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Macfarlane Burnet Centre for Medical Research and Public Health

Localisation of Vpr protein during HIV-1 infection

Supervisors: Associate Professor D McPhee, Dr M Bateson (Tel: 9282 2111)
Email: mcphee@burnet.edu.au / mbateson@burnet.edu.au
Location: AIDS Cellular Biology Unit, Macfarlane Burnet Centre for Medical Research and Public Health

The Vpr protein is a 96 amino acid "accessory protein" of HIV-1 which has been found to be essential to productive infection. Although the function of Vpr within the life cycle of the virus remains unclear, a number of functions such as G2/M cell cycle arrest, LTR transactivation and nuclear localisation of the viral genome have been reported. The Vpr protein is expressed late in the viral cycle and packaged within the nucleocapsid of maturing virus particles via an interaction with the p6 domain of Gag. Previously in our laboratory we have constructed a Vpr protein fused to the green fluorescence protein (GFP) of the jellyfish Aequorea victoria. This enabled the visualisation of the localisation pattern of this protein when it is expressed within human cells. The project will extend this work by co-transfecting cells with both the Vpr-GFP protein and a HIV-1 virus lacking native Vpr function. It is expected that by visualising the migration of the fusion protein during infection that more can be learnt about events and interactions that are important to the function of Vpr. This approach will lead to learning more novel ways to stop HIV-1 replication.

Interactions between the M and M-1 proteins of the human respiratory syncytial virus

Supervisors: Dr R Ghidhyal, Professor J Mills, Dr J Meanger (Tel: 9282 2111)
Email: mills@burnet.edu.au / jayesh@burnet.edu.au
Location: Children’s Virology Research Unit, Macfarlane Burnet Centre for Medical Research and Public Health

Respiratory syncytial virus (RSV) is the major cause of viral pneumonia in infants. It is also the cause of lower respiratory disease in the elderly and immunocompromised adults. Annual epidemics of RSV are well documented, occurring in winter in temperate climates and in the rainy season in tropical regions. These epidemics can cause substantial morbidity and mortality. There is no vaccine available for RSV and no efficacious therapy. We, at the Children’s Virology Research Unit are studying the assembly of RSV within the host cell with a view to identifying novel targets for therapy and possible vaccine candidates. RSV is an enveloped, negative strand RNS virus belonging to the family Paramyxoviridae and the genus Pneumovirus. Assembly of Paramyxovirus virions during the final stages of infection is initiated by the interaction of nucleocapsids with specific components of the viral envelope, leading to bud formation and subsequent viral release. The M (matrix) protein of paramyxoviruses facilitates assembly by associating with nucleocapsids and virion envelope glycoproteins (F and HN). Previous work in the laboratory has demonstrated a specific interaction of the RSV M protein with the envelope glycoproteins and with the nucleocapsid complex. The interaction with nucleocapsids is facilitated by a specific interaction of the M protein with the M2-1 protein of RSV.

In this project, we will attempt to define the domains of the interaction between these two proteins using in vitro binding assays. This will involve extensive cloning of truncated mutants of the two proteins followed by expression and purification from bacterial cells. The purified proteins will be used in ELISA and protein overlay protein binding assays.

Studies in international health

Supervisor: Dr M Toole (Tel: 9282 2111)
Email: toole@burnet.edu.au
Location: International Health Unit, Macfarlane Burnet Centre for Medical Research
and Public Health

Field study of perinatal interventions - hepatitis B vaccination, micronutrient supplementation growth monitoring, breast feeding (Indonesia).

Evaluation of community-based HIV prevention projects (India, Southern Africa, PNG).

Pilot study of communicable disease prevalence (and relevant knowledge, attitudes, and practices) in Victorian migrant communities.

Cloning, expression and interaction of the respiratory syncytial virus subgroup B G glycoprotein with M proteins

Supervisors: Dr J Meanger Dr R Ghidhyal, Professor J Mills (Tel: 9282 2111)
Email: mills@burnet.edu.au / jayesh@burnet.edu.au
Location: Children’s Virology Research Unit, Macfarlane Burnet Centre for Medical Research and Public Health

Respiratory syncytial virus (RSV) is the major cause of viral pneumonia in infants. It is also the cause of lower respiratory disease in the elderly and immunocompromised adults. RSV is an enveloped, negative stranded RNA virus belonging to the family Paramyxoviridae and the genus Pneumovirus.

