Campylobacter jejuni bacterial pathogen is among the primary causes of food-borne acute gastroenteritis in North America and the world. It has also been linked to severe post-infection sequelae such as Guillain-Barré syndrome. Previous studies identified C. jejuni surface capsular polysaccharide (CPS) as a target for creation of a carbohydrate based vaccine in which the CPS is conjugated to a carrier protein. In this thesis, following sample purification, aspects of C. jejuni HS:5 CPS structure were characterized using numerous analytical techniques such as NMR and GC-MS. CPS is comprised of α-DD-Heptoses linked at C2 to the anomeric carbons of glucose. The α-Glucose molecules are linked though C4 to the α-DD-Heptose anomeric carbon. The α-DD-Heptose structure also has an occasional ring structured amino acid modification. Following characterization the CPS was oxidized and developed into a prototype glycoconjugate vaccine using TEMPO oxidation and EDC-CRM197 coupling methods. / The Natural Sciences and Engineering Research Council of Canada (NSERC)

This thesis describes the production of adenovirus-based vaccines containing codon-optimized genes from Nipah virus and Crimean-Congo Hemorrhagic Fever virus. Genes encoding envelope proteins from Crimean-Congo Hemorrhagic Fever Virus and Nipah Virus were codon-optimized for translation in human cells and constructed using a modified method of non-gapped gene synthesis, while the entire M segment encoding the glycoprotein precursor for Crimean-Congo Hemorrhagic Fever Virus was commercially synthesized. Genes were cloned into recombinant human adenovirus serotype 5 and the resulting viral particles were amplified, titred and analyzed for in vivo efficacy. Results show that a modified method of non-gapped gene synthesis is an effective and efficient method of producing antigen-encoded DNA and at a fraction of the cost and time required for commercial synthesis. Furthermore, adenovirus-based vaccines induce both cellular and humoral immune responses providing for a highly efficacious vaccine during potential disease outbreaks, where time to completion is of utmost importance. This study has shown that recombinant adenoviral vaccines for Crimean-Congo Hemorrhagic Fever virus and Nipah virus can be produced rapidly and efficiently from virtual DNA sequence to optimized recombinant vaccines in just eight months.

The transfer of a desired gene to several types of target tissues has been accomplished successfully in the past using existing Adeno-associated viruses (AAVs). Also, it has recently been shown that AAV can stimulate robust antibody responses due to long-term transgene expression or abolishment of transgene product by cell-mediated immune responses, suggesting the potential use of AAVs as vaccines. Most humans already have pre-existing immunity to common AAV serotypes making novel AAVs of low seroprevalence attractive as gene transfer or vaccine vehicles. This thesis describes my primary research objectives that included the isolation of novel AAV serotypes based on AAV DNA sequences from porcine tissues, novel AAV vector production, and biological characterization of porcine AAVs in vitro and in vivo. This was followed by evaluating immune responses in mice vaccinated with porcine AAV vectors expressing the hemagglutinin (HA) from the avian influenza A/Hanoi/30408/2005 (H5N1) strain. These findings show that low seroprevalence porcine AAV vectors were able to efficiently transduce a wide range of cells and tissues. The porcine vectors also performed well as vaccine candidates and were efficient at stimulating host immune responses. Although porcine vectors were successful as vaccines, further studies involving long term gene expression by porcine AAVs is still necessary to confirm their role as gene therapy vehicles.

