The Wildlife Ark Trust News

Report to the Wildlife Ark Trust

Review of progress of squirrel pox vaccine development project at
Moredun Research Institute

January 2010

Summary and conclusion

A telephone discussion was held with Dr Colin McInnes on January 6th to review progress of the vaccine development programme against agreed objectives. It is concluded that the progress of the project is very satisfactory and that the objectives are generally on schedule according to the agreed timelines.

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:

To attenuate (weaken) the virus by repeated culture so that it will induce immunity but not cause disease.
To attenuate the virus by deleting (knock-out) one or two critical genes which confer pathogenicity while still retaining immunogenicity.
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.

The first 6-monthly review was completed in June 2009 and it was concluded that the project was on schedule, with all objectives having been progressed according to the timelines agreed in the contract.

Review of progress

Dr McInnes provided a brief written summary report of progress against the relevant objectives and this was used to complete the review below.

Objective 1.
Year 1 Q1 - Q2.
Adapt isolate 1296/99 to growth in vitro.

The purpose of Objective 1 is to ensure that the virus can be continually cultured in an in vitro system i.e. 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. It is also the virus that is currently being sequenced in its entirety (University of Liverpool). The virus has been adapted to growth in cell culture.

Conclusion: Objective 1 was achieved during the initial 6 months of the project and therefore this objective is complete.

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 is 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 was passaged in culture, although the passage history was not well-documented and hence largely unknown. In the project review carried out in June 2009, genomic DNA had been isolated from this virus in preparation for genomic characterisation. During the past 6 months, PCR analysis of the Sa/1984 genome has suggested that it is similar enough to the 1296/99 isolate to be regarded as the same species of virus.

Conclusion: Objective 2 is complete.

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 is to serially passage these virus isolates with the potential aim of producing an attenuated virus(es) which could be a possible candidate(s) for a live vaccine.

Both isolates (1296/99 and Sa/1984) have been undergoing continual passage in culture since the beginning of the project. This will be an ongoing activity up to the end of year 2. Passage histories are being kept and stocks of virus stored at various “landmark” passage stages.

A decision was taken to store two vials of virus at each passage, but to keep all virus produced at every 10th passage. Isolate 1296/99 has now been passaged 33 times in vitro. PCR analysis after 30 passages, using primers corresponding to a specific gene of the squirrel pox virus genome, confirmed the continued presence of the virus.

Isolate Sa/1984 has been passaged 31 times. Since the passage history of this virus was uncertain prior to this current work, PCR analysis was performed using a number of primer pairs. DNA was prepared after passage number 26 and analysed. Preliminary data suggest that all of the putative virulence genes at the left hand side (LHS) of the genome are missing. This is possibly not unexpected since genomic re-arrangements involving the deletion of blocks of genes at the LHS are well documented for the parapoxviruses. The deletion of the LHS genes from Sa/1984 will be confirmed by sequencing.

Conclusion: Objective 3 is an ongoing activity and is currently on track.

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 is to produce a single-gene-deleted virus that might be a potential candidate for an attenuated vaccine.

Two specific genes were targeted for individual removal from the squirrel pox virus genome to create the recombinant KO viruses. Removal of the genes will be achieved by homologous recombination. To achieve this, the flanking regions surrounding these two genomic areas require to be cloned initially into the virus KO shuttle vector. This has been achieved, but results are still awaited from the sequence analysis of these constructs, to ensure that they can be taken forward to the next stage of the process to produce a single KO virus.

Conclusion: Objective is not due to be completed until the end of quarter 2, but is currently making good progress and is on track.

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 is 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.

Conclusion: This part of the project is not scheduled to begin until the second quarter of 2010.

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 is to identify areas of the virus genome that are important in terms of producing proteins important for conferring immunity, with the eventual possibility of a sub-unit (individual specific antigen) vaccine approach.

At the time of the previous report, serum, collected from red squirrels previously identified as having been infected with the virus, had been used to probe western blots of SQPV proteins. At the same time proteomic mass-spectrometry analysis was performed on poly-acrylamide gel slices containing the SQPV proteins. In this way the proteins recognised by the squirrel sera were putatively identified. Eight candidate immunodominant genes were cloned, initially into sequencing vectors and thereafter into a mammalian expression vector. Three additional genes identified via comparative studies with other poxviruses were also cloned and inserted into the expression vector. Transient expression in COS-1 cells had been achieved but results were awaited on blotting studies to determine whether or not any of the immunodominant proteins had successfully been identified.