Viral assembly of paramyxoviruses during the final stages of infection is initiated by the interaction of the nucleocapsids with specific components of the viral envelope, leading to bud formation and subsequent viral release. The single M protein of paramyxoviruses facilitates assembly of virus particles by associating with virion glycoproteins (F and HN/G) on intracellular membranes followed by localisation to the plasma membrane or by forming patches of M protein-glycoprotein complexes at the plasma membrane. A similar role in viral assembly has been hypothesised for the two RSV matrix proteins, M and M2.

Two antigenic subgroups (designated A and B) have been described for RSV. Recent work in our laboratory has shown that the subgroup A, strain A2, G glycoprotein interacts with the M protein, and that the interaction is localised within the N-terminus of the G glycoprotein. The sequence of the subgroup B RSV G gene is slightly different than that of the A group, but interestingly there are only two substitutions in N-terminal amino acids. It is interesting that this maintains the charge balance pretty well and thus may not be critical to the interaction. In this project we would like to confirm if these changes have any effect on M-G interaction.

STUDIES IN THE EPIDEMIOLOGY OF INFECTIOUS DISEASES

Socioeconomic associations of sexually transmitted diseases: mapping the occurrence of genital chlamydial infection in Melbourne, using notifications of diagnoses and census data.

Measuring the social and economic costs of acute hepatitis in Victoria: we do not have any measure of the costs to Australia of viral hepatitis, either acute or chronic. To begin to rectify this lack, this project will follow up all notifications of acute viral hepatitis in Melbourne over a defined period of time, using a standardised instrument to measure social and financial costs thereof.

An audit of syphilis testing in Victoria: in 1999 there were at least 40,000 tests for syphilis (not including antenatal testing), for the discovery of two infectious cases. This project will review syphilis testing in Victoria with the aim of targeting it more efficiently.

Hepatitis C virus infection among attendees at a methadone general practice: a review of computerised records of all testing for hepatitis C at a large general practice specialising in methadone maintenance, and a file audit of records of those with new HCV infections, to monitor the incidence of hepatitis C in this population.

CENTRE FOR HARM REDUCTION

A review of development of policy relating to the control of hepatitis C virus infection among injecting drug users: why has it been so slow and ineffective?

HIV-1 Viral factors associated with delayed progression to AIDS

Supervisors: Associate Professor N Deacon, Dr M Churchill (Tel: 9282 2111)
Email: deacon@burnet.edu.au / churchil@burnet.edu.au
Location: AIDS Molecular Biology Unit, Macfarlane Burnet Centre for Medical Research and Public Health

A small percentage (<5%) of persons infected with HIV-1 have a significantly delayed progression to disease (AIDS). This may be due to host or viral factors, or a combination of both. Host factors include HLA haplotype, a strong immune response to HIV antigens or altered genotype of co-receptor molecule (e.g. CCR5D32 allele of chemokine receptor). Viral factors include mutations of a number of HIV-1 genes. We have demonstrated that deletions of the HIV-1 nef gene and the LTR region (viral gene promoter) are associated with slow/non-progression in 4 independent Australian strains of HIV-1. In one of these strains further deletion of LTR sequence has led to a progression to disease. In some other cases of slow progression without antiviral therapy nef/LTR deletions have not been found, suggesting that other factors are involved.

Current research is aimed at (1) investigating LTR sequence changes that are associated with the switch from non-progression to progression and the mechanism by which these changes may lead to a change in viral gene expression and (2) determining the genomic sequence of HIV-1 strains from therapy naïve slow/non-progressors to examine possible associations of sequence changes with non-progression.

Inhibition of phagocytosis by HIV-1 infection of human macrophages

Supervisors: Dr A Jaworowski & Professor Suzanne Crowe
Email: anthonyj@burnet.edu.au / crowe@burnet.edu.au
Location: AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research and Public Health

Our laboratory has shown that HIV infection inhibits the ability of professional phagocytes such as macrophages to ingest and destroy certain micro-organisms (particularly those responsible for HIV-related opportunistic infections in patients with AIDS). This is a major mechanism contributing to the development of opportunistic infections in AIDS patients.

