EPSTEIN BARR VIRUS (EBV)

The Epstein-Barr virus (EBV), also called human herpesvirus 4 (HHV-4) EBV is named after Anthony Epstein and Yvonne Barr, who together with Bert Achong discovered the virus in 1964. It is a member of the herpesvirus family and one of the most common human viruses

DEFINITION:

It occur in late adolescence or young adulthood, and primary infection with EBV occurs in childhood and is usually asymptomatic. is transmitted by close human contact, frequently with the saliva during kissing

EBV

Causes by infectious mononucleosis(glandular fever), a benign, self-limited lymphoproliferative disorder. Associated with the development of a number of neoplasms, like Burkitt Lymphomas and nasopharyngeal carcinoma.

INFECTIOUS MONONUCLEOSIS
known as the kissing disease comes from oral distribution known as mono. Mono is a sickness that cases extreme fatigue and general feeling of sickness usually lasting anywhere from 2 to 6 weeks.

EBV INFECTION:

Oropharynx
Viral Ingestion

B-Cells

Infectious Mononucleosis Lymphadenitis Splenitis Hepatitis Pneumonitis Menigitis Encephalitis

Burkitt Lymphoma

Classical Symptoms of Epstein Barr Virus Fever Sore Throat Lymphadenitis (Swollen Lymph glands) splenomegaly

Atypical Symptoms of Epstein Barr Virus Extreme fatigue, malaise Lymphoma Lymphadenopathy (disorder of Lymph nodes or vessels) Hepatitis Pneumonitis Menigitis Encephalitis

DIAGNOSIS OF INFECTIOUS MONONUCLEOSIS DEPENDS ON: 1. Lymphocytosis with the characteristic atypical lymphocytes in the peripheral blood.

2. A positive heterophile antibody reaction (monospot test) 3. Specific anti-bodies for EBV antigens

EPSTEIN BARR VIRUS IN BONE MARROW OF RHEUMATOID ARTHRITIS PATIENTS PREDICTS RESPONSE TO RITUXIMAB TREATMENT
Karla Tricia C. Sarmiento

INTRODUCTION

VIRUSES INVOLVED IN RHEUMATOID ARTHRITIS

EBV

Parvovirus B19
CMV HSV-1, HSV-2 Polyoma virus

 Sequence homology between gp110 of EBV and the shared epitope (SE) in the MHC Class II gene could be a link between RA and EBV EBV-infected RA patients have a diminished cytotoxic Tcell response to EBV as compared with non-RA controls, increased number of EBV-infected B cells and high prevalence of antibodies to different EBV antigens

EBV infection may transform infected B cells into antibody-secreting plasma cells

HOW VIRUSES MAY CONTRIBUTE TO ARTHRITIS

RA patients often stand on immunosuppressive treatment that may expose them to an increased risk of viral infection
immunosuppressive treatment that alters immune responses to pre-existing viral infections could theoretically influence response to treatment

OBJECTIVES
To determine the presence of parvovirus B19 and EBV before and 3 months after immunosuppressive treatment with rituximab
To determine the importance of specific viral infections for response to RTX

METHODOLOGY

 35 participants 33 on MTX 1 AZA 1 chlorambucil 34 non-responders to previous anti-TNF- tx

10 ml of heparinized bone marrow aspirates were collected from crista iliaca peripheral blood obtained by venipuncture from the cubital vein and placed in a sterile heparinized vacuum container

PARAMETER

EBV (+) (n=15)

Parvo B19 (+) (n=8)

Virus (-) (n=12)

Age, mean, years

Gender, female/male Dse duration, mean, yrs RF positive at baseline, n (%) DAS-28 at baseline, median DMARD tx MTX, mg/week Chlorambucil AZA HLA-DRB1 carriers, n (%) Anti-CCP median, U/ml

61.1
14/1 10.7 13 (87) 5.90 14 1 0 6/13 (46) 24,058

57.3
7/1 17.8 8 (100) 5.50 7 0 1 3/8 (3.75) 16,185

54.2
9/3 13.9 8 (67) 6.21 12 0 0 1/12 (8) 15,689

PARAMETER
Age, mean, years Gender, female/male Dse duration, mean, yrs RF positive at baseline, n (%) DAS-28 at baseline, median 61.1 14/1 10.7

EBV (+) (n=15)

Parvo B19 (+) (n=8)

57.3 7/1 17.8 8 (100) 5.50 7 0 1 3/8 (3.75) 16185 54.2 9/3 13.9

Virus (-) (n=12)

