Immunogenicity and protective efficacy of an anti-Streptococcus pyogenes vaccine candidate in multiple animal species

Streptococcus pyogenes, also known as Group A Streptococcus (GAS) has been associated with a range of diseases from the mild pharyngitis and pyoderma to more severe invasive infections such as streptococcal toxic shock. GAS also causes a number of non-suppurative post-infectious diseases such as rheumatic fever, rheumatic heart disease and glomerulonephritis. The large extent of GAS disease burden necessitates the need for a prophylactic vaccine that could target the diverse GAS emm types circulating globally. Anti-GAS vaccine strategies have focused primarily on the GAS M-protein, an extracellular virulence factor anchored to GAS cell wall. As opposed to the hypervariable N-terminal region, the C-terminal portion of the protein is highly conserved among different GAS emm types and is the focus of a leading GAS vaccine candidate, J8-DT/alum. The vaccine candidate J8-DT/alum was shown to be immunogenic in mice, rabbits and the non-human primates, hamadryas baboons. Similar responses to J8-DT/alum were observed after subcutaneous and intramuscular immunization with J8-DT/alum, in mice and in rabbits. Further assessment of parameters that may influence the immunogenicity of J8-DT demonstrated that the immune responses were identical in male and female mice and the use of alum as an adjuvant in the vaccine formulation significantly increased its immunogenicity, resulting in a long-lived serum IgG response. Contrary to the previous findings, the data in this thesis indicates that a primary immunization with J8-DT/alum (50ƒÊg) followed by a single boost is sufficient to generate a robust immune response in mice. As expected, the IgG response to J8- DT/alum was a Th2 type response consisting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. Intramuscular vaccination of rabbits with J8-DT/alum demonstrated that an increase in the dose of J8-DT/alum up to 500ƒÊg does not have an impact on the serum IgG titers achieved. Similar to the immune response in mice, immunization with J8-DT/alum in baboons also established that a 60ƒÊg dose compared to either 30ƒÊg or 120ƒÊg was sufficient to generate a robust immune response. Interestingly, mucosal infection of naive baboons with a M1 GAS strain did not induce a J8-specific serum IgG response. As J8-DT/alum mediated protection has been previously reported to be due to the J8- specific antibody formed, the efficacy of J8-DT antibodies was determined in vitro and in vivo. In vitro opsonization and in vivo passive transfer confirmed the protective potential of J8-DT antibodies. A reduction in the bacterial burden after challenge with a bioluminescent M49 GAS strain in mice that were passively administered J8-DT IgG established that protection due to J8-DT was mediated by antibodies. The GAS burden in infected mice was monitored using bioluminescent imaging in addition to traditional CFU assays. Bioluminescent GAS strains including the ‘rheumatogenic’ M1 GAS could not be generated due to limitations with transformation of GAS, however, a M49 GAS strain was utilized during BLI. The M49 serotype is traditionally a ‘nephritogenic’ serotype associated with post-streptococcal glomerulonephritis. Anti- J8-DT antibodies now have been shown to be protective against multiple GAS strains such as M49 and M1. This study evaluated the immunogenicity of J8-DT/alum in different species of experimental animals in preparation for phase I human clinical trials and provided the ground work for the development of a rapid non-invasive assay for evaluation of vaccine candidates.

