Expression and formulation of Bluetongue virus genes and proteins for development of effective vaccination strategies

Bluetongue virus (BTV) can infect all ruminants, including cows, sheep and goats, in most cases without causing disease. However, when the virus infects sheep, particularly the European breeds, it can cause severe disease, killing up to 70% of the infected animals. The virus is transmitted by biting midges (Culicoides species), particularly those species which prefer warmer climates. As a result of recent changes in the European climate that are thought to have been caused by global warming, midges that can transmit BTV are moving northwards. This places Europe at much greater risk from the disease than before. Indeed, since 1998, six different strains of BTV have invaded the Mediterranean region, on a total of seven different occassions, affecting all of the countries of Southern Europe and collectively causing the largest outbreak of the disease ever recorded (with over 1.5 million dead animals). The vaccines that are currently available to fight bluetongue are live strains of the bluetongue virus that have been weakened (attenuated) by growth for many generations in cell cultures. These are supposed to produce only mild symptoms but protect the animal from more dangerous virus strains. However, there is evidence that these vaccine strains are also dangerous, causing disease and being transmitted by the same biting midges as the wild type viruses. Alternative inactivated vaccines have recently become available, based on partially purified and chemically treated virus, although the nature of the response to these vaccines (protective or not) still has to be tested in the field. The bluetongue virus particle has an outer protein layer composed of two proteins, VP2 and VP5, and a core, with a surface composed of VP7, proteins which are recognised by the immune system of infected animals, generating a reponse that can protect against the disease. The project will make DNA copies of viral genes and use them to synthesise viral proteins in bacteria, or in insect cells. The proteins will then be purified and used to study their interactions with the animal's immune system, particularly the production of a protective response e.g. antibodies that can neutralise the virus. The project will also examine the structure of the outer surface proteins of BTV particles, to see which aminoacid sequences react with the immune system and lead to a neutralising response. The project will use DNA copies of the viral genes directly, or copied into another virus (vaccinia virus - MVA), to synthesise the BTV proteins in vaccinated animals and thus ativating their immune system. By incorporating the synthesised and purified viral proteins, DNAs or MVA into microscopic synthetic beads that breakdown over time in a controlled way. This should prolong the exposure of the animals to the vaccine, allowing more time to generate a higher level of protection against the virus, and therefore better protection against the disease. These new vaccination strategies are considered to be entirely safe, since they do not use live BTV to induce immunity/protection. They will therefore be used in different combinations to see which is most effective, and to design a better (and safer) vaccine. Because of the promise shown by these new types of vaccines, we will use this opportunity to study in more detail how they work. We want to find out if the position of the proteins on or in the beads affects the way the immune system responds to them. At the same time, we want to study which gives the best response : the use of each component on a single bead system, or beads containg different proteins and/or DNAs. Our ultimate goal being a vaccine that is 100% efficient after a single dose.

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