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Epstein-Barr Virus
Introduction
According to the Center for Disease Control and Prevention (CDC) “Epstein-Barr virus (EBV),
also known as human herpesvirus 4, is a member of the herpes virus family. It spreads most
commonly through bodily fluids, primarily saliva and is one of the most common human
viruses” and is suspected to have evolved from a nonhuman-primate virus [1,2].
The EB virus was discovered more than 50 years ago, and is known to cause infectious
mononucleosis as well as tumours of both lymphoid and epithelial origin [3].
Latest progress in EBV research links the virus to MS, suggesting it might be triggering the
neurodegenerative autoimmune disease. As it is reported, patients infected by EBV have 30
times higher probability to develope MS, compared to non-infected subjects [4].
As studied in this course, EBV infection triggers both innate and adaptive immune reactions,
which are aimed at the removal of the infection. EBV, though, is particular: exhibiting both latent
and lytic cycles, the former leads to residency of the virus in host memory B lymphocytes cells
(Figure 1) causing episodes of symptoms, often periodically [5, 6]. The infection cycle of EBV
is, however, not the focus of the present assignment.
The only known biomarker in latent infected cells it the Epstein-Barr virus nuclear antigen 1
(EBNA-1), which keeps the integrity of the viral DNA in the host’s genome [5].
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Figure 1. EBV transmits via the saliva and infects the epithelial cells of the oro-pharynx. The
lytic cycle of EBV includes the infection of the epithelial cells and their subsequent lysis. The
latent cycle of EBV includes infection of naive B cells found under the epithelial barrier and is
the reason of EBV persistence after primary infection is resolved. Source of image:
www.creative-biolabs.com/vaccine/epstein-barr-virus-vaccines.htm
In the sections that follow, the main points of the immunological response to EB virus infection
in human will be discussed. For this purpose, the slides provided under “Problem-solving tasks:
Session 9”(available at
https://learn.inside.dtu.dk/d2l/le/content/102803/viewContent/387793/View) were used. Note
that the slides are using the virus example of SARS-CoV-2 and here we are studying the EBV,
but the immunological features remain rouglhy the same. A big difference being the entry of the
virus.
Slide 1: Pathogen entry at tissue site
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1. Describe the inflammation process in the infected tissue, by naming the
cell types that initiate the immune response and the processes they
mediate.
Epstein-Bar virus enters the cell by recognizing and binding complement receptor 2 (CD21 or
CR2) and HLA class II molecules and then enters by fusion with the cell membrane. The virus
incorporates its envelope into the cell's plasma membrane and the capsid is transported through
the cytoplasm to a nuclear pore [6].
Macrophages secrete pro-inflammatory cytokines after sensing microbial products. These
cytokines produce vasodilatation, increase vascular permeability and induce the production
of adhesion molecules in the vascular endothelium, causing inflammation. All of these aid in
the entry of recruited cells and complement into the infected tissue and increase the flow of
lymph (lymph drainage). Also recruiting more monocytes from the bloodstream.
For this case, the secreted cytokines and their specific effects are the following:
TNF-a: activates vascular endothelium and increments vascular permeability.
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IL-1b: activates vascular endothelium and lymphocytes, tissue destruction, and aid in
the entry of effector cells to the site of infection.
IL-6: produces fever.
Infected Epithelial cells secrete CXCL8 a chemotactic factor that recruits neutrophils
(fast-acting phagocytes) from the bloodstream to the site of infection.
Dendritic cells uptake of infected apoptotic epithelial cells, via receptor-mediated
endocytosis, uptake of antigen process, and PRR activation.
2. Detail how the dendritic cells at the tissue site are activated (include
uptake process and PRR activation events + PAMPS/PRRs involved)
Activation of dendritic cells starts with the uptake of antigens. In the case of a viral infection,
the uptake of antigens could happen via 3 routes:
1. Viral infection of the dendritic cell, viral proteins synthesized inside of the dendritic cell are
processed to be presented as peptides loaded to MHC class I.
2. Macropinocytosis of viral particles. The degraded viral particles would be presented loaded
in MHC class II molecules. (CD4T cells stimulate antiviral antibodies production by B cell)
3. Cross presentation, uptake of virus particles or virus infected cells by macropinocytosis or
phagocytosis. Results in the presentation of processed viral peptides on MHC class I.
Any viral infection can lead to the activation of dendritic cells, whether the virus is directly
infecting the dendritic cell (route 1) or not (route 2 and 3).
