Periodic Reporting for period 1 - ADDovenom (ADDovenom: Novel Snakebite Therapy Platform of Unparalleled Efficacy, Safety and Affordability)
Reporting period: 2020-10-01 to 2021-09-30
Antivenom is the first-choice treatment of snakebite envenoming (SBE) since 1890 when the first antivenoms were developed. Nevertheless, this Neglected Tropical Disease still, annually, causes up to 138,000 deaths and 400,000 amputations and other permanent 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. Children often suffer more severe effects than adults, due to their smaller body mass. Current anti-venoms (AVs) are based on antibodies from hyperimmunised horses and sheep. These AVs are weakly effective: only 10-15% of the IgGs bind venom, and multiple vials are needed to effect cure, but each additional vial induces higher levels of adverse effects and increases treatment costs. An additional disadvantage of current AVs is that they cannot rationally incorporate the distinct immunogenicity or toxicity of the venoms’ proteins into the design (venoms comprise between 20 to >100 proteins that vary in molecular mass, bioactivity and pathogenicity). This is particularly problematic because snake venoms and their toxins vary significantly at every taxonomic level, and highly pathogenic, diverse and low-molecular mass toxins are notoriously poor immunogens. Thus, our ambition to generate a safe, affordable and efficient antivenom for Sub-Saharan Africa using innovative synthetic biology, biochemical and proteomics approaches aligns with WHO’s strategy to halve snakebite mortality and morbidity by 2030.
Within the first year of ADDovenom we have achieved all milestones and deliverables.
We have established an efficient management structure (WP1) by recruiting Dr Kathleen Sedgley (project officer) and Aggie Hewitt (project administrator) to the management team. All partners have recruited the required personnel to conduct the research. Management guidelines, a data management plan and the scientific advisory board are in place. Consortium meetings were organised for the kick-off of the project and year 1 and agreed on for year 2.
For mass spectrometric and bioinformatics characterisation of venoms (WP2) a global workflow has been established to characterize in detail the main toxins of venoms for nine African snakes. Venoms have been collected at LSTM and provided to ULiege. The overall quality of the workflow developed has been confirmed for venoms from one mamba, Dendroaspis polylepis and one viper, Echis ocellatus. Bioinformatics: integration of transcriptomics and proteomics data for an improved venom data base is ongoing.
LSTM and ULiege provided a list with most pathogenic toxins (based on abundance and genetic diversity) from viper and mamba for expression in WP3 as either recombinant whole toxins or epitope strings. A total of 38 toxins were selected for Echis spp. including SVMP, PLA2 and disintegrins, and 30 toxins were selected for mambas including 3FTx and Kunitz-like proteins. PLA2s and disintegrins could be successfully produced in Escherichia coli by using DsbC fusion proteins and co-expression of chaperones (Marseille). Bristol and Marseille established activity assays to confirm activity of the recombinant PLA2s and disintegrins. Bristol has designed, produced and quality controlled an ADDobody library with an estimated library size of ~2.7 x 1012. Furthermore, using a proof-of-principle selection strategy, we showed that in vitro selection of ADDobody binders by Ribosome Display is feasible.
In WP4 LSTM have established and refined toxin activity assays for use in this project, with SOPs generated and baseline data for (i) species-specific activity and (ii) neutralising ability (IC50 where possible) of a comparator gold-standard antivenom, to guide future comparison testing with ADDovenom. A cell-based assay for 3FTx has been developed, and assays for disintegrins and Kunitz-like proteins are in early development. Methods for pharmacokinetic measurements have been trialled to now initiate safety testing of ADDobodies and ADDomers in mice.
WP5 is due to start in month 25 at iBET and includes establishing ADDomer process optimisation and analytical methods for ADDomer quality control.
All partners engage in Communication, Dissemination and Exploitation Activities (WP6), including our ADDovenom webpage, social media (Twitter and LinkedIn), public understanding of science events and conference contributions. The ADDovenom Dissemination and Exploitation Plan sets out our targets for reporting periods 2 and 3.
We have established an efficient management structure (WP1) by recruiting Dr Kathleen Sedgley (project officer) and Aggie Hewitt (project administrator) to the management team. All partners have recruited the required personnel to conduct the research. Management guidelines, a data management plan and the scientific advisory board are in place. Consortium meetings were organised for the kick-off of the project and year 1 and agreed on for year 2.
For mass spectrometric and bioinformatics characterisation of venoms (WP2) a global workflow has been established to characterize in detail the main toxins of venoms for nine African snakes. Venoms have been collected at LSTM and provided to ULiege. The overall quality of the workflow developed has been confirmed for venoms from one mamba, Dendroaspis polylepis and one viper, Echis ocellatus. Bioinformatics: integration of transcriptomics and proteomics data for an improved venom data base is ongoing.
