Novel immuno-proteomic strategies to develop a polyspecific, non-cold chain liquid snake antivenom with unparalleled sub-Saharan African efficacy

Snakebite victims in Sub-Saharan Africa are therapeutically neglected (~32,000 deaths; 96,000 disabled yearly) because of the weak efficacy of current polyspecific antivenoms. In areas like Britain with a single venomous snake species, monospecific antivenom (antibodies (abys) purified from the blood of horses/sheep immunised with a single snake venom) are typically highly effective and safe. However, there are several venomous snake groups in sub-Saharan Africa, and polyspecific antivenoms, manufactured with venoms from several snakes, are therefore clinically preferred.

However, using many venoms for immunisation negatively impacts upon the efficacy of polyspecific antivenoms. Thus, the greater the genetic difference between the snakes whose venoms are used in antivenom manufacture, the more numerous and diverse the venom proteins (100+ proteins/venom), and the greater the number of distinct abys generated in the venom-immunised animals. This means that the proportion of total abys targeting the venom of any one snake is small - and consequently more vials of polyspecific antivenom are needed to achieve clinical cure. This significantly increases antivenom-induced adverse effects and often makes treatment unaffordable to the impoverished communities at greatest risk. There is therefore an urgent, compelling need for research to resolve this therapy deficit.

We will use an innovative strategy that greatly expands antivenom snake-species efficacy. Thus, we first add a 'base' antivenom, derived from an existing product that is safe and affordable but exhibits inadequate polyspecific efficacy, onto a laboratory matrix (a chromatography column). We next individually add venoms from all regional medically-important snakes to the column, and all proteins that are not bound by the antivenom are collected and their identity determined. Those found to be pathogenic (many venom proteins are not toxic) are isolated in sufficient amounts for immunisation and mixed with the venoms used to prepare the base antivenom. A new group of sheep is then immunised with the 'venom+supplement toxins' mixture to rationally generate antivenom with greatly expanded snake-species efficacy.

We will use this approach to first generate new antivenoms to treat either the (i) tissue destruction & bleeding, or the (ii) paralysis pathology syndromes: thereby covering the effects of envenoming by all medically-important sub-Saharan African snakes. We will use a suite of in vitro & in vivo tests to confirm that the two new antivenoms bind the venom proteins causing these pathologies and neutralise their lethal and tissue-destructive effects in mice, and compare these results with that of the base and existing polyspecific antivenoms. With this information as a guide, we will next develop a single pan-African antivenom using the first syndromic antivenom as the base to isolate non-binding proteins from all the African snake venoms for 'venom+supplement' immunisation of a new group of sheep. We will also test the efficacy of an alternate pan-African antivenom created by mixing abys of the two syndromic antivenoms in different ratios, and efficacy-testing the ratio that exhibits maximal binding of proteins from the African snake venoms.

We will also develop/test protocols enabling non-cold chain storage of liquid antivenom - greatly expanding the distribution potential of these improved antivenoms. We will also develop/test protocols to increase the amount and binding strength of antivenom abys to highly toxic venom proteins that are poor at stimulating potent aby responses.

Here, in the only research of its kind, we will deliver new non-cold chain antivenom with pan-African efficacy, and with an unparalleled dose-efficacy ensuring the product poses little adverse-effect risk and is affordable. These antivenoms will represent the most cost-effective investment in snakebite management ever available to governments in sub-Saharan Africa, and elsewhere.

To address the sub-Saharan African snakebite therapy deficit, we have devised an innovative 'antivenomic' strategy that maximally exploits the inter- & intra-generic immune cross-reactivity of venom proteins to greatly expand the snake-species efficacy of polyspecific antivenom.

We first immobilise a 'base' antivenom, derived from an existing antivenom that is safe & affordable but exhibits inadequate polyspecific efficacy, onto an IgG-affinity column, and venoms from target snakes added. All non-binding proteins are collected and proteomically identified. Those found to be toxic (BLAST search of gene/protein databases) are isolated in sufficient amounts for immunisation (preparative scale chromatography) and mixed with the venoms used to prepare the base antivenom. A new group of sheep is then immunised with the 'venom+supplement immunogen' mixture to rationally, and with minimal additional immunogens, generate antivenom with greatly expanded snake-species efficacy.