Dendritic cells can also be activated by PAMPs recognized by different PRRs present in the
dendritic cell. Epstein virus is a dsDNA virus, the specific PAMPs of this kind of virus and the
associated PRRs are the following:
1. Glycoproteins are recognized by CLRs. CLRs receptors influence the kind of cytokines that
will be secreted by the activated dendritic cell (signal 3).
2. dsDNA is recognized by TLR-9, AIM2, and DAI. TLR receptors are activators of signals 1 3 and induce the production of CCR7, a cytokine that allows the migration of the activated
dendritic cell to the lymph node.
The cytokine patterns that will be secreted by the dendritic cell after the activation depend on the
PAMPs recognized.
1. For TLR-9 the cytokines are INF-a IL12p70 and IL-10.
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2. For DAI the transcription factors IRF3, IRF7, and in some cases NF-kB, are activated.
3. For AIM2 caspase-1 transcription factor is activated.
3. Describe which molecules of importance for antigen presentation that
are up-regulated on the innate cell type(s) by interaction with the microbe.
Once dendritic cells have encountered and engulfed the antigen through MAMPs, expression of
CCR7 on the cell surface is increased, enhancing the sensibility of these cells to CCL21 —
migration into the local lymphoid tissue. CCR7 also boosts antigen presentation molecules by
up-regulating stable MHC class I and II molecules, as well as co-stimulatory proteins (CD80 and
CD86) that will boost naive-T-cell activation. Other up-regulated molecules upon antigen
encounter with related effects include DC-SIGN (adhesion, surface molecule) and CCL19
(attraction of naive T cells).
Slide 2: Migration to the lymph node
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4. Describe how the dendritic cell is entering into the draining lymph node
after activation and how naïve T and B cells will enter.
Upon infection, antigen-bearing and antigen-free dendritic cells actively migrate to the draining
lymph node by cytokine chemotaxis (CCL19). CCL19 and CCL21 release is thus enhanced,
which attracts naïve T and B cells to enter through the bloodstream (arteries).
5. Where is the activation of naïve T cells and B cells taking place, and
how?
In the lymph nodes, by dendritic cells. Naive T cells: stimulation through TCR x MHC-II and costimulation through CD28.
In the bloodstream, by regular cells through MHC-I/II (naive T cells go around looking for an
intracellular/extracellular antigen displayed on any cell, if they fail they go to the lymph nodes
where DCs reside to activate leukocytes which encountered an antigen, in which case the
aforementioned mechanism takes place.
Naive B cells, TH-dependent activation: if the B cell has antigen peptide on the surface (MHCII), it can then present it to a T cell. Co-stimulation takes place through CD40. TH-independent:
they go around in the bloodstream checking if by any chance their BCR is complementary to
anything out there.
Slide 3: Activation of naive T-cells in LN
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6. Detail how naïve T cells are activated, and which differentiation this
type of microbe is imposing (which cytokine signature in the activated ThX
cell, CD8+ (if activated) and in Tfh, and how)
(How: answered above). Main cytokine output: IL-12p70, IFN-g. Adaptive response type: Th1,
CTL (CD8+).
Slide 4: Activation of naive B-cells in LN
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7. Detail the B cell activation including the processes of SHM (somatic
hypermutation) and isotype switching (which isotypes and why?) Also
include the role of follicular DCs.
B cells can be activated in two fashions
1. Thymus independent activation:
Direct activation of B cells by the viral agents without the intervention of T cells, provides a
rapid response. However, antibodies produced in this way are only of IgM isotype, have thus
lower affinity, and are less functionally flexible. Signal 1 here is binding of polyvalent
antigen on BCR, signal 2 is the recognition of antigen TLR ligands by Toll-like receptors
(and activates the NFkB pathway) (will not be further discussed)
2. Thymus dependent activation:
B cells that interact with antigen-presenting T cells (will be discussed here) to become
activated belong in this category.
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7.1 B cell activation
Activation of the B cells takes part in secondary lymphoid tissues such as the lymph nodes,
spleen, and liver.
There, naive B cells encounter antigen displayed by FDCs or macrophages for the first time. The
events that follow are:
BCR on B cell recognizes and binds antigen (signal 1)
signaling by the BCR is enhanced by the co-receptors CD21 and CD19, which interact with
C3b on opsonized virus → the signal is transduced in the cell and the cascade that follows
activates the NFAT transcription factor (signal 2)
the antigen bound on BCR is engulfed by the cell and presented on MHC2
Activated B cells then move to take positions along the interface between B cell follicles and the
T cell zone.
