Intrinsic to these objectives has been refinement of PCR protocols for the accurate and efficient isolation from genomic DNA of members of large, highly homologous multigene families using PCR amplification.
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One research theme concerns DNA sequence analysis of germline immunoglobulin variable genes. We are investigating DNA sequence variation in a small, defined family of immunoglobulin heavy chain variable genes between inbred mouse lines originating from common genetic stock but bred separately for approximately 50 years. This project is providing insight into the rate of evolutionary change in such genes. Intrinsic to these objectives has been refinement of PCR protocols for the accurate and efficient isolation from genomic DNA of members of large, highly homologous multigene families using PCR amplification. |
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A related theme concerns somatic hypermutation of immunoglobulin variable genes in B lymphocytes. A number of molecular constructs are being produced which vary the location and composition of the intronic enhancer/matrix attachment region of the mouse heavy chain locus. These constructs will be used to produce single-copy transgenic mice so that the role of the intronic enhancer/matrix attachment region in the somatic hypermutation process can be elucidated, with particular focus on participation of pre-mRNA and reverse transcription as the primary agent of sequence variation.
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This laboratory also has an ongoing interest in the biological activity and chemistry of a number of toxins including the epipolythiodioxopiperazine (ETP) class of immunosuppressive fungal toxins. This has recently involved the resolution of optical isomers of a synthetic ETP compound using chiral HPLC A number of these ETP compounds have been shown to reversibly mobilize intracellular calcium by interaction with a putative cell surface molecule via mixed disulphide formation and attempts to identify this protein are currently underway. |
A major achievement of this year was the cloning of the entire genome of MVE as a cDNA copy into a plasmid vector, which allows the in vitro production of infectious viral RNA The "infectious" cDNA clone of MVE produces viral progeny with virulence characteristics comparable to that of the parental strain. It will be used in structure/function studies on the pathogenesis and replication of MVE
We are interested in the regulation of gene expression by proteolytic processing events on the viral polyprotein. The cleavages at the junction of the viral capsid-prM proteins, which occur in an obligatory but unusual order, have been studied. Using site-directed mutagenesis in the signal sequence of prM we could override the control of processing, in vitro, but found that the changes were detrimental for virus replication. An "infectious" cDNA clone of yellow fever virus was used in this study, which highlighted the importance in virus replication of a novel mechanism of polyprotein processing-mediated regulation of flavivirus gene expression..
One of our objectives is to explore the value of live vectors for the expression of recombinant structural and nonstructural flaviviral proteins for protection against flaviviral infection. We have tested the immunogenicity and protective efficacy of prM and E protein-based subunit vaccination using "naked" DNA-based and alphavirus replicon-vectored immunisation. Both methods fully protected mice from lethal challenge with MVE
An intriguing example of host/pathogen interaction is the up-regulation of MHC class I at the cell surface of flavivirus-infected cells. In collaboration with Dr A Müllbacher (Molecular Immunology Group) we have identified the mechanism of this virus-induced phenomenon. It involves the supply of peptides across the ER membranes for association with MHC class I molecules, and impinges on the immunological memory of cytotoxic T cell responses against flavivirus infection. A major advance in this project was achieved this year with the establishment (in collaboration with Dr. F Momburg, DKFZ, Heidelberg, Germany) of an in vitro peptide translocation assay. Results from this assay supported our previous finding of flavivirus-mediated augmentation of peptide transport into the ER, and demonstrated the requirement for some functional activity of the cellular peptide transporter in this process. Thus, flaviviral gene products appear to modulate the function of the peptide transporter for antigen processing.
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The Molecular Immunology and Immunopathology Group continues to investigate basic principles concerning immunology, cell biology and parasite/host interaction. Two main lines of investigation into T cell immunity are currently actively pursued by the Group together with local, National and International collaborators. Firstly, the group has a long standing interest in T cell memory. Immunological memory underpins all vaccine strategies. |
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Secondly, the group is pursuing its interest in the mechanism of action of cytotoxic T cells. This is a close collaboration with the group led by Markus Simon at the Max Planck Institute for Immunbiology in Freiburg, Germany. Tc cells are of primary importance in the recovery of mice from infection by viruses. We use mousepox ectromelia (ECT) as one disease model as ECT is a natural mouse pathogen. Tc cells exert their effector function by two very different mechanisms, one being mediated by cytokines such as gamma interferon and interleukins and the other by cytotoxic molecules. To date two major pathways of target cell killing by cytolytic leukocytes [ mainly natural killer (NK) and Tc cells] have been described. Firstly, the granule exocytosis pathway mediated by perforin or cytolysin and serine proteases or granzymes (gzm). This is generally believed to be the dominant mechanism by which Tc and NK cells eliminate virus-infected cells. The second mechanism, called Fas- mediated pathway , requires the interaction of the fas receptor on the target cell with the fas ligand on the killer cell and is supposedly involved in immunregulation and tolerance. We have a large panel of mouse strains genetically deficient in one or more of the cytolytic effector molecules and are analysing their role in the recovery from ECT and other virus infections.
