+100%-

Bhupesh Prusty is good at making waves. He and Bob Naviaux raised eyebrows in 2020 when their study showed that an HHV-6 infection might not only be damaging the mitochondria – putting the cell into a cell danger state – but also appeared to cause the cell to be pumping something into the serum that was doing the same thing to other cells.

Explaining ME/CFS? Prusty / Naviaux Study Ties Infections to Energy Breakdowns

Then last year, Prusty found that miRNAs produced by herpesvirus-infected cells were at least in part responsible for the mitochondrial fragmentation found. Plus in a small autopsy study he found evidence of widespread HHV-6/EBV activation across the brains of people with ME/CFS.

The Prusty Files: HHV-6 Found in the Brains of ME/CFS Patients

An enthusiastic researcher  Prusty primed the pump in the weeks leading up to the Charite and Invest in ME conference he recently presented at on his Twitter account stating:

“We will announce a biomarker for #MECFS and #LongCovid very soon. A very interesting piece of the puzzle to unfold in coming weeks. Knowingly I did not use the word ‘Novel Biomarker’ as a lot is known about it and that is actually a very good news. Fingers crossed!”

I saw Prusty’s presentation at the Charité conference, but it was so complex that I got completely lost halfway through. How good it was, then, to find (thanks to Jutta) the TLC Living with Long-COVID podcast where Prusty talked about his recent findings. Without that podcast, which focused on what appears to be a very large paper with multiple authors and multiple cohorts that was recently submitted, this blog would not exist.

A Focus on Herpesviruses

Prusty at the Charite Conference in Germany

Prusty at the Charité Conference in Germany.

Prusty has followed a somewhat different path than some other researchers. While some researchers have been gathering data that they hope will get at the cause of chronic fatigue syndrome (ME/CFS), Prusty, a molecular virologist, came at ME/CFS with a hypothesis – that herpesviruses were playing a huge role in it. Given that hypothesis, he tried to understand how – contrary to the evidence that had been gathered thus far – that was so.

Prusty was faced with a challenge. Herpesvirus reactivation, or primary infection in the form of infectious mononucleosis/glandular fever, appeared to be happening in ME/CFS, but studies had generally failed to find evidence of a high viral load. (A 2020 study did, however, find evidence of EBV reactivation in 25-66% of ME/CFS patients.) That left a question – how could a virus that wasn’t replicating and didn’t appear to be all that present (in the blood, at least) be causing something as devastating as ME/CFS?

Active Epstein-Barr Infections Found in Large ME/CFS Study

While Prusty was showing that, at least in lab cultures, herpesviruses could fragment the mitochondria in the cells they were found in – and possibly even in the cells they hadn’t infected, researchers at Ohio State University were digging deep into the EBV question in ME/CFS.

Over the past ten years or so, Marshall Williams and Maria Ariza had, in a long series of NIH-funded studies, had been showing that EBV didn’t need to replicate at all to potentially cause problems. All that was needed was to produce a kind of smoldering, aborted infection that got started but never made it to the replication phase.

They found that in a large subset of ME/CFS patients, EBV was producing a protein called EBV duTPase that: a) appeared to be capable of sending their immune systems into a tizzy; b) could be triggering an autoimmune reaction; and c) might even be contributing to neuroinflammation. Later, they added HHV-6 duTPase to the list.

The Epstein-Barr Virus, Neuroinflammation and Chronic Fatigue Syndrome (ME/CFS)

Prusty, Ariza, and Williams got together to see if an antibody response to the EBV and HHV-6 duATPases was present that would indicate that the immune system was fighting it off. Note that the immune response to a virus or protein can be a bigger problem than the presence of the virus or the protein itself.

The high antibody levels against every dUTPase tested (EBV, HHV-6, HSV-1) in ME/CFS suggested that aborted herpesvirus infections of all kinds (but particularly our old friend EBV) are present in this disease (!), in my mind at least, dramatically upping the potential role these enzymes are playing in ME/CFS.

