With an annual 5 million cases, 150,000 deaths, and about 400,000 amputations,snakebite envenoming is an ever-present threat in many parts of the rural tropical world. Parental administration of animal-derived, serum-based antivenoms remains the mainstay of snakebite envenoming therapy. However, the high level of immunogenicity of such heterologous medicines leads to severe side effects in human recipients. In order to bring antivenoms into the modern era of biopharmaceuticals, it is important to have a thorough understanding of snake venom toxins and to have an optimal antitoxin discovery strategy. In this thesis, a novel approach is presented on how to develop synthetic and recombinant antivenoms based on a range of different molecules,including peptides, nanobodies, antibodies, and antibody fragments. This approach is based on toxicovenomics and phage display selection.In the work behind this thesis, a systematic method for selecting keytoxins for antitoxin discovery was developed (the Toxicity Score). The combination of this approach with the venomics strategy was a central element inthe establishment of the new field of toxicovenomics – the study of snakevenom proteomes in relation to the pathophysiological effects of their toxins. Four toxicovenomics studies were performed on the venoms of Dendroaspispolylepis (Black mamba), Dendroaspis angusticeps (Eastern green mamba), Naja kaouthia (Monocled cobra), and Aipysurus laevis (Olive sea snake). These studies not only estimated the quantitative venom proteomes of these snakes and identified the medically most relevant toxins responsible for the pathophysiological effects of the venoms, but also revealed mechanistic differences between the venoms. As an example, the venoms from the black mamba, the green mamba, and the olive sea snake showed synergistic behaviors,while the venom from the monocled cobra displayed a dominance of non-synergistically-acting α-neurotoxins. α-neurotoxins played a major role in venom toxicity for all venoms, which cause flaccid paralysis in rodent models. Several drug discovery programs based on phage display selection were carried out, aiming at finding antitoxins against the medically relevant toxins identified in the toxicovenomic studies. A few hundreds of peptide displaying phages, dozens of nanobody-displaying phages, and over a thousand human scFv-displaying phages were selected and screened. Among these, dozens of promising peptidic antitoxins with inhibitory effects against elapid neurotoxins were identified. In two-electrode voltage clamp assays using Xenopus laevis (African clawed frog) oocytes, Peptide 33535 was capable of abrogating α-cobratoxin induced inhibition (at a concentration of 40µM peptide and 100 µM α-cobratoxin) of the nicotinic acetylcholine receptor, responsible for neuromuscular transmission. This peptide was shown by isothermal titration calorimetry to bind to α-cobratoxin with a Kd of 20 µM. However,despite these positive results, much more research is needed before recombinantor synthetic antivenoms may reach the clinic and benefit victims of snakebite envenoming.It is the hope that the work presented here will help enable that snakebite victims around the world will gain access to inexpensive and safe recombinant antivenom with high efficacy.
|Publisher||University of Copenhagen|
|Number of pages||143|
|Publication status||Published - 2016|