Education: About EBV Infection

A post for people who need to know more about EBV.




EBV is a member of the herpes family of viruses that are common in humans. 

The silent infection and the life-long persistence are the keys to the widespread infection of EBV in the human population. 

In most individuals, EBV infection occurs early in childhood and goes unnoticed.

In contrast, delayed primary infection during adolescence can lead to infectious mononucleosis (IM) = glandular fever. 

EBV primarily targets and remains latent in memory B cells. 

Depending on the type of latency, EBV expresses different sets of latent products. 

In the latency typically seen in IM, up to 12 viral products, including 6 EBV-encoded nuclear antigens (EBNA-LP, EBNA1, EBNA2, EBNA3A, EBNA3B, and EBNA3C), 3 latent membrane proteins (LMP1, LMP2A, and LMP2B) and large quantities of 2 non-coding RNAs (EBERs) are expressed 

A small percentage of latently infected cells intermittently enter the lytic cycle resulting in the release of viral particles

This transition from latency to lytic cycle is triggered by the expression of two immediate early viral proteins, BZLF1 and BRLF1

Almost all MS patients are infected with EBV compared to ~95% non-MS controls. 

Thus, EBV seronegative individuals have almost zero risk of developing MS. 

However, a dramatic upsurge in MS risk is seen when these individuals seroconvert following EBV infection. 

Furthermore, individuals who have a history of EBV-associated IM have also been shown to be at increased risk of developing MS.

In the paper below

Overall, 91/101 (90%) of MS cases were EBV positive by l compared to only 5/21 (24%) non-MS cases 

That damned, elusive EBV

Will this study get the field behind the EBV hypothesis once and for all? Or will it be another bun fight?




PLoS One. 2018 Feb 2;13(2):e0192109. doi: 10.1371/journal.pone.0192109. eCollection 2018.

Epstein-Barr virus is present in the brain of most cases of multiple sclerosis and may engage more than just B cells.

Hassani A, Corboy JR, Al-Salam S, Khan G.

Abstract


Multiple sclerosis (MS) is a chronic neuroinflammatory condition of the central nervous system (CNS). It is a major cause of neurological disability in young adults, particularly women. What triggers the destruction of myelin sheaths covering nerve fibres is unknown. Both genetic and infectious agents have been implicated. Of the infectious agents, Epstein-Barr virus (EBV), a common herpesvirus, has the strongest epidemiological and serological evidence. However, the presence of EBV in the CNS and demonstration of the underlying mechanism(s) linking EBV to the pathogenesis of MS remain to be elucidated. We aimed at understanding the contribution of EBV infection in the pathology of MS. We examined 1055 specimens (440 DNA samples and 615 brain tissues) from 101 MS and 21 non-MS cases for the presence of EBV using PCR and EBER-in situ hybridization (EBER-ISH). EBV was detected by PCR and/or EBER-ISH in 91/101 (90%) of MS cases compared to only 5/21 (24%) of non-MS cases with other neuropathologies. None of the samples were PCR positive for other common herpesviruses (HSV-1, CMV, HHV-6). By quantitative PCR, EBV viral load in MS brain was mainly low to moderate in most cases. However, in 18/101 (18%) of MS cases, widespread but scattered presence of EBV infected cells was noted in the affected tissues by EBER-ISH. Immunohistochemical analysis of EBV gene expression in the 18 heavily infected cases, revealed that the EBV latent protein EBNA1, and to a lesser extent the early lytic protein BZLF1 were expressed. Furthermore, using double-staining we show for the first time that astrocytes and microglia, in addition to B-cells can also be infected. To the best of our knowledge, this is the most comprehensive study demonstrating that EBV is present and transcriptionally active in the brain of most cases of MS and supports a role for the virus in MS pathogenesis. Further studies are required to address the mechanism of EBV involvement in MS pathology.

"We seek him here, we seek him there...Is he in heaven - Is he in hell?"

