Molecular and cellular mechanisms of synapse-mediated spread of Epstein Barr virus: overcoming the CD21-restricted cellular tropism.

Lead Research Organisation: University of Birmingham
Department Name: Cancer Sciences

Abstract

Epstein Barr Virus (EBV) is a common, orally-transmitted virus, infecting most of the human population. EBV maintains a silent infection for the lifetime of the host by hiding in a subset of lymphocytes called B cells. EBV infection of B cells in the laboratory has been extensively studied. In the laboratory, infected B cells are efficiently transformed into continuously growing cells with the potential to cause cancer. This happens only rarely in the body, but when it does can cause B cell cancers including Hodgkin lymphoma. One reason why B cell cancers are so rare is because infection is kept under control by immune responses involving other cells, including T and natural killer (NK) cells.
The mechanisms of EBV entry into B cells have been extensively studied. Proteins on the virus (gp350 and gp42) interact with virus receptors on the cell surface (CD21, HLA class II respectively) to bind the EBV to the B cell. Thereafter further virus proteins (gp82, gp25) trigger virus internalisation. However, infection of other cell types, including epithelial, T and NK cells that do not express the CD21 are also observed. These infections are associated with either virus transmission to a new susceptible host (epithelial), or are associated with diseases and malignancies of epithelial, T and NK cells.
Epithelial cells line the oral cavity (oropharynx) and create a barrier for entry of EBV into an uninfected host, and for escape of EBV from an infected host for transmission to a new susceptible host. Epithelial cells are thought to be the major site of EBV replication in the oropharynx; however they do not express CD21, and EBV infection of epithelial cells in the laboratory is poor. I have previously shown that infection of epithelial cells can be greatly enhanced by first binding the virus to B cells. EBV binding to CD21 triggers adhesion molecules and EBV on the B cell surface to polarise then 'adhere' to the epithelial cells, bringing EBV into close contact with epithelial cells and enabling efficient infection. This process is probably similar to the situation in the oropharynx, where epithelial cells and lymphocytes, including B cells, are in close contact with each other.
In our model of EBV infection of epithelial cells, we suggest EBV on the B cells may come from fully matured B cells, called plasma cells, which when infected with EBV produce new infectious virus. This EBV may be released from the plasma cell to interact with the resting B cell as above. Or, as increasingly observed with other viruses that infect lymphocytes (like HIV), the plasma cell may interact directly with the epithelial cell, cause EBV inside the plasma cell to relocate to the point of cell-to-cell contact and transmit directly from the plasma cell to the epithelial cell, thereby evading anti-EBV antibodies. Here, I intend to extend my studies to understand how EBV can infect epithelial cells using plasma cells as the virus donor, and determine the molecular requirements of epithelial cells and EBV for efficient infection, so we can understand how to disrupt EBV transmission with vaccines.
EBV associated T and NK cell diseases range from illnesses that appear to be a consequence of an over-active immune system (EBV-associated haemophagocytic lymphohistiocytosis, chronic active EBV), to aggressive malignancies (extranodal T/NK cell lymphoma, aggressive NK cell leukaemia).T and NK cells do not express CD21 and we do not understand how these cells can be infected with EBV. Using two different methods, I aim to determine how infection of these cells is achieved: (1) Using plasma cell interaction with T and NK cells, and (2) infecting CD21-positive immature T and NK cells. With this new information, we will be able to develop ideas of how the virus may be causing the T and NK cell diseases, and begin develop new treatments for these treatment-resistant diseases.

Technical Summary

The aim of this research is to understand one of the fundamental aspects of viral pathogenesis: How does Epstein Barr virus (EBV) gain entry to epithelial cells, T and NK cells which do not express CD21, the virus receptor?
(1) Efficient infection of epithelial cells requires EBV-loaded B cells to act as virus donors; physiologically this is likely to be lytic plasma cells. Using lytic B cell lines at different stages of plasma cell differentiation, or non-transformed B cells induced into plasma cell differentiation by CpG DNA and interferon alpha treatment, we will examine the formation of a virological synapse between plasma cells and uninfected epithelial cells. Viral synapse formation is triggered by viral and cellular molecules which polarise with adhesion molecules to the point of cell-cell contact. Using fluorescently-labelled recombinant viruses and cellular components, we will monitor relocalisation of virus capsid, adhesion molecules and intracellular transport machinery by live cell confocal microscopy and determine their individual roles in synapse formation.
(2) T and NK cells are refractory to EBV infection in vitro so may require a donor B cell for infection. Lytic plasma cells will be cultured with a panel of well characterised, HLA-matched CD4 and CD8 T cells and interferon alpha-induced NK cells to examine transmission of EBV via the virological synapse. Using a panel of recombinant viruses, we will determine the viral and cellular factors required. Finally, CD21 is temporally expressed on immature T and NK cells during differentiation from lineage negative haematopoietic stem cells into T or NK cells. Using the OP9-DL1 system to in vitro differentiate T and NK cells and using we'll take advantage of this temporal CD21 expression and infect the immature cells prior to lineage commitment. We will then be in a position to determine if EBV infection per se is sufficient to drive the T and NK associated pathologies.