13 (87) 5.90 14 1 0 6/13 (46) 24058

8 (67) 6.21 12 0 0 1/12 (8) 15689

DMARD tx MTX, mg/week Chlorambucil AZA

HLA-DRB1 carriers, n (%) Anti-CCP median, U/ml

RESULTS

 EBV and parvovirus B19 are frequently detected in bone marrow of RA px

 EBV positivity correlates to a better clinical response to RTX therapy

PARAMETER Age, mean, years Gender, female/male Dse duration, mean, yrs RF positive at baseline, n (%) at 6 mos followup, n (%) DAS-28 at baseline, median at 6 mos followup, median Change DAS28>1.3 DMARD tx MTX, mg/week Chlorambucil AZA 61.1 14/1 10.7

EBV (+) (n=15)

Parvo B19 (+) (n=8) 57.3 7/1 17.8 8 (100) 2 (25) 5.50 4.08 1 px (12.5%) 7 0 1 54.2 9/3 13.9

Virus (-) (n=12)

13 (87) 9 (60) 5. 90 3.95 12 px (80%) 14 1 0

8 (67) 5 (41) 6.21 4.86 5 px (42%) 12 0 0

HLA-DRB1 carriers, n (%) Anti-CCP median, U/ml

6/13 (46) 24058

3/8 (3.75) 16185

1/12 (8) 15689

PARAMETER Age, mean, years Gender, female/male Dse duration, mean, yrs RF positive at baseline, n (%) at 6 mos followup, n (%) DAS-28 at baseline, median at 6 mos followup, median Change DAS28>1.3 DMARD tx MTX, mg/week Chlorambucil AZA HLA-DRB1 carriers, n (%) Anti-CCP median, U/ml 61.1 14/1 10.7

EBV (+) (n=15)

Parvo B19 (+) (n=8) 57.3 7/1 17.8 8 (100) 2 (25) 5.50 4.08 1 px (12.5%) 7 0 1 3/8 (3.75) 16,185 54.2 9/3 13.9

Virus (-) (n=12)

13 (87) 9 (60) 5. 90 3.95 12 px (80%) 14 1 0 6/13 (46) 24,058

8 (67) 5 (41) 6.21 4.86 5 px (42%) 12 0 0 1/12 (8) 15,689

 RTX selectively depletes CD20-expressing B cells

PARAMETER Age, mean, years Gender, female/male Dse duration, mean, yrs RF positive at baseline, n (%) at 6 mos followup, n (%) DAS-28 at baseline, median at 6 mos followup, median Change DAS28>1.3 DMARD tx MTX, mg/week Chlorambucil AZA HLA-DRB1 carriers, n (%) Anti-CCP median, U/ml 61.1 14/1 10.7

EBV (+) (n=15)

Parvo B19 (+) (n=8) 57.3 7/1 17.8 8 (100) 2 (25) 5.50 4.08 1 px (12.5%) 7 0 1 3/8 (3.75) 16185 54.2 9/3 13.9

Virus (-) (n=12)

13 (87) 9 (60) 5. 90 3.95 12 px (80%) 14 1 0 6/13 (46) 24058

8 (67) 5 (41) 6.21 4.86 5 px (42%) 12 0 0 1/12 (8) 15689

 At baseline and at 3 months after treatment, there were no significant differences in the proportions of bone marrow immature, naive, unswitched memory and switched memory B cells between EBV-positive and EBVnegative patients. A population of CD19+CD95+ (Fas) expressing B cells in the bone marrow was significantly higher at baseline in EBV-positive than EBV-negative patients.

DISCUSSION

 B-cell depletion eradicated all traces of EBV infection in blood and bone marrow at 3 months post-RTX treatment
Carriers of EBV had a significantly better clinical response to B-cell depletion therapy than did patients without EBV infection

CYTOLYTIC T-CELL RESPONSE AGAINST EPSTEIN-BARR VIRUS IN LUNG CANCER PATIENTS AND HEALTHY SUBJECTS
Irene Kei Bariuad

ABSTRACT
Background
This study aimed to examine whether EBV seropositive patients with lung cancer have an altered virus-specific CTL response, as compared to age-matched healthy controls and whether any variation in this response could be attributed to senescence.

ABSTRACT
Methods
Peripheral blood mononuclear cells from lung cancer patients, age-matched and younger healthy individuals were used to measure EBVspecific CTLs after in vitro amplification with the GLCTLVAML and RYSIFFDYM peptides followed by HLA-multimer staining.

