Viral Immunology Research Group
Human cytomegalovirus (HCMV) is a clinically important pathogen with high prevalence worldwide. It is the leading infectious cause of congenital malformation, is associated with life-threatening disease in immunocompromised individuals (e.g. AIDS sufferers and transplant recipients), and is a causative agent of hepatitis, colitis, post-transplantation arteriosclerosis and infectious mononucleosis. As a result, the US Institute of Medicine has designated HCMV as a highest priority (Level I) vaccine target.
We have major interests in the basic biology of the virus, the development of therapeutics and diagnostics, the way in which the virus interacts with the immune system, and using the virus to understand how the immune system functions in both healthy and diseased states.
Immune evasion by HCMV
As a herpes virus, primary infection with HCMV is followed by lifelong persistence during which the immune systems must act to limit the consequences of infection. Through co-evolution, HCMV has developed an intimate relationship with our immune system, and the virus has been established as a paradigm for viral immune evasion.
Natural Killer (NK) cells are implicated in both the innate and adaptive immunity and make a crucial contribution in combating HCMV disease. Patients with defects in NK cell function exhibit extreme sensitivity to HCMV infection. We identified UL40 as the first virus gene definitively demonstrated to induce protection against NK attack, and have gone on to identify other novel NK evasion function including UL141, that contributes to immune evasion by at least three distinct pathways:
- Downregulation of natural killer cell-activating ligand CD155 by human cytomegalovirus UL141.
- Human cytomegalovirus UL141 promotes efficient downregulation of the natural killer cell activating ligand CD112.
- Human cytomegalovirus glycoprotein UL141 targets the TRAIL death receptors to thwart host innate antiviral defenses.
More recently we have been collaborating to use cutting edge proteomics techniques to dissect the specific ways in which HCMV influences the immune system. Through this work, we have identified an entire family of genes that act together to regulate NK cell activation through multiple mechanisms:
- Control of immune ligands by members of a cytomegalovirus gene expansion suppresses natural killer cell activation.
- Two novel human cytomegalovirus NK cell evasion functions target MICA for lysosomal degradation.
Not only has our work with HCMV provided information on how HCMV avoids being killed by the immune system, it has also improved our fundamental understanding of the way in which the immune system acts to recognise pathogen infected cells:
In vivo modelling of CMV infection
To examine the immune responses that control CMV infection in vivo, we utilise the murine CMV model (MCMV) of infection. Using this model system, we have investigated how soluble immune proteins called cytokines orchestrate antiviral cellular immune responses and have identified how certain immune inhibitory cytokines and other suppressive pathways suppress antiviral immunity and thus allow CMV to persist.
We now wish to understand the cellular mechanisms that regulate inflammatory versus inhibitory immune pathways. Our belief is that by understanding these complex mechanisms we will identify how, in cases of virus-induced inflammation, we can treat disease. Moreover, these studies will identify immune pathways that may be stimulated to enhance virus-induced immune responses, for example during vaccinations with viral-based vaccine vectors.
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Wellcome Trust Senior Research Fellow. Persistent and Resistant Infections Theme Lead, Systems Immunity Research Institute
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A recombinant Adenovirus (Ad) vector with Zero cloning steps
- A complete Ad5 vector is carried on a Bacterial Artificial Chromosome (BAC).
- The DNA insert is transformed directly into cells carrying the AdZ BAC.
The insert can be:
- Synthetic oligonucleotides (e.g. encoding shRNAs)
- PCR product
- Synthesized gene
- Conventional plasmid clone (e.g. from an expression library)
Recombineering is performed:
- The transgene replaces dual selectable markers
- Positive clones are identified without need for screening
- BAC DNA is purified & transfected into cells
- AdZ recombinant grows
- Cloning genes into the AdZ vectors and making virus - for inserting genes with any tag into the AdZ vectors.
- Growing RAds - for reconstituting RAds from the AdZ BACs, and growing them up.
- Titering Viruses - a simple immunofluorescence protocol to get titres.
- General Recombineering - for inserting the sacB cassette in order to modify the backbone.
Vector Maps for the AdZ vectors
The following maps are all just the expression cassettes - the remaining sequence is identical to pAdZ5-CV5 above.
- pAdZ5-CV5 for adding C terminal V5 tags
- pAdZ5-NV5 for adding N terminal V5 tags
- pAdZ5-NGFP for adding N terminal GFP tags
- pAdZ5-CGFP for adding C terminal GFP tags
- pAdZ5-CmCherry for adding C terminal mCherry tags
- pAdZ5-mIR155 for cloning shRNAs
- pAdZ5-CStrep2 for adding C terminal Strep-2 or strep-3 tags
- pAdZ5-CV5-NT for adding C terminal V5 tags, promoter lacks tet operators
- pAdZ5-CGFP-NT for adding C terminal GFP tags, promoter lacks tet operators
- pAdZ5-Ctrl is empty vector control, containing just a V5 tag inbetween the promoter & polyA