Following on from the previous report, it was demonstrated that a number of the individual genes cloned from the virus genome were indeed recognised by the red squirrel and grey squirrel sera. At least two of these genes have subsequently been used to create stable cell lines expressing the corresponding proteins, should it prove necessary to raise anti-sera against these proteins in the future.

The analyses of the immuno-dominant proteins have uncovered an interesting difference between red and grey squirrels. Side-by-side comparison of pooled grey squirrel sera and red squirrel sera on a 2-dimensional western blot of total virus proteins, showed that the grey squirrel sera blotted against more viral proteins than the red squirrel sera. Studies are ongoing to identify these proteins and to determine whether or not they represent quantitative or qualitative differences.

Conclusion: Objective 6 is on schedule.

Objective 7.
Year 1 Q3 – Year 2 Q2.
Clone 2/3 immunodominant genes into shuttle vector for transfer into MVA genome.

The purpose of Objective 7 is to insert important immune-dominant genes identified under Objective 6 so that they can be expressed as a sub-unit vaccine approach by the Modified Vaccinia Virus (MVA).

Individual immunodominant genes identified under Objective 6 have been cloned into general cloning vectors ready for transfer into the MVA shuttle vector. However we have delayed the actual cloning into the MVA shuttle vector to see if we can identify those proteins that are recognised by the pooled grey squirrel sera and not by the pooled red squirrel sera. These may represent better subunit vaccine candidates than proteins that we know are recognised by the red squirrel immune system.

Conclusion: Objective 7 is on schedule

Overall conclusions

During discussion it was agreed that progress to date has again been good with no major delays to any of the agreed milestones. The finding that there may be a qualitative difference between the major immuno-dominant proteins recognised by grey squirrels in comparison to red squirrels is important and is worth following up, as it could have relevance to differences in the disease characteristics between the two species.

Objectives 1 and 2 were completed on schedule and Objectives 3, 4, 6, and 7 are all being progressed on schedule. Work on Objective 5 is planned to begin as scheduled in the second quarter of this year.

Thus all milestones are on schedule with no alterations to content or timing required at this stage. There are no plans to make major changes to the programme of work outlined in the original proposal.

A R Peters
January 2010

_______________________

Research News

The first research project was carried out by the University of Liverpool to map the entire DNA structure of the squirrelpox virus. This will allow us to understand how the virus works and why it is more pathogenic in red squirrels than greys. Using this information the University is working on a more sensitive DNA test to identify the virus in all squirrels. Initial genetic results of the poxvirus have identified a unique molecule which hides the virus from the squirrel’s immune system. This work may have implications beyond the squirrelpox virus issue as it could have a bearing on the way virus infections affect human beings.
The second is research by the Moredun Research Institute into developing a vaccine against the squirrelpox virus which is now seen as the greatest threat to the survival of the reds. This research began on 19th January 2009 and is expected to take 3 years to complete after which there will be a further 2 years of safety and efficacy trials. In June we received our first progress report which confirmed that the objectives of the research were on schedule against agreed timelines. These were;

Objective 1 – Adapt isolate 1296/99 to growth in vitro – Obj 1 achieved

Objective 2 – Isolate genomic DNA from virus isolate Sa/1984 and determine
similarity to 1296/99 – Obj 2 is almost complete

Objective 3 – Serial passage of isolates Sa/1984 and 1296/99 in vitro and
stockpiling of defined virus stocks – Obj 3 has been achieved

Objective 6 – Sequence appropriate regions of the squirrelpox virus genome
containing the putative immunodominant genes – Obj 6 is on
schedule

A decision is to be taken shortly on whether to use an alternative poxvirus backbone e.g. Orf virus for the recombinant vaccine rather than the Modified Vaccinia Virus Ankera (MVA) as was originally planned. The report concludes that members of the research group are leaders in this scientific field and, whilst there is no guarantee of success, they stand a very good chance of achieving the end objective of developing an effective vaccine against squirrelpox. So, so far, so good.

