Persistence of SARS CoV-2 S1 Protein in CD16+ Monocytes in Post-Acute Sequelae of COVID-19 (PASC) ..., 2021, Patterson et al (in prep)

J

Jaybee00

Senior Member (Voting Rights)
https://www.biorxiv.org/content/10.1101/2021.06.25.449905v2


The recent COVID-19 pandemic is a treatment challenge in the acute infection stage but the recognition of chronic COVID-19 symptoms termed post-acute sequelae SARS-CoV-2 infection (PASC) may affect up to 30% of all infected individuals. The underlying mechanism and source of this distinct immunologic condition three months or more after initial infection remains elusive. Here, we investigated the presence of SARS-CoV-2 S1 protein in 46 individuals. We analyzed T-cell, B-cell, and monocytic subsets in both severe COVID-19 patients and in patients with post-acute sequelae of COVID-19 (PASC). The levels of both intermediate (CD14+, CD16+) and non-classical monocyte (CD14Lo, CD16+) were significantly elevated in PASC patients up to 15 months post-acute infection compared to healthy controls (P=0.002 and P=0.01, respectively). A statistically significant number of non-classical monocytes contained SARS-CoV-2 S1 protein in both severe (P=0.004) and PASC patients (P=0.02) out to 15 months post-infection. Non-classical monocytes were sorted from PASC patients using flow cytometric sorting and the SARS-CoV-2 S1 protein was confirmed by mass spectrometry. Cells from 4 out of 11 severe COVID-19 patients and 1 out of 26 PASC patients contained ddPCR+ peripheral blood mononuclear cells, however, only fragmented SARS-CoV-2 RNA was found in PASC patients. No full length sequences were identified, and no sequences that could account for the observed S1 protein were identified in any patient. Non-classical monocytes are capable of causing inflammation throughout the body in response to fractalkine/CX3CL1 and RANTES/CCR5.
 
This reads like someone who has read an immunology textbook but not understood it. I may be getting old but in the days when I was in immunology an abstract like this would get a straight rejection. The sentences aren't even in a sensible order.

'monocytes were sorted from PASC patients using flow cytometric sorting'. This is a bit like saying I opened the door by opening it. I don't understand how fifteen co-authors did not pick that up.
 
The authors claim the presence of spike protein fragments in CD16+ Monocytes is due to continued presence of viral genetic material up to 16 months later.

Unfortunately, they don't provide data for each of the patients (for the test of spike protein containing monocytes). They claim:

(...) we performed high parameter flow cytometry with antibodies that define B cell, T-cell, and monocytic subsets in addition to simultaneous staining of these cells with an antibody for the SARS-CoV-2 S1 protein (...)we found distinct subpopulations of SARS-CoV-2 containing cells in the CD14lo, CD16+ monocytic subset for 73% (19 out of 26) of PASC patients and 91% (10 out of 11) of severe COVID-19 patients. (...) the quantity of SARS-CoV-2 S1 containing cells were statistically significant in both the severe patients (P=0.004) and in the PASC patients (P=0.02). Neither classical monocytes nor intermediate monocytes expressed the SARS-CoV-2 S1 protein.
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They claim they double checked the findings with six patient samples using chromatography. (no discussion about how those samples were selected out of the overall group)

In addition, they sequenced samples from five patients (no discussion about how those samples were selected out of the overall group) and they state:

Full length sequencing of the five cases submitted for genomic analysis failed to identify any full-length sequence in the spike protein gene, or any other gene, that could account for the observed spike protein detected by proteomic analysis. In contrast, fragmented SARS-CoV-2 sequence was identified in all five of the cases.
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So there was unlikely to be replication competent virus persisting in patients.