In addition we are closely collaborating with the Viral Immunology group on the immunobiology of flavivirus infection and its effect on MHC class I expression and the role of viral genes on evasion of Tc cell mediated apoptosis.
| The Cell-Cell Interaction (CCI) group has been working for a number of years in two major areas of basic research, namely (i) the molecular basis of cell adhesion, cell migration and cell invasion, with a particular emphasis on the immune system and (ii) on mechanisms of immune regulation. In addition it has been possible to apply the group's basic research findings to the development of new drugs which inhibit inflammation, cancer spread and the growth of new blood vessels (angiogenesis). Considerable amounts of external research funds have been obtained to finance the drug discovery programs. Recent research highlights are : |
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The group has had considerable experience in designing sulfated oligosaccharide-based compounds as drug candidates, this part of the group's research being supported by a large R and D grant from Progen Industries, Brisbane. Sulfated oligosaccharide-based inhibitors of the heparanase enzyme have been synthesised and identified. Furthermore, based on a novel in vitro assay for human angiogenesis developed by the group, sulfated oligosaccharides have also been discovered which inhibit angiogenesis. An extensive drug screening program managed to identify a sulfated oligosaccharide, termed PI-88, which can simultaneously inhibit angiogenesis and heparanase activity.
Preclinical testing has shown that PI-88 can inhibit primary tumour growth and tumour metastasis and the drug has now entered phase I clinical trials.
The group has been studying the plasma protein, histidine-rich glycoprotein (HRG), for many years, particularly regarding the ability of the protein to inhibit cell adhesion by masking cell surface carbohydrates. Recently. however, it became clear that HRG plays an important role in the immune system by preventing the insolubilisation of complexes between antibody and antigen (termed immune complexes). In fact HRG also assists in the uptake of these complexes by phagocytic cells. Thus HRG is probably a key molecule in aiding the elimination of immune complexes from the circulation. In fact, deficiencies in HRG may lead to immune complex-associated diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosis (SLE). This is an extremely novel area of research which is being pioneered by the CCI Group.
A productive collaboration has developed with AMBRI, a CRC-based company that has pioneered a unique membrane system for measuring the cross -linking of molecules on the surface of cell membranes. In collaboration with Dr Joe Altin, BaMBi, ANU a procedure has been devised at ANU to tether the extracellular domains of membrane receptors to the AMBRI membrane and monitor spontaneous and ligand induced cross-linking of the receptors. This technology forms part of a proposed CRC and can be used to answer many questions about the oligomerisation of cell surface molecules, particularly cell adhesion molecules and hormone/cytokine receptors.
The group has a long standing interest in the molecular basis of lymphocyte migration/recirculation. A unique molecular basis for the entry of lymphocytes into the spleen has been identified. Much of this work is underpinned by novel tracking techniques, based on fluorescent dyes, that the group has developed. One of the fluorescent dyes used in these studies, CFSE, can be used to monitor cell division as well, a discovery that has revolutionised many studies in immunology.
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The underlying objective of this group is to understand the mechanisms of lineage determination in the haematopoietic system. To achieve this we are studying the molecular mechanisms by which cytokines transmit signals to the nucleus of progenitor cells causing them to proliferate, differentiate or die. We are also interested in how the functions of mature blood cells, particularly macrophages and dendritic cells are regulated.
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Virtually all of our work is done using recombinant cytokines that we make in this laboratory, and cell lines that we derive from yolk sac, fetal liver and adult bone marrow.
In order to generate reliable molecular data it is important to use homogeneous populations of cells. It is difficult to do this by isolating cells ex vivo so emphasis is placed on the generation of cell lines. Our primary experimental system involves the use of cell lines that we have made by transfecting progenitor cells that proliferate in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) with a retrovirus that contains a cDNA encoding a truncated form of c-Myb. Expression of this unregulated c-Myb allows the cells to grow indefinitely in GM-CSF Expression of Myb is a prerequisite for normal proliferation of myeloid cells. As far as we know our myb-transformed haematopoietic cell lines (MTHC) differ from “normal” cells only in the unregulated expression of Myb.
