Multimodal Molecular Imaging Reveals Tissue-Based T Cell Activation and Viral RNA Persistence for Up to Two Years Following COVID-19, 2023, Peluso +

Now published - link here

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Preprint
Multimodal Molecular Imaging Reveals Tissue-Based T Cell Activation and Viral RNA Persistence for Up to Two Years Following COVID-19
Michael J Peluso; Dylan M Ryder; Robert Flavell; Yingbing Wang; Jelena Levi; Brian H LaFranchi; Tyler-Marie M Deveau; Amanda M Buck; Sadie E Munter; Kofi A Asare; Maya Aslam; Walter Koch; Gyula Szabo; Rebecca Hoh; Monika Deswal; Antonio Rodriguez; Melissa Buitrago; Viva Tai; Uttam Shrestha; Scott Lu; Sarah A Goldberg; Thomas Dalhuisen; Matthew S Durstenfeld; Priscilla Y Hsue; J D Kelly; Nitasha Kumar; Jeffrey N Martin; Aruna Gambhir; Ma Somsouk; Youngho Seo; Steven G Deeks; Zoltan G Laszik; Henry F VanBrocklin; Timothy J Henrich

The etiologic mechanisms of post-acute medical morbidities and unexplained symptoms (Long COVID) following SARS-CoV-2 infection are incompletely understood. There is growing evidence that viral persistence and immune dysregulation may play a major role. We performed whole-body positron emission tomography (PET) imaging in a cohort of 24 participants at time points ranging from 27 to 910 days following acute SARS-CoV-2 infection using a novel radiopharmaceutical agent, [18F]F-AraG, a highly selective tracer that allows for anatomical quantitation of activated T lymphocytes.

Tracer uptake in the post-acute COVID group, which included those with and without Long COVID symptoms, was significantly higher compared to pre-pandemic controls in many anatomical regions, including the brain stem, spinal cord, bone marrow, nasopharyngeal and hilar lymphoid tissue, cardiopulmonary tissues, and gut wall. Although T cell activation tended to be higher in participants imaged closer to the time of the acute illness, tracer uptake was increased in participants imaged up to 2.5 years following SARS-CoV-2 infection. We observed that T cell activation in spinal cord and gut wall was associated with the presence of Long COVID symptoms. In addition, tracer uptake in lung tissue was higher in those with persistent pulmonary symptoms. Notably, increased T cell activation in these tissues was also observed in many individuals without Long COVID.

Given the high [18F]F-AraG uptake detected in the gut, we obtained colorectal tissue for in situ hybridization SARS-CoV-2 RNA and immunohistochemical studies in a subset of participants with Long COVID symptoms. We identified cellular SARS-CoV-2 RNA in rectosigmoid lamina propria tissue in all these participants, ranging from 158 to 676 days following initial COVID-19 illness, suggesting that tissue viral persistence could be associated with long-term immunological perturbations.

Link | PDF (Preprint: MedRxiv)

 

With the caveat that tissue biopsies were only looking at Long-Covid patients, this study looks very substantial. I don't recall a similar PET study in post-Ebola, if someone knows of one, could they please direct me to it to see how well they line up?

 

My own experience of Covid and continuing symptoms makes me think that residual virus maybe in gut is very plausible. I am not sure how it explains feeling terrible but it might have to do with T cells.

I am a bit worried about the methodology here though. It says pre-pandemic controls. For PET I think you need simultaneous control data and blinding because it is so easy for sensitivity to shift. I am also surprised by a T cell signal in brain stem and spinal cord, where T cells do not normally traffic in any number. Gut is always full of lymphocytes in comparison.

RNA is also maybe a tricky way to document persistent virus but techniques may be more reliable now.

 

Small study of 24 total individuals only 6 of which are pre pandemic controls - and whose images are obtained separate from the work in this paper. They say their dye [18F]F-AraG works by becoming phosphorylated by cytoplasmic deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) - markers of activated T cells - therefore getting trapped in the cells. They report big differences in uptake of [18F]F-AraG in lots of places eg brain stem and spinal cord, heart, gut wall. Everything is multiple test corrected with benjamini-hochberg, SUVmax means maximum Standardised Uptake Value of [18F]F-AraG:



They then compare those with and without long covid symptoms, and those pre and post 90 days since covid infection - these comparisons shouldn't be affected by the problems of differential sensitivity of the equipment over years as @Jonathan Edwards suggests could be the case for the above comparisons. There are no statistically significant differences in any of the regions of interest between long covid and non long covid patients, although across the board there appears to be slightly higher [18F]F-AraG uptake in long covid. Similarly just one significant result observed between pre and post 90 days (reduction in uptake in right colon wall at post 90 days), though with a possible slight reduction in uptake across the board in the post 90 days group:

 

Interesting why is that? I'm familiar with in situ hybridisation from developmental biology where it is a routine way of looking at expression domains. Why is it a problem with viruses? low copy number or maybe the RNA being bound up in viral assembly proteins? In the lab I formerly worked in we used single molecule FISH to get incredible sensitivity by using multiple probes each with different fluorophores that plaster the RNA along its whole length, allowing you detect even just a single molecule of RNA (as the name suggests). Again this was in a developmental biology context.

