Long COVID

Damaged endothelial cells from initial viral infection circulate the blood a month after symptom onset https://doi.org/10.7554/eLife.64909

These will activate monocytes and neutrophils, but are also thought to facilitate clotting which will add to symptoms.

Importantly, this late in the disease recovery it is not surprising to note that IL-2 and other pro-T cell cytokines are elevated which correlates with enhanced effector T-cells https://doi.org/10.7554/eLife.64909

These adaptive cells along with autoantibodies likely propagate the effects of COVID-19 long term.

C5a inhibition with IFX-1 appears to be safe in patients with severe COVID-19. The secondary outcome results in favour of IFX-1 are preliminary because the study was not powered on these endpoints, but they support the investigation of C5a inhibition with IFX-1 in a phase 3 trial using 28-day mortality as the primary endpoint https://doi.org/10.1016/S2665-9913(20)30341-6

Increased antibody dependent complement deposition and lower antibody dependent phagocytosis has been observed in hospitalised COVID-19 patients associated with higher inflammation https://doi.org/10.1101/2021.01.11.426209 . This is intriguing as perhaps the type of antibody produced against SARS-CoV-2 has a role in promoting disease severity (via complement activation). This could also potentially involve auto-antibodies.

The association between complement activation and the biomarkers of endothelial damage suggests that complement may contribute to tissue injury and could be the target of specific therapy https://doi.org/10.1016/j.jaut.2020.102560

Complement deposits in different organs of deceased COVID-19 patients caused by activation of the classical and alternative pathways support the multi-organ nature of the disease https://doi.org/10.1101/2021.01.07.21249116

Importantly, principal component analysis suggested that gene expression profiles in circulating leukocytes in COVID19 patients are unique to SARS-CoV-2 and distinct from profiles in sepsis and ARDS https://doi.org/10.1186/s40635-020-00361-9

HLA-DR and the co-stimulatory CD83, CD86, ICOSLG and ICAMI were found to be decreased in DCs. Also decreased in critical compared to mild-moderate COVID19 patients https://doi.org/10.21203/rs.3.rs-60579/v1 (preprint)

Migratory DCs in BAL fluid of COVID19 patients express TNF and IL12B which supports altered T cell polarisation https://doi.org/10.1101/2021.01.13.21249725 (preprint)

Suggested mechanism for this is via Spike protein binding TLR4, this can be blocked with NFkB and TLR4 inhibitors potentially offering a therapeutic strategy https://doi.org/10.1101/2020.12.18.423427

Additionally SARS-CoV-2 nucleocaspid can drive IL-6 in macrophages https://doi.org/10.3390/vaccines9010054

SLAMF7 'super activation' has been detected in lung infiltrating and monocyte derived macrophages of COVID19 patients resulting in significant upregulation of proinflammatory cytokines https://doi.org/10.1101/2020.11.05.368647 (preprint)

CD127 was upregulated in all monocyte subsets in COVID19 patients and other inflammatory conditions. These monocytes retained anti-inflammatory functions in pro-inflammatory environments. In COVID19 expansion of this subset was associated with mild disease https://doi.org/10.1101/2020.11.10.376277 (preprint)

Classical monocytes are proposed to be the main source of IL-1b, IL-18, CCL2, CXCL8 and TNF-a https://doi.org/10.21203/rs.3.rs-60579/v1 (preprint)

Circulating CD1QA/B/C+CD16+ monocytes express receptor-ligands that are predicted to interact with platelets and could be involved in thrombosis https://doi.org/10.1101/2021.01.13.21249725 (preprint)

Single cell analysis of BAL fluid cells from SARS-CoV-2 infected ferrets found 10 subsets of macrophages including resident and monocyte-derived. They revealed a diverse transcriptional response mainly of infiltrating monocyte-derived cells during the course of infection and in response to different treatments. The type I interferon signature was present in all https://doi.org/10.1101/2020.11.18.388280 (preprint)

https://www.biorxiv.org/content/10.1101/2021.02.03.429351v1 (preprint)

