PolyBio Fall 2024 Symposium

I mean evidence of COVID persisting in LC using different methods and in different cohorts is something, right? At least gives a general idea of where to go next.

If the huge difference in megakaryocyte persistence is found to be real or possibly made more sensitive, that seems like basically a biomarker of long COVID, possibly useful in treatment trials.

 

One thing I realized should be kept in mind, is a lot of these groups collaborate so there is some overlap in participants in different studies, for example John Wherry is apparently using the cohort from Iwasaki's lab and the cohort from Peluso/Henrich's lab, and these researchers all have talks here too.

Also, I definitely can't continue to make summaries of the talks at this rate. I had a pretty bad headache about an hour ago from doing this the past two days, and was barely able to focus on anything. (Though ibuprofen helped both, I wonder if it's just because the headache lessened or if it somehow helps brain fog.) Unfortunate because it's really interesting and forces me to try to understand the info as well as possible if I want to make as accurate a summary as I can for others.

 

No rush @forestglip and thank you for doing so many so well already.

 

An Overview of the Fall 2024 PolyBio Symposium: Part One
https://solvecfs.org/an-overview-of-the-fall-2024-polybio-symposium-part-one/

 

An Overview of the Fall 2024 PolyBio Symposium: Part 2
https://solvecfs.org/an-overview-of-the-fall-2024-polybio-symposium-part-two/

 

2:04:32 Michael Peluso–LIINC Updates, Expansion of LIINC to ME/CFS, Targeting the SCV2 Resevoir in Long COVID Clinical Trial

Main takeaways:

• Michael Peluso discusses LIINC, a program that is currently studying long COVID, especially in regards to viral persistence. They plan to expand to other infection associated chronic conditions, including ME/CFS.
• A previously published study on viral persistence is continuing to detect viral antigen at 2 years post infection.
• The LIINC program is studying a variety of tissues: gut, bone marrow, lymph node, spinal fluid. They are planning to include female reproductive tract.
• Mentions four ongoing clinical trials targeting long COVID: antiviral ensiltrevir, monoclonal antibody, IL-15 agonist N-803, and JAK/STAT inhibitor baricitinib
  • They hope to have preliminary results from the monoclonal study within the next couple months.

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Michael Peluso discusses the LIINC program (Long-term Impact of Infection with Novel Coranavirus). This began in April 2020, and studies long COVID through avenues such as acute infection, tissues, imaging, and clinical trials. They have worked with over 1200 participants so far.

Previous studies (Thread, Thread) on acute cohort showed that those with a higher measurable viral load in the nose are more likely to develop long COVID. Similarly, the more days after acute symptom onset that virus could be detected, the more likely they would develop long COVID. He says there is currently not enough focus on what can be done during acute infection to prevent long COVID.


Another previous study showed that viral antigen could be detected in blood up to 14 months post infection. (Thread) He says they are still detecting it over two years post infection. The study also showed an association between antigen persistence and long COVID symptoms, especially neurocognitive, cardiopulmonary, and musculoskeletal symptoms.

Discusses tissue program. Have collected many tissues samples, including gut, lymph node, spinal fluid, and bone marrow. They plan to sample female reproductive tract tissue starting in December 2024.

LIINC is expanding to other infection-associated chronic conditions (IACCs), including ME/CFS and Lyme associated conditions, in hopes of comparing all these different conditions. (Thread) Will start with clinical phenotyping and biospecimens, but will later expand to tissue and imaging to study possible viral persistence (enterovirus, EBV, etc.) and immune responses. Planned to launch in early 2025.

Clinical trials

• Hope to have initial results to report on monoclonal study in the next couple months. (Link)
• They have completed enrollment of the ensitrelvir antiviral study. (Link)
• Awaiting ethics review for study of IL-15 agonist called N-803 to stimulate immune system. (Previously discussed in a talk by Tim Henrich: Thread)
• Collaborating with NIH on baricitinib study. (Thread)

Discusses three potential forms of a viral reservoir, and how they may require different therapies

• Persistence of RNA, but no translation into proteins - Immunotherapy?
• RNA able to be translated into proteins, but new virus is not being created. - Monoclonals?
• Virus is capable of replicating, and possibly infecting other cells. - Antivirals?

