Thesis (Ph. D.)--Washington State University, May 2010. / Title from PDF title page (viewed on June 4, 2010). "College of Veterinary Medicine." Includes bibliographical references.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Thesis (Ph.D.)--Aberdeen University, 2008. / Title from web page (viewed on March 9, 2009). Includes bibliographical references.

Thesis (M.S.) in Biochemistry--University of Maine, 2003. / Includes vita. Includes bibliographical references (leaves 48-60).

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 103-111). Also available in electronic version.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 281-328).

No description available.

Campylobacter is the leading cause of bacterial food-borne diarrhoeal disease in the developed world and a significant cause of infant morbidity and mortality in developing countries. Epidemiological studies implicate poultry as a key source of infection, with up to 80% of human cases being attributable to the avian reservoir. An effective vaccine for broilers is predicted to limit the incidence of human campylobacteriosis. Vaccination of chickens with CjaA, either in recombinant form or vectored in live-attenuated Salmonella, has been reported to significantly reduce caecal colonisation by C. jejuni, with more invasive carriers eliciting greater protection. However, protection remains modest and is slow to develop. I therefore sought to improve such vaccines, first by vectoring codon-optimised CjaA in a licensed avian pathogenic E. coli ΔaroA vaccine. In two independent trials, White Leghorn birds were vaccinated subcutaneously on the day of hatch and 14 days later then challenged with C. jejuni M1 at 28 days post-hatch. No protection was observed despite significant induction of CjaA-specific serum IgY, however, a previously described S. Typhimurium ΔaroA vaccine vectoring CjaA also failed to protect. Owing to the variability observed with live CjaA-based vaccines in these and previous studies, other candidate antigens were sought and evaluated as subunits. Twenty-one candidate C. jejuni antigens were cloned and expressed as glutathione-S-transferase (GST) fusions. Nine of these could be purified in adequate soluble quantities to be tested in vivo. The intervals of vaccination and challenge were as above, with GST alone or GSTCjaA acting as negative and positive controls, respectively. Each antigen was administered subcutaneously in TiterMax Gold® adjuvant at the molar equivalent of the doses of GSTCjaA. Repeated testing of initially promising candidates revealed that, when averaged across three independent trials, GST-SodB and GST-FliD induced statistically significant reductions in caecal colonisation of 1-2 log10 colony-forming units of C. jejuni at 48 and 56 days post-hatch compared to negative controls. Induction of antigen-specific serum IgY was measured by enzyme linked-immunosorbent assays using maltose-binding protein fusions to each antigen. This revealed significant induction of antigen specific serum IgY for the majority of the antigens tested, even when no protection was observed. In the SodB- and FliD-vaccinated groups, the peak of antigen-specific serum IgY was not coincident with the onset of protection and the fold-change in specific IgY levels in individual birds did not correlate with caecal Campylobacter numbers. Furthermore, sera from SodB-vaccinated birds failed to detect SodB in the outer membrane or surface of Campylobacter cells, indicating that SodB-specific antibodies are unlikely to be neutralising. Taken together, these studies identified two novel protective antigens that, with further optimisation, could form part of an anti-Campylobacter vaccine for broilers. However further studies are required to define the nature and consequences of immune responses required for protection.

