Exploiting African swine fever virus surface proteins to develop rapid diagnostic tests and understand virus host interactions

Lead Research Organisation: The Pirbright Institute
Department Name: UNLISTED

Abstract

This project will investigate a virus disease of pigs, African swine fever (ASF), which causes major economic losses in affected countries. These are mainly in sub-Saharan Africa although ASF has recently been introduced to the Caucasus and Russia increasing the risk of further global spread. Most of the virus isolates cause an acutely fatal disease in pigs, although there are some less virulent isolates. ASF has potentially devastating effects on the commercial and subsistence pig production. In countries such as Cameroon or Madagascar, the introduction of ASF resulted in the loss of between 50 and 80% of the pig population. ASF has serious implications for food security. Pigs are often reared in areas where beef production is difficult. They are excellent converters of food waste and agricultural by-products into high quality protein and have a relatively short production cycle. ASF is difficult to control for several reasons. These include the presence of reservoirs of the virus, ASFV, in wild pigs and soft tick vectors, the lack of a vaccine and the stability of the virus in pork and in the environment. Most outbreaks of ASF are not reported and control measures are therefore not implemented. This is because samples have to be submitted to reference laboratories for diagnosis. In many African countries these are either not present or sending samples to them is costly and requires a long time. This project will aid control of ASF by development of penside diagnostic tests which can be used locally on farms. Similar penside tests have been used to great effect in the diagnosis and control of other animal diseases, such as in the campaign which has resulted in the global eradication of the disease known as cattle plague or Rinderpest. These penside diagnostic tests will be made available commercially in the short term. The approach taken will be to characterise the proteins that are exposed on the surface of the ASF virus particles and then to produce antibodies that recognise these proteins. ASFV is a large complex virus and it may be necessary to test antibodies against several proteins to identify those that are best to use in the diagnostic tests. The antibodies will be incorporated into simple devices, similar to pregnancy testing kits, which can be used on farms. These will be used to detect virus in samples from pigs that are suspected to be infected with ASFV thus ensuring rapid and accurate diagnosis. The virus surface proteins will be used for two other purposes. These proteins are expected to be important for the virus to bind and enter host cells to begin production of progeny virus particles. Activating the host's immune system to recognise these proteins may inhibit virus infection and protect pigs from ASF disease. The ability of combinations of these virus surface proteins to stimulate an immune response in pigs and protect them from ASF disease will be tested. Promising candidate proteins will be incorporated into programmes for vaccine development against ASFV. Finally, the reagents generated in the project will be used for basic scientific studies to improve our understanding of how ASFV virus particles bind to and enter host cells and how virus particles are assembled. This information will be of broad scientific interest in understanding the interaction of viruses with the host cell which in the longer term may lead to novel strategies to control virus infections.

Technical Summary

African swine fever virus (ASFV) causes an often acutely fatal disease of pigs, which results in major economic losses in many African countries. There is no vaccine and frequently disease is not diagnosed because of difficulties in sending samples to reference laboratories. ASFV particles are multi-layered and include more than 50 proteins. The two infectious forms of the virus include an intracellular mature form produced in cytoplasmic virus factories. This consists of a nucleoprotein core and shell surrounded by an internal envelope onto which the icosahedral virus capsid is assembled. The other infectious form of the virus is the extracellular virus, which contains an additional external membrane layer. The proteins on the surface of both of these virus particles will be characterised by tagging the proteins with biotin followed by affinity purification of biotinylated proteins and their identification using mass spectrometry. First, antibodies which recognise these virus surface proteins will be produced and used to develop penside diagnostic tests. Secondly, we will investigate the localisation of these proteins in virus particles and in infected cells and begin to characterise their roles in virus binding and entry and morphogenesis. These studies will include investigating if the antibodies or recombinant proteins can inhibit virus infection of cells. Thirdly, we will investigate the immune response to these proteins. This will involve analysis of the cellular and humoral immune response to these proteins in pigs which recover from infection with attenuated ASFV isolates. We will also vaccinate pigs using a DNA prime recombinant vaccinia virus boost strategy using pools of constructs expressing individual ASFV genes. The immune response following vaccination will be monitored and the ability to withstand lethal ASFV challenge tested. This will identify candidate protective antigens which can be included in vaccine development programmes.

