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ReoID Phylogenetic sequence analysis and improved diagnostic assay systems for viruses of the family Reoviridae
Edited by Peter. P. C. Mertens and Houssam Attoui |
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Development of a safe, efficacious bluetongue virus vaccination strategy for Europe TECHNICAL ANNEX:
QUALITY OF LIFE AND MANAGEMENT OF LIVING RESOURCES
FULL TITLE: Development of a safe, efficacious bluetongue virus vaccination strategy for Europe
ACRONYM: Bluetongue vaccination
KEY ACTION No AND TITLE: 1999/C361/06
CONTRACT NO: QLRT-2000-01722
TABLE OF CONTENTS
1. OBJECTIVES AND EXPECTED ACHIEVEMENTS 3
Work Package 6. To develop molecular epidemiological methodologies to enable 14
3. ROLE OF PARTICIPANTS 21
1. OBJECTIVES AND EXPECTED ACHIEVEMENTS Outbreaks of bluetongue (BT) have occurred several times in Europe in the past, resulting in over 200,000 dead sheep. In 1998-2001, BT is once again causing severe animal health and trade problems in much of southern Europe and the current outbreaks have already resulted the deaths of over 250,000 sheep. Throughout the current series of outbreaks the affected countries have attempted to control and eradicate the virus by restriction of animal movements and other traditional zoosanitary measures. Sadly this has not been sufficient to halt its spread.
BT is exceptionally difficult to control because it is transmitted by certain vector species of Culicoides biting midge. These are able to travel long distances on the wind so that appearance of the virus is difficult to predict and the origins of incursions difficult to identify. Furthermore, the major European vector species of Culicoides (C. imicola, and possibly species in the C. obsoletus and/or C. pulicaris gps.) have not been colonised so their respective efficiencies in transmitting the virus are unknown, and appropriate vector control measures yet to be devised. In this project methodologies will be devised and sequence data acquired, to enable backtracking of virus incursions to source (Objective 7). Attempts will be also be made to colonise European vector species of Culicoides and to test live vaccine viruses for possible reversion to virulence, subsequent to insect passage (Objective 2).
In the control of most infectious diseases vaccination is a vital element. However, in the case of BT in Europe this is an issue that is fraught with difficulties. The existing commercially available BTV vaccines are all live attenuated preparations that cause a viraemia in a proportion of vaccinated animals. Their use is associated with many concerns and limitations so that many countries prohibit their use. One major concern is that vaccine viruses may re-assort with wild-type viruses in the field leading to the development of new strains of virus with different or possibly enhanced virulence characteristics. A further major concern is that vector species of Culicoides may ingest vaccine viruses from vaccinated animals and after reversion to virulence on passage through the vector may transmit it in the field thus spreading disease. Scientific objectives 1 and 2 of this project address both of these important areas of concern.
The best long-term vaccination strategy for Europe would be to develop and use new inactivated whole virus or sub-unit vaccines that are inherently safe and confer enhanced protection to the target species of livestock. A further advantage in the use of non-replicating vaccines is that it is possible to develop tests to differentiate between vaccinates and naturally infected animals thus enabling the detection of field virus infections during the course of a vaccine campaign. Such candidate vaccines and tests will be developed by completing Objectives 3 - 6 and the most "promising" vaccines will be considered for commercial production. Consequently, the project will address all of the above areas by completing the following objectives:
2. PROJECT WORKPLAN
2.1 Introduction With the continuing spread of BTV across southern Europe and North Africa it is evident that application of traditional zoosanitary measures over the past 3-4 years has failed to contain the virus incursions, much less eliminate them. Clearly the absence of a safe and effective vaccination policy for Europe is one of the major causes of this failure. Even when the present series of BTV outbreaks is finally brought under control, the EU will remain under threat from future incursions of the virus. With the advent of global warming, which is increasing the range and seasonal incidence of the Culicoides vectors, accession of new Member States and enhanced animal movements into and within the Union, the risk of BTV incursions and their magnitude is likely to increase, year by year. What is needed for Europe is a series of BTV vaccines that are licenced for use here, are efficacious in a European context and are demonstrably safe. In order to fulfil these requirements this project will adopt three major approaches. Firstly, certain aspects of concern in the use of the existing commercially available BTV vaccines will be tested to assess the likelihood of reassortment with co-infecting field viruses and to determine whether passage through European vectors could result in the spread of disease. Secondly, the project will seek to develop and then test inactivated BTV vaccines. This will involve the production of inactivated whole viruses, synthetic virus like particles (VLPs) and recombinant sub-unit vaccines, and also the development of nucleic acid based vaccination methods, as well as novel (slow release) delivery systems and possibly selected adjuvants, to produce vaccines that are inherently safe and provide enhanced protection. The benefits accruing from this area of work will take significantly longer to reach fruition than those from the first approach but it is intended, as an integral and natural extension of project findings, that the best candidate vaccine(s) will be considered for commercial production. This intention is reflected by inclusion of Partner 3 in the project, who has a long and successful track record of inactivated-orbivirus vaccine production. As part of this work it is also intended to develop test(s) based upon one or more of the BTV non-structural proteins to enable differentiation between animals vaccinated with the new non-replicating vaccines, and those infected with live virus (field and/or live vaccine). Antibodies to the BTV non-structural proteins only develop in animals infected with live virus.
