Defining the reciprocal interaction between neutrophils and human cytomegalovirus

Human Cytomegalovirus (hCMV) is a highly prevalent opportunistic virus that infects up to 90% of the adult population and persists dormant in the body for life. The initial "primary" infection usually causes a mild glandular fever-like illness, but in certain at-risk populations the virus can cause substantial disease, or reactivate to cause "secondary" infection at a later date. Infection during pregnancy can result in congenital CMV infection in the newborn, leading to birth defects, and in immunosuppressed patients such as organ transplant recipients or HIV-infected individuals CMV is a major cause of disease, leading to increased hospitalisation, reduced survival of transplanted organs, and higher mortality.

The virus is believed to lie dormant in the bone marrow, and spreads by hijacking the hosts own cellular mechanisms to replicate, leading ultimately to rupture or lysis of the host cell and escape of large numbers of infectious virus particles. This "lytic" infection has been seen in certain white blood cell types such as the macrophage but not in the more common white blood cell, the neutrophil. This cell type protects the body from most bacteria and certain viruses, engulfing and killing the organisms then undergoing a process of self-destruction known as apoptosis, which triggers resolution of the inflammatory response. Recent research however has shown that certain viruses and bacteria can evade the neutrophil's defence mechanisms and survive its normally inhospitable internal environment, using it as a "Trojan horse" to replicate and spread. In this context, neutrophil apoptosis may even promote infection, allowing the transfer of live organisms to macrophages and other cell types, which remove apoptotic neutrophils from the inflamed site.

My preliminary work has shown that contact between hCMV and purified healthy neutrophils causes a profound increase in neutrophil survival by inhibiting apoptotic cell death. The surviving neutrophil does not produce new virus, but does release substances that quite profoundly affect its neighbouring cells, increasing the lifespan of other neutrophils and causing macrophages to alter their surface signals, making them potentially more susceptible to hCMV infection.

I will be investigating exactly how the virus produces this effect in the neutrophil, by defining its point of contact with the neutrophil surface and how this triggers a downstream effect to inhibit the cell death process. Additionally, I will determine the precise nature of the active substances that neutrophils produce following treatment with hCMV by analysing the material released by these cells and exploring exactly how these substances act to prolong their lifespan.

Finally, I plan to explore how the increased immune cell survival may be used by the virus for its own advantage, investigating whether the neutrophil can transfer intact virus to other cell types - the "Trojan Horse" model - or make surrounding macrophages more susceptible, producing more virus upon infection.

As an Infectious Diseases specialist trainee caring for HIV-infected patients I see first-hand the effects of this devastating opportunistic infection, and seek to find ways to better understand and treat CMV. This project has the potential to reveal new insights into the way hCMV spreads around the body, which in the future may enable us to combat its spread in those most at risk.

Human cytomegalovirus (hCMV) is a highly prevalent opportunistic infection and a major pathogen in immune-compromised patients including HIV-infected individuals and organ transplant recipients, causing disease through primary infection and secondary reactivation. The virus productively infects fibroblasts and macrophages, with detectable viral gene expression and the lytic release of new virions. However, hCMV is not thought to infect neutrophils, one of the main cells involved in innate immunity.

Preliminary data indicates that neutrophils in fact respond very effectively to incubation with hCMV. This manifests as profound inhibition of constitutive apoptosis, which does not involve hCMV uptake, lytic viral gene expression, or contaminating LPS or monocytes. hCMV also triggers the secretion of a highly bioactive secretome from neutrophils (but not monocytes), which induces a similar anti-apoptotic effect in fresh neutrophils, and stimulates monocyte chemotaxis and their differentiation to a phenotype permissive for hCMV infection.

This project aims to identify the bioactive components within the hCMV-induced neutrophil secretome responsible for the autocrine survival effect and paracrine effects on monocyte differentiation, through proteomic and lipidomic studies and the use of neutralising antibodies, and to define the receptor and cellular signalling mechanisms involved.

Additionally, I will investigate the hypothesis that the virus uses the neutrophil as a vehicle for dissemination. This will involve immunophenotyping of monocytes for markers of dendritic cell and macrophage lineage commitment, viral growth curve analysis to determine if monocyte infection is productive or abortive, and co-culture of neutrophils with permissive fibroblasts to look for evidence of infectious virus transfer. This work will shed light on the role of the neutrophil as an inadvertent promoter of hCMV disease, and offer new approaches to combating viral dissemination.

This training fellowship will produce a highly skilled research fellow with expertise in both the clinical aspects of hCMV infection and in the laboratory techniques required to investigate its pathogenesis. The project will provide training in a variety of laboratory techniques, complimented by formal teaching sessions in the University of Cambridge School of Graduate Studies. My honorary clinical contract will allow me to keep up-to-date with clinical advances in Infectious Diseases, and will provide access to patient groups most at risk of hCMV disease. This latter aspect may well be vital for future work where clinical samples from such patients may help to expand our understanding of hCMV dissemination in vivo, and, should this work lead to the discovery of future avenues for therapeutic manipulation, for recruiting into clinical trials.

This project will also provide an opportunity for me to support Clinical Medical and Natural Sciences students from the University of Cambridge undertaking short-term research placements, and potentially Academic Clinical Fellows, to enable them to have their first experience of research. Through this I hope to contribute to teaching and training on a broader front, encourage students to consider a career in research, and enhance my own teaching skills.

As outlined, this project aims to characterise a novel host-pathogen interaction thought to be involved in efficient viral dissemination in vivo. Through expanding understanding of this mechanism, I hope that there will be eventual impact on patient health and clinical outcomes for those most at risk of hCMV disease. Although it is not anticipated that this project will directly lead to the generation of new therapeutics, a greater understanding of the mechanisms of hCMV dissemination in the body will shed light on an area of viral pathogenesis not fully understood, and has the potential to further characterise signalling pathways that in the future may be identified as possible therapeutic targets. The results of this project would then inform colleagues in industry, supporting innovation and enterprise, and would hopefully lead to improvements in care for the patients most susceptible to severe hCMV infection - neonates, HIV positive patients and transplant recipients. These patient groups often have complex medical needs and are frequent users of the health system, therefore improvement in care and therapeutics has the potential to impact strongly on quality of life and has financial implications for both the individual and the healthcare system, and benefits for clinicians caring for immune-compromised patients who would become users of the research.

Additionally, an increase in our understanding of a previously underexplored area of viral interaction with the innate immune system may help to inform our knowledge of why certain patient groups with activated innate immunity are most at risk of hCMV disease and reactivation - it is known that certain patient groups such as the critically ill and those with severe sepsis have a higher risk of hCMV reactivation but the reasons for this are not known. I hope the results of this work may inform our understanding of the factors influencing this at a molecular and cellular level, opening up ideas for future avenues of investigation into protective mechanisms.