Periodic Reporting for period 2 - ADDovenom (ADDovenom: Novel Snakebite Therapy Platform of Unparalleled Efficacy, Safety and Affordability)
Okres sprawozdawczy: 2021-10-01 do 2023-03-31
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 even fewer neutralise venom toxins, 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. Our ambition is to generate a safe, affordable and efficient antivenom for Sub-Saharan Africa using innovative synthetic biology, biochemical and proteomics approaches. This aligns with WHO’s strategy to halve snakebite mortality and morbidity by 2030.
Within the second reporting period of ADDovenom we have achieved all milestones and deliverables. We have successfully reached three milestones and submitted 9 scientific deliverables on time. We have submitted deliverable D6.6 the White Paper, early based on the advice of our Expert Review Panel. This will feed into dissemination and exploitation plans. Our Data Management, Dissemination, Communication and Exploitation Plan, and Ethics licences have been reviewed and refreshed.
For mass spectrometric and bioinformatics characterisation of venoms (WP2) a global workflow has been established. Venoms have been collected at LSTM and provided to ULiege. The workflow has now been applied to characterize in detail the protein/ peptide content of venoms and the abundance of toxins for nine African snakes. Moreover, for two snakes, Echis ocellatus and Dendroaspis polylepis, the protein sequences (including mutations), post-translational modifications, isoelectric points and disulfide bonds were determined. Lists of identified toxins and their predicted pharmacological activity in the venoms were generated.
Based on this, 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.
ULiege and LSTM continue to work integrating the transcriptomics and proteomics data for an improved venom data base (ongoing).
In Bristol and Marseille, recombinant PLA2s, disintegrins, Kunitz-like proteins and 3FTX could be successfully produced. Escherichia coli production used DsbC fusion proteins and co-expression of chaperones (Marseille). PLA2s are produced using multibac/ insect cell expression which results in more reliable yields and activity of the toxins. Bristol and Marseille established activity assays to confirm activity of the recombinant PLA2s and disintegrins.
Bristol has designed, produced and validated a synthetic ADDobody library with an estimated library size of ~2 x 1012. Using a proof-of-principle selection strategy, Bristol showed that in vitro selection of ADDobody binders by ribosome display is feasible. In a next step, selections were performed against well established antigens (SARS-CoV-2 spike RBD) as well as disintegrins, epistring and PLA2. Resulting ADDobody binders are currently produced and characterised in Marseille to determine affinity constants.
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. LD50 values were determined for each snake species.
Cell-based assays for 3FTx and disintegrins have been developed, and a cell-based assay for Kunitz-like proteins is being developed. Prototype ADDobodies and ADDomers were injected at high concentrations into mice for safety testing, and no adverse effects could be detected.
In WP5, upstream and downstream process optimisation of ADDomer production and analytical methods for ADDomer quality control started. Optimal insect culture conditions were determined and different purification schemes are tested.
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 the next reporting period (RP3).
For mass spectrometric and bioinformatics characterisation of venoms (WP2) a global workflow has been established. Venoms have been collected at LSTM and provided to ULiege. The workflow has now been applied to characterize in detail the protein/ peptide content of venoms and the abundance of toxins for nine African snakes. Moreover, for two snakes, Echis ocellatus and Dendroaspis polylepis, the protein sequences (including mutations), post-translational modifications, isoelectric points and disulfide bonds were determined. Lists of identified toxins and their predicted pharmacological activity in the venoms were generated.
Based on this, 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.
ULiege and LSTM continue to work integrating the transcriptomics and proteomics data for an improved venom data base (ongoing).
In Bristol and Marseille, recombinant PLA2s, disintegrins, Kunitz-like proteins and 3FTX could be successfully produced. Escherichia coli production used DsbC fusion proteins and co-expression of chaperones (Marseille). PLA2s are produced using multibac/ insect cell expression which results in more reliable yields and activity of the toxins. Bristol and Marseille established activity assays to confirm activity of the recombinant PLA2s and disintegrins.
Bristol has designed, produced and validated a synthetic ADDobody library with an estimated library size of ~2 x 1012. Using a proof-of-principle selection strategy, Bristol showed that in vitro selection of ADDobody binders by ribosome display is feasible. In a next step, selections were performed against well established antigens (SARS-CoV-2 spike RBD) as well as disintegrins, epistring and PLA2. Resulting ADDobody binders are currently produced and characterised in Marseille to determine affinity constants.
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. LD50 values were determined for each snake species.
Cell-based assays for 3FTx and disintegrins have been developed, and a cell-based assay for Kunitz-like proteins is being developed. Prototype ADDobodies and ADDomers were injected at high concentrations into mice for safety testing, and no adverse effects could be detected.
In WP5, upstream and downstream process optimisation of ADDomer production and analytical methods for ADDomer quality control started. Optimal insect culture conditions were determined and different purification schemes are tested.
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 the next reporting period (RP3).
Progress beyond the state of the art: Marseille and Bristol plan to publish the first systematic analysis of snake venom phospholipases A2 (PLA2s) which so far could not be recombinantly produced. Therefore, a detailed enzymatic characterisation of the individual snake venom PLA2s is now possible.
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. These binders may be used to generate 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 two MSci students (4 month project) and 3 undergraduate internship students (6-8 weeks) 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. These binders may be used to generate 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 two MSci students (4 month project) and 3 undergraduate internship students (6-8 weeks) within the project.