Preface

Epstein-Barr virus (EBV) is one of the most common human viruses and the cause of pathologies such as infectious mononucleosis (IM) and certain cancers, namely immunodeficiency-related B cell lymphomas, Burkitt and Hodgkin, nasopharyngeal, and gastric carcinomas. Over the past two decades, the possibility of an association between EBV and other cancers and other chronic pathologies (i.e., multiple sclerosis (MS)) has also been put forward. One of the challenges facing researchers is the complicated life cycle of EBV, which goes through a phase of latent infection during which the virus induces the activation, proliferation, and differentiation of primary B cells into memory B cells. Additionally, EBV, like other human herpesviruses (HHV1-8), has co-evolved through a persistent viral infection in the host, and is then spread efficiently to others, generally without causing serious diseases. Symptoms of EBV infection vary widely based on the age and immune status of the patient. Most infections in younger children are benign and are often subclinical. EBV is also associated with autoimmune diseases, including rheumatoid arthritis, Sjogren's syndrome, systemic lupus erythematosus, and MS.

Classified as a herpesvirus (type IV), EBV encodes more than 80 genes. The core set of genes (minority) are involved in the latency phase. The other set comprises the genes of the lytic cycle. In addition to these gene-encoding proteins, there are gene-encoding microRNAs (regulatory RNAs), the functions of which are still poorly understood. For many years, researchers argued that only the products of the latency genes (i.e., LMP1 oncoprotein and EBNAs) were responsible for oncogenesis. It has recently been demonstrated that the proteins of the lytic cycle have also a role not only in cell transformation (the initial stage of the tumor process) but also in tumor progression. Certain viral proteins act as "transcription factors" capable of activating cellular genes involved in the regulation of cell survival or even in immunomodulation.

EBV-associated lymphomas are classically described as malignant proliferations of the lymphoid type but nonetheless group together a wide variety of histological and immunological types. In addition, this association with EBV, considered to be a group 1 carcinogen according to the International Agency for Research on Cancer (IARC) 2009, is highly variable for the type of lymphoma considered. For example, in Burkitt's lymphoma (BL), which was the first cancer associated with an infection and observed in Ugandan children thanks to the work of Denis Burkitt in 1958, we find it is the B lymphoma associated with EBV (discovered eight years later) that is present in more than 90% of cases. In contrast, in the same type of lymphoma but observed in a European subject, the association is only around 20%. This demonstrates that, in addition to the virus, there are environmental co-factors linked to each form of lymphoma, (regardless of whether it is type B or type T) or even a specific lymphoma, such as lymphoma by Hodgkin. The immune state is one of these cofactors linked to the host, and this explains the appearance of severe lymphoproliferative diseases in immunocompromised subjects (e.g., transplant recipients). In these patients, these lymphomas are called post-transplantation lymphoproliferative syndromes (PTLDs), bringing together several types of lymphomas (B lymphomas most often associated with EBV, but also T lymphomas, BLs, or Hodgkin's disease).

The chance of developing a lymphoma hangs over the heads of humans much like the sword of Damocles. Fortunately, we all have an internal immunosurveillance network capable of monitoring every cell infected with EBV. Over the long course of evolution spanning millions of years, a balance has been established between the virus and its host (primates and humans) so that we can live within this complex equilibrium. After initial exposure and infection with EBV (this frequently occurs in children and is asymptomatic), the virus persists in a form of latency with reactivations (periodic resumption of the activity of the virus, i.e., lytic cycle) that may go unnoticed. Under certain circumstances, the virus exerts uncontrolled oncogenic activity, which can sequentially (multi-step) lead to symptomatic tumors and cancer, such as lymphomas, stomach cancer (see Chapter 3), nasopharyngeal carcinoma (NPC), and leiomyosarcoma (LMS). For some of these tumors, EBV coinfection with malaria (African LB), ethnic and diet factors (NPC), and immune status (post-transplant lymphomas and lymphomas of the HIV-positive subject) are conducive to their development.