Thirdly, a database of BTV sequences will be set up using existing virus stocks of Partners 1 and 2, and other BTVs acquired during the course of project work. This will provide a comprehensive resource facilitating differentiation between vaccine viruses and field strains (ie. to enable the detection of vaccine breakdown), and for identifying the source of BTV incursions.
In addition to the three major approaches attempts will also be made to colonise one or more of the European vector species of Culicoides to facilitate the studies into the potential transmission of vaccine viruses. Prior to the establishment of such colonies "wild-caught" specimens will be used, with the known vector C. variipennis being available as a positive control.
The scientific objectives of the project, as stated in Section 1 (Objectives and Achievements) will be achieved via the following 6 Workpackages:
2.3 Project structure, planning and timetable List of participants Partner 1, Contact persons: Dr PS Mellor, Dr PPC Mertens The Institute for Animal Health, Pirbright Laboratory, Ash Rd., Pirbright, Woking, Surrey, GU24 0NF, UK. Tel.: +(44-1483) 232 441 Fax: +(44-1483) 232 448 Partner 2 Contact person: Dr J Paweska, Onderstepoort Veterinary Institute, PO Box X05, Old Soutpan Rd., Onderstepoort, South Africa. Tel: +(27-12) 5299 111 Fax: +(27-12) 5656 573 Partner 3 Contact person: Dr M Lombard, MERIAL Grande Prophylaxis Enterprise, 29, Avenue Tony Garnier, 69007 Lyon, France. Tel: +(33-4) 7272 3037 Fax: +(33-4) 7272 3181
Partner 4 Contact person: Dr D. Panagiotatos, Ministry of Agriculture, Directorate General of Veterinary Services, 2, Acharnon St., 10176 Athens, Greece. Tel: +(30-1) 883 5420 Fax: +(30-1) 822 9188 Partner 5 Partner 6 Contact person: Contact person: Prof P Roy, Prof HO Alpar, Pathogen Molecular Biology & Biochemistry Unit, Centre for Drug Delivery Research, Dept. of Infectious & Tropical Diseases, London School of Pharmacy, London School of Hygiene & Tropical Medicine, 29-39 Brunswick Square, Keppel Street, London, WC1E 7HT, UK. London, WC1N 1AX, UK. Tel: +(44-1865) 281 640 Tel: +(44-207) 753 5928 Fax: +(44-1865) 281 696 Fax: +(44-207) 837 5942
Table 1: Workpackage list
Partner 1 Dr PS Mellor/Dr PPC Mertens, The Institute for Animal Health, Pirbright Laboratory, Ash Rd., Woking, Surrey, GU24 0NF, UK E-mail philip.mellor@bbsrc.ac.uk peter.mertens@bbsrc.ac.uk Tel: +(44-1483) 232 441 Fax: +(44-1483) 232 448
Contractual links Partner 6 is an assistant contractor to partner 1.
