JCSMR Annual Report 2003

Division of
Immunology and Genetics

Professor Chris Parish, Head of Division

The Division of Immunology and Genetics pursues fundamental research into cellular, molecular and genetic processes of relevance to medicine. Common medical problems investigated by our researchers include infectious diseases, cancer, diabetes, autoimmune disease and mental illness, with a major interest being the immune system.

Specific research undertaken by the Division includes investigations of viral replication, analyses of the immune response to viral infections, development of HIV and cancer vaccines, molecular and physiological analysis of autoimmunity and its contribution to the pathogenesis of diseases such as diabetes and multiple sclerosis, and research on the processes of inflammation, blood vessel growth and spread of tumours.

Several of the groups with overlapping interests have formed collaborative research programs that focus on specific areas and generate synergies that could not be achieved independently. For example, the Integrative Genetics Program pools the expertise of several groups in order to address fundamental questions regarding the biological and clinical significance of genetic diversity. These studies have implications for the understanding of the genotype/environmental interactions that appear to have a role in many disorders such as common forms of mental illness that are characterized by anxiety and depression, Parkinson's disease and different forms of cancer.

The pursuit of long term basic science goals makes 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 negatively-charged 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 for the treatment of cancer.

Cancer and Vascular Biology Group
Leader: Professor C Parish

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.

Cellular Laboratory
Leader: Professor C Parish

A major interest of the Cellular Laboratory of the Cancer and Vascular Biology Group is to examine cellular aspects of cell migration and invasion, with the role of heparan sulfate and the heparanase enzyme in these processes being a key area of study.

The research of the laboratory includes an analysis of leukocyte entry into inflammatory sites, the metastatic spread of tumour cells, and angiogenesis. The ultimate aim of these studies is to generate, in the collaboration with chemists, heparan sulfate mimetics as inhibitors of cell migration and invasion that can be employed as novel anti-inflammatory, anti-metastatic and anti-angiogenic drugs.

The laboratory is also interested in harnessing normal inflammatory responses against pathogens to combat the growth of solid cancers. Recent research highlights are:

Recent Phase I and II clinical trials of PI-88 in melanoma and multiple myeloma patients have yielded promising results. Additional trials in other cancers, such as small cell lung carcinoma and hepatocellular carcinoma, are underway.

The group has had considerable experience in designing sulfated oligosaccharide-based compounds as drug candidates, this part of the group's research being supported for eight years 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.

A very productive collaboration has developed during the last few years with Professor Martin Banwell, Research School of Chemistry, ANU, in which sulfated pseudo-sugars have been synthesised as heparan sulfate mimetics. Although the initial aim of this collaboration was 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. Furthermore, some of the compounds may be anti-metastatic by interfering with the binding of platelets to tumour cells via blocking the leukocyte adhesion molecule P-selectin. Interestingly, although P-selectin also mediates leukocyte adhesion to endothelium, the sulfated pseudo-sugars are probably poor inhibitors of this interaction. Thus these drugs could be used to target the tumour cell-platelet interaction without blocking the development of normal inflammatory responses. Such drugs have potential as anti-angiogenic, anticoagulant, antiviral and antilipaemic agents.

The laboratory 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. Furthermore, recent studies indicate that HRG can bind to necrotic cells and facilitate necrotic cell uptake by macrophages. 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 laboratory in the future is to better understand the functional significance of this intriguing plasma protein.

In a collaboration with Dr Paul Foster's group in the Division of Molecular Biosciences, 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. These studies also imply that eosinophils may play a previously unrecognised role in tumour immunosurveillance. Data supporting this view have come from carcinogenesis studies. It has been found that eotaxin deficient mice are dramatically more susceptible to the development of methylcholanthrene-induced tumours, particularly when low doses of the carcinogen are used. Further analysis of the molecular and cellular basis of this system is underway.

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 using single chain antibodies as a means of developing better vaccines and to use the technology as a delivery vehicle for gene therapy.

The Cancer and Molecular Immunology Laboratory of the Cancer and Vascular Biology Group focuses on understanding the molecular basis of cell invasion, with particular interest in inflammation, tumour metastasis and new blood vessel growth (angiogenesis). The major barrier for invading tumour cells, migrating leukocytes, and growing blood vessels (endothelial cells) is the basement membrane (BM) that surrounds the vessels, and the extracellular matrix (ECM), which forms a scaffold in tissues to hold cells together. The BM and ECM are composed of an interlocking network of proteins and complex carbohydrates, and for cells to breach this barrier they deploy a battery of enzymes that break down these proteins and carbohydrate components. The major carbohydrate is heparan sulphate (HS), which acts as the glue to maintain the integrity of the BM and ECM. The enzyme responsible for cleaving HS, heparanase, has been shown to play a key role in the degradation of the BM and ECM, and its activity strongly correlates with the metastatic capacity of tumour cells and the migratory capacity of leukocytes and endothelial cells. HS in the ECM also binds a number of angiogenic growth factors, and the release of these by heparanase promotes angiogenesis and tumour growth. Following our recent cloning of mammalian heparanase, we have been able to develop the tools to investigate how heparanase functions at the molecular level and to directly determine the role of heparanase in cell invasion, angiogenesis and inflammation.

