Bovine herpesvirus-1 (BoHV-1) is associated with the syndrome bovine respiratory disease, which is the major cause of morbidity and mortality within feedlots in Australia and around the world. Currently there are no vaccines that completely prevent BoHV-1 infections and viral shedding. The most efficacious vaccines used are live attenuated which have the potential to revert to wild type and cause disease. DNA vaccines are ideal vaccine candidates as they not only induce humoral and cellular immunity, they are also inexpensive and easy to produce. However, DNA vaccines although efficacious in small animal models have not yielded similar success in large animals. The inconsistent translation of DNA vaccines to large animal models, including cattle, has been associated with poor delivery of the vaccine to the nuclei of cells which is required for antigen transcription. Recently, the human herpesvirus-1 protein VP22 (hVP22) was demonstrated to exhibit the uncommon capacity to spread intercellularly from the cell of expression to the nuclei of neighbouring cells in a golgi and energy independent process. This process was very efficient with hVP22 being identified in all cells of a monolayer after transfection. hVP22 was quickly used to promote the efficiency of DNA vaccines by fusing the hVP22 gene with antigen genes in the vaccine resulting in the increased delivery of the antigenic protein to neighbouring cells. The fusion protein was subsequently degraded and presented as peptides on the cell surface in association with major histocompatibility complex (MHC) class II molecules that lead to an increase in fusion protein specific antibody production. This pathway, although successful augmenting the humoral response, did not increase the amount of antigen presented on MHC class I molecules which is essential for protection against intracellular pathogens. This thesis describes the development of a methodology whereby VP22, fused to a DNA binding protein, was hypothesised to increase the number of cells the DNA vaccine was delivered to and then to facilitate the transport of the DNA vaccine to their nuclei. A homologue of hVP22 has been identified in BoHV-1 and the capacity of the BoHV-1 protein to spread intercellularly and localise in the nuclei of cells was determined in this thesis using a novel and definitive model. Although retaining similar translocation capabilities to hVP22 the BoHV-1 VP22 homologue could not be expressed in bacteria and was subsequently not able to be used to demonstrate the proposed vaccine concept. hVP22 instead was fused to the DNA binding protein, Gal4, for bacterial expression. The purified fusion protein was demonstrated to bind not only oligonucleotides encoding the Gal4 binding sequence but also to a model DNA vaccine encoding Gal4 binding sequences in vitro. However, application of the hVP22 fusion protein:vaccine complex alone or condensed with poly-L-lysine to mammalian cells did not promote the delivery of the DNA vaccine to the nuclei of cells. As part of the DNA vaccine development for BoHV-1 the first nucleotide sequence of the Unique Short region of the Australian BoHV-1 strain V155 (8925 nucleotides) was determined. The sequence information generated permitted insights into epitopes contained within BoHV-1 antigens, particularly glycoprotein D which has been identified as the most appropriate glycoprotein for the purpose of vaccination. Furthermore, comparison of the Unique Short sequence variations between different subtypes of BoHV-1 provided molecular data that may be associated with the observed variation in virulence. Further optimisation of the methodology described in this study is required to facilitate the delivery of the DNA vaccine into cells by the VP22 fusion protein. The future development of strategies that utilise polypeptides to augment delivery of DNA vaccines into cells and then to facilitate the transport of the vaccine to the nuclei of cells, resulting in increased antigen expression, may ultimately lead to the successful application of this vaccine technology in animal models.
| Identifer | oai:union.ndltd.org:ADTP/254135 |
| Creators | Kerri Clark |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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