REAP: A platform to identify autoantibodies that target the human exoproteome, 2021, Wang et al

I personally believe that ME/CFS is antibody driven and I therefore think this breakthrough technology could tell us alot about at least a subset of patients. Here you can identify antibodies to extracelluar proteins (non tissue degenerating) which relates to us as patients since there is no tissue damage. Who will be first to use this to screen a ME/CFS cohort?

Yale has spunoff a company around this called SeranovaBio https://www.seranovabio.com/

This is the same technique used to identify various antibodies in Covid 19 patients as described in New York Times and Nature.

https://www.biorxiv.org/content/10.1101/2021.02.11.430703v1

Abstract
Autoantibodies that recognize extracellular protein epitopes (the “exoproteome”) exert potent functional effects that underlie numerous disease processes. Identifying these antibodies can thus provide insights into the pathophysiology of a wide spectrum of illnesses and therapeutic strategies to treat them. Here, we developed Rapid Extracellular Antigen Profiling (REAP) as a technique for comprehensive and high-throughput discovery of exoproteome-targeting autoantibodies. With REAP, patient samples are applied to a genetically-barcoded library containing 2,688 unique members of the human exoproteome displayed on the surface of yeast. Antibody-coated cells are isolated by magnetic selection and deep sequencing of their barcodes is used to identify the displayed antigens, thereby converting an antibody:antigen binding event into a digital sequencing readout.

To benchmark the performance of REAP, we screened 77 patients with the rare monogenic autoimmune disease autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). REAP sensitively and specifically detected known autoantibody reactivities in APECED, including responses against type I interferons, IL-17, IL-22, and gastric intrinsic factor. REAP also identified highly prevalent reactivities that had not been previously described such as those against the glycoprotein hormone GPHB5. We additionally screened 106 patients with systemic lupus erythematosus (SLE) and identified novel autoantibody reactivities against a diverse set of antigens including growth factors, extracellular matrix components, cytokines, and immunomodulatory proteins. Several of these responses were associated with disease severity and specific clinical manifestations of SLE, including autoantibodies that target immunoreceptors, antagonize the pro-inflammatory cytokine IL-33, and recognize endosialin (CD248) and the chemokine CCL8. In summary, these findings demonstrate the utility of REAP to atlas the expansive landscape of exoproteome-targeting autoantibodies in patients.

 

It looks like a sensible technique.
It would make sense to screen an ME/CFS cohort like the ME Biobank.
The abstract does not say anything about rates of autoantibodies in normal healthy people - which of course is not zero. Quite a lot of healthy people have autoantibodies.

 

Is the idea that autoantibodies may be relevant in ME/CFS based on the idea that:

• rituximab only removes some antibody producing cells (i.e. B-cells) and that there are other cells which produce antibodies and are not removed by rituximab (plasma cells +?)?
• a subset of people with ME/CFS may have an autoimmune form of the disease?

I support the application of this technology to ME/CFS by the way.

Also, it reminds me of GWAS, OK this techniques is looking at a specific hypothesis, i.e. autoimmunity, but it's broad - not looking at autoantibodies to X.

The fact that they found "known" autoantibodies seems interesting; I assume that this means that they've managed to present the correct form of the protein, on the yeast surface [Post-translational modification form?].

I'm an interested amateur - rather than scientist with the relevant expertise - so ---

 

Hi all. Thanks for the positive feedback on this.

This technology has already been used in one Covid-19 study and has been featured as I said in Nature, NYT and Guardian. You might have come across those articles from somewhere else but I post here links to the actual study and news as a reference:

Diverse Functional Autoantibodies in Patients with COVID-19
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7743105/

Rogue antibodies could be driving severe COVID-19
https://www.nature.com/articles/d41586-021-00149-1
How the Coronavirus Turns the Body Against Itself
https://www.nytimes.com/2021/01/28/health/coronavirus-antibodies-immunity.html
'Autoantibodies' may be driving severe Covid cases, study shows
https://www.theguardian.com/science...may-be-driving-severe-covid-cases-study-shows

This is exactly the feeling I get as well when I speak to clinicians. It is understandable up to a certain extent but it find it interesting when doctors are not able to look out of the box and see the big picture. Measuring titers of one or two specific antibodies is not going to tell you anything when there thousands or millions different types of them out there.

