African swine fever outbreak containment strategy using small-molecule antiviral drugs

Lead Research Organisation: The Pirbright Institute
Department Name: African Swine Fever Virus

Abstract

African swine fever (ASF) is a frequently fatal disease of pigs with a very high socio-economic impact. The disease represents an increasing risk to the UK, EU and global pig industries due to its dramatic and increasing spread in a number of countries in the Trans Caucasus, Russian Federation and Eastern Europe, including EU member states and most recently China. The UK pig industry is valued at around £1 billion per year and represents 40% of the red meat market (Defra, 2008-2009; AHDB, 2014). Exports, currently valued at about £300 million, would be halted if ASF entered the UK.
ASF control relies on rapid diagnosis and implementation of quarantine, movement restrictions and stamping out policies. These measures have a high impact on the livelihoods of farmers and others involved in the pork industry and a severe effect on animal welfare. Several factors contribute to the difficulty in controlling ASF. Amongst these is the lack of a vaccine and presence of wild pig reservoirs of infection.
In this project we will tackle this emerging infectious disease of wild and domestic pigs by optimising small-molecule, antiviral lead compounds that specifically inhibit the replication the ASF virus (ASFV). We have already identified a class of molecules with potent anti-ASFV activity. Apart from elucidating their exact mechanism of action, the identified lead molecules will be optimised to increase (i) pan-genotype activity using a reference panel of ASFV isolates from 14 genotypes, (ii) selectivity and (iii) oral bio-availability prior to safety and efficacy testing in pigs. Once optimised, such antiviral drugs can be deployed quickly (e.g. following stockpiling) in case of an ASF incursion into a previously-free area by administering the drug as a prophylactic and metaphylactic measure to pigs in protection zones around infected premises.
Previous research on classical swine fever (CSF), another epizootic disease of pigs, has demonstrated that antiviral drugs are a valid supplement and/or alternative to emergency vaccination and stamping-out/pre-emptive culling from an epidemiological and economic point of view. Indeed, it has been shown that an antiviral drug administered to CSF-infected pigs (i) reduces the viral titre by a 1000-fold, (ii) shortens the period virus is detected in blood by 74% and (iii) reduces transmission from infected/treated pigs to untreated sentinel pigs by 85% (Vrancken et al., 2009a; 2009b). The proposed antiviral containment strategy for CSF was further validated using epidemiological modelling studies conducted in two pig-dense areas in the EU (Ribbens et al., 2012; Backer et al., 2013). CSF outbreaks were controlled at least as effectively with antiviral drugs as with more conventional strategies such as pre-emptive culling and emergency vaccination.
Although none have been registered for use in livestock, the merits of small-molecule, antiviral drugs are well established in human medicine against infectious diseases such as AIDS and hepatitis C, for which no vaccines are available. This is the first project to bring together a high-quality research organisation and a biotech company that combined have the technical skills, know-how and expertise to successfully introduce the use of antiviral drugs as an effective approach to ASF control. The academic-industry partnership will advance our understanding of how ASFV replicates including the role(s) of certain virus and/or cellular proteins by elucidating the mechanism of virus inhibition. These insights will help advance the rational development of complementary ASFV vaccine candidates and will help understand how to effectively combine an antiviral drug containment strategy with vaccination using a live-attenuated vaccine.

Technical Summary

African swine fever virus (ASFV) causes an acute haemorrhagic fever (ASF) in domestic pigs and wild boar resulting in high mortality. The disease has a high socio-economic impact in affected countries. The virus is a large cytoplasmic DNA virus that replicates primarily in macrophages in vivo. About one third of the 150 to 167 genes are predicted to code for proteins that help the virus to evade the host's defences and replicate rapidly following infection of animals. Hence, the development of an effective and safe vaccine has proven challenging. Therefore, alternative/supplementary control tools are needed.
Following a library screen of about 5,000 compounds, we have previously identified 13 small-molecule antiviral drugs that effectively inhibit ASFV replication in Vero cell culture by at least 90% without causing cytotoxic effects on the cells. Confirmation of their activity against a panel of different isolates representing different genotypes of ASFV will be carried out using primary cells. Moreover, drug-resistant ASFV variants will be generated and sequenced. Resistance-associated mutations will be identified using reverse genetics thereby elucidating the mechanism of action of the class of antiviral molecules. Following in vitro and in silico ADME-Tox studies (absorption, distribution, metabolism, extraction and toxicity studies), a maximum of six compounds will be selected for pharmacokinetic studies in pigs. Of these, a maximum of three compounds with a favorable oral exposure profile (i.e., plasma concentration above the in vitro 50% effective concentration for more than 6h) will be selected for single-dose and repeat-dose safety testing in pigs. A maximum 2 of the above candidate drugs will be tested for efficacy at the state-of-the-art BSL3+ animal facilities of the Pirbright Institute.
In this project, the academic-industry partnership will lead to the identification of a drug candidate to be taken forward in regulatory development studies.

