Serological profiling of EBV immune response in Chronic Fatigue Syndrome using peptide microarray

[Murph]

Serological profiling of the EBV immune response in Chronic Fatigue Syndrome using a peptide microarray

• Madlen Loebel,
• Maren Eckey,
• Franziska Sotzny,
• Elisabeth Hahn,
• Sandra Bauer,
• Patricia Grabowski,
• Johannes Zerweck,
• Pavlo Holenya,
• Leif G. Hanitsch,
• Kirsten Wittke,
• Peter Borchmann,
• Jens-Ulrich Rüffer,
• Falk Hiepe,
• Klemens Ruprecht,
• Uta Behrends,
• Carola Meindl,
• Hans-Dieter Volk,
• Ulf Reimer,
• Carmen Scheibenbogen

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179124

Abstract
Background
Epstein-Barr-Virus (EBV) plays an important role as trigger or cofactor for various autoimmune diseases. In a subset of patients with Chronic Fatigue Syndrome (CFS) disease starts with infectious mononucleosis as late primary EBV-infection, whereby altered levels of EBV-specific antibodies can be observed in another subset of patients.

Methods
We performed a comprehensive mapping of the IgG response against EBV comparing 50 healthy controls with 92 CFS patients using a microarray platform. Patients with multiple sclerosis (MS), systemic lupus erythematosus (SLE) and cancer-related fatigue served as controls. 3054 overlapping peptides were synthesised as 15-mers from 14 different EBV proteins. Array data was validated by ELISA for selected peptides. Prevalence of EBV serotypes was determined by qPCR from throat washing samples.

Results
EBV type 1 infections were found in patients and controls. EBV seroarray profiles between healthy controls and CFS were less divergent than that observed for MS or SLE. We found significantly enhanced IgG responses to several EBNA-6 peptides containing a repeat sequence in CFS patients compared to controls. EBNA-6 peptide IgG responses correlated well with EBNA-6 protein responses. The EBNA-6 repeat region showed sequence homologies to various human proteins.

Conclusion
Patients with CFS had a quite similar EBV IgG antibody response pattern as healthy controls. Enhanced IgG reactivity against an EBNA-6 repeat sequence and against EBNA-6 protein is found in CFS patients. Homologous sequences of various human proteins with this EBNA-6 repeat sequence might be potential targets for antigenic mimicry.

 

[Murph]

This picture seems to b where the rubber hits the road. The IgG response to one little peptide is different in CFS and controls.

Fig 4. Reactivity against EBNA-6, LPO and TPO in CFS patients.
A) Median intensity of IgG response against EBNA-6 peptide 740 in seroarray cohort (CFS, n = 93 and healthy, n = 50) and a subsequent validation cohort (CFS, n = 227 and healthy, n = 47) was analysed by multiwell assay.
B) Optical density (OD) and Interquartile Range (IQR) of IgG response against EBNA6_740, a negative control peptide, LPO, and TPO in healthy controls (n = 115) and CFS patients (n = 162) by peptide ELISA.
C) Correlation of ELISA OD values of EBNA6_740 and LPO for all patients and controls.
D) IgG responses against EBNA-6 protein in in healthy controls (n = 40) and CFS patients (n = 40) detected by Odyssey Infrared Imaging System as arbitrary fluorescence units (AFU), and correlation of EBNA-6 protein and EBNA-6 peptide as well as LPO peptide IgG in CFS patients. Statistical analysis by two-tailed Mann-Whitney-U test with * p<0.05, ** p<0.01, *** p <0.001, **** p<0.0001, r-spearman coefficient.

 

[Murph]

Here's the discussion section of the paper, wherein they talk about how that one peptide (ebna-6) might be relevant. Lots of hints - links to metabolism etc - but no smoking gun.
--
In this study we performed a comprehensive analysis of IgG responses against a peptide library of the major EBV proteins. In healthy controls we observed the strongest and broadest antibody responses against the latency proteins EBNA-1, -3, -4, and -6, and the lytic proteins BALF2, BALF5, and BZLF1. This finding is in accordance with studies analysing the IgG response against EBV proteins in which strong IgG responses against both EBNA proteins and BZLF1 are found and used for serodiagnosis [67–73].

The strong enhanced IgG response against various EBV proteins in MS and SLE shown in previous studies was confirmed in this and for MS in our prior study [8]. In contrast, although total IgG response against EBV peptides was higher in CFS the pattern of IgG responses was more similar to healthy controls. Further, we had performed a subgroup analysis in patients with Hodgkin lymphoma showing no difference in IgG EBV response pattern in patients with and without fatigue.