Discusses some considerations for trials

• Ideally, they would enroll/analyze based on measurable viral persistence levels, but they are not able to do so yet.
• Important to consider whether therapies are reaching tissues where there may be a reservoir. In monoclonal study, they plan to measure antibody levels in tissue.
• Longer trials may be required to eradicate a reservoir. Says cat models often require months of treatment to eliminate persistent coronaviruses.
• Need to figure out how to deal with reinfections during trials, which they are seeing in monoclonal study.
• Need to create more accessible measures of persistence, for example by comparing blood and tissue samples from the same individuals.

 

2:17:18 Tim Henrich–Molecular Imaging in Long COVID & Tissue Biopsy Program Updates

Main takeaways:

• Tim Henrich provides some updates about imaging and biopsy studies at LIINC. Their goal is to study viral persistence in long COVID using tissue.
• They have imaged 75 people and taken about 90 gut biopsies and 11 bone marrow biopsies.
• Finding high prevalence of EBV DNA in saliva in long COVID (~25%).
• Comparisons of markers in macrophages between LC and pre-COVID controls suggest immune system inhibition.
  
• In long COVID vs fully recovered, in peripheral blood, they find low proportion of mature NK cells, including a correlation of more LC symptoms to fewer mature NK cells. Also a lack of increased adaptive NK cells in the presence of SARS-CoV-2, suggesting impaired NK response to the virus. There is a planned IL-15 agonist trial (Interrupt-LC) which they hope will increase NK cell and cytotoxic T cell response.
• Planning to use imaging with radiolabeled monoclonal antibodies to detect virus directly in the body.

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Tim Henrich is providing more information about molecular imaging and tissue biopsy in LIINC (Long-term Impact of Infection with Novel Coronavirus) cohort.

Mentions previously published study which used ImmunoPET found T cell activation was higher up to 2+ years post COVID infection. Even those without long COVID symptoms had increased activation in some places, so it is important to take that into account when using pre-pandemic controls in studies.

They are continuing to probe the relationship between long COVID and fully recovered with an ongoing imaging study that has imaged 75 people so far with various clinical phenotypes. Have imaged over 15 people who have fully recovered from COVID. They have imaged over 30 people with LC before and after monoclonal antibody or antiviral intervention.

Reinfections during study present some challenges.

Also collecting blood and saliva at time of imaging. About a quarter of the cohort has detectable EBV DNA in saliva.

They previously found SARS-CoV-2 RNA years after infection in the body, including double stranded RNA up to two years after, which suggests either replication or endosomes containing the dsRNA persisting. RNA appears to cluster in the lamina propria tissue in the gut. They want to study the relationship of this to reactivation of other viruses like EBV.

They have processed around 90 gut samples, many of which are before and after monoclonal or antiviral interventions.

They are using high-dimensional digital spatial profiling to study individual cells. Some preliminary results for macrophages in long COVID vs never infected show upregulation of some markers of immune inhibition. At the same time, there is upregulation of interferon, suggesting the body senses virus.


In one participant, where they can detect large amount of virus in tissue, they find virtually no granzyme B production, a molecule secreted by natural killer cells and cytotoxic T cells to kill virus infected cells.


Also, some preliminary data on peripheral blood in long COVID vs fully recovered is showing that while total count of NK cells is similar (Figure E), they see a significantly lower proportion of CD56dim/CD16+ NK cells in LC (Figure F), which are thought to be more mature and cytotoxic versions of NK cells. This lowering is seen in different presentations of LC, including neuro, GI, and fatigue (Figure I). Further, they are seeing a correlation between levels of these mature NK cells and number of long COVID symptoms (Figure H).


No difference in levels of adaptive NK cells, which are more like a memory version of NK cells. I think he is also saying adaptive NK cells are increased in those with positive CMV antibodies, but not those with positive SARS-CoV-2 antigen (looks like decreased), which suggests there may be an insufficient NK cell response to COVID.


