CTA1-DD is an effective adjuvant for targeting anti-chlamydial immunity to the murine genital mucosa

Chlamydia trachomatis is a significant human pathogen with potentially severe disease sequelae in the genital tract, including infertility. A successful vaccine will need to effectively target immunity to the genital mucosa. Intranasal immunisation with cholera toxin (CT) can target immunity to the genital tract, but has the potential to cause neurological side effects. CTA1-DD is a non-toxic potent mucosal adjuvant which combines the enzymatic properties of CT, with a B cell targeting moiety. Here, we demonstrate that intranasal immunisation with CTA1-DD and chlamydial Major Outer Membrane Protein (MOMP) results in the induction of neutralising systemic and mucosal antibodies, and reduces the level of chlamydial shedding following intravaginal challenge with Chlamydia muridarum. Thus, CTA1-DD is an effective adjuvant for vaccine development against Chlamydia trachomatis, and possibly also a range of other genital pathogens.

Modeling the impact of potential vaccines on epidemics of sexually transmitted chlamydia trachomatis infection

Background. We investigated the likely impact of vaccines on the prevalence of and morbidity due to Chlamydia trachomatis (chlamydia) infections in heterosexual populations. Methods.An individual‐based mathematical model of chlamydia transmission was developed and linked to the infection course in chlamydia‐infected individuals. The model describes the impact of a vaccine through its effect on the chlamydial load required to infect susceptible individuals (the “critical load”), the load in infected individuals, and their subsequent infectiousness. The model was calibrated using behavioral, biological, and clinical data. Results.A fully protective chlamydia vaccine administered before sexual debut can theoretically eliminate chlamydia epidemics within 20 years. Partially effective vaccines can still greatly reduce the incidence of chlamydia infection. Vaccines should aim primarily to increase the critical load in susceptible individuals and secondarily to decrease the peak load and/or the duration of infection in vaccinated individuals who become infected. Vaccinating both sexes has a beneficial impact on chlamydia‐related morbidity, but targeting women is more effective than targeting men. Conclusions.Our findings can be used in laboratory settings to evaluate vaccine candidates in animal models, by regulatory bodies in the promotion of candidates for clinical trials, and by public health authorities in deciding on optimal intervention strategies.

Poly-immunoglobulin receptor-mediated transport of IgA into the male genital tract is important for clearance of chlamydia muridarum infection

Problem: Chlamydia trachomatis is the most common sexually transmitted infection worldwide. While infection in females requires a Th1 response for clearance, such a response in males may disrupt the immune privileged nature of the male reproductive tract, potentially contributing to infertility. Method of study: We investigated the role of IgA in protection against an intrapenile Chlamydia muridarum infection of C57BL/6 and pIgR−/− mice. Results: Here, we show that the poly immunoglobulin receptor is the main pathway for IgA transport into the male reproductive tract. The high levels of IgA seen in prostatic fluid of wild-type mice correlate with reduction in chlamydial infection both in vitro and in vivo. Conclusion: These findings indicate that a Chlamydia vaccine that induces neutralizing IgA in the prostate will aid in the protection against infection in males.

Morphologic and genomic characterisation of the koala Chlamydia pneumoniae strain

