venom-induced tissue destruction, unless administered within minutes after envenoming (rural tropical victims take hours getting to hospital treatment) • The high costs of antivenom explain why demand by governments is low, and why antivenom is rarely available in the rural hospitals that need it most • The poor safety profile of antivenoms necessitate additional medicines and specific clinical skills – often unavailable in remote rural hospitals

Effectively treating tropical snakebite is therefore very challenging and emphasises the urgent, compelling need for research to develop therapies that are:

• more effective against both local and systemic envenoming by all medically-important snakes in any given region of the world – a pan-specific treatment donating commercially attractive economies of scale to the manufacturers • safer than current antivenom treatment - enabling treatment to be administered at community level; before onset of severe life- and limb-threatening pathologies • affordable - costs of new treatments must be affordable to governments and victims to ensure they become available in the rural regions in greatest need

Research into new snakebite therapeutics is starting, including as a major research focus for LSTM, but does require models that provide more pathophysiological information, and

214 clinical relevance, than the current murine preclinical systems. My research therefore focussed upon the development and standardising of three new models to support this therapeutic research.

Figure 6.1: Summaries of the most significant findings in the current PhD, focusing on the pathologies induced by snake venom proteins. Four models were developed in this PhD; ex vivo ocular envenomation model, ex vivo human dermonecrosis model, in vivo systemic envenomation model and designing a novel therapy to treat snakebites. The findings revealed that the most significant pathologies were caused by direct or indirect mechanism of low molecular weight proteins present in the venom (3FTx and/ or PLA2). These proteins induced local and systemic damage,Widespread while tissue their necro poorsis, immunogenicity and associated had possibility direct effects of debridement on antivenom and efficacy.amputation (Hsu et al., New mols eye model – its utility ex vivo ocular envenomation model: Apart from some clinical observations studies, over the past 20 years venom-induced ocular damage has mainly been studied using live animals. This approach poses significant ethical concerns, even aside from the fact that some of these studies involve experiments lasting for more than a month. There is therefore an urgent need to develop an ex vivo model to identify the mechanistic causes and consequences of ocular envenoming. It is in this context that our study has developed an ex vivo bovine model to compare fresh venoms from spitting and non-spitting cobras in terms of their ocular envenomation effects. The model revealed that all the cobra venoms caused major injury to the cornea and epithelial necrosis. The venom of the African N. nigricollis caused complete

215 corneal opacification, while the venom of the non-spitting cobras, N. naja and N. nivea, from Asia and Africa, respectively, also caused corneas to become opaque. The present work is the first to discover that proteins from the fresh venom of both spitting and non-spitting cobras that were previously not known to induce eye damage, actually permeated the cornea within five minutes of their administration. Immunoblotting and mass spectrometry analyses identified these proteins as SVMP and NGF. This finding suggested that those venom proteins with low molecular weight such as 3FTx and PLA2 directly causes local corneal tissue damage. One of the very important finding in this study was flushing the eye with water immediately (less than five minutes following the accident) prevents the venom from causing corneal damage.

All previous studies that have been performed on animal models have reported that the venom of H. haemachatus, N. nigricollis, N. nivea and N. melanoleuca contains cardiotoxic fractions responsible for significant chemosis, blepharitis and opacification of the cornea, along with corneal and subconjunctival neovascularisation. Compared to the venom of N. nigricollis, which causes the cornea to lose epithelial cells and become opaque (Grüntzig et al. 1985; Ismail et al. 1993; Ismail & Ellison 1986), the venom of H. haemachatus induced the same effects during local application to rabbit eyes. The penetration research conducted by Ismail and Ellison (1986), using albino and pigmented eyes, revealed that, in the former case, labelled venom of N. nigricollis exhibited binding primarily to the corneal stroma, whilst in the latter case, there was suboptimal corneal binding and penetration. The association between our findings and all previous studies based on live animal models shows the benefits of developing an ex vivo model, for studying venom-induced ocular damage and for identifying the best intervention approach following ocular envenomation. ex vivo human dermonecrosis model – Venom contains toxic proteins that result in acute local effects. The tissue damage caused around the bite area by the venom of certain species of Elapidae and Viperidae initially takes the form of oedema, pain, redness and blistering. In severe cases, these manifestations may be followed by necrotic damage to the skin and muscle, which may necessitate debridement and even amputation. Phospholipases A2 and zinc-dependent metalloproteinases are the chief components responsible for these kinds of local effects, which occur quickly following envenomation and can degenerate into severe tissue damage and irreversible impairment if treatment is not administered as soon as possible. A new ex vivo model was devised in this work to investigate in detail over seven days the envenomation-induced skin necrosis and immune response. The major local effects manifested within an hour of envenomation, and oscillations over time were observed in the