Herpes simplex virus

HSV-1. While the immune responses to the herpesvirus dTUPases were not as pronounced in long COVID as in ME/CFS, a strong immune response was mounted against the HSV-1 dUTPase.

Things were a little calmer in the long-COVID world where a significantly higher IgG response was found against HSV-1 dUTPase as well as a higher, but not statistically significant, EBV response. One wonders if long-COVID patients simply need more time to develop the full range of aborted, but still active, herpesvirus infections seen in ME/CFS, but then came a weird finding – the kind of finding that doesn’t seem to make sense. The antibody response to HHV-6 dUTPAse was actually lower in the long-COVID patients. Prusty suggested that potential differences in T and B cell response in ME/CFS and long COVID might be responsible.

The Herpesvirus dUTPases – Mitochondrial Disrupters

We can now apparently add mitochondrial dysregulation to the increasing list of problems that these herpesvirus dUTPase proteins may be producing. When the researchers inserted dUTPase inside the endothelial cells, they found high levels of hyperpolarized, hyperfused mitochondria; i.e. the same types of mitochondria Prusty and Naviaux found in their earlier herpesvirus studies.

mitochondria

The herpesvirus dUTPase enzymes were like kryptonite to the mitochondria – each one caused them to clump, resulting in impaired autophagy.

All the dUTPases they assessed – whether they were from EBV, HHV-6, or HSV-1 – produced some major damage by disrupting the cytoskeleton or backbone of the mitochondria. That structural disruption, in turn, apparently changed the morphology or shape of the mitochondria. In biology, a simple thing like a change in shape can be determinative. The body may not, for instance, recognize it anymore, it may not function properly, and/or the body might try to attack it.

In this case, it appears it is at least having trouble trying to get rid of them. Prusty reported that clumped mitochondria are not being recycled properly. That’s an interesting finding, given the Simmaron Research team’s findings of reduced autophagy or mitochondrial clean-up in ME/CFS. He also noted that these clumped mitochondria are found in many different kinds of neurological diseases – which is a potentially good sign for the small ME/CFS field, which can’t begin to fully track down all its potential leads.

Cleaning Crisis? Defective Mitochondrial Cleanup In ME/CFS May Be Impairing Energy Production

Prusty noted that prolonged periods of herpesvirus reactivation are known to increase the risk of autoimmunity. Autoantibodies, he said, are increased during COVID-19 and long COVID (some research does not show this) and have been found in ME/CFS.

Profound Immune Dysregulation and Inflammation Found in Long COVID – but no Autoimmunity

IgM Antibodies to the Fore

The next step was to attempt to differentiate mild/moderate/severe ME/CFS patients from healthy controls using the IgG and IgM response against 120 autoantigens – bits of proteins known to trigger an immune (antibody) response. Plus, they looked for 120 associated autoantigens associated with pathogens. Prusty noted this was “a very small proof of concept study”.

There was no difference in the IgG response, but they found that high levels of autoantibodies associated with an IgM response against a variety of factors, including c-reactive protein (CRP), collagen 5,6; ss/ds DNA – (associated with lupus and multiple sclerosis) were found in the ME/CFS group, and that they did differentiate between the healthy controls and mild, moderate and severe ME/CFS patients. That broad-based difference was encouraging … as was the obvious potential autoimmune connection.

As interesting as those factors were, Prusty didn’t dwell on them. Instead, he focused on an IgM autoantibody called fibronectin that was low in ME/CFS and which he said correlated well with the severity of the disease; i.e. the more severe the patients, the lower the IgM fibronectin autobody levels.

Next, Prusty focused on the great question – how could the localized herpesvirus infections that he believes are present cause such widespread problems? The only possibility he could think of was that the viruses were dumping something into the blood or serum.

Several studies and findings – Prusty’s included – do indeed suggest something in the serum may be causing ME/CFS (and FM). That something – as has been suggested in fibromyalgia by Goebel – could be antibodies (immunoglobulins) that are reacting against something that has gone wrong in the cell – and are then getting pumped out of the cell – and disturbing other cells. It’s these antibodies that treatments like apheresis and immunoadsorption are seeking to affect.

