Epstein-Barr virus and immunity Elena Kashuba, PhD Associate Professor Department of Microbiology, Tumor and Cell Biology (MTC) Karolinska Institutet 1
Epstein-Barr virus EBV Everybody s virus 200 nm Enveloped Double-stranded linear DNA 172274 bp 82 open reading frames 2
Life style of Epstein-Barr virus Latent infection (Transformation/Immortalization) In B lymphocytes Lytic (productive) infection In epithelial/b cells Latent programs I-III 12 genes 80 genes
The EBV paradox Most common virus in the human population (>90% of all adults, life long persistence) Tumor-associated virus 80% of carriers secrete biologically active virus (saliva) Very efficiently immortalizes B-cells
EBV is the most prevalent virus 170,000 new cases of cancer/year Nasopharyngeal carcinoma (NPC) 58,000 100% Stomach cancer 50,000 5-10% Hodgkin lymphoma 30,000 40-50% T-cell lymphoma 20,000 30-40% Non Hodgkin lymphomas in AIDS 6,000 25-100% Burkitts lymphoma 3,000 98% Post-transplant lymphomas 500 99%
EBV cycle in vivo Virus replication in oropharyngeal epithelium Latent reservoir of EBV infected resting B cells IgA positive B cells? Infection of circulating B cells Ag? T cell mediated control of proliferation CTLs Proliferation of EBV activated blasts 6
50 years anniversary!!!! 1964, Antony Epstein and Yvonne Barr Cause of infectious mononucleosis (IM), glandular fever - Henle lab, 1967 EBNA-2 EBV causes heterophile-positive IM in young adults IM is characterized by fever, sore throat, lymphadenopathy, and atypical lymphocytosis 7
Kinetic analysis of immunologic and virologic changes during primary Epstein Barr virus (EBV) infection Balfour HH et al., Journal of Inf. Disease, 2013 8
During infectious mononucleosis: Lymphocytosis: 7.2 x 10 6 lymphocytes/ml 1.7 x 10 6 control 5 x 10 6 CD3 1 X 10 6 control) 4.4 x 10 6 CD8 0.48 x 10 6 control up to 40% are specific to lytic proteins 0.1-5% recognize latent proteins (EBNA3,4,6) 1.3 x 10 6 NK 0.24 x 10 6 control Cytokine storm - Th 1 type - IFN-γ, IL2 9
Changes over time following infectious mononucleosis Hislop AD et al., Annu. Rev. Immunol. 2007 10
Latent EBV infection of resting B cells converts them into transformed cells that are found in IM patients and can develop into tumors in immuno-compromised hosts B-cells Lymphoblasts 11
Adapted from Gratama and Ernberg, 1995. The latency-associated EBV proteins are the key factors for virusinduced cell transformation. EBNA-1, EBNA-2, EBNA-3, EBNA-5, EBNA-6 and LMP1 EBV-encoded proteins are essential for the efficient transformation process. 12
Maintains viral episome Regulates viral promoters Blocks BCR signaling. Blocks lytic cycle. Binds to MDM2, thus stabilizing and inactivating p53 EBNA-5 Enhances EBNA-2- dependent transcription EBNA-1 EBNA-2 Activates viral (LMP1) and cellular (CD23, c-myc, D1, HES) promoters as heterodimer with Notch1IC, RBP- Jk, TFIIB (H,E), TBP EBV Promotes cell proliferation LMP-2 LMP-1 EBNA-3,4,6 Binds to and targets to the nucleus XAP-2, UK/UPRT, AhR Mimics CD40 activation of NFkB (transmission of growth signal), anti-apoptotic (induces BCL-X), survival signaling (Akt activation). Represses RBP-Jk dependent transcription Binds to S18-2 and targets it to the nucleus. This inhibits binding of prb to E2F1 and prb repressor function 13
EBV induced CD8+ T cell responses Hislop AD et al., Annu. Rev. Immunol. 2007 14
Relative immunodominance in healthy virus carriers of representative proteins of lytic and latent cycle for CD8 + and CD4 + T cell responses Hislop AD et al., 2007, Annu. Rev. Immunol. 15
Extensive studies on CD8+ and CD4+ CTLs: epitopes were identified in most of the latent and many lytic proteins Hislop AD et al., 2007, Annu. Rev. Immunol. 16
Humoral responses in primary infection Antibodies directed to: Viral structural proteins Latent proteins 17
T and NK cell numbers return to normal after IM But symptomatic primary EBV infection leaves all T and NK cells in all post-im patients without IL-15R expression Sauce D et al., 2006, Blood 18
Symptomatic primary EBV infection leaves a long lasting scar on the immune system Sauce D et al., 2006, Blood 19
Surveillance has to function constantly, if it relaxes, EBV transformed B cells will proliferate immuno-suppressed (AIDS, post-transplant patients) The control of EBV infection is assessed to cytotoxic T cells 20
Epstein-Barr virus The accidents: Combined immunosuppression, immunodysregulation and chronic antigen stimulation Infection of the wrong cell type: T-cells, smooth muscle, epithelial cells? Infection in unfavourable genetic background (chinese, inuits, X-linked lymphoproliferative syndrome, ataxia-telangectasia) 21
EBV-associated tumors in man Lymphoid tissues Burkitt s lymphoma, endemic 98% Burkitt s lymphoma, sporadic 25% AIDS-immunoblastic lymphoma 60% -in CNS 100% Post-transplant lymphoma 100% Hodgkin s lymphoma 50% T-cell lymphomas 10-30% lethal midline granuloma >90% Epithelial tissues Oral hairy leukoplakia 100% Gastric adenocarcinoma 5-10% Nasopharyngeal carcinoma, undifferentiated 100% Salivary gland carcinomas <100% Muscle tissue Leiomyosarcoma in immunosuppressed 100%? 22
