Episode 248 : Autoimmunity and COVID part 2
In this second part, I will critically discuss
- various auto-immune phenomena in par 1,
- focus on the role of auto-immunity in thrombotic events during severe COVID in par 2
- summarize evidence about auto-immunity in rare adverse vaccine reactions in par 3
It is a very complex story, but I have tried to illustrate and explain it as good as I can….
PAR 1 AUTO-IMMUNE PHENOMENA: PROPOSED MECHANISMS and criticism
The paragraph is built on a recent review Ep 248-0 Jan Damoiseaux in Autoimmune Reviews March 2022, reporting on the 15th Dresden Symposium on Autoantibodies.
Ep 248-1: Dotan Autoimmune Reviews 2021 starts from the hypothesis that molecular mimicry and hyperstimulation in COVID contribute auto-immune disease
Autoimmunity in COVID-19-infected patients
Overview in Fig 3 of auto-antibodies and (potential) auto-immune diseases associated with COVID
Potential role of NETosis: As explained in Ep 248-2 by Navigantes, neutrophil extracellular traps, induced by infection, can also be instrumental in induction of auto-immune disease
Excessive NET formation has been involved in the autoinflammatory response in SLE, Rheumatoid Arthritis, myositis, type 1 diabetes and Multiple Sclerosis,
Fig. 2. COVID-19 and NETosis. SARS-CoV-2 viral particles invade the alveoli in the lung where they bind type 2 pneumocytes via angiotensin-converting enzyme 2
(ACE2), which is also present on the surface of many other cell types. As a result of the infection, neutrophils transmigrate into the alveoli, where NETosis is activated
leading to release of decondensed chromatin (and other nuclear, possibly modified, components) and granular contents to the extracellular space. This figure was
created using BioRender (https://biorender.com/).
A – SARS-CoV-2 invading the alveoli.
B – SARS-CoV-2 binding to the angiotensin-converting enzyme 2 of the type 2 pneumocytes.
C – Neutrophil transmigrating to the alveoli.
D – Neutrophil extracellular traps activation and release (NETosis).
E – Enhancement of platelet aggregation induced by NETosis.
F – Neutrophil cytokines and proteases degranulation.
G – Modification of self-proteins in the citrullination induced by peptidylarginine deiminases.
Potential role of molecular mimicicry between SARS-CoV-2 and human proteins
The viral versus human peptide overlaps involve human proteins that, if altered, mutated, deficient, or improperly functioning, can lead to severe pathologies.
- Cerebellin-2, alterations of which associate with MS ;
- Follistatin related protein 1 that protects against hypoxia-induced pulmonary hypertension ;
- Solute carrier family 12 member 6, alterations of which may associate with areflexia and severe progressive neuropathy often accompanied by psychiatric symptoms and
- Olfactory receptor 7D4, which is specific for smell
(It is not clearly stated whether auto-antibodies with these specificities have actually been found)
In addition, other possible identities may occur when the self- and viral proteins are folded in the secondary and tertiary structure
Criticism on this short-sequence-mimicry as a trigger for pathological auto-immunity (Bogdanos in Ep 248-0 p. 3):
- Sequential amino acid homologies is a frequent phenomenon and in most of the cases irrelevant to auto-immunity
- Only those mimics that are cross-recognized by antibodies (or T-cells) by COVID-19 sera or post-SARS-CoV-2 vaccinated sera are likely triggers of autoimmunity via molecular mimicry,
- Most mimicking sequences reported by BLAST-searches are stemming from short sequences of the viral or human autoantigen, despite the fact that anti-viral antibodies and autoantibodies in most cases are targeting conformational epitopes. Such targets are hardly recognized by bioinformatic protein-protein analyses.
Bogdanos argues that it is not enough to show antibodies in COVID patients that react with some auto-antigens in ELISA, one should show true cross-reactivity between SARS-CoV-2 antigens and auto-antigens.
He performed “absorption experiments”:
- Increasing amounts of the human autoantigen are used as solid phase competitors were used and incubated with sera from COVID patients: reactivity against SARS-CoV-2 viral protein was not decreased afterwards.
- Similarly, if SARS-CoV-2 antigens were used as solid phase, there was no decrease of auto-Ag reactivity in sera from a variety of patients with genuine auto-immune disease.
