Bluetongue virus reverse genetics: the way forward for Bluetongue vaccines

Lead Research Organisation: London School of Hygiene & Tropical Medicine
Department Name: Infectious and Tropical Diseases

Abstract

Bluetongue is a viral disease of sheep, cattle and goats that poses an economic threat to UK agriculture. The virus is spread by insects (flying midges), since 1998 outbreaks of Bluetongue have spread Northwards in Europe and in September 2007 it reached the UK. The current European situation is severe with widespread infection of sheep, goats and cattle causing many animal deaths, and there is significant risk that a similar scenario will develop in the UK. Current vaccines available against Bluetongue are considered neither safe nor effective enough to be used on a widespread basis, nor do they protect from more than one of the 24 different strains of the virus. As prior vaccination for one strain does not protect against other strains a vaccine that will protect against all currently circulating strains of the virus is a priority. This proposal will develop such a vaccine, while maintaining the possibility to distinguish between infected and vaccinated animals, which is important for trade considerations. The new vaccine is only possible due to recent discoveries made by UK researchers, and uses technology which is not yet available in other public or commercial sector organisations.

Technical Summary

Bluetongue virus (BTV) is an insect-vectored emerging animal pathogen which is currently having a severe economic impact in European agriculture. BTV causes high morbidity and mortality (up to 70%) in sheep. BTV is endemic in many tropical and sub-tropical countries, but since 1998 incursions of BTV into mainland Europe have been common events, reaching as far north as France, Belgium and Holland in 2006 and reaching the eastern counties of the UK in September this year. It is now likely that incursions will become regular events and plausible the BTV will become endemic in northern Europe. This proposal seeks to use a new technological advance, developed in the applicant's laboratory, to undertake proof of principle for a new type of vaccine for BTV. The vaccine will have improved safety over attenuated vaccines, be effective against multiple serotypes of the virus, compatible with current manufacturing processes and compliant with the DIVA principle. We will use a new reverse genetics system, currently only available in the applicant's laboratory, to generate a virus strain that is only capable of replication in tissue culture and not in vaccinated animals. This vaccine strain would be completely deficient in the production of key viral proteins and thus it will be possible to distinguish between vaccinated and infected animals on the basis of antibody responses to these proteins. The techniques and principles that are tested in this application will be transferable to other animal pathogens, for example African Horse Sickness virus.
 
Description Viruses can attack, multiply and establish an infection in organisms including human and animals. Vaccination constitutes a strong method to elicit the immune system and help to counteract this attack. The purpose of this study was to develop and test a new generation of vaccines for Bluetongue virus (BTV). BTV is an important pathogen of livestock and, although there are vaccines available, the complexity of this virus requires the development of new safer, more efficient vaccines. We developed a new generation of defective BTV viruses that cannot multiply in animals but can be used as vaccines. Several viruses were engineered and tested for their inability to multiply in normal cells. Some of these defective viruses were tested in sheep and showed that these vaccine strains could confer full protection against virulent virus challenge. Two types of vaccination schedules were assessed, a 2-dose (similar protocol as the commercial vaccines) and a 1-dose vaccination protocol which has a clear commercial advantage. In addition it was investigated whether BTV serotype can be easily changed using the same backbone of the vaccine strain. In each case the vaccination trials demonstrated complete protection in sheep against infectious BTV and none of the vaccinated animals had any trace of the virus infection. The data obtained is highly encouraging and has the potential to transfer the knowledge to a commercial partner. The output from this work should contribute to the development of safer vaccines with the potential to block virus replication helping to control not only BTV but also many related viruses.



The results and technology generated in this project constitute proof of concept for the development of a new generation of BTV vaccines. In two independent vaccine trials, DISC BTV1 and the reassortant BTV1/8 vaccines showed that both viruses were capable of conferring full protection in animals (sheep) after vaccination and challenge with a virulent strain. These results clearly demonstrated that the designer defective viruses are able to raise a protective immune response in vaccinated animals. Moreover, the safety of the vaccines was also tested and no virus replication was detected in the vaccinated animals. These results strongly support the use of DISC viruses as vaccines.
Exploitation Route Vaccine will directly relevant to farmers and other animal produce producers. To make new safe vaccines for BTV and related viruses
Sectors Other
 
