Improved vaccine manufacture to control foot-and-mouth disease: Production of recombinant vaccines by design

Lead Research Organisation: The Pirbright Institute
Department Name: Picornavirus Molecular Biology

Abstract

One of our biggest challenges will be to meet a growing demand for food especially in the developing world. Animal diseases have a significant impact on the productivity of the livestock industry and safeguarding animal welfare will be a major part of maximising food production. Foot-and-mouth disease (FMD) is endemic in many regions of the world and affects huge numbers of animals, both wild animals and livestock (e.g. cattle, sheep, goats, pigs) and is greatly feared due to the enormous economic losses associated with outbreaks. Thus improved control of FMD could sustainably improve the performance of livestock industries and have enormous economic and social value worldwide especially in developing regions where livestock are seen as a means to raise millions from poverty. In FMD-free regions, such as UK, the costs of FMD primarily result from the need to maintain a disease free status. However, FMD-free countries can face enormous losses as the result of periodic incursions and the estimated cost of the 2001 UK outbreak was £8-10Bn. Future incursions into the UK will undoubtedly occur with the potential to inflict substantial economic losses. Vaccination remains the most effective approach for controlling viral diseases and an estimated 2.35 billion doses of FMD vaccine are administered annually. Currently vaccination-led eradication campaigns are ongoing in India, South-East Asia and South America.

Two major constraints on FMD vaccination are (i) production capacity and (ii) the need to produce new vaccines from field isolates.

Production capacity: FMD vaccines are chemically-inactivated virus preparations produced in cell-culture and the highly contagious nature of FMD requires they are produced in expensive, high-containment facilities. Thus measure that improve vaccine yield from existing production facilities could have an enormous impact by increasing the amount of vaccine available globally for FMD control.

New vaccines: FMD is caused by FMD virus (MDV). A major problem for FMD control is the existence of seven FMDV serotypes, each formed by multiple and constantly evolving virus subtypes. Importantly, vaccination against one serotype does not protect against the others. Further, it is essential that within a serotype the vaccine offers protection against the circulating outbreak strain. This necessitates the periodic need to produce new vaccines, especially against emerging strains for which the current vaccines are a poor match. Vaccine production uses established cell lines (such as BHK cells) that are not normally targeted by FMDV. Thus, the production of new vaccines is critically dependent upon adaptation of a field virus for growth in cell-culture which can prove time consuming or impossible for some field viruses.

Thus the challenge is to produce sufficient quantities of vaccine for FMD control

We have shown that cell-culture adaptation of FMDV and improved cell-culture growth are achieved by specific mutations at either of two distinct sites in the viral capsid. It is our hypothesis that recombinant FMDV with such mutations will have in-built, improved cell-culture growth and can be used to overcome the delays in producing new vaccines from field viruses and, through improved cell-culture growth, increase vaccine yield from the existing manufacturing capacity. Here we will use reverse genetics to genetically engineer FMDV to include mutations in the viral capsid that result in improved virus growth and develop these viruses a vaccines. We will focus on four of the most prevalent serotypes and include viruses that are known to currently give a poor vaccine yield and have proven difficult to grow in cell-culture. Our ambition is to develop marketable products that could have enormous value by reducing the prevalence of FMD worldwide and the likelihood of further outbreaks of FMD in the UK and Europe.

Technical Summary

Our published results show that cell-culture adaptation and improved growth of foot-and-mouth disease virus (FMDV) can be achieved through specific mutations at either of two distinct sites on the viral capsid. It is our hypothesis that recombinant viruses with mutations that result in improved cell-culture growth can be used to overcome the delays in producing new vaccines from field viruses and, through improved cell-culture growth, increase vaccine yield from the existing manufacturing capacity. Thus the central goal of this proposal is to generate genetically engineered FMDV vaccine strains that overcome the need for cell-culture adaptation and give increased antigen yield under vaccine manufacturing conditions. We will focus on four of the most prevalent FMDV serotypes and include viruses that have proven difficult to grow in cell-culture and are known to give a poor vaccine yield. Guided by our extensive knowledge of cell-culture adaptation and high-resolution FMDV structures (including virus-receptor complexes), we will use an established reverse genetic system and synthetic biology to produce novel viruses with specific mutations in the capsid (Objective 1). Recombinant viruses will be characterised for genetic stability and growth (antigen yield) in batch fermenters (Objective 2) and for retained antigenicity (Objective 3). In Objective 4, we will establish if the best approaches to improve vaccine yield can be applied to viruses where cell-culture adaptation and vaccine yield are known to be poor. We will also determine the ability of the best candidate vaccine to induce neutralising antibodies and protect the natural bovine host from virulent virus challenge (Objective 5). The vaccines produced during this project could have enormous social and economic value by reducing the prevalence of FMD worldwide, and the likelihood of further outbreaks of FMD in the UK.