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of upper and lower respiratory tract infections. In more recent years there has been increasing evidence to suggest a link between C. pneumoniae and chronic diseases in humans, including atherosclerosis, stroke and Alzheimer’s disease. C. pneumoniae human strains show little genetic variation, indicating that the human-derived strain originated from a common ancestor in the recent past. Despite extensive information on the genetics and morphology processes of the human strain, knowledge concerning many other hosts (including marsupials, amphibians, reptiles and equines) remains virtually unexplored. The koala (Phascolarctos cinereus) is a native Australian marsupial under threat due to habitat loss, predation and disease. Koalas are very susceptible to chlamydial infections, most commonly affecting the conjunctiva, urogenital tract and/or respiratory tract. To address this gap in the literature, the present study (i) provides a detailed description of the morphologic and genomic architecture of the C. pneumoniae koala (and human) strain, and shows that the koala strain is microscopically, developmentally and genetically distinct from the C. pneumoniae human strain, and (ii) examines the genetic relationship of geographically diverse C. pneumoniae isolates from human, marsupial, amphibian, reptilian and equine hosts, and identifies two distinct lineages that have arisen from animal-to-human cross species transmissions. Chapter One of this thesis explores the scientific problem and aims of this study, while Chapter Two provides a detailed literature review of the background in this field of work. Chapter Three, the first results chapter, describes the morphology and developmental stages of C. pneumoniae koala isolate LPCoLN, as revealed by fluorescence and transmission electron microscopy. The profile of this isolate, when cultured in HEp-2 human epithelial cells, was quite different to the human AR39 isolate. Koala LPCoLN inclusions were larger; the elementary bodies did not have the characteristic pear-shaped appearance, and the developmental cycle was completed within a shorter period of time (as confirmed by quantitative real-time PCR). These in vitro findings might reflect biological differences between koala LPCoLN and human AR39 in vivo. Chapter Four describes the complete genome sequence of the koala respiratory pathogen, C. pneumoniae LPCoLN. This is the first animal isolate of C. pneumoniae to be fully-sequenced. The genome sequence provides new insights into genomic ‘plasticity’ (organisation), evolution and biology of koala LPCoLN, relative to four complete C. pneumoniae human genomes (AR39, CWL029, J138 and TW183). Koala LPCoLN contains a plasmid that is not shared with any of the human isolates, there is evidence of gene loss in nucleotide salvage pathways, and there are 10 hot spot genomic regions of variation that were previously not identified in the C. pneumoniae human genomes. Sequence (partial-length) from a second, independent, wild koala isolate (EBB) at several gene loci confirmed that the koala LPCoLN isolate was representative of a koala C. pneumoniae strain. The combined sequence data provides evidence that the C. pneumoniae animal (koala LPCoLN) genome is ancestral to the C. pneumoniae human genomes and that human infections may have originated from zoonotic infections. Chapter Five examines key genome components of the five C. pneumoniae genomes in more detail. This analysis reveals genomic features that are shared by and/or contribute to the broad ecological adaptability and evolution of C. pneumoniae. This analysis resulted in the identification of 65 gene sequences for further analysis of intraspecific variation, and revealed some interesting differences, including fragmentation, truncation and gene decay (loss of redundant ancestral traits). This study provides valuable insights into metabolic diversity, adaptation and evolution of C. pneumoniae. Chapter Six utilises a subset of 23 target genes identified from the previous genomic comparisons and makes a significant contribution to our understanding of genetic variability among C. pneumoniae human (11) and animal (6 amphibian, 5 reptilian, 1 equine and 7 marsupial hosts) isolates. It has been shown that the animal isolates are genetically diverse, unlike the human isolates that are virtually clonal. More convincing evidence that C. pneumoniae originated in animals and recently (in the last few hundred thousand years) crossed host species to infect humans is provided in this study. It is proposed that two animal-to-human cross species events have occurred in the context of the results, one evident by the nearly clonal human genotype circulating in the world today, and the other by a more animal-like genotype apparent in Indigenous Australians. Taken together, these data indicate that the C. pneumoniae koala LPCoLN isolate has morphologic and genomic characteristics that are distinct from the human isolates. These differences may affect the survival and activity of the C. pneumoniae koala pathogen in its natural host, in vivo. This study, by utilising the genetic diversity of C. pneumoniae, identified new genetic markers for distinguishing human and animal isolates. However, not all C. pneumoniae isolates were genetically diverse; in fact, several isolates were highly conserved, if not identical in sequence (i.e. Australian marsupials) emphasising that at some stage in the evolution of this pathogen, there has been an adaptation/s to a particular host, providing some stability in the genome. The outcomes of this study by experimental and bioinformatic approaches have significantly enhanced our knowledge of the biology of this pathogen and will advance opportunities for the investigation of novel vaccine targets, antimicrobial therapy, or blocking of pathogenic pathways.

