Division of Immunology and Cell Biology

Cancer and Vascular Biology | Vaccine Immunology | Immune Regulation and Vaccine Development | Synthetic Vaccines | Initiators and Regulators of Immunity | Immunology | Immunity and Immunopathology in Infectious Diseases | Immunopathology Research | Host Defence

The Division of Immunology and Cell Biology is comprised of a number of research groups that investigate fundamental aspects of cell biology, with a major interest being the immune system. Research undertaken by the Division includes investigations of viral replication, analyses of the immune response to viral infections, development of HIV and cancer vaccines, and research on the processes of inflammation, tumour angiogenesis and tumour metastasis.

The pursuit of long term basic science goals make up most of the Division's work but this is balanced by attempts to translate fundamental discoveries into clinical applications. The latter include the possible application of anionic sugar molecules as novel anti-inflammatory or anti-cancer drugs and naked DNA and recombinant poxviruses as vaccines for prevention of certain infectious diseases or treatment of cancer. More detail of these various themes is given in each Group or Laboratory report.

Professor Chris Parish, Head of Division

 

Cancer and Vascular Biology Group
(Formerly Cell-Cell Interaction Group)

The Cancer and Vascular Biology group has been working for a number of years on the molecular basis of cell adhesion, cell migration and cell invasion, with a particular emphasis on the immune system, tumour metastasis and the growth of new blood vessels (angiogenesis). Of particular interest has been the role of anionic carbohydrates, such as heparan sulfate, in these processes. In addition the Group aims to apply its basic research findings to the development of new drugs which inhibit inflammation, cancer spread and angiogenesis. Considerable amounts of external research funds have been obtained to finance the drug discovery programs.

Recent research highlights:
Following the recent cloning of the enzyme heparanase, a key enzyme involved in degradation of the extracellular matrix by invading tumour cells and by leukocytes entering inflammatory sites, the group has gained considerable information about the active site of the enzyme. This information will be invaluable for the design of more effective heparanase inhibitors. The development of tissue specific knock out mice is well underway and should provide information about the role of heparanase in a range of physiological processes.

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, with a sulfated oligosaccharide, termed PI-88, being found to be a potent inhibitor of angiogenesis and heparanase activity. Preclinical testing has shown that PI-88 can inhibit primary tumour growth and metastasis of a number of cancer types. By the end of 2000 the drug had successfully completed phase I clinical trials in healthy volunteers and cancer patients, with phase II trials in cancer patients commencing in 2001.

A very productive collaboration has developed during the last 3 years with Professor Martin Banwell, Research School of Chemistry, ANU in which sulfated pseudo-sugars are being synthesized as heparan sulfate mimetics. This project is also being funded by our commercial partner, Progen Industries. Although the initial aim of this collaboration is to produce better heparanase inhibitors, a number of sulfated pseudo-sugars have been identified that selectively inhibit certain protein-heparan sulfate interactions. Such drugs have potential as anti-angiogenic, anticoagulant, antiviral and antilipaemic agents.

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 interacting with the complement system and 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 regulating complement activity and 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). In a related study it has been shown that HRG can tether plasmin/plasminogen to the surface of cells and potentially aid cell invasion. Thus HRG represents a multifunctional protein that appears to play an important role in the immune system, inflammation and wound healing. A major focus of the group in the future is to better understand the functional significance of this intriguing plasma protein.

It has been known for decades that the alternative pathway of complement is being continually activated by a process termed C3-tickover. Recently we obtained conclusive evidence that a similar process occurs with the classical complement pathway both in mice and humans, the process being dependent on IgG and being particularly apparent following vascular stasis. The molecular basis of the activation process is still unclear but it seems likely that HRG is involved. We believe this spontaneous activation of the classical pathway may augment activation of the complement system at sites of inflammation and infarction.

A productive collaboration has developed with Dr Joe Altin, BaMBi, ANU in which a procedure has been devised to tether the extracellular domains of cell surface receptors to cell membranes. This technology has been used to graft costimulator molecules, such as CD40 and CD80, onto tumour cell surfaces to produce better cancer vaccines. The technology is also being used to target liposomes containing cytotoxic drugs to sites of angiogenesis in humans, such an approach being potentially a potent means of inducing tumour regression. Additional applications of the technology are to target antigens to dendritic cells as a means of developing better vaccines and to use the technology as a delivery vehicle for gene therapy.