As got this right. Rituximab only depletes the memory B cells in circulation with CD19 expression while we have abundance of plasma cells in bone marrow segregating antibodies and maybe alot of autoantibodies as well. SLE does not go into remission on Rituximab but interestingly there are some evidence that it does on Daratumumab that depletes plasma cells with CD38 expression as I recently posted:

Targeting CD38 with Daratumumab in Refractory Systemic Lupus Erythematosus, 2020, Ostendorf et al.
https://www.s4me.info/threads/targe...pus-erythematosus-2020-ostendorf-et-al.18865/

Based on all the above I think at least a subset of ME/CSF patients have autoantibodies that are continuously being produced by plasma cells in the bone marrow. These autoantibodies are towards important extracelluar proteins and that causes loss of homeostasis and sickness behavior. This correlates well with the fact that ME/CFS is in many cases constant and not with direct flare patterns like other autoimmune diseases.

 

This is also inline with the recent work and patent application from Fluge and Mella as discussed in another post. The application actually mentions Daratumumab as a possible therapeutic agent:

Patent: Method for the treatment of CFS using an inhibitory or cytotoxic agent against plasma cells, 2021, Fluge, Mella
https://www.s4me.info/threads/paten...-against-plasma-cells-2021-fluge-mella.20289/

 

Here you have the SLE patients vs controls from the actual pre printed study. I cannot see a pattern here for SLE but it is quite interesting anyway:


And here you can see APECED to controls. Of course APECED is know serious disease and here is a clear pattern:

 

As I noted above, if this technique had been tested against known autoimmune diseases and it identifies the known autoantibodies then we an have some degree of confidence that the technique has potential i.e. now. So yes, as @Jonathan Edwards has suggested, test this technique on ME/CFS samples from a biobank.

I'm wondering if this technique currently has all of the likely target proteins [possible self-antigens] i.e. proteins which could be the target of autoimmune antibodies in ME/CFS - @Jonathan Edwards ?

 

Aaron Ring at Yale University is very interested in running some ME/CFS samples on the REAP platform. He asked if I knew of someone who could provide samples. I contacted Solve to see if they
could help him. They failed to follow up with him. I then contacted Dr. Lipkin and asked if he could assist in anyway and he did not answer my email or contact Aaron.

 

Simmaron Research has provided ME/CFS samples to Dr Maureen Hanson's team in the recent past. It would be interesting if Cort Johnson could put Aaron Ring in touch with them.

The Stanford ME/CFS clinic comes to mind, too.

 

A bit out of my league to comment but might Wil de Vega be someone to contact for finding a suitable partner?

https://www.ficars.ca/news/meet-wil-de-vega

Wil finished his Ph.D. at the University of Toronto in 2017, with a focus on epigenomic modifications in the Myalgic Encephalomyelitis / Chronic Fatigue Syndrome patient population.

He is currently a Business Development Director with Mitacs, a national non-profit organization that advances Canada’s innovation agenda through supporting academic-industry R&D collaborations and international academic collaborations.

 

The CureME Biobank has previously shipped samples internationally, https://cureme.lshtm.ac.uk/

If he is looking for fresh samples then perhaps contacting the ME/CFS Clinician Coalition, https://mecfscliniciancoalition.org/, could be a good starting point?