216 levels of a number of cytokines, chemokines and growth factors. Interrogating immunoblots of the culture medium with SAIMR polyvalent antivenom and SAIMR Echis monovalent antivenom revealed that venom was continuously released from the tissue into the culture medium for the duration of the seven-day experiment. This finding suggested that, following injection, the venom causes local tissue damage and releases proteins that can cause systemic damage. The present work observed that PLA2 from the venom of N. nigricollis caused cell apoptosis, disrupted the integrity of the dermal structure, and triggered significant tissue inflammation. Although it is still unclear exactly how cytotoxins (e.g. cytotoxic PLA2) disrupt the membrane, there is a possibility that this occurs due to the attachment of toxin hydrophobic areas to membrane anionic phospholipids and subsequent membrane permeation (Bilwes et al. 1994; Forouhar et al. 2003). Although damage- associated molecular patterns (DAMPs) arise when the extra cellular matrix is subjected to the action of proteases and hydrolases originating from necrotic cells, there is so far little evidentiary support that the pathology of envenomation by Viperidae is significantly underpinned by DAMPs (Sunitha et al. 2015). It can be concluded that this ex vivo skin model provides researchers with a tool to investigate the toxicity and efficacy of new therapies (e.g. antivenom or small molecule therapeutics). in vivo systemic envenomation model – Envenomation by Viperidae manifests clinically as local oedema, haemorrhage and necrosis, as well as spontaneous haemorrhage, coagulopathy and cardiovascular shock if the venom enters the systemic circulation. Envenomation by Dendroaspis and Naja species of Elapidae also induces neurotoxicity, taking the form of ptosis followed by respiratory paralysis. The major concerns of existing studies are reports of envenomation in people and livestock and the local inflammatory effects of the venom at the bite site. So far, no study has established a correlation between inflammation and APR in vivo and only a limited range of snake genera have been used in empirical work on inflammatory processes. For instance, even though it is known that envenomation by Naja species causes inflammation the mechanisms of that inflammation have not been investigated. Since inflammation is also a confirmed effect of envenomation by the majority of vipers from Latin America, it is distinctly possible that envenomation by other species of snakes can trigger inflammation as well.

According to the findings of the present study, besides the direct action of venom toxins, stimulation of immune constituents can also promote systemic effects after envenomation. For example, the activation of acute phase and acute inflammatory response are linked to the occurrence of major heart and hepatic damage, and irregularities and increases in the

217 levels of white blood cells and platelets. The present work is the first to investigate the acute inflammatory and acute phase responses induced by the venom of African snake species of medical importance. In particular, it was noted that the proteins PLA2 and 3FTx from the venom of N. nigricollis and N. melanoleuca were mainly responsible for the systemic damage. Furthermore, it was observed that very severe local cytotoxicity was caused by cobra venom, leading to widespread swelling and local tissue injury. Such pathological effects are the outcomes of the combined action of four main venom toxins, namely, snake venom metalloproteinases (SVMPs), serine proteases (SVSPs), phospholipase A2 (PLA2), and three- finger toxins (3FTxs). As highlighted by several authors, however, these toxins do not occur at the same levels in the venom of different snake species, with SVMPs, SVSPs and PLA2 occurring in high levels in the venom of Viperidae, while the venom of Elapidae is rich in

3FTxs and PLA2 (Casewell et al. 2014a; Chippaux, Williams & White 1991; Tasoulis & Isbister 2017). The findings from this study help to arrive a better understating of systemic envenomation and thus are a step forward in the design of better therapies and intervention approaches.