In a collaboration with Dr Paul Fosters group in the Division of Biochemistry and Molecular Biology, JCSMR, a new approach to cancer immunotherapy has been developed. Currently most attempts at cancer immunotherapy involve the generation of CD8+ cytotoxic T lymphocytes (CTLs) against tumour-specific antigens. Recently we demonstrated that tumour-specific CD4+ T cells, that exhibit a cytokine secretion profile characteristic of Th2 cells, are capable of clearing established lung and visceral metastases of a B16 melanoma that is resistant to CTL lysis. Clearance of the lung metastases by Th2 cells was found to be dependent on degranulating eosinophils, with the eosinophil chemokine, eotaxin, playing an essential role. In contrast, tumour-specific CD4+ Th1 cells, that recruited macrophages into the tumour, had no effect on tumour growth. This work provides the basis for a new approach to cancer vaccination that is effective against CTL-resistant tumours and is, potentially, less susceptible to immune evasion.

Chris Parish

 

Vaccine Immunology Group
(formerly Viral Engineering & Cytokine Reserch Group)

There is an urgent need for an HIV/AIDS vaccine for developing countries. More than 16 million people have died of AIDS, more than 34 million people are living with HIV and nearly all will die of AIDS within the next two decades. Although other measures have slowed the spread of AIDS only an AIDS vaccine can end the HIV/AIDS pandemic. This year the US National Institutes of Health awarded a AUD$30 million contract to a consortium of Australian researchers to develop and trial an HIV/AIDS vaccine. This is the largest grant yet given to an Australian research team from an international agency. The members of the consortium include the John Curtin School of Medical Research, the University of Melbourne, the University of New South Wales, the University of Newcastle and the CSIRO. The HIV/AIDS vaccine approach involves both prime boost immunisation with DNA and recombinant fowlpoxvirus vectors with the co expression of immune-enhancing cytokine genes. It is gratifying that both technologies used in this novel vaccine approach were developed from fundamental research carried out at the JCSMR over the last decade. Although these vaccine strategies lead to unprecedented levels of immunity there is still a need to develop new approaches to cover the genetic diversity seen with HIV, this area will be a major focus of our future research.

Ian Ramshaw

Immune regulation and vaccine development laboratory

The focus of our research is to study the factors important in generating high levels of protective immunity to vaccination. In particular, we are concentrating on the use of prime boost immunisation to stimulate high levels of cell-mediated immunity. We have been studying why this vaccination strategy generates such a powerful response. Using a tetramer staining technique we have been able to show that DNA vaccines are able to induce very effective or high avidity T cells which are then expanded by boosting with a recombinant fowlpoxvirus vaccine encoding the same vaccine antigen. The quality of an immune response generated by a particular vaccine strategy may therefore be as important as the levels of immunity induced. Vaccines, that generate T cell populations of high avidity, optimising the early detection of infected cells, offer new hope for development of effective prophylaxis against pathogens such as HIV, which have presented major problems for vaccine development.

Ian Ramshaw

Synthetic Vaccines Laboratory

Research in the laboratory focuses on developing new vaccine technologies and therapeutic strategies for a range of human diseases including viral infections eg HIV and Hepatitis C, bacterial infections eg mycobacterium tuberculosis (TB) and cancer eg melanoma. With this goal the laboratory has been closely involved with the NIH funded Australian HIV Vaccine Consortium, which plans to carry out clinical trials in Australia and SE Asia in the near future. The HIV consortium work has involved modification of the HIV sequences from two HIV-1 strains and constructing two DNA vaccines, which will be used as the first component of the immunisations in these clinical trials.

Another main focus in the laboratory is the further development of the scrambled antigen vaccine (SAVINE) technology. This new technology (patented in 2001) will allow the development of more effective and safe vaccines using a totally synthetic approach to molecularly re-engineer whole pathogens or large numbers of antigens similtaneously. It can also be used to combine multiple cancer antigens safely into a single vaccine. Collaborations with Warwick Britton (Centenary Institute) and Rose Ffrench (Sydney Childrens Hospital ) to develop TB and Hepatitis C SAVINES respectively were established this year.

Further research into an older technology based on polyepitope vaccines, (synthetic antigens comprising just multiple contiguous T cell epitopes) is continuing through collaborations with the CRC for Vaccine Technology (Queensland Institute of Medical Research) and the Sir Albert Sakzewski Virus Research Center. These collaborations are developing therapies for nasopharyngeal carcinoma and cervical cancer.

The laboratory also has a keen interest in new vaccine vectors and delivery strategies that can augment the novel synthetic antigens being developed.