According to Bogdanos, the clearly negative results in these “cross-reactivity” studies argue against the presence of antibodies with sufficient affinity for auto-antigens to induce clinically significant auto-immunity.
PAR 2 COVID-19 IS A PROTHOMBOTIC CONDITION: what is the role of auto-immunity?
High frequencies of prothrombotic conditions in severe COVID:
- Severe COVID: venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary thrombosis (PE), up to 30%
- During intensive care: myocardial infarction up to 8 %; cerebrovascular accidents (CVA) 3 %; disseminated intravascular coagulation (DIC) 7 %
- In addition, at autopsy: microtrombi found in various organs, including lungs, liver and kidney
- Cytokine storm and hyperinflammation, including NETosis and endothelitis activates coagulation system.
- But what is the implication of auto-immunity ?
- Importance of antiphospholipid antibodies (APLA or aPL)?
Reminder Ep 247-4: review on the anti-phospholipid syndrome in tempore non suspect (2010) in Lancet. As can be see, there are several similarities with the extra-pulmonary hyper-coagulator COVID state.
Antibodies associated with anti-phospholipid syndrome (APS) are anticardiolipin (aCL), lupus anticoagulant (LAC) and beta2 glycoprotein I (β2GPI): leading to coagulation dysfunction and thrombus formation.
Several papers refer to occurrence of these antibodies in severe COVID e.g.
- 31 out of 66 (47%) severely-ill SARS-CoV-2-infected patients had produced β2GPI or/and aCL circulating autoantibodies .
- Patients with severe COVID-19 had significantly higher aCL autoantibody levels than patients with moderate disease .
- High concentrations of LAC among COVID-19 patients enduring coagulation disorders 
- A study of 172 hospitalized patients with SARS-CoV-2-infection reported that 24% carried aPS/PT IgG .
HOWEVER: it is far from sure that these antibodies are really crucial for thrombotic events during COVID hospitalization.
See Ep 248-3: Borghi in Front Imm Oct 2020: on aPL in COVID-19
- show a low prevalence of aPL and are not associated with major thrombotic events.
- are mainly directed against b2GPI with different epitope specificity from antiphospholipid syndrome.
See Ep 248-4: Gil-Etayo Biomedicines July 2021:
- No difference in prevalence of aPL in hospitalized COVID versus a carefully matched healthy population.
- Time to develop thrombotic event in aPL(+) patients LATER than in aPL(-) ones
DVT : deep venous thrombosis
PE: pulmonary emboly
AT = arterial thrombosis
- Nevertheless, there is a clear association between aPL and thrombosis….
DIFFICULT TO RECONCILE ALL THESE DATA:
- For sure, aPL in older COVID patients can be present, but are not 100 % linked with thrombosis
- However, they may play some pathogenic role:
Proposed hypothesis by Pier Luigi Meroni in Ep 248-0 p.4:
- COVID-19 patients suffer from a systemic inflammation with complement activation, which may be responsible for high density of β2GPI on the activated endothelium [24–27]. In this context, even low titers of aPL may become pathogenic, thus potentiating or even triggering thrombus formation, especially when anticoagulation is suspended.
- While transitory aPL are likely to be clinically irrelevant in COVID-19 patients as in other infections, detection of aPL may be useful for identifying patients potentially at risk of thrombosis after the hospital discharge.
- Heparin-induced thrombocytopenia (HIT) and anti-platelet factor 4 (aPF4)
Anti-heparin-PF4 (aPF4), a platelet-activating antibody that is used as a marker for heparin-induced thrombocytopenia (HIT), were identified in severely-ill COVID-19 patients. In some patients aPF4 had been recognized without a pre-exposure to heparin, thus strengthening the hypothesis that SARS-CoV-2 has the ability cause coagulation disorders though an autoimmune mechanism, particularly in severely-ill patients [52,53].
Ep 248-5: Bailly SAMJ Sept 2021: Heparin-induced thrombocytopenia: An update for the COVID-19 era
The interaction between heparin and antithrombin (AT) within the coagulation cascade
Anti-Thrombin is a serine protease inhibitor and major natural anticoagulant. Heparin binding to AT activates it into a potent inhibitor of thrombin (activated factor II) and activated factor X.
