West Nile virus (WNV) has a worldwide distribution, with this virus having been isolated on all continents except Antarctica. The recent emergence of highly pathogenic strains of WNV associated with increased rates of neurological disease is of great concern given this broad distribution of the virus. Although two vaccines have been licensed for veterinary use, no prophylactic measures have been approved for humans. Similarly, no antivirals are currently available for post-exposure treatment of WNV. Indeed, few therapeutic agents have shown promise when administered after WNV infection in animal models. KUNV is a highly attenuated, Australasian lineage 1 strain of WNV. This attenuation is mediated in part by the limited neuroinvasiveness of this virus. Phylogenetically, KUNV clusters with pathogenic lineage 1 WNV strains, including the isolates which have been associated with 997 deaths in North America since 1999. Recently, it was shown that mice exposed to KUNV were effectively protected from challenge with pathogenic WNV. The KUNV strain used in that study possessed a single amino acid substitution in NS1 protein that affected oligomerization of this protein, resulting in reduced virus replication in vitro and increased attenuation in mice. In the present study, further characterization of this attenuation marker in NS1 protein was undertaken to determine whether it is suitable for inclusion in a live-attenuated KUNV vaccine. Similarly, mapping of the residues that contribute to the dimerization domain surrounding NS1 protein was performed to identify other potential attenuation markers for stabilization of KUNV attenuation. The mutant viruses created in this study also were manipulated to characterize the role of NS1 protein dimerization in flavivirus replication. The results of this work indicate that NS1 protein dimerization is not absolutely required for virus replication or production of secreted oligomers of NS1 protein, which are important for eliciting protective humoral responses. Although replication of KUNV was found to be highly dependent on retention of the conserved amino acid sequence within the dimerization domain, two mutant viruses were generated by introducing substitutions at residue 250 of NS1 protein. The resultant viruses demonstrated reduced replication in vitro and attenuation in mice. Similarly, a non-conservative substitution in NS2A, which was previously shown to reduce the resistance of KUNV to the host interferon response, was able to attenuate KUNV in mice. Inoculation of adult mice with viruses containing mutations at either site afforded complete protection from lethal WNV challenge. However, the substitutions described in the dimerization domain of NS1 protein were unstable, with restoration of virulence being observed in mutant viruses after limited passaging in vitro. Concerns over the stability of attenuating mutations in KUNV and the time taken to characterize new attenuation markers prompted the evaluation of a novel approach to the development of rationally-designed flavivirus vaccines. The introduction of large complements of synonymous codon substitutions reduced KUNV replication in vertebrate cells. Escape mutations were not observed in a KUNV vaccine candidate containing 37 rare codons after repeated passaging in vertebrate cells at a low MOI. Replication of KUNV in C6/36 cells was unaffected by the introduction of large numbers of rare codons, indicating that this cell line exerts limited selective pressure on the codon composition of this virus. This observation indicates that C6/36 cells may be a useful cell line for the propagation of viruses containing this type of mutation. Finally, three monoclonal antibodies (MAbs) which bind to WNV envelope (E) protein were observed to potently neutralize the pathogenic NY99 strain of WNV. Passive administration of one of these antibodies was shown to afford mice protection even when administered seven days after challenge with WNV NY99 strain. Remarkably, this is the same time that mortality is first observed in control groups. These antibodies mapped to the putative receptor binding domain (domain 3) of E protein. However, these antibodies were found to block virus replication at a stage after receptor-binding. Homology modeling was used to propose a mechanism for the blockade of virus infection mediated by MAb binding. This study describes the development and characterization of a promising new vaccine as well as candidate immunotherapeutics for the prophylaxis and post-exposure treatment of WNV disease. This work described herein also has implications for the development of vaccines and antivirals for other flaviviral diseases.

