Development of rationally designed live-attenuated lumpy skin disease vaccines

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The poxvirus genus Capripoxvirus (CPPV) contains three viral species which each cause high consequence, transboundary disease in ruminants. Research into CPPVs is very timely as the three species are spreading from their traditional geographic ranges into new regions. In particular LSDV has spread since 2011 throughout Eastern Mediterranean and Western Asian countries into Turkey (2013) and in August 2015 entered Europe (Greece) for the first time. It is now present throughout the Balkans, Caucasus and south-eastern Russia and threatens other European and Asian countries. Despite their importance there has been little progress in the last 30 years in improving CPPV vaccines.The newest types of poxvirus vaccines currently being developed in human medicine, such as NYVAC, are genetically engineered. In brief, comparative analyses of viral genomes are used to identify specific target regions of DNA for manipulation in order to produce a safe and effective "live" vaccine. In contrast all currently available CPPV vaccines are randomly mutated and some contain a mix of viruses of variable genotypes and virulence. There have been reports from the field of poor efficacy and safety of these randomly mutated vaccines, indicating the need for better CPPV vaccines. This project will follow the approach used in human medicine to develop safer and more reliable genetically engineered CPPV vaccines. Full genome sequencing of wildtype and attenuated strains of CPPV by Pirbright researchers and others has enabled bioinformatic analysis of these strains to identify CPPV genes which associate with virulence. We have additionally identified orthologues of known virulence factors of other poxvirus genera and combined the datasets to single out four putative virulence factors of CPPV, genes most likely to encode proteins which contribute to CPPV virulence. Deletion of these genes is likely to result in attenuated CPPV strains.The initial work (objective 1) will develop CRISPR/Cas9 technology to overcome difficulties in manipulating the CPPV genome. Following on from this, the four putative CPPV virulence factors will be mutated in a wildtype strain of LSDV. The function of the virulence factors will be assessed in isolation (objective 3) and in the context of a viral infection (objective 4). The data will then be assessed. Each mutant strain that (i) replicates to normal or near normal levels in cell culture, and (ii) is supported by evidence of a function consistent with a role as a virulence factor from the in vitro studies in objectives 3 and 4, will be taken forward into animal trials to experimentally measure the influence of the protein on viral virulence (objective 5). Cattle will be infected with a mutant, wildtype or repaired LSDV strain and physiological, behavioural, virological and immunological measurements taken over the following 28 days to assess the severity of the resultant disease. In the final study of the project (objective 6) the data from objectives 3, 4 and 5 will be used to design two mutated LSDV strains which will be assessed as live-attenuated vaccines in a challenge model of LSD. The hypothesis is that the targeted mutation/s will substantially reduce the ability of the virus to cause disease, while retaining strong poxviral immunogenicity.The main outputs of this project will be (a) identification of virulence factors encoded by LSDV, (b) an improved method for genetically manipulating CPPVs, and (c) rationally designed, genetically engineered vaccines to prevent LSD.