Low molecular weight proteins- The pathological effects exhibited in both the ex vivo and in vivo models developed in this study might have been partly triggered by local and systemic damage and inflammatory events engendered by venom proteins with low molecular weight. The venom of Elapidae are primarily comprised of 3FTx and PLA2 toxins, whose neurotoxic or cytotoxic effects account for clinical outcomes of envenoming among both spitting and non-spitting cobras, as well as for the cardiac damage, systemic damage and inflammatory events shown in mouse models, and local tissue damage in the ex vivo human dermonecrosis models.

Hydrolysis of phospholipids from the cellular plasma membrane is undertaken by PLA2s. The precise mechanism through which membrane disruption is caused by cytotoxins remains uncertain, but it is likely that it is related to the binding of toxin hydrophobic sections to membrane anionic phospholipids, followed by penetration of the membrane, as claimed by Bilwes et al. (1994) and Forouhar et al. (2003). This process makes the production of antibodies difficult because the low immunogenicity of the proteins present in the venom of Elapidae mean that they are not suitable antigens. Despite possessing neutralisation potential, venom proteins that are poorly immunogenic have low glycosylation, which is adverse for cross-reactivity.

218

Neurotoxicity- The primary effect of envenomation by Elapidae is neurotoxic, with the facial bulbar, respiratory and limb muscles becoming paralysed due to the blockage of neuromuscular junctions, which can cause respiratory failure and death (Gutiérrez et al. 2010; Ranawaka, Lalloo & de Silva 2013)). To give an example, serious neurotoxic effects (e.g. paralysis, respiratory failure) can be caused by certain PLA2s and 3FTxs affecting pre- or postsynaptic junctions by antagonists of ion channels and nicotinic or muscarinic receptors (Casewell et al. 2013; Fry et al. 2003; Harris & Scott-Davey 2013; Lynch 2007; Tsetlin 2015). Antivenoms currently have only limited ability to counteract the neurotoxic effects of presynaptic toxins like PLA2, therefore. Local tissue damage is the primary issue related to the efficiency of antivenoms. More specifically, the neutralising antibodies present in antivenoms cannot keep up with the incredibly fast emergence of local pathological effects, so that permanent injury develops before the antibodies can reach the affected site.

Meanwhile, proteins from the venom of Elapidae are poorly immunogenic because they have a low molecular mass, which means they constitute suboptimal antigens, making the production of antibodies more challenging. Toxins can be made more immunogenic, and anti-toxin titres can be enhanced, by creating an adequate combination of immunogens, encompassing a range of key toxins with low molecular mass in a sufficient quantity, alongside, or in the absence of, small polypeptide cross-linking. A significant obstacle to the development of antivenom is that venoms are highly diverse biochemically and, implicitly, immunologically. This is because, even though there are only a few types of proteins in snake venoms, not all venoms have the same levels of every protein type and numerous iso- and proteo-forms with characteristic toxicological and immunological properties are encompassed in just one type of protein (Casewell et al. 2014b; Fernández et al. 2011; Petras et al. 2016)

In summary, several observations were derived from the empirical research conducted on the pathological and immunological reactions to the venom proteins. First, eye pathologies were caused by the venom of both spitting and non-spitting cobras, with corneal penetration by certain proteins. Secondly, inflammatory response and dermal cell apoptosis were caused by the venom of both Viperidae and Elapidae, with systemic effects arising due to the infiltration of the venom proteins into the circulation. Thirdly, systemic pathologies were caused by the venom of N. nigricollis and N. melanoleuca, as well as by stimulation of immune constituents in certain instances. Last, but not least, the development of antivenoms with cross-reactivity was challenging since the neurotoxic venom proteins with low molecular weight were poorly immunogenic.

219

6.2 Research priorities:

No methodical research has been dedicated to the complicated processes of protein formations in the venom of sub-Saharan African snakes, but this can be changed by technological innovations in proteomics underpinned by mass spectrometry. The proteomic analysis of venoms is known as venomics and constitutes the basis for the formulation of an approach known as “antivenomics”, which is geared towards the identification of all venom constituents by the antibodies contained in antivenoms. The antivenomics conducted in this work revealed that more in-depth knowledge of how venom proteins and antivenom interact can be derived by applying this technology. By separating and investigating the various toxins in different empirical models, it is possible to gain a better understanding of how they work and how the physiopathology of envenomation unfolds, thus enhancing the comprehension of snake venoms and related pathologies in the human body.

