Correlation of immunogenicity with microarray analysis of vector mutants to improve live recombinant poxvirus vaccines in poultry

How can we improve the development of better new vaccines to protect poultry (and other livestock) against major disease threats such as bird flu? Genetic manipulation (GM) is having an increasing beneficial impact on our lives, particularly in human and veterinary health care; nowhere more so than in vaccines, where many commercial products have been licensed and released for use in livestock and companion animals. These new vaccines are based on 'vectors', which can be regarded as carriers for the target vaccine, and are generally based on well-understood vaccines, such as poxviruses, with a long history of safe use against important diseases. The best-known example is Vaccinia virus, used in the only successful global eradication of a virus disease, Smallpox, and as a recombinant in the elimination of feral fox rabies from Belgium and France. Fowlpox virus vaccination since the 1920s has effectively eliminated fowlpox from poultry in developed countries in temperate climates. Spread by biting insects, it remains a major problem in tropical and sub-tropical countries where vaccination of chicks in hatcheries is common and extensive. Using GM, we can incorporate into the 'genome' (or chromosome) of the vector, a gene from a different disease-causing virus (or pathogen), such as bird flu H5N1, making a 'recombinant vector'. When that gene carries the instructions to make a structural protein of the pathogen, vaccination with the recombinant vector will induce an immune response in the vaccinated animal against the pathogen (and vector). Recombinant poxviruses have been licensed for veterinary use against West Nile fever, canine distemper, feral rabies and equine influenza. The most extensively used is a commercial recombinant fowlpox vector incorporating the H5 surface spike of bird flu. Two billion doses have been used to vaccinate poultry against H5 bird flu in Mexico since '95. There, the lethal form of bird flu was eradicated but a less dangerous form remained in circulation. The recombinant vaccine reduces shedding and transmission of bird flu but does not completely prevent infection of birds, possibly driving evolution of the virus by random mutation. There, therefore, remains considerable scope for improvement, particularly in terms of immunity that will clear birds of infection. The vectors are not just inert delivery systems. Poxviruses activate the immune system and have to survive in the presence of the host's immune response. To do so, the vector deploys tens of different gene products. Some of these will reduce the effectiveness of the vector as a recombinant vaccine. To improve the response we can use GM to remove such genes from the vector but, with so many candidates, our problem is identifying those which should be removed. Currently the only way to see if the vaccine has been improved is to test it in animals. We propose to look in detail our panel of fowlpox virus mutants, each defective in just 1 of the 250 genes of the vector. When the vector enters a host cell, it turns up (or down) the production of protein from about 1000 of the host's 30000 genes. We will look to see how the different mutations affect the control of these host genes by the vector virus, using the microarray technique (performed in tissue culture dishes in the laboratory). In this study, we will also need to see how each mutation affects the ability of the recombinant vector to induce an immune response (against structural proteins of H5N1 in chickens). We will then look for correlation between improved immune responses to the recombinant vector and changes in control of the host genes by the vector. This should then give us a profile, or a fingerprint, of gene control that we can associate with improved vaccines. In future, we would look for this profile in the laboratory as a first step. This will give us a way of predicting which new vaccines are likely to be improved, before testing them in animals.

The H5N1 panzootic emphasizes the need for effective vaccines, including recombinant vaccines, to combat this widespread and changing disease. Notable amongst recombinant vaccines is Fowlpox virus (FWPV) expressing avian influenza (AI) H5, with 2 billion doses used in Mexico ('95-06) against H5N2. The existing commercial vaccine does not induce sterilising immunity and reduces, but does not prevent, shedding or transmission. There remains considerable scope for improvement, particularly in terms of cell-mediated immunity. While this could be achieved by co-expression of host-derived immunomodulators there are doubts about the sustainability of such an approach for widespread use in live vectors. A safer and more acceptable alternative would be to delete ('knock-out') from the vectors genes that have an adverse affect on immunogenicity. It is the aim of this project to harness the power of genome-wide functional approaches (at the level of both virus and host) to develop our ability to predict which gene-knockouts will improve vector immunogenicity. A panel of 65 FWPV knockout mutants will be subjected to microarray analysis to identify changes in global infected cell expression attributable to each mutation. Expression data will be analysed to group mutants and to identify genes, groups of genes and pathways with differentially altered expression. About 50 of the mutants that induce significant relevant changes in expression patterns in infected cells will be chosen for immunogenicity studies in vivo, using an AI antigen model to assess induction of humoral and cellular immunity as well as induction of cytokine and chemokine responses. The two data sets will be compared to identify changes in host gene expression, attributable to individual FWPV gene knockouts, which correlate with changes in immunogenicity. The profile generated could be used in future to predict beneficial changes to vectors following expression analysis and before in vivo testing.

