Exploiting novel African swine fever virus virulence factors and a porcine macrophage cell line to develop a live attenuated vaccine

African swine fever (ASF) is a deadly disease of pigs and wild boar that has a very high socio-economic impact. The rapid spread of ASF in China and other Asian countries since 2018 has resulted in death or destruction of more than 7 million pigs, reducing the Chinese pig herd by about 40% and the global herd by 25%. As pork is the most widely consumed source of protein globally, this has had serious consequences on food prices and supply of protein for human consumption. Combined with continuing ASF spread in Africa and Europe, this represents an unprecedented threat to global food security. In Europe 1408 outbreaks in domestic pigs and 6003 detections in wild boars have been reported since July 2019. In S. E Asia ASF has spread to 11 countries and in Africa ASF is present in almost all countries south of the Sahara. There is no vaccine for ASF limiting disease control. A vaccine is urgently needed to limit ASF outbreaks in affected countries and prevent further global spread.
This project brings together a cutting-edge academic/industry partnership including two academic partners, The Pirbright Institute and Plymouth University and a leading international veterinary vaccine company, Zoetis, to rapidly progress vaccine development by exploiting our new research discoveries. The complexity of this large DNA virus (ASFV), which codes for up to 170 proteins, has hindered vaccine development. It is recognised that the fastest way to develop a vaccine is to produce a weakened ASFV (a live attenuated vaccine or LAV) which does not cause disease in pigs but induces an immune response that protects them from disease caused by subsequent infection with the deadly virus. We showed previously (7, BBSRC funded) that deleting ASFV genes that aren't required for the virus to replicate in cells but have important roles in helping the virus to evade the host's defences in infected animals, can produce LAV candidates. Now, we have identified novel genes, which, when deleted, can eliminate any disease signs caused by the LAV and induce good levels of protection against infection with the deadly virus. However, moderate disease signs and virus replication are observed after challenge. In this project we will improve on these results by deleting different combinations of these novel virulence factor genes to produce LAV strains causing only mild or no clinical signs following immunisation. We aim to protect 80 to 100% of pigs from disease and prevent the replication of the deadly virus so that it doesn't spread to other pigs. The lack of a cell line to grow the LAV candidates to high titres and in large volumes has prevented commercial production of this type of ASFV vaccine. Now we have developed a cell line that closely resembles the macrophage cells that ASFV naturally replicates in. Infection of this cell line produces high amounts of ASFV. In this project, the growth of this cell line will be optimized and scaled up for commercial production. The production of the selected LAV vaccine strain will also be scaled up using the cell line and pre-licensing tests of the vaccine completed. We will also use this cell line to investigate how ASFV modulates the functions of its target cell, the macrophage, thus avoiding detection and activation of the host's defences. The project will deliver high-quality science as well as providing a major step forward to commercial vaccines for ASFV.

The dramatic spread of African swine fever through Africa, Europe and Asia has resulted in the death of more than 7 million pigs in 2019 and reduced the global pig herd thus limiting pork supply and driving up prices. Live-attenuated vaccines (LAVs) produced by gene deletions are recognized to be the fastest route to vaccine development. However, the availability of a gene-deleted ASFV which meets the stringent safety criteria required for vaccine registration and the lack of a continuous cell line to scale up the production of the vaccine candidates impede the commercial development of ASFV LAVs. We will build on new developments from our team to overcome both barriers. Firstly, we have developed a porcine macrophage cell line, pMPI, that accurately represents the target cells that ASFV naturally replicates in. These cells support high levels of ASFV replication without any requirement for adaptation of virus to the cells. We will optimize the growth of pMPI cells to achieve levels compatible with large scale vaccine production and confirm that our LAV candidates do not undergo genome rearrangements during passage in these cells. Secondly, we have identified novel ASFV virulence factors, MGF 360-12L, MGF 505-1R and both EP153R and EP402R and showed that deletion of these genes in combination with others can prevent clinical signs post-immunization but still induce good levels of protection. Moderate clinical signs and virus replication are observed after challenge. We will improve these results by testing different combinations of these gene deletions by immunization and lethal challenge of pigs. This will identify optimized candidates to take forward for vaccine development. The project will deliver a pMPI master cell stock and a master seed virus of the candidate LAV produced in the cells for pre-licensing. Using these cells, high quality science will be delivered by further characterizing how ASFV evades the host's defences.