Scott Thomson

Initiators and Regulators of Immunity Laboratory

The nature of work in the Initiators and Regulators of Immunity Laboratory aims to further our understanding of how immune responses are initiated or avoided by cytokines, pathogens, and vaccine agents. This knowledge may be used to design more effective vaccines and to identify immunoregulatory molecules that may be used as vaccine adjuvants, or immunotherapeutics that re-direct inappropriate or enhance immune responses to pathogens.

Dendritic cells (DC) are the immuno-interpretors of the innate immune cell system that identify, process, and present antigens from micro-organisms to adaptive immune cells. Whether the DC initiates an adaptive cell mediated immune (CMI) or humoral immune (HI) response is dependent on the nature of the pathogen and the types of cytokines that are released into the microenvironment by innate immune cells at the site of infection. These combinations of factors act on DCs to develop immunoregulatory properties which communicate to adaptive immune cells the identity of the pathogen and the type of immune response which should be generated against the pathogen. How the DC communicates this information and regulates adaptive immunity is not completely understood. To address this issue our laboratory is investigating the effects of CMI and HI cytokines on the immunoregulatory properties of DCs. Our approach utilises a combination of biological and molecular analysis of ex vivo DCs and DC cell lines to identify immunoregulatory molecules that are differentially regulated by these cytokines.

Interactions between the pathogen and the DC provide the perfect opportunity for the pathogen to modify the type of immune response that develops. Pathogens such as HIV and Hepatitis C utilize these interactions to reduce the immunostimulatory properties of DCs thereby disrupting clearance of the virus and supporting viral persistence. The immune avoidance mechanisms elicited by these viruses are also not completely understood. Our laboratory is investigating these interactions using Hepatitis C infection of human monocyte derived DCs as a model system. Previous studies of chronically infected patients have indicated Hepatitis C persistently infects and disrupts the immunoregulatory function of these cells. To establish how the virus achieves this we are targeting the initial interaction between the virus and non-infected DCs utilising both biological and molecular analysis.

Many vaccines are based on the use of pathogen-derived vectors and some pathogen molecules, such as CpG sequences, are used as vaccine adjuvants to enhance immunoresponsiveness to vaccines. The nature of these vaccine agents, however, also influences the type of immune response that a DC directs. In addition, some of these vaccine agents have been shown to elicit immune avoidance mechanisms that target the immunoregulatory properties of DCs. To further understand the effects of vaccine agents on the immunoregulatory properties of DCs our laboratory has examined the effects of DNA and viral vaccine vectors in combination with CMI and HI associated cytokine stimulus of DCs. This work established that in the presence of CMI-associated cytokines DCs are more efficient at taking up and processing these vaccine agents. Also, in combination with these cytokines the vaccine agents can enhance the immunoregulatory properties displayed by the DCs and therefore are likely to trigger stronger immune responses. Similar to other recent studies, we found that particular viral vaccine agents, vaccinia and fowl-pox, effect the viability of the DC, however we established that particular CMI associated cytokines enable the DC to process and present the viral antigen prior to cell death. In addition, preliminary studies in our laboratory suggest that DCs utilise other mechanisms, such as cross-presentation, to present virally encoded antigen. These studies have highlighted the important role of cytokines that enable DCs to handle pathogen-derived antigen appropriately and thereby support their potential use in effective immunization strategies.

Design and engineering of highly effective vaccines in the future will benefit from a more thorough understanding of the immune stimulatory and inhibitory properties of vaccine agents so these properties may be altered to better support generation of strong and appropriate immune responses.

Joanne Banyer

 

Immunology Laboratory

In adult humans, almost all of the B lymphocytes responsible for memory and secondary antibody responses display mutations in the genes encoding the protein chains of the antibody molecule. These mutations contribute to the phenomenon of affinity maturation of antibody responses in which secondary and later responses show increased affinity over early responses, thus improving the protective potential of the antibodies.

Conditions under which hypermutation of antibody genes is induced in B lymphocytes and the mechanisms of selection of B cells with mutated antibodies of high affinity against the immunizing antigen have been well defined.

However the molecular mechanism of somatic hypermutation remains one of the major unsolved mysteries in immunology. We are testing an hypothesis involving error-prone transcription of pre-mRNA from rearranged variable regions of antibody genes and reverse transcription to produce mutated cDNA, which replaces the original gene by homologous recombination. Predictions of this molecular model are being tested using transgenic and knock-out mice.

Bob Blanden

Immunity and Immunopathology in Infectious Disease

Eva Lee

The general aims of the program are first, to generate new knowledge relevant to our understanding of the fundamental properties of immune responses at the molecular, cellular and whole system level with particular emphasis on immune responses against viruses and second, to study virus/host interactions at the cellular and molecular level and through this devise strategies for the prevention of viral disease.