To investigate how the toxic effects of venom develop in empirical animal models, methods such as histology, immunohistochemistry, immunofluorescence and the proteomic analysis of inflammatory exudates must be applied to complete venoms and their individual constituents. The novel models that have been proposed minimise reliance on the use of live animals; for example, the ocular model can contribute to the formulation of better treatments by improving knowledge not only of the pathological effects of both spitting and non-spitting cobra venom, but also of the pathological effects of proteins isolated from species of medical significance. Meanwhile, the progress of necrosis and inflammation can be examined for up to one week based on the new dermonecrosis human skin model, which has potential for use in the investigation of proteins from the venom of various Viperidae and Elapidae from sub-Saharan Africa and their pathological effects.

Ex vivo or in vitro models should be created to improve knowledge of how the venom- induced neurotoxic, hemotoxic and coagulopathic effects manifest. The outcomes of such models can then aid the formulation of better treatments for deprived areas of sub-Saharan Africa as well as for other parts of the world. In the short-term, it is impractical to develop antivenoms based on therapeutic biotechnology products like recombinant human antibodies or related fragments (e.g. scFv) generated in different prokaryotic or eukaryotic expression systems because the industrial production of components that meet the specifications of an injectable product is expensive and because the complexity of venom composition would necessitate the creation of a mixture of recombinant antibodies or

220 fragments. Nevertheless, these kinds of antidotes still have potential for use in neutralising the effects of the venoms of some species of Elapidae.

Based on the findings of this research, antivenoms, as a gold standard of treatment, will likely be refined progressively rather than abruptly. The techniques through which immunoglobulins are processed, purified and stabilised demonstrate ever-greater efficiency, so these techniques are the most probable source of antivenom improvement in the short term. Meanwhile, immunisation approaches capable of addressing the immunogenicity shortcomings of certain venom toxins of clinical importance can contribute to make antivenom treatment more efficient. Furthermore, the geographical range in which antivenoms are efficient can be expanded through in-depth investigations of the constituents of venoms from snake species in different regions coupled with the production of novel antigenic formulas. As a result, antivenom treatment can become more broadly available in areas where it is currently not, leading to improvement in the high mortality and morbidity rates associated with envenomation in such areas. Last but not least, in spite of the lack of funding and backing from big international pharmaceutical organisations, there is potential to introduce certain non-immunological blockers targeting particular toxins into clinical practice, as long as the medical community dedicates more effort to the performance of controlled clinical studies.

221

6.3 References:

Bilwes, A., Rees, B., Moras, D., Menez, R. & Menez, A. (1994) 'X-ray Structure at 1· 55 Å of Toxin γ, a Cardiotoxin from Naja nigricollis Venom: Crystal Packing Reveals a Model for Insertion into Membranes', Journal of molecular biology, vol. 239, no. 1, pp. 122-136. Casewell, N.R., Wagstaff, S.C., Wüster, W., Cook, D.A.N., Bolton, F.M.S., King, S.I., Pla, D., Sanz, L., Calvete, J.J. & Harrison, R.A. (2014a) 'Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms', Proceedings Of The National Academy Of Sciences Of The United States Of America, vol. 111, no. 25, pp. 9205-9210. Casewell, N.R., Wagstaff, S.C., Wüster, W., Cook, D.A.N., Bolton, F.M.S., King, S.I., Pla, D., Sanz, L., Calvete, J.J. & Harrison, R.A. (2014b) 'Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms', Proceedings of the National Academy of Sciences, vol. 111, no. 25, pp. 9205-9210. Casewell, N.R., Wüster, W., Vonk, F.J., Harrison, R.A. & Fry, B.G. (2013) 'Complex cocktails: the evolutionary novelty of venoms', Trends in ecology & evolution, vol. 28, no. 4, pp. 219-229. Chippaux, J.-P., Williams, V. & White, J. (1991) 'Snake venom variability: methods of study, results and interpretation', Toxicon, vol. 29, no. 11, pp. 1279-1303. Fernández, J., Alape-Girón, A., Angulo, Y., Sanz, L., Gutiérrez, J.M., Calvete, J.J. & Lomonte, B. (2011) 'Venomic and antivenomic analyses of the Central American coral snake, Micrurus nigrocinctus (Elapidae)', Journal of proteome research, vol. 10, no. 4, pp. 1816-1827. Forouhar, F., Huang, W.-N., Liu, J.-H., Chien, K.-Y., Wu, W.-g. & Hsiao, C.-D. (2003) 'Structural basis of membrane-induced cardiotoxin A3 oligomerization', Journal of Biological Chemistry, vol. 278, no. 24, pp. 21980-21988. Fry, B.G., Wüster, W., Kini, R.M., Brusic, V., Khan, A., Venkataraman, D. & Rooney, A.P. (2003) 'Molecular evolution and phylogeny of elapid snake venom three-finger toxins', Journal of molecular evolution, vol. 57, no. 1, pp. 110-129. Grüntzig, J., Lenz, W., Berkemeier, B. & Mebs, D. (1985) 'Experimental studies on the spitting cobra ophthalmia (Naja nigricollis)', Graefe's archive for clinical and experimental ophthalmology, vol. 223, no. 4, pp. 196-201.

