Risultati finali
It is important to be able to distinguish between animals that have been vaccinated and those that have been infected with field strains of virus so that the effectiveness of the control campaign can be assessed and so that continuing presence of field strains can be monitored.
An ELISA, that detects antibodies to the BTV non-structural protein NS2, has now been developed that allows discrimination between animals vaccinated with most non-replicating �second generation� vaccines (i.e. VLP, CLP or purified protein-based) and those infected with or vaccinated with live virus. A disadvantage of the assay is that animals vaccinated with partially purified but inactivated vaccines (i.e. first generation inactivated vaccines), which may contain small amounts of NS2, can develop antibodies to this protein, which will confound the ELISA results.
A reference collection of BTV isolates has been established at IAH-Pirbright. Details of the viruses are available on the website:
www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/viruses-at-iah.htm
Vaccine strains of BTV 1, 2, 9 and 16 originally generated at OVI plus a Turkish vaccine strain are also in the collection. Sequences for genome segments 2, and 6 of these viruses have been determined see: www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/btv_sequences.htm
Sequence comparisons of genome segments 2 from multiple strains of BTV 1, 2, 4, 9 and 16 have been completed and phylogenetic trees, which indicate the likely geographic origins of the European BTV strains are shown on a project website at:
www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/orbivirus-phylogenetic-trees.htm
The databases also contain sequence analyses of S 10 for many Greek BTV isolates showing how they group into clusters. In addition, sequencing studies of genome segments 2 and 6 from strains of all 24 serotypes of BTV provide a database to support typing of all BTV isolates by sequence comparisons alone. Using such information primers have been designed to distinguish between genome segments from the live attenuated vaccines and European field strains and is listed at:
www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm
These databases will form a basis for future molecular epidemiological studies of BTV isolates and are freely available as an international resource.
During the course of this project a small laboratory stock of the European potential BTV vector, Culicoides nubeculosus, was upgraded to a maximum weekly production level of some 5,000 flies. This allowed several commercially available live vaccine viruses to be tested for ability to infect, replicate in and be transmitted by vector Culicoides species. It also allowed vaccine viruses, subsequent to their passage through vector Culicoides, to be tested for evidence of increasing virulence in sheep.
The existence of laboratory colonies of vector species of Culicoides allows experiments involving all aspects of virus-vector interactions to be carried out throughout the year (even in the winter) and in countries where wild vector populations are absent, difficult to acquire and manipulate, or unknown.
C. imicola is the major vector of BT V in southern Europe and Africa. Its breeding sites can now be broadly classified according to soil type, pH and carbon percentage. However, no correlation between any of these characteristics and abundance was detected. Moisture content and the speed with which a location dries are the controlling factors. It is suggested that C. imicola occupies temporary breeding sites that are peripatetic within the suitable areas according to moisture content and drying rate.
In relation to colonisation of C. imicola, a protocol has been established whereby adult midges can be collected from the wild and induced to blood feed in the laboratory. A larval breeding medium has been developed that has allowed larvae from eggs laid in the laboratory to be raised to pupae and then to adults. The adult midges deriving from these pupae have also been induced to blood feed in the laboratory and then to lay eggs but so far the numbers of colony midges have been insufficient to establish a permanent colony.
The existence of laboratory colonies of vector species of Culicoides allows experiments involving all aspects of virus-vector interactions to be carried out throughout the year (even in the winter) and in countries where wild vector populations are absent, difficult to acquire and manipulate, or unknown.
Live attenuated vaccine viruses have long been used to alleviate disease in sheep in various parts of the world and they are now being used in the face of BT V incursions into southern Europe. However, many authorities consider that there are a number of risks associated with the use of such vaccines.