Planned Impact

African swine fever (ASF) causes high mortality of pigs and major economic losses in many African countries. The lack of a vaccine and accurate diagnosis in most countries contributes to difficulties in controlling ASF. This project will characterise proteins on the surface of virus particles and develop antibodies against these for use in penside diagnostic tests. These will be available in the short term. We will also investigate if these surface virus proteins are targets for generating a protective immune response in pigs. This will identify candidate protective antigens which can be incorporated into vaccine development programmes. Vaccine development is likely in the medium term. Knowledge gained from the project will include information on the role of the surface proteins in virus binding and entry and their interactions with bystander cells which do not become infected. Beneficiaries from the research will include the commercial partners who will have the opportunity to develop and market penside diagnostic tests for ASFV. We expect to have developed prototype devices and produced these on a larger scale for validation to OIE standards during the course of the project. An initial training course in use of the devices will be held during the project for selected participants from several African countries. Development on a commercial scale is expected soon after completion of the project. Implementation of the use of these will involve more widespread training of staff in veterinary services from different countries. The development of these diagnostic tests will be disseminated through publication in peer reviewed open access journals and on IAH and OVI websites. The information will also be disseminated via presentations at meetings involving scientists, veterinary authorities and by press releases. Use of these devices will provide more and better information concerning the prevalence of ASF and enable diagnosis at the local level. This will improve the implementation of control measures both by providing more information rapidly and involving stakeholders at the local level more directly. Outputs from the research will also contribute to development of vaccines against ASF in the medium term. Availability of vaccines is essential for effective control particularly in areas where the tick vector and other wildlife reservoirs of the virus are present. Beneficiaries of the improved control will include governments which will gain from the reduced costs of disease control and increased food security. Farmers in the commercial, rural poor and peri-urban sectors will also benefit as will other stake-holders in the food supply chain. These benefits will impact in countries where ASF is endemic and will also be available to ASF free countries if outbreaks did occur. The research will also contribute to basic knowledge on the interaction of ASFV with host cells which will be of interest generally to those studying virus host interactions. The reagents generated, including antibodies, will be publicised in articles in peer reviewed Journals, on IAH and OVI websites and in presentations at meetings of scientists and veterinarians. The staff employed on the project will benefit from training in molecular and cell virology, diagnostic test development and immune assays. Through interaction with commercial partners they will gain experience of the Biotechnology Industry. The project team is multidisciplinary and involves experts in ASFV and other economically important animal viruses with experience ranging from the molecular and cellular study of virus host interactions to studies on immune responses and pathogenesis at the whole animal level. Several of the PIs have experience in the commercial exploitation of antibodies and in interacting with potential beneficiaries outside of the immediate academic community in both Europe and African countries. These include government bodies, farmers and veterinary authorities.

Publications

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Description African swine fever is a devastating disease of domestic pigs which can result in very high mortality. The diseases is endemic in many sub-Saharan African countries and has a high socio-economic impact for small-scale rural and peri-urban farmers as well as larger commercial farms. ASF has serious implications for food security. Pigs are often reared by women and in areas where beef production is difficult. They are excellent converters of food waste and agricultural byproducts into high quality protein and have a relatively short production cycle. The lack of a vaccine limits options for disease control. Rapid and effective diagnosis is required together with implementation of control measures including movement restrictions and quarantine.

One of the project aims was to develop new tests that could be used for diagnosis and surveillance of African swine fever virus. A novel test for detection of antibodies against African swine fever virus was developed. This was achieved by screening a proprietary phage library expressing chicken single chain scFv antibodies with recombinant ASFV protein P30 (encoded by CP204L) to idenfify clones expressing scFV antibodies that recognised recombinant protein P30 and also detected the presence of ASFV specific antibodies in pig serum when used in an inhibition ELISA. The ASFV P30 protein is very immunogenic and is one of the proteins against which antibodies are first detected following infection of pigs.