We have recently shown that HIV infection of human monocyte-derived macrophages drastically inhibits their ability to phagocytose both complement- and IgG-opsonised particles. A project is offered to investigate the mechanism by which HIV infection of human monocyte-derived macrophages inhibits phagocytosis. Specifically, we are interested in (a) whether HIV infection inhibits phagocytosis by elevating intracellular [cAMP] and (b) whether inhibition is at the level of target binding, or phagosome formation and maturation. The project involves a wide variety of techniques including cell culture, growing and maintenance of HIV-1 virus stocks, measurement of intracellular cAMP levels and confocal microscopy.

Importance of tat in HIV infection in macrophages

Supervisor: Dr Con Sonza
Email: sonza@burnet.edu.au
Location: AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research and Public Health

When compared to infection in CD4+ T lymphocytes, HIV infection in macrophages does not kill the cells and they remain infected for their lifetime. In vivo, tissue macrophages are thought to be important reservoirs of virus and their infection contributes to the pathogenesis of HIV infection. We have shown that even long-term treatment with highly active antiretroviral drugs, which reduce circulating virus to undetectable levels, does not prevent blood monocytes, which home to various tissues in the body, from remaining or becoming infected. When blood monocyte-derived macrophages are infected with HIV in the laboratory, the virus replicates to reasonable levels over a 2 to 3 week period but then the levels of virus produced decline and remain low for the life of the culture. We have shown that this decline in virus production from the macrophages is coincident with a specific down-regulation of the production of the messenger RNAs which code for an important viral regulatory protein, Tat. Tat's main role in replication is as a transactivator of viral transcription. Supplying the infected macrophages with an exogenous source of Tat produces a burst of virus production from them.

In this project we are interested in further characterising the role of Tat in the regulation of HIV infection in macrophages. Areas to be explored include: the characterisation of Tat protein production in macrophages during long-term infection determining whether the tat gene becomes defective over the course of infection and determining the levels of a variety of cellular proteins known to interact with Tat protein or RNA and to affect its function to see which may explain the observed decline in tat mRNA.

The impact of HIV-1 protease mutations on the arrangement of HIV-1 RNA

Supervisors: Dr J Mak & Professor S Crowe
Email: mak@burnet.edu.au / crowe@burnet.edu.au
Location: AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for
Medical Research and Public Health
The HIV-1 encoded protease (PR) protein is an essential participant in the assembly process of HIV-1. The recent successful use of PR inhibitors (PIs) as part of highly active antiretroviral therapy (HAART) in patients with HIV infection highlights both the importance of viral assembly in the HIV-1 replication cycle and its potential as an anti-retroviral target for drug development. The emerging clinical evidence of drug failure in HAART suggests that development of safe, novel, and effective antiretroviral agents, such as those which target HIV-1 assembly, is highly desirable.
The focus of this proposal is to closely examine the molecular biology of how the HIV protease enzyme is involved in HIV-1 assembly, in particular the formation of virion RNA dimers, and to identify important events in HIV-1 assembly for the development of new drugs. The clinical significance of HIV-1 virion RNA dimerisation will be examined using patient isolates of HIV-1 that are resistant to PIs. We predict that mutations conferring HIV-1 PI resistance will inhibit the formation of stable virion RNA dimers, and that multiple or increasing numbers of PI resistance mutations will further destabilise the virion RNA dimers.

Functional and structural requirement of sphingomyelin in the assembly of HIV-1

Supervisors: Dr J Mak & Professor S Crowe
Email: mak@burnet.edu.au / crowe@burnet.edu.au
Location: AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research and Public Health

The envelopes of retroviruses are derived from the plasma membrane of the virion-producing cell. Analysis of HIV-1 and other retroviruses has shown that an unusually high cholesterol:phospholipid ratio is found in the retroviral envelope in comparison of the host cell membrane. Cholesterol and sphingomyelin are two lipids that are enriched in the HIV-1 envelope, and they represent approximately 15% and 4% of the total mass to HIV-1, respectively. We have evidence that removal of cholesterol from HIV-1 envelope alters the virion structure and inhibits virion infectivity, but the contribution of sphingomyelin in HIV-1 replication is less well understood. The focus of this proposal is to examine the structural and functional importance of sphingomyelin in HIV-1, and to identify important events in HIV-1 assembly for the development of new antiretroviral drugs. Various forms of fractionation procedures will be used in combination with different lipases to delineate the roles of sphingomyelin in the HIV-1 replication cycle and to identify new targets for anti-HIV-1 therapy.