Planned Impact

As a pathogenic, oncogenic virus, Epstein Barr Virus is the subject of much research worldwide. However, despite this there is a great deal of uncertaintly surrounding the mechanisms by which it infects non-B cells, namely epithelial cells, T and NK cells. This is relevant not only in the study of cancer, but, at a more basic level, in our understanding of how the virus is shed from epithelial cells into the saliva to infect the next host. This is particularly relevant not just to understand the basic biology of the virus, but also in the investigation of EBV-associated malignancies such as nasopharyngeal carcinoma and NK/T cell lymphoma. The identification of the mechanisms by which EBV enters cells not expressing the virus receptor, namely epithelial cells and particularly T and NK cells will (1) increase the academic knowledge base in the UK. (ii) Will be of interest to biotechnology companies and academic groups involved in generating anti-herpesvirus vaccines. This study will indicate what virus proteins are essential in transmission and indicate whether, during cell-cell transmission the virus can be accessible to the neutralising antibodies. (iii) Will be of interest to clinicians and scientists working on EBV infection of T and NK cells. Nothing is currently known of how the virus enters T and NK cells, so equally little is known of the pathogenic mechanisms by which EBV causes disease in these cells. Furthermore, malignancies associated with EBV in T and NK cells are highly resistant to conventional treatment and the only model we currently have to examine EBV in these cells are cell lines, therefore reliable infection of primary cells will also be of interest to biotechnology companies in the development of improved treatment. (iv) Will be of interest to charities, such as one set up by the family of a patient who died from EBV-associated NK/T cell lymphoma.
Given the potential interest of biotechnology companies in development of new treatments, and in vaccine developemnt, this could be an advantage economically to the UK.
Diagnosis of EBV-associated disease in non-B cells is devastating, not least because the prognosis is invariably poor. Therefore, in the future this may benefit people who have EBV-associated cancers that are refractory to current treatments. This is particularly applicable outside the UK given the worldwideincidence of nasopharyngeal carcinoma, haemophagocytic lymphohistiocytosis, chronic active EBV, extranodal NK/T cell lymphoma etc.
The project will provide valuable experience in state of the art microscopic techniques, particularly real time confocal microscopy. It will also provide experience in many molecular biology and cell biology techniques which are transferable skills and can be taken to any laboratory environment.

Publications

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Shannon-Lowe C (2014) Epstein Barr virus entry; kissing and conjugation. in Current opinion in virology

 
Description Project grant
Amount £386,241 (GBP)
Funding ID MR/N023781/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 11/2016 
End 10/2019
 
Description Research Grant
Amount £100,000 (GBP)
Organisation Gregor Mckay Fund 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2015 
End 06/2017
 
Description Wellcome Trust ISSF
Amount £4,800 (GBP)
Organisation University of Birmingham 
Sector Academic/University
Country United Kingdom
Start 03/2014 
End 10/2014
 
Description Wellcome Trust ISSF
Amount £20,000 (GBP)
Organisation University of Birmingham 
Sector Academic/University
Country United Kingdom
Start 03/2013 
End 09/2013
 
Description Wellcome Trust ISSF
Amount £5,200 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2015 
End 04/2016
 
Title Plasma cell differentiation 
Description Primary resting memory B cells can be infected with EBV and differentiated into plasma cells. 
Type Of Material Biological samples 
Year Produced 2014 
Provided To Others? Yes  
Impact I have identified how, by silencing viral gene transcription using CD40 ligation and Notch ligation, we can differentiate memory B cells into plasma cells and initiate viral lytic cycle. We can now determine what factors are required to initiate viral replication in a primary cell system. I am currently writing a manuscript detailing this and redefining what we currently know about primary infection with EBV in vivo. I am currently collaborating on two further different pieces of work: 1. Differentiation of KSHV-infected B cells into plasma cells as a model of primary effusion lymphoma with Dr Andrew Hislop. 2. Factors that control viral replication with Prof Alison Sinclair. 
 