IMPACT PLAN Beneficiaries of this research would be: Commercial vaccine producers. See letter of support for this project from Prof J W Almond, VP Discovery Research & External R&D at Sanofi Pasteur (one of the world's largest vaccine manufacturers). The work, though directly relevant to producers of poultry vaccines will have ramifications for the vaccine industry in mammalian livestock, companion animals and even human sectors. Benefits of the work, if it leads (directly or indirectly) to improved recombinant vaccines will be across the board: increased food security, wealth creation (vaccine producers, poultry producers), quality-of-life (reducing risk of pandemic flu, protecting supply of poultry meat and eggs - a key source of nutrition worldwide, protecting from infection). Poultry Farmers. Farmers will benefit from access to improved recombinant vaccines. Initially this will be via relatively high technology commercial producers. However, propagation of fowlpox vaccines can involve relatively low technology, as evidenced by the proliferation of small veterinary vaccine producers that produced conventional fowlpox vaccines on the 'garage scale' in the mid 1900's. Such technology could therefore become accessible to more marginal poultry production in developing countries (fowlpox itself is second only to Newcastle disease as a problematic virus to back-yard poultry in Africa). Those involved in promoting Public Perception of Science. The results will offer positive indications of efforts being made to make specific recombinant GM vaccines that are even more useful, and safer, than the first generation products that are now being licensed for wider use. BBSRC (see below). Results will be made accessible via conferences, standard publication, data repositories (for MIAMI-compliant data) and websites (as well as more specialist trade publications). MAS and CB both have experience in presentations to the media (TV interviews on Avian Influenza). MAS has experience in Public Perception of Science, in the agricultural sector (farm walks, agricultural shows), in schools and in Cafés Scientifique (Science Oxford). The work, which has a good chance of generating IP, is not funded by any beneficiary but we have good links with industry via IAH's poultry sector contacts and via MAS's contacts with broader vaccine sectors (as evidenced by recent commercial funding on basic aspects of recombinant FWPV vector performance, which led to a publication - Cottingham, 2006). MAS and PK are experienced in the patent process in the vaccine and immunomodulator sectors. IP protection and any commercialisation would be via Imperial Innovations, which combines the activities of technology transfer, company incubation and investment, bringing valuable ideas to market either by building businesses or licensing to industry. Innovations has recently sold Thiakis, an ICL-derived spin-out, to Wyeth. Innovations already advise on and handle MTAs on fowlpox technology for MAS, on behalf of ICL and IAH. BBSRC. As well as addressing the developing priority of 'Food Security', the project is a clear example of the goal in BBSRC's '04 'Bioscience for Society: A ten-Year Vision' document subtitled 'Towards predictive biology'. In using microarrays to predict improved vaccines it fits squarely with the intentions of the BBSRC Delivery Plan '08-'11 to harness systems biology to predictive biology. The project also aligns with the BBSRC's current Strategic Plan ('03-'08 update '05), meeting all 6 strategic objectives: + 'Excellent Science' - 'Understanding Diseases' and 'Innovative Agricultural Practices', 'Pathogenesis'. 'Food Safety' in 'Bioscience for Industry' + 'Tools and Technology' and 'People' - genetic modification; strengthens capacity in influenza research, vaccinology and recombinant vector technology (in which IAH/BBSRC has an IP interest) + 'Knowledge Transfer' + 'Partnership' + 'Effective Organisation'