<br/>Ian Lipkin on Long Hauling with the Coronavirus, a Possible B-cell Subset, and Protein Biomarkers for ME/CFS

Dr. Ian Lipkin, known to much of the world as the "virus hunter", has throughout his career developed new technologies for discovering viruses and has uncovered many new viruses. Recently, Lipkin uncovered the apparent cause of a puzzling and devastating disease affecting children called acute flaccid myelitis (AFM). He's helped to uncover several potential subsets in ME/CFS including Dr. Peterson's "atypical subset" and a gut subset. Dr. Lipkin leads the NIH funded Center for Solutions for ME/CFS at Columbia University's Center for Infection and Immunity (CII). He, Dr. Mady Hornig and their colleagues there have contributed important insights into our understanding of ME.Simmaron Research has had a special collaboration with Lipkin and CII over the years, especially through its signature studies on spinal fluid of ME/CFS patients. The Simmaron Research Foundation has collaborated with Dr. Lipkin and/or Hornig on 7 peer reviewed publications. Dr. Hornig, one of the co-authors of the proteome study, is a member of the Simmaron Research Foundations Scientific Advisory Board.Between the coronavirus pandemic and publication of groundbreaking proteomics results, there was a lot to discuss with Dr. Lipkin.

Coronarvirus

It's not surprising that Ian Lipkin, one of the top virologists in the world, has been featured again and again in media stories about the coronavirus. Lipkin was hailed for flying to China in 2003 while it was in the midst of the first coronavirus outbreak (SARS) and playing a key role in helping the Chinese build a pathogen response system. Lipkin quickly flew to China during the present Sars-Cov-2 outbreak, and then, ironically, became infected with the virus in March while back in New York City.I asked him a series of questions, including how he was doing almost six months later. Ian Lipkin, unfortunately, has joined the long-hauler community. Fortunately for him and us, his cognitive faculties have not been affected.

I have not recovered completely. My capacity for exercise is reduced. Fortunately, there is no cognitive impairment. The experience has given me a deeper appreciation for the challenges faced by people with ME/CFS. However, my level of disability does not compare with what many people with ME/CFS suffer.

Q: What can the U.S. expect from the coronavirus over the fall/winter?

I am concerned that we will see a surge in disease as people move indoors and schools and businesses re-open. Until vaccines are readily available it will continue to be important to maintain physical distancing and to wear masks. These are are the best tools we have for reducing virus transmission.

Q: KCRW reported that you've seen many COVID-19 patients with long term fatigue. The COVID-19 long haulers obviously present a real opportunity to catch a post-infectious illness in its tracks. A year from now do you believe we are going to have some answers to how these mysterious post-infectious illnesses occur?

It is too early to know how the fatigue associated with COVID-19 relates to ME/CFS. Some COVID-19 survivors have severe lung and/or heart damage that is clearly different from what is observed in ME/CFS. I nonetheless think we need to be open to the possibility that COVID-19 may provide clues that will help us understand and develop new approaches to ME/CFS.

Q: A vaccine or rather vaccines are coming. I'm sure much will depend on how effective the vaccines are, but do you have any idea what percentage of the US population needs to be vaccinated to really stop the virus?

This point is controversial. Most of my colleagues who model the spread of COVID-19 have estimates in the range of 60-80%.

Q: As bad as the coronavirus has been, it could have been much worse. A highly contagious and highly deadly virus could have produced a disaster unlike anything we could imagine. Are you seeing signs that the U.S., China and other countries are bulking up their pathogen detection capabilities?

China has been building capacity for infectious disease surveillance since 2003 when SARS devastated its health care system and economy. There is nonetheless room for improvement in both China and the US. I am strongly advocating for GIDEoN, a global surveillance system, where countries commit to sharing information. This is our best hope for preventing future pandemics.

Q: You were just starting or about to start a study using the same Serochip analysis on people with ME/CFS that you used to understand acute flaccid myelitis - a mysterious illness affecting young children - which had defied attempts to understand it. The Serochip analysis could uncover a pathogen or pathogens which initiated ME/CFS or an autoimmune reaction underway. What is the status of that study, and how has the coronavirus shutdown affected your ME/CFS work?