Several types of latent EBV- host cell interactions: - various combinations of EBV encoded genes EBNA2 - NPC - GC In malignancies with non-proliferative latencies additional factors contribute for proliferation 23
Latency: III II I 24
Lymphomas in immuno-compromised individuals Latency III (all latent genes) Role of EBV: proliferation Lack of virus specific functional T cell is the cause AIDS patients: Immunoblastic Diffuse large cell lymphomas CNS lymphomas Transplant patients: Post-transplant lymphoproliferative disease (PTLD) Intervention: lowering immunosuppression adoptive transfer of EBV specific cytotoxic T cells (CTL) 25
Liver CT scan pre and post CTL adoptive transfer Khanna et al., PNAS, 1999 26
HIV infected patients primary central nervous system lymphoma (PCNSL) Absolute CD4+ T cell counts versus EBV-Specific CD4+ T cell function Irrespective of absolute CD4+ T cell counts, HIV-positive patients who subsequently developed PCNS lymphoma lacked EBV-specific CD4(+) T cell function. Gasser O et al., Plos Med 2007
Malignancies in immuno-competent individuals Burkitt lymphoma EBER Endemic: Africa, Papua New Guinea - is nearly 100% EBV associated Sporadic: worldwide, 5-10% EBV positive -Ig/c-myc translocation is the primary event -> constitutive activation of c-myc -Ig/myc translocation occurs in rare cells as an accident in normal B cell differentiation Role of EBV: rescue from apoptosis
Plasmodium falciparum co-infection: stimulates EBV+B cell compartment -> c-myc translocation high EBV load was found in malaria infected children - compromises EBV specific immune control, leading to immune escape - T cell responses against EBNA1 were significantly decreased - while CD4+ T cell responses against malaria antigens and CD8+ T cell responses against other EBV antigens were intact Moormann AM et al., Int. J. Cancer 2009
Nasopharyngeal carcinoma (NPC) frequent epithelial cell cancer with a high incidence rate in Southeast China: especially the Guangdong province and Hong Kong - 100% associated with EBV - Latency II: EBNA1, LMPs LMP1
NPC local immune suppression, rather that systemic deficiency in EBV specific immune control seems to contribute to NPC development Li J et al., Plos One 2007
Hodgkin s lymphoma - The most common EBV associated lymphoma in the US and Europe. - Around 40% of Hodgkin s lymphomas are EBV associated. Role of EBV: rescue from apoptosis - Latency II: EBNA1, LMPs LMP1
Relative risk of EBV positive Hodgkin s lymphoma increases 3-10 times after infectious mononucleosis Hjalgrim et al. New England J Med 349, 2003
Hodgkin lymphoma malignant H/RS cells 1% interaction with the surrounding cells (NF-κB constitutive activation) Has managed to generate an immunosuppressive environment that allows the tumor cells to grow despite extensive homing of immune cells to the tumor site. S. Amit and Y. Ben-Neriah 2002
Immunosuppressive functions of the HRS cells: -produce immunosuppressive cytokines like IL-10, IL-13 and TGF-b -galectin-1 secretion shown to inhibit EBV specific T cell proliferation and cytokine secretion. of the infiltrating lymphocytes: -T (Treg) cell populations enriched in Hodgkin s lymphoma tissues -Tr1 cells and CD4+CD25+ natural Treg cells suppress peripheral blood cell proliferation and cytokine secretion (trough IL-10) -LAG-3 positive CD4+ T cells suppress LMP specific T cell responses
Mechanism of HL immunoevasion
Systemic impairment of EBV specific T cell responses in HL - Hodgkin s lymphoma patients have - diminished EBNA1 specific CD4+ T cell responses, - they maintain CD8+ T cell responses against other latent and lytic EBV antigens Heller KN et al., Int.J Cancer 2008
Adoptive transfer: Successful in PTLD Not so much in HL, BL, NPC - generation of CTLs with LCL stimulation primarily expand T cell responses specific for the latent EBNA3, 4, 6 antigens, not expressed in these tumors. To solve the problem: T cells are expanded to EBNA1, LMP1 and LMP2 antigens, with recombinant viruses (adenovirus) encoding for these EBV products. Tested in vitro on T cells from HL patients. LMP2: CLG, FLY LMP1: YLG Smith C et al., J. Immunol 2006
EBV antigen loaded DCs: LMP2 specific CD8+ T cells were expanded after peptide pulsed DC injection in NPC patients, these responses were too weak or transient to achieve clinical effects. Lin CL et al., Cancer res, 2002
Conclusions There is now mounting evidence that EBV associated malignancies in otherwise immune competent individuals escape immune control by either - immune compromising co-infection - or conditioning of their microenvironment. The consensus at the moment: EBNA1 as a promising CD4+ T cell antigen, should be combined with LMP1 and LMP2 for CD8+ T cell generation in a vaccine for both - prevention of symptomatic EBV infection as well as - immunotherapy against EBV associated malignancies.