Seems like they're using standard in situ hybridisation here on colon biopsies from control and post covid ( not necessarily long covid) patients:

Spike protein RNA lighting up in green - CD68 and CD3 are markers I believe for macrophages and T cells respectively. They say they see Spike protein RNA in all 5 of the patients they looked at and none of the controls:

 

SUV is Standard Uptake Value.

*That ref is SARS-CoV-2 infection and persistence in the human body and brain at autopsy (2022, Nature)

ETA: a quick hunt around gives a couple of recent papers that discuss T Cells in the CNS —

Peripherally induced brain tissue–resident memory CD8+ T cells mediate protection against CNS infection (2020, Nature Immunology)
Tissue-resident memory T cells populate the human brain (2018, Nature Communications)

 

Also posted a thread for Tissue-resident memory T cells invade the brain parenchyma in multiple sclerosis white matter lesions (2020, Brain)

 

Just that in the days when I was involved in chasing nucleic acids with PCR specificity was a big issue. Specificity with fluorescence staining is also a big issue if you are not careful. I realise that these things can work well when done carefully but these days so much data seems to turn out to be substandard, even if techniques have improved.

If there is a bandwagon, people will get on.

 

Appreciating this UCSF group's work. We have most already in threads —

• A common allele of HLA is associated with asymptomatic SARS-CoV-2 infection (2023, Nature)
• Autoantigen profiling reveals a shared post-COVID signature in fully recovered and long COVID patients (2023, JCI Insight)
• Reduced Exercise Capacity, Chronotropic Incompetence, and Early Systemic Inflammation in Cardiopulmonary Phenotype Long Coronavirus Disease 2019 (2023, The Journal of Infectious Diseases)
• Chronic viral coinfections differentially affect the likelihood of developing long COVID (2023, The Journal of Clinical Investigation)
• Long COVID manifests with T cell dysregulation, inflammation, and an uncoordinated adaptive immune response to SARS-CoV-2 (2023, Preprint: BioRxiv)
• Use of Cardiopulmonary Exercise Testing to Evaluate Long COVID-19 Symptoms in Adults: A Systematic Review and Meta-analysis (2022, JAMA Network Open)
• SARS-CoV-2 and Mitochondrial Proteins in Neural-Derived Exosomes of COVID-19 (2022, Annals of Neurology)
• Long-term SARS-CoV-2-specific immune and inflammatory responses in individuals recovering from COVID-19 with and without post-acute symptoms (2021, Cell Reports)
• Characterization and Biomarker Analyses of Post-COVID-19 Complications and Neurological Manifestations (2021, Cells)

 

Michael Peluso interviewed on The Long Covid Sessions podcast, discusses this pre-print at 35:36.

 

Only scanned a few posts above, cool technology, seems like a new way to look for hidden viral (pathogen) reservoirs - a persistent theory in ME/CFS! Has this technology/opportunity featured in any of the NIH ME Roadmap webinars?

 

Good point - I was wondering if they'd used the technique on other known diseases e.g. shingles where the pathogen persists?

 

I'd missed this preprint when it popped up last year. It looks like it is still a preprint.

https://pubmed.ncbi.nlm.nih.gov/37577714/

Is that not a bit weird? Seems odd that it's not been peer reviewed by now.

It's been cited 24 times.

 

A substantial chunk of papers are taking a year or longer to pass peer review. From what I recall the Iwasaki, Klein et al paper also took longer than a year to pass per review and by then already had beyond hundred citations.

 

Merged thread
Now published

Tissue-based T cell activation and viral RNA persist for up to 2 years after SARS-CoV-2 infection

Authors (formatted by AI):

Editor’s summary
The term “Long Covid” covers a diverse array of symptoms that an individual might experience weeks to years after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many drivers of Long Covid have been proposed, with supporting data for each. Here, Peluso et al. provide compelling evidence for two potential contributors: persistent SARS-CoV-2 and aberrant T cell activation. The authors used whole-body positron emission tomography imaging with a tracer that selectively tags activated T cells to show that those with Long Covid had certain tissues that were enriched for activated T cells in comparison with those without Long Covid. Moreover, because the gut was one of the sites of activated T cell enrichment, the authors analyzed gut biopsies from a subset of individuals with Long Covid. In these samples, the authors were able to identify the presence of SARS-CoV-2 RNA; this feature was consistent across all five samples analyzed. These data suggest that viral persistence and sustained immune activation are linked to Long Covid. —Courtney Malo