Planned Impact

Academic Impact
The academic impact will be to improve understanding of how the small-molecule antiviral drugs inhibit ASFV replication by identifying viral target proteins (e.g. viral polymerase PolX or DNA polymerase). Knowledge of the targeted viral proteins contributes to a better overall understanding of the viral replication mechanisms and how these might differ in continuous cell lines (e.g. Vero cells) and primary cells. The class of drugs may also target virus cell attachment proteins which will open up new approaches for vaccine development efforts. Moreover, an improved understanding of the mode of action of the antiviral drugs and of the ASFV replication mechanisms will lead to new strategies for ASFV control (e.g, the possibility of combining an antiviral drug strategy with vaccination using a live-attenuated vaccine. Such a combination strategy has never been put to the test for ASF).
Economic and societal impacts
The present research is pre-competitive in nature. If successful, the economic and societal impacts are huge. Globally, there are an estimated 1.3 billion swine. The EU is the second largest pork producer with ca. 186 million head. The proportion of world meat consumption equals 40% pork, 29% chicken, 24% beef and 7% other. The potential economic impact of an ASF introduction in the USA has been estimated at US$ 4,500M; ca. 5% of the value of total sales of pork products. Estimates are that the pig industry will be one of the fastest growing agriculture sectors to meet growing demand for meat from developing economies. Further spread of ASF would threaten this growth and global food security. Pork producers need tools to respond to the pressure to supply increasing quantities of meat using fewer natural resources, and to combat highly infectious emerging diseases such as ASF, especially in the absence of a safe and effective ASF vaccine. The here proposed interdisciplinary approach (i.e., chemistry expertise combined with ASF know-how and an industrial development perspective) is expected to deliver a solution to safeguard this industry by developing an antiviral drug to contain outbreaks of ASF, thereby reducing the economic and death losses that characterize this devastating disease. Following regulatory approval, such drugs can be taken up in EU Directives (e.g. Council Directive 2002/60/EC) and national ASF contingency plans since there is no impediment to their use according to the current EU Animal Health Law. Antiviral drugs alone or in combination with vaccines have the potential to reduce if not completely eliminate the occurrence of ASF in Europe (Zakaryan & Revilla, 2016).
Beneficiaries
The beneficiaries from the research will include the research community, animal health companies and organizations involved in national and global control of epizootic virus diseases. These include other UK Government Departments including DEFRA and DFID, veterinary authorities in the UK, EU and other countries and International Organizations including the World Animal Health Organization (OIE) and United Nations Food and Agriculture Organization (FAO). The research will also benefit farmers and others involved in food production chains and the public. The latter will benefit from improved control of an important livestock disease and resulting stabilization of supplies and price of pork. Outreach and knowledge exchange activities carried out during the project will help to inform the public at large, schools and farming communities of the threats imposed by ASF and other livestock diseases and how we can develop methods to control these diseases.
Academic beneficiaries will benefit from new cutting edge knowledge on ASFV replication mechanisms and alternative control tools. The use of this knowledge to construct modified viruses which do not cause disease and induce a protective response will benefit both academic beneficiaries and animal health companies involved in vaccine production.

