Sir Peter Gwynn-Jones

It is with great sadness that we belatedly announce the death of one our patrons, Sir Peter Gwynn-Jones. Sir Peter had only been knighted when he retired. He expressed a keen interest in doing voluntary work on behalf of the red squirrels in his retirement but unfortunately died 5 months later. The reds have lost one of their staunchest allies.

Report to the Wildlife Ark Trust Review of progress of squirrel pox vaccine development project at Moredun Research

Institute January 2011

Summary A meeting was held with Dr Colin McInnes on January 7th to review progress of the vaccine development programme against agreed objectives. It is concluded that the progress of most of the project is satisfactory and that the objectives are generally on schedule according to the agreed timelines. The issue of conventional vs. recombinant approach is still an important consideration. It is recommended that Merial are approached again to request the use of the ALVAC® system, in the event of a conventional live attenuated approach not being successful. During the next 6 months it is suggested that interested parties convene to discuss the way forward for field testing of a vaccine, assuming that one or more vaccine candidates is found suitable in the planned protection experiments.
The project
The project was designed to evaluate and develop one of three possible approaches to a vaccine for squirrel pox disease in red squirrels. These are: i) To attenuate (weaken) the virus by repeated culture so that it will induce immunity but not cause disease. ii) To attenuate the virus by deletion (knock-out) of one or two critical genes which confer pathogenicity while still retaining immunogenicity. iii) To identify one or more immuno-dominant genes which could be cloned and expressed in a vaccine vector e.g. Modified Vaccinia Virus (MVA). The project officially started on January 20th 2009. There had been an initial payment at the beginning of the project with subsequent payments scheduled at 6-month intervals. These subsequent payments are contingent on a satisfactory review of progress against objectives also at 6-month intervals. Previous reviews were completed in June 2009, January 2010 and July 2010 and it was concluded that the project was on schedule, with all objectives having been progressed according to the timelines agreed in the contract.

