EBV Infection and Immunity. Andrew Hislop Institute for Cancer Studies University of Birmingham
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1 EBV Infection and Immunity Andrew Hislop Institute for Cancer Studies University of Birmingham
2 EBV Introduction Large ds DNA virus Spread by saliva contact Lifelong infection Predominantly B-lymphotropic via CR2 also infects epithelial cells rarely T cells and NK cells
3 EBV infection and disease Infection with EBV usually occurs early in life usually asymptomatic if delayed onset may result in Infectious mononucleosis Malignancies
4 Symptomatic primary infection 1 o infection epith B Tonsil
5 and Latent Infection by EBV EBV Latent infection EBNA 1, 2, 3A, 3B, 3C, LP LMP1, LMP2 Transformed B-cell B-lymphocyte
6 and Latent Infection by EBV EBV Latent infection EBNA 1, 2, 3A, 3B, 3C, LP LMP1, LMP2 Transformed B-cell B-lymphocyte infection Immediate x2 e.g. BZLF1 Early x30 e.g. BMLF1 Late x30 e.g. BLLF1 Virus replication
7 Symptomatic primary infection 1 o infection epith B Tonsil? Lat I/II
8 ! " # $%"&
9 Infectious mononucleosis tonsil; EBNA2 staining
10 Symptomatic primary infection 1 o infection epith B Tonsil? Lat I/II
11 Symptomatic primary infection Persistence 1 o infection epith B Expression of LMP1 and LMP2? Lat I/II Tonsil Germinal centre transit? Lat I/II Lat 0 Memory B cell
12 Symptomatic primary infection Persistence 1 o infection epith B Expression of LMP1 and LMP2? Lat I/II Tonsil Germinal centre transit? Lat I/II Lat 0 Memory B cell B
13 Symptomatic primary infection Persistence 1 o infection epith B? Lat I/II Tonsil Germinal centre transit? Lat I/II Lat 0 Memory B cell B T T T T T T T T T T T Latent Ag specific Ag specific Ag specific 1 o CTL response Latent Ag specific Memory CTL response
14 Recognition of viral peptides by CD8 T cells CD8+ T cell CD8 T cell CD8 TcR Peptide Heavy chain β2 microglobulin Plasma membrane Class I MHC Virus infected cell Endogenous antigen processing pathway Viral protein
15 Quantitation of EBV-specific responses Class I MHC tetramers CD8+ T cell Phycoerythrin CD8 TcR Peptide Heavy chain β2 microglobulin Avidin Plasma membrane Biotin Stain PBMCs with tetramer and anti-cd8 Analyse by flow cytometry
16 Symptomatic primary infection Persistence 1 o infection epith B? Lat I/II Tonsil Germinal centre transit? Lat I/II Lat 0 Memory B cell B T T T T T T T T T T T Latent Ag specific Ag specific Ag specific 1 o CTL response Latent Ag specific Memory CTL response
17 High virus throat loads are maintained over time in IM patients 10 7 Throat wash 10 6 Viral load per 10 6 PBMC or per ml of throat wash PBMC Normal limit of replication in throat Normal limit of latent infection in blood Days post onset symptoms
18 Infectious mononucleosis large numbers of atypical mononuclear cells in the blood
19 EBV-specific responses in IM are expanded in PBMC compared to tonsil PBMC IM % CD8 RAK lytic
20 EBV-specific responses in IM are expanded in PBMC compared to tonsil PBMC IM-5 Tonsil 46.71% 13.13% CD8 3 to 4-fold dilution of lytic response in tonsil v blood RAK lytic
21 EBV-specific responses in IM are expanded in PBMC compared to tonsil PBMC IM-5 Tonsil 46.71% 13.13% CD8 3 to 4-fold dilution of lytic response in tonsil v blood RAK lytic 2.16% 1.61% CD8 QAK latent
22 EBV-specific responses in IM are expanded in PBMC compared to tonsil Very high levels of EBV-specific CD8 T cell seen in blood 3 to 4-fold dilution of lytic response in tonsil versus blood Cells highly activated Cells loose lymphoid homing markers Cells loose mucosal epithelium attachment molecules
23 Symptomatic primary infection 1 o infection epith B Tonsil? Lat I/II T T T T T T T T T T T Ag specific 1 o CTL response Latent Ag specific
24 High virus throat loads are maintained over time in IM patients 10 7 Throat wash 10 6 Viral load per 10 6 PBMC or per ml of throat wash PBMC Normal limit of replication in throat Normal limit of latent infection in blood Days post onset symptoms
