Development of rationally designed live-attenuated lumpy skin disease vaccines

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.

The three viral species in the capripoxvirus (CPPV) genus are Sheeppox virus (SPPV), Goatpox virus (GTPV) and Lumpy skin disease virus (LSDV). They cause severe disease in ruminants particularly in low and middle income countries.

Live-attenuated vaccines which target CPPV disease have been available for decades however they have not been modernised in parallel with other poxvirus-based vaccines and remain poorly characterised with variable efficacy. The recent spread of lumpy skin disease (LSD) through the Middle East and into Europe has highlighted the problems with the currently available CPPV vaccines, for example the RM65 vaccine was ineffective at controlling LSD in Israel in 2012. This project aims to develop improved live-attenuated CPPV vaccines.

The programme of work will target four putative CPPV virulence factors identified by genomic analysis of wildtype and attenuated strains of CPPV. The functions of these four CPPV genes can be predicted by extrapolation from previous studies carried out into orthologues from other poxvirus genera. This prior information will be used to support our studies into their function. The putative LSDV virulence proteins will be studied in isolation using appropriate recombinant systems, and in the context of a viral infection in cell culture by characterising mutagenised strains of LSDV lacking each of the genes. If evidence of a function as a virulence factor is gained from these in vitro studies, the virulence of the mutated LSDV strains will be tested empirically using an experimental bovine model of LSD. In the final objective of the programme of work, mutated LSDV strains which show evidence of severe attenuation coupled with robust immunogenicity will be tested for suitability as vaccines in a bovine LSD challenge model.

What is the benefit of this research?
The main benefit of this research is better control of LSD through the use of a safe and effective, rationally designed LSD vaccine.

Who will benefit and how?
For each beneficiary the timescale of the impact has been estimated as immediate (I, during the time frame of the project), medium term (M, 1-5 years after the project has been completed) or long term (L, >5 years after the project has been completed).

1. Countries in Europe and Asia. Vaccination is a crucial component of LSD control programmes, as evidenced in the current Euroasian epidemic. When LSD entered Europe in Greece in 2015 and Bulgaria in 2016 initial stamping out and quarantine measures did not stop the spread of the virus. Both countries enacted widespread emergency vaccination campaigns to slow the spread of the virus and reduce disease incidence. Countries in southwest Europe and the Balkans are now vaccinating millions of cattle annually and likely to do so for at least the next 4-5 years. If the virus spreads further into Europe even wider vaccination programmes will be required, as recognised by the European Commission who have recently placed a tender for 2 340 000 LSD vaccine doses over the next four years to stock their LSD vaccine bank (https://etendering.ted.europa.eu/cft/cft-documents.html?cftId=2209). The improved vaccine produced in this project will therefore benefit all countries in Europe and Asia which have or are threatened with LSD. I, M, L.

2. Countries in Africa with endemic LSD. Vaccination against LSD is even more important in resource-poor countries such as many parts of Africa. Movement controls and slaughter campaigns are not able to be implemented in these areas therefore LSD control depends almost entirely on vaccination. A safe and effective vaccine will reduce LSD, and thereby increase the efficiency and sustainability of animal production and ensure safe and nutritious diets leading to improved global food security. I, M, L.

3. Rural communities with sheeppox and goatpox. This project will also promote control and eradication of sheeppox and goatpox. This will specifically benefit the poorest subsistence farmers who cannot afford cattle, and women who are often given responsibility for raising small ruminants. Sheep and goat farming in low and middle income countries (LMICs) occurs mostly in smallholdings with the animals providing high quality protein food source (meat and milk), wool, fibre and skins for clothing, a means of accumulating capital and a ready source of emergency funds as well as socio-cultural wealth. Controlling and preventing sheeppox and goatpox will improve the productivity and stability of sheep / goat farming in LMICs and thereby reduce rural poverty through raising farm income, expanding the labour market, and reducing food prices. M and L.

4. Non-governmental agencies. CPPV diseases are priorities for charities working in low and middle income countries such as GalvMed and the Bill and Melinda Gates Foundation. They will likely seek to implement the outputs from this research. I and M.

5. Vaccine manufacturers. This project will develop a new vaccine and new tools to construct genetically modified poxvirus vaccines which will be of benefit to poxvirus vaccine manufacturers. I and M.

6. UK government, farmers and rural communities. LSDV is an emerging threat to the UK as it spreads through southeast Europe. The risk of LSDV reaching the UK will be reduced as a result of this research, therefore avoiding the loss of lucrative UK trade markets. The main trade-related economic consequence of a LSDV outbreak in the UK is loss of semen and embryo markets (there is very little live cattle trade from the UK). In addition the outcomes from this work will strengthen the UK CPPV research base, and provide broader capabilities and new tools for use in a future LSDV outbreak in the UK. I, M and L.