There were issues:
The sequencing coverage for the five samples was consistent with low viral titer samples or samples with high Ct values. Average coverage was between 24.17-592.87x and percent bases covered at 10x and 20x was between 10.81-19.18% and 7.69-15.24% respectively (Table 2). This is well below the expected threshold to eliminate stretches of Ns > 99 for consensus sequence submission to GenBank and > 90% genome coverage at 10x for accurate lineage determination and sequence submission to GISAID (www.gisaid.org). Evaluation of the reads revealed predominantly short reads (<100bp). To address poor quality reads, primer-dimers or reads that could possibly map to multiple loci, reads < MAPQ 10 were filtered resulting in the removal of 3.63-18.99% of total reads per sample.
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I don't want to speculate about this too much since I am not an expert in the methodology, but could there be a risk of contamination leading to these results?

Some additional speculation by the authors:

Interestingly, a number of papers have been written discussing the increased mobilization of CD14lo, CD16+ monocytes with exercise (27). These data support the reports of worsening PASC symptoms in individuals resuming pre-COVID exercise regimens.
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https://www.healthrising.org/blog/2021/07/21/patterson-cracked-long-covid/
Cort's analysis 'Has Bruce Patterson cracked Long Covid?'
Bruce Patterson MD is a former Stanford researcher with quite a record. The former Medical Director of Diagnostic Virology at Stanford University Hospitals and Clinics, Patterson has co-authored around 90 papers – most prior to 2011 – at about the time he left the University and created the incellDx diagnostic laboratory. Over the past ten years, incellDx has focused mostly on cancer screening and has produced products to test for HPV, CMV, antibodies, and others. Over the past two years, though, Patterson has jumped back into the publication field, co-authoring 7 papers on COVID-19 with more to come.
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Having watched a video: Dr Patterson seems to be connected with a network of labs testing Long Covid patients, and clinicians treating them according to his protocol. Even Cort showed skepticism in his write-up, including a question-mark in his article's title. Patterson certainly has attracted a lot of attention.

I'm a bit concerned by Patterson's suggestion that they can fix the immune system and that is what they are primarily aiming to do - rather then eliminating symptoms. Those patients with ongoing symptoms after treatment are waved away as just suffering the effects of having been sedentary for a long time. So, there's talk of slowly ramping up their activity levels after treatment. With a paradigm like that, it is hard to know if the supposed success of the treatment (i.e. the return of the 'immune system' to something that looks normal) is just the markers of infection declining over time. It sounds as though the patients who don't recover will be assured that the treatment was successful in fixing the immune system and so any failure to recover will be either their fault for not becoming active again, or perhaps will be due to some permanent damage done to the body.
 
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Figure 1

So there are three types of monocytes: classical, intermediate and non-classical. The paper shows three charts (below) relating to each of those subsets, in that order. One key finding from the paper was that populations of intermediate and non-classical monocytes were increased in the long-haulers. Actually, the populations of the monocyte subsets looked quite similar in both of the non-healthy groups.

Screen Shot 2021-07-22 at 3.23.50 PM.png

This isn't particularly surprising, given that the intermediate and non-classical monocytes are involved in antigen-presentation. Increases in these two monocyte populations seems to be associated with infections. The healthy participants didn't have an infection, so, yeah... The chart does not show the level of monocyte subsets in people who have had Covid-19 but don't have Long Covid - we need to see that in order to evaluate the idea that levels of monocytes subsets are part of the story of Long Covid pathology.
 
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Figure 3
(as best I can work out - the charts in Figure 2 are just leading up to the conclusion presented in Figure 3)

So, here again, there are three charts, with classical monocytes (defined as CD14++ CD16-) on the left;
intermediate monocytes (defined as CD14+ CD16+) in the middle; and
non-classical monocytes (defined as CD14lo CD16+) on the right.

The y axis is a measure of the expression of the CoV-2 S1 protein on the monocyte cells. The three columns in each chart on the x-axis are for healthy controls (HC); patients with severe Covid-19 (severe); and patients with Long Covid (LH).

Screen Shot 2021-07-22 at 3.35.39 PM.png

So, it doesn't feel as though we have got to anything ground-breaking yet, as the people who are having or have recently had Covid-19 are the ones whose antigen-presenting monocytes are presenting a CoV-2 antigen. Again, there is no data for people who have had Covid-19 and recovered, or any examination of the effect of time since the illness.
 
