Rapid HIV-1 Tropism Testing Using Novel, Soluble Mimics Of The HIV-1 Coreceptors CCR5 And CXCR4
Funder
National Health and Medical Research Council
Funding Amount
$163,426.00
Summary
This proposal seeks to develop an inexpensive assay to determine whether HIV patients will benefit from treatment with new drugs referred to as CCR5 antagonists. These are effective against HIV strains that use the CCR5 coreceptor, therefore a patient�s HIV coreceptor usage must be assessed before commencing therapy. Current assays are complicated, slow and expensive. Using novel, soluble mimics of the coreceptors we will develop an ELISA based test that can be operated using standard equipment.
Kunjin Replicons For Gene Therapy And Protein Manufacture
Funder
National Health and Medical Research Council
Funding Amount
$310,000.00
Summary
This grant seeks to provide proof of concept (PoC) for the use of the Kunjin replicon technology for gene therapy and protein production. (A) Protein production. Two Kunjin replicon constructs expressing green fluorescent protein (GFP) and secreted alkaline phosphatase (SEAP) are to be constructed and protein production monitored using FACS and SEAP bioactivity reporter kit (Roche), respectively. Protein production and biological activity of the proteins will be monitored in transient transfecti ....This grant seeks to provide proof of concept (PoC) for the use of the Kunjin replicon technology for gene therapy and protein production. (A) Protein production. Two Kunjin replicon constructs expressing green fluorescent protein (GFP) and secreted alkaline phosphatase (SEAP) are to be constructed and protein production monitored using FACS and SEAP bioactivity reporter kit (Roche), respectively. Protein production and biological activity of the proteins will be monitored in transient transfections and over an extended time period. Several cell lines, culture conditions and Kunjin replicon vector modifications will be tested. Arrangements have also been made to send the constructs to Roche, GSK, Eli Lilly, and Exelixis for side by side comparisons of this system with existing proprietary protein production echnologies. (B) Gene therapy. Two PoC gene therapy systems are proposed to be used for evaluation of Kunjin replicon vectors. (i) Tumours expressing granulocyte macrophage colony stimulating factor (GMCSF) by transfection cause the generation of anti-tumour CD8 T cells and subsequent tumour rejection. Current approaches include adoptive transfer of adeno-GM-CSF transfected tumour cells, a costly and laborious process resulting in only transient expression (Can. Imm. Immunother 2001 50:373). We intend to inject Kunjin replicon virus like particles into growing s.c. B16 melanomas and expect to see a high infection rate, a sustained high-level expression of GMCSF, and rejection of the tumour. In contrast to Kunjin, nearly all humans have antibody responses to adenovirus, and very high titres of adenovirus are required to obtain high infection and GM-CSF expression. Both factors limit adenovirus use in vivo. (ii) Transplant rejection can be inhibited by expression in the graft of CTLA4-Fc a reagent that blocks T cell co-stimulation enhancing allo-graft acceptance (Transplantation 2000 69:1806). High-level expression for over 100 days is expected to correlate with optimal graft acceptance. Our ability to use Kunjin to express beta galactosidase for several months in vivo without inflammation illustrates the potential for this approach (CIB ref 15). Initially we intend to use P815 cells injected i.p. into C57BL-6, where they are usually rejected within a few days. In contrast, P815 cells with Kunjin replicon-mediated CTLA4-Fc expression should survive for an extended period. Graft survival is easily monitored using FACS and anti-H-2d antibodies.Read moreRead less
Construction And Immunogenic Evaluation Of Recombinant HBsAg-S Virus-like Particles Containing B And T Cell Epitopes Of
Funder
National Health and Medical Research Council
Funding Amount
$170,000.00
Summary
Helicobacter pylori is a significant human pathogen impacting on the health and well being of not only thousands of Australians, but also millions of people world-wide. However, the task of developing a vaccine against H. pylori remains important. Vaccination is the most effective mechanism to prevent disease associated with this infection, particularly gastric cancer, one of the most common causes of cancer death world-wide. However, current attempts to develop an effective vaccine for humans h ....Helicobacter pylori is a significant human pathogen impacting on the health and well being of not only thousands of Australians, but also millions of people world-wide. However, the task of developing a vaccine against H. pylori remains important. Vaccination is the most effective mechanism to prevent disease associated with this infection, particularly gastric cancer, one of the most common causes of cancer death world-wide. However, current attempts to develop an effective vaccine for humans has been limited by the non-availability of an effective and safe adjuvant. The aim is to construct a recombinant Virus-Like Particle which can be used as a safe and effective vaccine against Helicobacter pylori infections. We specifically aim to: · determine the most efficacious singular or combinatorial route-s of delivery of Virus-Like Particles (VLPs) which will induce the desired Th2 and B cell responses in mice · define the Th2 and B cell epitopes of H.pylori Kat A carboxyl terminus that can be used to construct chimeric HBsAg-S-Kat A VLPs · determine if the induction of desired immunological responses in mice are protective against wild type challengeRead moreRead less
Inhibitors Of Bacterial Protein Synthesis - A New Class Of Antibiotics
Funder
National Health and Medical Research Council