The antigenic variability of the Influenza virus hinders our ability to develop new therapeutic and vaccine strategies which provide a broad protection against all influenza strains. It has been previously suggested that a means to approach this challenge is to identify conserved sequences within viral proteins and use these for future therapeutic targets. Although such conserved sequences are plentiful amongst the internal viral proteins, their lack of exposure to the host immune system makes mounting an immune response against these regions difficult. Alternatively, the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) have been shown to provide host protection against a limited number of influenza strains when used as vaccine targets; however conserved regions within these proteins which are also antibody accessible are extremely rare. My Ph.D. thesis project is focused on investigating the functional role of a conserved region within the NA protein and to further determine the protection afforded by a monoclonal antibody to this region. In a comprehensive bioinformatics analysis, the only universally conserved sequence amongst all influenza A and B viral NA has been previously identified as being located between amino acids (a.a.) 222-230 (dubbed the HCA-2 region). However, the potential role of this region remains largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics and growth kinetics, I have found that substitutions in this sequence significantly affect viral replication by impairing the catalytic activity, substrate-binding and thermostability of NA. These findings prompted me to further investigate if antibody to this region may provide protection against influenza infection. Indeed, universal monoclonal antibody (HCA-2 MAb) against this peptide provided broad inhibition against all nine subtypes of NA in vitro and heterosubtypic protection in mice challenged with lethal doses of mouse-adapted viruses. I further demonstrated that residues within this peptide that are exposed on the surface of NA and located in close proximity to the active site, I222 and E227, are indispensable for antibody-mediated inhibition. These data are the first to demonstrate a monoclonal antibody against the NA protein which provides heterosubtypic protection. Since I observed that the HCA-2 antibody provided a broad inhibition against all nine subtypes of influenza A NA, I decided to investigate whether this inhibitory effect could be extended against Influenza B. Here, I have further reported that HCA-2 MAb provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. I also demonstrate that the growth and NA enzymatic activity of two drug resistant influenza B strains are also inhibited by the HCA-2 antibody. The findings of my Ph.D. thesis project have thus demonstrated that the HCA-2 region is paramount to optimal viral function. Additionally, my data show that antibodies generated against this region provide heterosubtypic protection both in vitro and in vivo and against drug resistant strains. These results indicate that this universally conserved epitope should be further explored as a potential target for future antiviral intervention and vaccine-induced immune responses.

This thesis describes the production of adenovirus-based vaccines containing codon-optimized genes from Nipah virus and Crimean-Congo Hemorrhagic Fever virus. Genes encoding envelope proteins from Crimean-Congo Hemorrhagic Fever Virus and Nipah Virus were codon-optimized for translation in human cells and constructed using a modified method of non-gapped gene synthesis, while the entire M segment encoding the glycoprotein precursor for Crimean-Congo Hemorrhagic Fever Virus was commercially synthesized. Genes were cloned into recombinant human adenovirus serotype 5 and the resulting viral particles were amplified, titred and analyzed for in vivo efficacy. Results show that a modified method of non-gapped gene synthesis is an effective and efficient method of producing antigen-encoded DNA and at a fraction of the cost and time required for commercial synthesis. Furthermore, adenovirus-based vaccines induce both cellular and humoral immune responses providing for a highly efficacious vaccine during potential disease outbreaks, where time to completion is of utmost importance. This study has shown that recombinant adenoviral vaccines for Crimean-Congo Hemorrhagic Fever virus and Nipah virus can be produced rapidly and efficiently from virtual DNA sequence to optimized recombinant vaccines in just eight months.

The transfer of a desired gene to several types of target tissues has been accomplished successfully in the past using existing Adeno-associated viruses (AAVs). Also, it has recently been shown that AAV can stimulate robust antibody responses due to long-term transgene expression or abolishment of transgene product by cell-mediated immune responses, suggesting the potential use of AAVs as vaccines. Most humans already have pre-existing immunity to common AAV serotypes making novel AAVs of low seroprevalence attractive as gene transfer or vaccine vehicles. This thesis describes my primary research objectives that included the isolation of novel AAV serotypes based on AAV DNA sequences from porcine tissues, novel AAV vector production, and biological characterization of porcine AAVs in vitro and in vivo. This was followed by evaluating immune responses in mice vaccinated with porcine AAV vectors expressing the hemagglutinin (HA) from the avian influenza A/Hanoi/30408/2005 (H5N1) strain. These findings show that low seroprevalence porcine AAV vectors were able to efficiently transduce a wide range of cells and tissues. The porcine vectors also performed well as vaccine candidates and were efficient at stimulating host immune responses. Although porcine vectors were successful as vaccines, further studies involving long term gene expression by porcine AAVs is still necessary to confirm their role as gene therapy vehicles.

Vaccination is one of the major strategies available for combating viral infections in humans. However, currently available vaccines are not without pitfalls; they are laborious to produce, could potentially be unsafe, and in the case of the highly variable influenza virus need to be reformulated each season. The use of synthetic peptides thus represents an exciting alternative to traditional vaccines. However, these synthetic peptides are not highly immunogenic without the use of potent adjuvants. The lack of immunogenicity might be addressed by conjugation between T or B cell epitopes with universal or immunodominant T-helper epitopes. The construction of branched peptides, lipidated peptides, or designs combining both of these elements might also enhance the immunogenicity, as they might target Toll-like receptors and/or mimic the 3-dimensional structure of epitopes within the native protein. In this study, a recognized T-B peptide based on the hemagglutinin protein of the A/Puerto Rico/8/34 influenza virus was chosen as a backbone and modified to evaluate if the construction of branched peptides, lipidation, the addition of cysteine residues, or mutations could indeed alter reactivity. Screening the different designs with various antibody binding and cellular assays revealed that combining a branched design with the addition of lipid moieties leads to a greatly enhanced activity as compared to other similar T-B diepitope constructs.