Over the last year our major research achievements include: (i) using antisense RNA technology to demonstrate that heparanase plays a critical role in tumour metastasis; (ii) the generation of a heparanase gene targeted mouse embryonic stem cell line for the inactivation of the heparanase gene in mice; (iii) characterisation of a novel cell surface receptor for human heparanase; and (iv) the identification of the transcription factor Early growth response gene 1 as a key regulator of inducible heparanase gene expression. We are currently working towards (i) further understanding the molecular basis of heparanase function at the structural level using mutagenesis and computer modelling, (ii) identifying the protease(s) responsible for processing the enzyme to its active form, (iii) further characterising the molecular basis of heparanase gene transcription, and (iv) generating gene targeted mice that lack heparanase in specific cells and tissues to further define its role in cell invasion and angiogenesis.

Matrix Biology Laboratory
Leader: Dr C Freeman

Research within the Matrix Biology Laboratory complements that of the Molecular Mechanisms Laboratory by focusing on the biochemical basis of cell invasion during cancer spread (tumor metastasis), inflammation, and during new blood vessel growth (angiogenesis). We are also investigating the roles that the sulfated carbohydrate heparan sulfate (HS) and its degradative enzymes play in health and disease.

HS plays a vital role in many biological processes, including cell growth and development, cell attachment to the surrounding matrix, the breakdown of triglycerides and the entry of viruses and other pathogens into cells. Many biologically important proteins, enzymes and growth factors bind to cell surface HS which can regulate their physiological actions. To ensure the specificity of their actions, many of these proteins recognise unique sugar sequences within the HS molecule, which is quite variable in its structure. We are therefore developing procedures to determine these particular sugar sequences, which will allow us to design novel drugs (HS-mimetics) that mimic that specific HS sugar sequence. These drugs may then be used to specifically inhibit various physiological and pathological processes involving HS-protein interaction. For example, since tumor growth is critically dependent upon the development of a new blood supply, the selective blocking of angiogenic growth factor binding to cell surface HS has become a novel way to interfere with tumor development.

HS is also a key component of the extracellular matrix (ECM) and the vascular basement membrane that surrounds the blood vessels and acts as a barrier to cell invasion during tumor metastasis. Malignant tumor cells have elevated levels of the enzyme heparanase, which degrades HS, causing breakdown of the ECM structure and allowing tumor cell invasion. Heparanase is normally involved in embryonic development, angiogenesis, wound repair and inflammation, permitting cell migration through the ECM and the release of growth factors stored within the ECM that stimulate cell growth. However, heparanase activity secreted by a growing tumor may also release these growth factors, stimulating further tumor growth and new blood vessel growth that can allow subsequent tumor cell escape. Similarly, the uncontrolled invasion of leukocytes into the ECM can lead to inflammatory diseases such as inflammatory bowel disease and the progression of autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis.

Within the Cancer and Vascular Biology Group we are collaborating with Professor Chris Parish and Dr Mark Hulett to investigate the roles of heparanase in both health and disease, including the factors that regulate its normal activity. Using a novel enzyme assay, we were one of the first to purify and clone human heparanase, demonstrating that only one heparanase activity is expressed. Therefore heparanase represents an excellent target for the development of anti-cancer and anti-inflammatory drugs. We are currently characterising the proteases that activate the enzyme as well searching for the presence of endogenous inhibitors to determine if control of these factors can also be used to inhibit heparanase activity. We are also studying the interaction between tumor cells and blood platelets which is occurs during tumor metastasis as well as identifying important ligands involved in this interaction.

Previously, our group identified the HS-mimetic PI-88 which is both an effective inhibitor of heparanase activity and angiogenenic growth factor binding to HS. In animal models PI-88 prevented the growth and spread of cancer and it is currently undergoing clinical trials in cancer patients. We have developed a series of sulfated oligosaccharide HS-mimetics. Some of these compounds exhibit potent and selective inhibitory activity against heparanase activity and the binding of various growth factors, chemokines and proteins to HS. Such compounds may lead to the development of a new series of drugs to prevent cancer, inflammation, viral infection and to lower blood triglyceride levels.