Title PrimeFlowRNA 
Description Fluorescence in-situ hybridisation for viral mRNA transcripts in combination with cell surface staining. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The PrimeFlow RNA has enabled us to identify the lymphocyte subset infected by EBV in a very small number of PBMC directly from patients suffering from EBV-associated T cell and NK cell lymphoproliferations or malignancies. Because we are using very small cell numbers, we can perform experiments we have not previously been able to perform, including: Isolation and culture of cells containing EBV and perform drug sensitivity assays using novel drugs. Immunophenotype the EBV-infected cells and the non-infected cells to determine the difference between the two. Perform RNAseq on EBV-infected vs non-infected T cells or NK cells to determine how EBV changes the cellular gene expression profile. Determine why the immune system does not recognise EBV infected cells. Viral gene expression profile. This assay has really opened up the field of research to enable us to determine exactly how EBV is causing these diseases and if we can identify potential new treatments. 
URL http://eu.ebioscience.com/knowledge-center/application/flowrna/primeflow-rna-assay-principle.htm
 
Description Chromatin remodelling by Epstein Barr virus 
Organisation University of Sussex
Country United Kingdom 
Sector Academic/University 
PI Contribution The West research team research has focused on the molecular mechanisms involved in the regulation of transcription initiation and elongationby EBV and cellular factors. This work has primarily used cell lines. However I have provided the team with a primary cell model of infection which provides a meaningful context to their work on lymphomagenesis. I have previously identified how EBV infects primary resting B cells, the changes in dynamics of EBV and cellular gene expression over time and identified appropriate controls to mimic EBV infection. These infections were then used at appropriate times, such as following cMyc upregulation, to determine how EBV manipulates the expression of cMyc, amongst others.
Collaborator Contribution The West team employ their expertise in performing the analysis of the regulation of cellular genes by EBV. They have pioneered the use of 4C to identify how long range enhancers and promoters are regulated by the EBV-encoded transcription factors (EBNAs).
Impact 10.7554/eLife.18270 We have achieved one publication, with a further publication at the point of submission and are continuing this work.
Start Year 2015
 
Description EBV infection of human gastric organoids 
Organisation University of Wurzburg
Country Germany 
Sector Academic/University 
PI Contribution We have recently set up this collaboration. One postdoc from the laboratory of Sina Bartfeld will be coming to my lab to learn how to infect gastric organoids with EBV.
Collaborator Contribution I was taught by Dr Sina Bartfeld how to make human gastric organoids in the laboratory of Prof Hans Clevers.
Impact None yet.
Start Year 2015
 
Description EBV-associated Haemophagocytic lymphohistiocytosis 
Organisation National Taiwan University
Country Taiwan, Province of China 
Sector Academic/University 
PI Contribution EBV-associated Haemophagocytic Lymphohistiocytosis is a life-threatening, lymphoproliferative disease where EBV ectopically infects T cells and NK cells. It is a rare disease in developed countries, but is still common in South East Asia. In Children's Hospital No 1, Ho Chi Minh City, up to 6 patients are diagnosed every month. We have taken blood from HLH patients at time of diagnosis to determine exactly which cells the virus is in and what is the virus gene expression profile. We then extracted DNA for deep sequencing to be performed in Taiwan.
Collaborator Contribution Children's Hospital No 1, Ho Chi Minh City diagnose the patients, collect and send us the HLH blood samples. The whole study is run by Prof Ih-Jen Su in Taiwan. The deep sequencing will be performed in the laboratory of Prof Su.
Impact None yet
Start Year 2013
 
Description EBV-associated Haemophagocytic lymphohistiocytosis 
Organisation University Children's Hospital, Basel
Country Switzerland 
Sector Hospitals 
PI Contribution EBV-associated Haemophagocytic Lymphohistiocytosis is a life-threatening, lymphoproliferative disease where EBV ectopically infects T cells and NK cells. It is a rare disease in developed countries, but is still common in South East Asia. In Children's Hospital No 1, Ho Chi Minh City, up to 6 patients are diagnosed every month. We have taken blood from HLH patients at time of diagnosis to determine exactly which cells the virus is in and what is the virus gene expression profile. We then extracted DNA for deep sequencing to be performed in Taiwan.
Collaborator Contribution Children's Hospital No 1, Ho Chi Minh City diagnose the patients, collect and send us the HLH blood samples. The whole study is run by Prof Ih-Jen Su in Taiwan. The deep sequencing will be performed in the laboratory of Prof Su.
Impact None yet
Start Year 2013
 
Description Epstein Barr virus and epigenetics 
Organisation Bellvitge Biomedical Research Institute
Department Cancer Epigenetics and Biology Programme
Country Spain 
Sector Public 
PI Contribution I provided all the material (Viruses, Cells and infection time courses) that were crucial for these studies, without which the studies would not have proceeded. So far we have achieve d two publications and one further about to be submitted. Furthermore, Henar Hernando successfully secured an EMBO travel award to come and work with me to learn the technology involved in the virus infection part of these studies. Epstein-Barr virus-mediated transformation of B cells induces global chromatin changes independent to the acquisition of proliferation. The B cell transcription program mediates hypomethylation and overexpression of key genes in Epstein-Barr virus-associated proliferative conversion.
Collaborator Contribution Professor Esteban Ballestar is a world-leader in cancer epigenetics. His group provided the intellectual and technical expertise wrt epigenetics to the study, without which the study would not have proceeded.
Impact 24097438 23320978 25200074 These papers are listed in the publications section. This collaboration is multidisciplinary. My lab provides the virology and B cell expertise and the Ballestar lab provides the epigenetic expertise.
Start Year 2011
 
