epstein-barr virus group

8/3/2019 Epstein-Barr Virus GROUP

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EPSTEIN BARR VIRUS (EBV)



DEFINITION:• 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

• 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

http://en.wikipedia.org/wiki/File:Epstein_Barr_Virus_virions_EM_10.1371_journal.pbio.0030430.g001-L.JPG





EBV•

Causes by infectious mononucleosis(glandular fever), abenign, 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 distributionknown 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:

Viral

Ingestion

Oropharynx

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



Karla Tricia C. Sarmiento

EPSTEIN BARR VIRUS IN BONE MARROW OFRHEUMATOID ARTHRITIS PATIENTS

PREDICTS RESPONSE TO RITUXIMAB

TREATMENT



INTRODUCTION



• EBV

• Parvovirus B19

• CMV

• HSV-1, HSV-2

• Polyoma virus

VIRUSES INVOLVED IN RHEUMATOID

ARTHRITIS



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 T-

cell 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



• RA patients often stand on immunosuppressive treatmentthat 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

HOW VIRUSES MAY CONTRIBUTE TO

ARTHRITIS



• To determine the presence of parvovirus B19 and EBVbefore and 3 months after immunosuppressive treatment

with rituximab

• To determine the importance of specific viral infections for

response to RTX

OBJECTIVES



METHODOLOGY



35 participants◦ 33 on MTX◦ 1 AZA◦ 1 chlorambucil◦

34 non-responders to previous anti-TNF-α tx

10 ml of heparinized bone marrow aspirates were collectedfrom crista iliaca

peripheral blood obtained by venipuncture from the cubital veinand placed in a sterile heparinized vacuum container



PARAMETER EBV (+)

(n=15)

Parvo B19 (+)

(n=8)

Virus (-)

(n=12)

Age, mean, years 61.1 57.3 54.2

Gender, female/male 14/1 7/1 9/3

Dse duration, mean, yrs 10.7 17.8 13.9

RF positive

at baseline, n (%) 13 (87) 8 (100) 8 (67)DAS-28

at baseline, median 5.90 5.50 6.21

DMARD tx

MTX, mg/week

Chlorambucil AZA

14

10

7

01

12

00

HLA-DRB1 carriers, n (%) 6/13 (46) 3/8 (3.75) 1/12 (8)

Anti-CCP median, U/ml 24,058 16,185 15,689



PARAMETER EBV (+)

(n=15)

Parvo B19 (+)

(n=8)

Virus (-)

(n=12)

Age, mean, years 61.1 57.3 54.2



RF positive

at baseline, n (%) 13 (87) 8 (100) 8 (67)DAS-28

at baseline, median 5.90 5.50 6.21

DMARD tx

MTX, mg/week

Chlorambucil AZA

14

10

7

01

12

00


Anti-CCP median, U/ml 24058 16185 15689



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 EBV (+)

(n=15)

Parvo B19 (+)

(n=8)

Virus (-)

(n=12)

Age, mean, years 61.1 57.3 54.2



RF positive

at baseline, n (%)

at 6 mos followup, n (%)

13 (87)

9 (60)

8 (100)

2 (25)

8 (67)

5 (41)

DAS-28at baseline, median

at 6 mos followup, median

Change DAS28>1.3

5. 90

3.95

12 px (80%)

5.50

4.08

1 px (12.5%)

6.21

4.86

5 px (42%)

DMARD tx

MTX, mg/weekChlorambucil

AZA

141

0

70

1

120

0





PARAMETER EBV (+)

(n=15)

Parvo B19 (+)

(n=8)

Virus (-)

(n=12)

Age, mean, years 61.1 57.3 54.2

Gender, female/male 14/1 7/1 9/3Dse duration, mean, yrs 10.7 17.8 13.9

RF positive

at baseline, n (%)


13 (87)

9 (60)

8 (100)

2 (25)

8 (67)

5 (41)



Change DAS28>1.3

5. 90

3.95

12 px (80%)

5.50

4.08

1 px (12.5%)

6.21

4.86

5 px (42%)

DMARD tx

MTX, mg/week

Chlorambucil

AZA

14

1

0

7

0

1

12

0

0


Anti-CCP median, U/ml 24,058 16,185 15,689



• RTX selectively depletes CD20-expressing B cells



PARAMETER EBV (+)