-The Scarlett Pimpernel by E Orczy

No really! The cause of MS is as elusive as the Pimpernel was to the French. Despite mounting evidence, no research group has yet successfully managed to nail EBV to MS. This work by Hassani and colleagues is hope that it may yet happen!

Epstein-Barr virus (EBV) is a member of the Herpes family of viruses and is common in humans. It's target is B cells and can remain dormant or latent in memory B cells for their lifespan. A small percentage, however, do reactivate in what is called the lytic phase.

EBV is associated with more than one disorder, but is thought in MS at least it causes a virus-host immune system response imbalance, with the immune response to the virus to the virus itself being disrupted (Sundström P, Juto P, Wadell G, Hallmans G, Svenningsson A, Nyström L, et al. An altered immune response to Epstein-Barr virus in multiple sclerosis: a prospective study. Neurology. 2004;62: 2277–2282). Almost all PwMS demonstrate evidence of previous EBV infection, and those who have not been exposed (i.e. seronegative) have almost zero risk of developing MS. The spanner in the works is that ~95% of those without MS have also had previous EBV exposure (Ascherio A, Munch M. Epstein-Barr virus and multiple sclerosis. Epidemiology. 2000;11: 220–224).

The question then becomes can you place EBV successfully in the brain where it can cause MS? And that is what the authors have done. Hassani et al, examined the presence of EBV in 122 post-mortem MS brains and non-MS brains. Others have tried to do this in the past but not successfully for any number of reasons - techniques, sampling etc. They used a PCR technique and a highly sensitive and specific EBER- in situ hydridization (EBER-ISH) technique to localise EBV infected cells in brain tissues.

Surprisingly, they found that EBV was present in 90% of MS cases, but also found no presence of other viruses, such as HSV-1, CMV and HHV-6 that have been previously implicated in MS. Of note, they also found that not only did EBV target B cells, but also infected ~10-15% activated microglia and astrocytes (the innate immune system, see Figure). Therefore, the role of EBV in the inflammatory cascade needs to be looked at in greater detail.

Overall, a very interesting paper and others will now have to re-examine EBV in MS brains using the more sensitive techniques in order to identify scattered and low level EBV infection.

Figure: Double staining for EBV and cellular markers.
Double staining for EBV (EBER-in situ hybridization: dark blue staining) and different cellular markers (immunohistochemistry: brown) in the white matter of 3 different heavily infected MS cases. The pattern of double-staining seen in these 3 cases is representative of that seen in other double-positive cases. (A) EBV and CD20 (B-cell marker), (B) EBV and GFAP (astrocyte marker), (C) EBV and Iba1 (microglia marker). Double positive cells are indicated by the arrows.

Is the problem of MS due to EBV in the B cells?

What genes are active in MS? 

In this study they looked at lesions and surrounding white matter and found a load of CD8 T cells, a lot of CD20 B cells and a few CD4 T cells and plasma cells (antibody making cells).

The B cells contained EBV 


In this study they take brain tissue and then cut it into very thin slices (eg. a 100th of a mm) and then you look to see if you can find a lesion. You use a laser to cut out the cells from the section (laser capture) and then you remove the nucleus to see what proteins the cells were making. 

There were CD4 T cells in all the lesions, CD8 T cells in all of the lesions and CD19 B cells in all the lesions. Not many CD4 T cells and only 1-3% lesions contained IL-17/IL-22 (Th17 cytokines). If if there is more IL-17 in the blood (see beleow) how come there 

So are these few cells causing autoimmunity as the EAEers would have us believe There were 70% of lesions with gamma interferon, which could be Th1 derived or could come from CD8.

There was about 70-95% of lesions with clear B cell growth and differentiation factors like IL-6, IL-10 (yes it is a B cell factor and IL-10 does not have to be a regulatory cytokine), BAFF, lymphotoxin etc etc.

There are CXCR3 and CXCL12 chemokines in most lesions ,which recruit B cells 

However the paper is open access and they do vastly different association for these products 

One or more EBV genes were detected in immune infiltrates from 9 of 11 MS cases and in 41.3% of the samples. 