There, CD4+ T follicular helper cells (Tfh) that have seen antigen (priming) of the same
specificity will recognize the antigen:MHC2 complex on B cells. This interaction is mediated via
the CD40L on TCR binding to CD40 on B cell (linked recognition) and further stabilized by
SLAM adhesion molecules. In this way, the non-canonical NFkB signaling pathway is activated,
which uses the NFkB-inducing kinase (NIK), and leads to the expression of vital signals such as
Bcl-2 for the B cell-by the B cell (signal 3). Moreover, Tfh’s produce various cytokines, one of
them being IL-21. IL-21 activates the STAT3 activator of transcription and enhances
proliferation and differentiation into plasma and memory cells.
After B cells have received T cell help they move. These movements are orchestrated by changes
in expression of CCR7 - to leave the B, T cell zone boundary- and EBI2 - to migrate back into
the interfollicular regions and subcapsular sinus in the lymph nodes, or to the splenic bridging
channels in the spleen.
At this point, some B cells will go to medullary cords of lymph nodes or in the splenic red pulp
in the spleen to form the primary focus (an area of differentiating B cells). The proliferation in
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the primary focus takes place for several days and produces short-lived plasmablasts which
secrete low affinity IgM antibodies.
B cells that don't go to the primary focus will move into the lymphoid follicle together with their
associated Tfh. Proliferation is continued in situ and the germinal center is formed. In contrast
to plasmablasts of the primary foci, B cells in germinal centers undergo somatic hypermutation
(V regions alter) and affinity maturation. This process produces higher affinity antibodies.
Moreover, class switching takes place, with the help of IFN-gamma cytokine produced by the
Tfh. This regulates the type of antibody produced, giving the distinct effectors functions to the
antibodies.
7.2 Somatic Hypermutation
Somatic Hypermutation takes place in the dark zone of germinal centers during B cell
proliferation. It is the process by which high-rate mutations of one to few residues happen in the
already-rearranged V region genes. It is mediated by the enzyme activation-induced cytidine
deaminase (AID) and results in the production of B cell clones with fine-drawn changes in
antigen specificity.
Many of these mutations will produce non-functional immunoglobulin and the B cell will be
forced to undergo programmed cell death. Nearby macrophages (tingible body macrophages)
will clear the cellular waste released in the cytoplasm. Cells that produce higher affinity
receptors will exit the dark zone, capture antigen from FDC, process it for presentation on
MHC2. These cells will receive survival signals from Tfh (through CD40L and IL21 as
described above) and thus are positively selected (affinity maturation). Most changes
accumulate in the CDRs of the V region genes.
During EBV immune responses following ab are produced (in order of secretion)
1. viral capsid antigen- IgM
2. viral capsid antigen- IgG
3. Epstein Barr nuclear antigen - IgG (produced once the infection is resolved)
4. little is known about IgA production during EBV infection
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Figure 2. Responde to EBV (antibody production levels) during the course of the infection.
The mechanism by which the production of different isotypes is orchestrated, will be further
discussed in Section 7.3 .
7.3 Class Switching
The first BCR expressed are IgM and IgD, and the first antibody to be secreted is IgM. After
activation of B cells by antigen, irreversible DNA recombination will lead to the expression of
IgG by a process initiated by the AID enzyme. During this process switch regions (repetitive
DNA regions) that are placed between the J-heavy chain gene and upstream of each heavy-chain
isotype (M, G, A, E), except Cdelta, are joined together by a machinery known as double strand
break repair (DSBR). The intervening DNA between rearranged VDJ and the C isotype chosen
(G, A, E) is deleted and thus this process is non-reversible.
Selection of the particular C region during class switching is mediated by the production of
cytokines from Tfh cells and other cells during the immune response. The production of IFN-
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gamma by the Tfh in response to the EB virus will induce class switch to IgG1, IgG2, IgG3, and
IgA. This is achieved because IFN gamma induces the transcription from promoters that lie
upstream of these particular switch regions. For the production of IgG2, in example, promoters
on C gamma2 start transcription.
Activated B cells will give rise to plasma cells. Long lived plasma cells migrate to the bone
marrow where they produce antibodies continuously.