The transformation of banana with potential virus resistance genes

One approach to reducing the yield losses caused by banana viral diseases is the use of genetic engineering and pathogen-derived resistance strategies to generate resistant cultivars. The development of transgenic virus resistance requires an efficient banana transformation method, particularly for commercially important 'Cavendish' type cultivars such as 'Grand Nain'. Prior to this study, only two examples of the stable transformation of banana had been reported, both of which demonstrated the principle of transformation but did not characterise transgenic plants in terms of the efficiency at which individual transgenic lines were generated, relative activities of promoters in stably transformed plants, and the stability of transgene expression. The aim of this study was to develop more efficient transformation methods for banana, assess the activity of some commonly used and also novel promoters in stably transformed plants, and transform banana with genes that could potentially confer resistance to banana bunchy top nanovirus (BBTV) and banana bract mosaic potyvirus (BBrMV). A regeneration system using immature male flowers as the explant was established. The frequency of somatic embryogenesis in male flower explants was influenced by the season in which the inflorescences were harvested. Further, the media requirements of various banana cultivars in respect to the 2,4-D concentration in the initiation media also differed. Following the optimisation of these and other parameters, embryogenic cell suspensions of several banana (Musa spp.) cultivars including 'Grand Nain' (AAA), 'Williams' (AAA), 'SH-3362' (AA), 'Goldfinger' (AAAB) and 'Bluggoe' (ABB) were successfully generated. Highly efficient transformation methods were developed for both 'Bluggoe' and 'Grand Nain'; this is the first report of microprojectile bombardment transformation of the commercially important 'Grand Nain' cultivar. Following bombardment of embryogenic suspension cells, regeneration was monitored from single transfom1ed cells to whole plants using a reporter gene encoding the green fluorescent protein (gfp). Selection with kanamycin enabled the regeneration of a greater number of plants than with geneticin, while still preventing the regeneration of non-transformed plants. Southern hybridisation confirmed the neomycin phosphotransferase gene (npt II) was stably integrated into the banana genome and that multiple transgenic lines were derived from single bombardments. The activity, stability and tissue specificity of the cauliflower mosaic virus 358 (CaMV 35S) and maize polyubiquitin-1 (Ubi-1) promoters were examined. In stably transformed banana, the Ubi-1 promoter provided approximately six-fold higher p-glucuronidase (GUS) activity than the CaMV 35S promoter, and both promoters remained active in glasshouse grown plants for the six months they were observed. The intergenic regions ofBBTV DNA-I to -6 were isolated and fused to either the uidA (GUS) or gfjJ reporter genes to assess their promoter activities. BBTV promoter activity was detected in banana embryogenic cells using the gfp reporter gene. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that generated by the maize Ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter (BT6.1) was restricted to the phloem of leaves and roots, stomata and root meristems. The activity of the BT6.1 promoter was enhanced by the inclusion of intron-containing fragments derived from the maize Ubi-1, rice Act-1, and sugarcane rbcS 5' untranslated regions in GUS reporter gene constructs. In transient assays in banana, the rice Act-1 and maize Ubi-1 introns provided the most significant enhancement, increasing expression levels 300-fold and 100-fold, respectively. The sugarcane rbcS intron increased expression about 10-fold. In stably transformed banana plants, the maize Ubi-1 intron enhanced BT6.1 promoter activity to levels similar to that of the CaMV 35S promoter, but did not appear to alter the tissue specificity of the promoter. Both 'Grand Nain' and 'Bluggoe' were transformed with constructs that could potentially confer resistance to BBTV and BBrMV, including constructs containing BBTV DNA-1 major and internal genes, BBTV DNA-5 gene, and the BBrMV coat protein-coding region all under the control of the Ubi-1 promoter, while the BT6 promoter was used to drive the npt II selectable marker gene. At least 30 transgenic lines containing each construct were identified and replicates of each line are currently being generated by micropropagation in preparation for virus challenge.

Gender equality in national politics : the views of Australian male politicians

Studies of gender and politics have typically been studies of women and politics. In contrast, this paper places men at the centre of its inquiry by drawing on interviews with 15 current federal male politicians. Of concern is exploring the ways in which men conceptualise the question of gender equity in the Australian parliament. Three frameworks are identified in the men's narratives. These are that the parliament is a masculinised space but that this is unavoidable; that the parliament is now feminised and women are advantaged; and that the parliament is gender neutral and gender is irrelevant. It is argued that collectively these framing devices operate to mask the many constraints which exist to marginalise women from political participation and undermine attempts to address women's political disadvantage as political participants. The paper concludes by highlighting the significance of the paper beyond the Australian context and calling for further research which names and critiques political men and their discourses on gender and parliamentary practices and processes.

