Centre for Virus Research 

HIV molecular pathogenesis

The HIV molecular pathogenesis group’s primary focus is to understand the very early interactions of the HIV virus with host cells of the body, predominantly human dendritic cells (DCs). These are the first cells to come into contact with HIV within the genital tract.

Our group has utilised microarrays to assess the global effects of HIV on the DC transcriptome. In doing so we have gained an understanding of the many ways that HIV shapes the intracellular environment and manipulates these cells to facilitate its own transport and subsequent transfer to CD4-T lymphocytes, its primary target cell. We have shown that HIV triggers two distinct phases of gene expression in DCs, one early in infection (6hrs) corresponding to viral entry and second burst later in infection (48hrs) corresponding to viral replication. Based on this data we are currently following three main avenues of research.

In 2009 we published in Blood journal (PMID: 19436054) that HIV is able to down regulate lysosomal enzyme activity in DCs after infection. We are currently investigating the functional consequences of this i.e. enhanced viral survival within the DC and reduced HIV antigen presentation toT-cells.

Our microarray studies have also and indicated that HIV is able to induces the expression of discrete subset of interferon stimulated genes (ISG) in absence of any interferons. We are currently investigating the mechanism by which HIV i) inhibits the production of interferon by DC and ii) how the virus induces ISG expression. We are specifically focussing on the role the interferon regulatory factor family in the process .

The global gene changes that HIV induces in dendritic cells continue to be investigated using DNA microarray technology, and investigations are now being broadened to include the effects that HIV has on the proteins regulating the expression of interferons, subverting the effect of these antiviral substances and inducing a subset of interferon stimulated genes, probably to boast its own replication.

In addition to microarray studies we are conduction proteomic studies on DCs. We are particularly interested in the oligomeric structures of the DC surface receptors (C-type lectin receptors) that bind HIV. We have published two papers in the Journal of Biological Chemistry (PMID: 15385553 and 19224860) looking at DC-SIGN and the mannose receptor respectively. We are currently conducting similar studies on Langerin.

We are also investigating ways to prevent initial infection of the female genital tract via DCs in skin/genital mucosa.The methods employed include using soluble receptors as decoys binding to the virus as well as direct antagonism of the HIV binding receptors by blocking. Preliminary studies indicate that these can be effective.

Cytomegalovirus

Cytomegalovirus (CMV) is a medically important virus which infects a vast majority of the world’s population. Although infection is usually mild in healthy individuals, it is a frequent cause of serious, life-threatening disease in neonates and immunosuppressed individuals such as solid organ and bone marrow transplant recipients, and in people with HIV AIDS.

After initial (primary) infection, the virus is not completely cleared by the host’s immune system, but rather has the remarkable ability to remain inside the body for the life of an individual in a dormant (or latent) form. Periodically, the virus may reawaken from this latent state by a process called reactivation, resulting in the production of new infectious virus. It is this reactivation from the latent state in immunosuppressed individuals which is thought to be the major cause of serious, often fatal disease in these people. Despite the critical importance of latency to resulting disease, this phase of infection remains very poorly understood.

The CMV Research Group is working to discover the fundamental basis by which CMV is able to remain hidden in the human host in a latent form. The group has applied a variety of molecular and cell biology and proteomics approaches to identify both viral genes and human genes that play important roles during CMV latent infection of human cells. These studies include identification of a viral gene active latent infection which renders latently infected cells “invisible” to the immune system. Identifying the critical virus and host cell components of latency and reactivation will provide a rational basis for the design of drugs and therapies to limit the consequences of CMV disease in immunosuppressed individuals.

There is currently no vaccine against CMV, so the Group is also seeking to develop a CMV vaccine with enhanced capacity to stimulate protective immunity to infection.

Retroviral genetics

The Retroviral Genetics Laboratory focuses on several different aspects of HIV pathogenesis aimed at unveiling genomic and proteomic interactions of HIV with its host.

We are working on the innate and adaptive immune factors, which are found in untreated HIV+ patients with non- progressive disease, especially the ones who have naturally controlled HIV disease for >20 years with strength of their immune system. One such combination of factors has been characterized and recently patented, which will be the subject of in vitro human clinical trial. Our main goal is to understand what guides the path to non-progressive HIV disease in a subset of HIV+ individuals and whether these natural factors, which provide them protection against HIV, can find some use as therapeutic agents/vaccines. We are adopting a variety of immunological, virological, proteomic and genomic techniques to identify and characterize novel immune factors, which underlie non-progressive HIV disease.

Recently we have extended our studies to cell transcriptome level, where we are investigating the whole human genome (>47,000 genes) in different cell types, such as CD4+/CD8+ T cells, Monocytes and NK Cells derived from viremic and aviremic HIV+ therapy naïve and therapy experienced individuals in order to understand how HIV guides different stages of HIV disease by differentially and systematically subverting the cellular gene machinery within the same host.

