Application of next generation phage display technology to development of a Differentiation of Infected from Vaccinated Animals (DIVA) strategy

Incursions of hitherto exotic or unknown viral diseases affecting livestock animals appear to have been increasing in frequency in recent years. Climate change and increasing globalisation are thought to be contributing factors; therefore it seems likely that this trend will continue. The most recent example was the emergence of Schmallenberg virus, which causes birth defects and abortions in cattle and sheep. Diagnostic tests, including assays to measure antibodies against the virus, were rapidly developed and an inactivated whole virus vaccine was granted an emergency license in a relatively short time-frame. However, economic losses as a result of Schmallenberg virus have been substantial, particularly as a result of countries that have remained free of the disease imposing trade restrictions on animals (and products from them such as semen) from affected regions.

A differentiation of infected from vaccinated animals (DIVA) strategy refers to an approach in which a diagnostic assay detects antibodies that are present in naturally-infected animals but not in animals that have been vaccinated. Such a test allows demonstration that animals are free of infection even if they have been vaccinated to protect them from disease. In the case of some virus diseases, the lack of such a strategy can mean that international trade restrictions and killing animals are seen as the best option for controlling an outbreak.

It is difficult to distinguish between antibodies raised to an inactivated whole virus vaccine because all of the same structural virus components that generate an antibody response in infection are present. However, we hypothesise that the chemical treatments used to inactivate the virus in the preparation of vaccines will modify the structure of viral proteins and lead to subtle changes in the antibody repertoire.

Next generation phage display (NGPD) is a powerful emerging technology that we propose to apply in a novel way to identify any regions that represent linear epitopes or mimotopes of conformational epitopes of the virus proteins that differ sufficiently to allow antibodies from naturally-infected cows to be distinguished from those from vaccinated cows. These peptides could then be applied in a DIVA test. Schmallenberg virus has been chosen to provide 'proof-of-principle'. If successful, the approach could be applied to other emerging viruses that pose a threat to UK livestock now or in the future allowing a rapid response to include not only an inactivated vaccine but an accompanying DIVA test thereby providing a comprehensive control strategy without any associated product export restrictions.

Schmallenberg virus (SBV) is a recently emerged virus that results in fetal malformation in ruminants and can cause reduced milk yield in dairy cattle. However, the main economic impact of SBV has been as a result of international trade restrictions, particularly in live animals and semen. Despite being a completely novel virus, an ELISA using recombinant nucleoprotein for detection of antibodies was rapidly developed and an inactivated virus vaccine was granted an emergency license in a short time-frame. Mass vaccination with inactivated virus vaccines can be effective in bringing an emerging virus outbreak under control, but has potential implications for international trade with virus-free countries that ban import of antibody-positive animals. In order to limit the financial implications of vaccinating animals, a DIVA strategy is required that enables 'differentiation of infected from vaccinated animals'. A DIVA strategy can be readily employed with recombinant vaccines but inactivated virus vaccines are likely to be the first available for emerging viruses. We hypothesise that the chemicals used to inactivate virus for vaccine production will affect viral protein structure resulting in the stimulation of a subtly altered antibody repertoire. We have been applying the next generation phage display (NGPD) technique, panning panels of serum samples against a phage-display library and applying next generation sequencing and bioinformatics techniques, to determine peptide sequences uniquely recognised by sera from SBV seropositive cows. The aim of this proposal is to use SBV for proof-of-concept to establish whether NGPD can be used to identify peptides that could be used in an assay to differentiate antibodies from infected and vaccinated cows. If successful, this would enable a DIVA test to be established for use in conjunction with vaccination to control or even eradicate future emergent viruses with reduced impact on the export economy and animal welfare.

The UK has one of Europe's largest livestock herds with over 32 million sheep and 10 million cattle, worth >£6 billion annually to the UK's economy. The potential adverse social, economic and welfare impact of emerging viruses such as Schmallenberg virus is immense. The prospect of further emerging viruses affecting livestock production is a source of fear and anxiety in the farming community. If successful, this project would provide a framework for developing a differentiation of infected from vaccinated animals (DIVA) strategy to enable the use of inactivated virus vaccines in the control of emerging virus outbreaks without the potential implications for international trade or the requirement to impose culling of potentially infected animals. The main beneficiaries of the knowledge generated from the project are therefore:

1. Academic scientists. Researchers, including those more generally involved in studying the immune response to infectious diseases, not just in the development of viral vaccines and diagnostics for emerging viral vaccines. Awareness of the power of the next generation phage display technique for understanding immune responses to pathogens and the development of multi-peptide vaccines and diagnostic arrays could lead to development of other applications.
2. Pharmaceutical sector. The potential for a DIVA strategy to be applied when inactivated virus vaccines are used would give the manufacturers of livestock viral vaccines the confidence to invest in development of vaccines against emerging viruses.
3. Veterinary and livestock sectors. Emerging viral disease outbreaks cause considerable economic losses to the livestock sector worldwide and pose a major threat to global food security. Outcomes of this research could empower farmers with the option of vaccinating their animals against an emerging viral disease without the fear of suffering the economic consequences of trade restrictions or of having their animals destroyed as an outbreak control measure. For veterinary surgeons accustomed to putting animal welfare first, the implications of being unable to administer preventive vaccines in the face of an outbreak could be distressing.
4. Government authorities. In the face of an emerging virus threat, government authorities have to make difficult decisions regarding the best measures to implement to prevent spread of disease. A decision to impose compulsory vaccination (even if the cost was subsided) could be unpalatable to farmers if it resulted in economic losses due to trade restrictions. Compulsory slaughter (with or without vaccination, which might be required to buy time to implement a culling programme) would be unpopular with a number of stakeholders including farmers, veterinary surgeons and the public.
5. General public. Although the average UK consumer would profess to preferring to buy ethically-farmed products, their choices are also driven be price. In the event of a major emerging viral disease outbreak, members of the public would be appalled by a repeat of the mass slaughter seen during the 2001 foot-and-mouth disease outbreak. Conversely, if effective control measures were not initiated to curtail the spread of an emerging viral disease, reduced output from the livestock industry would raise prices for the consumer