There is an abundance of information available on the molecular mechanisms of antigenic variation in Plasmodium falciparum. The variant antigen PfEMP1, which mediates antigenic variation as well as cytoadherence and rosetting, has been extensively characterised. Genes coding for the antigen belong to the gene family var, and several var genes have been cloned and characterised. The rodent malaria parasite P. chabaudi is a widely studied in vivo model for P. falciparum. The P. c. chabaudi AS parasite strain has been shown to exhibit antigenic variation and the variant antigen has been detected by surface fluorescence. As with P. falciparum, there is a link between antigenic variation and cytoadherence, however genes coding for the variant antigen in P. chabaudi have not been cloned to date. Therefore, potentially useful in vivo experiments on antigenic variation are restricted. In this thesis it is shown for the first time that the P. c. adami DS parasite strain also exhibits antigenic variation. / Chapter 3 describes efforts to locate genes coding for variant antigens in P. c. adami DS. The main strategy involved a genome survey, by sequencing and analysing randomly selected clones from a P. c. adami DS genomic library. DNA sequences were compared to Plasmodium spp. sequence databases to look for similarity to var genes or other genes encoding variant antigens. Of the 297 clones analysed none had significant sequence similarity to genes coding for variant antigens. However, in a small proportion of sequences some similarity to var genes was noted. Several genes of potential interest were identified, most importantly the gene coding for the vaccine candidate rhoptry associated protein 1 (RAP1), which was subsequently cloned and characterised. Further attempts to locate var gene homologues in P. c. adami involved amplification of P. c. adami genomic DNA using degenerate oligonucleotide primers corresponding to conserved regions of var genes. This strategy proved to be unsuccessful, most likely due to lack of sequence similarity between P. falciparum and P. c. adami genes. In several vaccination studies with the apical membrane antigen 1 (AMA1) of P. c. adami DS, mice were significantly protected against homologous parasite challenge. However, some mice developed late, low-level breakthrough parasitaemias. In Chapter 4, the characterisation of two such breakthrough parasitaemias is described. The ama1 genes of the breakthrough parasites were found to be identical to the ama1 gene of the parental parasites. Similarly, no alteration in AMA1 expression was observed. However, the breakthrough parasites were found to be more resistant than the parental parasites to the effects of passive immunisation with rabbit antisera to AMA1, RAP1 and possibly also MSP119. P. chabaudi infections in mice have been previously shown to consist of a primary parasitaemia followed by a short period of subpatency, and a recrudescent parasitaemia. In surface immunofluorescence studies with P. c. chabaudi, parasites of the recrudescence were shown to be distinct from parasites of the primary parasitaemia, with respect to antigens expressed on the surface of late trophozoite- and schizont-infected erythrocytes. / Chapter 4 describes similar surface immunofluorescence assays carried out with P. c. adami infected erythrocytes, and quantitation of fluorescence by flow cytometry. As with P. c. chabaudi, the recrudescent parasites were found to be antigenically distinct from the primary parasitaemia, indicating that antigenic variation had taken place. Because breakthrough parasites from the AMA1 vaccination trial were similar to recrudescences in peak and duration, we hypothesised that breakthrough parasitaemias, like recrudescent parasitaemias, occur as a result of antigenic variation. In Chapter 4 it was shown by surface immunofluorescence and flow cytometry using hyperimmune sera raised against different parasite populations, that breakthrough parasites express antigens on the surface of late trophozoite- and schizont infected erythrocytes that differ from those expressed by the parental and recrudescent parasites. These results support the hypothesis that switching of the variant antigen on the infected erythrocyte surface enables parasites to evade protective antibody responses directed against merozoite antigens. / Chapter 5 describes the cloning and characterisation of P. c. adami RAP1 which was identified in the process of the genomic survey described in Chapter 3, as well as P. berghei RAP1. Both rodent parasite orthologues of RAP1 were found to have 30% sequence similarity to P. falciparum RAP1, and 6 of 8 cysteines were conserved in the rodent parasite orthologues. However the three polypeptides vary significantly in size. P. c. adami RAP1 and P. berghei RAP1 consist of 691 aa and 604 aa respectively, whereas P. falciparum RAP1 consists of 783 aa residues. These size differences reflect very different N-terminal sequences prior to the first cysteine, whereas the cysteine-rich C-terminal regions are more conserved. Both P. falciparum RAP1 and P. c. adami RAP1 contain N-terminal repeats, however they bear no sequence similarity to each other. P. berghei RAP1 lacks N-terminal sequence repeats that are characteristic of P. falciparum and P. c. adami RAP1. The large cysteine-rich C-terminal region P. c. adami RAP1 (PcRAP1 C3) was expressed in E. coli as a hexa-his fusion protein. Rabbit antiserum to recombinant PcRAP1 C3 was used to characterise the expression and sub-cellular localisation of the RAP1 antigen. P. c. adami RAP1 was found to have a Mr of approximately 80,000 and was shown by immunofluorescence to localise to the merozoite rhoptries. Passive immunisation of mice with rabbit anti-RAP1 serum was shown to protect against fulminant parasitaemia and mortality. In a mouse vaccination trial using the recombinant PcRAP1 C3 polypeptide partial protection was conferred against homologous parasite challenge.

