Interferon, virus vaccines and antiviral drugs
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The emergence of viruses with zoonotic potential, i.e. with the potential ability to cross species barriers to infect unnatural hosts, poses a huge threat to humans. It is therefore essential to develop new methodologies to rapidly and efficiently generate attenuated virus vaccine candidates to attempt to control the threat. Viruses need to be able to at least partially inhibit the host’s innate defence mechanism, known as the interferon (IFN) system, to replicate efficiently in vivo and establish a productive infection. It has been previously reported that viruses that have lost their ability to circumvent the host’s IFN response, or IFN-sensitive viruses, are promising candidates for live attenuated virus vaccines. Here we report on the development of a cell-based method to attempt to rapidly select IFN-sensitive viruses that can not block IFN signalling, from wild-type virus populations. Lentivirus vectors containing selection markers (HSV-tk – Herpes Simplex virus thymidine kinase gene and pac – puromycin resistance gene) under the control of a tight IFN-inducible promoter (the murine Mx1 promoter) were generated and used to specifically engineer HEp2 cell lines, termed Mx GIPSE and Mx TIPSE, for this purpose. The developed methodology relies on the engineered cell lines and a selection procedure using exogenous IFN-α and puromycin: if a cell is infected with IFN-resistant virus, it will die in the presence of IFN-α and puromycin because IFN signalling will be blocked, thereby blocking the activation of the Mx1 promoter and consequent expression of pac; if a cell is infected with an IFN-sensitive virus, it will survive in the presence of IFN-α and puromycin because the Mx1 promoter will become activated through the IFN signalling pathway, leading to the expression of pac. IFN-sensitive viruses can then be rescued from the surviving cells, and amplified using IFN-permissive cell lines expressing viral IFN antagonist proteins (proteins that block the host’s IFN response). When tested on PIV5 strains CPI- (an IFN-sensitive virus) and CPI+ (an IFN-resistant virus), the developed method allowed the survival and amplification of cells infected with CPI-, whilst cell death was observed for cells infected with CPI+. Whilst the developed methodology seems promising, further developments of the system are required. The possibilities of using the developed methodology in combination with other techniques, such as FACS sorting and immune selection, to rapidly select IFN-sensitive mutant viruses from wild-type and mutagenised virus populations are discussed. The potential to use Mx TIPSE cells to select IFN-resistant revertant viruses from IFN-sensitive virus populations is also discussed. In addition, a high throughput screening assay has been developed using the engineered Mx GIPSE and Mx TIPSE cell lines to search for compounds that block IFN signalling or that block the action of viral IFN antagonist proteins. Compounds that block IFN signalling would potentially be useful as anti-inflammatory drugs whilst compounds that block the action of viral IFN antagonist proteins would be valuable as antiviral drugs.
Thesis, PhD Doctor of Philosophy
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