(n=15)

Parvo B19 (+)

(n=8)

Virus (-)

(n=12)

Age, mean, years 61.1 57.3 54.2

Gender, female/male 14/1 7/1 9/3Dse duration, mean, yrs 10.7 17.8 13.9

RF positive

at baseline, n (%)


13 (87)

9 (60)

8 (100)

2 (25)

8 (67)

5 (41)



Change DAS28>1.3

5. 90

3.95

12 px (80%)

5.50

4.08

1 px (12.5%)

6.21

4.86

5 px (42%)

DMARD tx

MTX, mg/week

Chlorambucil

AZA

14

1

0

7

0

1

12

0

0





• At baseline and at 3 months after treatment, there wereno significant differences in the proportions of bone

marrow immature, naive, unswitched memory and

switched memory B cells between EBV-positive and EBV-

negative patients.

• A population of CD19+CD95+ (Fas) expressing B cells in

the bone marrow was significantly higher at baseline inEBV-positive than EBV-negative patients.



DISCUSSION



• B-cell depletion eradicated all traces of EBV infection inblood and bone marrow at 3 months post-RTX treatment

• Carriers of EBV had a significantly better clinicalresponse to B-cell depletion therapy than did patients

without EBV infection



Irene Kei Bariuad

CYTOLYTIC T-CELL RESPONSE AGAINST

EPSTEIN-BARR VIRUS IN LUNG CANCER

PATIENTS AND HEALTHY SUBJECTS



ABSTRACTBackground

This study aimed to examine whether EBV

seropositive patients with lung cancer have an

altered virus-specific CTL response, as comparedto 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 EBV-specific CTLs after in vitro amplification with the

GLCTLVAML and RYSIFFDYM peptides followed

by HLA-multimer staining.



ABSTRACTResults

Lung cancer patients and aged-matched controls

had significantly lesser EBVspecific CTL than

younger healthy individuals. Multimer positivepopulations from either group did not differ with

respect to the percentage of multimer positive

CTLs and the intensity of multimer binding.



ABSTRACTConclusions

This study provides evidence that patients with

lung cancer exhibit an EBV-specific CTL response

equivalent to that of age-matched healthycounterparts. These data warrant the examination

of whether young individuals have a more robust

antitumor response, as is the case with the anti-

EBV 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 elicitsystemic immune deficiency and are sometimes

associated with antigen-specific tolerance

•

Generalized immunodeficiency can arise duringthe 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 virus-

specific CTL response, as compared to age-matched 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.





• 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





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 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 HLA-

multimer 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 byHLA-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 thespecific 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• 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 stainwith 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 complexescould 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 cellimmunosenescence



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 alsoappeared 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 aninefficient anti-tumor response.



EBV PROMOTES HUMAN CD8+ NKT CELL

DEVELOPMENT

Karen Cas



INTRODUCTION

• NKT cells

• unconventional T cells

• recognize CD1d-presented glycolipids



[MICE]

αβT cell development in THYMUS proceeds through 3

major stages:

• CD4- CD8- (DN)

• CD4+ CD8+ (DP)

• CD4+ CD8- or CD4- CD8+ (SP)



[MICE]

CORTICAL

THYMOCYTES

THYMIC

DENDRITIC CELLS

Semi-invariantαβTCR DP

NKT

precursors

CD1dligand

complex

POSITIVE

SELECTION

NEGATIVE

SELECTION



Final NKT-

differentiation

step

THYMUSPERIPHERY

(liver, spleen,lymph nodes,

bone marrow,

lung, gut)



[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



PATIENTS, CELLS, TETRAMERS AND OTHER

REAGENTS

• Latent EBV-infected [EBV+(La)] - healthy EBV

seropositive individuals

• Normal control subjects (NS) – healthy seronegative

individuals





REAGENTS

Patients with EBV-associated acute infectious

mononucleosis [lytic phase] ]

Diagnosed by a monospot test; and

Detection of capsid-specific serum IgM

Followed up at 1 year [latent phase]

EBV+(IMa)

EBV+(IMy)





REAGENTS

Patients with EBV-associated

Hodgkin

lymphomaEBV+(HL)

Diagnosed according

to WHO criteria; and

Staged according to

the Ann Arbor

classification





REAGENTS

• in- or out-patients in different hospitals in Hubei

Province in China.