(AT = antithrombin; TF = tissue factor; FVIIa = activated factor VII; Factor Xa = activated factor X; Factor Va = activated factor V.)
Pathogenesis of clinically significant heparin-induced thrombocytopenia
Simplified model, explaining both thrombocytopenia and thrombosis: complexes between platelet factor 4 (PF4) and heparin elicit auto-antibodies, resulting in immune complexes that bind to platelets. This has a dual effect
- Platelet activation, resulting in thrombus formation
- Platelet removal (via Fc receptors on macrophages) resulting in thrombocytopenia
- Plasma cells produce and secrete specific IgG antibodies in response to circulating PF4/heparin complexes. The antibody/PF4/heparin immune complexes then bind platelets, monocytes and neutrophils via their Fcγ receptors, resulting in platelet and immune system activation.
- Procoagulant mediators such as Tissue factor (TF)-rich microparticles are released, and NETosis occurs.
- NETosis due to neutrophil extracellular trap formation, and the release of TF-rich microparticles result in activation of the coagulation cascade and thrombin formation.
- Thrombin, a potent activator of platelets, monocytes and endothelial cells, further potentiates their activation and TF production. Widespread activation of the coagulation cascade results in thrombus formation in numerous organ systems and tissues, resulting in ischaemia and localised necrosis.
- The activation and incorporation of platelets within the thrombi, as well as increased peripheral sequestration by the spleen, result in thrombocytopenia.
(IgG = immunoglobulin G; PF4 = platelet factor 4; TF = tissue factor; MPs = microparticles; GAGs = glycosaminoglycans.)
IT mostly due to unfractionated heparin (UF) > low molecular weight heparin (LMWH)
Clinical spectrum and management of HIT:
The 4Ts scoring system: looking at
- Thrombocytopenia degree
- Timing of platelet drop
- Thrombosis extent
- Likelihood of other causes for thrombocytopenia
→ Likelihood of HIT: scores > 6
HIT and COVID:
- Both conditions predispose to thrombosis and thrombocytopenia;
- In COVID-19, heparin is the mainstay of thromboprophylaxis and therapy, whereas immediate discontinuation of heparin is absolutely essential in the management of HIT.
- An increased frequency of anti-PF4/heparin antibodies has been shown in COVID, but true prevalence uncertain
- Thrombocytopenia and disseminated intravascular coagulation (DIC) may occur in severe fatal COVID-19, and moderate thrombocytopenia is found in HIT
- Monitor platelets: baseline and alternate-day platelet count monitoring for hospitalised COVID-19 patients receiving any form of heparin therapy (UFH or LMWH; prophylactic, intermediate or therapeutic dosing; dialysis catheter flushing).
- If suspicion of HIT: Check the clinical 4Ts and determine HIT antibodies (see Table 5).
Ref 248-6 and -7: Two complementary papers by Favorolo, discussing the various types of HIT and the laboratory tests used.
To make the diagnosis of HIT, not only the anti-PF4 Ab should be shown, but also their functional activity
Figure 1. Platelet responses induced by HIT antibodies and functional assay targets. (Tardy J Clin Med 2020)
Donors’ platelets are stimulated in whole blood (WB), platelet-rich plasma (PRP), or after washings (washed platelets, WP).
Gold Standard =
- Serotonin release assay (SRA), which measures the release of serotonin from dense granules when platelets are activated,
- Heparin-induced platelet aggregation (HIPA), based on a visual inspection of platelet aggregates,
The oldest but probably the most used assay is light transmission aggregometry (LTA), classically performed with PRP, but which can also be carried out with WP. Platelet aggregation can also be assessed in whole blood using heparin-induced multiple electrode aggregometry (HIMEA), which considers the possible contribution of monocytes, neutrophils, and red blood cells (RBC) to platelet activation induced by
HIT antibodies. Dense granules secretion may also be evaluated by measuring the ATP released using chemiluminescence. Several assays based on flow cytometry (FC) have also been proposed, most of them assessing P-selectin (CD62P) expression or Annexin V binding to phosphatidylserine on the surface of activated platelets.
Three diagnostic categories
Only HITT = really pathogenic
True significance of HITT in COVID pathology?