Drawing by Lyndy Bew	The Wildlife Ark Trust News Report to the Wildlife Ark Trust Review of progress of squirrel pox vaccine development project at Moredun Research Institute January 2010 Summary and conclusion A telephone discussion was held with Dr Colin McInnes on January 6th to review progress of the vaccine development programme against agreed objectives. It is concluded that the progress of the project is very satisfactory and that the objectives are generally on schedule according to the agreed timelines. 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: To attenuate (weaken) the virus by repeated culture so that it will induce immunity but not cause disease. To attenuate the virus by deleting (knock-out) one or two critical genes which confer pathogenicity while still retaining immunogenicity. 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. The first 6-monthly review was completed in June 2009 and it was concluded that the project was on schedule, with all objectives having been progressed according to the timelines agreed in the contract. Review of progress Dr McInnes provided a brief written summary report of progress against the relevant objectives and this was used to complete the review below. Objective 1. Year 1 Q1 - Q2. *Adapt isolate 1296/99 to growth in vitro.* The purpose of Objective 1 is to ensure that the virus can be continually cultured in an in vitro system i.e. 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. It is also the virus that is currently being sequenced in its entirety (University of Liverpool). The virus has been adapted to growth in cell culture. Conclusion: Objective 1 was achieved during the initial 6 months of the project and therefore this objective is complete. 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 is 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 was passaged in culture, although the passage history was not well-documented and hence largely unknown. In the project review carried out in June 2009, genomic DNA had been isolated from this virus in preparation for genomic characterisation. During the past 6 months, PCR analysis of the Sa/1984 genome has suggested that it is similar enough to the 1296/99 isolate to be regarded as the same species of virus. Conclusion: Objective 2 is complete. 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 is to serially passage these virus isolates with the potential aim of producing an attenuated virus(es) which could be a possible candidate(s) for a live vaccine. Both isolates (1296/99 and Sa/1984) have been undergoing continual passage in culture since the beginning of the project. This will be an ongoing activity up to the end of year 2. Passage histories are being kept and stocks of virus stored at various “landmark” passage stages. A decision was taken to store two vials of virus at each passage, but to keep all virus produced at every 10th passage. Isolate 1296/99 has now been passaged 33 times in vitro. PCR analysis after 30 passages, using primers corresponding to a specific gene of the squirrel pox virus genome, confirmed the continued presence of the virus. Isolate Sa/1984 has been passaged 31 times. Since the passage history of this virus was uncertain prior to this current work, PCR analysis was performed using a number of primer pairs. DNA was prepared after passage number 26 and analysed. Preliminary data suggest that all of the putative virulence genes at the left hand side (LHS) of the genome are missing. This is possibly not unexpected since genomic re-arrangements involving the deletion of blocks of genes at the LHS are well documented for the parapoxviruses. The deletion of the LHS genes from Sa/1984 will be confirmed by sequencing. Conclusion: Objective 3 is an ongoing activity and is currently on track. 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 is to produce a single-gene-deleted virus that might be a potential candidate for an attenuated vaccine. Two specific genes were targeted for individual removal from the squirrel pox virus genome to create the recombinant KO viruses. Removal of the genes will be achieved by homologous recombination. To achieve this, the flanking regions surrounding these two genomic areas require to be cloned initially into the virus KO shuttle vector. This has been achieved, but results are still awaited from the sequence analysis of these constructs, to ensure that they can be taken forward to the next stage of the process to produce a single KO virus. Conclusion: Objective is not due to be completed until the end of quarter 2, but is currently making good progress and is on track. 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 is 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. Conclusion: This part of the project is not scheduled to begin until the second quarter of 2010. 