Objectives The coordinator, partner 1, is directly involved in all of the workpackages except the detailed commercial procedures devised by partner 3 for the development, validation and production of whole particle inactivated vaccines, and will ensure that the overall project work plan is followed and the objectives achieved. Partner 1 has extensive experience in managing international projects, with identifying, colonising and infecting vector species of Culicoides, and with a wide range of molecular technologies appertaining to BTV including, virus growth and purification, reagent production, cloning and sequencing, and establishment of sequence databases to ascertain relatedness between virus isolates. All of these areas of expertise will be used to ensure the development and selection of the most appropriate BTV vaccines for Europe. Partner 1 will also seek to ensure that the most appropriate vaccines are selected for commercial production and are made available to European farmers and veterinary authorities as a primary line of defence in future BTV incursions. In addition, partner 1 will take the leading part in establishing the BTV RNA database to enable backtracking of BTV incursions into Europe and to facilitate the detection of (live) virus vaccine breakdowns.
Workplan
Partner 2 Dr J Paweska, Onderstepoort Veterinary Institute, PO Box X05, 0110 Onderstepoort, South Africa. E-mail janusz@moon.ovi.ac.za Tel: +(27-12) 5299 111 Fax: +(27-12) 5656 573
Contractual links None
Objectives Partner 2 will manufacture and supply specific attenuated vaccine viruses for use in the work described in Workpackages 2 and 3 and for use in comparative testing of these and novel BTV vaccines throughout the 3 years of the project. In addition, partner 2 will lead the colonisation attempts of potential European vector species of Culicoides concentrating in the major confirmed vector, C. imicola. Partner 2 will also assist in generating cDNA clones and providing inactivated antigens for use as reagents in the development of novel vaccine formulations, and will conduct sequence analyses in order to help establish a database.
Workplan
Partner 3 Dr M Lombard, MERIAL Grande Prophylaxis Enterprise, 29, Avenue Tony Garnier, 69007 Lyon, France. E-mail michel.lombard@merial.com Tel: +(33-4) 7272 3037 Fax: +(33-4) 7272 3181
Contractual links None
Objectives Partner 3 will establish a collection of European BTV serotypes and strains using isolates acquired by partners 1 and 4. Suitable vaccines strains will be selected and Master Seed Vaccine strains prepared. Parameters for BTV growth in cell culture, for inactivation and for concentration and purification will then be elucidated and measured. Potency and safety tests in the target species of animals will then be established and the dose-effect relationship determined, to select the antigen payload according to virus type and adjuvant. All vaccines will be tested for efficacy and duration of protection in the target animals in comparison with existing live virus vaccines and novel sub-unit vaccines. Finally, ability to differentiate between live virus infections and inactivated virus vaccination will be assessed by testing for the presence of BTV non-structural antibodies in the sera of vaccinates.
Workplan
Partner 4 Dr D. Panagiotatos, Ministry of Agriculture, Directorate General of Veterinary Services, 2, Acharnon St., 10176 Athens, Greece. E-mail vetserv@ath.forthnet.gr Tel: +(30-1) 883 5420 Fax: +(30-1) 822 9188
Contractual links None
Objectives Partner 4 will collect and supply BTV isolates made on its territory to other partners so that appropriate novel inactivated vaccines can be formulated and produced. Partner 4 will then test the efficacy of the inactivated vaccines and of the recombinant expressed vaccines in local breeds of sheep and possibly in cattle and goats. The antibody responses will be evaluated in comparison with similar work, plus challenge, to be carried out by partners 1, 2 and 3. Partner 4 will also assist in establishing the BTV sequence database and will participate in the testing and validation of the ELISA to distinguish between naturally infected animals and animals vaccinated with non-replicating vaccines.
Workplan
Partner 5 Prof P Roy, Pathogen Molecular Biology & Biochemistry Unit, Dept. of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine,, Keppel Street, London, WC1E 7HT, UK, E-mail polly.roy@lshtm.ac.uk Tel: +(44-1865) 281 640 Fax: +(44-1865) 281 696
Contractual links None
Objectives Partner 5 has pioneered the development of baculovirus expression systems as used in the production of VLPs and CLPs of BTV. The main task of this partner will be: 1) To prepare cDNA of the relevant gene segments of various European serotypes. 2) To manipulate each DNA such that they can be used to make recombinant viruses. 3) To isolate recombinant baculovirus and to generate VLPs and CLPs. This partner will also be responsible for the production of sufficient amounts of VLPs and CLPs, which will be supplied to other partners, for the formulation of vaccines based upon them and for comparative testing of these vaccines with live virus vaccines and other inactivated vaccines. Partner 5, in company with other partners will then assist in drawing up recommendations for vaccine selection and use. In addition, partner 5 will assist in developing the BTV RNA database to enable backtracking of virus incursions and (live) virus breakdowns.