Our objective is to define genomic and proteomic imprints of HIV in HIV-infected progressing and non-progressing individuals. Also, since the majority of HIV+ individuals receive highly active antiretroviral therapy (HAART), our lab is most interested in unveiling the pharmaco-genomic imprints of drug toxicity in HIV+ individuals and genetic mechanisms 1. Which can partially restore the host immune system during treatment and 2 which can the success and failure of HAART treatment.

All the aforementioned genomic and proteomic studies using microarray will be complimented by analysing microRNA in each of the cell types discussed above, which associate with HIV virus infection in humans. This detailed mapping of cellulartranscriptomes of HIV-infected cells will guide the development of new generation of biomarkers/prognostic and diagnostic tools, along with providing a detailed snapshot of virus-host interaction at the genomic and proteomic levels.

The group is also highly focused on current trends in viral epidemiology (such as emerging viruses and HIV-HCV co- infections) and developing novel diagnostic and prognostic molecular technologies for HIV, influenza, Hepatitis C virus (HCV), Human Papilloma virus (HPV), SARS virus and most recently Influenza Swine Flu (H1N1) virus, etc.The Retroviral Genetics lab also runs services for drug resistance and epidemiologic testing of HIV in Australia.

HIV Biology

The HIV Biology team aims to improve our understanding of how HIV can spread rapidly between cell types which are known to be important for HIV transmission. HIV transmission begins as a chain of events that enables a critical infection threshold to be reached. By researching this spread, a greater understanding can be attained with respect to mechanisms of HIV migration and amplification. In doing so strategies designed specifically at breaking any part of the chain of transmission can be used in future scenarios for HIV prevention and/or therapy.

Current studies of the group include the genetic manipulation of HIV vectors to spy on HIV in live cells using fluorescent microscopy and the generation of HIV vectors for visualization at the electron microscopy level. It is through the generation of such tracking devices, that the group has observed cellular “needle like” projections are used by the virus to initiate the process of jumping from cell to cell. The work would not be possible, if it were not for two criteria: Firstly the genetic engineering of viruses that permits visualisation over time in living cells and secondly the use of cutting edge microscopy to capture these events. For the latter, the HIV biology group has been instrumental in acquiring this technology in collaboration with chief investigators from the Westmead Research Hub.

Recently the group has aligned with several collaborators from the University of Sydney (School of Chemistry) and CMRI, to develop new therapeutics that act at blocking viral entry and spread throughout the body. For the latter the HIV Biology group has been successfully awarded a translational grant through the Australian Centre for HIV and Hepatitis Virology Research scheme, to study the role of a common cellular protein, Dynamin II, in HIV entry and spread. This collaboration with Professor Phil Robinson at CMRI, will potentially short-list existing drugs targeted towards this cellular protein for use in future HIV therapeutics and prevention strategies.

Molecular viral transport and assembly

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are important human pathogen, causing encephalitis, blindness and severe neonatal infection. They also enhance the acquisition of HIV three-fold. The transport of HSV-1 and HSV-2 to and from the periphery within the axons of nerve cells is a key component of their life cycle.

The group aims to define a molecular interaction network or infectome at the protein level during the course of infection of a host cell by HSV-1. This will unlock the mechanism(s) involved in the entry, axonal transport, assembly and exitof HSV-1 from nerve cells. In addition, this will assist in a general understanding of host cell function.

Recent work has identified crucial molecular interactions, involving viral and host cell proteins, required for viral assembly and transport. Such information on viral replication will allow development of inhibitors of key steps within this process. These may then be used as antivirals for control of recurrent herpes simplex. A similar approach is also being undertaken with HIV.

Varicella zoster virus

Despite its significant impact on the community, little is known about the molecular basis of Varicella zoster virus (VZV) infection, due in part, toVZV only infecting humans. To more closely examine the interaction of VZV with host cells, the group has established several models of infection using human cell-types which are targets for infection and are relevant to those that suffer from either Varicella or herpes zoster/PHN because each of these cell types are likely to play different, but essential roles in the disease process.These include human fibroblasts (skin cells), neurons (nerve cells) and specialized immune cells (T cells and dendritic cells).

The group has shown that human nerve cells infected withVZV do not undergo programmed cell death (apoptosis). This is an important finding because it suggests the nerve cell damage observed when VZV reawakens from its "silent" state in nerve cells to cause shingles is not due to programmed cell death.

Another implication from this observation is that VZV encodes a function to interfere with the death response in human nerve cells, thus providing a possible mechanism by which the virus can establish and maintain its life-long dormant infection.

We went on to identify the first VZV encoded anti-apoptoticgene ORF63. To further our understanding of VZV with human nerve cells the group has developed and recently published a novel model of VZV infection of intact human explant ganglia.

The group has shown for the first time that VZV can infect intact human ganglionic cells and this is a novel way of studying the interaction of this virus with human nerve cells. These features of intact ganglionic infection can now be studied in further detail to better define the molecular mechanisms that underlie VZV infection of ganglionic cells.

For example, this model provides a means to rapidly test viral gene mutant viruses and new candidate vaccine strains containing targeted gene disruptions to define viral genes that may play critical roles in VZV neurotropism and to examine in detail the outcome of infection of both neurons and non-neuronal cells with respect to apoptosis and cell function.