The pandemic did force us to temporarily table this work. Only last week, we began analyzing samples from Derya Unematz of the Jackson Laboratories using the SeroChip.

http://simmaronresearch.com/2020/01/lipkin-serochip-chronic-fatigue-syndrome/

The Proteomics Paper

Proteomics has kind of been the lost "omics" field in ME/CFS. Genomics (the study of genes), transcriptomics (the study of gene expression), metabolomics (the study of metabolites) and even epigenomics (the study of epigenetic modifications of the genome) have all had their moments but proteomics (the study of proteins) has been mostly left behind.Only four proteomic ME/CFS studies have been done - none of which, interestingly, has been done on the blood. The first on cerebrospinal fluid (CSF) occurred back in 2005, the second in 2011 (CSF), one seven years ago in 2013 (saliva), and one in 2016 (saliva). Each has had interesting results. The last study - a saliva study - pointed a finger at mitochondrial proteins. (Update - two proteomics studies were recently published: a New Zealand study using PBMC's and an Australian study examining immortalized lymphocytes.)

Proteomics has the potential to reveal much. Genes, after all, code for proteins, and it's the proteins that do the actual work of the cell. Because they play that crucial role, proteomics has particularly been used to identify biomarkers, produce diagnostic tests, and drug targets.

Studying proteins, however, is in some ways more difficult that studying genes. Genes, at least, are constant and unchanging. Their expression may be modified but they themselves remain the same. Proteins, on the other hand, change from cell to cell and in the same cell over time. Many of them undergo processes such as phosphorylation and ubiquination that modify their structure. Assessing proteomics, then, is a potentially very fruitful but nonetheless complex endeavor.

The Study

Plasma proteomic profiling suggests an association between antigen driven clonal B cell expansion and ME/CFS, Milica Milivojevic , Xiaoyu Che , Lucinda Bateman, Aaron Cheng, Benjamin A. Garcia, Mady Hornig, Manuel Huber, Nancy G. Klimas, Bohyun Lee, Hyoungjoo Lee, Susan Levine, Jose G. Montoya, Daniel L. Peterson, Anthony L. Komaroff, W. Ian Lipkin. Plos One : July 21, 2020. https://doi.org/10.1371/journal.pone.0236148

Our work, whilst exploratory in nature, shows that the plasma proteome is a viable and untapped source of potential biomarkers in ME/CFS, and can provide insight into disease pathophysiology. The authors

This present study - using "ultra-performance liquid chromatography-tandem mass spectrometry" - is the first done on plasma. It assessed the plasma of 50 ME/CFS cases and 50 controls from the practices of ME/CFS experts (Dr. Peterson, Dr. Klimas, Dr. Bateman, Dr. Levine) across the country. Various self-report questionnaires were used including one to assess if the symptoms of irritable bowel syndrome (IBS) were present.The team at CII ploughed through hundreds of proteins. While a linear model didn't pick up any proteins, a linear plus quadratic model appears to have struck proteomic gold - at least for a subset of patients.

Twelve ME/CFS patients had "extremely high" levels of a protein called IGHV3-23/30 - an immunoglobulin protein.Immunoglobulins refer to antibodies produced by B cells which bind to pathogens, neutralize toxins, regulate immune system functioning and help kill cells infected with bugs. The immunoglobulin system wards off infections but when it goes wrong it can produce autoimmune diseases and contribute to cancer.The region of the genome IGHV3-23/30 comes from is associated with Non-Hodgkins lymphoma's (NHL), Waldenstrom’s macroglobulinemia, anti-myelin associated glycoprotein neuropathy and monoclonal gammopathy. The lymphomas have been linked to a bacterial driven immune response or an autoimmune response. One study has suggested an increased risk of NHL in ME/CFS.

So, why did massive amounts of one particular immunoglobulin appear to be present in about a quarter of the ME/CFS patients? The authors speculated that an antigen - something that was activating the immune system - was driving the expansion of a set of B-cells associated with these immunoglobulins.