EBV genes were detected more frequently in meningeal than in WM perivascular infiltrates (55.6 vs 28% of the samples) and genes expressed during viral latency were detected more frequently (i.e. present in memory cells) than genes associated with the viral lytic which is where the virus kills the cell and releases the cells contents which is live virus). 

LMP1 and LMP2A are viral genes, deliver surrogate B cell survival and differentiation signals (CD40 and B cell receptor, LMP1 was detected more commonly than LMP2 suggest that the B cells may be more T cell-independent .

The authors suggests that "EBV could be the main antigenic trigger of an immunopathological, CD8+ T cell-mediated response that damages the brain/spinal cord in MS. This model is consistent with the notion that CD8+ T cells are the main drivers of bystander tissue damage in EBV-associated immunopathologic diseases"

So are CD8 cells in the brain to destroy EBV infected B cells? 

The authors suggest that "the use of drugs that, by directly targeting the virus and its cellular reservoir, could be more effective in normalizing an altered EBV-host interaction in MS. For example, B cell-depleting therapies could lower EBV load and hence the burden of EBV-induced immunopathology in MS more efficiently than other drugs". 

Is this what we are doing?

Veroni C, Serafini B, Rosicarelli B, Fagnani C, Aloisi F. Transcriptional profile and Epstein-Barr virus infection status of laser-cut immune infiltrates from the brain of patients with progressive multiple sclerosis. J Neuroinflammation. 2018;15:18.

BACKGROUND:It is debated whether multiple sclerosis (MS) might result from an immunopathological response toward an active Epstein-Barr virus (EBV) infection brought into the central nervous system (CNS) by immigrating B cells. Based on this model, a relationship should exist between the local immune milieu and EBV infection status in the MS brain. To test this hypothesis, we analyzed expression of viral and cellular genes in brain-infiltrating immune cells.

METHODS:Twenty-three postmortem snap-frozen brain tissue blocks from 11 patients with progressive MS were selected based on good RNA quality and prominent immune cell infiltration. White matter perivascular and intrameningeal immune infiltrates, including B cell follicle-like structures, were isolated from brain sections using laser capture microdissection. Enhanced PCR-based methods were used to investigate expression of 75 immune-related genes and 6 EBV genes associated with latent and lytic infection. Data were analyzed using univariate and multivariate statistical methods.

RESULTS: Genes related to T cell activation, cytotoxic cell-mediated (or type 1) immunity, B cell growth and differentiation, pathogen recognition, myeloid cell function, type I interferon pathway activation, and leukocyte recruitment were found expressed at different levels in most or all MS brain immune infiltrates. EBV genes were detected in brain samples from 9 of 11 MS patients with expression patterns suggestive of in situ activation of latent infection and, less frequently, entry into the lytic cycle. Comparison of data obtained in meningeal and white matter infiltrates revealed higher expression of genes related to interferonγ production, B cell differentiation, cell proliferation, lipid antigen presentation, and T cell and myeloid cell recruitment, as well as more widespread EBV infection in the meningeal samples. Multivariate analysis grouped genes expressed in meningeal and white matter immune infiltrates into artificial factors that were characterized primarily by genes involved in type 1 immunity effector mechanisms and type I interferon pathway activation.

CONCLUSION: These results confirm profound in situ EBV deregulation and suggest orchestration of local antiviral function in the MS brain, lending support to a model of MS pathogenesis that involves EBV as possible antigenic stimulus of the persistent immune response in the central nervous system.

However, without wanting to upset the authors too much, they are the group that have proposed that B cell follicles ares associating with, and implicated in, the cause of progression. However as in their own words

"several groups have reported absence or paucity of EBV in postmortem MS brain samples"....whilst....

"we have repeatedly shown not only presence of EBVinfected B-lineage cells but also EBV latency disruption and reactivation in the MS brain"

"RNA/protein nor deregulated EBV infection was detected in brain tissues from patients with other infectious and non-infectious neuroinflammatory diseases"..."ruling out the possibility that an active EBV infection in the CNS is the general consequence of immune cell invasion and local activation."