They then purified IgG immunoglobulins from 30 healthy controls and ME/CFS patients and put them into a variety of different cells. Most of the cells were not affected by the ME/CFS patient serum, but interestingly – very interestingly – the mitochondria in the endothelial cells that line the blood vessels became fragmented.

They were even able to link the mitochondrial fragmentation to a decrease in a mitochondrial protein that keeps the mitochondria intact – a nice sign that the finding is correct. Prusty cautioned that other factors than IgG may be fragmenting the mitochondria in ME/CFS, but we clearly have to add IgG to the list.

Because the IgG purification process is not completely effective, they then crossed their I’s and dotted their T’s by using a mass spectrometer to see if the ME/CFS immune complexes that made up the immunoglobulins contained other factors that might account for the changes they’d seen. Three proteins showed up – all of which were decreased in the ME/CFS patients.

Two Fibronectin Diseases?

They then zeroed in on fibronectin-1, one of the more severely decreased proteins. This protein is incorporated into the complement pathway where it plays a major role in the fight against infections. Low levels of fibronectin found in these immune complexes could potentially constitute a kind of immune hole that leaves patients vulnerable to pathogens. They could also, Prusty suggested, produce the symptoms of sickness behavior we see in ME/CFS even if no virus is present.

The next question was why such low levels of fibronectin were present in these immune complexes. Next came a big surprise. ME/CFS patients had higher – not lower – but significantly higher levels of fibronectin levels in the serum compared to the healthy controls. That suggested that the protein was abundant in the serum but for some reason was not being taken up by the immune complexes.

It got even more interesting. Using the functional Bell assessment as a measure of health, they found that more severe patients had even higher levels of fibronectin in their serum than the mild/moderate patients. (As patients got worse and worse, were their cells  fruitlessly calling out for more and more fibronectin?)

However, while fibronectin levels did differentiate 80% of the severe ME/CFS patients from the healthy controls, Prusty noted that was not a significant enough difference to constitute a biomarker. Fibronectin, he thought, though, probably played a bigger role in ME/CFS than in long COVID.

Cellular fibronectin is a marker of inflammation, is found in many immune cells, and is very important in wound healing. Plasma fibronectin, on the other hand, plays a role in immune modulation, complement, mast cells, and anti-inflammation. High levels of plasma fibronectin could result in increased clotting, platelet, and mast cell activation, etc. Both cellular and plasma fibronectin were increased in ME/CFS serum. (Immune complex fibronectin, on the other hand, was decreased.)

Moving to long COVID, the same fibronectin finding showed up but not to the same extent, and Prusty suggested that as long COVID progresses over time it might become more substantial.

Gender differences are being explored more and more in ME/CFS and they are showing up in spades – and so they did in this study. Males, interestingly, tend to have lower fibronectin levels at baseline than females. That low fibronectin baseline combined with the much higher fibronectin levels in the ME/CFS patients made fibronectin a very significantly distinct factor in men.

In women, with their higher baseline fibronectin, the changes in fibronectin were not as distinct, but Prusty felt the naturally higher levels of fibronectin put females more at risk as they needed less fibronectin than males to reach “a pathological level” of the factor. If he’s right, he’s found a nice way to explain the gender divide in ME/CFS. It also suggests that it might take a more severe infection to tip men over into ME/CFS.

Depleted Natural IgM Antibodies – Could They Tell the Tale?

Natural IgM antibodies participate in a wide variety of processes in the body. Their levels were low in long COVID and may be low in ME/CFS. (Image rom Panda, 2015 – Journal of Immunology)

Prusty noted early infections in mice cause fibronectin in the blood to go up while IgM response to fibronectin goes down, and that old papers found that healthy individuals have high levels of IgM against fibronectin. This kind of pattern only shows up, he said, in natural antibodies that play a healthy role. These antibodies apparently function as a kind of cleanup or scavenger crew that removes dead cells before an autoimmune process can begin.

