Development of novel vaccines to control infections with newly emerging highly pathogenic Marek's disease viruses
A fundamental prerequisite of the whole MADIVAC programme was the evaluation of the causes of Marek’s disease (MD) problems in Europe and to investigate whether novel more virulent MD virus (MDV) strains are circulating in Europe. About 65% of the samples tested were found to be positive for MDV and CIAV. Only samples which were negative for CIAV were used in the pathotyping experiments. Three of these samples exhibited a pathotype, which was defined as very hyper-virulent and certainly representing a novel class of MDV present within the European Union. These strains differ from those designated vv+ in the United States, because they are able to efficiently break Rispens (MDV-1) vaccination. In another aspect of this proposal, subunit vaccines were tested with regard to protection against in infections with hyper-virulent MDV. After the generation of a number of DNA and Sindbis virus vaccines and their testing in various cell culture systems, these constructs were used in animal experiments. The results of experiments applying DNA vaccines and Sindbis virus vaccines to prevent MD in vaccinated birds were very disappointing and no protection whatsoever could be observed. These results were especially discouraging, because preparations were used that contained viral proteins (e.g. glycoprotein B), for which good protection in other herpes viral systems had been shown. The reason for the malperformance of the subunit vaccines, however, became increasingly clear during the course of the project, when the efficacy of other vaccine preparations was tested. Importantly, the MADIVAC consortium succeeded in generating a bacterial artificial chromosome (BAC) clone of an attenuated vaccine MDV strain, which had been derived from a vv+ strain that had been attenuated by serial passage in chicken embryo cells. This BAC clone was used to: a) generate replication-competent and -deficient modified live vaccines and b) was used as a DNA vaccine. The results applying the BAC-based vaccines, be it modified live vaccines or BAC-based DNA vaccines, provided conclusive evidence that protection against MD in the domestic chicken - at least MD that is caused by novel hyper-virulent strains circulating in the European Union - requires the in vivo replication of the vaccine virus, because a replication-deficient virus was completely unable to induce any form of protection against subsequent challenge infection even under laboratory conditions. Therefore, any subunit vaccine designed to prevent MD - under the present circumstances - is bound to fail. This is one of the most important conclusions that can be drawn from this project. Another primary objective of the project was to further characterize key structures of the chicken immune system and to investigate their roles in Marek’s Disease. A focus was put on the role of cytokines in MD infection and their application as vaccine adjuvant. Using different approaches we were able to clone chicken IL-6, IL-18 and BAFF as new cytokines. Bioassays were established for each of these cytokines and neutralising reagents were developed. In addition, quantitative PCR assays (TaqMan) and RNase protection assays were established for these and most other chicken cytokines cloned in previous years. Using the new tools, the functional characteristics of these cytokines were intensively studied in vitro and in vivo. IL-6 production was not induced by MDV (RB1B, CVI 988, EU1) infection in vitro and in vivo. However, IL-6 was strongly induced by LPS, CpG motives and by dsRNA in vitro and by LPS in vivo. These and other data strongly support the view that chickens have developed a fully functional Toll like receptor (TLR) system to sense pathogen associated molecular patterns of a large number of pathogens. Interestingly, MDV does not seem to be a potent inducer of this early immune response.