The members of the program have wide expertise in immunology, immunopathology and molecular virology. Our current investigations focus on the different functions of cytolytic effector molecules, MHC class I antigen presentation, T lymphocyte responses against infection with viruses, bacteria and fungi, the cytotoxic T memory response, virus/host interactions in flavivirus assembly and replication, viral immune evasion strategies, and apoptotic cell death, including death induced by T cells. A large number of virus models including flaviviruses, poxviruses, influenza and parainfluenza viruses, alphaviruses, herpes viruses and adenoviruses are employed in these studies. The availability and establishment by our laboratories of gene targeted mice defective in immune effector molecules including perforin, the granzymes, and Fas receptor/ligand has allowed us to elucidate important host/parasite relationships in the context of the host immune response. Another important approach which is currently applied widely in the group is that of reverse genetics using full-length cDNA copies of flavivirus RNA genomes. These allow the in vitro synthesis of infectious viral RNA and thus structure/function studies in flavivirus replication and pathogenesis.

Mario Lobigs

Progress in our research in the last year Substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role of glycosaminoglycans in entry The flavivirus receptor-binding domain has been putatively assigned to a hydrophilic region (FG loop) in the envelope (E) protein. In some flaviviruses this domain harbors the integrin-binding motif, Arg-Gly-Asp (RGD). One of us has shown earlier that host cell adaptation of Murray Valley encephalitis virus (MVE) can result in the selection of attenuated variants altered at E protein residue Asp390, which is part of an RGD-motif.

Here, a full-length, infectious cDNA clone of MVE was constructed and employed to systematically investigate the impact of single amino acid changes at Asp₃₉₀ on cell tropism, virus entry, and virulence. Each of ten different E protein 390 mutants was viable. Three mutants (Gly₃₉₀, Ala₃₉₀ , and His₃₉₀) showed pronounced differences from an infectious clone-derived control virus in growth in mammalian and mosquito cells. The altered cell tropism correlated with (1) a difference in entry kinetics, (2) an increased dependence on glycosaminoglycans (determined by inhibition of virus infectivity by heparin) for attachment of the three mutants to different mammalian cells, and (3) the loss of virulence in mice. These results confirm a functional role of the FG loop in the flavivirus E protein in virus entry and suggest that encephalitic flaviviruses can enter cells via attachment to glycosaminoglycans. However, it appears that additional cell surface molecules are also used as receptors by natural isolates of MVE and that the increased dependence on glycosaminoglycans for entry results in the loss of neuroinvasiveness.

Mechanism of virulence attenuation of GAG-binding variants of Japanese encephalitis and Murray Valley encephalitis viruses

Arno Müllbacher

The in vivo mechanism for virulence attenuation of laboratory-derived variants of two flaviviruses in the Japanese encephalitis virus (JEV) serocomplex was analysed. Host cell adaptation of JEV and Murray Valley encephalitis virus (MVE) following serial passage in human adenocarcinoma (SW13) cells selected for variants characterised by (i) a small plaque phenotype, (ii) altered attachment/entry properties reflected in an increase in affinity to heparin, and (iii) loss of neuroinvasiveness in mice.

Despite the close genetic relatedness of JEV and MVE, the SW13-passaged attenuated variants have single amino acid substitutions at different residues in the E protein (Glu₃₀₆ to Lys for JEV, and Asp₃₉₀ to Gly or His for MVE). Given the close spatial proximity of residues 306 and 390 in the predicted E protein structure, it is likely that these residues define a receptor-binding domain involved in virus attachment to glycosaminoglycans (GAGs) on host cells. The SW13 cell-adapted variants showed increased binding to GAGs in vitro and reduced capacity to produce disease in mice, including mice defective in the type I interferon antiviral response, which are highly susceptible to peripheral infection with JEV serotype flaviviruses. The attenuated viruses were rapidly removed from the blood stream and failed to spread from extraneural sites of inoculation into the brain, hence the loss of neuroinvasiveness is most likely the consequence of enhanced affinity of attenuated variants for sulfated proteoglycans on cell surface and in extracellular matrix.

