Since 1998, five serotypes of bluetongue virus (BTV) have invaded southern and central Europe, killing over 1.5 million sheep. The virus is transmitted by biting midges (Culicoides spp.) which are most active and abundant in warm climates. As a result of climate change, both vectors and virus have expanded progressively northwards and westwards within Europe. The live attenuated vaccines currently available (developed in South Africa) are not suitable for European breeds of sheep (causing disease, abortion, foetal malformation) and are not recommended for cattle or goats (alternative host species). The other potential vaccine candidates already available, include baculovirus-expressed virus like particles (VLP) and inactivated virus preparations, although questions remain concerning their relative cost, efficacy in the field or safety of inactivation processes. We will use established technologies to express BTV capsid proteins (VP2, VP5 and VP7) in bacteria and insect cells (recombinant baculovirus). The open reading frames of relevant genome segments (Seg-2, -6 and -7) of representative European BTV strains will be converted to full length cDNA using terminal primers. These will be cloned into IPTG-inducible T7 promoter plasmids and expressed as hexa-His-tagged fusion proteins, initially in the Origami E.coli strain (for optimal disulphide bond formation believed important for VP2). The His-tagged recombinant proteins will be purified using affinity chromatography on 'nickel' columns (AKTA purification system). The BTV cDNAs will also be inserted into i) the pFastBac vector (Invitrogen) to facilitate the generation of recombinant baculoviruses, ii) a recombinant vaccinia virus (Ankara strain - MVA), considered safe in humans and other animals (viral DNA vaccine) and iii) the high-yield transgene-producing plasmid gWIZ (Gene Therapy Systems) harbouring an optimised human cytomegalovirus (CMV) expression cassette and intopCI-neo ( Promega), (for non- viral DNA vaccine). The expressed fusion proteins will be purified using affinity chromatography on 'nickel' columns (AKTA purification system). The pCI-neo Vector contains the neomycin phosphotransferase gene, a selectable marker for mammalian cells and can be used for transient expression or for stable expression by selecting transfected cells with the antibiotic G-418. The BTV outer capsid and cell attachment protein VP2, is also the major neutralisation antigen. It can be cleaved into a number of polypeptides by proteinases, generating infectious subviral particles (ISVP). However, ISVP retain the ability to interact with neutralising antibodies and full infectivity for mammalian cells, but also have an enhanced infectivity (x1000) for Culicoides cells. VP2 cleavage products will be characterised by mass spectroscopy, to identify cleavage sites; recombinants will also be expressed and purified as described above, and used to study their interaction with neutralising antibodies. The expressed outer capsid proteins (VP2 and VP5), and the cleavage products of VP2 and VP7, will be used to raise an immune response, initially in mice. The project will analyse the contribution of each arm of the immune system in protection. We will incorporate recombinant BTV proteins and/or DNA vaccines into polymeric carrier systems to generate non-viral particulate vaccines. Controlled release of antigen or DNA, will be used to achieve prolonged exposure of the immune system to the vaccine, stimulating a secondary immunisation and, improving immunity. During these studies we will investigate the influence of specific polymeric carrier systems, and the effect of i) protein localisation on/in the polymer matrix on immune response bias and ii) formulation of protein and/or DNA-encoded antigens or co-administration of these and/or MVA vaccines.