Utilising proteomics to develop anti-HCMV immunotherapy

Human cytomegalovirus (HCMV) is a herpesvirus that infects almost everyone worldwide. Infection is lifelong, during which time it is controlled by the immune system. While the majority of infections do not cause symptoms, HCMV is nevertheless a major cause of disease in certain groups of people. If caught during pregnancy, the virus can pass to the foetus and can result in deafness, mental retardation, or even death of the unborn child. In the UK up to 1000 babies per year have severe permanent disabilities due to HCMV, more than Down's syndrome or foetal alcohol syndrome. HCMV is a major cause of severe life-threatening disease in individuals with poor immune systems, notably transplant patients and people with HIV/AIDs. HCMV has also been associated with the most common form of brain cancer, hardening of the arteries, premature aging of the immune system, hepatitis and inflammatory bowel problems. Antiviral drugs have toxic side effects, and viruses rapidly become resistant to them. There is an urgent need for better therapeutic options.

CMV is the most complex human virus. Unfortunately we, and others, have shown that the virus changes whenever it is grown in the laboratory. This means that researchers have not been able to study the actual virus that causes clinical disease, and this has limited the development of vaccines and therapy. To solve this problem we took a clinical virus, sequenced its genome and designated it strain Merlin. This has been adopted as the worldwide HCMV reference strain. My work has characterised how this virus changes when grown in the lab and enabled me to devise a unique system to stop these changes happening. For the first time, scientists can perform experiments with a virus that accurately mimics the virus that causes disease. I will now use this virus, along with state-of-the-art technology, to define all HCMV proteins that can be targeted for vaccination or therapy.

To date vaccines have aimed to prevent initial infection with the virus. These studies need to target proteins present in the virus, yet the entire set of proteins present in clinical virus is completely unknown. Using our viruses we can now define the complete set of proteins available for targeting.

Although these vaccine strategies may reduce the chances of becoming infected, they cannot stop infection completely. Once a person is infected, a different strategy is required - one that enables cells to be killed after they are infected. The immune system can only kill infected cells if it can recognise them, yet we don't know how the virus changes infected cells. Using our state-of-the-art techniques and viruses, we will now determine exactly how HCMV changes the surface of an infected cell. We will determine which of these changes are the best targets for killing infected cells, and engineer antibodies that can be given to patients to enable their immune system to recognise these cells, and kill them - thereby controlling the HCMV infection.

Although we are in a unique position to develop and test these therapeutic reagents against infected cells, our novel viruses still suffer from a limitation. Virus is naturally spread by being secreted in bodily fluids (e.g. urine, saliva). To understand and control the process by which the virus spreads between people, we must be able to mimic this process in the lab. Yet when we grow clinical virus in the lab, very little virus is secreted. We have shown that this is because of a viral gene called RL13. We will investigate how RL13 stops secretion of virus, how this can be circumvented in the lab, and how it is circumvented when virus is secreted in patients. This will enable the production of large amounts of secreted virus that, as closely as possible, represents virus secreted from patients. This is crucial to enable laboratories to investigate the way HCMV spreads between people, and to develop ways of stopping it happening.

Development of vaccines and therapy requires that research uses viruses that mimic the agent of disease. We have developed the only HCMV BAC containing a complete wildtype genome, and can prevent in-vitro mutation by tet-repressing the unstable regions UL128L and RL13, enabling growth of high titre, genetically wildtype virus. During subsequent infection without repression, the entire virion proteome is expressed for analysis. We will use this virus to define and validate the entire proteome available for therapeutic targeting.

For the production of neutralising antibodies and inhibitors of viral entry, the virion proteome of wildtype virus will be defined. These strategies can reduce transmission, but spread in vivo is cell-to-cell, for which antibody dependent cellular cytotoxicity (ADCC) antibodies may be more effective. We will use Stable Isotope Labelling of Amino Acids in Cell Culture (SILAC) of surface proteins to define all potential targets for ADCC antibodies on the infected cell. We will determine which are targets during natural infection, and the functional consequences of viral encoded Fc binding proteins in circumventing ADCC. We will engineer chimeric antibodies against targets, and test their ability to promote ADCC killing of infected cells for therapy.

Our viruses permit the study of wildtype HCMV infected cells, but it remains hard to produce high titre cell free wildtype virus for studying virus entry, because RL13 (an Fc binding protein) inhibits virus release. We have defined the binding partners of RL13 using SILAC-IP and will determine, and circumvent, the interactions responsible for this inhibition. This will enable the production of high titre wildtype virus (with reference to our virion proteome). Since RL13 inhibits virus release, we will determine whether clinical virus lacks gpRL13 when secreted at high titres into urine. Finally we will begin determining whether RL13 promotes persistence of Rhesus CMV based vaccine vectors.

Successfully preventing HCMV disease has huge health and financial implications - other than genetic defects, HCMV accounts for more cases of congenital malformation and deafness than any other, and in the USA, the lifelong costs associated with congenital HCMV infection is estimated to be around $4 billion per year. It is clear that work leading to treatment or vaccination is of major importance and will see benefits worldwide.

Our development of a clonal, wildtype HCMV genome, has already had clear benefit to companies developing diagnostics for HCMV, with Merlin being designated the first international diagnostic standard for HCMV. The BAC clone is also being used for drug discovery in the field of peptide based drugs and virus derived from the BAC is also being used as a vaccine vector to vaccinate against TB. However these vaccine vectors have, to date, had to lack important components of the virion envelope, potentially compromising their effectiveness. The work in the project will enable us to produce the first clonal virus that stably expresses the complete wildtype HCMV proteome. This virus will be the definitive virus with which to test and develop diagnostics and therapeutics. Companies that are using HCMV as a vaccine delivery agent will also benefit from the development of wildtype virus, and the knowledge of whether and which tropism modifiers in the genome of a vaccine strain are beneficial in terms of attenuating virus while stimulating an immune response.

Using wildtype virus to define the complete set of therapeutic targets available within the virion itself, or on the surface of the infected cell, and developing chimeric antibodies to take advantage of this information, has the potential to completely change the landscape of HCMV therapy. Chimeric antibodies will offer the chance to readily control HCMV in vivo, and this work will define which proteins within the virus are ideal for targeting in a vaccine. Together these advances will bring health and financial benefits to both patients and the pharmaceutical industry.

Under all of these scenarios, the companies involved will benefit from the use of the reagents, technologies and principles developed in this grant. The staff member employed on the grant will obtain expertise in a wide range of scientific techniques, including molecular biology, proteomics, virology and immunology. They will obtain experience of presenting at both national and international meetings, and writing scientific papers. Through the development of chimeric antibodies, they will also work with our research and commercial division (RACD) to ensure we have patent protection for any reagents we successfully produce. Thus they will obtain a wide range of skills that can be applied in multiple job sectors.