Title BTV DISC viruses 
Description BTV DISC viruses were able to grow in cells that provide the VP6 protein in trans (BSR-VP6 cell line) but were not able to go through a full replication cycle in normal cells. The restriction imposed by this replication deficiency suggests that these viruses will not represent a risk of wild type virus release. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact These viruses has a potential for vaccine 
 
Title BTV S9 and S10 mutant segments 
Description Series of BTV S9 and S10 mutated segments, with truncations and/or insertion of marker genes (eg. EGFP) were also generated. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact These mutations were regenerated in order to develop vaccine candidates 
 
Title Complementary cell lines 
Description BSR-VP6 and BSR-NS3 cell lines were generated by transfecting the expression vector pCAG-VP6 or pCAG-NS3 available in the lab to BSR cells. Cells with integrated copies of the plasmid were selected by addition of 7.5mg/ml of puromicyn to the cell medium. Several independent colonies for each cell line were tested for protein expression and the best expressing clones were selected. 
Type Of Material Cell line 
Year Produced 2013 
Provided To Others? Yes  
Impact The BSR-NS3 cell line supports the growth of some mutant viruses and can be used to study the release of BTV during replication 
 
Title DISC 
Description Disable infectious single cycle (DISC) BTV1 viruses carrying mutated segments S9. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact A range of mutations in the BTV segment S9 (encoding VP6) were designed in order to generate disabled viruses that can be potentially used as vaccine. 
 
Title DISC BTV reassortant strains 
Description DISC BTV reassortant strains with the backbone (all core proteins) of BTV-1 and L2 and M5 (outer capsid proteins) from a different serotype: BTV1/2, BTV1/4, BTV1/8, BTV1/10, BTV1/13, BTV1/21, BTV1/23 and BTV1/24. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2009 
Provided To Others? No  
Impact Understanding restriction in reassortment in multi segmented virus has the potential for development of antiviral reagents, in this case a new generation of BTV vaccines 
 
Title DISC viruses as vaccines 
Description These DISC viruses are compatible with virus entry and protein expression in cells. The expression of viral proteins upon virus entry, synthesise the viral proteins (antigens) in sufficient amount that are available for the immune system, thus reducing the dose required compared to the killed vaccines. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact These mutant viruses were regenerated in order to develop vaccine candidates 
 
Title Generation of replication deficient virus strains 
Description A series of BTV DISC viruses targeting an essential viral protein VP6, involved in virus replication have been made. These also include a series of eight DISC reassortant viruses, that have the outer capsid proteins from BTV-2, -4, -8, -10, -13, -21, -23, and -24. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact These viruses have the potential to be used as vaccine. Some of them were tested showing complete protection in challenged animals 
 
Title Mutant viruses with changes in the S10 
Description Some of the mutant viruses with changes in the S10 showed growth in normal cells although in some cases the growth was severely affected. The mutant viruses that were still capable of replication in normal cells were not selected as potential vaccine candidates. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact These mutant viruses were regenerated in order to develop vaccine candidates 
 
Title Mutations to study VP2 and VP5 entry mechanism 
Description Two BTV outer capsid proteins VP2 and VP5 are responsible for BTV passing through the cell membrane and endosomal membrane barriers to deliver the viral core into the cytoplasm for viral replication. Virus must sense the cellular environment, often through changes in pH to achieve this. How this happens is poorly understood for a complex non-enveloped virus like BTV. To understand this process, a series of mutations based on our high-resolution atomic structure of BTV was generated for exploiting how VP2 and VP5 sense low pH for conformational change to facilitate membrane penetration. Mutations made to all of conserved histidine residues within VP2, including H38A, H95F, H426Y, H640A, H756Y, H866F, H900A, H925F and H947F showed no impact on virus recovery by reverse genetics and viral growth. We found any single mutation to the unique zinc finger CCCH motif, including H164C, C162H, C617H and C851H, or double mutation to change the position of the zinc finger, including C162H+H164C, C617H+H164C and C851H+H164C, result in non-rescuable virus by reverse genetics. This suggests that zinc finger plays a vital pH sensor role in the VP2. We also express recombinant mutant protein C162H using baculovirus expression system to confirm that the mutant protein is no longer able to respond to low pH for conformational change in a thermal stability assay. In addition, in the absence of VP2, certain histidine residues at the critical locations in VP5, identified from mutations we generated, including H272A, H319A, H365F, H384A, H385A, H386A, H412F and H465F, are critical for sensing low pH in the late endosome and coordinate the conformational changes that facilitate membrane penetration. We confirmed that the recombinant mutant protein H385A has lost its membrane fusion activity in a liposome assay. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2019 
Provided To Others? Yes  
Impact Our comprehensive molecular analysis reveals key amino acids of VP2 and VP5 required for detecting cellular pH, leading to an irreversible change that propels the virus through the cell membrane. Our data illustrate a novel function of zinc finger in sensing pH and identify for the first time the key residues essential for cell entry by BTV, also highlight the dynamic nature of the multi-conformational process required for virus infection and indicate a potential mechanism that may be shared by other similar viruses, and may be targets for future therapies. 
 