They suggested that identifying that antigen or the use of kinase inhibitors that could effect the functioning of IGH3-23/30 could be helpful in these patients.If this finding holds up, it should be noted that dozens of kinase inhibitors are available or are under development for the treatment of cancer and inflammatory diseases.

Additionally, another immunoglobulin IGKV3(D)-11 was significantly associated with ME/CFS patients without IBS. Increased levels of this immunoglobulin, interestingly, enough have shown up with IGH3-30 in response to a cytomegalovirus (CMV) or Streptococcus pneumoniae infection.

Every study Lipkin does seems to further differentiate ME/CFS patients with IBS from those without and this study was no exception. In the past he's found different set of metabolites and distinctive gut flora in this group. This time the ME/CFS+IBS group was distinguished by a protein - IGLC7 - another B-cell associated immunoglobulin protein - this time associated with antibody regulation.

Biomarkers

Next the group looked for a biomarker. Biomarker identification is a burgeoning field in proteomics. Just in the past couple of weeks, studies have used proteomics to determine how sick a COVID-19 patient is likely to become, how sick people with bacterial infections will get, which athletes will have more trouble recovering from a concussion and potential protein biomarkers in type II diabetes.

Using a variety of techniques the Lipkin group attempted to identify proteins that have high diagnostic efficacy using "Area under the curve" (AUC) analyses. These analyses identify how well a result identifies a patient group. A value of 0 indicates a perfectly inaccurate test and a value of 1 reflects a perfectly accurate test; i.e. every patient is correctly identified.Eight plasma proteins with high AUC's (up to .838) were identified. While no particular pathways were distinguished, a strong focus on the immune dysregulation with several proteins involved in B-cell regulation was seen throughout.

cathelicidin antimicrobial peptide (CAMP) - an antimicrobial peptide increased during both bacterial and viral infections, high CAMP levels have been found in several autoimmune/inflammatory diseases
Ig lambda variable region 1–47 (IGLV1-47) - associated with B cells and decreased in ME/CFS
Fc receptor-like protein 3 (FCRL3) - found in low levels in ME/CFS FCRL3 has been shown to inhibit B-cell signaling. Reduced levels of this and IGLVI-47 suggest problems with B-cell regulation may be present in ME/CFS.
leucin-rich glycoprotein 1 (LRG1) - lower levels of this pro-inflammatory protein could reflect problems with neutrophil functioning which have cropped up in ME/CFS in the past
gelsolin (GSN) - decreased levels of this actin scavenger could result in inflammation and oxidative stress. Lower serum GWN levels have been found in inflammatory conditions
cartilage acidic protein 1 (CRTAC1) - the presence of a connective tissue protein is intriguing
insulin-like growth factor 1 (IGF1) - The low levels found in ME/CFS are interesting given that low levels of IGF1 have been associated with infection, trauma, and aging. This protein has shown up in ME/CFS before.
IGF-binding protein acid labile subunit (IGFALS) - an IGFI binder low levels were also found in ME/CFS

Breaking Up ME/CFS?

Next - in a step which showed how serious Lipkin is about assessing the possible differences between the IBS+ and IBS- group, they attempted to see if they could use proteomics to differentiate the ME/CFS+IBS group from the ME/CFS group.Five proteins (LRG1, CRTAC1, IGLC7, KNG1, PON3) differentiated the ME/CFS-IBS group. One of them in particular (KNG1), with it's impact on inflammation (bradykinin), blood vessels and pain, appears to fit ME/CFS well. Six proteins differentiated the ME/CFS+IBS group (IGF1, CRTAC1, and CAMP, SERPINA3, ITIH2, IGHV1-18), with B-cells, inflammation and oxidative stress providing a familiar theme. Very high levels of CAMP — an antimicrobial peptide found in several autoimmune diseases and in the mucosa of inflammatory bowel disease (IBD) — particularly distinguished the ME/CFS+IBS group. IBD is a broad term used to describe diseases like ulcerative colitis and Crohn's disease.