Meta-analysis showed IL-6 was higher in severe COVID-19 than in sepsis/non-COVID ARDS, hence has the potential to be a good biomarker or indicate a different disease mechanism https://doi.org/10.1016/S2213-2600(20)30404-5

IL-6 measured in 1484 patients was shown again to predict severity/death, median level was 67.7pg/mL https://doi.org/10.1038/s41591-020-1051-9

Meta analysis of 50 studies showed significant increase in IL-2, IL-4, IL-6, IL-8, IL-10, TNF-a, and IFN-y in severe patients compared to non-severe patients https://doi.org/10.1016/j.lfs.2020.118167

Another open-label trial NCT02735707 showed that both IL-6 receptor antagonists improved recovery and survival compared to standard care https://doi.org/10.1101/2021.01.07.21249390 (preprint)

Reduced innate lymphoid cells with age and in males may account for increased risk of severe COVID-19 https://doi.org/10.1101/2021.01.14.21249839

In addition, a study has shown that a combined reduction in circulating NK cells in the blood (as well as T cells) alongside increased IL-6 and IL-10 was observed in non-survivors of COVID-19 until death https://doi.org/10.1186/s12879-021-05792-7

Blockade of NFkB/TLR4 pathway may also be beneficial due to the SARS-CoV-2 activation of TLR4 https://doi.org/10.1101/2020.12.18.423427

Anti-viral neuraminidase inhibitors such as Oseltamivir or Zanamivir reduced neutrophil hyper-activation (via inhibition of host neuraminidase) and may present a new targeted therapeutic strategy https://doi.org/10.1101/2020.11.12.379115

IL-18 blockade may represent a therapeutic option for COVID-19 as it may participate in hyperinflammation and tissue damage https://doi.org/10.1002/jcp.30008

Increasing evidence that NK cells could be used as therapies for COVID-19 https://doi.org/10.1186/s40164-021-00199-1 as well as evidence for targeting specific mutants of COVID-19 with ‘off-the shelf’ CAR-NK cells https://doi.org/10.1101/2021.01.14.426742

An ongoing clinical trial (NCT04371367) investigating Avdoralimab, an antibody for C5a receptors, which may disrupt C5a engagement with NK cells in COVID-19 https://doi.org/10.3389/fimmu.2020.586765

As NK cells do not express the COVID-19 host entry receptor ACE2 the authors suggest NK cell loss unlikely due to infection induced apoptosis that is observed in influenza infection https://doi.org/10.1038/s41586-020-2922-4 https://doi.org/10.3389/fimmu.2020.586765

Further evidence for functionally competent CXCR3+, CXCR6+ and/or CCR5+ NK cell migration to the lungs in moderate COVID-19. Lung homing receptor expression is biased towards phenotypically activated NK cells. https://doi.org/10.1101/2021.01.13.426553

In Healthy donor NK cells, in vitro stimulation of the soluble IL-6 receptor by IL-6 reduced NK cell degranulation markers perforin and granzyme https://doi.org/10.1002/art.39295 which suggests that IL-6 may hasten the downregulation of granzyme expression on NK cells reported in COVID-19 https://doi.org/10.1016/j.ajpath.2020.08.009

HLA-C, the dominant ligand for KIR receptors on NK cells, has also been observed as a novel genetic predictor of clinical course in COVID-19 https://doi.org/10.1101/2020.12.21.20248121

HLA-A/B types, which bind T cells, are correlated with population responses to COVID-19 https://doi.org/10.3389/fimmu.2020.586765 https://doi.org/10.3389/fimmu.2020.565730

In addition, another study shows the genetic variants associated with NKG2C loss were significantly overrepresented in hospitalised patients with COVID-19, particularly those requiring intensive care compared to those with mild symptoms https://doi.org/10.1038/s41436-020-01077-7

The uniform reduction in CD56bright NK cells suggests that peripheral NK cells do not directly contribute to cytokine storm in COVID-19 https://doi.org/10.1016/j.ajpath.2020.08.009