Funding Amount
$120,000.00
Summary
Pioneering work by CSIRO scientists has identified specific peptide motifs in the DNA replication machinery of bacteria that are critical for the correct functioning of the organism. In collaboration with CI Alewood potent (Kd ~ nM) lead compounds that inhibit bacterial DNA replication have been designed and synthesised. Through the application of a number of novel bioinformatics approaches to the analysis of the complete genome sequences of bacteria, the key sites of interaction of a number of ....Pioneering work by CSIRO scientists has identified specific peptide motifs in the DNA replication machinery of bacteria that are critical for the correct functioning of the organism. In collaboration with CI Alewood potent (Kd ~ nM) lead compounds that inhibit bacterial DNA replication have been designed and synthesised. Through the application of a number of novel bioinformatics approaches to the analysis of the complete genome sequences of bacteria, the key sites of interaction of a number of protein families (DNA synthesis and repair enzymes) with the beta subunit of bacterial DNA Polymerase III have been identified. The nature of the sites, and preliminary experimental data, suggests that the approach will be generally applicable to all species of bacteria. In addition a wide range of novel assays for the identification of inhibitors of the interaction of proteins with the beta subunit have been developed. In this proposal we wish to demonstrate that our in vitro nanomolar inhibitors of the beta subunit can inhibit bacterial cell growth. The development program proposes to develop methods and strategies to gain bacterial cell entry of inhibitors of the interaction of proteins with the beta subunit of bacterial DNA Polymerase III. Proof of concept will be demonstrated by inhibition of bacterial cell growth. Stable compounds with good binding characteristics and able to be taken up by cells will be developed based on structure-function assay results, structural studies and modelling of inhibitors bound to the target. Antimicrobial activity of compounds will be demonstrated in standard FDA approved NCLLS (National Centre of Clinical Laboratory Standards USA) tests. Spectrum of activity will be demonstrated by testing compounds against bacterial species representative of the range of pathogenic organisms in standard FDA assays.Read moreRead less
Oxidised Mannan As A Novel Adjuvant To Vaccinate Against Mucosal Infections
Funder
National Health and Medical Research Council
Funding Amount
$150,000.00
Summary
Most pathogens invade via the mucosal surfaces. However, current vaccines, which are delivered by injection, are poor at inducing mucosal immunity. An ideal vaccine would comprise a defined protein antigen combined with a suitable adjuvant which could be administered intranasally or orally. Protective antigens have been defined for a number of infections but suitable adjuvants have been elusive. We showed that mannan, a complex carbohydrate from yeast, oxidatively linked to protein antigens can ....Most pathogens invade via the mucosal surfaces. However, current vaccines, which are delivered by injection, are poor at inducing mucosal immunity. An ideal vaccine would comprise a defined protein antigen combined with a suitable adjuvant which could be administered intranasally or orally. Protective antigens have been defined for a number of infections but suitable adjuvants have been elusive. We showed that mannan, a complex carbohydrate from yeast, oxidatively linked to protein antigens can be used as an adjuvant for mucosal IgA and other classes of antibody. Given to mice intranasally, antigen coupled to mannan markedly enhanced production of IgA, IgG1 and IgG2a in serum, and IgA in lung, tears, vaginal secretions, saliva and gut. We have confirmed this for a number of known or putative protective antigens. In addition, both the Th1 and Th2 arms of the lymphocyte response were activated. We have demonstrated protection against P. gingivalis (cause of periodontitis and associated with premature birth and cardiovascular disease) in a mouse lesion model. However, before commercial interests will commit themselves, we need to demonstrate protection against viral infections and in other sites like lungs and gut. Three infection models where IgA has been shown to protect are already set up and can realistically produce results in 1 year. 1. Rotavirus is the major cause of severe infantile gastroenteritis in humans and animals world wide. The latest (live) vaccine was withdrawn because of side effects. We have established a model with Simian rotavirus causing an acute self-limiting disease in infant mice. Adult females will be immunised with mannan linked to killed virus preparations, mated and passive protection of their offspring will be assessed. Preliminary evidence links rotavirus infection with the onset of type 1 diabetes. If this is confirmed, there will be an opportunity to test the vaccine against diabetes. 2. Influenza: IN infection of mice with flu virus is a well established model. Mice will be immunised IN with mannan coupled to haemagglutinin-neuraminidase purified from egg-grown virus. They will be challenged IN with influenza virus and virus titrated in lung homogenates. Neutralising antibody in serum and lung washings will essayed. 3. Respiratory syncytial virus: RSV is the commonest cause of bronchiolitis and pneumonia in infants for which there have been unsuccessful attempts to produce a vaccine. F and G membrane glycoproteins have been shown to protect mice against IN infection, and they will be used coupled to mannan to vaccinate mice against intranasal challenge.Read moreRead less
Mechanically-restricted Percutaneous Gene Therapeutic Solutions For Heart Failure.