Australian Cancer Research Foundation Genetics Laboratory
Leader: Professor C Goodnow

A one million dollar grant from the Australian Cancer Research Foundation in 1997 made possible the establishment of the ACRF Genetics Laboratory in the Medical Genome Centre. The laboratory's focus is on developing genome-wide mutagenesis resources for studying candidate human cancer genes and novel laboratory mouse models to accelerate cancer research at both the basic and clinical ends of the spectrum. Four new mouse strains to illuminate cancer genes have been obtained from the ENU1 forward genetics library. One of these strains, Plastic, carries a single dominant gene defect that results in acute T cell lymphoblastic leukemia, providing a valuable model for understanding this important form of childhood leukemia. Peter Papathanasiou has mapped and identified the mutation in this strain, which creates a single change in a protein that appears to be frequently defective in human T cell lymphoblastic leukemia. The cancer protein normally regulates expression of many other genes and plays an essential role in forming all the major blood cell types. The Plastic mutation also has broad significance for the field of mammalian genetics, because it highlights an important, unrecognised class of gene variant we have dubbed 'recessive niche-filling alleles'.

Adele Loy has mapped the mutation in another cancer-prone strain, Bblast, which carries a semi-dominant gene defect that results in leukemia, lymphoma, osteosarcoma, chondrosarcoma and teratocarcinoma. These cancers arise because the mice carry a single misspelling of a protein called Tumor suppressor p53. Tsp53 is the most frequent defect in a wide range of human cancers, and controls a range of tumour and ageing processes. The Bblast strain therefore provides a valuable model for investigating cancers of many types. A second Tsp53 mutant strain with a different missense change corresponding to one found in human cancer has been characterized in collaboration with Dr Keats Nelms in his colleagues at Phenomix Corp. Progress continued on a separate discovery project aimed at illuminating genes regulating solid tumours of the skin and cervix. This project involves a consortium with Dr Douglas Hanahan at the University of California in San Francisco, who has developed a sensitized mouse strain for skin and cervical cancer research, and Dr Simon Foote at the Walter and Eliza Hall Institute. The project has mapped cancer inhibitory genes between two inbred mouse strains, C57BL/6 and FVB.

Immunogenomics Laboratory
Leader: Professor C Goodnow

Our research aims to understand how immune cells make a fundamental decision: either to fight or to disarm. The process of deciding which immune cells should fight and which should disarm is key to our ability to resist infection and parasitism. Mistakes in this process result in autoimmune diseases, allergy, lymphoma, and leukemia. Moreover, drugs and other ways to alter fight or disarm decisions are sorely needed to improve the success of organ transplantation and treatment of autoimmune diseases and metastatic cancer.

The immune system is made up of billions of immune cells called lymphocytes. By a remarkable gene shuffling process akin to a poker machine, each lymphocyte carries a unique receptor, enabling every lymphocyte to detect a different set of molecules termed antigens. Some lymphocytes have receptors for foreign antigens that are unique parts of the molecular makeup of different infectious organisms. When these rare lymphocytes bind a foreign antigen during an infection, they receive a signal to fight. The lymphocyte first multiplies to make many clones of itself, and then the cells elaborate destructive compounds that neutralize the infectious antigen.

By chance, other lymphocytes carry receptors for self antigens, ie. parts of our own normal tissues and body fluids. When a lymphocyte binds a self antigen it normally receives a signal to disarm. Instead of multiplying and producing destructive compounds, the lymphocyte either commits cell suicide by apoptosis or the cell disarms itself by becoming functionally tolerant, ie. less responsive to antigens and less able to multiply or produce destructive compounds.

For a long time it was not possible to see how self-reactive lymphocytes disarm themselves. Our laboratory has developed ways to visualize this process in genetically modified laboratory mice called transgenic mice. By studying cells in the transgenic mice, we have discovered that each immune cell must run through a complex series of fight or disarm checkpoints before it can be fully launched into an immune response. In some ways, the process resembles the sequence of fight/disarm decisions in a military missile launch, which serve a similar purpose of preventing friendly fire.

Members of the laboratory are deciphering different fight/disarm checkpoint processes, using a combination of biochemistry, cellular immunology, genetic analysis, and transgenesis. At each of these checkpoints, we are focusing much of our work on elucidating how it is that antigen receptors on lymphocytes can trigger several different cell fates ranging from cell proliferation to cell death. Three examples of our work that have been published this year are summarized below.