In addition, crosstalk with monocytes and macrophages via cytokines which may mediate NK cell dysfunction and impair SARS-CoV-2 clearance https://doi.org/10.1016/j.ajpath.2020.08.009

Neutrophil to lymphocyte ratio as a cost-effective biomarker of COVID-19 severity https://10.1016/j.ajem.2021.01.006

Rebendenne A, Valadão ALC, Tauziet M, Maarifi G, Bonaventure B, McKellar J, Planès R, Nisole S, Arnaud-Arnould M, Moncorgé O, Goujon C. SARS-CoV-2 triggers an MDA-5-dependent interferon response which is unable to control replication in lung epithelial cells. J Virol. 2021 Jan 29:JVI.02415-20 https://doi.org/10.1128/JVI.02415-20

Bayati A, Kumar R, Francis V, McPherson PS. SARS-CoV-2 infects cells following viral entry via clathrin-mediated endocytosis. J Biol Chem. 2021 Jan 18;296:100306 https://doi.org/10.1016/j.jbc.2021.100306

Zhou, Q., Chen, V., Shannon, C. P., Wei, X.-S., Xiang, X., Wang, X., Wang, Z.-H., Tebbutt, S. J., Kollmann, T. R., & Fish, E. N. (2020). Interferon-α2b Treatment for COVID-19. Front Immunol, 11(1061) https://doi.org/10.3389/fimmu.2020.01061

Baker SA, Kwok S, Berry GJ, Montine TJ. Angiotensin-converting enzyme 2 (ACE2) expression increases with age in patients requiring mechanical ventilation. PLoS One. 2021 Feb 16;16(2):e0247060 https://doi.org/10.1371/journal.pone.0247060

A M58R mutation in ORF6 inhibited the binding of orf6 to the nuclear core protein Nup98-Rae1 complex, abolishing its IFN antagonistic function https://doi.org/10.1073/pnas.2016650117

D614G mutation not associated with higher mortality, but is associated with a higher viral load and affecting younger patients https://doi.org/10.1016/j.cell.2020.11.020 . D614G mutation proposed to allow increased epitope exposure and greater neutralisation, thus should not affect vaccine efficacy https://doi.org/10.1016/j.chom.2020.11.012

Additionally a N501Y mutation in the S1 has been reported. This mutation is already present in the UK SARS-CoV-2 variant (20B/501Y.V1, B1.1.7 lineage), and is associated with high higher rates of transmission through increased receptor binding https://doi.org/10.1101/2021.01.04.425316 and potentially higher viral loads https://doi.org/10.1101/2021.01.12.20249080 . However, post vaccination sera can neutralise this variant and thus current vaccines in circulation should protect against this strain https://doi.org/10.1101/2021.01.19.21249592 .

Interestingly, examination of the global effects of the N501Y mutation revealed that MHCII presentation was poorer than wild type controls. This implicates the N501Y mutation in hindering immune cell cooperation, resulting in immune escape https://doi.org/10.1101/2021.02.02.429431

and also possibly by enhancing the lysosomal trafficking of the SARS-CoV-2 spike protein https://doi.org/10.1101/2020.12.08.417022

Another mutation in the receptor binding domain of the S protein, E484K, has been found in the South African and Brazilian variants of the virus. The glutamate to lysine substitution switches the charge on the flexible loop region of the RBD resulting in the formation of novel favourable contacts https://doi.org/10.1101/2021.01.13.426558 . Early studies indicate that higher antibody titres will be required post vaccination to neutralise the variant https://doi.org/10.1101/2021.01.26.21250543

Indeed, a recent study indicates that diversity of ACE2 expression amongst those of different ethnicities impacts selection pressures for mutations in SARS-CoV-2, for example, the D614G mutation has become dominant in North America, Europe and Africa where ACE2 expression amongst the population is low in comparison with those from China, where D614G is not the dominant form https://doi.org/10.3390/genes12010016