Rural Poultry in the Developing world

In 2007 I travelled to Mozambique and Tanzania to photographically document the work undertaken by a group of Australians who are working to bring a self sufficient lifestyle back to the HIV Aids stricken communities of Africa. University of Queensland veterinary researchers have developed a vaccination that can eradicate disease from local rural poultry. The Kyeema Foundation is working in country to supply this vaccine to families battling HIV AIDS and to teach local residents how to successfully implement it. QUT, AusAID and the Kyeema Foundation funded the trip.

Pandemic (H1N1) 2009 Influenza Outbreak in Australia: Impact on Emergency Departments.

Executive summary Objective: The aims of this study were to identify the impact of Pandemic (H1N1) 2009 Influenza on Australian Emergency Departments (EDs) and their staff, and to inform planning, preparedness, and response management arrangements for future pandemics, as well as managing infectious patients presenting to EDs in everyday practice. Methods This study involved three elements: 1. The first element of the study was an examination of published material including published statistics. Standard literature research methods were used to identify relevant published articles. In addition, data about ED demand was obtained from Australian Government Department of Health and Ageing (DoHA) publications, with several state health departments providing more detailed data. 2. The second element of the study was a survey of Directors of Emergency Medicine identified with the assistance of the Australasian College for Emergency Medicine (ACEM). This survey retrieved data about demand for ED services and elicited qualitative comments on the impact of the pandemic on ED management. 3. The third element of the study was a survey of ED staff. A questionnaire was emailed to members of three professional colleges—the ACEM; the Australian College of Emergency Nursing (ACEN); and the College of Emergency Nursing Australasia (CENA). The overall response rate for the survey was 18.4%, with 618 usable responses from 3355 distributed questionnaires. Topics covered by the survey included ED conditions during the (H1N1) 2009 influenza pandemic; information received about Pandemic (H1N1) 2009 Influenza; pandemic plans; the impact of the pandemic on ED staff with respect to stress; illness prevention measures; support received from others in work role; staff and others’ illness during the pandemic; other factors causing ED staff to miss work during the pandemic; and vaccination against Pandemic (H1N1) 2009 Influenza. Both qualitative and quantitative data were collected and analysed. Results: The results obtained from Directors of Emergency Medicine quantifying the impact of the pandemic were too limited for interpretation. Data sourced from health departments and published sources demonstrated an increase in influenza-like illness (ILI) presentations of between one and a half and three times the normal level of presentations of ILIs. Directors of Emergency Medicine reported a reasonable level of preparation for the pandemic, with most reporting the use of pandemic plans that translated into relatively effective operational infection control responses. Directors reported a highly significant impact on EDs and their staff from the pandemic. Growth in demand and related ED congestion were highly significant factors causing distress within the departments. Most (64%) respondents established a ‘flu clinic’ either as part of Pandemic (H1N1) 2009 Influenza Outbreak in Australia: Impact on Emergency Departments. the ED operations or external to it. They did not note a significantly higher rate of sick leave than usual. Responses relating to the impact on staff were proportional to the size of the colleges. Most respondents felt strongly that Pandemic (H1N1) 2009 Influenza had a significant impact on demand in their ED, with most patients having low levels of clinical urgency. Most respondents felt that the pandemic had a negative impact on the care of other patients, and 94% revealed some increase in stress due to lack of space for patients, increased demand, and filling staff deficits. Levels of concern about themselves or their family members contracting the illness were less significant than expected. Nurses displayed significantly higher levels of stress overall, particularly in relation to skill-mix requirements, lack of supplies and equipment, and patient and patients’ family aggression. More than one-third of respondents became ill with an ILI. Whilst respondents themselves reported taking low levels of sick leave, respondents cited difficulties with replacing absent staff. Ranked from highest to lowest, respondents gained useful support from ED colleagues, ED administration, their hospital occupational health department, hospital administration, professional colleges, state health department, and their unions. Respondents were generally positive about the information they received overall; however, the volume of information was considered excessive and sometimes inconsistent. The media was criticised as scaremongering and sensationalist and as being the cause of many unnecessary presentations to EDs. Of concern to the investigators was that a large proportion (43%) of respondents did not know whether a pandemic plan existed for their department or hospital. A small number of staff reported being redeployed from their usual workplace for personal risk factors or operational reasons. As at the time of survey (29 October –18 December 2009), 26% of ED staff reported being vaccinated against Pandemic (H1N1) 2009 Influenza. Of those not vaccinated, half indicated they would ‘definitely’ or ‘probably’ not get vaccinated, with the main reasons being the vaccine was ‘rushed into production’, ‘not properly tested’, ‘came out too late’, or not needed due to prior infection or exposure, or due to the mildness of the disease. Conclusion: Pandemic (H1N1) 2009 Influenza had a significant impact on Australian Emergency Departments. The pandemic exposed problems in existing plans, particularly a lack of guidelines, general information overload, and confusion due to the lack of a single authoritative information source. Of concern was the high proportion of respondents who did not know if their hospital or department had a pandemic plan. Nationally, the pandemic communication strategy needs a detailed review, with more engagement with media networks to encourage responsible and consistent reporting. Also of concern was the low level of immunisation, and the low level of intention to accept vaccination. This is a problem seen in many previous studies relating to seasonal influenza and health care workers. The design of EDs needs to be addressed to better manage infectious patients. Significant workforce issues were confronted in this pandemic, including maintaining appropriate staffing levels; staff exposure to illness; access to, and appropriate use of, personal protective equipment (PPE); and the difficulties associated with working in PPE for prolonged periods. An administrative issue of note was the reporting requirement, which created considerable additional stress for staff within EDs. Peer and local support strategies helped ensure staff felt their needs were provided for, creating resilience, dependability, and stability in the ED workforce. Policies regarding the establishment of flu clinics need to be reviewed. The ability to create surge capacity within EDs by considering staffing, equipment, physical space, and stores is of primary importance for future pandemics.