 

The collection of samples that Columbia had have now been moved, as described here,
"The second new tool, searchMECFS, is an online interactive search tool that allows researchers to find biospecimens from ME/CFS studies that are available for research purposes. Researchers can identify and request biospecimens—such as DNA, RNA, urine, plasma, and peripheral blood cells—based on specific demographic and clinical characteristics. The initial cohort providing ME/CFS biospecimens was the Chronic Fatigue Initiative, a research study supported by the Hutchins Family Foundation that collected biospecimens and associated data from 201 individuals with ME/CFS and 200 matched controls. Additional cohort biospecimens and datasets will be available in the future. The specimens are housed at BioSEND, NINDS’ biospecimen repository at Indiana University in Bloomington, and the data are hosted by RTI International. Researchers who access biospecimens through searchMECFS will be able to upload completed assay results into mapMECFS to make the information widely available to other investigators."
https://www.ninds.nih.gov/News-Even...ages/New-resources-large-scale-MECFS-research

 

To be precise, rituximab depletes all CD20+ B cells from pre-B cells onwards to early plasmablasts. At least in some cases it depletes in tissue as well as blood although tissue depletion is variable.

Virtually all antibody is made by plasma cells, which are in both spleen and marrow, with the longer lived ones in marrow. Many cases of SLE remit very well with rituximab (as first reported by our group in around 2000) but trials were done badly initially, with inappropriate entry criteria and assessment. The most recent trial shows a clear positive effect. The anti-CD38 study is a small study showing possibly some benefit but does not amount to a formal trial. Plasma cell targeting is an obvious option, as I pointed out in my lecture to the British Society for Rheumatology AGM a few weeks back, but the pharmacodynamics are really pretty complex. Unfortunately, I am not convinced that any of the groups currently working in the area really appreciate what is involved - other than perhaps Andreas Radbruch.

Antibodies to protein-based nuclear antigens tend to be made by long lived plasma cells and so their clinical correlates do not remit well (e.g. anti-Sm, anti-RNP). It is possible that some people with ME have antibodies to auto antigens produced by long lived plasma cells. I do not see why these particularly need to be extracellular. Nuclear antigens are our best example of fatigue-inducing autoantibodies.

My main reservations about this are that ME does in fact fluctuate quite a lot in many cases and we do not yet have any very plausible immune effector mechanism that would be as invisible in terms of things like CRP or complement malfunction that it seems to be. It is possible but what I have seen in terms of suggested autoantibodies does not look convincing. I am not very impressed by findings in Covid either. Odd antibody responses to cytokines and receptors turn up on assays in lots of situations and turn out not to mean much.

 

Has anyone looked for these in ME/CFS? Also, would an association turn up via the GWAS study?

 

Yes, antinuclear antibody rates have been reported and the consensus is that there is nothing specific to find. I don't have any references bt I think Lipkin and ME Biobank amongst others have looked. Venables looked in the 1990s and found anti-phospholipid antibodies but not ANA I think.

 

I don't have enough knowledge to have a view on immune issues, I defer to those who understand better.

However it is not safe to assume previous studies have ruled anything out in ME. The fukuda definition is not only nonspecific for ME it was very open to interpretation depending on how much weight was given to each part and PEM was not a necessary component. The emphasis on fatigue has distorted a lot of work even by the likes of Lipkin as it is not known how many of the trial subjects really had ME

Also, it was not uncommon for the findings of papers to not show that patients were the same as controls but that some patients had more of the thing and the rest had less.

 

When patients are studied as group, do any autoimmune diseases exist which involve only autoantibodies from long lived plasma cells?

Or is there a mix of short and long lived one?

I was wondering about this because the null response to Rituximab appears to leave only two possibilities: either there is very little autoimmunity in a sample of ME patients or there is autoimmunity but it only involves long lived plasma cells.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289281/

Pemphigus is cited as example of disease driven by short lived plasma cells, while Sjögren’s syndrome is believed to be driven by long lived plasma cells, and lupus involves a mix.

 

Thanks for sharing the article. Very interesting and in line with my thoughts. There is going to be major developments on this on the coming years.