Epstein–Barr virus (EBV) deletions as biomarkers of response to treatment of chronic active EBV
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Evaluation Summary:
The author present a large amount of sequencing data of EBV present in a variety of individuals, including a few with the interesting and unusual disease manifestation known as Chronic Active EBV (CAEV). The data show that there is a great deal of heterogeneity in the EBV genomes among people and overtime in some individuals, but the data do not reveal any particular mechanistic insights about the importance of this heterogeneity and the sample size is too small to warrant use of these deletions as therapeutic biomarkers.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Chronic active Epstein–Barr virus (CAEBV) disease is a rare condition characterised by persistent EBV infection in previously healthy individuals. Defective EBV genomes were found in East Asian patients with CAEBV. In the present study, we sequenced 14 blood EBV samples from three UK patients with CAEBV, comparing the results with saliva CAEBV samples and other conditions. We observed EBV deletions in blood, some of which may disrupt viral replication, but not saliva in CAEBV. Deletions were lost overtime after successful treatment. These findings are compatible with CAEBV being associated with the evolution and persistence of EBV + haematological clones that are lost on successful treatment.
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Evaluation Summary:
The author present a large amount of sequencing data of EBV present in a variety of individuals, including a few with the interesting and unusual disease manifestation known as Chronic Active EBV (CAEV). The data show that there is a great deal of heterogeneity in the EBV genomes among people and overtime in some individuals, but the data do not reveal any particular mechanistic insights about the importance of this heterogeneity and the sample size is too small to warrant use of these deletions as therapeutic biomarkers.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This manuscript by Venturini & colleagues identifies EBV deletions as a potential biomarker for CAEBV. The authors identify the same large deletion in the blood but not saliva of CAEBV patients. No mechanism linking deletion with clonality is identified so the utility of the study would be purely based on using these deletions as biomarkers. To this end, loss of large deletions is shown overtime in 3 treated patients (2 with PBSCT & one with Rituxan). The sequencing work and statistical comparisons are rigorous.
While these findings are certainly of interest and raise an interesting hypothetical way to track treatment of CAEBV, the generalizability is limited by small sample size and lack of clarity in presentation. The study raises hypotheses for subsequent mechanistic work in animal models and could serve …
Reviewer #1 (Public Review):
This manuscript by Venturini & colleagues identifies EBV deletions as a potential biomarker for CAEBV. The authors identify the same large deletion in the blood but not saliva of CAEBV patients. No mechanism linking deletion with clonality is identified so the utility of the study would be purely based on using these deletions as biomarkers. To this end, loss of large deletions is shown overtime in 3 treated patients (2 with PBSCT & one with Rituxan). The sequencing work and statistical comparisons are rigorous.
While these findings are certainly of interest and raise an interesting hypothetical way to track treatment of CAEBV, the generalizability is limited by small sample size and lack of clarity in presentation. The study raises hypotheses for subsequent mechanistic work in animal models and could serve as a pilot for future formal, hypothesis-driven biomarker studies.
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Reviewer #2 (Public Review):
EBV infections are widespread among humans and have been associated with a variety of diseases, including CAEBV. The pathogenesis of this rare syndrome is mysterious but presumably is a result of unusual host and/or viral factors. This paper explores whether variation among EBV strains might somehow play a role in CAEBV. The main conclusions the author propose - that unique genomic deletions are found in EBV strains in CAEBV patients and are lost after recovery - is intriguing, but not convincingly supported by the data.
Strengths of the study: The authors did a large amount of sequencing and many analyses of the EBV genomic sequence data in this heterogenous group of samples. Among the interesting findings is that salivary samples (which were available for CAEBV patients only) were considerably more diverse …
Reviewer #2 (Public Review):
EBV infections are widespread among humans and have been associated with a variety of diseases, including CAEBV. The pathogenesis of this rare syndrome is mysterious but presumably is a result of unusual host and/or viral factors. This paper explores whether variation among EBV strains might somehow play a role in CAEBV. The main conclusions the author propose - that unique genomic deletions are found in EBV strains in CAEBV patients and are lost after recovery - is intriguing, but not convincingly supported by the data.