Therefore not all people agree on this and so requires replication by other groups who do not have a vested interest in this story.

What do others find?

Trenova AG, Slavov GS, Draganova-Filipova MN, Mateva NG, Manova MG, Miteva LD, Stanilova SA. Circulating levels of interleukin-17A, tumor necrosis factor-alpha, interleukin-18, interleukin-10, and cognitive performance of patients with relapsing-remitting multiple sclerosis. Neurol Res. 2018 Jan 3:1-7. doi: 10.1080/01616412.2017.1420522. [Epub ahead of print]

Multiple sclerosis (MS) is associated with cytokine imbalance and high rate (40-70%) of cognitive impairment. The objective of this study is to investigate the relationship between serum concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-17A, IL-18, IL-10, and cognitive performance in patients with relapsing-remitting MS (RRMS). 
Methods The study comprised 159 patients with RRMS (mean age 40.08 ± 8.48 years) in remission phase and 86 age-, gender-, and education-matched healthy controls. Paced Auditory Serial Addition Test (PASAT), Symbol Digit Modalities test (SDMT), and Isaacs test were used for assessment of working memory, attention, visuo-perceptual abilities, information processing speed, and executive functions. Serum cytokine concentrations were measured by enzyme-linked immunosorbent assay (ELISA). 

Results Patients had significantly increased serum concentrations of TNF-alpha and IL-17A and decreased levels of IL-10 compared to the controls (p < 0.05). Negative correlation was found between serum TNF-alpha and SDMT score in patients with disease evolution longer than 10 years (rxy = -0.258 p = 0.033); PASAT and SDMT scores were in negative correlation with concentration of IL-17A (rxy = -0.229 p = 0.004; rxy = -0.166 p = 0.041). Cognitive impairment was established in 46.5% (n = 74) of the patients. Cognitively impaired patients had significantly higher serum IL-17A than cognitively preserved individuals (p = 0.007). Multiple linear regression analysis revealed IL-17A as a significant predictor of cognitive performance in RRMS patients. 

Conclusion The results from this study suggest that pro-inflammatory cytokines IL-17A and TNF-alpha simultaneously with decreased IL-10 are involved in cognitive deterioration in RRMS.

When I was rreally interested in cytokines, we looked for cytokines like TNF in the blood and didn't find any. Now it could be sensitivity of the assay.Alternatively it mean what was produced was used, so that if excess is found, it suggests more is produced than needed. 

However it had been reported that TNF levels in blood in MS, correlated with disease activity and the the results appeared to good. Indeed when the person writing the paper was asked to repeat the analysis with coded and blinded samples, so they wouldn't know what was what...They ran a mile never to be heard off again. 

So you can guess what I thought of the data. This report may suggest the neuro was correct. They find what you expect if you follow dogma...an increase in pro-inflammatory cytokines and a decrease in regulatory cytokines. 

Who Needs T cells..Not EBV infected Memory B cells

To finish off the debate...a prominent T cell immunologist in the red corner said B cells need T cells.....However, do some reading and listening and as said with EBV in the equation...maybe not.

Did people listen to the Evidence with an open mind and no preconceptions?
Epstein–Barr virus latent membrane protein 1 (LMP1) is an Epstein–Barr virus (EBV) protein. 

Rastelli J, Hömig-Hölzel C, Seagal J, Müller W, Hermann AC, Rajewsky K, Zimber-Strobl U.LMP1 signaling can replace CD40 signaling in B cells in vivo and has unique features of inducing class-switch recombination to IgG1. Blood. 2008;111:1448-55.