Tissue engineered prefabricated vascularized flaps

BACKGROUND.: Microvascular free tissue transfer has become increasingly popular in the reconstruction of head and neck defects, but it also has its disadvantages. Tissue engineering allows the generation of neo-tissue for implantation, but these tissues are often avascular. We propose to combine tissue-engineering techniques together with flap prefabrication techniques to generate a prefabricated vascularized soft tissue flap. METHODS: Human dermal fibroblasts (HDFs) labeled with fluorescein diacetate were static seeded onto polylactic-co-glycolic acid-collagen (PLGA-c) mesh. Controls were plain PLGA-c mesh. The femoral artery and vein of the nude rat was ligated and used as a vascular carrier for the constructs. After 4 weeks of implantation, the constructs were assessed by gross morphology, routine histology, Masson trichrome, and cell viability determined by green fluorescence. RESULTS: All the constructs maintained their initial shape and dimensions. Angiogenesis was evident in all the constructs with neo-capillary formation within the PLGA-c mesh seen. HDFs proliferated and filled the interyarn spaces of the PLGA-c mesh, while unseeded PLGA-c mesh remained relatively acellular. Cell tracer study indicated that the seeded HDFs remained viable and closely associated to remaining PLGA-c fibers. Collagen formation was more abundant in the constructs seeded with HDFs. CONCLUSIONS: PLGA-c, enveloped by a cell sheet composed of fibroblasts, can serve as a suitable scaffold for generation of a soft tissue flap. A ligated arteriovenous pedicle can serve as a vascular carrier for the generation of a tissue engineered vascularized flap.

In vitro characterization of natural and synthetic dermal matrices cultured with human dermal fibroblasts

The ideal dermal matrix should be able to provide the right biological and physical environment to ensure homogenous cell and extracellular matrix (ECM) distribution, as well as the right size and morphology of the neo-tissue required. Four natural and synthetic 3D matrices were evaluated in vitro as dermal matrices, namely (1) equine collagen foam, TissuFleece®, (2) acellular dermal replacement, Alloderm®, (3) knitted poly(lactic-co-glycolic acid) (10:90)–poly(-caprolactone) (PLGA–PCL) mesh, (4) chitosan scaffold. Human dermal fibroblasts were cultured on the specimens over 3 weeks. Cell morphology, distribution and viability were assessed by electron microscopy, histology and confocal laser microscopy. Metabolic activity and DNA synthesis were analysed via MTS metabolic assay and [3H]-thymidine uptake, while ECM protein expression was determined by immunohistochemistry. TissuFleece®, Alloderm® and PLGA–PCL mesh supported cell attachment, proliferation and neo-tissue formation. However, TissuFleece® contracted to 10% of the original size while Alloderm® supported cell proliferation predominantly on the surface of the material. PLGA–PCL mesh promoted more homogenous cell distribution and tissue formation. Chitosan scaffolds did not support cell attachment and proliferation. These results demonstrated that physical characteristics including porosity and mechanical stability to withstand cell contraction forces are important in determining the success of a dermal matrix material.