Cytotoxic T cell response to Flaviviruses

We have investigated the reactivities of cytotoxic T (Tc) cells against the two immunodominant, H - 2K^k-restricted determinants from the flavivirus Murray Valley encephalitis virus (MVE), MVE₁₇₈₅ (REHSGNEI) and MVE₁₉₇₁ (DEGEGRVI). The respective Tc cell populations cross-reactively lysed target cells pulsed with determinants from the MV E₁₇₈₅- and MV E₁₉₇₁-corresponding positions of six other flaviviruses, despite low sequence homology in some cases. Notably, anti-MV E₁₇₈₅ Tc cells recognized a determinant (TDGEERVI) which shares with the determinant used for stimulation only the carboxy-terminal amino acid residue, one of two H - 2K^k anchor residues. These reactivity patterns were also observed in peptide-dependent interferon-gamma production and the requirements for in vitro restimulation of memory Tc cells. However, the broad cross-reactivity appeared to be limited to flavivirus-derived determinants, as none of a range of determinants from endogenous mouse-derived sequences, similar to the MVE-determinants, were recognized. Neither were cells infected with a number of unrelated viruses recognized. These results raise the paradox that virus-immune Tc cell responses, which are mostly directed against only a few “immunodominant” viral determinants, are remarkably peptide cross-reactive.

Virus-infected cells trigger cytolysis, but not interferon-gamma production in cytotoxic T cells

Cytolysis and interferon-gamma (IFNgamma) production are two independent effector functions of activated cytotoxic T (Tc) cells. We have used the Tc cell response against the flavivirus, Murray Valley encephalitis virus (MVE), to investigate the requirements for inducing these two functions with regard to antigen-concentration and CD8 co-receptor involvement. Cognate peptide-pulsed target cells triggered cytolysis by primary ex vivo MVE-immune as well as in vitro peptide-restimulated splenocytes at lower peptide concentrations than IFN-gamma production (100-fold lower in the case of primary ex vivo effectors). Little difference was observed in CD8 dependency. Importantly, neither of the effector populations were triggered to produce IFNgamma by virus-infected target cells, although cytolysis occurred. This result raises the possibility that the levels of presentation of cognate antigen on virus-infected cells in vivo may be below the threshold required for IFNgamma-production.

Cross-protective and infection-enhancing immunity in mice vaccinated against flaviviruses belonging to the Japanese encephalitis virus serocomplex

The Japanese encephalitis virus serocomplex is a group of closely related mosquito-borne flaviviruses that cause severe encephalitic disease in humans. The recent emergence of several members of this serocomplex (Japanese encephalitis and West Nile viruses) in geographic regions where others are endemic has raised urgent human health issues. Thus, the impact of vaccination against one of these neurotropic virus on the outcome of infection with a second, serologically related virus is unknown. We have found that the immunity elicited in vaccinated mice can cross-protect but also augment disease severity due to an enhancement of infection following heterologous virus challenge. A key determinant in disease outcome in vaccinated animals infected with a second flavivirus belonging to the Japanese encephalitis virus serocomplex is the magnitude of the virus-specific antibody response raised against the first virus.

Modulation of transporter associated with antigen processing (TAP)-mediated peptide import into the endoplasmic reticulum by flavivirus infection
In contrast to many other viruses that escape the cellular immune response by downregulating major histocompatibility complex (MHC) class I molecules flavivirus infection can upregulate their cell surface expression. Previously we have presented evidence that during flavivirus infection peptide supply to the endoplasmic reticulum is increased (A Müllbacher and M. Lobigs, Immunity, 3:207-214, 1995). We have further investigated this phenomenon using in vitro assays to detect peptide transport from the cytoplasm into the lumen of the endoplasmic reticulum in semi-permeabilised infected and uninfected cells. We found that during the early phase of infection with different flaviviruses the transport activity of the peptide transporter associated with antigen processing (TAP) is augmented by up to 50.

Virulence of mousepox virus is independent of serpin mediated control of cellular cytotoxicity
We have investigated whether the differential virulence seen of two Ectromelia (Ect) strains, Ect_Moscow and Ect_Hampsteadegg, is due to mutation or differential regulation of their serpins (SPI). Poxvirus encoded serine proteinase inhibitors (serpins) have been shown to interfere with cytolytic activity of leukocytes and can also determine virulence. We show that the deduced amino acid sequences of SPI-1, 2 and 3 are identical for the highly virulent Ect_Moscow and the low virulent Ect_Hampstead strains and that the two viruses express similar potential to inhibit T cell cytotoxicity, in particular Fas-mediated target cell lysis, by allorective effectors. Virus titres in wild type B6 mice were effectively controlled very early after inoculation with Ect_Hampstead as compared to Ect_Moscow, but lack of perforin renders B6 mice similarly susceptible to both virus strains.