In relation to these risks the work of this project has shown that:
1. Vaccine viruses themselves may cause clinical signs of disease in some breeds of sheep.
2. Vaccine viruses in vaccinates may elicit a viraemia of sufficient titre and duration to infect vector midges.
3. Vector midges can support vaccine virus replication to levels confirming transmission will take place.
4. There was no evidence of increasing virulence on vaccine virus passage through vector midges � but see 1.
5. �Vaccine-like� viruses can be isolated in the field suggesting that natural reassortants between vaccine and field strains may occur in dually infected hosts.
During the course of this project various carrier systems & adjuvant were investigated to generate immune responses to recombinant virus like particles (VLP) in a mouse model. In vitro serum neutralization antibody titres from the sera collected from guinea pigs immunized with purified BTV particles were also evaluated.
For the first time it was demonstrated that Oligodeoxy nucleotides (ODN) containing immuno- stimulatory CpG motifs either alone or with combinations of adjuvant or with carrier systems not only enhanced immune responses but also developed a mixed type (Th1/Th2) immune response to recombinant VLPs of BTV when delivered subcutaneous or nasally. A very strong synergistic effect was observed between alum & CpG. Adsorption/association of VLP & CpG to the positively charged microparticles & liposomal & dimethyldioctadecylammonium bromide (DDA) formulations have shown to further improve adjuvant activity.
Microencapsulated BTV-1 ISVPs along with alum co-encapsulated microspheres enhanced serum neutralization antibody responses compared to the group of guinea pigs that received BTV-1 ISVPs alone. Guinea pigs immunized with Montanide & non ionic block co-polymers or in combination with chitosan glutamate resulted in a serum neutralization antibody immune response similar to that obtained with Freud�s complete adjuvant (FCA), which is a significant finding as FCA is a potent adjuvant but limited in clinical application due to its unsuitability for use in humans. Alum in the presence of chitosan, enhanced the titre of serum neutralization antibodies produced.
We have utilized novel technology to create several new vectors. These vectors are designed to be used in the Baculovirus Expression System to create Virus Like Particles (VLPs) of BTV.
We generated a Baculovirus that expresses all 4 capsid proteins and self assembles to form a VLP within the insect cells. We have then used existing technologies and have purified these VLPs from insect cells. This preparation has the potential to be used as a vaccine for sheep.
The VLPs generated in this project have been shown to contain no genetic material and none of the other BTV proteins making them ideal as a basis for vaccines. This, along with the work of Professor Mertens, will allow us to differentiate our vaccine from all other vaccines on the market.
One of the concerns related to the use of live attenuated virus vaccines is that in dually infected hosts (vertebrate or insect vector) such vaccine viruses may reassort with field strains producing progeny viruses of uncertain virulence - though evidence of the occurrence of such natural reassortants in the field has not hitherto been discovered.
In South Africa during a search for such natural reassortants, 8 of 130 random isolates of BTV taken from the field were found to be �vaccine-like� on sequencing of genome segment 2. Seven of these isolates came from sheep and one from a bovine. As bovines are not vaccinated in South Africa this suggests that this infection with a �vaccine-like� virus may have been midge transmitted.
Strains of virulent bluetongue virus were isolated from the field.
Based on previous experience, the African Horse Sickness (AHS) vaccine production process has been taken as a reference to develop inactivated bluetongue vaccine production at industrial scale.
Merial developed a bluetongue virus serotype 2 (BTV2) and serotype 4 (BTV4) production process and analytical tools.
Efficacy and safety of these vaccines were verified by vaccination ¿ challenge studies.
Industrial bivalent vaccine BTV2 / BTV4 was produced at industrial scale and delivered to France, Italy, Spain and Portugal under temporary authorizations of use. Populations of ruminant have been vaccinated.
The development of vaccines to BTV serotypes 9 and 16 was started, to reach Merial's objective of including all the European serotypes to the range of vaccines available to fight the disease.
The inactivated vaccine is not only an effective, innocuous and easily tolerated vaccine it also meets the practical and economic demands of mass prevention. These are prerequisites for an efficacious strategy of control and prevention of the disease, which can lead to the eradication of the bluetongue in the southern European Union.
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