LSTM and ULiege provided a list with most pathogenic toxins (based on abundance and genetic diversity) from viper and mamba for expression in WP3 as either recombinant whole toxins or epitope strings. A total of 38 toxins were selected for Echis spp. including SVMP, PLA2 and disintegrins, and 30 toxins were selected for mambas including 3FTx and Kunitz-like proteins. PLA2s and disintegrins could be successfully produced in Escherichia coli by using DsbC fusion proteins and co-expression of chaperones (Marseille). Bristol and Marseille established activity assays to confirm activity of the recombinant PLA2s and disintegrins. Bristol has designed, produced and quality controlled an ADDobody library with an estimated library size of ~2.7 x 1012. Furthermore, using a proof-of-principle selection strategy, we showed that in vitro selection of ADDobody binders by Ribosome Display is feasible.
In WP4 LSTM have established and refined toxin activity assays for use in this project, with SOPs generated and baseline data for (i) species-specific activity and (ii) neutralising ability (IC50 where possible) of a comparator gold-standard antivenom, to guide future comparison testing with ADDovenom. A cell-based assay for 3FTx has been developed, and assays for disintegrins and Kunitz-like proteins are in early development. Methods for pharmacokinetic measurements have been trialled to now initiate safety testing of ADDobodies and ADDomers in mice.
WP5 is due to start in month 25 at iBET and includes establishing ADDomer process optimisation and analytical methods for ADDomer quality control.
All partners engage in Communication, Dissemination and Exploitation Activities (WP6), including our ADDovenom webpage, social media (Twitter and LinkedIn), public understanding of science events and conference contributions. The ADDovenom Dissemination and Exploitation Plan sets out our targets for reporting periods 2 and 3.
Progress beyond the state of the art: Marseille and Bristol plan to publish the first systematic analysis of toxin expression for snake venom PLA2 and SVMPs which so far could not be recombinantly produced.
The ADDobodies show excellent expression yields and thermostability. A first publication describing the structure and thermostability of the ADDobodies is in preparation.
LSTM and Liege will publish their analysis of the Dendroaspis polylepis and Echis ocellatus venoms.
Expected results until the end of Project: We are confident that we will be able to generate neutralising ADDobodies/ ADDomers or nanobodies against the most pathogenic toxins of the medically most relevant venomous snakes in Sub-Saharan Africa, generating a first completely synthetic antivenom.
Impacts: New collaborative projects emerged within the ADDovenom consortium which already now led to additional funding for LSTM (lead) and UnivBris. A Wellcome Trust Collaborator Award supports the establishment of additional international academic (Nigeria, India) and private company (IAVI, USA) collaborations and the establishment of long-term collaborations. It also helps to accelerate ADDovenom impacts.
LSTM, Bristol, Marseille and Liege are actively participating in the European Venom Network (EUVEN), EU COST Action (CA19144) which aims at consolidating collaboration and knowledge exchange between European researchers working on venoms creating additional synergies. Moreover, EUVEN financed early career researcher training visit (ULiege) at the Walther-Straub-Institute for Pharmacology and Toxicology - LMU/Munich-Germany.
ADDovenom is included in the synthetic biology teaching curriculum at UnivBris; it is popular project for Biochemistry students, and we have already trained 3 internship students within the project.
The ADDobodies show excellent expression yields and thermostability. A first publication describing the structure and thermostability of the ADDobodies is in preparation.
LSTM and Liege will publish their analysis of the Dendroaspis polylepis and Echis ocellatus venoms.
Expected results until the end of Project: We are confident that we will be able to generate neutralising ADDobodies/ ADDomers or nanobodies against the most pathogenic toxins of the medically most relevant venomous snakes in Sub-Saharan Africa, generating a first completely synthetic antivenom.
Impacts: New collaborative projects emerged within the ADDovenom consortium which already now led to additional funding for LSTM (lead) and UnivBris. A Wellcome Trust Collaborator Award supports the establishment of additional international academic (Nigeria, India) and private company (IAVI, USA) collaborations and the establishment of long-term collaborations. It also helps to accelerate ADDovenom impacts.
LSTM, Bristol, Marseille and Liege are actively participating in the European Venom Network (EUVEN), EU COST Action (CA19144) which aims at consolidating collaboration and knowledge exchange between European researchers working on venoms creating additional synergies. Moreover, EUVEN financed early career researcher training visit (ULiege) at the Walther-Straub-Institute for Pharmacology and Toxicology - LMU/Munich-Germany.
ADDovenom is included in the synthetic biology teaching curriculum at UnivBris; it is popular project for Biochemistry students, and we have already trained 3 internship students within the project.