Epstein-Barr-Virus / Basic Research

Epstein-Barr virus (EBV) is a human pathogen that belongs to the large family of herpes viruses. It is the most prominent representative of several known related viruses, which are endogenous to apes and monkeys of the Old and New World. Very likely, EBV has co-evolved with its host, Homo sapiens and its ancestors, for millions of years. EBV is a very prevalent virus and infects more than 90% of the human population. EBV’s success is probably linked to its adaptation to and co-evolution with its human host. In fact, most of us, who carry EBV for a lifetime, do not suffer from the viral infection though EBV can also cause severe acute diseases and is associated with very different types of life-threatening malignancies. It was recognized as the first human tumor virus about 50 years ago and is now categorized as a type 1 carcinogen (i.e. directly involved in causing cancer) according to the definition of the World health organization (WHO). In addition to EBV-associated tumors, acute infections with EBV can also cause disease, which together have a large impact on global health. The diversity of EBV-associated diseases mirrors the complexity of the interactions between EBV and its human host.

We know now that EBV has acquired many genes and other genetic elements from its cellular host during co-evolution. Over time the cellular predecessors have been extensively modified and altered by adaptive selection and do not resemble their original counterparts any more but primarily suit viral needs. It is remarkable that EBV has not only adopted cellular genes but lives on cellular principles that, for example, are key to gene regulation, signal transduction, cell transformation, or immune control. EBV is a prime model to unravel paradigmatic molecular mechanisms and a tool to discover previously unknown (cellular) molecules and their functions in the context of the normal and the infected cell. EBV is also an ideal model to study immune responses of the infected human being. Thus, in studying EBV we use its versatile processes as a window to the cell and its host organism.

From a basic perspective, EBV constitutes a platform to investigate fundamental mechanisms and principles of an important viral pathogen at the level of the single cell and at the level of the organism. From a clinical point of view, understanding the principles of EBV-linked and -associated diseases is a prerequisite to develop novel therapeutic concepts. In order to address both basic and clinical perspectives, we dedicate our research to EBV’s biology and pathogenic mechanisms focusing on particular aspects of cell biology, molecular genetics and epigenetics, and immune biology. We are interested in how the virus-host balance is established and maintained in the healthy virus carrier, how it is undermined when EBV causes disease, and how it may be restored in order to reinstate health.

EBV shares certain fundamental aspects of its biology with other members of the large herpes virus family. Human herpes virus 6 (HHV6), human cytomegalovirus (CMV) and Kaposi’s sarcoma-associated herpes virus (KSHV) all aberrantly activate cellular pathways and contribute to immunological diseases including B-cell lymphomas. Studies on selected aspects of these herpes viruses complement our research on EBV.

EBV and its diseases

EBV is an infectious agent with a considerable disease burden. It is a human oncovirus that is associated with various malignant diseases throughout the world. EBV is present in about 50% of Hodgkin's lymphoma and up to 10% of gastric carcinoma, which are important malignancies in developed countries. EBV is part of the etiology of nasopharyngeal carcinoma, which occurs very frequently in China and Southern Asia (incidence up to 20 per 100.000)[1] , and of Burkitt's lymphoma, a major childhood cancer in malaria-afflicted areas of Central Africa, termed the ‘lymphoma belt’ (Table 1).

Infectious mononucleosis (IM) is another major EBV disease, which is clearly a risk factor for Hodgkin's lymphoma later in life. In immunocompromised patients, EBV may lead to post-transplant lymphoproliferative disease (PTLD), a life-threatening condition occurring in up to 20% of organ recipients[2]  and 1 - 10% of stem cell recipients[3].

Table 1. EBV-associated diseases and their incidence

Disease

Incidence 2008

(Germany)

Incidence 2008

(worldwide)

% EBV infected

Immunocompetent patients

Hodgkin's lymphoma

1535[4]

2-4 / 100,000[5]

50

Gastric carcinoma

15,263[4]

17 / 100,000[5]

5-10

Nasopharyngeal carcinoma (sporadic)

321

0.3 / 100,000[5]

>95

Nasopharyngeal carcinoma (endemic)

 

1.7-15 / 100,000[5]

100

Infectious Mononucleosis

8000[6]

60-100 / 100,000[7]

100

Burkitt's Lymphoma (sporadic)

100[4]

 

30

Burkitt's Lymphoma (endemic)

 

2.9-7.0 / 100,000[8]

85-100

Immunocompromised patients

Post-transplant lymphoproliferative disorder

100-200[9]

 

>90

AIDS-related lymphoma

100[10]

 

>95


Different strategies may be used for a targeted therapy of these diseases. Adoptive transfer of EBV-specific T cells is a powerful means to prevent or treat EBV malignancy especially in different transplant settings. Small molecules that inhibit key steps in EBV's oncogenic signaling have a great potential to fight cancer. Finally, EBV-specific vaccination is a promising approach to prevent severe IM in EBV-negative healthy adults or EBV-associated malignancies in transplant recipients.