25 Contrasting patterns of localisation of EBV-specific CD8s in recovered IM patient PBMC Post IM % Tonsil UMs 0.27% CD8 YVL lytic
26 Contrasting patterns of localisation of EBV-specific CD8s in recovered IM patient PBMC Post IM % Tonsil UMs 0.27% CD8 YVL lytic 0.13% 0.59% CD8 CLG latent
27 Contrasting patterns of localisation of EBV-specific CD8s in recovered IM patients Lower levels of EBV-specific CD8 T cells seen 4 to 10-fold dilution of lytic response in tonsil versus blood 4-fold enrichment of latent responses in tonsil versus blood Cells highly activated in tonsil NOT in blood Enriched cells gaining lymphoid homing markers Cells gaining mucosal epithelium attachment molecules
28 High virus throat loads are maintained over time in IM patients 10 7 Throat wash 10 6 Viral load per 10 6 PBMC or per ml of throat wash PBMC Normal limit of replication in throat Normal limit of latent infection in blood Days post onset symptoms
29 EBV-specific responses in healthy carriers are expanded in tonsil compared to PBMC PBMC T-26 Tonsil UMs 1.31% 3.87% CD8 RAK lytic
30 EBV-specific responses in healthy carriers are expanded in tonsil compared to PBMC PBMC T-26 Tonsil UMs 1.31% 3.87% CD8 RAK lytic 1.16% 12.18% CD8 FLR latent
31 Symptomatic primary infection Persistence 1 o infection epith B? Lat I/II Tonsil Germinal centre transit? Lat I/II Lat 0 Memory B cell B T T T T T T T T T T T Latent Ag specific Ag specific Ag specific 1 o CTL response Latent Ag specific Memory CTL response
32 Contrasting patterns of localisation of EBV-specific CD8s in healthy carriers Low yet substantial levels of EBV-specific CD8 T cells seen in blood Large expansions can be found in tonsil 3 to 10-fold expansion of responses in tonsil versus blood Cells not activated in tonsil or blood Enriched cells gaining lymphoid homing markers Cells gaining mucosal epithelium attachment molecules
33 Applications of EBV cellular immunology research Model antigen Vaccine design Therapeutic interventions Bone marrow transplant / solid organ transplant where immunosuppression is induced
34 Healthy Carrier Persistence B Latent Ag specific Ag specific Memory CTL response
35 Immunosuppression B Latent Ag specific Ag specific Memory CTL response
36 Immunosuppression? Lat I/II? Lat I/II? Lat I/II? Lat I/II B Latent Ag specific Ag specific Memory CTL response
37 Applications of EBV cellular immunology research Therapeutic interventions: adoptive transfer of EBV-specific CTL Lung transplant patient Developed EBV-positive post transplant lymphoproliferative disease Had numerous lymphomas In vitro stimulation and amplification of remaining EBV-specific T cells Re-infuse amplified cells
38 Applications of EBV cellular immunology research CT scan analysis of liver Presentation Khanna et al PNAS 1999, 96:
39 Applications of EBV cellular immunology research CT scan analysis of liver Presentation 2 Weeks post CTL infusion Khanna et al PNAS 1999, 96:
40 Applications of EBV cellular immunology research CT scan analysis of liver Presentation 2 Weeks post CTL infusion 20 Weeks post CTL infusion Khanna et al PNAS 1999, 96:
41 Lessons from the study of EBV immunity A persistent B lymphotropic virus elicits strong T cell responses Primary infection as infectious mononucleosis causes: massive expansions of EBV-specific T cells in blood poor trafficking ability these cells to tonsil/site of viral replication Resolution of disease sees some improved trafficking to site of viral replication Healthy carrier state associated with substantial levels of EBV-specific T cells in blood good trafficking high levels of these cells to tonsil/site of viral replication EBV responses can be mapped and applied for therapy
42 Suggested papers 1. Thorley-Lawson, D.A Epstein-Barr virus: exploiting the immune system. Nature Reviews Immunology 1: Moss, D.J., S.R. Burrows, S.L. Silins, I. Misko, and R. Khanna The immunology of Epstein Barr virus infection. Philosophical Transactions of the Royal Society of London Series B-Biological Sciences 356:
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