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 is to identify areas of the virus genome that are important in terms of producing proteins important for conferring immunity, with the eventual possibility of a sub-unit (individual specific antigen) vaccine approach. At the time of the previous report, serum, collected from red squirrels previously identified as having been infected with the virus, had been used to probe western blots of SQPV proteins. At the same time proteomic mass-spectrometry analysis was performed on poly-acrylamide gel slices containing the SQPV proteins. In this way the proteins recognised by the squirrel sera were putatively identified. Eight candidate immunodominant genes were cloned, initially into sequencing vectors and thereafter into a mammalian expression vector. Three additional genes identified via comparative studies with other poxviruses were also cloned and inserted into the expression vector. Transient expression in COS-1 cells had been achieved but results were awaited on blotting studies to determine whether or not any of the immunodominant proteins had successfully been identified. Following on from the previous report, it was demonstrated that a number of the individual genes cloned from the virus genome were indeed recognised by the red squirrel and grey squirrel sera. At least two of these genes have subsequently been used to create stable cell lines expressing the corresponding proteins, should it prove necessary to raise anti-sera against these proteins in the future. The analyses of the immuno-dominant proteins have uncovered an interesting difference between red and grey squirrels. Side-by-side comparison of pooled grey squirrel sera and red squirrel sera on a 2-dimensional western blot of total virus proteins, showed that the grey squirrel sera blotted against more viral proteins than the red squirrel sera. Studies are ongoing to identify these proteins and to determine whether or not they represent quantitative or qualitative differences. Conclusion: Objective 6 is on schedule. Objective 7. Year 1 Q3 – Year 2 Q2. Clone 2/3 immunodominant genes into shuttle vector for transfer into MVA genome. The purpose of Objective 7 is to insert important immune-dominant genes identified under Objective 6 so that they can be expressed as a sub-unit vaccine approach by the Modified Vaccinia Virus (MVA). Individual immunodominant genes identified under Objective 6 have been cloned into general cloning vectors ready for transfer into the MVA shuttle vector. However we have delayed the actual cloning into the MVA shuttle vector to see if we can identify those proteins that are recognised by the pooled grey squirrel sera and not by the pooled red squirrel sera. These may represent better subunit vaccine candidates than proteins that we know are recognised by the red squirrel immune system. Conclusion: Objective 7 is on schedule Overall conclusions During discussion it was agreed that progress to date has again been good with no major delays to any of the agreed milestones. The finding that there may be a qualitative difference between the major immuno-dominant proteins recognised by grey squirrels in comparison to red squirrels is important and is worth following up, as it could have relevance to differences in the disease characteristics between the two species. Objectives 1 and 2 were completed on schedule and Objectives 3, 4, 6, and 7 are all being progressed on schedule. Work on Objective 5 is planned to begin as scheduled in the second quarter of this year. Thus all milestones are on schedule with no alterations to content or timing required at this stage. There are no plans to make major changes to the programme of work outlined in the original proposal. A R Peters January 2010 _______________________ Research News The first research project was carried out by the University of Liverpool to map the entire DNA structure of the squirrelpox virus. This will allow us to understand how the virus works and why it is more pathogenic in red squirrels than greys. Using this information the University is working on a more sensitive DNA test to identify the virus in all squirrels. Initial genetic results of the poxvirus have identified a unique molecule which hides the virus from the squirrel’s immune system. This work may have implications beyond the squirrelpox virus issue as it could have a bearing on the way virus infections affect human beings. The second is research by the Moredun Research Institute into developing a vaccine against the squirrelpox virus which is now seen as the greatest threat to the survival of the reds. This research began on 19th January 2009 and is expected to take 3 years to complete after which there will be a further 2 years of safety and efficacy trials. In June we received our first progress report which confirmed that the objectives of the research were on schedule against agreed timelines. These were; Objective 1 – Adapt isolate 1296/99 to growth in vitro – Obj 1 achieved Objective 2 – Isolate genomic DNA from virus isolate Sa/1984 and determine similarity to 1296/99 – Obj 2 is almost complete Objective 3 – Serial passage of isolates Sa/1984 and 1296/99 in vitro and stockpiling of defined virus stocks – Obj 3 has been achieved Objective 6 – Sequence appropriate regions of the squirrelpox virus genome containing the putative immunodominant genes – Obj 6 is on schedule A decision is to be taken shortly on whether to use an alternative poxvirus backbone e.g. Orf virus for the recombinant vaccine rather than the Modified Vaccinia Virus Ankera (MVA) as was originally planned. The report concludes that members of the research group are leaders in this scientific field and, whilst there is no guarantee of success, they stand a very good chance of achieving the end objective of developing an effective vaccine against squirrelpox. So, so far, so good.	Drawing by Phillip Allder
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