The data demonstrate that in Ect infection the perforin-mediated cytolytic pathway is not the primary target of serpins and suggest that the apparent attenuation of Ect_Hampstead seen in B6 mice is due to control elements distinct from SPI-1,2 and 3.

Cell Death Mediated by Alloreactive Cytotoxic T Cells via the Granule Exocytosis or the Fas Pathway is Independent of p34^cdc2 Kinase Fas Dependent Killing of Cells Arrested in the Cell Cycle.
Inappropriate activation of p34^cdc2 kinase has been shown to occur during apoptosis induced by cytotoxic T cell derived perforin and fragmentin. We analyzed the effect of two inhibitors of p34^cdc2 kinase on alloreactive Tc cell mediated lysis and DNA fragmentation of P815 and L1210 target cells. Olomoucine, a specific inhibitor of cyclin dependent kinases, did not affect DNA fragmentation in the target cells. Lysis of olomoucine treated target cells as assessed by ⁵¹Cr release over a typical 8 hour period was also unaffected. We also examined the effects of thapsigargin on target cell death. This toxin causes increased intracellular calcium rises which then result in irreversible inhibition of cyclin dependent kinases including p34^cdc2 kinase. The same extent of specific cell lysis was induced by cytotoxic T cells from perforin (-/-), granzyme B (-/-), granzyme A (-/-), perforin (-/-) X granzymeB (-/-) X granzymeA (-/-) KO mice or normal mice in untreated target cells or target cells treated with either olomoucine or thapsigargin. Similarly DNA fragmentation measured by release of tritiated DNA was also unaffected. Thus inhibition of p34^cdc2 kinase affects neither the Fas nor perforin/granzyme pathways of alloreactive cytotoxic T cell killing as measured by DNA fragmentation or chromium release. P815 cells treated with olomoucine were arrested in the cell cycle after 12-16 hours exposure to the toxin. After cell cycle arrest, target cells now showed enhanced ⁵¹Cr release induced by effector CTL derived from perforin (-/-) mice compared to untreated cells. This lysis was accompanied by increase in cell surface Fas expression. Olomoucine induced cell cycle arrest and expression of Fas was reversible and when cells re-entered the cell cycle, surface expression of Fas was lost.

Simultaneous activation of the Fas/FasL and perforin/granzymeA,B pathways is mandatory for the development of viral hepatitis, but not for recovery from viral infection
Cytotoxic T lymphocytes play a major role in the recovery from primary viral infections and the accompaning tissue injuries. However, it is unclear to what extent the two main cytolytic pathways, perforin/granzyme exocytosis and Fas-Fas ligand (FasL) interaction, contribute to these processes. Here we have employed mouse strains with either spontaneous mutations or targeted gene defects in one or more components of either of the two cytolytic pathways to analyse the molecular basis of viral clearance and induction of hepatitis during lymphocytic choriomeningitis virus infection. Our results reveal that perforin-mediated viral clearance is similarly effective in mice deficient in either FasL/Fas or in granzymes A and B. In contrast, virus-induced liver damage only occurred in mice expressing both, FasL/Fas and perforin as well as the two granzymes. The observation that antibodies to FasL prevent hepatocyte damage, but allow viral clearance presents an exciting possibility for therapeutic treatment of hepatitis during viral infections.

Eva Lee, Mario Lobigs and Arno Müllbacher

 

Immunopathology Research Group

Brett Charlton

Work in the Immunopathology Research Group is focused on developing novel treatments for and understanding key biochemical processes underlying debilitating cell-mediated disease processes. As the term immunopathology suggests, the Group is interested in the pathological (tissue damaging) changes that occur as a direct result of either normal or abnormal immunological processes. Autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis are examples of pathologies that occur as the result of an abnormal immunological process because in these cases cells of the immune system appear to attack what seems to be healthy tissue rather than performing their intended role of dealing with invading pathogens.

Such cell-mediated autoimmune diseases are characterised by an accumulation of leucocytes, white blood cells that are the mainstays of the immune system, in the affected tissue. Thus, in the case of MS an abnormal accumulation of leucocytes is found in the brain and spinal cord while in arthritis this build-up occurs in the diseased joint.

The usual role for these cells is in fighting infection but in the case of autoimmunity the cells appear to recognise normal tissue as being foreign and as a consequence of this misinterpretation these cells attack the affected tissue. This results in damage to the tissue and this is reflected in clinical symptoms. In the case of MS, symptoms range in type and severity depending upon the areas of the brain affected. Thus, transient mild weakness or tingling in the limbs may be the only symptom in some MS patients while others may suffer paralysis or blindness. In the case of arthritis symptoms are invariably directly associated with the inflamed joint and once again the clinical signs range in severity from mild discomfort to crippling disfiguration.