As is the case for many myelomonocytic cell lines we have tested,myb-transformed cell lines grown in GM-CSF can be induced to differentiate into macrophages in the presence of tumour-necrosis factor alpha (TNF-alpha) and interleukin-4 (IL-4), while the use of interferon gamma (IFN-gamma) instead of IL-4 forces the cells into the dendritic cell lineage. We are examining the novel genes that are expressed during commitment to these two different lineages using the technique of cDNA subtraction. This project is being conducted by Dr. Joanne Banyer, a post-doctoral fellow in the group. Already we have isolated a large number of genes that are selectively involved in either macrophage and/or dendritic cell differentiation and are now identifying and characterising these genes as a prelude to investigating their role in differentiation and cell function.
Instead of differentiating in response to TNF-alpha some of our cell lines undergo programmed cell death or apoptosis. Since the apoptotic (A) and differentiating (D) cell lines were sub-cloned from the same cultures we are trying to determine the key differences between them that result in such different outcomes of TNF signaling. During her thesis work in this group Ms. Peta O'Connell showed that one difference between the A and D cells was that when apoptosis occurred it did so as a result of ligand binding to both type I and type II TNF receptors whereas differentiation required binding to the type I receptor only. Another student, Mr. Hayden Henry, is now identifying differences in the signaling pathways downstream from the receptors, and in collaboration with Dr. Banyer will attempt to identify genes that are selectively expressed or silenced in the A vs D cell lines. In parallel with these projects we are working with Dr. Waring's laboratory on cAMP regulated pathways activated during apoptosis, proliferation and differentiation, and with Dr. Crouch's laboratory on the role of Gia in proliferation and differentiation of myeloid cells.
At the level of cell biology we have shown that macrophages that differentiate in vitro from myb transformed cell lines are effective at phagocytosis but are poor presenters of antigen. In contrast, cells of the dendritic phenotype, induced by IFN-gamma have potent antigen presenting capacity. In an attempt to induce immune responses against tumour antigens we are transfecting selected genes into our progenitor cell lines and inducing differentiation with cytokines. Differentiated cells can then be used to immunise mice or to induce immune responses in vitro. This cell system provides a model for examining antigen processing and presentation, and for identifying phentypic differences between antigen handling cells that influence immune class regulation.
Another program of work involves the application of gene targeting technology toward the analysis of certain aspects of cytokine signaling. Dr. Hapel has cloned the gene for c-fms (the receptor for macrophage colony stimulating factor, an important blood cell growth factor) in the mouse and has used this to make a targetting construct containing a selectable marker (Neo) and a green fluorescent protein (GFP) tag from A victoria. Mice in which the gene for c-fms has been targetted and functionally deleted are now available for the study of fms and its interaction with its cognate ligand M-CSF (or CSF-1). Cells that would normally have expressed c-fms are identifiable by the expression of GFP These cells can be isolated and reconstituted with engineered versions of fms allowing analysis of different parts of the fms molecule in cell signalling.
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Several years ago our Group made the discovery that cytokines expressed by genetically engineered viruses, could greatly alter the pathogenicity and immunogenicity of the virus. Cytokines are hormone-like signalling molecules that influence the class of immune response induced. This concept was further developed to show that the efficacy of recombinant vaccines could be markedly enhanced by encoding specific cytokine genes. |
As well as developing an immunotherapeutic vaccine strategy we have made considerable progress towards a preventive vaccine for HIV-1. In collaboration with Dr Stephen Kent at the Macfarlane Burnet Centre, Melbourne we have shown that by first priming with a DNA vaccine followed by boosting with a recombinant fowlpoxvirus vector encoding the same HIV-1 antigens we can protect monkeys against HIV-1 challenge. The strategy of priming with DNA and boosting with FPV was found to generate primarily Th1 and CTL responses rather than antibodies, a potentially desirable property of an HIV-1 vaccine regimen given previous observations on the immune correlates of the control of HIV-1 in humans.
These new vaccine strategies have important implications not only for control of HIV-1 but also for a variety of other infectious diseases, cancer and auto-immunity.