Planned Impact

One of our biggest challenges will be to meet a growing demand for food especially in the developing world. Animal diseases have a major impact on food production and safeguarding animal welfare will be a major component of protecting food security. Foot-and-mouth disease (FMD) is one of the most economically important viral diseases of domestic livestock and is recognised as a global threat to sustainable food security. Vaccination remains the most effective approach for controlling FMD and a major challenge is to produce sufficient quantities of vaccine to meet the needs of eradications campaigns in the developing world. Here we will produce novel viruses with improved cell-culture growth during vaccine manufacture which could (i) significantly increase the amount of vaccine available for FMD control, and (ii) reduce the time taken for producing new vaccines in response to novel emerging field strains. These outcomes are expected to improve vaccine yield from existing production facilities which could have enormous economic and social value worldwide by reducing the prevalence of FMD in developing regions and the likelihood of further outbreaks in the UK. The impact of this work will not be limited to FMDV as our approach could be used to improve vaccine yield for important, related picornaviruses of humans such as poliovirus, human rhinovirus and EV71.
The results of the project will be communicated to Pirbright stakeholders, such as the BBSRC and DEFRA. Other key beneficiaries will be international bodies with an interest in animal health and food security (e.g. The Food and Agriculture Organization of the United Nations, The European Commission for the Control of Foot-and-Mouth Disease (EuFMD), the OIE (World Organisation for Animal Health, and the Directorate-General for Health and Consumers [DG SANCO]). The Pirbright Institute (TPI) is fully committed to engage the public's attitudes to science and the impact of the project will also be publicised by communication with the farming community and the public through our web page, TV and radio, workshops, training courses and exhibitions. Our results will also be of great interest to the academic community studying mechanisms of virus cell-culture adaptation, virus-receptor interactions and cell-entry mechanisms, and, where possible, will be made available through peer review publications and scientific meetings.
TPI fully embraces the need for its science to underpin the needs of a range of activities undertaken by commercial/industrial Science Companies. The commercial exploitation and intellectual-property potential of the research will be maximised through regular interactions between the Business Development Managers at TPI and Merial. If our technologies are found to be commercially viable, a strategy for intellectual-property rights will be implemented.
TPI undertakes high quality research and is committed to the development of highly skilled scientists. The research investigators will receive excellent training in the fields of virology, reverse genetics, and vaccine manufacture and vaccine characterisation, thereby contributing to the wider training, innovation, skills and capability of the UK science base with consequent boosting to science-based industries and the UK economy.

Publications

10 25 50
 
Description SB- Improvement of growth of wild type Foot-and-mouth disease viruses in the cell lines required for growth of virus for vaccine production, by cloning of capsid proteins in to a reverse genetic system and the introduction of targeted mutational changes to the capsid to enhance cell entry.

We have shown for the four most prevalent serotypes of FMDV (O, A, Asia, SAT-2), that virus can be rapidly produced by cloning of capsid protein sequences (produced by synthetic biology) in to a single reverse genetics system, and recovering virus. Virus produced recombinantly in this way is free from extraneous and contaminating agents potentially present in viruses derived from field samples. We have also demonstrated that by introducing targeted capsid changes associated with cell culture adaptation, speed of growth and yield of these viruses in the cell lines required for vaccine production could be improved. This rational approach reduces the need for time consuming adaptation by cell culture passage. The introduced changes did not negatively affect virus stability or antigenicity.

Lastly, the techniques described here were used to produce vaccine, and this vaccine was effective in protecting cattle from challenge with FMDV virus.
Exploitation Route These findings could be utilised by vaccine companies to greatly speed up the development of vaccines in response to new FMDV strains. This would be achieved be streamlining the process of adaptation to cell culture and reducing the testing required for extraneous agents
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The nature of this project, as a link grant with industry, mean that the impact of the findings naturally expand beyond academia. Our partners at Merial (now Boehringer Ingelheim - BI), have established reverse genetics systems as a direct result of this project, and we have demonstrated together that effective FMDV vaccines can be produced by recombinant methods. BI are now evaluating how to push this forward and apply these systems in future. Additionally, we have presented our results at EUFMD, a forum for not just scientists but policy makers and the industry as a whole, therefore giving influence beyond the academic world. The recombinant methods used in this project will reduce the time required to produce vaccines against emerging strains, and potentially make vaccines cheaper, by reducing the need for expensive and time consuming extraneous agent testing and cell culture adaptation. This can only aid the fight against this socially and economically important disease.
 
Title Use of Incucyte Imaging system to measure rate of CPE development 
Description We previously developed a CPE assay during the course of this grant using the minimax imaging plate reader. This utilises an imaging plate reader to assess the proportion of the well covered by cells over time. As CPE develops and cells die this figure reduces, giving a measure of how quickly a virus spreads through cell culture. We have now adapted this assay for use on the incucyte imaging system. This allows assay plates to be kept at optimal temperature and CO2 conditions during assay, and gives much cleaner and more accurate data than the minimax assay. 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? No  
Impact This method allows the easy and rapid comparison of the rate of spread through cell culture of different viruses or the same virus under different conditions (e.g with and without drug). This allows a reduction in the number of laborious plaque assays or TCID50 assays which need to be carried out, reducing the use of plasticware and reagents required 
 
Description LMB 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration
Collaborator Contribution Collaboration
Impact Collaborative research
Start Year 2016
 
Description Merial 
Organisation Sanofi
Department Merial Plc
Country Global 
Sector Private 
PI Contribution BBSRC Link award
Collaborator Contribution BBSRC Link award
Impact BBSRC Link award in progress
Start Year 2016
 
Description Diamond (TT) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Diamond Light Source Open Day - explaining to general public the importance of structural biology and microscopy for understanding viruses and designing improved vaccines.
Year(s) Of Engagement Activity 2019
 
Description Europic Conference (5-8/09/16) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Europic Conference (5-8/09/16)
Year(s) Of Engagement Activity 2016
 
Description GFRA (TT) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Global Foot-and-mouth disease Research Alliance (GFRA) meeting, presentation on virus packaging and implications for vaccine production, initiated collaboration discussions between international academic and industry partners.
Year(s) Of Engagement Activity 2019
 
Description SB - Talk at EUFMD 2018 Conference (29th-31st October 2018) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact A talk was given about the work being carried out in this grant to produce vaccine strains of FMDV by reverse genetics, thus potentially speeding up production of new vaccines. The main impact was to make policymakers and industry in general aware of the approaches being taken, and the potential for this to speed up production of vaccines against emerging strains.
Year(s) Of Engagement Activity 2018