Rational design of rapidly translatable, highly antigenic and novel recombinant immunogens to address deficiencies of current snakebite treatments

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Background: Snakebite envenomation (SBE) kills 138,000 and maims >400,000 people annually. Antivenom (IgG purified from animals hyper-immunized with mixtures of venoms) is the only assured therapy for SBE, is manufactured using expensive, century-old protocols of immunizing horses/sheep with crude venoms. Current protocols make no attempt to account for variant venom protein immunogenicity or toxicity during design or manufacture. Consequently, antivenoms often have poor dose-efficacy, which results in the administration of large volumes (often 200-400 ml in India) to neutralize pathology, often leading to severe adverse reactions and unaffordable costs for already impoverished victims. Furthermore, due to inter-species venom diversity, crude venom immunisation results in therapy that is snake species-specific, resulting in physicians having to make difficult diagnostic and antivenom-selection decisions when the offending snake species is unknown. There is therefore an urgent and compelling need to drastically improve the venom-neutralizing scope and efficacy of antivenom therapy. Rationale: Antivenoms for treating neurotoxic envenoming (a common global pathology often resulting in rapid fatal respiratory paralysis) are especially weakly-effective because of the weak immunogenicity and large diversity of the neurotoxins in the venoms used for immunisation. However, despite this diversity, examination of toxin sequence datasets demonstrates that neurotoxins possess commonly conserved features. This project will replace the use of crude neurotoxic venoms in antivenom manufacture with rationally engineered, synthetic particles displaying only the conserved regions of neurotoxins. By focusing the immune response to regions of only the most pathology-important toxins that are conserved in venoms of all the neurotoxic sSA snakes, I anticipate generating an antivenom which is (i) able to neutralize neurotoxic snake envenoming throughout sSA, regardless of species, and (ii) highly potent, resulting in smaller antivenom doses being needed to effect cure and improved safety. Approach: 1 First, I will computationally and experimentally investigate sequences encoding neurotoxins from the most medically important snakes of sSA to identify regions that are conserved among all neurotoxins.2 Identified regions will then be engineered for display on highly immunogenic antigen delivery vehicles (ADVs) such as Virus Like Particles (VLPs) or Fc fusions, which have inherent immune system modulating characteristics. Each approach can be easily manipulated to display foreign antigens, therefore allowing efficient display and enhanced recognition of the identified conserved neurotoxin regions by the immune system.3 I will test these approaches by immunizing mice to identify optimal configurations of ADVs displaying neurotoxin antigens, determined by examining (i) the extent of immune-responses and (ii) the ability of the antibodies generated to prevent neurotoxin activity using in vitro assays. The two optimal configurations of ADVs displaying neurotoxin antigens will then be used to immunise antivenom manufacturing animals (sheep) to generate experimental antivenom. 4 Finally, I will demonstrate the superior efficacy of the experimental sheep-generated antivenom in vitro, prior to in vivo neutralisation of lethality studies. Through these pre-clinical murine studies, I will determine whether the ADV generated antivenom exhibits superior venom neutralisation potential compared to existing commercial, crude venom produced antivenoms. Implications: As this project will improve the initial immunizing material only, with no changes to downstream antivenom manufacturing processes or product formulation, I anticipate that ADV-generated antivenoms will not require extensive regulatory approval. This will allow rapid translation of positive results into clinical trials and an immediate reduction in SBE burden in the short to medium term.