EPSTEIN BARR VIRUS - the ability to persist in the human immune-system and to immortalize B cells in culture
Young Researcher's Corner News@ESID - Spring 2013, by Stefania Giannelli (HSR-TIGET Milano, Italy)
The Epstein-Barr –Virus (EBV), also called human herpesvirus-4 (HHV-4), is a double strand DNA virus belonging to the herpes family, and is one of the most common viruses in humans.
EBV takes his name from the Professor Michael Anthony Epstein and Yvonne Barr, who discovered and documented the virus. Virus particles were identified in cultured infected cells, and the results were published in the Lancet journal in 1964 by Epstein MA, Anchor B and Barr Y. In the following years serological markers were identified in cell lines and in 1968 mimicking some forms of EBV–related infections, was discovered that EBV can directly immortalize B cells after infection. The human EBV preferentially infects B cells, but occasionally infects other cell types, especially epithelial cells. EBV is usually rapidly cleared by healthy human immune system.
Human herpes viruses have a unique capacity to establish a life-long latent infection in the host, whereby the virus can persist within specific host cells, and protects itself from immune recognition by limiting viral gene expression. The establishment of EBV latency is the final phase of the four different EBV infection stages: the growth phase in which the virus activates resting B cells to became proliferating lymphoblasts, the default phases in which EBV provides survival signals to infected lymphoblasts to induce their differentiation into memory B cells and the maintenance of persistently infected memory cells and finally the latency phase that allows persistence of the virus in resting recirculating memory cells in a way that is non-pathogenic and not detectable by the immune system.
The human immune response is generally very successful at controlling infections and minimizing symptoms during primary and persistent infections; however, herpes viruses are responsible for several diseases including conditions associated with primary infections. These clinical problems are more common and severe in immune-compromised individuals such as transplant patients on immunosuppressive medication and human immunodeficiency virus (HIV)-infected individuals, because of an impaired adaptive immune system. In particular, the most benign transient infection that affects adolescent or adult is the infectious mononucleosis, in which 50% of T cells and 25% of the memory B cells are specific for the virus during peaks of infection. From this infection EBV will latently persist in the individual’s B cells life-long and can wake up in the moments of immune weakness.
However, there is a dark side for this seemingly inoffensive virus. A simple mutation in small and signaling molecule, the SLAM-associated protein (SH2D1A/SAP) diverts EBV infection from a benign persistence to an acutely aggressive disease, X-linked lymphoproliferative disease (XLP), which rapidly kills the infected individual. The EBV latency phase in combination to environmental and genetic cofactors could links EBV with neoplastic malignancy.
EBV is present in malignant pathologies such as the Burkitt’s lymphoma (the tumor in which the virus was discovered, but without a clear role in the pathogenesis of the tumor), the nasopharyngeal carcinoma, the Hodgkin’s disease and the immunoblastic lymphoma. It is also present in heterogeneous group of B-cell tumors that arise in immunocompromised individuals unable to mount an effective cytotoxic T lymphocyte response to these infected cells.
After primary infection, EBV can be detected in the peripheral blood of healthy seropositive individuals. Spontaneous outgrowth of virus carrying B cells from peripheral blood gives rise to B lymphoblastoid cell line in culture and PCR analysis of peripheral blood cell DNA can detect viral genomes.
Also in our lab we can generate immortalized B cell lines. The technical procedure used to establish EBV transformed B cell lines have not substantially changed over the last 25 years. Free EBV particles are produced by maintaining an EBV infected marmoset cell line (e.g. B95.8), which is overgrown and subsequently lysed. Human lymphocyte cultures are inoculated with free virus that enters into B-lymphocytes via their CD2I (CR2) cell surface molecules. As the virus becomes integrated into the B cell, cytotoxic T lymphocytes can be generated, which subsequently kill the infected B cells, leading to transformation failure. A range of techniques has been developed to avoid this. These include removal of T lymphocytes following resetting with sheep red blood cells or immune suppression of T cells using cyclosporin A. An alternative strategy is the incubation of lymphocyte cultures with T cell mitogens such as phytohaemagglutinin (PHA), which induces T cells to rapidly transform into blast cells and die before cytotoxic T cells can be generated.
Current techniques are based on variations of these procedures and use isolated B lymphocytes from blood samples. These are either transformed as fresh cells or following cryo-preservation and storage in liquid nitrogen. Fifteen days after infections in transformed B cell lines viral genomes are detected by PCR or in alternative by FACS is possible to check for CD20, 30, 38 and 27 B cell marker.
We can take advantage from EBV transformed B cell lines considering that the somatic mutation rate of DNA is low (0.3%) so EBV cell lines is largely representative of a wide range of metabolic pathways specific for the individual/patient from whom the cell line was generated. The generation of EBV cell lines is thus especially suitable to study pathologies in which B cells pathways are impaired or to expand the poor B cells pool from immunodeficiency’s patients or from tissues not easily available like cerebrospinal fluid of multiple sclerosis or the synovial fluid of rheumatoid arthritis patients. B cell immortalization can be also a valuable tool for the production and characterization of auto-reactive antibodies from patients with autoimmune disease. Furthermore, EBV cell lines could be used for the isolation of therapeutic antibodies for passive vaccination, but also to analyze the antibody repertoire in immune or vaccinated individuals to identify neutralizing, enhancing, or irrelevant epitopes, thus guiding the formulation of candidate vaccines. This “analytic vaccinology” will be particularly useful in the case of highly variable viruses, such as hepatitis C virus or HIV, or highly complex pathogens.
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