A new role of primer binding site (PBS) in HIV-1 replication

Supervisors: Dr J Mak & Professor S Crowe
Email: mak@burnet.edu.au / crowe@burnet.edu.au
Location: AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research and Public Health

The primer binding site (PBS) of HIV-1 is found near the 5’end of the HIV-1 RNA genome, and it is essential for the annealing of tRNALys3 onto the RNA genome to initiate HIV-1 replication via reverse transcription. The HIV-1 PBS is part of the 5’ untranslated region (5’UTR) which has been shown to be important in multiple parts of HIV-1 replication, eg stability of RNA genome, mRNA transcription, mRNA splicing, protein translation, genomic RNA dimerisation and cDNA synthesis of genomic RNA via reverse transcription. The 5’UTR has extensive RNA secondary structures, and it is probable that PBS is involved in other aspects of the HIV-1 replication cycle. The focus of this project is to explore the potential importance of PBS mRNA stability, transcription, and protein expression.

We have constructed a series of 12 HIV-1 PBS deletion mutants, which consist of various deletions within PBS. Preliminary data have shown that a PBS _25 deletion expresses wild type level of HIV-1 protein whereas a PBS _13 deletion does not express HIV-1 protein with similar efficiency. These preliminary data suggest for the first time that the HIV-1 PBS sequence has a role in the regulation of viral protein production, and the precise contribution of the PBS to HIV-1 viral protein production will be initially mapped using the existing mutants. The precise RNA secondary structure and RNA sequences that are required for viral protein production will also be delineated via site-directed mutagenesis.

Role of disulphide interactions in hepadnaviral envelope stability and entry into

the host cell
Supervisors: Dr E Grgacic (Tel. 9282 2109) and Dr D Anderson
Email: grgacic@burnet.edu.au
Location: Hepatitis Research Unit, Macfarlane Burnet Centre for Medical Research and Public Health

Hepatitis B virus (HBV) is responsible for most hepatocellular carcinomas worldwide. Despite increasing use of an effective vaccine, there remain 350 million carriers who will continue to progress to severe disease for the next 30-40 years. There is thus a need to develop effective antiviral drugs, for which a detailed understanding of viral replication is essential.

A major obstacle to HBV research is the lack of a cell culture infection system for human HBV, precluding studies of the early events of the virus life cycle. The best system to study these events are animal models of the related members of the family, hepadnaviridae, of which the duck hepatitis B virus (DHBV) model has been a major source of our understanding of hepadnaviral replication. We have been using this model to study the two viral envelope proteins, their topology, interactions and modifications and their role in the entry process.

The small (S) and the large (L) envelope proteins are multi-spanning transmembrane proteins, synthesised from the same open reading frame. Both proteins contain three highly conserved cysteine residues, at least two of which are involved in intramolecular disulphide bonding which may serve to stabilise the entire envelope. We also have evidence that reduction of these disulphide bonds may play a role in conformational changes in envelope proteins associated with viral fusion. In this project, we will determine the role of these disulphide bonds in stabilising the virus envelope by examining the temperature sensitivity of native and reduced viral particles, and we will examine the role of the cysteine residues in promoting correct protein folding and S:L interactions in the membrane during virus assembly and disassembly/fusion.

Interactions of hepatitis B virus with polarised hepatocytes

Supervisors: Dr D Anderson (Tel: 9282 2239) and Dr E Grgacic
Email: anderson@burnet.edu.au
Location: Hepatitis Research Unit, Macfarlane Burnet Centre for Medical Research and Public Health

In order to interact with both external (lumenal) and internal compartments of the body, epithelial cells are highly polarised, with discrete protein and lipid compositions in the surfaces facing the lumen (apical domain, AP) and the serosa and neighbouring cells (basolateral domain, BL). This polarity is established through the formation of tight junctions (TJs) at the points of contact between cells, and the network of TJs between neighbouring cells provides an effective barrier to simple diffusion of small molecules and viruses.