2 Review of progress to December 2010 A meeting was held with Dr Colin McInnes on January 7th. He provided a brief verbal report of progress against the relevant objectives and this was used to complete the review below. There is some repetition here of earlier reports in order to present a comprehensive update.
Objective 1.
Year 1 Q1 - Q2. Adapt isolate 1296/99 to growth in vitro. The purpose of Objective 1 was to ensure that the virus can be continually cultured in an in vitro cell culture. Isolate 1296/99 was the virus isolated from an outbreak of squirrel pox in Gateshead in 1999 and that was the subject of the characterisation papers published in 2003 and 2006. This is also the virus that was sequenced recently by the University of Liverpool. The virus has been adapted to growth in cell culture. Conclusion: Objective 1 was successfully completed achieved during the initial 6 months of the project.
Objective 2.
Year 1 Q1 - Q2. Isolate genomic DNA from virus isolate Sa/1984 and determine similarity to 1296/99. The purpose of Objective 2 was to determine the genomic similarity of two separate isolates of the squirrel pox virus. Isolate Sa/1984 was the original isolate of SQPV that was reported in 1984. Since then it had been passaged in culture, although the passage history was not well-documented and hence largely unknown. In the first 6 months of this project, genomic DNA was isolated from this virus in preparation for genomic characterisation. Subsequent PCR analysis of the Sa/1984 genome had suggested that it is similar enough to the 1296/99 isolate to be regarded as the same species of virus. Conclusion: Objective 2 was completed during the first 12 months of the project.
Objective 3.
Year 1 Q2 – onwards. Serial passage of isolates Sa/1984 and 1296/99 in vitro and stockpiling of defined virus stocks. The purpose of Objective 3 was to serially culture (passage) these virus isolates with the potential aim of producing one or more attenuated viruses which could then be possible candidates for a live vaccine. Both isolates (1296/99 and Sa/1984) have been undergoing continual passage in culture since the beginning of the project. Passage histories have been kept and stocks of virus stored at various “landmark” passage stages. Two vials of virus have been stored at each passage, and all virus produced at every 10th passage has been kept. These strains are now considered ready for initial non-virulence testing. 3 Careful analysis of the entire virus genome will probably be required of if any of the continually passaged viruses selected for potential use as a vaccine. Conclusion: Objective 3 has been completed.
Objective 4.
Year 1 Q3 – Year 2 Q2. Produce single ‘knockout’ (KO) recombinant squirrelpox virus lacking specific virulence gene(s). The purpose of Objective 4 was to produce a single-gene-deleted virus that might be a potential candidate for an attenuated vaccine. This has now been completed and a candidate could be available for testing within 2 months. Conclusion: Objective 4 has been completed.
Objective 5.
Year 2 Q2 – Year 3 Q2. Produce double ‘knockout’ (KO) recombinant squirrel pox virus lacking specific virulence genes. The purpose of Objective 5 was to produce a double-gene-deleted virus that might be a potential candidate for an attenuated vaccine. This could have advantages over a single KO virus in that it would be much less likely to revert to virulence. Under Objective 4 both right and left hand sides of a putative virulence gene have been deleted. Work is continuing to determine whether these can potentially be combined to produce a double KO virus. Conclusion: This objective has continued on schedule.
Objective 6.
Year 1 Q1 – Q3. To sequence appropriate regions of the squirrel pox virus genome containing the putative immuno-dominant genes. The purpose of Objective 6 was to identify areas of the virus genome that are relevant in terms of producing proteins important for conferring immunity, with the eventual possibility of a sub-unit (individual specific antigen) vaccine approach. Previously there had been a suggestion of differential antibody responses to virus proteins between grey and red squirrel sera. However these differences were subsequently not found to be qualitative. Two specific immunoreactive proteins that are recognised by both red and grey squirrels have been identified. Conclusion: Objective 6 is complete.
Objective 7.
Year 1 Q3 – Year 2 Q2. Clone 2/3 immunodominant genes into shuttle vector for transfer into MVA genome. 4 Originally the purpose of Objective 7 was to insert important immuno-dominant genes identified under Objective 6 into a vector so that they can be expressed as a sub-unit vaccine approach by the Modified Vaccinia Virus (MVA). However during 2010 there were discussions between the Moredun group and the Wildlife Ark Trust regarding the regulatory implications of different approaches to an eventual vaccine. It became apparent that selection of a recombinant approach would necessitate a more complex and expensive regulatory pathway. It was thus suggested that Wildlife Ark Trust’s preference would be for Moredun to concentrate on non-recombinant methods of producing a vaccine. This request was taken on board, but work on a recombinant vaccine was continued in parallel. At a previous review meeting, Dr McInnes had proposed possible access to ALVAC®, the recombinant virus vector vaccine system that is widely used in veterinary vaccines already registered and on the market. This would have the advantage of being a well-characterised system that is already in approved use in the UK and elsewhere and has a well-established safety profile. Whilst this would not solve all of the regulatory issues it could prove to be an acceptable and pragmatic way forward. However there has been no further contact with Merial Animal Health, the owners of ALVAC® and therefore there has been no further progress on this Objective. Conclusion: Objective 7 has not been progressed since July 2010 but the approach proposed by Dr McInnes, still has potential as an attractive and pragmatic way forward to obviate some of the regulatory issues associated with recombinant products. However it is recognised that persuasion of a global animal health pharmaceutical company to collaborate in this way will be a challenge. Objective 8.
Year 2 Q2 – Q4 Produce recombinant MVA containing immuno-dominant genes from squirrelpox virus. This objective has been delayed pending discussions on the availability of the ALVAC® system for creating a recombinant virus vectored vaccine. Thus there has been no progress since July 2010

Overall conclusions It is clear that most of the key objectives of the project have been achieved. However work on the recombinant aspects particularly on the potential use of the shuttle vector has not progressed as originally planned. It is recommended that contact is re-established with Merial to ask if they would consider allowing the ALVAC system to be used. The group now has 3 strains of virus that have been passaged and are available for virulence testing. Also the single knock-out virus could be ready for testing within 2 months.
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A R Peters January 2011