Chlamydia muridarum major outer membrane protein-specific antibodies inhibit in vitro infection but enhance pathology in vivo

PROBLEM Chlamydia trachomatis is a significant worldwide health problem, and the often-asymptomatic disease can result in infertility. To develop a successful vaccine, a complete understanding of the immune response to chlamydial infection and development of genital tract pathology is required. METHOD OF STUDY We utilized the murine genital model of chlamydial infection. Mice were immunized with chlamydial major outer membrane protein, and vaginal lavage was assessed for the presence of neutralizing antibodies. These samples were then pre-incubated with Chlamydia muridarum and administered to the vaginal vaults of immune-competent female BALB/c mice to determine the effect on infection. RESULTS The administration of C. muridarum in conjunction with neutralizing antibodies reduced the numbers of mice infected, but a surprising finding was that this accelerated the development of severe oviduct pathology. CONCLUSION Antibodies play an under-recognized role in chlamydial infection and pathology development, which possibly involves interaction with Th1 immunity.

Rodent blood-stage Plasmodium survive in dendritic cells that infect naive mice.

Plasmodium spp. parasites cause malaria in 300 to 500 million individuals each year. Disease occurs during the blood-stage of the parasite’s life cycle, where the parasite is thought to replicate exclusively within erythrocytes. Infected individuals can also suffer relapses after several years, from Plasmodium vivax and Plasmodium ovale surviving in hepatocytes. Plasmodium falciparum and Plasmodium malariae can also persist after the original bout of infection has apparently cleared in the blood, suggesting that host cells other than erythrocytes (but not hepatocytes) may harbor these blood-stage parasites, thereby assisting their escape from host immunity. Using blood stage transgenic Plasmodium berghei-expressing GFP (PbGFP) to track parasites in host cells, we found that the parasite had a tropism for CD317+ dendritic cells. Other studies using confocal microscopy, in vitro cultures, and cell transfer studies showed that blood-stage parasites could infect, survive, and replicate within CD317+ dendritic cells, and that small numbers of these cells released parasites infectious for erythrocytes in vivo. These data have identified a unique survival strategy for blood-stage Plasmodium, which has significant implications for understanding the escape of Plasmodium spp. from immune-surveillance and for vaccine development.

Transcutaneous immunization with novel lipid-based adjuvants induces protection against gastric Helicobacter pylori infection

The development of vaccines to combat pathogens that infect across mucosal surfaces has been a major goal of vaccine research. Successful mucosal vaccination requires the co-administration of adjuvants that can overcome the state of immune tolerance normally associated with mucosal application of proteins. In the case of oral immunization, delivery systems are also required to protect vaccine antigens against destruction by gastric pH and digestive enzymes. Furthermore, adjuvants used for mucosal delivery must be free of neurotoxic effects like those induced by the commonly used experimental mucosal adjuvant cholera toxin. Maintenance of the "cold chain" is also essential for the effectiveness of any vaccine and adjuvants/delivery systems that enhance the stability of a vaccine would offer a significant advantage. Needle-free methods of vaccination that induce protective immunity at multiple mucosal surfaces are also desirable for rapid vaccination of large populations. In the present study we show that transcutaneous immunization (TCI) using Lipid C, a novel lipid-based matrix originally developed for oral immunization, containing soluble Helicobacter sonicate significantly reduces the gastric bacterial burden in mice following gastric challenge with live Helicobacter pylori. Protection is associated with the production of splenic gamma interferon and gastric IgA and was achieved without the co-administration of potent and potentially toxic adjuvants, although protection was further enhanced by inclusion of CpG-ODN and cholera toxin in the lipid delivery system.