Interestingly, we observed a significantly enhanced IgG response against EBNA-6 peptides spanning a repeat region in CFS patients compared to healthy controls. A 21 aa long peptide comprising the repeat region of EBNA-6 was already described by Rajnavölgyi et al. as a HLA-DR restricted T cell epitope [74]. Further we could show a close correlation between IgG responses against EBNA-6 peptide and a recombinant EBNA-6 protein suggesting that this peptide is recognized in the protein and is an immunodominant epitope.

If the enhanced IgG response against EBNA-6 may be of diagnostic relevance in a subgroup of patients needs to be analysed in a longitudinal study. Cross-reactivity of EBV-specific IgG with human antigens due to antigenic mimicry is known to trigger pathogenic immune responses in SLE and MS [75–77]. We thus performed a sequence comparison of the EBNA-6 repeat region with human proteins and identified a 7 amino acids homologous sequence in the LPO protein, an enzyme producing oxidants and secreted by mammary, salivary and mucous glands of the bronchia. LPO is involved in immune defence with broad activity against bacteria and viruses [78–80]. Interestingly, we detected a correlation of EBNA-6 peptide and protein IgG with LPO peptide IgG levels.

However, we observed no elevated levels of LPO protein IgG in patients with CFS suggesting that the peptide is not recognized in the recombinant protein. Interestingly, human TPO shows a sequence homology with 5 (comprising 4 identical aa) of the 7 amino acids of the EBNA-6 repeat region as well. Autoantibodies against TPO and hashimoto's thyroiditis are detected in 10–20% of patients with CFS and lead to thyroid destruction through antibody dependent cellular cytotoxicity and complement activation [81].

We could, however, not observe elevated IgG responses against the TPO peptide in our study. Two other proteins showing homology to the EBNA-6 repeat region, the enzymes OTC and PFK arouse our interest because of their metabolic function (Table 3). Data from Yamano et al. revealed higher ornithine/citrulline ratio in CFS patient which could be explained by reduced OTC activity [66]. The PFK catalyses the rate limiting step of glycolysis and inhibition of the enzyme could play a role in metabolic alterations in CFS [65, 66]. However, as for LPO protein, we observed no reactivity of CFS patients against these two proteins.

 

[Snow Leopard]

As I posted in the other thread that mentioned this study:


Overall: a null result. But it is an interesting, high quality study definitely worth reading.


Overall similar patterns to healthy controls (similar patterns of immune response to EBV and reactivation patterns). Some types of reactivity in patients were notable, with a hypothesis of molecular mimicry, but when immune responses were tested against those endogenous proteins directly, no unusual immune response (that could be explained by molecular mimicry) was found.

 

[Londinium]

But it is an interesting, high quality study definitely worth reading.

Agreed. Possibly a little OT but one of my main bugbears I've had while getting smart on ME/CFS research is the number of papers that find something statistically significant but don't either correct their p-value for multiple comparisons or try to replicate in a validation dataset (the latter I imagine if often tricky due to the low funding levels for ME). That, and excessive post-hoc subgroup analysis which is a rant I'll save for another day

It was good to see here that once the team had found the IgG respone to EBNA-6 that was statistically significant in the original (n=93/hc=50) cohort they then tested again for this in a validation cohort (n=227/hc=47). This is exactly the kind of approach we want to see taken to give robust results that this isn't just a fluke.

The enhanced IgG response against an EBNA-6 repeat sequence may point to a potential antigenic mimicry and requires further studies.

Part of my wife's job is to translate scientific research into more understandable language to be read by the general public, and she jokes she's never seen a scientific paper that didn't end with 'more research is required'. However, in this case, I do think the fact that this immune response was found in both a test and validation cohort indicates the team may, just may, be onto something - even if they couldn't then link this to an autoimmune response against specific human proteins. More research like this please!

 

[Wonkmonk]

I have zero knowledge about immunology, so please excuse me if this makes no sense, but my first thought was: Shouldn't they look into Early-antibodies (EBV-EA diffuse) instead of IgG antibodies according to the theory proposed by Dr. Lerner that early gene products and non-permissive replication is to blame for most symptoms in a subset of EBV patients?

If I remember correctly, Drs. Lerner and Montoya found IgG antibodies often normal, but additionally a presence of EA antibodies in the absence of complete viral DNA (tested with PCR). In a state of latency, no EA antibodies should be detected, in case of reactivation, complete viral DNA would have to be detected.