The authors speculated that gut dysbiosis (dysregulated gut flora) results in increased inflammation and gut permeability resulting in higher plasma levels of CAMP and a protein named PIGR. That was encouraging given that it should (hopefully) be possible at some point to repopulate the gut with a better floral mix.

Despite the fact that Lipkin found gut dysbiosis in all ME/CFS patients, he found a distinctive gut flora in ME/CFS+IBS vs ME/CFS-IBS patients. He also found dysregulated metabolic pathways that were common to both groups. With the two groups sharing some similarities and differences, it's not clear how closely allied these two groups are.

For those thinking the gut couldn't possibly be a major issue, Lipkin found in a separate study that the severity of symptoms such as pain, fatigue, and reduced motivation were correlated with distinct gut species and the metabolic pathways they impacted. http://simmaronresearch.com/2018/07/subset-maker-lipkin-chronic-fatigue-syndrome-study-highlights-gut-subset/

I asked Lipkin about the two ME/CFS groups:
Q: Your work has now shown a divergence between ME/CFS+IBS and ME/CFS-IBS patients with regards to metabolites, the microbiome and now proteins. Some commonalities between these two groups have been seen but also some distinct differences. How close would you speculate the two groups are? Is IBS a kind of add-on to the general group of ME/CFS patients, or are ME/CFS with IBS patients fundamentally different?

There are many paths to ME/CFS. One is associated with intestinal dysbiosis and may respond to dietary and pre- and probiotic interventions.

I also asked Lipkin about the overall study results:

Q: This was an exploratory study. Besides the IgG finding, it uncovered a set of potential biomarkers that might differentiate the ME/CFS group as a whole and between ME/CFS+IBS and ME/CFS-IBS patients. Were the results good enough to pull for a larger study, and how much larger would a proteomics study need to be to produce really robust results?

As you note, this was an exploratory study. We will need to examine samples from a minimum of another 100 people with ME/CFS and 100 matched controls. The results are nonetheless intriguing and support the hypothesis that at least some patients have immunological abnormalities.

The study was funded by an NIH grant and the Solve ME/CFS Initiative.

Conclusion

Ian Lipkin, Mady Hornig, and the CII team produced the first proteomics study done on plasma in ME/CFS, in collaboration with clinical experts including Dr. Peterson and Simmaron. Since proteins do the work in cells they represent a particularly direct way of getting a biological snapshot of what's happening in a disease. Because proteins change over time and from cell to cell, and can be affected by different processes, proteomics studies are complex endeavors. Proteomics has, however, been used successfully many times to uncover biomarkers.

A linear plus quadratic approach in this preliminary study uncovered a possible subset of patients with high levels of an immunoglobulin (antibody) which suggested that B-cells in this group of patients have responded to a pathogen or are engaged in an autoimmune reaction.

Validating this finding could open to the door to use of kinase inhibitors in ME/CFS.A search for a biomarker yielded eight possibilities, most of which were involved in the immune response - and some of which were associated with B-cells.

While some similarities were seen, this study also added to a string of studies which suggest that ME/CFS+IBS patients have different proteomic, metabolomic and transcriptomic profiles; i.e. that they differ biologically in significant ways. Very high levels of one protein (CAMP) suggested that an autoimmune process may be underway, and that ME/CFS+IBS patients may have something in common with people with inflammatory bowel disease.

The authors proposed that gut dysbiosis (altered gut flora) in these patients was resulting in increased gut permeability (leaky gut) and inflammation. The authors warned that larger studies are needed to validate the findings in this study. With a B-cell subset of patients appearing, eight candidate protein biomarkers showing up, and with ME+IBS patients being further differentiated from ME/CFS-IBS patients, one would hope, though, that this study proves to be a stepping stone to a much bigger study.

This study furthers Simmaron's work to collaborate in scientific investigations that have the potential to identify immune-based biomarkers and characterize ME/CFS subsets, so we can discover effective treatments for patients.

Ian Lipkin on Long Hauling with the Coronavirus, a Possible B-cell Subset, and Protein Biomarkers for ME/CFS