ABSTRACT
Results
Lung cancer patients and aged-matched controls had significantly lesser EBVspecific CTL than younger healthy individuals. Multimer positive populations from either group did not differ with respect to the percentage of multimer positive CTLs and the intensity of multimer binding.

ABSTRACT
Conclusions
This study provides evidence that patients with lung cancer exhibit an EBV-specific CTL response equivalent to that of age-matched healthy counterparts. These data warrant the examination of whether young individuals have a more robust antitumor response, as is the case with the antiEBV response

INTRODUCTION
Cancer patients present with a compromised immune response of multifactorial origin, including the tumor itself. Early stages of tumor growth appear not to elicit systemic immune deficiency and are sometimes associated with antigen-specific tolerance Generalized immunodeficiency can arise during the late stages of tumor development

INTRODUCTION
This study was scheduled in order to examine whether, at diagnosis, EBV seropositive patients with lung cancer, have a compromised virusspecific CTL response, as compared to agematched healthy controls.
A group of younger healthy individuals was also examined to ascertain whether a possible reduction in the anti-EBV CTL responses of the above patients and age-matched controls could be attributed to senescence.

SUBJECTS AND METHODS

Patients and controls
1. PBMC were isolated from whole blood collected at diagnosis from 19 patients with primary lung cancer.

SUBJECTS AND METHODS

Patients and controls
Thirteen- (+) NSCLC (non small cell lung carcinoma)

mean age: 66.8 +/- 11.8 years; 3 females, 10 males

Six- (+) SCLC (small cell lung carcinoma) mean age: 67.0 +/- 7.4 years; 1 female, 5 males

SUBJECTS AND METHODS

Patients and controls
2. PBMC were also collected from 14 age-matched healthy Individuals

mean age: 58.2 +/- 5.8 years; 4 females, 10 males

3.PBMC were also collected from 7 healthy younger individuals mean age: 26.7+/- 1.0 years; 4 females, 3 males

All PBMC were kept frozen till required

SUBJECTS AND METHODS

Patients and controls
Subjects expressed HLA-A2 and/or -A24 There were positive for IgG antibodies against the EBV nuclear antigen 3C (EBNA3C).

DETECTION OF EBV-SPECIFIC CTLS

Peptide-specific CTLs were detected using HLAmultimer flow cytometry after a previous step of in vitro amplification of MLPCs with peptides under limiting dilution conditions
Two EBV peptides GLCTLVAML(BMLF1.A2 presented by HLA-A2) and RYSIFFDYM (EBNA3C.A24 presented by HLA-A24) were used.

DETECTION OF EBV-SPECIFIC CTLS

Specific multimers labelled with APC and control multimers with PE were used to stain MLPC.
Each MLPC was considered to contain a multimer positive population, only if staining with the specific HLA-multimer resulted in a distinct cell cluster that did not stain with control HLA-multimers of different specificity.

RESULTS
EBV-specific CTL responses were detected in the peripheral blood of 8/19 lung cancer patients (42%) and 5/14 (36%) aged-matched controls (p=0.713).
Both of these proportions were statistically significantly different than 86% (6/7) of younger healthy individuals (p=0.048 and p=0.031, respectively) that presented with an EBV-specific CTL response

RESULTS

RESULTS

RESULTS

RESULTS
Each MLPC was considered to contain a multimer positive population, only if staining with the specific HLA-multimer resulted in a distinct cell cluster that did not stain with control HLA-multimers of different specificity.

RESULTS

RESULTS
This indicates that all antiviral T cells had TCR with a similar avidity towards the peptide/MHC complex and no difference in the kinetics of interaction between TcR and multimer complexes could be observed

DISCUSSIONS
This study provides direct evidence that lung cancer patients dispose an EBV-specific CTL response equivalent to that of age-matched healthy counterparts
the EBV-specific CTL response mounted by subjects of this age group, either with cancer or not, was twice as less than that elicited by younger healthy individuals

DISCUSSIONS
Regarding the healthy individuals, results demonstrated an inverse correlation between age and the percentage of circulating EBV-specific CTLs.
Most likely, these observations can be explained in the context of the complex process of T cell immunosenescence

DISCUSSIONS
With respect to cancer patients, it is interesting that they present with the same age-related alteration of EBV-specific CTL response as their healthy counterparts
Beyond differences observed in the specific pCTL frequency related to age, cancer patients also appeared with a decreased proliferative capacity of virus specific pCTL

CONCLUSIONS
this study provides evidence that lung cancer patients dispose an EBV-specific CTL response equivalent to that of age-matched healthy counterparts
study suggests that possibly the poor outcome of cancer immunotherapeutic approaches in lung cancer can be a result of the underlying effects of senescence on the immune system rather than an inefficient anti-tumor response.