• newly diagnosed

• no previous treatment

• Informed consent were provided




Voluntarily electivepregnancy terminations

(<24 wk gestation; HLA

typing matched HLA-A2

& HLA-DRB1)


REAGENTS

Human fetal

thymic cells

Bone marrow

(BM) cells

Peripheral bloodmononuclear cells

(PBMC)

Liver cells





REAGENTS

Thymic

cellsBM cells PBMCs

ISOLATED

ALIQUOTED

Maintained in the vapor phase of liquid

nitrogen for further use





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 andmature NKT cells

• Transplanted into the thymus of anaesthetized SCID

mice.



HUMAN THYMUS/LIVER SCID CHIMERAS

• Human fetal liver tissue – simultaneously implantedunder the mouse kidney capsule

• The chimeras were then intrathymically challenged withEBV 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 asingle-cell suspension -> stained and analyzed



FETAL THYMIC ORGAN CULTURE (FTOC)

• DP thymocytes – obtained by gently grinding freshly fetalthymus lobes

• resulting suspensions -> sorted using CD8 and CD4

labeling

REAGGREGATED THYMIC ORGAN CULTURE



REAGGREGATED THYMIC ORGAN CULTURE

(RTOC)

• Reaggregates were formed by mixing together thedesired thymic stromal cells and DP thymocytes at

1:1 ratio with other stimuli



FLOW CYTOMETRY

• α-GalCer-loaded CD1d tetramer and αβTCR

• Used to define total NKT cells

• In intracellular staining for detection of perforin,

different cells were resuspended in cold Dulbecco’s PBS -

> permeabilized -> stained with mAb specific for human

perforin -> analyzed



1 EBV-INDUCED CD8+ NKT CELLS IN



1. EBV-INDUCED CD8+ NKT CELLS IN

VARIOUS EBV-INFECTED INDIVIDUALS

177 eligible patients and subjects

128 healthy latent

EBV-infectedsubjects

16 newly-diagnosedEBV-associated

Hodgkin Lymphoma

patients

16 EBV-

negative normalcontrol subjects

17 newly-onsetacute infectious

mononucleosis

patients

EBV+(IMa)

EBV+(HL)

EBV+(La)

NS

EBV+(IMy)






Figure 1. Frequency of total NKT cells






Figure 2. Frequency of co-receptor expressing NKT cells

2 EBV INDUCES INTRATHYMIC CD8+ NKT



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



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



3. EBV INDUCED CD8+ NKT CELL

DEVELOPMENT DEPENDS ON THYMIC DCS

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






Figure 6. Frequency of co-

receptor expressing NKT cells in

thymus and liver







receptor expressing NKT

cells in thymus and liver

3. EBV-INDUCED CD8+ NKT CELL






receptor expressing NKT cells in

thymus and liver




3. EBV INDUCED CD8 NKT CELL


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






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





DEVELOPMENT IS IL-7-DEPENDENT

Figure 11.

Frequencies of

total NKT cells

and co-receptor-

expressing NKT

cells in different

FTOCs.





DEVELOPMENT IS IL-7-DEPENDENT

Figure 12. Development

of co-receptor-

expressing NKT cells in

thymus or livers from

hu-thy/liv SCID chimerachallenged 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

• Certain pathogens are important contributors to CD8-lineage commitment of NKT cells

• Infection by HIV and HTLV-1 results in a decrease inNKT cells

• EBV-infection promotes generation of perforin-biasedCD8+ NKT cells



DISCUSSION

• Protective role of CD8+ NKT against EBV-associatedmalignancies has been verified

• Any co-receptor-expressing human NKT cells detectedin the mice

• developed and differentiated post-cell-

transplantation



DISCUSSION

• EBV-challenge chimeras – reflects viral effects on thedifferentiation 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 Bcells in patients with rheumatoid arthritis infected with

EBV.

•

Thus rheumatoid arthritis patients have better clinicalresponses to Rituximab when they are infected with EBV



CONCLUSION

• Lung cancer patients have EBV-specific CTLsequivalent 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 increasednumber of NKT cells

• Amount of increase in NKT cells is proportional to duration of

infection with EBV

• EBV infection promotes generation of IFN-γ- and perforin-

biased CD8+ NKT

epstein-barr virus group

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