We will use this approach to generate antivenoms to treat the (i) dermal necrosis & haemotoxic or the (ii) neurotoxic pathology syndromes: thereby covering the effects of envenoming by all medically-important sub-Saharan African snakes. Thus informed, we will next develop a single pan-African antivenom using the first syndromic antivenom as the base to isolate supplement immunogens from all the African snake venoms for 'venom+supplement' immunisation. We will also develop an alternate pan-African antivenom created by mixing IgGs of both syndromic antivenoms in different ratios, and test the ratio exhibiting maximal polyspecific venom protein binding. Serological and murine venom-neutralising efficacy results of the new antivenoms will be assessed against that of the base and existing polyspecific antivenoms.

We will also develop protocols enabling non-cold chain storage of liquid antivenom, and to increase antivenom IgG titre & avidity to highly toxic venom but weakly immunogenic proteins.

OUTPUTS RELEVANT TO BENEFICIARIES
- Distinct strategies to design polyspecific antivenom with extensive snake-species efficacy.
- The novel toxoiding process to improve immunogenicity of venom toxins.
- Additives to impart thermostability to liquid biological therapeutics (antisera; vaccines, proteins).
- Snake venom Transcriptomic and Proteomic datasets.

POTENTIAL BENEFICIARIES & HOW THEY WILL BENEFIT

SNAKEBITE VICTIMS: We designed this pre-clinical project to ultimately save the lives and livelihoods of the medically-neglected snakebite victims of sub-Saharan Africa.

COMMERCIAL SECTOR: We will deliver the design of non-cold antivenoms with pan-African efficacy and unparalleled dose efficacy and safety - significant, previously missing, incentives to commercial manufacturers. As the antivenoms are delivered during the project, we will pursue translation grants with antivenom manufacturers with a proven record of delivering quality antivenoms to Africa: eg, MicroPharm, Wales; Instituto Clodomiro Picado, Costa Rica.

The above antivenom manufacturers will gain financially through collaborative translation grants and government contracts and through capacity strengthening. Manufacturers of antivenoms for other regions could also benefit from these incentives. Albeit less certainly, manufacturers of other biological therapeutics may benefit from the thermostability and 'toxoiding' systems developed by this project.

The venom transcriptomic/proteomic data provide an exciting cardiovascular & neurological drug-discovery resource for Biotech/Pharmaceutical industries. We have budgeted for a meeting of relevant individuals from industry and academia to devise a new drug-/reagent-discovery research program.

GOVERNMENT POLICY MAKERS: Efficacious and safe antivenoms retailed at affordable prices (relative to existing polyspecific antivenoms) will represent the most cost-effective investment in snakebite management ever available to governments in sub-Saharan Africa - enabling the dislocation of the vicious cycle that has limited the commitment of manufacturers and public health authorities to increase the availability of antivenoms to this region. The health benefits are likely to translate to political advances, both nationally and internationally.

INTERNATIONAL HEALTH AGENCIES: The current lack of government demand/action and therapeutic tools has limited WHO's ability to motivate other agencies in resolving Africa's snakebite burden. The tools provided here, coupled with renewed manufacturing and government interest is likely to attract the fiscal and health-promoting support of agencies such as DFID, USAID, EU and the Bill and 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 so sorely neglected for so long.

PROJECT STAFF AND STUDENTS: The project involves many different scientific disciplines and activities that will enable staff and students from our 3 institutes to improve their technical skills portfolio and subsequent marketability. The project's collaborative nature provides many opportunities for information exchange, science discussion, cultural and scientific environments - all empowerments for young scientists.

PUBLIC SECTOR: The project's potential to have such a positive impact upon the lives of deeply disadvantaged African populations will have wide appeal to the lay public and media - by demonstrating how UK research funds benefit human health. We will incorporate our progress within our public engagement activities. Furthermore, the collaborative success of three groups from countries from different cultural, economic and geographical backgrounds will strengthen the principle that north-north and north-south international partnerships have great potential for confronting pressing global health issues.