Malaria causes almost half a million deaths each year. Existing interventions will almost certainly not be enough to tackle this enormous public health problem on their own. An effective vaccine is urgently needed. The leading malaria vaccine, RTS,S, confers suboptimal protective efficacy, and in addition targets only Plasmodium falciparum and not the other major species of human malaria, P. vivax. This thesis investigates the potential of combining pre-erythrocytic malaria vaccines as a means of enhancing protective efficacy. A novel mathematical model was developed which expresses probability of protection as a function of vaccine-induced humoural and cellular responses. The model predicts that combining partially effective vaccines should result in more than additive improvements in protective efficacy. This was supported by an experiment combining Rv21, a P. vivax circumsporozoite virus-like particle, with viral vectored P. vivax TRAP, the two leading pre-erythrocytic malaria vaccine antigens; this combination raised protective efficacy from 50% and 0%, respectively, to 100% sterile protection. It was also found that antigenic interference, a reduction in anti-CSP titres when Rv21 and PvTRAP are combined, occurred only in the presence of Matrix M adjuvant, and not when using alum, AddaVax or no adjuvant. With a view to creating a single-component multi-antigen vaccine, which would be more cost-effective than a multi-component vaccine, experiments were carried out to establish the virus-like particle Qβ as a platform capable of eliciting protective immunity via the display of short peptides derived from the CSP repeat region of both P. vivax and P. falciparum. For the first time, a tetramer peptide derived from the CSP repeat region of P. vivax VK210, AGDR, was shown capable of eliciting protective immunity alone. Finally, five novel linear B-cell epitopes were discovered, one from P. falciparum CSP, three from P. vivax TRAP and one from TRSP, each capable of conferring partial protection on mice. These epitopes were identified using novel screening methods, using sera from whole-protein vaccinated mice or by exploiting conservation within invasion protein sequences. Two of the protective epitopes, (NANP)6 and (ADGN long) were combined and found to enhance protective efficacy as predicted by the mathematical model. Thus this thesis lays the groundwork for the development of a single-component multi-epitope malaria vaccine with enhanced protective efficacy.

Piscirickettsia salmonis is the aetiological agent of salmonid rickettsial septicaemia (SRS), an economically devastating rickettsial disease of farmed salmonids. SRS responds poorly to antibiotic treatment and no effective vaccine is available for its control. A molecular biology approach was used to characterize and identify antigens of P. salmonis that would be suitable to use as a recombinant subunit vaccine to aid in the control of SRS. A system for routine and reliable growth of P. salmonis was established using a chinook salmon (Oncorhynchus tshawytscha) embryo cell line. A purification protocol to separate P. salmonis from host cell material was devised using a combination of differential and Percoll density gradient centrifugation. Purified P. salmonis was used to generate polyclonal rabbit antisera. Indirect immunofluorescence microscopy, immunogold transmission electron microscopy, and biotin labeling of intact P. salmonis confirmed that P. salmonis was effectively separated from host cell debris and that immunoreactive antigens identified by rabbit antisera were surface associated. Rabbit anti- P. salmonis sera recognized the lipooligosaccharide component of bacterial lipopolysaccharide, and 7 protein antigens with relative mobilities of 27, 24, and 16 kDa and 4 migrating between 50–80 kDa. P. salmonis lipopolysaccharide was observed to be predominantly low m.w., but less abundant high m.w. species containing O-antigen were present. Genomic DNA was isolated from purified P. salmonis and used to construct an expression library in lambda ZAP II. In the absence of preexisting DNA sequence, rabbit polyclonal anti-P. salmonis serum was used to identify immunoreactive clones. A lambda clone encoding an immunoreactive 17 kDa outer surface protein (OspA) of P. salmonis was identified. The 4,983 by insert contained a high molar percentage of adenine and thymine, encoded four intact ORF's, and represented the first non-ribosomal DNA sequence data from P. salmonis. OspA is modified as a bacterial lipoprotein in Escherichia coli and is most closely homologous to a rickettsial 17 kDa surface lipoprotein previously only observed within the genus Rickettsia. A codon optimized version of ospA was constructed and the lipoprotein nature of OspA was determined to be a limiting factor in its production in E. coli. High level production of immunoreactive OspA targeted to inclusion bodies was achieved in E. coli by combining OspA with an N-terminal fusion protein. The OspA fusion was recognized by convalescent salmon sera thereby identifying OspA as an excellent candidate for a recombinant vaccine against P. salmonis. Vaccine preparations using P. salmonis bacterins were found to elicit variable immune responses in coho salmon (Oncorhynchus kisutch) that resulted in either protection or immunosuppression of vaccinates which varied with antigen dosage. Recombinantly produced OspA elicited an astonishing level of protection in vaccinated coho salmon with a relative percent survival (RPS) as high as 59%. In an effort to further improve the efficacy of the OspA recombinant vaccine, T cell epitopes (TCE's) from tetanus toxin and measles virus fusion protein which are universally immunogenic in mammalian immune systems were incorporated into an OspA fusion protein. Addition of the TCE's dramatically enhanced the efficacy of the OspA vaccine, reflected by a 3-fold increase in the number of coho salmon protected (83% RPS). These results represent an effective monovalent recombinant subunit vaccine for the rickettsial pathogen, P. salmonis. / Graduate