Mapping of virulence markers in Respiratory Syncytial Virus (RSV)
Generally differences in the virulence of a virus are encoded within the genes of that virus. To test if we can predict if an infection with RSV is likely to be virulent or less virulent, we are sequencing the surface glycoproteins of Australian respiratory syncytial virus (hRSV) which have known virulence characteristics. We are looking for nucleotide changes within the viral genome which correlate with virulence characteristics. When we have identified potential sites we will concentrate on this/these sites to see if the predictive properties are conserved amongst different isolates. This project is currently being funded by The Royal Children’s Hospital Foundation for which we are indebted.
Back
Cloning and expression of Respiratory Viral antigens
Efforts to improve the availability of cheap, rapid and specific immunologically based tests for important respiratory viruses which circulate in Australia is an area in which our laboratory is concentrating most of our research efforts. The viruses which are of particular concern to us and to health authorities are hRSV, hMPV, hBoV and hCoV (human respiratory syncytial virus, metapneumovirus, Bocavirus and Coronavirus, respectively). We are currently cloning important surface antigens as well as the viral capsid proteins from each of these viruses and inserting them into bacterial as well as yeast expression systems. At present we are trialing various expression systems to optimize the yield as well as the correct conformational aspects of the recombinant proteins. This important aspect of the work needs to be done before we immunize animals with these antigens to raise polyclonal antibodies for eventual use in immunologically based detections systems such as ELISA. These recombinant antigens as well as the mono-specific antibodies are also very important aids for research into the morphogenesis (life cycle) of these viruses. At present such reagents from Australian viral isolates are not available in Australia. The recombinant antigens are useful not only to raise mono-specific antisera but to prime immunized animals to prepare messenger RNAs coding for the antibodies against these antigens. We are then able to clone the variable regions of the heavy and light chains of these messenger RNAs and insert them into bacterial expression systems to make soluble recombinant antibodies (scFvs). These antibodies can be genetically manipulated to make a range of reporter and binding molecules which when combined with the recombinant antigens gives us the opportunity to make a range of newer immunological detection systems for these respiratory viruses.
At present all of the viral proteins have been cloned and expression systems are being trialed and optimized. We have commenced preliminary cloning of chicken immunoglobulin variable light and heavy chain regions to make scFvs. We are also in negotiation with an overseas company to trial a first generation llama antibody library for its usefulness with our recombinant antigens in ELISA tests.
Back
Cloning and expression of Hepatitis B surface antigen in yeast
In collaboration with Professor Robert Tindle and his researchers we have cloned and expressed the small surface antigen from Hepatitis B virus into yeast. We have looked at its expression as a secreted protein as well as one produced in the cytoplasm of the cells and found that internally synthesized protein accumulates 500 fold higher when compared to that secreted by the yeast cells. The production of virus-like particles by this yeast system was done by using a monoclonal antibody which is capable of detecting a conformational epitope only present on viral particles. This aspect is currently being confirmed by electron microscopy.
The yeast expressed clone described above is being used as a molecular frame-work to add on other viral proteins of interest to see if the yeast expressed hepatitis B VLPs are capable of being ‘decorated’ with this antigens which in turn are capable of eliciting a protective immune response.
Back
Cloning and expression of capsid proteins from Bocavirus
Recently in 2005, a new Parvovirus was detected in humans in the Netherlands which was capable of producing severe respiratory disease in children. After its discovery it was quickly detected in respiratory cases in Australian children by the use of a PCR assay. However this virus is unable to be replicated in tissue culture or in animals and thus there is no laboratory method for producing large quantities of the virus for diagnostic assays or potential vaccines. To address these issues we are at present amplifying and cloning the two genetic regions of this virus which encode the proteins responsible for encapsidation of the viral genome. These two constructs will be expressed individually and together in a yeast expression system to see if viral like particles (VLPs) can be produced in vivo. The VLPs will be analysed for their ability to react with human antisera in diagnostic assays as well as their ability to produce protective antibodies when injected into mice or other species. This project is currently the focus for an Honours student within the laboratory.
Back |