Educating our T cells to recognize our own organs

One of the main classes of lymphocyte, T cells, are generated and exported by the thymus gland. A long standing mystery has surrounded how T cells that might attack unique parts of other organs in our body are disarmed, since the T cells would not have the chance to encounter those unique parts within the thymus. In the last year, Adrian Liston and colleagues in the lab have made a major advance to illuminate how the body solves this problem. The starting point was a rare, devastating human disorder called Autoimmune Polyendocrine Syndrome 1 (APS1), where the T cells attack and destroy a range of vital organs in the body. Collaborators in Finland had identified a mutation that inactivated a single gene, Autoimmune Regulator (AIRE), as the cause of APS1, and shown that inactivation of the corresponding mouse AIRE gene also caused an autoimmune polyendocrine syndrome. The sequence of the AIRE gene nevertheless provided no clues to its vital role in disarming T cells. By analysing transgenic mice lacking the AIRE gene, Adrian found that the key function of AIRE is to display parts of other organs within the thymus. He showed that the insulin gene, which normally produces its products abundantly in the pancreatic islets, is switched on in the thymus by AIRE in order to disarm T cells that might otherwise attack the pancreas and cause diabetes. Ongoing work is continuing to decipher how other genes cooperate in this vital pathway.

A central component of the fight/disarm switch.

How are lymphocytes triggered into diametrically opposite responses to fight or disarm, when both responses are triggered through a single receptor type? Previous work in the lab found that the receptors on B lymphocytes can switch between two different signal modes, activating a different set of signals and genes in cells that need to fight as opposed to cells that are disarmed. A central component for switching into the fight mode has now been discovered by Jesse Jun and a group of collaborators in the lab and at Phenomix Corp. A mutant mouse strain from our ENU mutagenesis libraries, unmodulated, inherited a single nucleotide DNA sequence change, altering one amino acid of a novel protein called Carma-1 or Card-11. The consequence of this change is that the receptors on B and T lymphocytes cannot switch into the fight mode. The Carma-1 protein is related to a class of proteins that serve as intracellular scaffolds, gathering many other proteins in cells together into complex circuits. Based on the changes in receptor signalling in unmodulated mice, we have hypothesized that Carma-1 switches receptors on lymphocytes into the fight mode by gathering many other proteins together into an immunosome - a complex that provokes the immune response.

Preventing our DNA from provoking autoimmune disease

In the human disease systemic lupus erythematosus (SLE), the normal processes for disarming self-reactive lymphocytes fail in the most extreme way by allowing antibodies to be made against the core of our body's fabric - our own DNA. Recent findings by others have shown that particular CG-rich sequences in our DNA, when unmasked and released from inside cells, can serve as a potent trigger of the B lymphocyte fight response. In the last year, Lixin Rui and colleagues in the lab have revealed an important checkpoint to counteract the ubiquitous autoimmune threat from our CG DNA. By analysing cells from transgenic mice, and employing retroviral gene therapy technology, he showed that B lymphocytes that react with ubiquitous parts of our body disarm the DNA response in two ways. First, the cells shut down the fight-response pathway from their receptors. Second, the cells keep active a separate blocking pathway involving the enzyme, ERK, which actively inhibits B lymphocyte differentiation into plasma cells. By blocking this final step in the process of antibody secretion, the immune system avoids SLE. By identifying this final blocking pathway, it provides a starting point to search for the genetic changes that allow SLE to develop in some people.

Molecular Virology Group
Leaders: Dr M Lobigs and Dr E Lee

Our aim is to study flavivirus host/pathogen interactions at the cellular and molecular level and through this devise strategies for the prevention of flaviviral disease. Flaviviruses are small, enveloped, positive-strand RNA viruses. They are transmitted to vertebrates by the bite of arthropod vectors and include some of the most important viral pathogens for humans (e.g. dengue, yellow fever, Japanese encephalitis, and West Nile viruses). Our studies focus on flavivirus replication, the molecular basis of flavivirus virulence, mechanisms of flavivirus pathogenesis, and the role of cellular and humoral immune responses in the recovery from flavivirus infection. Our expertise in these fields has been critical in the elucidation of unique, flavivirus-mediated, cellular and immunological phenomena.

Recent research highlights

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 mosquito-borne flaviviruses that cause severe encephalitic disease in humans. The recent emergence of several members of this serocomplex in geographic regions (including Australia) where other closely related flaviviruses 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 shown that immunity against Murray Valley encephalitis virus in vaccinated mice can cross-protect but also augment disease severity following challenge with Japanese encephalitis virus. Immunepotentiation of heterologous flavivirus disease was apparent in animals immunized with a 'killed' virus preparation when humoral antiviral immunity of low magnitude was elicited.