This leads into IL-15 trial, Interrupt-LC, which they hope will boost NK cell and cytotoxic T cell response and clear virus. Nearing approval.

Also previously saw high T cell activation in bone marrow. They have biopsied 11 people, looking at markers of viral persistence, inflammation, platelet function, and more.

Continuing to develop techniques to directly measure viral proteins throughout the body, using imaging and radiolabeled monoclonal antibodies, which they hope to begin applying to cohort soon.

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Edit: Data on NK cells now published as a research letter:

Long COVID is associated with lower percentages of mature, cytotoxic NK cell phenotypes, 2024, Tsao et al (Thread)

 

2:40:22 Michael VanElzakker–Understanding Long COVID & ME/CFS brain fog, from cells to circuits

Main takeaways:

• Michael VanElzakker's lab is studying both pre-COVID ME/CFS and long COVID, analyzing participants with MRI, PET, saliva, blood, pupillometry, and sleep testing.
• In both groups, they see inflammation in the same area of the brain that is involved in performing cognitive tasks (anterior mid-cingulate) and they think the inflammation here may be causing brain fog.
• They see worse quality sleep in both groups.
• Using tests of light flashing on pupils, they found that long COVID group has lower diameter pupils before and after flashes compared to both other groups (ME/CFS and HC), but ME/CFS has a longer pupil response time compared to both other groups (LC and HC).
  
• Also looking for fibrin accumalation using imaging in these groups, plus CSF flow and/or vagus nerve involvement in cranio-cervical instability.

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Michael VanElzakker says most of the work in his lab is both on people with long COVID and people with pre-COVID ME/CFS.

Their work includes MRI-PET neuroimaging, looking for neuroinflammation, and MRI-PET whole body scans, looking for fibrin accumulation. Before scans, patients do two nights of at-home sleep monitoring, as well as pupillometry tests (measure of autonomic function and intracranial pressure) and blood/saliva samples on day of imaging.

Also looking at patients with cranio-cervical instability. They use a vagus nerve stimulator to see how the brain responds under MRI. They also measure cerebrospinal fluid flow rate to see if there are differences in patients.

For blood and saliva analysis, they make use of collaborations through PolyBio to send samples to different institutions for different analyses (SARS-CoV-2 RNA, neutrophils, saliva microbiome, microclots, spike protein, immune, vascular, endocrine, antibody function).

Gave an example of seeing neutrophils being much more activated in a person with ME/CFS versus a healthy person.

Discusses meta-analysis (written by others) that looked at MRIs of brain responses to inflammation. One region shown to be involved in inflammation, the anterior mid-cingulate, stood out to VanElzakker because it is involved in being able to focus through distractions, an area that people with cognitive dysfunction say is difficult.

An earlier paper from this lab on neuroinflammation measured using PET: Neuroinflammation in post-acute sequelae of COVID-19 (PASC) as assessed by [11C]PBR28 PET correlates with vascular disease measures, 2024, VanElzakker et al

They have imaged LC and ME/CFS patients who were doing cognitive tasks, and found overlap of inflammation (orange) and task activation (blue) in that same region, the anterior mid-cingulate. They think inflammation in this region may cause cognitive dysfunction.


In regards to the sleep study portion, they are looking at a measure called Sleep Quality Index, which combines several metrics, including sleep fragmentation, stability, etc. They see low sleep quality in both ME/CFS and LC. This graph includes 10 HC, 17 ME/CFS, and 25 LC.


For pupillometry, where a quick flash of light is aimed at the eye, LC, but not ME/CFS, has a lower initial pupil diameter than HC. After light, LC has a lower pupil diameter than HC and ME/CFS.


Pupils of those with ME/CFS take longer to respond to light compared to the other two groups, "which is really what you'd expect in something a little bit more like concussion."

 

This part is interesting. I wonder why ME/CFS but not long COVID. Maybe specific to ME/CFS and some in the LC group don't have it? But at least some should have it, and their group level isn't higher at all. Develops over time?

I can't find past ME/CFS studies that measured latency of pupil response.