Workplan
Partner 6 Prof HO Alpar, Head of Centre for Drug Delivery Research, The School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK. E-mail: oya.alpar@ams1.ulsop.ac.uk Tel: +(44-207) 753 5928 Fax: +(44-207) 837 5942 Contractual links Partner 6 is an assistant contractor to Partner 1 Objectives Partner 6 will provide slow release delivery systems (possibly including selected adjuvants) specifically for use with and to enhance the efficacy of the non-replicating BTV vaccines (based upon whole virus particles, VLPs, CLPs, and expressed proteins). Partner 6 will also participate in the experimental design of the comparative testing studies of the various live, inactivated and sub unit vaccines, and in the analysis of the results and in the formulation of recommendations to the Commission.
Workplan
4. PROJECT MANAGEMENT AND COORDINATION
The project coordinator will be responsible for the overall project management, will issue calls for meetings, will be responsible for ensuring achievement of all tasks and with the assistance of the other participants will prepare and produce all reports. A project management team, usually consisting of the team leaders of each partner will be formed that will be responsible for achievement of all Work Packages and project aims. At the commencement of the project a meeting will be held to discuss the first year work plan in detail and to ensure that all partners are aware of their part in it. Additional project meetings will be held every 12 months to coordinate the organisation of the work, to exchange results, evaluate the work carried out and to prepare the reports. Visits between partners will be actively promoted to provide access for all groups to the different areas of expertise and to ensure the exchange of technology and data. Figure 1 shows the relationships between the partners involved in this project. The major communication flows will be between the coordinator and each of the partners. However, those partners directly involved in the development and production of the inactivated vaccines (partners 3, 5 and 6) will be expected to interact together during the course of this work both formally and informally to ensure that their efforts are complementary. Similarly, partners involved in testing candidate inactivated vaccines on animals (partners 2 and 4) will also be expected to liaise with each other and with the vaccine producers (partners 3 and 4) to ensure that their testing protocols are comparable and that vaccine production is co-ordinated with animal availability. Details of the relationships between the various Work Packages comprising the work of the project and the responsibilities of each of the partners in respect of these is set out in Figure 2. The time spans for each of the Work Packages, broken down into Tasks, together with the number and the times of the various coordination and other meetings that will be necessary for the efficient management of the project have been set out in Figure 3. Timing of the annual and other reports, and of the milestones is also shown in Figure 3. In some circumstances the Commission may request to perform a mid-term review of the contract. In this case a Mid-Term Review Meeting, organised by the co-ordinator, will take place between representatives of all the participants and the Commission. The Commission may be represented by independent experts who shall be subject to confidentiality agreements. The meeting will take place between the 18th and the 24th months of the project. The co-ordinator shall agree with the Commission the date, the agenda and the participants of the meeting at least two months in advance of the meeting. At the meeting progress in each of the completed and of all ‘under-way’ actions will be assessed by delegates from each involved partner in the light of relevant milestones. The track record of each partner is respect of their individual areas of responsibility will also be assessed. Shortfalls in achievement will be examined in detail to determine cause. Joint project meetings with other EU-funded and work-related projects (eg. Arbovirus vectors and disease QLK2-CT-2000-00611, Phylogenetic sequence analysis and improved diagnostic assay systems for viruses of the family Reoviridae QLRT-1999-30143) may be held at intervals to ensure that maximum value is extracted from all findings.
5. EXPLOITATION AND DISSEMINATION ACTIVITIES
The findings of this proposal will be forwarded to the Commission in the form of annual progress reports and a final report drawing together all of the main conclusions and results. As appropriate the findings will also be presented at national and international scientific meetings, trade conventions, via the media and as scientific publications or popular articles. During the course of the project further dissemination on the progress of the work will be encouraged by contribution to such of the Commission’s own organs of publication as may be deemed appropriate to ensure that findings of likely practical benefit reach the end users (farmers, national veterinary services, legislators, government departments, international animal health organisations) with the minimum of delay.