Description Epstein Barr virus vaccine study 
Organisation Uniformed Services University of the Health Services
Country United States 
Sector Academic/University 
PI Contribution Epstein Barr virus is associated with a number of malignancies of B cell origin (Burkitt Lymphoma, Hodgkin Lymphoma, Post-Transplant Lymphoproliferative Disease), of epithelial cell origin (Nasopharyngeal Carcinoma, Gastric carcinoma) and of T cells and natural killer cells (Extranodal NK/T cell lymphoma, Acute NK leukaemia). This has highlighted the need for the development of a prophylactic vaccine. Professor Clifford Snapper has developed novel protein expression technology to express EBV glycoproteins in the conformation they are found in the viral envelope. He vaccinated rabbits with the purified recombinant proteins and has given me the pre-immune, 31 day and 52 day post immune rabbit sera. I have used these sera to determine if the antibodies produced by the rabbits block EBV entry into B cells and epithelial cells, the natural target cells of EBV. We have so far identified one of the antibodies functions extremely well in blocking entry of EBV into primary resting B cells, anti gH/gL.
Collaborator Contribution Professor Clifford Snapper has developed novel protein expression technology to express EBV glycoproteins in the conformation they are found in the viral envelope. He vaccinated rabbits with the purified recombinant proteins and has given me the pre-immune, 31 day and 52 day post immune rabbit sera. Prof Snapper is continuing to make all the glycoproteins identified as being involved in viral entry into both B cells and epithelial cells and inoculate rabbits. We also submitted a grant application to the NIH, USA to develop a potential vaccine.
Impact None yet
Start Year 2014
 
Description An introduction to scientific research for new science undergraduates. 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact All the new intake of students for the BMedSci course at The University of Birmingham attended this event. I presented a poster of my research to discuss careers options for graduates.
Myself and a colleague run a scheme called 'Step into Research" where we actively recruit second year undergrads to undertake a research project at the School for Cancer Sciences, UoB. This scheme gives the students an excellent oportunity to undertake a real research project, whilst giving the supervisors the opportunity to identify exceptional students who could be trained as the next generation of researchers.

I was asked to give a presentation to the BMedSci undergraduates to discuss Step into Research and the type of research they could potentially be involved in. We already have applications from at least 10 students, with the closing date being mid January.
Year(s) Of Engagement Activity 2013
URL http://www.birmingham.ac.uk/schools/cancer/study-here/step-into-research/index.aspx
 
Description Cancer Research public engagement tour 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Supporters
Results and Impact We regularly have groups of 15-30 regional fundraisers come to the labs to have a talk on our research activities and have lab tours. During these tours, we demonstrate some of the research we are doing and chat about our own work. The visitors are not scientists, making the lab tours extremely challenging to pitch at a level where our work can be understood by all.

We always have positive feedback.
Year(s) Of Engagement Activity 2013
 
Description Public engagement laboratory tours for Cancer Research UK 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Supporters
Results and Impact These events are excellent because many fundraisers are involved in these activities because they have either had cancer, or they know someone who has. The groups of visitors are split up so they see several aspects of the research. We always have several questions during the demonstrations and talks. We often also have long discussion following the tours over coffee. It is very difficult for people to visualise what research is, how it is performed, how researchers use the money raised. The vast majority of the public have no idea about how much research costs. These are therefore excellent tours to answer many of these questions.

After these events, we are always asked for further information and sometimes if we can give talks outside the laboratory.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014
 
Description Talk to the Upper GI Support Group, Queen Elizabeth Hospital, Birmingham 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact I gave a research talk to the members, families and healthcare supporters of the Upper Gastro Intestinal Support group based at the QEH, Birmingham. At least 20 people attended. The talk was on Epstein Barr virus and its role in cancer, particularly gastric carcinoma. The support from the group was amazing. The majority of patients asked questions and volunteered to help in future studies. Below is the email I received following the talk.

When you kindly contacted Mr Griffiths and volunteered to talk to us, I must admit I did wonder whether it would be appropriate for our group, would it be a bit above me and perhaps some of the group. I had no need for these questions. It was excellent and interesting. The applause, interest shown and questions asked is truely proof of an excellent presentation. Thankyou. Also, at a suitable date in the future we would certainly like a return visit for an update.
Year(s) Of Engagement Activity 2016