Polarised cells can provide a barrier to viral entry by diffusion, yet the respiratory, genital and intestinal mucosa are also the primary sites of infection for many viruses. In such cases cell polarity can modulate the pathogenesis of viruses, many viruses being released in a vectorial fashion, predominantly to either the AP or BL domain. This has been extensively studied for many viruses such as influenza A and poliovirus. In addition, it is now clear that some viruses, such as the human immunodeficiency virus (HIV), can penetrate the epithelial barrier by the process of "transcytosis" (trans-cellular transport), gaining access to internal sites of the body without infecting the epithelial cells.

Nothing is known of the interactions between the viruses which infect the liver (hepatitis A, B, C, and E viruses) and the polarised hepatocytes of the liver. Such studies have not been possible in the past because polarised hepatocytes have a complex cellular architecture which prevents experimental access to the separate AP and BL domains of the cell.

We have recently developed a subline of liver-derived HepG2 cells which exhibit cell polarity but with the simple cellular architecture more typical of kidney, intestinal and respiratory epithelia which have been used to study virus/polarised cell interactions in the past.

In this project, interactions between the human and duck hepatitis B viruses (HBV and DHBV) and polarised HepG2 cells will be studied, focussing initially on three main questions. (1) Is the expression of viral receptors and virus binding restricted to either AP or BL domains? (2) Is the export of virus from infected cells similarly (or differently) restricted? (3) Is such export affected by the presence or absence of virus receptors and the morphological state of the viral particle?

This study will involve cell culture, virus growth, various molecular biology techniques and immunofluorescence and confocal laser scanning microscopy. These studies will have direct implications for understanding the pathogenesis of hepatotropic viruses.

Drug resistance in hepatitis B

Supervisor: Dr D Anderson (Tel: 9282 2239)
Email: anderson@burnet.edu.au
Location: Hepatitis Research Unit, Macfarlane Burnet Centre for Medical Research and Public Health

Hepatitis B virus (HBV) is responsible for most hepatocellular carcinomas worldwide. Despite increasing use of an effective vaccine, there remain 350 million carriers who will continue to progress to severe disease for the next 30-40 years. Chronic hepatitis B virus (HBV) infection is increasingly being treated with the use of antiviral drugs (nucleoside analogues), but drug-resistant viruses are rapidly emerging with subsequent treatment failures. Improved therapies, including new targets with less cross-resistance, are required.

The mutations in HBV associated with resistance to different drugs and the pattern of cross-resistance between drugs has been largely evaluated, but it is not known how these drug-resistant viruses spread through the liver. Because almost every cell in the target organ is infected with HBV, and HBV infection of cells normally prevents "superinfection" by another (eg drug-resistant) HBV, the question arises of how the drug-resistant virus becomes "fixed" in the patient, with important implications for treatment.

The closely related virus of domestic ducks, DHBV, has a replication cycle, structure, genome, and profile of drug sensitivity and resistance almost identical to that of human HBV. Using infectious cDNA clones of DHBV, drug-resistant viruses will be constructed in which an antigenic epitope "tag" will allow us to detect the spread of drug-resistant virus through the liver in the presence or absence of ongoing wild-type virus infection, and the presence or absence of antiviral therapy (applying selective pressure in favour of the tagged virus). These studies will directly contribute to an improved understanding of drug resistance and ultimately to improved treatment strategies for chronic hepatitis B.

Antigenic structure of hepatitis E virus

Supervisor: Dr D Anderson (Tel: 9282 2239)
Email: anderson@burnet.edu.au
Location: Hepatitis Research Unit, Macfarlane Burnet Centre for Medical Research and Public Health

The enterically transmitted hepatitis E virus (HEV) causes acute and often severe hepatitis, with a mortality of around 30% during pregnancy. HEV is the most common cause of acute hepatitis in many developing countries, accounting for as much as 70% of hepatitis in Nepal, India and similar countries. Infection is rare in developed countries, although there is evidence that zoonotic (eg pig) strains cause some disease. We have developed improved serological assays for diagnosis of HEV infection and an experimental subunit vaccine which is currently being trialed in animals. These advances have been based on the unique structure of one antigen derived from the virus, known as ORF2.1. Monoclonal antibodies (MAbs) to ORF2.1 have allowed us to dissect its antigenic structure in some detail, showing that it is highly conformational and immunodominant.