A major focus has been the establishment and use of models in which to study antiviral and allergic immune regulation in vivo. A key molecular approach to this work has involved the establishment, largely through international collaboration, of a number of strains of mice deficient for the production of individual immuno-modulatory factors, particularly cytokines or combinations of these factors. A further major approach has been the design and construction of different vector systems engineered to express genes encoding cytokines and other immuno-modulatory molecules for the delivery of these factors in vivo in attempts to study and selectively modulate immune responses. These vector systems include vaccinia virus, fowlpox virus, DNA plasmids, adenovirus and Salmonella. We have used these approaches to investigate the key roles played by a variety of immuno-regulatory molecules, such as cytokines, in the pathogenesis of virus infection and in allergy.
A particular focus has been HIV infection, and our work, as members of the National Centre for HIV Virology, has contributed to the development of a prime-boost vaccination protocol which produces both strong systemic T cell-mediated immunity and disseminated mucosal immune responses against the virus. This work is directed towards the development of HIV vaccine strategies for use in under-developed countries where the AIDS pandemic is devastating the population.
In the case of allergic disease, we have recently shown that DNA and fowlpoxvirus vectors encoding allergens along with particular cytokines, including interleukin-12, are able to suppress the symptoms of allergic airways disease in animal models. Of particular interest, these model vaccines when administered at the neonatal stage are protective against the development of disease in adult life. We have also shown, using genetically-deficient mice, that inducible nitric oxide plays a significant role in the development of allergic airways pathology. Together, these findings have significant implications for the treatment and control of atopic diseases, including asthma.
A further research interest has been the development of novel, vectored fertility vaccines for some of Australia's feral pest species (mice, rabbits and foxes). As members of the Vertebrate Biocontrol CRC, we have, through a wide network of collaborators both internationally and throughout Australia, developed DNA and virally-vectored vaccines encoding fertility antigens, such as the ovarian zona pellucida glycoprotein, ZP3, which are of proven immunogenicity and are currently being tested in fertility trials.
Several of these studies have utilised mice whose gene encoding the molecule of interest (e.g. cytokine, chemokine receptor or leukocyte subset) has been disrupted and rendered non-functional, i.e., Ôgene knock out miceÕ. We have also undertaken a study of the kinetics of expression of a large panel of cytokines and chemokines during the course of viral infections in an attempt to associate these with the class of the immune response generated and disease outcome. We have found distict differences in profiles of cytokines made by mice which are either susceptible or resistant to infection and disease. There is a strong association between the cytokine type and the class of immune response generated, i.e., cell-mediated immunity or antibody. We have also found that distinct chemokines are produced under conditions of Th1 or Th2 responses. We recently demonstrated that two interferon inducible chemokines, i.e., MuMig and Crg-2, have antiviral activity. This antiviral activity is mediated through recruitment of NK cells to sites of infection as well as enhancement of their cytolytic activity.
The genetics of resistance to viral infection is another aspect our laboratory in interested in. In collaboration with Dr. Tony Scalzo of the University of Western Australia, we have been able to demonstrate that a particular gene on the mouse chromosome 6 which confers resistance against murine cytomegalovirus may also serves as a resistance gene against a murine poxvirus. We now know that this gene product confers resistance through the activity of NK cells. We are currently investigating whether the same gene confers resistance against to two different viruses. These collaborative studies complement our ongoing studies on the mechanism(s) through which CD8 and CD4 T lymphocytes clear virus in vivo.
More recently we have shown that IL-10, a cytokine largely believed to be important in the generation of antibody responses is also critical for the generation of optimal cell-mediated immune response against viruses. We have also established that IL-10 is critical for primary and recall IgA production in the lung.
The role of antibody and cell-mediated immunity in recovery from primary viral infection has also been an important aspect of our studies. We have found that certain poxviruses (which are cytopathic) require both both antibody and CD8+ T-cell-mediated cytolysis for virus clearance.
The laboratory is also interested in proteins which modulate immune responses for the purpose of enhancing and/or deviating the immune responses to vaccine antigens. We are currently, in collaboration with Drs Andrew Hapel and Joanne Banyer, characterising the mouse homologue of a chemokine called I-TAC Chemokines are molecules whose role is to enhance recruitment of specific immune cells. This particular chemokine is thought to attract a subpopulation of T cells which is important for the development of cell-mediated immunity and can also in some circumstances enhance the progression of some autoimmune diseases.
Viral Immunology Group
Immunology Laboratory
Apoptosis Laboratory
Molecular Virology Laboratory
Antigen Presentation Laboratory (until February)
Molecular Immunology and Immunopathology Group
Transcriptional Regulation Group (until July)
Cell-Cell Interaction Group
Viral Engineering and Cytokine Research Group:
Experimental Haematology Group