Female genital mutilation : Australian law, policy and practical challenges for doctors

As long ago as 1994, the Family Law Council accepted it was likely that female genital mutilation (FGM) was being conducted in Australia. In 2010, doctors and hospitals reported that it is being conducted and that they are seeing female patients who have experienced FGM. It is impossible to obtain precise data about the extent to which it is performed in Australia, but data indicates that FGM is a relevant issue for Australian medical practitioners. The medical profession has an interest in this topic because its members may be asked to conduct FGM, advise those considering it, or treat female patients with effects from the practice. This article provides a background on the practice of FGM, explains the relevant Australian law, considers whether the current legal prohibition on FGM is justified, and discusses the practical challenges facing individual practitioners and the profession. To inform further discussions about methods of responding to demand for FGM, reference is made to strategies being promoted in African nations to abolish this cultural practice.

Throwing frame and performance of elite male seated shot-putters

Seated shot-putters rely on a customized assistive device called a throwing frame. Currently, the construction of each individual throwing frame is mainly driven by an empirical approach. One way to refine the conception is to improve the basic understanding of performance of seated shot-putters. The relationship between performance and throwing technique has been well described. Remarkably, the relationship between performance and throwing frame characteristics has received limited attention. The primary objective of this study was to present a cataloguing of characteristics of throwing frames used by seated shot-putters. This cataloguing consisted of defining and grouping 26 characteristics into three main categories (i.e., whole body, foot and upper limb specific characteristics) and seven sub-categories. The secondary objective of this study was to provide raw characterisations of the throwing frames for a group of athletes who participated in a world-class event. The characterisation consisted of describing the characteristics of each throwing frame. Potential relationships between characteristics, performance and classification were also identified. The cataloguing was achieved using a 6-step heuristic approach, involving expert opinions and the analysis of 215 attempts produced by 55 male athletes during the 2006 IPC Athletics World Championships. The distribution of samples across characteristics suggested a relevant level of comprehensiveness for the proposed cataloguing. The raw data, the profile of best athletes and the frequency of characteristics provided key benchmark information for construction of a throwing frame as well as coaching, classification and officiating. Analysis of data sets relating to characteristics, performance and classification were inconclusive.

Chlamydial infection of immune cells : altered function and implications for disease

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects the genital and ocular mucosa of humans, causing infections that can lead to pelvic inflammatory disease, infertility, and blinding trachoma. C. pneumoniae is a respiratory pathogen that is the cause of 12–15% of community-acquired pneumonia. Both chlamydial species were believed to be restricted to the epithelia of the genital, ocular, and respiratory mucosa; however, increasing evidence suggests that both these pathogens can be isolated from peripheral blood of both healthy individuals and patients with inflammatory conditions such as coronary artery disease and asthma. Chlamydia can also be isolated from brain tissues of patients with degenerative neurological disorders such as Alzheimer’s disease and multiple sclerosis, and also from certain lymphomas. An increasing number of in vitro studies suggest that some chlamydial species can infect immune cells, at least at low levels. These infections may alter immune cell function in a way that promotes chlamydial persistence in the host and contributes to the progression of several chronic inflammatory diseases. In this paper, we review the evidence for the growth of Chlamydia in immune cells, particularly monocytes/macrophages and dendritic cells, and describe how infection may affect the function of these cells.

A mathematical model of chlamydial infection incorporating movement of chlamydial particles

We present a spatiotemporal mathematical model of chlamydial infection, host immune response and spatial movement of infectious particles. The re- sulting partial differential equations model both the dynamics of the infection and changes in infection profile observed spatially along the length of the host genital tract. This model advances previous chlamydia modelling by incorporating spatial change, which we also demonstrate to be essential when the timescale for movement of infectious particles is equal to, or shorter than, the developmental cycle timescale. Numerical solutions and model analysis are carried out, and we present a hypothesis regarding the potential for treatment and prevention of infection by increasing chlamydial particle motility.