Role of type I and type II interferon responses in the recovery from infection with an encephalitic flavivirus

We have investigated the contribution of the interferon (IFN)- system, IFN-g, and nitric oxide to recovery from infection with Murray Valley encephalitis virus, using a mouse model for flaviviral encephalitis where a small dose of virus was administered to 6-week-old wild-type and gene knockout animals by the intravenous route. We have shown that a defect in the IFN-a/b responses results in uncontrolled extraneural virus growth, rapid virus entry into the brain, and 100% mortality. In contrast, mice deficient in IFN-g or nitric oxide production display an only marginally increased susceptibility to infection with the neurotropic virus.

Lack of both Fas ligand and perforin protects from flavivirus-mediated encephalitis in mice

The mechanism by which encephalitic flaviviruses enter the brain to inflict a life-threatening encephalomyelitis in a small percentage of infected individuals is obscure. We have investigated this issue in a mouse model for flavivirus encephalitis in which the virus was administered to six-week-old animals by the intravenous route, analogous to the portal of entry in natural infections, using a virus dose in the range experienced following the bite of an infectious mosquito. In this model infection with 0.1-10⁵ PFU of virus gave mortality in ~50% of animals despite low or undetectable virus growth in extraneural tissues. We have shown that the cytolytic effector functions play a crucial role in invasion of the encephalitic flavivirus into the brain. Mice deficient in either the granule exocytosis- or Fas-mediated pathways of cytotoxicity showed delayed and reduced mortality. Surprisingly, mice deficient in both cytotoxic effector functions were resistant to a low dose peripheral infection with the neurotropic virus.

Mechanism of virulence attenuation of glycosaminoglycan-binding variants of Japanese encephalitis virus and Murray Valley encephalitis virus

We have investigated the in vivo mechanism for virulence attenuation of laboratory-derived variants of two flaviviruses in the Japanese encephalitis virus (JEV) serocomplex. Host cell adaptation of JEV and Murray Valley encephalitis virus (MVE) by serial passage in adenocarcinoma cells selected for variants characterized by (i) a small plaque phenotype, (ii) increased affinity to heparin-Sepharose, (iii) enhanced susceptibility to inhibition of infectivity by heparin, and (iv) loss of neuroinvasiveness in a mouse model for flaviviral encephalitis. We previously suggested that virulence attenuation of the host cell-adapted variants of MVE is a consequence of their increased dependence on cell surface glycosaminoglycans (GAGs) for attachment and entry (E. Lee and M. Lobigs, J. Virol. 74:8867-8875, 2000). In support of this proposition, we find that GAG-binding variants of JEV and MVE were rapidly removed from the bloodstream and failed to spread from extraneural sites of replication into the brain. Thus, the enhanced affinity of the attenuated variants for GAGs ubiquitously present on cells and extracellular matrices most likely prevented viremia of sufficient magnitude and/or duration required for virus entry into the brain parenchyma.

Inefficient signalase cleavage promotes efficient nucleocapsid incorporation into budding flaviviral membranes

We have investigated the mechanism for efficient nucleocapsid (NC) uptake into flaviviral particles which form by budding through the membranes of the endoplasmic reticulum (ER). Budding of flaviviral membranes is driven by the viral transmembrane proteins, prM and E, independent of NC interaction. We have shown that control of a signalase cleavage in the multi-membrane-spanning flavivirus polyprotein by the catalytic function of the viral protease is critical for efficient virus morphogenesis. In wild-type virus signalase cleavage of prM remains inefficient until cleavage of capsid at the cytosolic side of the signal sequence separating the two proteins has occurred. This obligatory sequence of cleavages was uncoupled in a mutant virus with the consequence of greatly reduced incorporation of NC into budding membranes and augmented release of NC-free virus-like particles. Efficient signalase cleavage of prM in the mutant virus resulted in partial inhibition of cleavage of capsid by the viral NS2B-3 protease. Our results support a model for flavivirus morphogenesis involving temporal and spatial coordination of NC assembly and envelopment by regulated cleavages of an ER membrane-spanning C-prM intermediate.

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 down-regulating major histocompatibility complex (MHC) class I molecules flavivirus infection can up-regulate 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). Our recent studies show 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%. TAP expression is unaltered during infection and viral but not host macromolecular synthesis is required for enhanced peptide transport. This is the first demonstration of transient enhancement of TAP-dependent peptide import into the lumen of the endoplasmic reticulum as a consequence of a viral infection. We suggest that the increased supply of peptides for assembly with MHC class I molecules in flavivirus infected cells accounts for the up-regulation of MHC class I cell surface expression with the biological consequence of viral evasion from natural killer cell recognition.