Publications

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Description The overarching objective of the project is to develop a small-molecule antiviral drug for African swine fever (ASF) that can be deployed in a safe and effective way to contain outbreaks of ASF even in the absence of an effective vaccine. During the initial 6 months of the project, ViroVet has provided a number of reference compounds to TPI. In parallel, ViroVet's medicinal chemist designed 50 closely related analogues, 20 of which have been synthesised at our custom synthesis provider. Synthesis of the remaining compounds is ongoing. Moreover, the 5 reference compounds and 6 of the related analogues were tested in porcine microsomal stability studies in vitro. All 11 compounds proved stable with a half-life greater than 60 minutes. The microsomal stability assay is important in understanding the metabolisation and intrinsic clearance of molecules. Hence, the promising in vitro stability data are a good indicator to proceed with pharmacokinetic and tolerability studies in pigs for a selection of compounds.
The antiviral activity of five lead compounds identified by ViroVet were examined in primary porcine macrophages against wild type ASFV (Georgia 2007/1). All of the compounds inhibited ASFV replication in a dose dependent manner. Cell viability was measured following continuous incubation with serial dilutions of the compounds in uninfected cells. From this, the IC50 and CC50 were calculated and the selectivity index for each compound determined. VV03-0035 and VV03-0054 displayed high selectivity indices (>100) and were chosen for further interrogation. Both compounds display pangenotype antiviral activity, significantly inhibiting replication of ASFV genotypes from each of the four clades (A-D). The data suggest VV03-0054 inhibits the DNA polymerase, as only late gene expression and protein synthesis was inhibited by the compound and administering VV03-0054 after genome replication in a single synchronised replication cycle removed its antiviral effect. ViroVet has completed pharmacokinetic studies in pigs. This showed that plasma levels of the compounds above the IC50 value could be achieved by using oral administration. The half life of the compounds in plasma was measured and used to calculate the dosing frequency. A further safety study in pigs has been carried out. An experiment is planned in pigs to measure the ability of one of the selected compounds to inhibit clinical signs and replication induced following infection with the ASFV genotype II virulent strain. This will use the selected dose and frequency of dosing.
Report March 2021- February 2022
The stage of ASFV replication at which VV03-0035 acts was further investigated by comparing levels of mRNA and protein expression for selected early and late ASFV genes. These results indicated the drug acted to inhibit late gene expression. This was further suggested by varying the time of adding the drug. Addition of the drug at times up to 9 hours post-infection was effective at inhibiting virus replication whereas at 11 hours no effect was observed. Since VV03-0035 is from a family of nucleoside analogues our results strongly indicate that the compound acts to inhibit the ASFV DNA polymerase and also late gene expression which depends on the onset of DNA replication. Thus late genes including most virus structural proteins are not expressed and virion production is inhibited. Passage of the virus in suboptimal concentrations of the drug did not have a significant effect on levels of virus replication indicating that mutation to avoid the drug effects did not occur to a significant extent.
Two in vivo ASFV challenge experiments were carried out using a drug concentration selected from initial in vivo safety studies. In the first experiment drug dosing was twice a day for 3 days from day -1 to +1 after challenge and daily between days 2 and 6. In the second experiment pigs were dosed once daily for 4 days from day -1 to +2 after challenge and thereafter every 2 days. In the first experiment from the 6 drug treated pigs two survived until the end of the experiment and two until 19 or 20 days post-challenge. The other two were culled at the humane end point at the same time that control non-treated pigs were culled on day 6 post-challenge Analysis of antibody responses and levels of virus in blood showed that surviving pigs had a higher level of antibodies to ASFV by day 14 compared to the pigs culled on days 19 and 20. Thus induction of an earlier immune response by low levels of virus replication may have contributed to survival of these pigs. In the second experiment treated pigs were culled on day 8 post-challenge showing a significant delay compared to control pigs which were culled between days 4 and 6. Levels of virus in blood and tissues were also significantly lower in the treated pig group. In the first experiment treated pigs experienced diarrhoea and reduced eating during treatment whereas in the second experiment no toxic effects of drug treatment were observed.
This is the first in vivo demonstration of an antiviral compound which is effective at inhibiting ASFV replication and disease. It is expected that further refinement of the drug and comparison of different treatment regimes could produce an effective drug that is well tolerated. The drugs identified will be useful tools to investigate the ASFV replication cycle by arresting replication at specific stages.
Exploitation Route ViroVet may commercially develop the compounds
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology

 
Description The overarching objective of the project is to develop a small-molecule antiviral drug for African swine fever (ASF) that can be deployed in a safe and effective way to contain outbreaks of ASF even in the absence of an effective vaccine. During the initial 6 months of the project, ViroVet has provided a number of reference compounds to TPI. In parallel, ViroVet's medicinal chemist designed 50 closely related analogues, 20 of which have been synthesised at our custom synthesis provider. Synthesis of the remaining compounds is ongoing. Moreover, the 5 reference compounds and 6 of the related analogues were tested in porcine microsomal stability studies in vitro. All 11 compounds proved stable with a half-life greater than 60 minutes. The microsomal stability assay is important in understanding the metabolisation and intrinsic clearance of molecules. Hence, the promising in vitro stability data are a good indicator to proceed with pharmacokinetic and tolerability studies in pigs for a selection of compounds. ViroVet has scheduled a pharmacokinetic study in pigs with two analogues to be initiated in April 2020. Pirbright has tested antivirals supplied by ViroVet by infecting primary porcine macrophages with genotype I or genotype II field isolates circulating in Europe, Asia and Africa and treating with different concentrations of antivirals. This data was used to calculate the concentration of the drugs that inhibited virus production by 50%.. In combination with data on the cellular cytotoxicity this allowed the effectiveness (selectivity index) of the antivirals to be calculated. These results have so far identified three compounds suitable for further analysis in pharmacokinetic studies in pigs. In parallel studies have been carried out to determine the stage in virus replication that the compounds act by testing for expression of early or late ASFV proteins.
First Year Of Impact 2019
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description Collaboration with Nesya Goris and colleagues from ViroVet Belgium on development of small molecule inhibitors to control African swine fever 
Organisation ViroVet Belgium
Country Belgium 
Sector Private 
PI Contribution Screening activity of antiviral compounds in inhibiting filed isolates of African swine fever virus grown in macrophages
Collaborator Contribution Provided small molecule inhibitors of African swine fever virus identified from a of a screen library of compounds
Impact Multidisciplinary, medicinal chemistry and virology
Start Year 2019
 
Description Animal Health Investment Forum. Panel Discussion on African swine fever 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Panel discussion on African swine fever impact and control
Year(s) Of Engagement Activity 2020,2022
 
Description Article in Animal Pharm 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Description of the collaboration between Pirbright and ViroVet to develop antivirals against ASFV
Year(s) Of Engagement Activity 2019
URL https://animalpharm.agribusinessintelligence.informa.com/AP015772/ViroVet-Pirbright-collaborating-to...
 
Description Interview with journalist and provided input to article on African swine fever vaccine development 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interviews with Katya Zimmer journalist with the Scientist Magazine and help with preparation of figures
Year(s) Of Engagement Activity 2019
URL https://www.the-scientist.com/features/can-a-vaccine-save-the-worlds-pigs-from-african-swine-fever--...