Inconsistent results in MS. [1][2]

Also seeing evidence for increased latency in Parkinson's [3][4], autism [5] and as he said in the video, concussion. [6]

Latency wasn't associated with COVID-19 mortality in one study. [7]

 

2:50:17 Petter Brodin–Sex Hormone Regulation of the Immune System

Main takeaways:

• Peter Brodin's lab is interested in genetics of long COVID, particularly genes involved in antiviral immunity. They hope to report results of a whole genome sequencing study of 110 people with severe long COVID (LC) at the next symposium.
• They are also studying how the immune system is influenced by sex hormones. This may help explain what causes LC, as it is known that females have a higher risk of developing LC.
• A study they published this year showed that testosterone therapy causes downregulation of type I interferon genes, which are important for effective immune response against viruses, and upregulation of pro-inflammatory genes, such as TNF and IL-6 [1].
  
• They found that plamacytoid dendritic cells (pDCs) may be key players in the relationship of sex hormones and immune function as they have high numbers of sex hormone receptors compared to other immune cells.
• Further study of the interaction of sex hormones and immune function may help in understanding the pathophysiology of long COVID.

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Peter Brodin's lab is interested in genetics of long COVID. So far, they have done whole genome sequencing on 110 people with well-characterized, severe long COVID which includes objective organ dysfunction. They hypothesize that they will find variants involved in antiviral immunity pathways, such as T cell or NK cell cytotoxicity or type-I interferon. They hope to present results from this at the next PolyBio symposium.

The present talk is mainly about the influence of sex hormone differences on immune responses. He repeats what is known: that infection severity is typically greater in males, but vaccine response and vaccine side effects are more intense, and autoimmune diseases are more frequent in females.

In virtually all countries, before vaccines, COVID killed more males than females. Part of the reason is known to be that a quicker type I interferon (IFN) response helps prevent severe infection, and this type of response is more typically seen in females.


Delayed type-I IFN leads to low T cell response, but a higher B cell response. With delayed type-I IFN, a proinflammatory response driven by TNF and IL-6 may occur, sometimes leading to cytokine storm, severe disease, and/or death.


Several publications have found increased risk of long COVID in females, as well as increased risk in those who are younger.

So Peter's lab is interested in how differences in sex chromosomes or sex hormones may be influencing this risk. A study which they designed in 2016 reported their first of several planned reports in September 2024 [1]. They studied the immune systems of people assigned female at birth and taking testosterone therapy in the setting of gender affirming care; this allowed them to separate the influences of sex genes and sex hormones on the immune system. In these individuals, testosterone levels were raised, and estradiol and progesterone were decreased, to levels typical of males.

Using whole blood transcriptomics, they found that genes associated with type-I IFN response were downregulated, while those associated with a pro-inflammatory response (TNF, IL-6, etc) were increased.


Part of this change could be explained by changes in numbers of cells, for example there were fewer plasmacytoid dendritic cells (pDC) after testosterone therapy, and these are key producers of type-I IFNs.


But cell count does not fully explain lower type-I IFN response because individual pDCs produced less IFN when stimulated by a viral mimic, and this was the most significant change seen in these cells after 3 months of testosterone therapy.


Similarly, monocytes produced less type-I IFN when stimulated by a viral mimic, and they also produced more TNF. The increase in TNF after testosterone therapy was even more pronounced when stimulated with LPS, a chemical produced by bacteria.


pDCs stand out among immune cells as having the highest levels of sex hormone receptors - androgen receptor (AR) for testosterone and ESR receptor for estradiol - in both males and females, as if they are the main sensors of sex hormones, and then modulate the immune system based on hormone levels.


So this study is showing the cross-regulation of IFN-I and TNF. There has previously been evidence of this: when patients are given anti-TNF drugs, their IFN-I response is sometimes increased, which can lead to a lupus-like disease.