Bayesian methodology for genetics of complex diseases

Genetic research of complex diseases is a challenging, but exciting, area of research. The early development of the research was limited, however, until the completion of the Human Genome and HapMap projects, along with the reduction in the cost of genotyping, which paves the way for understanding the genetic composition of complex diseases. In this thesis, we focus on the statistical methods for two aspects of genetic research: phenotype definition for diseases with complex etiology and methods for identifying potentially associated Single Nucleotide Polymorphisms (SNPs) and SNP-SNP interactions. With regard to phenotype definition for diseases with complex etiology, we firstly investigated the effects of different statistical phenotyping approaches on the subsequent analysis. In light of the findings, and the difficulties in validating the estimated phenotype, we proposed two different methods for reconciling phenotypes of different models using Bayesian model averaging as a coherent mechanism for accounting for model uncertainty. In the second part of the thesis, the focus is turned to the methods for identifying associated SNPs and SNP interactions. We review the use of Bayesian logistic regression with variable selection for SNP identification and extended the model for detecting the interaction effects for population based case-control studies. In this part of study, we also develop a machine learning algorithm to cope with the large scale data analysis, namely modified Logic Regression with Genetic Program (MLR-GEP), which is then compared with the Bayesian model, Random Forests and other variants of logic regression.

Elevated plasma fibronectin levels associated with venous thromboembolism

Elevated plasma fibronectin levels occur in various clinical states including arterial disease. Increasing evidence suggests that atherothrombosis and venous thromboembolism (VTE) share common risk factors. To assess the hypothesis that high plasma fibronectin levels are associated with VTE, we compared plasma fibronectin levels in the Scripps Venous Thrombosis Registry for 113 VTE cases vs. age and sex matched controls. VTE cases had significantly higher mean fibronectin concentration compared to controls (127% vs. 103%, p<0.0001); the difference was greater for idiopathic VTE cases compared to secondary VTE cases (133% vs. 120%, respectively). Using a cut-off of >90% of the control values, the odds ratio (OR) for association of VTE for fibronectin plasma levels above the 90th percentile were 9.37 (95% CI 2.73-32.2; p<0.001) and this OR remained significant after adjustment for sex, age, body mass index (BMI), factor V Leiden and prothrombin nt20210A (OR 7.60, 95% CI 2.14-27.0; p=0.002). In particular, the OR was statistically significant for idiopathic VTE before and after these statistical adjustments. For the total male cohort, the OR was significant before and after statistical adjustments and was not significant for the total female cohort. In summary, our results suggest that elevated plasma fibronectin levels are associated with VTE especially in males, and extend the potential association between biomarkers and risk factors for arterial atherothrombosis and VTE. © 2008 Schattauer GmbH.

A comparison of the effects of a chlamydial vaccine administered during or after a C. muridarum urogenital infection of female mice

There are approximately 92 million new chlamydial infections of the genital tract in humans diagnosed each year, costing health care systems billions of dollars in treatment not only of acute infections, but also of associated inflammatory sequelae, such as pelvic inflammatory disease (PID) and ectopic pregnancy. These numbers are increasing at a steady rate and, due to the asymptomatic nature of infections, the incidence may be underestimated and the costs of treatment therefore higher. Over the previous few decades there has been a large amount of research into the development of an efficacious vaccine against genital tract chlamydial infections. The majority of this research has focused on females, due to the high rate of development of associated diseases, including PID, which can lead to ectopic pregnancy and infertility. In light of the increasing infection rates that have occurred despite the availability of antibiotics, and the asymptomatic nature of chlamydial infections, it is imperative that an efficacious vaccine that protects against infection and associated pathology be developed.

Ovarian steroid hormones: effects on immune responses and Chlamydia trachomatis infections of the female genital tract

Female sex hormones are known to regulate the adaptive and innate immune functions of the female reproductive tract. This review aims to update our current knowledge of the effects of the sex hormones estradiol and progesterone in the female reproductive tract on innate immunity, antigen presentation, specific immune responses, antibody secretion, genital tract infections caused by Chlamydia trachomatis, and vaccine-induced immunity.