Ester Gea-Mallorquí is co-corresponding author, email: ester.gea-mallorqui@ndm.ox.ac.uk

Link to part II https://academic.oup.com/ooim/article/1/1/iqaa005/6026120

Corresponding email: ester.gea-mallorqui@ndm.ox.ac.uk

Corresponding author: ester.gea-mallorqui@ndm.ox.ac.uk

https://clinicaltrials.gov/ct2/show/NCT04320238

Wang, K., Chen, W., Zhang, Z. et al. CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells. Sig Transduct Target Ther 5, 283 (2020). https://doi.org/10.1038/s41392-020-00426-x

Zhang, L., Jackson, C.B., Mou, H. et al. SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity. Nat Commun 11, 6013 (2020). https://doi.org/10.1038/s41467-020-19808-4

Lee, I.T., Nakayama, T., Wu, CT. et al. ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs. Nat Commun 11, 5453 (2020). https://doi.org/10.1038/s41467-020-19145-6

Studies have shown that neutrophils taken from severe COVID-19 patients are immunosuppressed, with a lack of ability to phagocytose and kill bacteria https://doi.org/10.21203/rs.3.rs-142927/v1 , https://doi.org/10.1101/2020.12.01.406306

It is unclear at this stage whether this defect is programmed into the cells (inherent) or whether it is a result of the in-situ environment – i.e. already activated cells are likely to have a refractory loss of function. This activation has been heavily reported and shown again here with neutrophil elastase deposition in the blood of severe COVID-19 patients, which can predict organ damage https://doi.org/10.1111/all.14746

However, what is clear is that these cells are not as functional against new threats and this may explain the reported secondary bacterial infections in COVID-19. (Note: this will be covered in a future update)

Link to part I https://academic.oup.com/ooim/article/1/1/iqaa004/6026109

Another study was unable to find a correlation between severe COVID-19 and polymorphisms that result in loss-of-function in type-1 IFN signalling (TLR3- and IRF7-dependent) https://doi.org/10.1101/2020.12.18.20248226

Monocyte and neutrophil aggregates with platelets have been identified in COVID-19 patients and correlate with disease severity https://doi.org/10.1055/s-0040-1718732

Von Willebrand’s factor and ADAMTS13 are also implicated in NET-associated immunothrombosis https://doi.org/10.3389/fimmu.2020.610696 , https://doi.org/10.1002/ctm2.268

Additionally, NETs have been shown to accumulate and persist in the lungs of severe COVID-19 patients https://doi.org/10.1093/infdis/jiab053 and recruits coagulation Factor XII to promote clotting https://doi.org/10.1101/2020.12.29.424644 .

This persistence is attributed to poor DNase clearance of NETs https://doi.org/10.1111/all.14746

A range of autoantibodies is now thought to be promoted in severe COVID-19 https://doi.org/10.1101/2020.12.10.20247205

It is not thought that circulating immune cells (i.e. monocytes) become infected with SARS-CoV-2 to a large extent https://doi.org/10.1101/2021.01.19.427282

Additionally, a study showed that SARS-CoV-2 does not readily infect human monocyte derived macrophages nor drive significant cytokine production https://doi.org/10.1101/2020.12.22.423940

However, this is not seen in other studies and the cells primarily thought to be infected would rather be lung alveolar macrophages. These have different receptors, origins (i.e. embryonic vs monocyte perhaps), and are in an in situ setting unlike studies with in vitro-derived monocytic macrophages (6-day culture with only one growth factor). Therefore, this is still unclear.

In contrast low C3 and C4 have been observed, though it is unclear whether this is decreased production or increased utilisation https://doi.org/10.21203/rs.3.rs-127493/v1

SARS-CoV-2 N protein is thought to bind MASP-2 as part of the lectin pathway.

The lectin pathway is thought to be generally increased in COVID-19 https://doi.org/10.1016/j.trsl.2020.11.008

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https://clinicaltrials.gov/ct2/show/NCT04320238

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