Immunity and Immunopathology in Infectious Disease
Leaders: Dr A Müllbacher and Dr M Regner

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.

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 infections, and viral immune evasion strategies. A large number of virus models including flaviviruses, poxviruses, influenza and parainfluenza viruses, alphaviruses 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.

CD8⁺ T cells mediate recovery and immunopathology in West Nile virus encephalitis

C57BL/6J mice infected intravenously with Sarafend strain of West Nile virus (WNV) develop a characteristic central nervous system (CNS) disease, including an acute inflammatory reaction. Dose response studies indicate two distinct kinetics of mortality. At high doses of infection (10⁸ PFU) direct infection of brain occurred, resulting in 100% mortality with a 6 day mean survival time (MST), and minimal destruction of neural tissue. A low dose (10³ PFU) of infection resulted in 27% mortality (MST 11 days), and virus could be detected in the CNS 7 days post-infection (p.i.). Virus was present in the lymph nodes and spleens at days 4 to 7 p.i.. Histology of brains revealed neuronal degeneration and inflammation within leptomeninges and brain parenchyma. Inflammatory cell infiltration was detectable from day 4 p.i. onwards in the high dose group, and day 7 p.i. in low dose group with severity of infiltration increasing over time. The cellular infiltrates in brain consisted predominantly of CD8⁺ but not CD4⁺ T cells. CD8⁺ T cells in brain and spleen expressed the activation markers CD69 early, and CD25 at later time points. CD8⁺ T cell deficient mice infected with 10³ PFU WNV showed increased mortalities but prolonged MST and early infection of the CNS. Using high doses of virus in CD8 deficient mice leads to increased survival. These results provide evidence that CD8⁺ T cells are involved in both recovery and immunopathology in WNV infection.

Exocytosis and Fas mediated cytolytic mechanisms exert protection from West Nile virus induced encephalitis in mice

Infection of mice with the flaviviruses West Nile virus (WNV) and Murray Valley encephalitis (MVE), induces cytolytic T cell responses which are highly cross-reactive on target cells infected with heterologous flaviviruses. Thirty to forty percent of C57Bl/6 mice infected with low doses (10²-10⁶ PFU) of either virus develop encephalitis and die within 10-12 days. Mice with defects in the Fas or granule exocytosis (perforin and granzymes A and B) pathway of cellular cytotoxicity display reduced mortality and increased survival time when infected with MVE and are protected from encephalitis when deficient in both pathways. This contrasts with infection with WNV where defects in these cytolytic mechanisms increase the percentage of mice that succumb to encephalitis. Thus, no generalizations as to protective or detrimental effects of cytolytic effector functions in recovery from closely related flavivirus infections can be made. Virus-host immune interactions have to be assessed individually and cannot be generalized.

Mice with deficiencies in Fas or in perforin plus granzymes succumb to Trypanosoma cruzi infection by distinct lethal processes

Cytotoxic T cells (CTL) are critical for acute resistance to acute Trypanosoma cruzi infection, but are also implicated in the pathology induced by this protozoan parasite. Here we explore to what extent the two main cytolytic pathways of CTL, i.e. the granule exocytosis and Fas ligand/Fas pathways, are responsible for these pathogen-induced disease processes. We have employed mouse strains with targeted gene defects in one or more components of either of the two cytolytic pathways, including perforin, granzyme A and granzyme B, and Fas, to analyse the molecular basis of parasite clearance and disease susceptibility during infection with the highly pathogenic Tulahuen strain of T. cruzi strain. We show that parasites are effectively cleared from infected tissues by the concerted action of perforin and the two granzymes and independent of Fas. However the presence of both cytolytic pathways is mandatory for survival of infected mice. The finding that both mouse strains with deficiencies in either Fas or perforin plus the two granzymes similarly suffer from early death as compared to C57BL/6 wildtype mice, independent of their potential to eliminate parasites, clearly indicates that the two cytolytic pathways control distinct but vital processes during infection with T. cruzi.