He provides some speculation on why sex hormones may cause higher IFN-I and lower TNF in females, and the opposite in males: In females, where reproductive function is a top priority, TNF has been associated with complications in IVF and pregnancy, while IFN-I is an important defense against infections transmitted to a fetus. In contrast, TNF plays an important role in muscle repair and growth, while in the autoimmune condition dermatomyositis, IFN-I is associated with greater muscle wasting, and muscle function may have evolutionarily had a higher priority in males.

In conclusion, Peter says further study of differences in immune function influenced by sex differences may be important for understanding long COVID.

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[1] Immune system adaptation during gender-affirming testosterone treatment, 2024, Lakshmikanth, Brodin et al.

 

The problem with those PET and MRI pictures is that they make use of highly enhanced differences in values that may be absolutely tiny in reality. Cognitive tasks are not pinpointed to any particular place. They involve vast networks all over the brain. And the PET signal is not 'inflammation'. It is presumably uptake of a tracer in resident microglia or something similar and they are showing as. tiny difference from the rest of the brain.

And this is just one picture - presumably the one that fits the theory best.

 

3:03:17 Lael Yonker–Updates for Pediatric Long COVID

Main takeaways:

• Lael Yonker's lab is focused on long COVID in children.
  
• She describes earlier research which has shown that children more frequently report a wide variety of symptoms long after COVID infection.
• Her lab is looking at how long COVID in children compares to MIS-C, a severe inflammatory disorder that can occur in children after COVID infection. She finds that both long COVID and MIS-C show increased spontaneous NET formation by neutrophils. Neutrophils are cells of the innate immune system, and NETs are released by neutrophils to kill pathogens.
• They are also continuing to look at other features of these groups, such as cytokines, microclots, and endothelial function.
  
• She believes MIS-C and long COVID may be similar conditions that mainly differ in severity.
  
• Her lab is running the only drug trial for children in LC that is currently recruiting, a trial of larazotide.
  
• She argues that more observational research needs to be focused on children with long COVID because they have fewer comorbid inflammatory conditions - these may complicate studies - and evidence of long COVID in children will make it make it more likely they will be included in clinical trials.

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Lael Yonker's talk is about pediatric long COVID. She shows that currently a very small proportion of long COVID papers are focused on children.


She discusses the study Characterizing Long COVID in Children and Adolescents, 2024, Gross et al which compared symptoms between children who had confirmed COVID infection vs who had not been infected. The median time after infection these groups were surveyed (split into two age groups) was over 500 days for both groups. Numerous symptoms were more common in the infected children. (Red boxes are just around the symptoms she thought were worth highlighting.) From paper: "The index emphasizes neurocognitive, pain, and gastrointestinal symptoms in school-age children but change or loss in smell or taste, pain, and fatigue/malaise–related symptoms in adolescents."


Mentions studies which have found orthostatic intolerance, differences in autonomic cardiac function, and less capacity during cardiopulmonary testing in children with long COVID.

Her lab surveyed pediatric medical specialists to ask what signs, symptoms, or tools they rely on to suspect that a child has long COVID. They found a wide variety of responses, with no single measure being universally used by all providers.


They also asked how likely they were to diagnose a child with long COVID based on different features. Practitioners were more likely to diagnose with LC for children older than 10, than younger. More likely for females than males. More likely with fewer vaccinations. And small differences with regard to race, where asian children may have been the least likely to be diagnosed with long COVID.


Some other differences in diagnosing included those with significant school disruption, or those with family that asked about long COVID, being more likely to get a long COVID diagnosis.


Another project (Alvarez-Carcamo B, et al, in preparation) is looking at neutrophil profiles of healthy children, children with long COVID, and children with MIS-C (Multisystem Inflammatory Syndrome in Children). Overview of this last condition from Yale Medicine:

She says they are finding that MIS-C and long COVID appear to be similar conditions along a spectrum, with MIS-C being the extreme form, and long COVID being "a more indolent inflammatory response".