Prusty said “autoimmunity is clearly there” in ME/CFS, long COVID and SAR-CoV-2 patients. Not everybody would agree with that, but the team’s next task was to develop an assay that could assess the IgG and IgM antibody response against fibronectin. Did the high levels of fibronectin found in the plasma and serum of ME/CFS and long-COVID patients mean that the IgG/IgM response to it was reduced?

What they found, Prusty said, was “very, very interesting”. Only the severe ME/CFS patients had a reduced IgM response to fibronectin. In contrast, the mild, moderate, and severe long-COVID patients all had a significantly reduced IgM response to fibronectin. Even the mild patients – who had few symptoms – had significantly reduced IgM responses to fibronectin.

Prusty posited that the loss of the natural IgM response in long COVID to fibronectin doesn’t tank things immediately, but as the scavenging processes are lost, the dead cells and cellular debris build up and the body eventually produces an autoimmune response to them.

Akiko Iwasaki asked him what about the other IgM natural responses. Are they all going down? In what may be a novel finding in long COVID, further study suggested that the natural IgM population goes way down after a COVID-19 infection.

Natural IgM responses are produced by B-1 B cells. They play a variety of roles including “scavenger, protector, and regulator“. They play a big role in the complement arm of the immune system, help to knock down pathogens, and clean up dead cells. Because they bind to self-antigens, they provide a bulwark against an autoimmune response.

This suggests that the plasma B cells that produce these natural IgMs are being dramatically affected by the coronavirus. Over time, the B-1 cells in the recovered COVID-19 patients return to normal, but they do not in the long-COVID patients – the reduction in the natural IgM responses persist. The effect appears to be dramatic indeed: a comparison of the natural IgM levels in the long COVID to the recovered patients results in a 99% accurate diagnostic rate.

Prusty has also found very low IgM levels to fibronectin in ME/CFS but hasn’t checked the levels of other lgM natural antibodies. He noted that EBV duATPase could be producing a similar effect in the B-1 plasma cells in ME/CFS.