Significantly, we have now obtained rare naturally infected ganglia samples at autopsy from patients who suffered from herpes zoster close to the time of death, but who had died from unrelated causes. We are now uniquely placed to extend our earlier work by examining the interaction of VZV with ganglionic cells in both experimentally and naturally infected settings. These studies will be important for the development of better therapies to lessen the impact of VZV disease on the community.

Given the importance of the skin as a site of VZV infection and the role skin DC play in the induction of anti-viral immunity, there is good reason to study infection and modulation of DC in human skin during VZV infection. To date, the group has determined the DC subsets that may participate in VZV pathogenesis by immunostaining sections of chickenpox and shingles skin lesions for immune cell markers. InVZV infected skin, Langerhans cells (LC), were decreased and plasmacytoid DC (PDC), DC that produces high levels of IFN-alpha, were increased in frequency compared to uninfected skin.

We investigated whether these DC subsets support VZV infection in vivo by dual immunofluorescently staining sections of VZV infected skin lesions for LC/PDC markers and VZV proteins. Notably, a proportion of LC and PDC were positive for VZV proteins, suggesting these cells can be infected. Further assessment of skin DC infection, immune function and viability will define the mechanisms underlying cutaneous infection.

These studies will enable a critical definition of the mechanistic basis of VZV modulation of host cell functions which is needed for development of a “second generation” vaccine to lessen the impact of VZV disease on the community.

Herpes Neuropathogenesis

Transmission of Herpes simplex viruses (HSV) occurs from close contact with an individual who is actively shedding virus. Viral shedding generally occurs from lesions but can occur even when lesions are not apparent. Herpes infection can be treated but not cured, as the virus infects nerve cells and lies dormant in these cells during the lifetime of the human host.

HSV can awake or reactivate frequently and its reactivation can be triggered by a number of factors including stress or illness. Our laboratory has established several models for culture and HSV-1 infection of primary human fetal and rat nerve cells in vitro.

Our studies aim to understand how HSV-1 invades and replicates in nerve cells and the mechanisms used by the virus to travel along nerves for efficient virus spread during primary infection and after reactivation.

We have used fluorescent-tagged viruses to visualize how the virus enters, travels along axons (nerves) and exit nerve cells using fluorescence and real time imaging. We have also used electron microscopy in order to elucidate, at an ultra structural level, the mechanisms used by the virus to travel along nerves and for virus assembly and exit from nerve endings.

Our findings have led to the establishment of a model in which virus components are transported separately along nerves and the virus is assembled at axonal swellings and nerve endings before the virus exit the nerve cell. Our studies have shown that the virus has evolved to utilize existing neuronal vesicles and secretory pathways for transport of its components and for its exit from nerve cells.

Understanding the mechanisms used by HSV for entry, transport along nerves, and assembly in nerve cells will assist in the development of new strategies for antivirals for control of recurrent herpes disease. In addition, elucidation of the mechanisms of how HSV-1 utilizes the existing transport pathways in nerve cells for virus spread from nerve cell to nerve cell will further assist in the use of HSV as gene therapy vector to deliver drugs to the nervous system.

HSV Immunopathogenesis

Herpes Simplex Virus (HSV) causes two common infections, cold sores (HSV 1) and genital herpes (HSV2). Infection can sometimes result in fatal encephalitis or neonatal herpes. Genital herpes enhances HIV acquisition up to threefold. A partially effective vaccine candidate has been developed (partly based on our previous work) but is not yet licensed.

The HSV Immunology group is focussed on discovering and developing new vaccine candidates and has also been studying the interaction between HSV and immune cells like dendritic cells (DC) and T lymphocytes.

Dendritic cells (DC) are the most potent antigen presenting cells to stimulate T lymphocytes and are classified as myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC). Langerhans cells are another type of DC residing in epidermis, which are presumed to play a primary role in herpes lesions. Such Langerhans cells have now been shown to be infected in herpes lesions. How HSV infects these cells in vitro and alters their function is now being studied. pDCs are supposed to be restricted to blood and lymph nodes. However during 2008, the group used confocal microscopy, to identify pDC in the skin of genital herpes lesions and demonstrated they were resistant to HSV infection but produced much antiviral interferon, preventing virus spread deeper into the skin. They also interact with other immune cells, probably to eradicate the virus.

In herpes lesions CD4 lymphocytes are the first to enter and initially control HSV infection, preparing the way for later infiltration by CD8 lymphocytes which clear up the infection. \We have now identified key peptides in a major herpes simplex viral protein glycoprotein , (which is a vaccine candidate) which stimulate these ‘CD4 lymphoytes’. They are recognised broadly by most people infected by HSV1 and HSV2 a rationale for why infection with one of these two viruses can protect against the other.

Some of these peptides were conjugated to lipid (in collaboration with Professor David Jackson, University of Melbourne). The immunogenicity of the lipopeptides was improved 2-3 fold. We are currently examining the mechanism of these effects and hope to further develop these peptide stimulators for both CD4 and CD8 lymphocytes as potential vaccines.