So according to that theory, if I understood correctly, CFS in that subset of patients is caused by a state of permanent non-permissive replication, which produces early gene products, but not complete virions that could cause mono-like illness.

But then, isn't it unsurprising that Scheibenbogen et al. didn't find abnormal results with respect to IgG?

 

[pattismith]

we can find more studies about association between EBV and RA, maybe it can enlight this question:

Decreased T cell precursor frequencies to Epstein-Barr virus glycoprotein gp110 in peripheral blood correlate with disease activity and severity in patients with rheumatoid arthritis

CONCLUSION These results suggest that patients with RA have a decreased T cell response to EBV gp110. Since gp110 is an important protein in the control of EBV replication, this might lead to a poor control of EBV infection, chronic exposure to other EBV antigens, and thus to a chronic inflammatory response in patients with RA.

Extract:

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease of unknown cause.
Both genetic and environmental factors are thought to have a role in its development. Indeed, RA is strongly associated with certain HLA-DR alleles containing in the third hypervariable region of their β chains the QK/RRAA sequence or shared epitope.1
Among environmental factors, an association between a virus and RA has long been suspected.2
For 15 years Epstein-Barr virus (EBV) has been suspected to be a causative factor for RA and to contribute to its pathogenesis.2-4
Indeed, RA sera contain increased titres of antibodies precipitating a nuclear antigen present in EBV infected cells and patients with RA have a higher percentage of EBV infected peripheral blood B cells than controls.3 4

In addition, it was shown that T cell control of the outgrowth of EBV infected cells was impaired in patients with RA.5
Furthermore, it was recently found that a large fraction of T cells infiltrating the synovium from patients with RA recognised EBV proteins of the replicative cycle6 7 and EBV DNA was detected in the synovial membrane of patients with RA. 8
Moreover, the EBV genome encodes a protein in its BamHI C restriction fragment open-reading frame or viral interleukin 10 that shares protein homology and biological properties with human interleukin 10. 9

An explanation for the link between an infectious agent and autoimmune or inflammatory disease might be the molecular resemblance between a microbial antigen and a host antigen, a mechanism usually called molecular mimicry.
EBV is a good candidate for RA pathogenesis as several EBV proteins share similar sequences with normal human proteins3: the EBV nuclear antigen EBNA-1 shares sequence identity with human type II collagen,3 4 the EBNA-6 protein contains a sequence found in the HLA-DQB1*0302 molecule 3 4 and the EBV glycoprotein 110 (gp110) carries the QK/RRAA sequence or shared epitope.4 10
GP110 is a glycoprotein of the replicative cycle11 and constitutes a B and T cell epitope in normal controls.10 GP110 is the EBV equivalent of gB proteins of other herpes viruses (herpes virus and cytomegalovirus) and these proteins have an important role in the control of the virus infection.12

 

[pattismith]

Two other proteins showing homology to the EBNA-6 repeat region, the enzymes OTC and PFK arouse our interest because of their metabolic function (Table 3). Data from Yamano et al. revealed higher ornithine/citrulline ratio in CFS patient which could be explained by reduced OTC activity [66]. The PFK catalyses the rate limiting step of glycolysis and inhibition of the enzyme could play a role in metabolic alterations in CFS [65, 66]. However, as for LPO protein, we observed no reactivity of CFS patients against these two proteins.

maybe the phosphofructokinase guy will apreciate this finding

@alex3619

 

[pattismith]

I have zero knowledge about immunology, so please excuse me if this makes no sense, but my first thought was: Shouldn't they look into Early-antibodies (EBV-EA diffuse) instead of IgG antibodies according to the theory proposed by Dr. Lerner that early gene products and non-permissive replication is to blame for most symptoms in a subset of EBV patients?

If I remember correctly, Drs. Lerner and Montoya found IgG antibodies often normal, but additionally a presence of EA antibodies in the absence of complete viral DNA (tested with PCR). In a state of latency, no EA antibodies should be detected, in case of reactivation, complete viral DNA would have to be detected.

So according to that theory, if I understood correctly, CFS in that subset of patients is caused by a state of permanent non-permissive replication, which produces early gene products, but not complete virions that could cause mono-like illness.

But then, isn't it unsurprising that Scheibenbogen et al. didn't find abnormal results with respect to IgG?

EBN 1 EBN 2...EBN 6 are latent proteins ( http://virology-online.com/viruses/EBV.htm ). If I understand right, this means that antibodies against EBN 6 will be noticed in latent infections (non lytic), either IgM or IgG...