The Epstein-Barr virus (EBV) protein LMP1 is considered to be a functional homologue of the CD40 receptor. However, in contrast to the latter, LMP1 is a constitutively active signaling molecule. To compare B cell-specific LMP1 and CD40 signaling in an unambiguous manner, we generated transgenic mice conditionally expressing a CD40/LMP1 fusion protein, which retained the LMP1 cytoplasmic tail but has lost the constitutive activity of LMP1 and needs to be activated by the CD40 ligand. We show that LMP1 signalling can completely substitute CD40 signaling in B cells, leading to normal B-cell development, activation, and immune responses including class-switch recombination, germinal center formation, and somatic hyper-mutation. In addition, the LMP1-signaling domain has a unique property in that it can induce class-switch recombination to IgG1 independent of cytokines. Thus, our data indicate that LMP1 has evolved to imitate T-helper cell function allowing activation, proliferation, and differentiation of EBV-infected B cells independent of T cells.

The season of MS: Glandular fever may not be linked to time of year

Vitamin D (as measured by time of year) and glandular fever are not linked. Should we be surprised?

I guess it boils down to how you think it is all working. 

If the month of birth theory holds any water then the major risk period is when you're in the womb or shortly after birth. That the vitamin D hypothesis is also evident in type I diabetes suggests the risk factor is early in life. The biology suggests that vitamin D can shape your immune repertoire (range of different things your immune system responses to). So your die is cast as to whether you are at risk of autoimmunity. But this does not give you autoimmunity. Your other genes also put you at risk.

Whether vitamin D exhibits a major influence once the condition appears, remains to be seen. The trials are being done. 

Migration studies from low to high incidence countries suggest you acquire the trigger before you are 15 years for risk of MS. One suggestion is that this trigger is Epstein Barr Virus. This shapes your B cell (antibody making cells) repertoire. Which in turn may determine whether you get MS or other autoimmune conditions associated with EBV. 

About half the western population become infected with EBV in infancy. Are these the diabetes-prone people?  I guess infection goes unnoticed (undiagnosed) as one of the many episodes of fever that a child has. 

In adolescent life the naive/mature B cell gets infected by EBV because the virus enters the B cell via CD21 and an action via HLA-DR. Is this where the number one autoimmune genetic MS risk factor occurs? Is HLA-DRB1*1501 a gene that is good at getting B cells infected by EBV?

The mature B cell is then triggered to proliferate by the virus, and their killing by anti-EBV CD8, cytotoxic T cells cause a cytokine storm and sickness behaviour...that is glandular fever. 

The virus causes the B cells to mature into memory B cells and not antibody-producing plasma cells by a protein called EBNA3. 

The virus then hides in the memory B cells out of sight of the immune system, but switching off virus production. And as memory B cells are like buses that go round the body, the virus hitches a ride all round the body. 

However, the virus does other things, including making the memory B cell independent of the requirement of T cell help. So they do not need to get stimulated by a molecule called CD40. This means, for example, if a memory B cell enters the brain and sees its target it will become activated and so does not need a T cell of the same specificity to be present.

As more damage occurs, more antigens capable of stimulating B cells are liberated. You don't have to postulate that it is one antigen causing the problem and these can change over time. However there is specificity because the B cell would have to see a brain derived signal to trigger release of cytokines to start the lesion.

This would be a great immune-evolution because it makes us more able to fight infection quickly, creating a survival advantage for the human population.

On the down-side, autoimmunity could occur. However, EBV has been with us for thousands of years (co-evolving with us). As humans used to have children earlier in life - and died earlier than we do now - autoimmunity would arguably not be selected against. Why? Because most autoimmunity occurs later in life (old-age and after child birth for our ancestors). 

So, evolution-wise, this would not be selected against. Furthermore, even if it did occur in our ancestors, it is at such a low frequency that it does not affect the overall survival of the population.  

What do you think of the idea?

Please post refuting (& supporting) work

Downham C, Visser E, Vickers M, Counsell C. Season of infectious mononucleosis as a risk factor for multiple sclerosis: A UK primary care case-control study. Mult Scler Relat Disord. 2017 Oct;17:103-106.