EBV PROMOTES HUMAN CD8+ NKT CELL DEVELOPMENT

Karen Cas

INTRODUCTION
NKT cells unconventional T cells recognize CD1d-presented glycolipids

[MICE]
T cell development in THYMUS proceeds through 3 major stages: CD4- CD8- (DN) CD4+ CD8+ (DP) CD4+ CD8- or CD4- CD8+ (SP)

[MICE]

POSITIVE SELECTION CORTICAL THYMOCYTES

Semi-invariant TCR DP NKT precursors

CD1d ligand complex THYMIC DENDRITIC CELLS NEGATIVE SELECTION

PERIPHERY
(liver, spleen, lymph nodes, bone marrow, lung, gut)

Final NKTdifferentiation step

THYMUS

[MICE]
It is believed that there are no CD8+ NKT cells

[HUMAN]
CD8 is expressed on a minor proportion of human NKT cells CD8 marker is usually acquired after egress from the thymus

MATERIALS AND METHODS

PATIENTS, CELLS, TETRAMERS AND OTHER REAGENTS

Latent EBV-infected [EBV+(La)] - healthy EBV seropositive individuals Normal control subjects (NS) healthy seronegative individuals

PATIENTS, CELLS, TETRAMERS AND OTHER REAGENTS

Patients with EBV-associated acute infectious mononucleosis [lytic phase] ] EBV+(IMa) Diagnosed by a monospot test; and Detection of capsid-specific serum IgM

Followed up at 1 year [latent phase] EBV+(IMy)

PATIENTS, CELLS, TETRAMERS AND OTHER REAGENTS

Patients with EBVassociated Hodgkin lymphoma

EBV+(HL)

Diagnosed according to WHO criteria; and Staged according to the Ann Arbor classification

PATIENTS, CELLS, TETRAMERS AND OTHER REAGENTS

in- or out-patients in different hospitals in Hubei Province in China. newly diagnosed no previous treatment

Informed consent were provided

PATIENTS, CELLS, TETRAMERS AND OTHER REAGENTS

Human fetal thymic cells Bone marrow (BM) cells

Peripheral blood mononuclear cells (PBMC)

Liver cells

Voluntarily elective pregnancy terminations (<24 wk gestation; HLA typing matched HLA-A2 & HLA-DRB1)

PATIENTS, CELLS, TETRAMERS AND OTHER REAGENTS

Thymic cells

BM cells

PBMCs

ISOLATED ALIQUOTED Maintained in the vapor phase of liquid nitrogen for further use

PATIENTS, CELLS, TETRAMERS AND OTHER REAGENTS

THYMIC DENDRITIC CELLS were separated from thymocytes by adhesion onto plastic culture dishes

HUMAN THYMUS/LIVER SCID CHIMERAS

8 wk-old female SCID mice irradiated (300cGy/mouse) prior to cell-transplantation Human fetal thymic cells depleted of immature and mature NKT cells Transplanted into the thymus of anaesthetized SCID mice.

HUMAN THYMUS/LIVER SCID CHIMERAS

Human fetal liver tissue simultaneously implanted under the mouse kidney capsule The chimeras were then intrathymically challenged with EBV or HTLV-1 Repeated after 6 days Maintained for 4wks

FETAL THYMIC ORGAN CULTURE (FTOC)

Fetal thymus dissected into pieces of ~2mm3 3 pcs of tissue placed into 24-well plates with culture medium containing various stimuli (Day 7) cultured thymus fragment was dispersed into a single-cell suspension -> stained and analyzed

FETAL THYMIC ORGAN CULTURE (FTOC)

DP thymocytes obtained by gently grinding freshly fetal thymus lobes resulting suspensions -> sorted using CD8 and CD4 labeling

REAGGREGATED THYMIC ORGAN CULTURE (RTOC)

Reaggregates were formed by mixing together the desired thymic stromal cells and DP thymocytes at 1:1 ratio with other stimuli

FLOW CYTOMETRY
-GalCer-loaded CD1d tetramer and TCR Used to define total NKT cells

In intracellular staining for detection of perforin, different cells were resuspended in cold Dulbeccos PBS > permeabilized -> stained with mAb specific for human perforin -> analyzed

RESULTS

1. EBV-INDUCED CD8+ NKT CELLS IN VARIOUS EBV-INFECTED INDIVIDUALS

177 eligible patients and subjects
EBV+(La)

