To enhance the global control of encephalitis and hepatitis caused by rabies virus (RABV), Japanese encephalitis virus (JEV), enterovirus 71 (EV71) and hepatitis B virus (HBV), novel immunisation strategies are needed. All four diseases particularly affect low income countries with marginal health services – an affordable combined vaccine strategy could alleviate the large burden of disease. Therefore, we aimed to construct a multipathogen vaccine assessing the immunising activity of a recombinant modified vaccinia Ankara (MVA), expressing key antigens (RABV-glycoprotein, JEV pre-membrane & envelope protein, EV71-P1 protein and large hepatitis B surface antigen) from the various pathogens. Successful delivery of the pathogen sequences into non-essential sites (deletion site I, II, VI) of MVA via homologous recombination with a transfer plasmid, was demonstrated by transient color selection (LacZ-marker) in vitro. The stable insertion of the expression cassettes was validated over ten virus passages by PCR with specific primer sets, targeting the pathogen sequence. Two recombinants, one carrying the EV71 and JEV pathogen sequence and one carrying the RABV-HBV pathogen sequence were generated and validated by PCR.To ensure similar expression of the key antigens, a T7-promoter was linked to the expression cassettes of all pathogen sequences. Direct regulation of this promoter was achieved through co-infection with a second T7-polymerase expressing MVA under the control of a vaccinia p7.5 promoter. Protein expression from recombinant MVA using the co-infection model of expression in vitro, was further characterised by Western blot, dot blot and immunocytochemistry. All inserted transgenes were expressed using an avian (chicken embryo fibroblasts) or mammalian (human fetal lung fibroblasts) cell culture system. To investigate the co-infection model of antigen delivery in vivo, a pilot murine immunogenicity study was performed in six Balb/c mice using the MVA-RABV-HBV recombinant in a homologous prime-boost regimen two weeks apart. To detect antibodies against the expressed pathogen sequences in the mouse serum an antibody-capture assay was performed (Western blot, dot blot). The antigen (used to capture murine antibodies) was purified RABV-glycoprotein or large hepatitis B surface antigen expressed from a baculovirus. The murine antibodies were detected by a secondary anti-mouse antibody, conjugated with horseradish peroxidase for a chemiluminescent reporter assay. Although, serum antibodies against MVA were induced in all mice, no serum antibodies against RABV or HBV could be detected. In summary, we were able to demonstrate that two transgenes, when inserted into one or two different loci in the MVA genome, can be expressed in vitro when using the co-infection model of gene expression with a T7-expression system. This project has provided new insights into a novel group of vaccines, the multipathogen viral vector vaccines, employing MVA as a vector. Future studies will be needed to further explore this vaccine-group, as well as the co-infection model of expression.

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The development of a safe and effective HIV vaccine remains challenging due to its high antigenic variability. Poxviruses are large, stable, and have a track record of use as human vaccine candidates. Recombinant fowlpox virus 9 (rFP9), a highly attenuated host range-restricted poxvirus strain, has been safely administered to humans with no ill effects, and is known to be immunogenic. This thesis describes the construction of complex rFP9 encoding various sequences of SIVmac239. The SIVmac239/macaque model is widely used for HIV vaccine development. The ultimate aim of this work was to combine the advantages of FP9 with those of live attenuated SIV to produce a safe yet hopefully effective model HIV vaccine candidate. Transfer plasmids for five different insertion sites within the FP9 genome were designed and constructed. Homologous recombination (HR) of adjacent FP9 sequences was employed to facilitate the integration of SIVmac239 sequences into the FP9 genome. Positive rFP9 were identified by blue colouration in presence of X-gal using a transient colour selection (TCS) technique, and the final markerless pure recombinants were confirmed by PCR. Expression of the target SIV proteins in the presence of T7 polymerase has been demonstrated by immunocytochemical (ICC) staining and Western blotting (WB) assays. Expression was also quantified by enzyme-linked immunosorbent assay (ELISA) in various cell lines at multiple time points. Five different unique rFP9 have been constructed through this project. All SIVmac239 open reading frames (ORFs) save nef have been integrated into the FP9 genome, and protein expression demonstrated where possible. Moreover, a single rFP9 vector expressing the defective SIVmac239 genome driven by T7 RNA polymerase has been successfully constructed and validated using a green fluorescent protein marker.rFP9 showed appropriate transgene expression in both avian and mammalian cells, although at different levels. The expression efficiency of rFP9 was finally compared to another attenuated poxvirus vector, modified vaccinia Ankara (MVA). Comparing the protein expression levels between rFP9 and rMVA was quite difficult because different poxvirus promoters (early/late in rFP9; intermediate in rMVA) were used to direct the transcription of the T7 RNA gene. Given this limitation, although generally higher levels of expression were seen with rFP9, this cannot be attributed to the FP9 with any certainty.