Part of the testing involved looking at isolated neutrophils and seeing how many neutrophil extracellular traps were spontaneously formed (NETosis). NETs are clumps of proteins and DNA which are released by activated neutrophils in order kill extracellular pathogens. NETosis was very significantly increased above healthy controls (unclear if these are uninfected or recovered children) in MIS-C, as well as long COVID groups positive for spike protein or negative for spike protein (fig B). Comparing levels of extracellular DNA, a marker of NETosis, showed a significant increase in children with MIS-C and spike-positive long COVID, but not spike-negative LC (fig C).


I don't really know what the "plasma" row on this graph means, but she says there is a relationship where the more spike-antibody immune complexes there are, the greater the levels of NETosis, and speculates that this may play a part in the differing severities of MIS-C and LC.


High expression of many cytokines in MIS-C, and not as much in spike-positive LC, though some.


Their other ongoing work includes detection of spike antigen, more neutrophil profiling, including RNA sequencing, microclots, endothelial function, cytokines, and profiling of antibodies. They want to further compare LC with MIS-C as well as compare children with adults. An advantage of studying children is that they are less likely to have confounding comorbid inflammatory conditions. Evidence for LC in children is also important for including them in clinical trials.

She points out that there are only 10 clinical trials for LC in children which are currently recruiting, only one of which is a drug trial, with the rest being behavioral or device trials. Yonker's lab is running this drug trial, which will test larazotide (a drug currently being studied for celiac disease - it decreases intestinal permeability). They are recruiting participants from age 7 to 50 who have long COVID and spike detected in the blood.

From the university's description of the study (click link if interested in enrolling as well):

 

That's a very stark difference in NETosis shown in Lael Yonker's presentation. Pretty much no overlap. Looking forward to the paper to see what the cohorts are like. Were the healthy controls ever infected? How long after infection for the two groups? If the healthy controls were never infected, this probably isn't very related to long COVID, as one of the studies below shows NETosis is just increased for a long time after COVID. (Edit: actually I think that study is looking at people with persisting symptoms.)


I tried searching for activated neutrophils or NETosis in ME/CFS but couldn't find much. But here are some neutrophil related findings in long COVID:

NETosis induction reflects COVID-19 severity and long COVID: insights from a 2-center patient cohort study in Israel, 2023, Krinsky et al

Chronic inflammation, neutrophil activity, and autoreactivity splits long COVID, 2023, Woodruff et al

Neutrophil extracellular traps and long COVID, 2023, Shafqat et al (Review)

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Also, not the same thing, but related to neutrophils from Ron Davis's lab. This is from a summary I made of one of their videos.

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Edit: Actually, I think the study from the review was on people with at least one persisting symptom. Still doesn't necessarily mean NETosis is associated with persisting symptoms since there's no comparison with fully recovered.

 

I asked Dr. Yonker about that NETosis plot. So the healthy controls were COVID recovered. Interesting...

 

A clinical trial of repurposed HIV antivirals in Long Covid

Polybio is supporting several trials of potential Long Covid treatments. Working with @PutrinoLab and CoRESinai, Polybio President Dr. Amy Proal is spearheading an investigation of repurposed HIV antivirals Truvada & Maraviroc for treatment of LC, targeting viral reservoirs

2/SCV2 reservoirs have been identified in LC gut tissue months after initial infection & EBV reactivations have been observed by the trial team in LC patients, potentially contributing to symptoms. Treatment with repurposed antiviral medications may help mitigate these viruses. Read more
LINK
https://twitter.com/user/status/1880026772592275805

 

As far s I know NETosis is a mirage. Everyone is now working on it so you will find fascinating papers about increased NETosis in every possible condition being published, but as someone trained in inflammation science I have no idea what they actually think this means. If neutrophils were clumping together and spilling out DNA traps or whatever there would be some clinical signs - funny red spots under the skin maybe. There aren't any either in Long Covid or ME/CFS. Presumablyy the assays are showing some sort of artefact like the 'micro clots'.

I am afraid I am sick to death of hearing presentations about NETosis this last year.

 

That was my guess. The difference between groups seems too good to be true. I remember in the video by Ron Davis linked above, he said neutrophils are hard to work with because they get activated very easily once out of the body, so maybe the procedure wasn't exactly the same between groups and all the LC neutrophils went boom.

 

Very likely so!!