Title Non-structural protein NS3 
Description The non-structural protein NS3 interacts with cellular trafficking proteins (Tsg101 and S100A10). Mutant viruses with changes in the S10 segment that disturb this interaction showed different levels of trafficking disruption indicating that NS3 is an essential viral protein responsible for intracellular trafficking of particles. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact Understanding the mechanisms involved in virus release can have a major impact in the development of antiviral strategies. 
 
Title Reassortant defective viruses 
Description All reassortant viruses were tested for their growth in complementary cells (BSR-VP6) and their inability to grow in normal cells (BSR) as a tested for vaccine candidate. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This tool was developed to test if BTV-1 can be used as a platform for a rapid generation of vaccines against different BTV serotypes. It was successful. 
 
Title Reassortant virus strain 
Description Reverse Genetics was used to recover viruses with reassortant segments S2 and S6 (enconding VP2 and VP5 respectively) in order to test their potential used as vaccine. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact In an animal trial a reassortant virus strain, BTV1/8 with the backbone of BTV1 and the outer capsid proteins from BTV-8, was tested. As with the parental virus, this reassortant replication-deficient virus was also able to protect vaccinated animals against a challenge with the virulent BTV-8. This was essential to prove that the same deficient virus backbone can be used as scaffolding and only the outer capsid proteins (VP2 and VP5) are needed to be changed to generate a vaccine against a different serotype. Overall these results showed that the replication-deficient BT viruses can be successfully used as vaccine. 
 
Title S10 defective viruses 
Description Defective viruses with mutations in S10 were generated and their ability to grow in normal or complentary cell lines. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact Understanding the mechanism of virus release has the potential for development of antiviral reagents 
 
Title Templates for T7 transcripts for different serotypes 
Description Vectors carrying exact copies of segments 2 (L2) and 5 (M5) from several serotypes were generated. pUCT7BTV-2L2, pUCT7BTV-2M5, pUCT7BTV-4L2, pUCT7BTV-4M5, pUCT7BTV-8L2, pUCT7BTV-8M5, pUCT7BTV-13L2, pUCT7BTV-13M5, pUCT7BTV-21L2, pUCT7BTV-21M5, pUCT7BTV-23L2, pUCT7BTV-23-M5, pUCT7BTV-24L2, pUCT7BTV-24M5. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact This reagents were generated as a proof of concept to development of new set of vaccines against different BTV serotypes. 
 
Title Dose Response analysis 
Description In the second trial, a dose-response analysis was undertaken, and was demonstrated that one dose was sufficient to elicit neutralizing responses that afforded protection from infection of a virulent BTV-1. 
Type Of Material Database/Collection of data 
Provided To Others? No  
 
Title METHOD FOR PRODUCING VACCINAL VIRAL STRAIN OF A VIRUS OF THE REOVIRIDAE FAMILY 
Description The invention relates to a method for producing a modified viral strain of a virus which is a member of the Reoviridae family and, in particular, relates to vaccinal viral strains of the Orbivirus genus. 
IP Reference WO2009068870 
Protection Patent granted
Year Protection Granted 2009
Licensed No
Impact This will allow potentially for future BTV vaccine manufacturing and marketing. This discovery also lead to make similar vaccines for all strains of African Horse sickness virus, that are now currently ready for potential manufacturer . The technology has opened up understanding of virus life cycle and its impact on immune response and pathogenicity that are currently being studied by orbivirus virologists around the world.