Funder
National Health and Medical Research Council
Funding Amount
$187,000.00
Summary
We have developed a novel system for the localized delivery of specialised genes to the heart in order to improve contractility and function of a failing heart. Many genes, for reasons of toxicity, clearance, or uptake, require direct delivery to the target region without spillover to the systemic circulation. Our system addresses these issues by isolating the local circulation of the target organ and directly delivering the agent with minimal systemic loss and improved delivery and uptake effic ....We have developed a novel system for the localized delivery of specialised genes to the heart in order to improve contractility and function of a failing heart. Many genes, for reasons of toxicity, clearance, or uptake, require direct delivery to the target region without spillover to the systemic circulation. Our system addresses these issues by isolating the local circulation of the target organ and directly delivering the agent with minimal systemic loss and improved delivery and uptake efficiency, while minimizing potentially dangerous and toxic systemic effects.Read moreRead less
Development Of Chimeric Hepatitis B Virus Like Particles As A Vaccine Delivery Platform For Multiple HIV-1 Epitopes
Funder
National Health and Medical Research Council
Funding Amount
$139,500.00
Summary
The small envelope protein of hepatitis B virus (HBsAg) can self-assemble into highly organised viruslike particles with about 150 HBsAg-proteins forming a virus-like particle (VLP). VLPs induce an effective immune response, mainly against the exposed major antigenic site, the hydrophilic ‘a’- determinant region. To create a novel HBsAg-specific vaccine vector, foreign epitopes were inserted into the major antigenic site allowing surface orientation of the inserted sequence. Pilot studies involv ....The small envelope protein of hepatitis B virus (HBsAg) can self-assemble into highly organised viruslike particles with about 150 HBsAg-proteins forming a virus-like particle (VLP). VLPs induce an effective immune response, mainly against the exposed major antigenic site, the hydrophilic ‘a’- determinant region. To create a novel HBsAg-specific vaccine vector, foreign epitopes were inserted into the major antigenic site allowing surface orientation of the inserted sequence. Pilot studies involving the vaccination of mice with VLPs containing an epitope derived from the AIDS-virus (human immunodeficiency virus 1, HIV-1) or various hepatitis C virus-specific epitopes resulted in high titre antibody responses. This project aims for the development of a multi-component vaccine targeting a non-structural HIV-1 protein and therefore, avoiding the selective pressure directed against the structural proteins. The non-structural HIV-1 tat-protein is a multi-functional protein with an extracellular mode to sensitise uninfected cells for HIV-1 infection and to reactivate HIV-1 from quiescently infected cells. The use of eight tat-sequences is sufficient to provide coverage against 99% of HIV-1 sequences. We will develop hybrid particles that are composed of different sets of chimeric HBsAg proteins each containing a distinct tat-epitope. With this application, we aim to develop hybrid particles for the delivery of the complete set of tat-epitopes. The hybrid particles will be used for vaccination studies in mice, and the antibodies assessed by an in-vitro assay. This will lead to the development of a therapeutic and-or prophylactic HIV-1 vaccine, which could be used either for mass immunisation or in support of combination drug therapy and would have all the cost and production advantages of the widely used hepatitis B vaccine.Read moreRead less
A Novel Vaccine Platform For Trimeric Envelope Proteins: HIV-1 Envelope
Funder
National Health and Medical Research Council
Funding Amount
$139,250.00
Summary
Vaccines are urgently needed for the prevention of HIV/AIDS. The design of this vaccine candidate is based on the display of HIV-1 envelope spikes using a related primate retrovirus envelope with a more stable assembly to anchor the the spikes in a particle.
Production Of A Novel Humanised Anti Dendritic Cell Therapeutic Antibody For Graft Versus Host Disease
Funder
National Health and Medical Research Council
Funding Amount
$202,500.00
Summary
A transplant of bone marrow or other source of blood stem cells from a donor is often used to treat leukaemia patients whose disease has failed to respond to chemotherapy. The Mater Medical Research Institute has developed a world first dendritic cell depleting therapeutic antibody which may open a new strategy for the control of acute graft versus host disease, which is a very common and often fatal complication of bone marrow transplantation. The new antibody treatment is also likely to be use ....A transplant of bone marrow or other source of blood stem cells from a donor is often used to treat leukaemia patients whose disease has failed to respond to chemotherapy. The Mater Medical Research Institute has developed a world first dendritic cell depleting therapeutic antibody which may open a new strategy for the control of acute graft versus host disease, which is a very common and often fatal complication of bone marrow transplantation. The new antibody treatment is also likely to be useful for the prevention of rejection in solid organ transplantation. If successful, it will selectively control graft versus host disease, without compromising the essential anti-viral immunity and desired anti-leukemia activity of the graft.Read moreRead less
A New Non-invasive Diagnostic Technique Based On Detection Of Exhaled Respiratory Pathogens.
Funder
National Health and Medical Research Council
Funding Amount
$179,300.00
Summary
We developed a special collection mask and showed that the breath of people with colds or flu contains a tiny amount of virus. Currently, diagnostic samples are collected by putting a tube into the airways - this is very uncomfortable. Our masks may provide a new and more comfortable way to diagnose lung infections. We want to build better masks and ways to detect viruses and bacteria to test out this method. This may create a new test that will improve diagnosis and treatment.