The causative agents of plague (Y. pestis), glanders (B. mallei) and melioidosis (B. pseudomallei) are included in critical agents of bioterrorisim. They belong to the most intensively studied agents during cold war, specially in the former Soviet Union (FSU). Mostly what is known about these agents, particularly (Y. pestis ) is not available in English language publications. Many of the studies are written in Russian language and published in Russian scientific journals. Thus, the work is designed to evaluate, published and unpublished Russian language written data obtained, in comparisions to the current state of global knowledge on the pathogens in concern.

Fowlpox viruses (FPV) are attractive platform vaccine vector candidates because their capacity for insertion of multiple heterologous genes makes them favourable for genetic modification. They also have strong adjuvant activity in their own right. As FPV does not replicate in mammalian cells, there is significantly less opposition associated with their clinical application, with a number already in use. However, a thorough understanding of the immunological relationship between FPV and the mammalian immune system is still lacking. The aim of this thesis was to construct a series of recombinant FPV vectors that co-expressed the nominal antigen chicken ovalbumin (OVA), (FPV[subscript]OVA), and/or murine interleukin-4 (mIL-4). These constructs were used for the characterisation of the relationship between FPV and the mammalian immune system and how this is altered by the co-expression of mIL-4. Immunisation with FPV[subscript]OVA resulted in rapid and highly localized OVA expression which induced strong CD8⁺ cytotoxic T cell (CTL) activity but only weak CD4⁺ T helper and antibody responses. In addition, presentation of FPV-derived antigen and the priming of antigen-specific CTL responses required a permissive bone marrow (BM)-derived cell as the antigen presenting cell (APC). Co-administration with FPV[subscript]mIL-4 resulted in a dramatic reduction in CTL activity that remained largely non-functional throughout the infection and a skewing of the T helper (Th) response towards Th2 with a reduction in interferon (IFN)-γ production by OVA-specific Th cells. These findings provide a sound basis for further characterization of how FPV interacts with the innate and adaptive arms of the immune system, how these can be manipulated via the co-administration of cytokines, and discovering if future rationally designed modifications result in FPV vectored vaccines that induce durable cellular and humoral immunity. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1352466 / Thesis (M.Med.Sc.) - University of Adelaide, School of Medicine, 2009

Fowlpox viruses (FPV) are attractive platform vaccine vector candidates because their capacity for insertion of multiple heterologous genes makes them favourable for genetic modification. They also have strong adjuvant activity in their own right. As FPV does not replicate in mammalian cells, there is significantly less opposition associated with their clinical application, with a number already in use. However, a thorough understanding of the immunological relationship between FPV and the mammalian immune system is still lacking. The aim of this thesis was to construct a series of recombinant FPV vectors that co-expressed the nominal antigen chicken ovalbumin (OVA), (FPV[subscript]OVA), and/or murine interleukin-4 (mIL-4). These constructs were used for the characterisation of the relationship between FPV and the mammalian immune system and how this is altered by the co-expression of mIL-4. Immunisation with FPV[subscript]OVA resulted in rapid and highly localized OVA expression which induced strong CD8⁺ cytotoxic T cell (CTL) activity but only weak CD4⁺ T helper and antibody responses. In addition, presentation of FPV-derived antigen and the priming of antigen-specific CTL responses required a permissive bone marrow (BM)-derived cell as the antigen presenting cell (APC). Co-administration with FPV[subscript]mIL-4 resulted in a dramatic reduction in CTL activity that remained largely non-functional throughout the infection and a skewing of the T helper (Th) response towards Th2 with a reduction in interferon (IFN)-γ production by OVA-specific Th cells. These findings provide a sound basis for further characterization of how FPV interacts with the innate and adaptive arms of the immune system, how these can be manipulated via the co-administration of cytokines, and discovering if future rationally designed modifications result in FPV vectored vaccines that induce durable cellular and humoral immunity. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1352466 / Thesis (M.Med.Sc.) - University of Adelaide, School of Medicine, 2009