Strengths of the study: The authors did a large amount of sequencing and many analyses of the EBV genomic sequence data in this heterogenous group of samples. Among the interesting findings is that salivary samples (which were available for CAEBV patients only) were considerably more diverse than most other blood and tissues EBV sequences. They also found considerable variation in the sequences among patients as well as changes over time within an individual. For example, they detected many point mutations and large and small deletions. One large region of the genome was deleted in all CAEBV samples, so a specific association with CAEBV is possible, but the same region was deleted In one other non-CAEBV sample. At a minimum, these data will be provide a useful compilation of EBV genomic variation that will aid future studies of possible causal links to differing EBV-associated diseases. The authors appropriately only allude to 'associations." Many viruses generate defective progeny, so quite possibly some or many of the variants detected in these samples are not able to replicate. Nonetheless, they might be useful marker of disease status.
Weaknesses of the study.
Although the authors sequenced EBV from many samples of blood, saliva and tissues, the complexity and variety of these patients limits the power of even this large effort to generate firm conclusions. The three well-studied CAEBV patients all had very complex medical histories, including treatments such as rituximab (all 3) and hematopoietic stem cell transplantation (2 of 3). The comparison groups were quite diverse as well. Thus, unless a specific common variation is a major contributor to CAEBV, these data set are unlikely to have the power to reveal any specific genotypic association with EBV diseases.The potentially very interesting conclusion that deletions disappear over time and with resolution of CAEBV seems to be quite an over-simplification of the results. EBV in patient 1 has multiple deletions at times 1-3 and 5, but not 4, 6 and 7. EBV in patient 2 is not very informative about any patterns. The statement on line 153 that the deletions in patient 2 "were stable overtime but then lost ..." is particularly unconvincing since it is based on just two time points. It is not clear how frequent of these deletions in all these patients are in the samples at these times points. The comparison to changes over time in non-CAEBV samples (i.e. IM and PTLD) is hard to evaluate without more information - for example how far apart in time these samples were collected. The conclusion that deletions disappear following treatment such as stem cell transplantation is inconsistent with the results from Patient 3, whose 1st post-transplant sample still had the deletions (although it may be that this time point was relatively early after the transplant and so the EBV genome might still reflect the pre-transplant viruses).
As is common in these kinds of studies, a considerable amount of filtering of the primary data is necessary to limit the data to a reliable set. However, these steps limit the strength of the conclusions. For example, discarding sequences with "less than 90% genome coverage" (line 71) could eliminate some potentially important large deletions. Perhaps there were not any samples that had high read depth but low genomic coverage, which would somewhat mitigate this concern. What is the rationale for considering deletion <30 bp to be artifacts (Figure 2 - Supplementary 4)? Figure 3- Supplementary 1 shows only one sample for each patients - is this a fair representation of the whole set?
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Reviewer #3 (Public Review):
This study confirms previous data on the detection of deletions in the Epstein-Barr virus (EBV) genome associated with chronic active infection (CAEBV). The rare persistence of unresolved EBV replication in CAEBV is associated with significant morbidity and increased risk of developing lymphoproliferative disease. This study further implicates deleted forms of the EBV genome in contributing to this process and indicates that these deleted virus genomes appear to resolve upon treatment. Novel data shows that specific large deletions are found in the blood but not the saliva of CAEBV patients and that deletions are also found in the blood of individuals with the acute manifestation of EBV infection, infectious mononucleosis. While implicating deleted forms of EBV in the aetiology of CAEBV, there is no direct …
Reviewer #3 (Public Review):
This study confirms previous data on the detection of deletions in the Epstein-Barr virus (EBV) genome associated with chronic active infection (CAEBV). The rare persistence of unresolved EBV replication in CAEBV is associated with significant morbidity and increased risk of developing lymphoproliferative disease. This study further implicates deleted forms of the EBV genome in contributing to this process and indicates that these deleted virus genomes appear to resolve upon treatment. Novel data shows that specific large deletions are found in the blood but not the saliva of CAEBV patients and that deletions are also found in the blood of individuals with the acute manifestation of EBV infection, infectious mononucleosis. While implicating deleted forms of EBV in the aetiology of CAEBV, there is no direct casual evidence linking these phenomena. Given the small size, it is difficult to make firm conclusions. Deleted forms of EBV are also found in patients in some patients with either post-transplant lymphoproliferative disease (PTLD) or Hodgkin's lymphoma (HL). The relevance of these forms of EBV to the pathogenesis of these conditions remains unknown.
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