THE GIST

  • Bhupesh Prusty is a dynamic researcher who has contributed enticing findings in ME/CFS in the past, including herpesvirus-infected cells which not only damage their own mitochondria but appear to do so in cells adjacent to them.
  • Prior to his talks at two ME/CFS conferences recently, Prusty raised expectations with his announcement that he’d found a long-elusive biomarker for both ME/CFS and long COVID.
  • This blog resulted from the TLC long COVID podcast in which Prusty talked about the findings from a large paper that was recently submitted.
  • Prusty, a molecular virologist, came to ME/CFS with a hypothesis – that herpesviruses were playing a large role in it. The problem was, though, that the viral loads of herpesviruses in ME/CFS have never been particularly high.
  • Enter the Williams/Ariza team at Ohio State University, which for the last decade has been showing that a smoldering EBV infection is present in ME/CFS that’s pumping out an enzyme called EBV dUTPase. Their studies suggest that this enzyme may be causing an immune reaction, producing autoimmunity, and even may be involved in neuroinflammation.
  • Prusty and the Ohio State group teamed up to show that an immune response to all three herpesvirus (HSV-1, EBV, HHV-6) dUTPase enzymes was found in ME/CFS. In contrast, only an immune response to HSV-1 was present in long COVID. Either the long-COVID patients had yet to get all their herpesviruses involved or something very different was happening in that disease.
  • We can add mitochondrial disruption to the rather long list of problems these enzymes may be producing. When the researchers inserted dUTPase inside the endothelial cells, they found it produced high levels of damaged, clumped mitochondria; i.e. the same type of mitochondria Prusty and Naviaux found in their earlier herpesvirus studies.
  • These strangely shaped mitochondria – which Prusty said are commonly found in neurological diseases – are resistant to getting cleaned up and recycled. Interestingly – and encouragingly for both teams (and for ME/CFS), Simmaron’s research team has recently found evidence of impaired “autophagy”; i.e. mitochondrial cleanup in ME/CFS as well.
  • Next, the Prusty group attempted to differentiate between ME/CFS, long COVID, and healthy controls by assessing the levels of pathogenic antigens (proteins that evoke an immune response) and antibodies (the immune response) to those antigens. Note that it’s often the antibody response that’s the most harmful.
  • Several IgM antibodies most of which were associated with autoimmune diseases popped up, but the Prusty team was more interested in the reduced levels of an immune factor called fibronectin. This factor plays a role in pathogen defense, mast cell activation, inflammation, clotting, and platelet activation.
  • Putting the antibodies (immune complexes) from ME/CFS patients into the endothelial cells that line the blood vessels added yet another way (miRNA, herpesvirus dUTPases) to fragment the mitochondria in ME/CFS.
  • Because women typically have higher levels of fibronectin than men, Prusty questioned whether they may more easily reach “pathogenic” levels of the protein in the blood during an infection – leaving them more vulnerable to ME/CFS.
  • Next, they did a deep dive into the immune antibody complexes in ME/CFS to find, lo and behold, low levels of fibronectin. Checking the blood, they found high levels of fibronectin there. Something was clearly interfering with fibronectin intake into the immune complexes. While fibronectin levels were altered in long COVID, they were clearly lower in ME/CFS.
  • Next came a potentially crucial finding. The IgM antibodies to fibronectin are “natural” antibodies; i.e. they are naturally produced by the body to regulate fibronectin levels. Did the increased fibronectin levels in ME/CFS in particular mean that these natural antibodies were reduced? Yes, but only in the severe ME/CFS patients. Meanwhile, the entire cohort of long-COVID patients had reduced IgM antibodies.
  • That set up an experiment where they looked at other natural IgM and found, in what appears to be a unique finding, that they were also depleted in long COVID. This set up a possible scenario where a widespread depletion of natural IgM antibodies causes long COVID.
  • Because these antibodies specialize in removing dead and damaged cells, Prusty proposed that as these scavenging processes are lost, the dead cells and cellular debris build up and the body eventually produces an autoimmune response to them. They also suggest that the B-1 B cells that produce these antibodies are damaged in long COVID. Interestingly, the dUTPases found in ME/CFS may be able to damage these very cells as well.
  • While Prusty found some differences between long COVID and ME/CFS, the alterations in fibronectin in both diseases constitute the biomarker he believes he’s found. The good news is that measuring fibronectin is a cheap and easy thing to do.
  • One sore spot stuck out – the interviewer’s apparent belief that in contrast to ME/CFS in where symptoms slowly build over time, long COVID occurs more quickly. It’s true there’s a gradual onset subset in ME/CFS – and there’s one in long COVID as well – but that’s certainly not the experience of many people with ME/CFS who can remember the day their illness abruptly began.
  • With regard to treatments, Prusty, who is not a clinician, mentioned things like IVIG – which will excite no one probably – as well as immunoadsorption, apheresis, and some others. Prusty, though, noted that these are complex multifactorial diseases that will likely require complex, multifactorial treatments.
  • Prusty has done a lot of work, but his next step is looking for a new place for his lab as his current location is not providing sufficient support.
  • The findings do not appear to be based on large studies and will need to be replicated. With his dUTPase results, Prusty has built on and enlarged past findings, and in his fibronectin and natural IgM antibody results, he’s introduced a new concept of how these diseases occur.
  • Time, of course, will tell how this all works out. Hope that a new understanding of them has emerged, along with a suitable dose of caution as we await replication, would seem to be a good approach to take :).

ME/CFS vs Long COVID

Prusty noted the many clinical similarities between long COVID and ME/CFS. An analysis found that with regard to circulating fibronectin and low IgM responses to fibronectin, the ME/CFS patients, in particular, the severe ME/CFS patients and long-COVID patients, look more or less similar. The total similarity between all the long-COVID and all the ME/CFS patients is less but still significant, and there’s essentially no difference between the severe ME/CFS and severe long-COVID patients with regard to these factors.

In the long COVID patients, the decrease in natural IgM in response to the coronavirus is so clear that Prusty is not willing to bring herpesviruses into the equation. If he was to make a guess, though, he said he believes that herpesvirus reactivation does play something of a role. After all, he found antibodies to the herpesvirus duATPase protein in more than 50% of long-COVID patients and evidence of a strong reactivation of EBV and HSV-1.