Recent studies have revealed a seamlessness between latent and lytic proteins and the types of infections to which they contribute. Some lytic proteins can be expressed in the context of latent infections as seen in some cancers and in prelatent infection of B lymphocytes. This raises the possibility that these lytic antigens (specifically the ZEBRA protein encoded by the EBV BZLF1 gene) might be useful therapeutic or vaccine targets for the prevention of EBV-induced cancers. In addition, advances in high-throughput next-generation DNA sequencing have made it possible to analyze a growing number of EBV isolates (see Chapters 1 and 2). It appears that different isolates of EBV vary in their ability to infect lymphocytes and epithelial cells. It can thus be suggested that certain specific variants of EBV are more oncogenic than others, and we can therefore establish a clear link between EBV-induced oncogenesis and that of human papillomavirus (HPV). It is also plausible that a particular variant is more prone to reactivate towards the lytic cycle, causing an increased viral load or an increase in the circulating ZEBRA antigen. This protein could act as a tumor progression factor and increase the occurrence of EBV-induced cancers. Recent clinical studies demonstrate a causal role of EBV in MS and in myasthenia gravis (an autoimmune disease characterized by intrathymic B-cell activation; see Chapter 5). Once again, it is not yet clear why MS only develops in a small fraction of people infected with EBV. The role of gene variants is worth investigating in future studies.

Usually, the diagnosis of an EBV primary infection is established through serological testing (detection of antibodies specific to EBV). This is particularly so regarding EBV whose symptoms are found in adolescents suffering from IM. In certain cases (e.g., NPC), this serology can also be applied to consolidate a diagnosis or a follow-up therapy. In contrast, in the case of lymphomas, clinicians have chosen molecular biology techniques (viral load by quantification of circulating EBV DNA in the blood). This technique (exploring the virus in its latency form) is routinely used to monitor transplant patients and to intervene at an early stage with drugs (rituximab, an anti-B lymphocyte monoclonal antibody) to prevent the onset of PTLD. Unfortunately, this technique (Rituximab or its derivatives) has many side effects (among them, hypogammaglobulinemia) and therefore lacks specificity as a potential treatment. However, techniques based on the detection of the lytic cycle have been developed to improve the specificity of the diagnosis of PTLD and will soon be available to everyone.

**V**

specific therapies.

Despite the discovery of EBV more than 50 years ago, immune control of the virus is not very well understood and there is still no vaccine available. This knowledge gap is due, in part, to the lack of a preclinical small animal model that can realistically recapitulate EBV infection and immune control and therefore allows testing of EBV-specific vaccine candidates (see Chapter 6). With the advent of mice with reconstituted human immune system compartments during the past decade, this is now changing. The complex interplay between host and virus has also made it difficult to elaborate useful vaccine strategies to protect against EBV-associated diseases (including chronic diseases like MS) or to find efficient drugs that specifically target EBV malignancies. Recently, the incorporation of immunotherapeutic strategies as first-line therapy has provided a better long-term outcome for patients. On the other hand, new predictive biomarkers have been found for patient follow-ups.

EBV is present in many pathologies, thus there is a need to encourage further research in this domain, which could lead to the discovery and development of new

**Emmanuel Drouet, PharmD, Ph.D.**

(Complement, Antibodies and Infectious Diseases Group),

Institut de Biologie Structurale,

Université de Grenoble-Alpes,

Full Professor,

Grenoble, France

Despite the discovery of EBV more than 50 years ago, immune control of the virus is not very well understood and there is still no vaccine available. This knowledge gap is due, in part, to the lack of a preclinical small animal model that can realistically recapitulate EBV infection and immune control and therefore allows testing of EBV-specific vaccine candidates (see Chapter 6). With the advent of mice with reconstituted human immune system compartments during the past decade, this is now changing. The complex interplay between host and virus has also made it difficult to elaborate useful vaccine strategies to protect against EBV-associated diseases (including chronic diseases like MS) or to find efficient drugs that specifically target EBV malignancies. Recently, the incorporation of immunotherapeutic strategies as first-line therapy has provided a better long-term outcome for patients. On the other hand, new predictive biomarkers have been found for patient follow-ups.

EBV is present in many pathologies, thus there is a need to encourage further research in this domain, which could lead to the discovery and development of new specific therapies.

## **Emmanuel Drouet, PharmD, Ph.D.**

Full Professor, Institut de Biologie Structurale, (Complement, Antibodies and Infectious Diseases Group), Université de Grenoble-Alpes, Grenoble, France

## **Chapter 1**