BACKGROUND:Infectious mononucleosis (IM) and vitamin D deficiency are both risk factors for multiple sclerosis (MS).
OBJECTIVE:We wished to establish if IM in the winter months when vitamin D levels are low may be a greater risk factor for MS than IM in the summer months.
METHODS:We identified all patients with MS diagnosed aged 16-60 in a large primary care database in the United Kingdom and matched each by age, sex, general practice and observation period with up to six controls. We identified a coded diagnosis of IM prior to the index date (date of diagnosis). Logistic regression was used to calculate the odds ratio for prior IM exposure in cases versus controls and for winter versus summer exposure in cases and controls with prior IM exposure.
RESULTS:Based on 9247 cases and 55,033 matched controls (246 and 846 with prior IM respectively), IM was associated with the development of MS (OR 1.77, 95%CI 1.53-2.05) but there was no evidence that IM in the winter as opposed to summer was associated with developing MS (OR 1.09, 95%CI 0.72-1.66).
CONCLUSION: We found no evidence that the season of IM influences the risk of subsequent MS.

Programmed Death 1 disappears and leaves EBV to trigger Activity

Now that we know what PD-1 is, it's time to look at this paper

Cencioni MT, Magliozzi R, Nicholas R, Ali R, Malik O, Reynolds R, Borsellino G, Battistini L, Muraro PA. Programmed death 1 (PD1) is highly expressed on CD8+ CD57+ T cells in patients with stable multiple sclerosis and inhibits their cytotoxic response to Epstein-Barr virus. Immunology. 2017. doi: 10.1111/imm.12808. [Epub ahead of print]

We know that virtually everybody with MS is infected with Epstein Barr Virus. Most people are as it is very common in humans.

We get infected through saliva. It then infects B cells and makes them proliferate like crazy and turn into memory B cells. The virus then shuts down and hides in the B cell, where it is not going to do much damage. But every now and then the virus activates, and sheds live virus. The immune response recognises the virus and kills the infected B cells and things all quieten down.

In Australia they are making T cells that will kill EBV, based on a very small, unreproduced study stating that it may influence the activity of MS.

MS and the CD8 that attack anti-EBV viral T cells 

A subset of CD8 cells express CD57  - which is
an enzyme - (Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1-B3GAT1) that recognises sugar molecules and is involved in the destruction of chemicals, and I guess viruses.

In healthy individuals they killed EBV infected cells but in stable MS these cells also expressed PD-1, suggesting that they had become immunologically exhausted so they were not killers, but in active MS the CD8 cells had PD-1 and were killing. 

This suggested that when MS is stable they do not control the EBV infection, maybe because it is at a silent stage, but this may enable the re-activation of virus, which could trigger disease. 

Is the disease caused by the attack of virus?. 

The question is, is it a cause or really a consequence?

ABSTRACT

Growing evidence points to a deregulated response to Epstein-Barr virus (EBV) in the central nervous system of patients with multiple sclerosis (MS) as a possible cause of disease. We have investigated the response of a subpopulation of effector CD8+ T cells to EBV in 36 healthy donors and in 35 patients with MS in active and inactive disease. We have measured the expression of markers of degranulation, the release of cytokines, cytotoxicity and the regulation of effector functions by inhibitory receptors, such as programmed death 1 (PD-1) and human inhibitor receptor immunoglobulin-like transcript 2 (ILT2). We demonstrate that polyfunctional cytotoxic CD8+ CD57+ T cells are able to kill EBV-infected cells in healthy donors. In contrast, an anergic exhaustion-like phenotype of CD8+ CD57+ T cells with high expression of PD-1 was observed in inactive patients with MS compared with active patients with MS or healthy donors. Detection of CD8+ CD57+ T cells in meningeal inflammatory infiltrates from post-mortem MS tissue confirmed the association of this cell phenotype with the disease pathological process. The overall results suggest that ineffective immune control of EBV in patietns with MS during remission may be one factor preceding and enabling the reactivation of the virus in the central nervous system and may cause exacerbation of the disease.