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

Iron is a trace element required for a range of metabolic reactions in virtually all living organisms. Studies of prokaryotes, plants, yeast, and vertebrates have established detailed information on iron uptake and the role iron plays in metabolic processes. Iron is an essential growth requirement of schistosomes in vitro and schistosomes also express the highly conserved iron storage protein ferritin. However, studies into how this iron is taken up by the parasite have been neglected. This study aims to identify molecules involved in iron uptake and homeostasis in the human parasite Schistosoma japonicum. I have characterised two isoforms of a divalent metal transporter (DMT), a membrane bound protein of schistosomes. These DMTs have significant homology to the mammalian DMT1, the primary ferrous iron uptake protein of the intestinal brush border. Both schistosome isoforms displayed functional iron uptake by rescuing growth in a yeast strain deficient in iron uptake (fet3fet4). Interestingly schistosome DMT1 was localised to the tegument and not the gastrodermis of adult parasites, suggesting surface mediated iron uptake across the tegument. In physiological conditions, iron is abundant as largely insoluble ferric iron and hence ferric reductases are an essential component of iron uptake, reducing iron to the soluble ferrous form. Cytochromes b561 (Cyts-b561) are a family of ascorbate reducing transmembrane proteins found in most eukaryotic cells. Recent observations that Cyts-b561 may be involved in iron metabolism have opened new perspectives for their physiological function. Here, I have identified a new member of the cytochrome b561 family in Schistosoma japonicum that localises to the tegument of this trematode. Expression of the SjCytb561 in a Saccharomyces cerevisiae mutant that lacks plasma membrane ferrireductase activity (fre1fre2) was able to rescue the growth defect in iron deficient conditions, suggesting involvement in iron metabolism. Plasma membrane ferrireductase activities were also quantified using intact transformed yeast cells. These data further support the hypothesised tegumental uptake of host iron. Further, I have identified a putative schistosome transferrin. In mammals, transferrin is a glycoprotein responsible for binding and transporting iron in the bloodstream and delivering iron into cells via a specific transferrin receptor. Preliminary characterisation of the schistosome transferrin sequence has revealed it does not contain all the conserved amino acid residues associated with iron binding, with conservation seen only in the C-terminal lobe, not in both the N and C-lobes observed in mammalian transferrins. This difference makes it unclear whether the schistosome transferrin shares functional homology with its mammalian counterpart. In addition, no transferrin receptor has been identified to support an iron trafficking and uptake function, nor would this function be expected in an acoelomate organism. Further characterisation and localisation of this protein is required to elucidate its biological significance and function. The tegumental location of both the SjDMT1 and the SjCytb561 for the uptake of host iron make it possible to consider these proteins as potential vaccine candidates. A preliminary vaccination study with these proteins elicited only low to moderate protection from infection, and further studies are required to fully assess their potential. The data presented in this thesis provide evidence for surface-mediated uptake of iron by adult schistosomes, and represent the first characterisation of iron uptake proteins in any helminths. These studies show a novel method of iron uptake in schistosomes, and contribute to our understanding of how these parasites are able to survive and thrive by scavenging nutrients, in this case iron, from the host organism.

Swine dysentery (SD) is an important endemic infection in many piggeries, and control can be problematic. In this study, the gene encoding a 29.7 kDa outer membrane lipoprotein of the causative intestinal spirochaete Brachyspira hyodysenteriae, was identified and sequenced. An 816 bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site, and putative –10 and –35 promoter regions upstream from the start of the ORF. The 29.7 kDa outer membrane lipoprotein was designated Bhlp29.7 and the encoding gene named bhlp29.7. The amino acid sequence of Bhlp29.7 included a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. In silico analysis of this protein together with lipidation studies further supported its probable outer membrane localisation. Comparison of the Bhlp29.7 sequence with public sequence databases showed that it had up to 40% similarity with the D-methionine substrate-binding outer membrane lipoprotein (MetQ) of a number of bacterial pathogens. The Bhlp29.7 gene was detected in all 48 strains of B. hyodysenteriae examined, and in Brachyspira innocens strain B256T, but not in 10 other strains of B. innocens or in 42 strains of other Brachyspira spp. The gene was sequenced from B. innocens strain B256T and from 11 strains of B. hyodysenteriae. The B. hyodysenteriae genes shared 97.9-100% nucleotide sequence identity and had 97.5-99.5% identity with the gene of B. innocens strain B256T. The Bhlp29.7 gene was subsequently cloned and expressed as a histidine fusion protein in an Escherichia coli expression system. An ELISA test using recombinant his-tagged Bhlp29.7 (His6-Bhlp29.7) as the detecting antigen was developed and evaluated. The threshold value of the test was chosen to provide a highly stringent assessment of the disease status of a herd. The sensitivity and specificity of the test was 100%. When the test was applied to sera from eight herds with suspected SD, four gave ELISA values indicating that the herds were diseased. The remaining four herds gave ELISA values below the threshold value. These results indicated that the Bhlp29.7-ELISA was useful as an indirect test for exposure of a herd to B. hyodysenteriae and may be a helpful complement to current methods of SD diagnosis. Recombinant His6-Bhlp29.7 was evaluated as a vaccine subunit for prevention of SD. The His6-Bhlp29.7 was shown to be immunogenic in mice following two intramuscular injections. Vaccination of mice with His6-Bhlp29.7 provided full protection after oral challenge with B. hyodysenteriae. In two experiments, intramuscular and oral vaccination of pigs with the His6-Bhlp29.7 resulted in a 50% reduction in incidence of SD compared to unvaccinated control pigs (P=0.047). This is the first subunit vaccine shown to provide pigs with protection from SD. Further work is needed to optimise delivery routes and adjuvants for commercial development of the vaccine.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Adelaide, Dept. of Paediatrics, 1989. / Includes bibliographical references (leaves [177-194]).

Thesis (PH.D.)--University of Wollongong, 2004. / Typescript. Bibliographical references: leaf 231-250.

Senior honors Thesis--University of Michigan, 1999. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (p. 103-106).

Thesis (M.Med.Sc.)--University of Hong Kong, 2002. / Includes bibliographical references. Also available in print.