[1] Globocan (2008) Word Health Organization (http://www.iarc.fr)
[2] Everly et al. (2007) Posttransplant lymphoproliferative disorder. Ann Pharmacother 41:1850-1858; Taylor et al. (2005) Post-transplant lymphoproliferative disorders (PTLD) after solid organ transplantation. Crit Rev Oncol Hematol 56:155-167
[3] Landgren et al. (2009) Risk factors for lymphoproliferative disorders after allogeneic hematopoietic cell transplantation. Blood 113:4992-5001
[4] Krebs in Deutschland, Häufigkeiten und Trends (2005/2006) Edited by the Robert Koch Institute, Berlin ISBN 978-3-89606-207-9
[5] Globocan (2008) Word Health Organization
[6] hospitalized patients
[7] Human Herpesviruses. Biology, Therapy and Immunoprophylaxis. (2007) Edited by Ann Arvin et al. Cambridge University Press ISBN-13: 978-0-521-82714-0
[8] Orem et al. (2007) Burkitt's lymphoma in Africa, a review of the epidemiology and etiology, African Health Sciences. 7:166-175
[9] incidence 1-20% of solid organ transplants depending on EBV serostatus, age and type of transplant. Taylor et al. (2005) Post-transplant lymphoproliferative disorders (PTLD) after solid organ transplantation. Critical Reviews in Oncology/Haematology 56, 155-167
[10] incidence 0.7 – 1.7 / 1000 of HIV-infected patients (cerebral lymphoma, immunoblastic lymphoma, Burkitt's lymphoma).

Biology of infection

EBV readily infects human B cells in vitro. When isolated from peripheral blood or lymphoid tissue, these cells are predominantly quiescent, do not proliferate and die rapidly in vitro. Upon infection with EBV, they are rescued from cell death, acquire an activated phenotype and start proliferating. Immortalized, so-called lymphoblastoid cell lines (LCLs) emerge, which are latently infected with EBV.

EBV also infects other human cells like epithelial cells and T cells among others. Primary epithelial cells in a particular state of differentiation can be infected with EBV in vitro but infection is abortive and the cells die. Currently, there are no easily accessible models to study the facets of EBV’s biology in T cells or epithelial cells in vitro.

Infection with EBV results in viral latency. During this phase the virally infected cell does not synthesize progeny virus but resumes a stable relationship with its host cell. Viral latency in LCLs is preceded by a so-called pre-latent phase that is characterized by the expression of a subset of lytic viral genes in conjunction with latent genes of this virus (Fig. 1). In latently infected cells, virus de novo synthesis can be induced giving rise to new virus several weeks post infection.

Latently infected cells and primary (tumor) cells infected with EBV express different sets of EBV’s latent genes. The latent genes are grouped into three classes: EBNAs (viral proteins expressed in the nucleus of infected cells), LMPs (viral proteins predominantly located to cell membranes), and non-coding RNAs. The different sets of latent viral genes are termed latency programs 0, I, II, or III.

 

Figure: EBV has a dynamic life style; AGV
A. The pre-latent phase is characterized by the expression of latent genes (blue line) and a very restricted subset of lytic genes of the so-called immediate early and early lytic class (red line). No new virus is synthesized because expression of other early lytic genes and EBV’s structural genes is blocked during this initial latent phase. EBV’s genome is established as an extrachromosomal plasmid in the nucleus of infected cells supported by latent genes that are expressed during latency (continuous blue line). B. Exogenous signals such as antigen encounter or expression of the viral transcription factor BZLF1 can initiate the switch to the lytic phase, during which early and late lytic genes are expressed. They encode viral enzymes that support amplification of viral genomes (red line) and structural proteins of the viral particle (grey line) yielding viral progeny.

Perfect adaptation to its main host cell type, the B lymphocyte, is a hallmark of EBV's biology. Upon entry into the cell, EBV addresses, reinterprets, and co-opts key mechanisms of B cell biology, resulting in cell survival, proliferation, and massive phenotypic alterations. A set of EBV-encoded proteins mimics essential B cell activation and differentiation signals, providing surrogates for antigen recognition, T cell help, and innate co-activation. EBV's DNA genome is maintained in the cell in the form of extrachromosomal plasmids, also called episomes that replicate in synchrony with the cell cycle, which is made possible by the integrated action of viral and cellular factors. Consequently, we make use of EBV in order to study both viral and cellular aspects of DNA replication. Distinct epigenetic states govern different phases of EBV's life cycle in the course of infection: initial activation of resting cells, an intermittent phase of stable latent infection, and finally productive virus synthesis. Thus, the epigenetic state of the EBV genome is a crucial regulator of viral gene expression programs and viral DNA replication.

A recurring characteristic of all these processes is the sophisticated viral mimicking of cellular molecules and their functions, which can be explained by EBV's co-evolution with its primate and human hosts over millions of years. Co-evolution is documented by the observation that the virus acquired a number of cellular genes and modified them to its benefit. To mention an example, the multifunctional EBNA1 protein, encoded by the viral latent gene BKRF1, consists of different functional domains. One prominent domain resembles the cellular chromatin protein HMGA1a, which is involved in the definition of origins of DNA replication. EBNA1 combines this function with a sequence-specific DNA binding motif and a transcriptional transactivation domain to provide at least four very different functions: In the context of the virus, EBNA1 promotes DNA replication of the viral genome, tethers it to cellular chromatin, regulates transcription of latent genes, and inhibits its own proteasomal degradation in order to avoid mobilizing virus-specific T cells. This extended set of EBNA1 functions vastly exceeds those of its cellular ancestor, HMGA1a.