Abstract
The mechanisms of postacute medical conditions and unexplained symptoms after SARS-CoV-2 infection [Long Covid (LC)] are incompletely understood. There is growing evidence that viral persistence, immune dysregulation, and T cell dysfunction may play major roles. We performed whole-body positron emission tomography imaging in a well-characterized cohort of 24 participants at time points ranging from 27 to 910 days after acute SARS-CoV-2 infection using the radiopharmaceutical agent [18F]F-AraG, a selective tracer that allows for anatomical quantitation of activated T lymphocytes. Tracer uptake in the postacute COVID-19 group, which included those with and without continuing symptoms, was higher compared with prepandemic controls in many regions, including the brain stem, spinal cord, bone marrow, nasopharyngeal and hilar lymphoid tissue, cardiopulmonary tissues, and gut wall. T cell activation in the spinal cord and gut wall was associated with the presence of LC symptoms. In addition, tracer uptake in lung tissue was higher in those with persistent pulmonary symptoms specifically. Increased T cell activation in these tissues was also observed in many individuals without LC. Given the high [18F]F-AraG uptake detected in the gut, we obtained colorectal tissue for in situ hybridization of SARS-CoV-2 RNA and immunohistochemical studies in a subset of five participants with LC symptoms. We identified intracellular SARS-CoV-2 single-stranded spike protein–encoding RNA in rectosigmoid lamina propria tissue in all five participants and double-stranded spike protein–encoding RNA in three participants up to 676 days after initial COVID-19, suggesting that tissue viral persistence could be associated with long-term immunologic perturbations.

Link (Science Translational Medicine, Paywall)

 

I can't access the actual paper, but the figures are available to view in a PDF linked on the page above.



Figure S4. Differences in [18F]F-AraG SUVmean uptake in certain tissues were observed when grouped by time from initial COVID-19 symptom onset to PET imaging and by Long COVID symptoms. (A) SUVmean values in tissue ROIs in post-acute COVID-19 participants imaged <90 days or >90 days from acute infection onset and control volunteers are shown. (B) SUVmean values in tissue ROIs in post-acute COVID-19 participants with or without Long COVID symptoms reported at the time of imaging and control volunteers are shown. (C to E) SUVmean values in tissue ROIs in post-acute COVID-19 participants with or without pulmonary symptoms (C), neurocognitive symptoms (D), and gastrointestinal symptoms (E) are also shown. Bars represent mean SUVmean and error bars represent 95% confidence interval. Adjusted P values <0.05, <0.01 and <0.001 represented by *, **, and *** respectively from two-sided non-parametric Kruskal–Wallis tests using a Benjamini-Hochberg adjustment for false discovery rates across multiple comparisons (q value = adjusted P value). All data points are shown.


Figure S5. Inverse relationship observed between gut wall [18F]F-AraG uptake and days from initial infection to PET imaging but not number of reported LC symptoms. (A and B) Scatter plots and two-sided Spearman rank correlation results between colonic and rectal wall [18F]F-AraG uptake and the days from COVID-19 symptom onset and PET imaging (A) and tracer uptake and the number of Long COVID-19 (LC) symptoms reported at the time of imaging (B).

 

I don't think you have to have access to the paper. The majority of it, if not everything, will be the same as in the preprint which was discussed here (it makes sense for someone to check how big the changes between preprint and publication are).

 

Team famous for HIV breakthroughs demonstrates both persistent COVID virus and widespread immune activation in long COVID; provides clear targets for treatment

Key points:

• SARS-CoV-2 double-stranded RNA indicative of viral replication was found in long COVID gut tissue up to 676 days post-infection
• T cell immune activation was documented across long COVID body sites including the spinal cord and gut wall
• Clinical trials of drugs to target persistent virus or immune activation in long COVID must be urgently accelerated

LINK

 

STAT News: '‘Visionary’ study finds inflammation, evidence of Covid virus years after infection"

'In people with long Covid symptoms, like brain fog and fatigue, the study found the gut wall and spinal cord lit up..'

“The data are striking," said Akiko Iwasaki, a professor of immunobiology and long Covid researcher at Yale University.

"The findings also suggest a need for more aggressive evaluation of immunomodulating therapies, and treatments that target leftover virus."

“I can now see with my own eyes the kind of dysfunction going on throughout my own body,” said Spier, who created a website for long Covid patients to more easily find clinical trials near them."

"..Peluso and Vanbrocklin said imaging could be a major tool in figuring out long Covid."