The availability of mutant and gene-targeted knock-out mice with defects in components of either the Fas or the exocytosis mediated pathways of cellular cytotoxicity permitted an analysis of their role in recovery from poxvirus infections. Ectromelia (EV), a natural mouse pathogen causing mouse pox, the closely related orthopoxviruses cow pox (CPV) and vaccinia virus (VV), encode serpins which inhibit Fas mediated apoptosis and lysis of target cells. A lack of the perforin gene renders the highly resistant C57Bl/6 mice susceptible to low doses of EV. Lack of perforin has the opposite effect and increases resistance to CPV and finally deletion of perforin has no effect on VV infections. Absence of the cytolytic granule associated granzymes (gzm) A and B renders C57Bl/6 mice increasingly more susceptible to EV infections. Lack of both gzms renders them as susceptible as perforin deficient mice, this despite the presence of functionally active perforin. Elevated EV titres in liver and spleen of gzmAxB deficient mice, early after infection, strongly suggests that these two gzms exert an anti-viral effect by a mechanism distinct from effector molecules of NK and cytotoxic T cells.

Perforin does not express anti-viral activity by itself, but facilitates Fas-mediated recovery from lymphocytic choriomeningitis virus infection

We have analysed the response of mice that lack Fas as well as granzyme (gzm)A and gzmB (FasxgzmAxB^-/-), but possess normal levels of functional perforin and CTLs, to infection with lymphocytic choriomeningitis virus (LCMV). FasxgzmAxB^-/- mice are indistinguishable in their composition of lymphocyte subsets in their lymphoid compartments at an early age (up to 6-8 weeks) when compared to Fas-deficientmice. At greater than 8 weeks of age they exhibit an increased accumulation of the unusual CD4^-CD8^-Thy1.2⁺B220⁺ T cells in lymph nodes and spleen. This indicates a role for granzymes, in addition to Fas ligand/Fas, in the generation and/or maintenance of mature resting T cells. In contrast to Fas^-/-mice, FasxgzmAxB^-/- mice are unable to control a primary infection with LCMV. Moreover, compared to Fas^-/- mice, ex vivo-derived virus-immune FasxgzmAxB^-/- CTL lack perforin-mediated nucleolytic activity and express reduced cytolytic activity in vitro. The additional finding that virus-immune CTL from mice which express FasL and perforin (gzmAxB^-/-) exhibit higher nucleolytic potential in vitro than those from mice expressing FasL alone (perfxgzmAxB^-/-) suggests that Fas-mediated apoptosis is enhanced in the presence of perforin. We conclude that perforin on its own is functionally inert in vivo and strictly depends on either both gzmA and gzmB and/or FasL/Fas for optimal effector function of CTL in the recovery from viral infections and induction of apoptosis in vitro.

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 research 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.

Human Genetics Group
Leader: Professor S Easteal

Professor Simon Easteal

My research is aimed at understanding human genetic variation as it relates to disease. I take a broad view, recognising the importance of the evolutionary forces that have shaped the structure of the human genome and the nature of human genetic diversity. Our investigations fall into two areas: Mechanisms of gene and genome evolution, and the nature of human genetic and genomic variation.

Gene and genome evolution

In mammals genetic novelty arises through genetic and evolutionary processes such as mutation, gene duplication, gene conversion, genetic drift and natural selection, occurring in large and structurally complex genomes. Information relating to genome and protein function, the processes by which functional novelty has arisen and patterns of species evolution are contained within DNA and protein sequences. We use a comparative approach to the interpretation of this information, investigating differences in genomes, genes and the proteins they encode, among related species, particularly humans and other primates.

Human genetic and genomic variation

Medical genetics has largely been focused on understanding single gene defects. Less attention has been given to genetic disorders that have a polygenic basis, such as mental illness, cardiovascular disease and diabetes. Although these are more important from a public health point of view, they are technically more difficult to investigate. This situation is changing largely through the technological advances of the human genome project. Associated with this change is an increased recognition of the importance of the normal range of human genetic variation, and its importance in relation to disease prevention. Understanding the relationship between molecular genotypes and diseasephenotypes is becoming more dependent on population-based rather than family-based investigations. Our research is directed at understanding the basis of a number of genetic disorders, and of the patterns of normal genetic variation in human populations.One particular focus is the genetic basis of personality variation, which is associated with a predisposition to common forms of mental illness involving depression and anxiety. This work is done in collaboration with the NHMRC Centre for Mental Health Research.

There is increasing evidence that successful vaccination against HIV-1 will require the induction of strong, specific Cell Mediated Immunity (CMI), particularly cytotoxic CD8+ T-cell responses. Such CTL may attack cells early after infection through their recognition of specific peptides in association with class I molecules of the major histocompatibilty complex (MHC) on their surface and may also secrete a variety of soluble factors that help to control infection. DNA vaccines and recombinant virus vectors induce CMI responses but on their own have proved disappointing in the level of immunity elicited. We have pioneered the consecutive use of these vectors, prime-boost immunisation, and have shown greatly enhanced T-cell immune responses that in non-human primate models protect against HIV challenge. These vaccine approaches have now entered phase I clinical trials through a NIH funded consortium involving the Australian National University, University of Melbourne, University of New South Wales, University of Newcastle and CSIRO. The trial involves immunisation of volunteers with a DNA vaccine which has been designed with optimised immunostimulatory CpG motifs and encoding HIV genes. The vaccine boost is delivered through a non-replicating fowlpox virus vector encoding the same HIV-1 genes. The first volunteers were immunised June 2003 and further trials are planned for Thailand in 2004.

