Immunity to FMDV following combined DNA and inactivated virus antigen.

Despite being clinically protected from FMD vaccinated ruminants replicate virus in the upper respiratory tract and become persistently infected carriers with consequential impact on control measures. However, evidence supports a relationship between vaccine potency, magnitude of response, and incidence of virus replication in the oropharynx. It has been conclusively shown that higher antigen payload vaccines are capable of inhibiting local virus replication, persistence and the carrier state. The effector mechanisms behind this are unclear. Antibody mediated protection is a possibility since increasing the antigen payload promotes a more rapid and greater systemic response. However, the magnitude of this inevitably reaches a ceiling where additional payload shows little beneficial affect, and there are many observations where persistence occurs in the face of high antibody titre. Local antibody may influence persistence, but there is little evidence to support this, even with increased payload, being promoted or even eliciting, such a response. It is therefore extremely likely that other factors play a major role, like cell mediated and innate responses. High potency FMD vaccines are capable of eliciting durable systemic cytokine responses in pigs, such as IL-6, IL-8 and IL-12 , which is indicative of monocytic cell activity. This is an important part of an innate defence that contributes to early protection of the host and absence of persistence. This has led to a similar focus in the ruminant. IFN gamma is a potent inhibitor of FMD virus in persistently infected bovine epithelial cells in-vitro and alpha and beta interferons also inhibit FMDV replication. IFN gamma can be increasingly stimulated systemically in FMD vaccinated sheep, by augmented payload, resulting in no persistence following challenge. Thus, a vaccination regime which consistently enhances a diverse repertoire of cellular and humoral responses, over and above those afforded by traditional FMD vaccines, should have direct beneficial attributes to the inhibition of persistence and the carrier state. This could have major benefit both endemically and in emergency use. The proposal will use DNA vaccine, to examine these hypotheses. An FMD DNA vaccine encoding the 'empty capsid' along with the non-structural proteins 2A, 3C and 3D has been used with an adjuvant plasmid expressing granulocyte macrophage colony stimulating factor (GM-CSF), which induced extremely strong humoral and cellular responses and conferred protection in pigs and sheep. Further optimisation amplified the specific and important neutralising antibody response further, and resulted in cellular immune responses, detected as T cell proliferation. This was more dramatically enhanced when FMD inactivated homologous virus antigen and 3D recombinant protein was used as a final boost and the production of IFN-?, IFN-a, IL-2, 4, 6, 8, and 10 confirmed both innate and specific immune responses are activated. Indeed DNA vaccines are now considered to be an excellent tool for priming an extensive immune repertoire as part of a prime boost strategy and there are many examples where this has been successful in hosts such as cattle, goats and horses. Use of this strategy against bovine herpesvirus-1 led to enhanced and protective cellular and humoral immunity and a significant reduction in virus shedding. Recently, a two dose regime of DNA prime- protein boost or vice versa, 14 day apart, was sufficient in stimulating enhanced immunity against FMDV in cattle. There is therefore potential for shorter intervals between prime and boost and in examining simultaneous vaccination of DNA with the protein antigen for emergency purpose. The ability of DNA vaccination regimes to augment immune responses will be exploited to provide a more comprehensive understanding of those important to protection which can be utilized or better promoted by newer vaccines to prevent persistence and the carrier state.

EU recognises vaccination as a principle means for FMD control, despite its inefficiencies including lack of a strong cellular response. DNA vaccines, in contrast, elicit a repertoire of effective immune responses as the encoded antigen is endogenously synthesised and processed, mimicking natural infection and targetting DC's and thus is presented via the MHC class I and class II pathways to generate durable humoral and cellular responses. Innate immunity is also triggered, partly through unmethylated CpG motifs. Other practical advantages include non-infectiousness, ease of manipulation, inexpense and thermostability, providing potential for emergency and endemic use, with scope to incorporate marker genes and cover 'difficult' isolates. Cattle are the principle target of the susceptible domestic species and this programme will utilise FMD DNA vaccination to augment bovine immune responses and study their importance to both protection and persistence. Successive dosage with FMD and GMCSF DNA plasmids,will be compared with a prime-protein boost regime alongwith simultaneous DNA and protein immunisation to mimic emergency use. Humoral, cellular and innate parameters will confirm the optimal regime. T cell proliferation and IFN? ELIspot assays will estimate the memory T cells primed by DNA regimes and Th1 responses and the degree of recall antigen responding cells, and the phenotype of the cells will define the induced lymphocyte subset(s). Cytotoxic assays will establish whether a polarized Th1 or Th2 response is promoted. The ability of dendritic cells to present antigens to lymphocytes will be analysed to understand the mechanism by which DNA vaccine works in vivo. Protection studies, will be monitored for at least 28 days post challenge, to assess persistence, the various immune parameters and the efficacy of the DNA vaccine regime. Key non-structural protein antibody responses will be examined to differentiate vaccinates supporting viral persistence.