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Hutan said:
Having watched a video: Dr Patterson seems to be connected with a network of labs testing Long Covid patients, and clinicians treating them according to his protocol. Even Cort showed skepticism in his write-up, including a question-mark in his article's title. Patterson certainly has attracted a lot of attention.
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Patterson also seems to have been involved in a group called FLCCC, which produced this "treatment protocol" - he is listed as one of the co-authors - which is full of unevidenced claims about MCAS and ivermectin.
 
Hutan said:
The chart does not show the level of monocyte subsets in people who have had Covid-19 but don't have Long Covid - we need to see that in order to evaluate the idea that levels of monocytes subsets are part of the story of Long Covid pathology.
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The 2022 Klein, Iwasaki paper did a similar analysis, but crucially also with a group of healthy people who had recently had Covid-19 (healthy convalescents).
[Preprint] Distinguishing features of Long COVID identified through immune profiling, 2022, Klein, Iwasaki et al

They found a similar increase in non-classical monocytes in people with Long Covid, and there was no increase in the healthy convalescents. So, this looks a lot more interesting, as does the finding of Covid S1 protein in the non-classical monocytes many months after the Covid-19 infection.
 
Cort Johnson (in the discussion linked above) says
Cort said:
Nor have altered monocyte levels been found in ME/CFS. Out of the many gene expression studies, only one found evidence of increased monocyte expression.
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The first link is to
Cellular immune function in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) (2019) Cliff, Nacul et al.
and it's true that they didn't find different levels of monocytes. But they only looked at CD14+ monocytes, so perhaps they weren't looking specifically at the non-classical monocytes which don't express CD14 much. Possibly we can discount that as evidence against the idea that non-classical monocytes are also altered in ME/CFS (?)
 
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Hutan said:
But they only looked at CD14+ monocytes, so perhaps they weren't looking at the non-classical monocytes which don't express CD14 much.
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The classification of 'non-classical monocytes' leaves me pretty sceptical. There are lots of these so called subset designations. But the crucial work done for me by Vikki Abrahams that led to our identification of targeting mechanisms in RA was on maturation of CD14+CD16- monocytes into CD16+ cells - which become capable of responding to small immune complexes. As far as we were concerned CD16+ cells were just a bit more streetwise having hung around and got some signals for a bit.

If that is true then we are back to the problem of circulating cell populations possibly showing maturation shifts simple from lasting longer in the blood with general factors like activity levels. Shifts might genuinely reflect some unknown stimuli but this sort of shift in monocyte populations is about as non-specific as you could get I think.
 
There was the finding of the incorporation of virus, or viral particles, in that group of monocytes, and the idea that that made the monocyte live a lot longer (15 months after infection, so, unless there was a viral reservoir or the particles survived upon cell death, at least that long).

There was also the finding reported in the Iwasaki paper that this non-classical monocyte group was expressing HLA-DR and CD15, unlike those of the healthy controls.
 
Hutan said:
There was the finding of the incorporation of virus, or viral particles, in that group of monocytes, and the idea that that made the monocyte live a lot longer (15 months after infection, so, unless there was a viral reservoir or the particles survived upon cell death, at least that long).

There was also the finding reported in the Iwasaki paper that this non-classical monocyte group was expressing HLA-DR and CD15, unlike those of the healthy controls.
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CD16 monocytes are more likely to pick up virus simply by being older and having the immunoglobulin receptor (CD16) to bind virus complex. They are unlikely to continue in the circulation for more than a few days. All blood monocytes are young cells. They may live for years once they have got to tissue but that is another issue. So as monocytes they must be picking up virus fresh from some other reservoir.

If our experience is anything to go by people may be getting recurrent re-infection with Covid every few months so presence of virus may not tell us much?

CD16+ monocytes normally express some DR. Not sure of the relevance of CD15.
 
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