In this project, we will use molecular biological methods to gain a more detailed understanding of the ORF2.1 antigenic epitope. Sequence differences between animal and human strains of HEV will be reconstructed in the ORF2.1 clones by site-directed mutagenesis, and the reactivity of these mutant proteins with MAbs, patient and animal sera will be determined. Domains of the protein which have been implicated in protein dimerisation will also be targeted to determine the role of dimerisation in formation of the epitope. Finally, panning of phage display peptide libraries with MAbs will be used to isolate "mimotopes" which mimic the antigenic structure of this immunodominant epitope. These studies will contribute to further improvements in the diagnosis and control of HEV infection.

The cysteine loop of the large envelope protein of duck hepatitis B virus Role

in hepadnaviral infectivity
Supervisors: Dr Elizabeth Grgacic (Tel 9282 2109)
Dr David Anderson (Tel 9282 2239)
Email: grgacic@burnet.edu.au
Location: Hepatitis Research Unit, Macfarlane Burnet Institute for Medical Research and Public Health (Burnet Institute), AMREP, Prahran

Description: Hepatitis B virus is still a major human pathogen, responsible for most hepatocellular carcinomas worldwide. Despite an effective vaccine, the 350 million carriers remain a major stumbling block to curtailing this long-term disease, calling for a need to identify new antiviral targets for the development of effective therapies. We have been using the Duck Hepatitis B Virus (DHBV) model to study the assembly of the viral envelope proteins and their role in the poorly understood process of viral entry and fusion. The hallmark of enveloped viral entry is a major conformational change of an envelope protein allowing fusion with the cell. For the hepadnaviruses, this has been observed for the first time in the DHBV large envelope protein, where the reduction of disulphide bonds in the L protein is implicated in the entry process.

Aims: The aim of this project is to study the role of 3 critical cysteines of the L protein in viral particle stability, export and entry into the host cell. Characterisation of these highly conserved residues will make an important contribution to understanding the entry mechanism of the hepadnaviruses.

Techniques:

  • molecular biology for DNA cloning and site-directed mutagenesis
  • cell culture for transfection of cell lines with mutant plasmids and infection of primary duck hepatocyte cultures
  • biochemistry for analysis of viral proteins by SDS-page and Western blotting
  • basic virology, such as sucrose gradient purification of viral particles

Transmission of hepatitis viruses through epithelial barriers

Supervisor: Dr David Anderson (tel 9282 2239)
Email: anderson@burnet.edu.au )
Location: Hepatitis Research Unit, Macfarlane Burnet Institute for Medical Research and Public Health (Burnet Institute), AMREP, Prahran

Description: In order to interact with both external (lumenal) and internal compartments of the body, epithelial cells are highly polarised, with discrete protein and lipid compositions in the surfaces facing the lumen (apical domain, AP) and the serosa and neighbouring cells (basolateral domain, BL). Polarized epithelia provide an effective barrier to simple diffusion of small molecules and viruses, but it is now clear that some viruses, such as the human immunodeficiency virus (HIV), can penetrate the epithelial barrier by the process of "transcytosis" (trans-cellular transport), gaining access to internal sites of the body without infecting the epithelial cells. Hepatitis A virus (HAV) is enterically transmitted and must gain access to the liver after first entering the gut. While hepatitis B and C are often considered as "blood borne", HBV is in fact readily transmitted by sexual or other close contact which implies that it must be able to cross epithelial barriers. In contrast, HCV is almost always transmitted by direct blood to blood contact, and may therefore be less efficient in crossing the epithelial barrier. Although these interactions are a key step in viral pathogenesis, this area has never been explored for the hepatitis viruses.

Aims: This project will examine the efficiency of virus transmission through epithelial barriers. Polarized epithelial cells representing the mucosal and hepatic epithelia will be grown in semi-permeable Transwell culture vessels, providing a model of intact epithelia. HAV or HBV will be added to the apical side of such cultures, and the efficiency of virus release at the opposite face of the monolayer will be measured using cell culture infectivity assays for each virus. The effects of factors such as antibody, cell-associated versus free virus, the presence of viral receptors, and differences between mucosal and hepatic epithelial cells will be examined, leading to a greater understanding of viral pathogenesis.