7.4 The role of FDCs
FDCs in the follicles trap antigens, coming into the lymph tissue, on their surface via CR1 and
CR2 receptor mediated recognition of opsonized antigen and produce abundant BAFF ( B cell
activating factor belonging to TNF family) a signal necessary for B-cell survival. FDCs are
specialized to capture antigens and exhibit them on their surface for B cell receptor mediated
recognition. B cells express 3 receptors for BAFF : BAFF-R ,BCMA, TAC1. BAFF promotes
survival mainly through BAFF-R receptor on B cells, which signals through TRAF3 and
activates the non-canonical pathway (like CD40 does) leading to Bcl-2 expression. TAC1 and
BCMA bind to the APRIL cytokine and lead to B cell activation
Slide 5: Effector functions in infected tissue
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8. Describe how effector T cells and B cells/antibodies will enter the site of
infection.
EBV is commonly transmitted by infected saliva and primary infection involves epithelial cells
of the oropharynx and parotid gland, and infection of resting B cells under the epithelium
(which is not part of the curriculum and thus will be not discussed further here).
B cells leave the secondary lymphatic tissue via the blood and circulate in the body, reaching
infected tissues. IgG2 and cytotoxic T cells are the main responses that act during viral
inflammation.
IgM abs are transported across the epithelium and mainly activate the complement
system (in pentameric formation). They are found mainly in blood. Rapid IgM secretion is
vital for the clearance of infected blood
IgG2 enter through the blood into tissues and diffuse easily throughout the
extracellular fluid inside the tissue. They have mainly neutralizing functionality however
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can activate complement to some less degree. IgG acts in extracellular spaces in tissue
where it protects cells from viral infection (e.g the lower respiratory tract).
small quantities of IgG1 and IgG3 are expressed, diffuse into extravascular sites in the same
way as IgG2, and acts to neutralize, opsonize, activates complement and attracts NK cells.
IgA expression exists but is not typical for EB virus infection. Nevertheless, IgA antibodies
neutralize viruses at the mucosal surfaces of the body, in a similar manner to IgG in tissues.
IgA secreting plasma cells are found below the surface epithelia, from there IgA
antibodies are transported across the epithelium to its external surface. There they act
to neutralize the virus, thus protecting epithelial cells from viral infection.
Changes in the expression of surface molecules on T cells that have differentiated into
effector cells, redirect them from the lymphoid tissue to the infected tissues. This
happens after:
CCR7 and L-selectin stop being expressed
S1PR1 and a glycosylated PSGL-1 are expressed on their surface
Expression of integrins that guide the effector cells to the infected tissue
other adhesion molecules
Inflammatory cytokines TNFa, IL-1b and IL-6, acting on the blood vessels attract
effector T cells to the inflammation site
Cytotoxic CD8+ T cells exit the secondary lymphoid tissues via efferent lymphatics and
enter the bloodstream. From there they are targeted toward infected tissues. This
movement is guided by differential expression of surface molecules on the CTLs.
9. Which effector cell types/soluble components will clear off the microbe?
The expression of interferon-α and -γ by monocytes in the infected tissues induces the Th1
response and inhibits the outgrowth of EBV-infected cells [2].
Also, IFN-gamma secretion, by NK cells, in response to the viral infection induces
macrophages to kill the intracellular virus, before Th1 cells are present at the infection site.
Those macrophages will present peptides on MHCII molecules which will be recognized by the
Th1 cells. Once Th1 cells are stimulated they express membrane protein CD40L and secrete
cytokine IFNgamma and lymphotoxin that enhance the macrophage’s antimicrobial features
(called the classical macrophage activation, macrophages are called M1 macrophage). The
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M1 macrophage is an antimicrobial effector cell and secrete TNF-a which positively reinforces
their function.
Th1 response also helps to activate CD8 cytotoxic cells (through the activation of M1
macrophages which produce IL-12 and consequently more IFN-gamma).
Clearance of virus correlates with the presence of CD8+ T cells (CTL) with virus-specific
cytolytic activity; they are attracted by the MHCI: antigen complex on the surface of the infected
cells, which interacts with the TCR on the CTL. In response the complex produces cytotoxic
elements- releases Perforin and Granzymes- that leads to lysis of the infected cell or apoptosis.
Macrophages then engulf the dead epithelial cell and release it as waste. CTLs also produce
IFN- gamma to further activate M1 macrophages.