Phage displayed scfv E12 which consisted of only a light chain was converted to full length (VH + VL) scFvs. The adjusted library was subsequently panned on ASFV P30 and 12 full length scFvs with different sequences were isolated. In contrast to the parent E12, these scFvs recognized P30 in the phage displayed format as well as soluble format. Similar to the parent, all the full length scFvs could also detect the presence of ASFV-specific antibodies in pig serum when used in an inhibition ELISA. During an earlier stage of this activity an ASFV P30-specific phage displayed scFv (E12) was isolated. This phage displayed scFv which has only a variable light chain (VL) was converted by chain shuffling to an scFv sub-library that contains full length scFvs consisting of variable light (VL) chains and variable heavy (VH) chains. ASFV P30-specific scFvs selected from this sub-library can, similar to the E12 parent, also be used in an inhibition ELISA to detect the presence of ASFV P30-specific antibodies in a small panel of field sera collected from domestic pigs. Large batches of ASFV 30 and P30-specific scFv E2 were prepared and will be used in the near future for a preliminary validation of an inhibition ELISA based on recombinant ASFV P30 and ASFV P30-specific scFv E2. This will improve the implementation of control measures both by providing more information rapidly and involving stakeholders at the local level more directly. Farmers in the commercial, rural poor and peri-urban sectors will benefit as will other stake-holders in the food supply chain. These benefits will impact in countries where ASF is endemic and will be available to ASF free countries if outbreaks did occur.

As part of the training course held from 27-30 May 2013 at the ARC-OVI, southern Africa participants were educated on development of new skills, including a pipetting training course presented by AEC-AMERSHAM SOC Ltd. Most of the laboratories in SADC are not equipped to do basic surveys of diseases. Using easy and fast detection methods and collaborating with researchers from these countries makes it possible to empower female researchers. Research on ASF and improved detection techniques helps rural pig farmers (mostly women) to detect and control spread of diseases.
Trainnig booklet and CD:
A training course on African swine fever (ASF), Rift Valley fever (RVF), peste des petits ruminants (PPR) and lumpy skin disease (LSD) prevention, recognition and control presented from 27-30 May 2013, supported from two externally-funded projects: the UK BBSRC project titled "Exploiting proteins on the surface of African swine fever virus to develop rapid diagnostic tests and understand virus host interactions (Grant no: BB/H008969/1)" and the Canadian IDRC-CIDA CIFSRF project "New vaccines to control livestock diseases in sub-Saharan Africa (Grant no: 106930-001)". A training booklet and CD was given to each participant after completion of the course.

Oral presentation:
"Molecular characterisation of African swine fever outbreak viruses in Africa can assist in ASF control", van Heerden J, Malan K and Heath L at The 6th annual meeting Epizone, 12th - 14th June 2012.
"Application of a rapidvaccine discovery system to African swine fever virus" Chapman D., Takamatsu H., Jankovich J., Robida M., Goatley L.C., Netherton C., Dixon L., Jacobs B., Sykes K. XIX International Poxvirus, Asfarvirus, Iridovirus Conference, Salamanca Spain "June 2012 and poster EPIZONE 2012
"High throughput insertion of foreign antigens into vaccinia virus for vaccine development" Jankovich J., Conwell K., White S., Sykes K., Dixon L. and Jacobs BL. XVIII International Poxvirus, Asfarvirus, Iridovirus Conference,Sedona USA June 2010
"African swine fever virus host interactions and vaccine development". Korean Society of Veterinary Science October 10th 2013. Dixon LK, Abrams C., Chapman DAG, Netherton C., Goatley LC., Takamatsu H., Taylor G.

Seminar:
"Molecular characterisation of African swine fever outbreak viruses in Africa can assist in ASF control", van Heerden J at the Young Scientist Meeting, ARC-OVI, South Africa, June 2012.