The Commission will also be presented with clear science-based recommendations concerning the efficacy of the existing live virus vaccines in cattle and goats. A sequence database of the BTV types and strains circulating in the European theatre will also be established and will be made available to the Commission, the partners and other organisations concerned with controlling this disease. In addition, details of the best candidate inactivated BTV vaccines, including assessments of their suitability for commercial production will also be supplied to the Commission. Should commercial production of one or more of the candidate vaccines be shown to be a viable option then it is the intention of the partners to exploit this opportunity. The partners may adopt a consortium policy on the sharing of any benefits that derive from the work of the project.
Contractors shall through the co-ordinator also submit at, or before, the end of the project a technology implementation plan acceptable to the Commission. This plan should indicate all potential foreground rights and exploitation intentions (including a timetable) taking into account Community policies, including those for transfer of technology to SMEs, and promoting the use of generic technology.
Not later than the first report, the co-ordinator shall provide to the Commission a publishable poster targeted to a non specialist audience and summarising the main features of the entire project. In addition, the co-ordinator shall provide, by contract signature, a one-page publishable summary of the project which can be easily disseminated and distributed to the public.
6. ETHICAL ASPECTS AND SAFETY PROVISIONS
The work of this project is designed specifically to alleviate or prevent an OIE List A disease in European and other livestock by enabling the source of incursions to be determined, by investigating aspects of the efficacy of existing vaccines and by developing where possible, improved vaccines. It will thereby significantly promote animal health and welfare. In the context of vaccine development and testing some animal usage will be essential since no other method has been devised that will provide the necessary safety and efficacy data. Nevertheless, throughout this work the number of animals used will be the minimum that is consistent with acquiring reliable results and the earliest (humane) end points necessary to confirm the performance of the candidate vaccines will be selected. Throughout the work, the cost benefit equation will be applied to each and every experiment and all experimental animals will be placed under the direct supervision of experienced veterinary surgeons. All animal work to be carried out in the UK will be performed under the British Government’s Animal Scientific Procedures Act (ASPA) which is internationally recognized as being the strictest legislation of its type in the world. The use of animals in Greece and South Africa will similarly be undertaken under existing national legislation. The use of animals in the UK, in the ways described in the project document, has already been authorized by the ethical review process of Partner 1 and will be carried out under existing Project Licences (PPL 70/5028, PPL 70/5029, PPL 70/4329, PPL 70/4733) with amendments where necessary. In this context, the co-ordinator shall implement the research project in full respect of the legal and ethical national requirements and code of practice. Wherever authorisations have to be obtained from national bodies, these authorisations shall be considered as documents relevant to the project under Article 27 of Annex II of the contract. Copies of all relevant authorisations shall be submitted to the Commission prior to commencement of the relevant part of the research project. This project does not involve work on modification of the human genome, cloning of individuals or any form of transgenic animal work. This project does not involve the release into the environment of genetically modified organisms. The live viral agents that may be used in Europe during the course of this project will be conveyed to the laboratories of Partner 1 under MAFF approved procedures, and under the authority of the British Government’s Animal Health Act (Importation of Animal Pathogens Order 1980, Licence No. AHZ/1309/A) or under similar appropriate national government regulations. All envisaged virological work to be carried out in the high security laboratories of Partner 1 is covered by the MAFF Specified pathogens Order 1998. In relation to safety, the co-ordinator shall take all measures to assure that appropriate environmental safety provisions are fulfilled in the course of the project by all contractors, particularly those related to the deliberate release into the environment of genetically modified organisms. In addition the co-ordinator shall take all measures to assure for all contractors that, when dealing with biological material, strict safety procedures are in place in compliance with national and EU regulations on biosafety. All work must be carried out in compliance with national and EU regulations on safety.
7. ONGOING EC FUNDED PROJECTS
The present project is designed to provide information on aspects of the efficacy of existing live BTV vaccines and to develop novel, inactivated virus vaccines of equal or improved efficacy in order to provide, for the first time, a coherent BTV vaccination policy for the EU. It is also intended to establish a database of BTV sequences in order to enable backtracking of virus incursions into the EU. As such this project is a unique concept. However, the co-ordinator of the project (Partner 1 – IAH) has an established and successful track record of co-ordinating EU-funded projects dealing with a variety of aspects of BT and related orbiviruses. These are as follows:
The African horse sickness epidemic in Morocco:
Arboviruses in southern Africa
BT and other Culicoides-borne disease threatening the EU: Identification of vulnerable areas by surveillance & GIS modelling to aid risk assessment.
family Reoviridae.
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