128 healthy latent EBV-infected subjects

16 EBV- NS negative normal control subjects

17 newly-onset acute infectious mononucleosis patients

16 newly-diagnosed EBV-associated Hodgkin Lymphoma patients EBV+(HL)

EBV+(IMa)

EBV+(IMy)

1. EBV-INDUCED CD8+ NKT CELLS IN VARIOUS EBV-INFECTED INDIVIDUALS

Figure 1. Frequency of total NKT cells

1. EBV-INDUCED CD8+ NKT CELLS IN VARIOUS EBV-INFECTED INDIVIDUALS

Figure 2. Frequency of co-receptor expressing NKT cells

2. EBV INDUCES INTRATHYMIC CD8+ NKT CELL DEVELOPMENT

Figure 3 . Frequencies of NKT cells in thymus and liver from hu-thy/liv-SCID chimeras challenged with EBV or HTLV-1

2. EBV INDUCES INTRATHYMIC CD8+ NKT CELL DEVELOPMENT

Figure 4. Frequencies of co-receptor-expressing NKT in the unchallenged (Nil) or EBV challenged (EBV) hu-thy/liver SCID chimeras

3. EBV-INDUCED CD8+ NKT CELL DEVELOPMENT DEPENDS ON THYMIC DCS

Figure 5. Frequency of co-receptor expressing NKT cells in thymus and liver

3. EBV-INDUCED CD8+ NKT CELL DEVELOPMENT DEPENDS ON THYMIC DCS

Figure 6. Frequency of coreceptor expressing NKT cells in thymus and liver

3. EBV-INDUCED CD8+ NKT CELL DEVELOPMENT DEPENDS ON THYMIC DCS

Figure 7. Frequency of coreceptor expressing NKT cells in thymus and liver

3. EBV-INDUCED CD8+ NKT CELL DEVELOPMENT DEPENDS ON THYMIC DCS

Figure 8. Frequency of coreceptor expressing NKT cells in thymus and liver

3. EBV-INDUCED CD8+ NKT CELL DEVELOPMENT DEPENDS ON THYMIC DCS

Figure 9. Frequency of total NKT cells in different RTOCs.

3. EBV-INDUCED CD8+ NKT CELL DEVELOPMENT DEPENDS ON THYMIC DCS

Figure 10. Frequency of co-receptor-expressing NKT cells in different RTOCs.

4. EBV-INDUCED CD8+ NKT CELL DEVELOPMENT IS IL-7-DEPENDENT

Figure 11. Frequencies of total NKT cells and co-receptorexpressing NKT cells in different FTOCs.

4. EBV-INDUCED CD8+ NKT CELL DEVELOPMENT IS IL-7-DEPENDENT

Figure 12. Development of co-receptorexpressing NKT cells in thymus or livers from hu-thy/liv SCID chimera challenged i.t. with EBV

5. EBV-INDUCED CD8+ NKT CELLS PRODUCE ABUNDANT PERFORIN

Figure 13. Perforin expressions in human and chimeric CD8+ NKT cells

DISCUSSION
CD8+ fraction (up to 25%)

generated in vivo after EBV-challenge

development of CD8+ NKT cells Promoted Thymus

DP precursor stage
thymic DCs

DISCUSSION
Certain pathogens are important contributors to CD8lineage commitment of NKT cells Infection by HIV and HTLV-1 results in a decrease in NKT cells EBV-infection promotes generation of perforin-biased CD8+ NKT cells

DISCUSSION
Protective role of CD8+ NKT against EBV-associated malignancies has been verified Any co-receptor-expressing human NKT cells detected in the mice developed and differentiated post-celltransplantation

DISCUSSION
EBV-challenge chimeras reflects viral effects on the differentiation of CD8+ NKT cells. IL-7 enhancer of EBV-induced development of thymic CD8+ NKT cells in vivo, in the hu-thy/liv-SCID chimeras in vitro in FTOCs

CONCLUSION
Rituximab treatment eradicated CD20-expressing B cells in patients with rheumatoid arthritis infected with EBV.
Thus rheumatoid arthritis patients have better clinical responses to Rituximab when they are infected with EBV

CONCLUSION
Lung cancer patients have EBV-specific CTLs equivalent to their age-matched healthy counterparts.
Cancer does not predispose a patient to decrease their CTL response to EBV infection.

EBV-specific CTLs decrease with age only when healthy individuals of differing age groups are compared.

CONCLUSION
Epstein-Barr Virus is implicated in the increased number of NKT cells
Amount of increase in NKT cells is proportional to duration of infection with EBV

EBV infection promotes generation of IFN-- and perforinbiased CD8+ NKT