Since an essential component of these diseases is the accumulation of inflammatory cells, a potential approach to treating them is to reduce or prevent the accumulation of leucocytes in the affected tissue (inflammatory site). White blood cells normally circulate through the body, along with erythrocytes (red blood cells), inside the vascular system (blood vessels). The mechanism by which leucocytes arrive at the inflammatory site is both complex and incompletely understood. There are, however, many potential points at which this process may be interfered with. Work carried out in collaboration with others within the School and at the Neurosciences Research Group at The Canberra Hospital has identified a promising biochemical target for preventing cell migration. Thus, research in our Group is currently targeting one of the final biochemical steps involved in the migration of the leucocytes from blood vessels into the adjacent tissue. The importance of translating basic findings into clinical outcomes is central to our efforts and since experimental results have been promising we hope to trial the potential treatment in the clinic. Therefore, one of our goals over the next 12 months is to initiate and facilitate a multi-centre clinical trial in the treatment of relapsing-remitting MS.

Another major interest of the Group is the role of a molecule, called nitric oxide, in autoimmune processes. This molecule is produced in large amounts by certain leucocytes that have been stimulated by invading pathogens, especially bacteria and protozoan parasites (malaria for example) that live and replicate inside host cells. Infection with some viruses also elicits the production of nitric oxide. This molecule is chemically reactive and this attribute may be responsible for its ability to inhibit the growth and spread of certain infectious agents. This property may also contribute to its widely reported immunopathological properties in both infectious and in autoimmune diseases. Another way of looking at this is that cells of the immune system produce nitric oxide in order to damage invading micro-organisms but in the process, normal tissue in the immediate vicinity can also be damaged by this chemically reactive molecule. On the other hand, nitric oxide is also produced in small quantities by cells other than those of the immune system. In this situation, nitric oxide has some roles that are directly related to normal, non-immunological processes such as the control of high blood pressure and neurotransmission. Other groups within the School study some aspects of these activities.

The Immunopathology Group has investigated the potential pathological role of nitric oxide in some autoimmune diseases such as type-1 or juvenile-onset insulin-dependent diabetes and in a multiple sclerosis-like disease. In contrast to many reports in the medical and scientific literature, our group has discovered that in some autoimmune disease settings, nitric oxide has a down-regulating effect on the disease. In other words, in addition to possibly having a localised tissue damaging role in these diseases, it may, in contrast, actually slow down or reverse the disease process. In this regard our group has found that in the absence of nitric oxide production by the immune system, some autoimmune diseases are actually more severe and protracted than would otherwise be the case. This finding implies that nitric oxide may function in a feed-back manner to signal the immune system to reduce or halt its attack. This was further supported by our discovery that by increasing systemic levels of nitric oxide, either through immunological manipulation or drug treatment, these diseases were less severe. This finding is perhaps important from a therapeutic perspective, and because of this the group is actively studying potential means by which nitric oxide production can be elevated in the hopes that such therapy may be of benefit in the treatment of autoimmune diseases.

Bill Cowden and Brett Charlton

 

Host Defence Group

Overview
Our research interests are in the broad area of virus-host interactions. The immune response has several strategies to combat infectious disease. These include innate and adaptive components, all of which are regulated by a network of critically important cellular interactions and soluble mediators. Interestingly, however, the effector mechanisms that are generated to control and clear virus instead often cause immunopathology that has serious, sometimes lethal, consequences for the host. The objective is to modulate the response in order to direct the outcome towards efficient virus clearance and to minimize pathology. For this, a clear understanding of the host’s response to a virus infection is essential. These studies are being carried out in parallel with others that attempt to reveal the many strategies that viruses have evolved to subvert the host immune response. An integral component of our research program, therefore, involves basic studies that attempt to dissect the roles of leukocyte subsets, cytokines, chemokines, effector proteins and some signaling molecules in viral infection and disease. We pursue this goal using a range of viral and animal models.

Ongoing Research Areas

Nitric oxide and immunopathology
We have studied the antiviral role of nitric oxide (NO) using nitric oxide synthase (NOS) inhibitors and NOS2 deficient mice. In addition, we are currently investigating the role of NOS2 and NO in influenza pneumonia. Intriguingly, we have found that NO has no antiviral activity in this virus model and that virus clearance occurred via other interferon (IFN)-gamma-mediated pathways. This work demonstrates for the first time, 1) the definitive role of NO in the generation of viral immunopathology, and 2) the existence of a novel IFN-gamma-mediated antiviral pathway in NOS2 GKO mice not obvious in wildtype animals.