After a thorough review of literature it is evident that auto-immunity is at the base of thrombosis in a minority of COVID patients: Favorolo
BUT: question over the role of heparin in some of the cases reported in the literature, and instead, a “HIT-like” event may be occurring in some patients without heparin exposure.
This may be linked to theoretical concepts around PF4/Heparin mimicry in infectious disease, although additional confounders may exist in relation to COVID-19.
The binding of SARS-CoV-2 spike protein to platelet surface receptor(s), leading to platelet activation, is another potential mechanism that deserves further scrutiny in the pathogenesis of thrombosis in COVID-19.
It is evident that a syndrome, closely resembling heparin-induced thrombocytosis and thrombocytopenia (HITT) occurs in a low proportion of severe COVID patients.
To make this diagnosis, three elements are needed:
- The clinical definition 4Ts
- Presence of anti-PF4 antibodies
- Functional activity of these antibodies (serotonin release assay, platelet aggregation assay).
According to the thorough analysis of Favorolo, only a minority of the cases, reported in the literature, concur with these prerequisites.
Remarkably also, heparin is apparently not always involved in the syndrome.
All in all, neither anti-phospholipid nor anti-PF4 antibodies could explain the majority of frequent thrombotic phenomena in severe COVID, which seems “multifactorial” in pathogenesis….
PAR 3 Vaccine induced auto-immune phenomena
Ep 248-8 : Very nice summary of new-onset autoimmune phenomena post-COVID-19 vaccination by Yue Chen et al in Immunology Oct 2021
These are all very rare conditions: the prevalence is not exactly known (only case reports in the literature), with the exception of VITT that has a prevalence of about 1 in 100,000 Adenoviral vaccinations.
Out of this list, the former five (VITT, ITP, auto-immune liver disease, GBS and IgA nephropathy) are most clearly linked to vaccination.
Pathogenic mechanisms have not been fully elucidated, but may include molecular mimicry, role of adjuvants and stimulation of (pre-existing) auto-antibody B cell clones.
Potential mechanisms for adjuvant-induced allergic and auto-immune reactions as well as VIIT are illustrated in Fig 1 and 2 (and further detailed in the text).
Principles of treatment
VITT: no-heparin anticoagulant and intravenous immunoglobulin therapy
ITP and GBS: also intravenous immunoglobulins
(Intravenous normal immunoglobulins should occupy Fc receptors on macrophages to prevent binging of the pathological auto-immune Ig)
Auto-immune hepatitis and SLE: prednisone
Arthritis: non-steroidal anti-inflammatory agents or glucocorticosteroids
3.2 More details on vaccine-induced thrombocytosis and thrombocytopenia (VITT)
Clearly, this syndrome is very reminiscent of the heparin-induced HITT, but without involvement of heparin !!
There are numerous papers about VITT and I will just briefly discuss some overviews.
Ep 248-9: Sakar Ann Thorac Med Jan 2022 analyzes the first series of cases in Europe and US
Mostly young female subjects of thrombosis, with low platelets, evidence consumption of coagulation factors (low fibrinogen, high D-dimer) and anti-PF4. Platelet-activating Ab in most Astra-Zeneca cases
Proposed diagnostic approach:
Treatment: anticoagulants (other than heparin seev below) and intravenous immunoglobulins
Ep 248-10: Elberry thoroughly analyzes 26 papers (21 Astra-Zeneca with 157 cases and 5 J&J with 15 cases).
Summary of clinical findings:
Presenting symptoms = vague: headache, eye symptoms, fever, back pain, epigastric pain, and nausea/vomiting
Clear evidence of thrombocytopenia, coagulation and anti-PF4 in most investigated cases.
- There is very clear evidence for a role of auto-antibodies to type 1 interferon as a predisposing factor for severe COVID and for anti-platelet factor 4 Ab in Adenoviral vaccine induced thrombocytosis and thrombocytopenia (VITT).
- The role of Spike protein “superantigenic sites” in genetically predisposed (young) individuals to super-activate particular T cell clones in MISC-C seems very likely
- Various auto-immune phenomena may play a role in some complications of severe COVID and in some rare reactions to vaccines, but this needs to be further investigated
There is (much) more literature on auto-immunity, but I leave it here for now….
5 Oct 2022 Episode 289: Omicron BA.2.75 revisited and the outlook for new variants, including BQ.1.1
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