222

Gutiérrez, J.M., Williams, D., Fan, H.W. & Warrell, D.A. (2010) 'Snakebite envenoming from a global perspective: Towards an integrated approach', Toxicon, vol. 56, no. 7, pp. 1223-1235. Harris, J. & Scott-Davey, T. (2013) 'Secreted phospholipases A2 of snake venoms: effects on the peripheral neuromuscular system with comments on the role of phospholipases A2 in disorders of the CNS and their uses in industry', Toxins, vol. 5, no. 12, pp. 2533-2571. Harrison, R.A., Hargreaves, A., Wagstaff, S.C., Faragher, B. & Lalloo, D.G. (2009) 'Snake envenoming: a disease of poverty', Plos Neglected Tropical Diseases, vol. 3, no. 12, pp. e569-e569. Ismail, M., Al-Bekairi, A.M., El-Bedaiwy, A.M. & Abd-El Salam, M.A. (1993) 'The ocular effects of spitting cobras: I. The ringhals cobra (Hemachatus haemachatus) venom- induced corneal opacification syndrome', Journal of Toxicology: Clinical Toxicology, vol. 31, no. 1, pp. 31-41. Ismail, M. & Ellison, A.C. (1986) 'Ocular effects of the venom from the spitting cobra (Naja nigricollis)', Journal of Toxicology: Clinical Toxicology, vol. 24, no. 3, pp. 183-202. Lynch, V.J. (2007) 'Inventing an arsenal: adaptive evolution and neofunctionalization of snake venom phospholipase A 2 genes', BMC evolutionary biology, vol. 7, no. 1, p. 2. Petras, D., Heiss, P., Harrison, R.A., Süssmuth, R.D. & Calvete, J.J. (2016) 'Top-down venomics of the East African green mamba, Dendroaspis angusticeps, and the black mamba, Dendroaspis polylepis, highlight the complexity of their toxin arsenals', Journal of proteomics, vol. 146, pp. 148-164. Ranawaka, U.K., Lalloo, D.G. & de Silva, H.J. (2013) 'Neurotoxicity in snakebite--the limits of our knowledge', Plos Neglected Tropical Diseases, vol. 7, no. 10, pp. e2302-e2302. Sunitha, K., Hemshekhar, M., Thushara, R.M., Santhosh, M.S., Sundaram, M.S., Kemparaju, K. & Girish, K.S. (2015) 'Inflammation and oxidative stress in viper bite: an insight within and beyond', Toxicon, vol. 98, pp. 89-97. Tasoulis, T. & Isbister, G. (2017) 'A review and database of snake venom proteomes', Toxins, vol. 9, no. 9, p. 290. Tsetlin, V.I. (2015) 'Three-finger snake neurotoxins and Ly6 proteins targeting nicotinic acetylcholine receptors: pharmacological tools and endogenous modulators', Trends in pharmacological sciences, vol. 36, no. 2, pp. 109-123.

223

WHO (2019) Neglected tropical diseases [Online], World Health Organization, Available from: http://www.who.int/neglected_diseases/diseases/en/ (Accessed: 19th September).

224