Additional examples are two multifunctional membrane proteins encoded by EBV’s latent genes. They pervert major signaling pathways and govern the fate of infected cells: LMP2A and LMP1 mimic the antigen receptor of B cells (BCR) and the co-activatory receptor CD40, respectively, provide autonomy to the cell, making it independent of exogenous signals and its microenvironment. It comes to no surprise that these viral mediators regulate differentiation of infected cells and even have transforming potential. Several groups (Research group EBV Genetics and Vectors; Research group Signal Transduction; Research group Kempkes) study both functions in infected B cells and a variety of engineered cellular models in their biochemical components, signaling pathways, regulated target genes and their consequences on cellular fate. Our activities (Research group B cell development and activation; Research group MHV68) logically extend these cell-based studies to the level of the organism by devising appropriate mouse models for the effects of EBV factors on cellular differentiation, transformation and tumorigenesis.

EBV also directly manipulates the cell it infects. This herpesvirus encodes 44 viral microRNAs (miRNAs), a large number considering that only about 1000 cellular miRNAs are known although the human host cell has a far superior coding capacity than EBV. miRNAs are key regulators of many biological processes. There is emerging evidence that EBV’s miRNAs repress gene products, which critically support immune recognition or limit survival and proliferation of a virus-infected cell. During co-evolution of virus and host, EBV’s miRNAs have selectively identified those cellular genes that can restrict the success of viral infection. This is an ideal scenario to hunt for these pivotal cellular genes.

Thus, cellular functions are either directly targeted or creatively reinterpreted but also exploited by EBV. Effects mediated by several different cellular molecules may be triggered by a single viral mediator, ligand-dependent signals may become constitutive, and signaling intensities may be enhanced. EBV offers us genes that are most attractive to study because the virus taps the most important cellular pathways and regulatory circuits. The study of essential viral genes provides us with unique opportunities to identify and decipher fundamental cellular functions and their mediators. As EBV is amenable to targeted genetic analysis in all its components whereas the cell is not, viral recombinant techniques offer fundamental biological insights that cannot be straightforwardly obtained by any other means.

Immunology of EBV infection

As a complex virus that codes for multiple immunogenic proteins and has adapted to a life cycle in the immune cell compartment itself, EBV relies on sophisticated strategies to either inhibit or circumvent the immunological efforts of its host to abort infection. In healthy carriers, these efforts result in a stable virus host-balance, in which a multitude of immune effector cells and mechanisms restrict the virus to immunologically silent compartments, but never completely eliminate infection. Two groups (Research Group HOCOVLAR; Research group CCG Pediatric Tumor Immunology) study EBV-specific cellular immunity in healthy carriers in order to understand the essential features and components of this balance. The aim of these studies is to find ways how a stable immune control can be (re-)installed in patients who have been unable to develop such a balance or have lost it in the course of massive clinical interventions such as transplantations or infections with massively immunosuppressive agents such as HIV. We also develop cellular and animal models (Research Group HOCOVLAR; Research group Zeidler) of how EBV antigens are presented by infected cells or accessory cells, how EBV-specific effector cells are mobilized, and how they control infection. Our final goal is to offer an adequate immunotherapeutic option for different groups of individuals. EBV-non-immune adults at risk of infectious mononucleosis and prospective transplant recipients will benefit from a prophylactic EBV vaccine based on virus-like particles. For immunosuppressed patients without EBV-specific cellular immunity, adoptive transfer of virus-specific T cells from immunocompetent donors or genetic engineering of patient T cells with appropriate antigen receptors will be efficient therapeutic strategies.

Therapy of EBV infection

Therapeutic options to treat a latent infection in otherwise healthy individuals are not available (and probably not needed) and a causal therapy of patients suffering from EBV-associated tumors does not exist to-date.

Diseases linked to EBV infections are treated according to their clinical symptoms. Antiviral compounds, which interfere with the viral DNA replication during a productive, lytic infection are used in the clinics in the course of Infectious Mononucleosis (IM). They are not very effective because IM patients suffer from an abundant antiviral reaction of the immune system rather than from symptoms that arise from virus-producing cells.

EBV-associated tumors in immune competent individuals cannot currently be addressed by specific antiviral compounds. Instead, immunotherapy is an option that has reached the state of clinical trials.

PTLD, a life-threatening tumor that can arise as EBV-positive B cell lymphomas in immunosuppressed patients, can be treated in certain cases by alleviating immunosuppressive regimens. PTLD has also been successfully treated or prevented in clinical research studies by the infusion of EBV-specific T cells (Research Group HOCOVLAR; "Immunotherapy”).

The scarcity of therapeutic options led us to pursue the development of an EBV vaccine .

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