Poster:
Detection of African Swine Fever Virus-specific antibodies in pig serum using ASFV P30 and a P30-specific phage displayed chicken scFv. Wouter van Wyngaardt, Juanita van Heerden, Jeanni Fehrsen and Livio Heath. The 7th EPIZONE Annual Meeting "Nothing permanent, except change", will be held in Brussels, Belgium, 1-4 October 2013.
A training course on African swine fever (ASF), Rift Valley fever (RVF), peste des petits ruminants (PPR) and lumpy skin disease (LSD) prevention, recognition and control were presented from 27-30 May 2013 at the ARC-OVI, South Africa. Twelve participants from ten of the neighbouring Southern African Development Community (SADC) countries (Angola, Botswana, Lesotho, Malawi, Mauritius, Mozambique, Namibia, Tanzania, Zambia and Zimbabwe) attended the course. All of them work in Veterinary Research Institutes in their respective countries. Each day of the training course started with lectures which provided information on new tools and strategies for the diagnosis, prevention and control of African swine fever (ASF), Rift Valley fever (RVF), peste des petits ruminants (PPR) and lumpy skin disease (LSD), as recommended by the Office International des Epizooties (OIE). Each afternoon was dedicated to practical training of the participants on techniques used at the ARC-OVI for diagnosis of African swine fever (ASF), Rift Valley fever (RVF) and lumpy skin disease (LSD). The participants were given unknown samples to analyse and results for each test completed were discussed to ensure all participants understand the methods.
Two groups of 6 pigs each were immunised with a pool of 42 or 43 ASFV antigens by a DNA prime and recombinant vaccinia virus boost. The groups included 12 predicted surface proteins and were identical except that one group did not include the p30 protein which we had previously shown to be immunodominant and generated an antibody response much stronger than other antigens. This was a larger pool of antigens than we had previously tested. A control group of pigs were immunised with 3 irrelevant antigens by the same regime. Pigs were challenged with virulent ASFV strain Georgia 2007/1. As expected control pigs developed clinical signs specific for ASFV from 3 to 4 days post-immunisation and were euthanized by day 5 to 6. Post-mortem signs were typical of acute ASFV. Unexpectedly the experimental groups also developed clinical signs by day 3 including fever and loss of apetite but not typical ASF signs. The pigs were euthanized as humane end-points were reached although some pigs showed reduced signs of disease. Post-mortem signs in all except one pig were not typical of ASF. Thus spleens were normal in size and haemorrhages were not detected in organs. Measurement of ASFV load by quantitative PCR showed that immunised pigs had reduced levels of virus in blood and tonsils by about 10 to 100 fold compared to control pigs and no virus was detected in spleen in pigs from the experimental groups. In contrast high levels of virus were detected in spleen in control pigs. The data indicate that partial protection was achieved. Experimenst are in progress to measure immune response to individual proteins. A further experiment will take place starting in September using smaller groups of 21 or 23 antigens in pools. This is the number of antigens we had previously optimised.
Exploitation Route The monoclonal antibodies generated could be incorporated into a diagnostic kit for detecting antibodies against African swine fever virus in infected pigs. The antigens identified which induced an immune response and evidence of partial protection in pigs may be incorporated into design of vaccines. The genome sequence of the highly virulent Georgia 2007/1 isolate may be used for vaccine design,
Sectors Agriculture, Food and Drink,Education,Pharmaceuticals and Medical Biotechnology

 
Description Monoclonal antibodies have been selected from a chicken immunoglobulin library and their use in diagnostic assays to detect antibodies in pigs against African swine fever virus has been demonstrated. This may lead to development of a commercial diagnostic kit. The complete genome sequence of a highly virulent African swine fever virus isolate introduced to the Trans Caucasus and Russian Federation has been determined. This provided information for developing approaches for vaccine development. Immune responses in pigs immunized with vectors expressing ASF virion surface proteins were measured following delivery of the genes to pigs by DNA prime and recombinant vaccinia virus boost. Antigens inducing potential protective responses were identified fllowing immunization and challenge of pigs. This may lead to development of effective vaccines for African swine fever virus. Further work has been funded and through this work we have identified a pool of 8 ASFV antigens that can induce 100% protection in pigs.
First Year Of Impact 2010
Sector Agriculture, Food and Drink,Education,Manufacturing, including Industrial Biotechology
Impact Types Societal,Economic

 
Description GALVmed DFID Funding
Amount £216,000 (GBP)
Organisation GALVmed 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 09/2017
 
Description Zoetis Research Funding
Amount £550,000 (GBP)
Organisation Zoetis 
Sector Private
Country United States
Start 04/2017 
End 10/2018