Since he’s seeing similar fibronectin findings in ME/CFS and long COVID – and the ME/CFS patients haven’t been exposed to the SARS-CoV-2 virus – then it’s possible that herpesvirus reactivation is causing it, but that’s conjecture at this point. Nevertheless, the end product – problems with fibronectin – appears to be the same in both diseases.

A Slow Burn Disease?

The interviewers were great – they asked great questions and seemed able to follow along in a very complicated discussion quite well but at the end, one of them asked a strange question. Referencing the changes in IgM response to natural antibodies, she compared the almost immediate effect of the SARS-CoV-2 virus – which produced long COVID within 2 months of suffering – to the “much longer slower burn” of ME/CFS where their symptoms get worse over a longer period of time.

I have never heard ME/CFS referred to in that way before. While there is a gradual onset subset in ME/CFS (there is also one in long COVID), many people with ME/CFS can date their illness to the specific day they came down with the devastating cold they never got over. Their onset was dramatic and unforgettable.

In fact, given that we now know that long COVID can manifest without producing any symptoms during the initial infection, one could make the case that ME/CFS might, in some cases, actually manifest itself more quickly.

I don’t know where that idea came from, but I wonder if it speaks to some ideas in the long-COVID community that ME/CFS is somehow less damaging and less real.

Treatments

Prusty is not a clinician but feels potential treatment strategies exist. The most relevant, he thought, were IVIG and immunoadsorption, and mentioned apheresis, plasma B-cell therapy, bone marrow-derived cell transfusion, and recombinant B-cell therapy – among other things. That said, he also said that these diseases were complex and multifactorial and likely would require similarly multifactorial treatments.

A case certainly can and has been made for IVIG in ME/CFS, but it has been tried and my understanding is that the general results are pretty moderate.  It is apparently possible, though, to create natural IgM-enriched IVIG – and the results of that could be different.

There was some good news regarding testing, however. Because a fibronectin assay can be readily and cheaply produced in any lab, we could quickly get a sense of its levels in ME/CFS. While a test to assess natural IgM antibodies is not commercially available, it could be produced quickly and easily by many labs.

Prusty said the next phase for him is not jumping into more study but, citing the lack of manpower, professional stability, lack of lab space, etc. in his current digs, he’s looking for a new home for his lab.

A paper with numerous co-authors including Ariza and Williams, and Carmen Scheibenbogen, and which includes multiple cohorts of ME/CFS and long COVID, may be out soon.

Conclusion

It was exciting to see Prusty join hands with the longstanding Ohio State University ME/CFS effort and discussing his work and getting recommendations from immunologists like Akiko Iwasaki and Tim Henrich. The Ohio State University effort pretty much flies below the radar in ME/CFS circles, but it’s consistently produced positive results and boasts what is easily the longest-standing NIH grant award – probably over ten years at this point – in the ME/CFS field.

Seeing increased antibody responses to all three herpesvirus dUTPases in ME/CFS was striking indeed, as was the finding that these early herpesvirus enzymes may be damaging the mitochondria. That said, the high immune responses to HSV-1 dUTPase, but reduced immune responses to HHV-6 dUTPase found in long COVID, suggested the two diseases might be parting ways at least early in the illness.

It was nice to see Prusty follow the thread – to find the low fibronectin levels in the immune complexes, then the high fibronectin levels in the serum, then the reduced IgM response to fibronectin, the possible autophagy connection, and then the biggie – the possible widespread depletion of natural IgM antibodies in COVID-19, and ultimately long COVID, which implicates the plasma B-1 B cells.

Prusty said he believes he has found a biomarker (others, he noted, disagree) but does not claim that the loss of the natural IgM responses is the complete cause of long COVID.

Time, of course, will tell whether Prusty has produced a beautiful hypothesis that gets dashed down or one which stands the test of time and provides a new opening for understanding both ME/CFS and long COVID. We should note that these initial studies appear to be quite small and replication is essential. We’ll know more when the paper comes out. Both hope and caution would seem to be in order.

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