Interferon-inducible chemokines in virus clearance
IFN-g mediates its antiviral effects through the induction of a number of proteins. We have looked at two IFN-inducible chemokines, namely Mig and Crg-2, and found that they are prominently upregulated during infection of mice with vaccinia virus (VV). This led us to speculate that Mig and Crg-2 may, in fact, mediate some of the antiviral effects of IFNs. Using the recombinant VV approach, we have now shown that Mig and Crg-2 mediate antiviral activity in vivo through the rapid activation and recruitment of NK cells and T lymphocytes to the site of virus replication. Indeed, this is the first study that has established a role for these chemokines in antiviral immunity.

Cytokines, cell-mediated immunity and resistance to disease
Some strains of mice (e.g. C57BL/6) are genetically resistant, while others (e.g. BALB/c and A/J) are genetically susceptible, to mousepox. We were interested in determining whether or not these differences in disease susceptiblity correlated in vivo with cytokine profiles and cell-mediated immune responses. We have shown that resistance is associated with the capacity of mice to produce the type 1 cytokines IL-2, IFN-g, IL-12 and TNF-a and to generate a potent CTL response early after infection. The type 2 cytokine, IL-4, is produced by both resistant and susceptible mice. Further, the cytokine response is compartmentalised; there are clear differences in the cytokines that are produced in the lymph node (priming site) compared with the spleen (effector site). These studies indicate that susceptibilty to mousepox is associated with the lack of type 1 cytokine synthesis and a delayed cell-mediated immune response.

Cytokines, cytotoxic T lymphocytes and antibody in antiviral immunity
We remain interested in determining the in vivo effector function of CD8 T lymphocytes that is critical for virus clearance. There is still some question of whether it is their cytolytic potential or their ability to produce cytokines which is important. Indeed, it has been proposed that recovery from infection with cytopathic viruses requires cytokines like IFN-g or antibody but not CTL whereas non-cytopathic viruses require CTL but not antibody or cytokines. Our investigations using ectromelia virus (EV), a cytopathic virus, indicate that recovery from a primary infection with this virus clearly requires CTL (perforin-mediated cytolysis), IFNs (IFN-a, -b and -g), and most intriguingly, antibody. In addressing the role of these immune parameters in recovery from a secondary EV infection, we have found that only antibody is critical for protective immunity. Our data show that the requirement for cell-mediated immunity, cytokines and antibody for recovery from a primary viral infection is unrelated to whether the virus is cytopathic or non-cytopathic. However, it may be related to virulence, replication properties or mode of spread of the virus within the host.

The innate immune response in antiviral immunity
We are also interested in the link between innate and adaptive immune responses to viral infections. Components, such as neutrophils, not only provide the first line of defence, but also have the ability to profoundly influence and direct the adaptive immune response. In addition, we are also studying the role of B cells and antibody in the control of virus replication in a primary infection.

Role of Membrane TNF in host response to virus infection
One unique aspect of this study involves the assessment of the membrane form of this cytokine in vivo, during the host immune response to a replicating infectious pathogen. The initial studies in TNF gene knockout mice have been promising and we are in the process of extending these to utilize the recently developed membrane TNF knockin mouse (Jon Sedgwick, DNAX, USA).

Genetic Control of Early Immune Responses to Poxviruses and Herpesviruses
Cmv1 controls replication of murine cytomegalovirus (MCMV) in the spleen via regulation of NK cells. This gene is linked to a region on the mouse chromosome 6 designated Natural killer complex (NKC). One of 3 known genes that confer resistance to mousepox (designated Rmp1) is also linked to the NKC region and Cmv1. The B6 mouse is resistant to MCMV and ectromelia virus (the causative agent of mousepox) infections whereas the BALB/c mouse is susceptible to both. Dr. A.A. Scalzo (UWA) has generated BALB/c congenic mice with B6 NKC (BALB/C.Cmv1r) and several intra-NKC recombinants. He has also established a colony of B6 congenic mice with the BALB/c NKC. These congenic mice are unique tools and are not available elsewhere. The purpose of this study is to establish (i) whether Cmv1 also confers resistance against mousepox, (ii) whether Rmp1 and Cmv1 are the same gene, and (iii) the mechanism(s) through which Cmv1 and/or Rmp1 regulate NK cell activity and confer resistance.