As described in the previous section, secreted antibodies that have entered the infected tissue will
activate the classical complement system (mainly IgG1, IgG3 and to a lesser extent IgG2) and
attract NK cells to the infection site. How:
IgM and IgG antibodies specific to viral particles will bind to the virus present in the
extracellular fluid. This leads to a slight change in the conformation of the ab. C1
complex can now recognize and bind to the Fc region of the antibodies. This initiates the
classical complement cascade which will finally lead to the recruitment of the
membrane attack complex (MAC). MAC forms a pore on the target cell's membrane,
resulting in the lysis of the cell.
Another important mechanism for the clearance of the virus is the activation of
accessory effector cells expressing Fc receptors: an IgG-coated virus that enters a
host cell will be sensed by the Fc receptor TRIM21, which binds to the antibody and
ubiquitinates the viral proteins. This leads to the destruction of the virus before it can
encapsulate and spread from the host cell.
Moreover, although natural killer cells (NK cells) are part of the innate response, they
can also recognize antibody-coated infected host cells. This is called the antibodydependent cell-mediated cytotoxicity (ADCC) and is triggered by binding of Fc
receptor FCgRIII on NK cells to IgG1and IgG3 antibody coated cells infected with
the virus. The infected cell is subsequently destroyed by the release of Granzymes and
Prerforin, just as the CTL mediated lysis happens.
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10. Finalize by describing how protective immunity (memory) against the
microbe is established?
The primary infection is followed by the creation of immunological memory in both B and T
cells.
Both memory B and T cells are distinguished from non-memory cells due to the changed
expression of surface molecules.
Memory B cells can arise from both germinal-center-resident B cells, that have undergone
somatic hypermutation and class switching, and short lived-plasma cells produced during the
primary response. Memory B cells express higher MHCII and B7.1 ligand which gives them the
ability to interact faster with Tfh cells. Plasma cells are triggered to produce IgG way quicker
during re-exposure to the virus. Also, memory B cells have the capacity to undergo somatic
hypermutation and affinity maturation again in the germinal centers, thus producing higher
affinity antibodies in the secondary responses.
After the primary infection, the population of effector T cells drops gradually and enters the
memory phase where lower but stable numbers of memory T cells are present. At this point, the
population of memory T cells is higher than the naive. Memory T cells, display different patterns
of surface markers compared to naive and effector T cells (eg. IL7-Ra) and express more Bcl-2.
Moreover, cytokines IL-7, IL-5 and IL-15 are important for the survival of these cells. For the
reactivation of memory cells, interaction with MHC:antigen is needed but they react faster than
naive cells, as well as secrete IFN-gamma faster. Memory T cells can be distinguished into three
populations: central memory cells (which can circulate in secondary lymphoid tissues), effector
memory cells (which access inflammation sites rapidly) and tissue-resident memory cells (that
anchor at multiple different epithelial sites).
Cell-mediated immunity by populations including tissue-resident memory CD8 + and infiltrating
or resident memory CD4+ T cells is an important factor in local viral control [6].
However, neither the successful termination of virus replication during primary infection nor the
establishment of immunological memory prevents the reactivation of the latent virus and the
development of recurrent lesions [6].
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References
The main resource for this hand-in has been the curicculum book Murphy, Kenneth, Paul
Travers, Mark Walport, and Charles Janeway. Janeway's Immunobiology. New York: Garland
Science, 2008. Print.
[1]
“About Epstein-Barr Virus (EBV).” CDC, accessed on 01 March 2022,
www.cdc.gov/epstein-barr/about-ebv.html.
[2] Cohen, Jeffrey I. "Epstein–Barr virus infection." New England journal of medicine 343.7
(2000): 481-492.
[3] Young, Lawrence S., Lee Fah Yap, and Paul G. Murray. "Epstein–Barr virus: more than 50
years old and still providing surprises." Nature Reviews Cancer 16.12 (2016): 789-802.
[4] Brian, Doctrow. “Study suggests Epstein-Barr virus may cause multiple sclerosis.” NIH,
accessed on 01 Mar.2022,
www.nih.gov/news-events/nih-research-matters/study- suggests-epstein-barr-virus-maycause-multiple-sclerosis.
[5] Westergaard, Marie Wulff, et al. "Isotypes of Epstein-Barr virus antibodies in rheumatoid
arthritis: association with rheumatoid factors and citrulline-dependent antibodies."
BioMed research international 2015 (2015).
[6] Padgett, D. A., et al. “Herpesviruses.” Encyclopedia of Stress, Elsevier, 2007, pp. 305–311.
Crossref, doi:10.1016/b978-012373947-6.00193-8.
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