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

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.

This project has the potential to impact a diverse range of stakeholders.

Snakebite victims: Ultimately this project is designed to reduce burden suffered by the rural, impoverished tropical snakebite victims through reducing the number of deaths and disability inflicted by poorly effective treatments.

Antivenom/Immunotherapy Manufacturers: Although focused on a treatment for neurotoxic venoms, this project will deliver a methodology applicable for the generation of more efficacious antivenoms/immunotherapies for other regions, venom-induced pathologies and other diseases. As this method does not alter the core manufacturing process or formulation of the final antivenom product, it should not be subject to extensive new regulatory approvals. I anticipate that this will provide significant commercial incentive to antivenom manufactures. The potential multi-regional utility of pathology-specific antivenoms will attract significant interest from governments and aid agencies active in affected regions, increasing demand, thus driving manufacturing capacity.

Government Policy Makers: More efficacious and safer snakebite treatments available at overall lower treatment costs (relative to existing polyspecific antivenoms) will represent a cost-effective investment in snakebite management, which is simply not currently present in many countries, being available to governments in tropical regions of the world - enabling the dislocation of the vicious circle that has limited the commitment of manufacturers and public health authorities to increase the availability of antivenoms to this region.

International Health Agencies: The current lack of (i) solid government demand/action and (ii) therapeutic tools has limited WHO's ability to interest other agencies in resolving the world's snakebite burden. The tools provided from this project, coupled with new demand for effective and safe snakebite treatments by African and Asian governments, are likely to attract the financial and health-promoting support of agencies such as DFID, USAID, EU and the Bill & Melinda Gates Foundation - providing them with additional evidence of cost-effective success of using public and philanthropic funds to instigate substantial health benefits in communities whose snakebite victims have been neglected for so long.

Clinicians: Due to the current restricted-species efficacy of current antivenoms, clinicians in sSA are faced with significant treatment challenges when deciding on the appropriate antivenom to use. The availability of an anti-neurotoxic generic antivenom will sidestep these issues, allowing clinicians to simply use this antivenom when patients present with easily identifiable neurotoxic pathology.

Academics: A diverse variety of academics will benefit from the outputs of this application, including toxinologists, immunologist, vaccinologists, etc. Please refer to "Academic Beneficiaries" for further details.

Project staff: This project will enable myself and associated staff to improve technical skills portfolio and subsequent employability. The project's translational nature will provide many opportunities for information exchange and scientific discussion in various cultural and scientific environments.

Public Sector: The project's potential to have such a positive impact upon the lives of deeply disadvantaged tropical populations will have wide appeal to the lay public and media - by demonstrating how UK government funds benefit human health. We will incorporate our progress within our public engagement activities defined in 'Pathways to Impact'.