Techniques: This study will involve cell culture, virus growth, SDS-PAGE and western blotting, molecular biology techniques (cloning and preparation of DNA, transfection), ELISA, immunofluorescence and confocal laser scanning microscopy. These studies will have direct implications for understanding the pathogenesis of hepatotropic viruses.

References:

  1. Blank CA, Anderson DA, Beard M, Lemon SM. 2000. Infection of polarized cultures of human intestinal epithelial cells with hepatitis A virus: vectorial release of progeny virions through apical cellular membranes. J Virol. 74: 6476-6484.
  2. http://www.burnet.edu.au/Internet/research/anderson/HepG2.html
  3. Hocini H, and Bomsel M. (1999). Infectious human immunodeficiency virus can rapidly penetrate a tight human epithelial barrier by transcytosis in a process impaired by mucosal immunoglobulins. J Infect Dis 179 Suppl 3:S448-53.

Therapeutic DNA vaccines for hepatitis B – studies in an animal model

Supervisors: Dr Fan Li (tel 9282 2121) & Dr David Anderson (tel 9282 2239)
Email: lifan@burnet.edu.au (Dr Li), anderson@burnet.edu.au (Dr Anderson)
Location: Hepatitis Research Unit, Macfarlane Burnet Institute for Medical Research and Public Health (Burnet Institute), AMREP, Prahran

Description: DNA vaccines hold enormous promise for the prevention and/or therapy of many diseases where conventional vaccine approaches have failed. In particular, the ability of DNA vaccines to express native antigens in the cells of the host, and thus elicit both antibody and cellular immune responses to these antigens, are key theoretical advantages of the DNA vaccine approach. However, the magnitude of immune responses to most DNA vaccines has been disappointing. A variety of complementary methods have been explored to enhance such responses, such as the inclusion of immunostimulatory sequences (CpGs), costimulatory cytokines, prime-boost protocols etc. A further method is the targeting of vaccine antigens by fusion with proteins which have defined routes in the cell, eg ubiquitin leading to rapid degradation, or LAMP-1 leading to ER translocation, or TPA signal sequences leading to excretion from the cell, or CTLA4 leading to binding of antigen presenting cells.

We have developed a novel strategy for targeting of vaccine antigens, which is being explored in our laboratory for a number of viral diseases. Hepatitis B is a major human health problem, for which there is already a good preventative vaccine but a therapeutic vaccine would have enormous benefit for the 350 million carriers of the virus worldwide. Domestic ducks carry a closely related virus, the duck hepatitis B virus (DHBV) and will be used as a model of chronic HBV disease in this project.

Aims: This project will examine the intracellular processing and subsequent antibody responses to a number of candidate DHBV DNA vaccines, in which the DHBV antigens are targeted using different fusion proteins (including our novel strategy). DNA sequences encoding DHBV antigens will be cloned into plasmids, and the expression and intracellular processing of the antigens will be examined in transfected cell cultures. Subsequently, the most promising DNA vaccine candidates will be used to immunise ducks which are already infected with DHBV, and the level of immune suppression of viral replication will be measured by standard techniques (infectivity assay for virus, dot blot and southern blot for viral DNA, immunohistochemistry for viral antigens in liver). We expect that one or more targeting strategies will lead to an enhanced immune response and control of DHBV infection compared to previous studies of "untargeted" DHBV DNA vaccines (2), which will form the basis for further studies on therapeutic vaccines for human HBV.

Techniques: This study will involve cell culture, virus growth, SDS-PAGE and western blotting, molecular biology techniques (cloning and preparation of DNA, transfection, southern and dot blots), ELISA, indirect immunofluorescence, and some animal studies. These studies will have implications for the development of therapeutic vaccines for HBV.

References:

  1. Jilbert A R, and Kotlarski I. 2000. Immune responses to duck hepatitis B virus infection. Dev Comp Immunol. 24:285-302.
  2. Triyatni M, Jilbert AR, Qiao M, Miller DS, Burrell CJ. 1998. Protective efficacy of DNA vaccines against duck hepatitis B virus infection. J Virol. 72:84-94.
  3. Vickery K, Cossart Y, Dixon R. 1999. Comparison of the kinetics of the specific cellular immune response to duck hepatitis B virus in infected and immune ducks. Vet Microbiol. 68:157-69.