Cytokines and co-stimulatory molecules play a critical role in the regulation of immune responses and have been found to significantly affect the immune response to vaccine vectors, when co-expressed with HIV antigens. We have used a subtractive hybridisation approach to study the genes expressed by dendritic cells cultured under differing immunostimulatory conditions (Dr Joanne Banyer). We have demonstrated that distinct gene profiles are expressed under differing culture conditions including the expression of many novel genes. These are being investigated further to determine their role as vaccine adjuvants.

A wide range of live, attenuated viral and bacterial vectors have been used to deliver vaccine antigens, with alternative strategies focusing on the use of protective vehicles (including liposomes, microspheres and immunostimulatory complexes) or mucosal lectins (including cholera toxin and E. coli enterotoxin), but each has given mixed results. Alternative lines of research into mucosal vaccination for immune responses in the genital and/or intestinal tissues have shown that intranasal delivery may induce specific IgA antibodies and CD4+ T cell responses, both locally and in the reproductive tract, when antigen is conjugated to the beta-subunit of cholera toxin. More recently, work by ourselves and others has demonstrated that intranasal immunization with different types of recombinant vaccine vectors may also prime for mucosal antibody and T cell-mediated responses in the female reproductive tract. These include DNA vaccines, adenovirus vector and human papillomavirus-like particles. In addition, despite the almost axiomatic view that direct stimulation of MALT is necessary to induce good mucosal immunity, parenteral administration of certain antigens, particularly DNA vaccines, may, under certain circumstances, prime for immune responses at mucosae. The capacity of vaccines to produce, in this way, effective mucosal immunity in the reproductive tract and other mucosal sites following intranasal or parenteral delivery, is a highly attractive feature and may overcome the difficulties of mucosal priming that are particularly problematical for oral vaccination. However, there is little information available on the key issue of vaccine-induced T cell avidity. We now have data to show that intranasal immunisation with DNA vaccines and poxvirus boosting generates both mucosal and systemic CTL responses of high avidity. The combination of DNA and viral vectors appears to be critical, since a variety of other approaches, including multiple doses of peptides, DNA vaccines or other recombinant vectors, do not appear to produce this outcome. With both mucosal and systemic responses it is essential that priming with DNA occurs first followed by boost with recombinant virus. This project forms the basis for developing vaccine strategies that elicit CMI responses at mucosal surfaces, where HIV is first encountered.

We have recently reported that the MCP-1/CCR2 signaling pathway plays an important role in the rejection of pig islet tissue xenografts, by regulating the recruitment of macrophages and CD4 T cells. In that model, unlike allograft rejection, host antigen presenting cells (APCs) such as macrophages are essential for the processing of xenoantigens, presentation of xenopeptide(s) via class II MHC and the selective activation/expansion of xenoreactive CD4 T cells. In contrast, the present study has demonstrated that while the MCP-1/CCR2 pathway appears to regulate the initial recruitment of macrophages into allograft sites, this signaling mechanism plays only a minor role in allograft rejection, a finding consistent with no absolute requirement for alloantigen processing by host APCs. Thus, in the transplantation of pancreatic islet tissue, the histocompatibility barrier strongly influences the repertoire of chemokines, their interaction with chemokine receptors and, in association with the cytokines produced during rejection, the panel of leukocyte populations recruited to the foreign tissue transplant. In view of the known capacity for chemokines to bind to several different chemokine receptors (i.e. chemokine promiscuity) and to the clinical observation that renal allografts show prolonged survival in humans with a homozygous mutation in the CCR5 gene, our study suggests that targeting CCR5 and IP-10 -signaling chemokine receptors (e.g. CXCR3), rather than individual chemokines, may be useful for clinical islet tissue allotransplantation and the treatment of Type 1 diabetes.

Although much is known about NK cell activating receptors, little is known about the cellular ligands they bind. We have embarked on a programme of preparing extracellular domains of a number of these NK cell activating receptors, with the aim of using these recombinant proteins in a sensitive binding assay to identify the cells, and eventually to identify the ligands for these receptors.