It has become clear that the type 1 interferon responses to SARS-CoV-2 have a great deal to do with the outcome of infection. Patients with benign courses of infection yield high levels of expression of interferon-α (IFNα), while critical cases of COVID-19 show low levels of its expression. Paradoxically, in severe disease, expression of INF receptors is elevated, while expression of INF sensitive genes is depressed, suggesting that things are awry downstream from the receptors. It is not clear if this is because of the activity of virally encoded gene products or the lack of adequate INF-α or both. Variants of genes involved in INF activity have been identified in severe cases of Covid-19 patients. And the virus seems to have multiple proteins that are able to antagonize aspects of INF signaling. We will consider these points separately.
Coronavirus infections in general appear to involve perturbations in INF activities. A good overview of the role of INF in coronavirus infections shows some of the involved signaling pathways(1). Innate immunity is abnormal in severe disease. Early expression of INF-α appears to correlate with a favorable outcome in controlling SARS-CoV-2 infection, whereas INF-β expression appears to be negligible(2). While mild infections are correlated with continued high levels of expression of INF-α, severe disease is marked by low levels of INF-α expression(2). In contrast, inflammatory cytokine expression, as evidenced by IL-6 and TNFα, is high. Although INF-α and inflammatory cytokines are both part of innate immunity, the former is regulated by the transcription factors IRF3 and 7, while the latter are regulated by NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), a protein complex controlling transcription of DNA, cytokine production and cell survival. This suggests that NF-κB signaling is relatively unperturbed by the virus. Thus, its IFN perturbation could be important for antagonizing host defense system.
Some clues as to which factors involved in regulating INF activities may be affected in Covid-19 have been learned by whole exome sequencing of patients with severe disease genomes, particularly those that lack obvious risk factors. In light of this, it is instructive to look at a study of two pairs of young adult brothers, one pair from the Netherlands and the other from West Africa, who had severe Covid-19 (one died)(3). The pairs each had different loss-of-function mutations in TLR7, an intracellular receptor for viral RNA. Primary mononuclear cells showed downregulation of downstream INF signaling, including that of IRF7. The genes are X-linked; this means that men who inherit the defective gene are at greater risk than females, who would need to be homozygous for risk. While loss of function mutations in TLR7 are rare, so is severe Covid-19 in young people, and the congruency of these two rare phenomena is telling. Another study showed loss of function variants in 3.5% (out of 659) of severely ill patients of 13 genes involved in TLR3 and IRF7-dependent INF activities(4). Cells with IRF7 variants produced no type 1 IFN when infected in vitro with SARS-CoV-2.
There is also a report that some patients with severe Covid-19 have neutralizing autoantibodies to IFN-α, -ω, or both(5). These were present in 101 of 987 patients with life threatening disease but in none of the 663 patients with mild or asymptomatic infection. The autoantibodies existed prior to infection. Oddly, most of the patients with autoantibodies were male, whereas generally women have a higher incidence of autoimmunity. This suggests an X-linked genetic basis.
The above studies (and others for which space constraints preclude detailed consideration) point to a failure of INF-related activities as a critical risk factor for the development of severe Covid-19 and suggest that INF represents a potentially serious block to replication of SARS-CoV-2. We might then ask how the virus deals with this problem.
It appears that the virus encodes a number proteins that potentially antagonize cellular signaling pathways involved in INF activity. As a convenient reference to the proteins encoded by SARS-CoV2, we commend you to a website maintained by the Zhang lab at the University of Michigan (https://zhanglab.ccmb.med.umich.edu/COVID-19/). Several studies have shown an effect on IFN activity in vitro(6-10), but these should be interpreted with caution as they use artificially high levels of viral gene expression. In fact, it is not clear at what levels these proteins are expressed in vivo. Moreover, these studies do not completely agree on which are the most relevant viral proteins.
Yuen et al.(10) transfected expression constructs for 27 different genes for their ability to inhibit RIG-I-dependent induction of an IFN-β promoter. RIG-I (retinoic acid-inducible gene I) is a cytosolic pattern recognition receptor (PRR) responsible for the type 1 interferon (IFN1) response. They found that four viral proteins (nsp13, 14 and 15 and orf6) were strongly inhibitory. However, the papain-like protease (PLpro) of SARS-CoV-2 was not an inhibitor, even though the SARS-CoV-1 PLpro is known to inhibit type I IFN by inhibiting IRF3 phosphorylation, thus blocking downstream Interferon induction. This suggests interference with activities downstream from RIG-I. Orf6 also inhibited IFN α-2, β, and λ secretion induced by Sendai virus infection. Using similar methodology, Lei et al.(7) found similar activities with nsp1, 3, 12, 13 and 14, ORFs 3 and 6, and the membrane (M) protein leading to RIG I or MDA5 activation. In contrast, ORF6 overexpression inhibited nuclear translocation of STAT1, which is required for IFN-stimulated gene expression. Li et al.(8) looked at the ability of selected viral proteins to inhibit Sendai virus-induced IFN activity. ORF 6 strongly inhibited the activity of IFN-β, interferon-sensitive response element (ISRE), and NF-κB promoters, as assessed by reporter gene activity. ORF8 and the nucleocapsid protein (N) showed less dramatic inhibitory activity. ISRE related activity was inhibited less markedly by ORF6 when cells were stimulated by IFN-β. From the above studies, there appears to be general agreement that ORF6 is a critical component of IFN antagonism by SARS-CoV-2 infection, with the caveat that the studies all involve overexpression.
Liu et al.(9) show that PLpro has IFN antagonistic activity, but by a different mechanism than those reported above. ISG15 is a multi-function protein that serves as a cytokine but also as a ubiquitin-like intracellular protein conjugate. ISGylation activates the viral RNA sensor MDA5, which is involved in IFN activity. MDA5 activity is antagonized by removal of ISG moieties by PLpro, thus inhibiting IFN expression.
Konno et al.(6) reported that ORF3b, a 22 amino acid protein that is truncated by introduction of a stop codon, antagonizes IFN activity. This is contradictory to SARS-COV-1 homolog. In spite of its small size, ORF3b suppressed Sendai virus-mediated stimulation of the IFNβ1 promoter more strongly than did the larger size of SARS-CoV-1 protein. Interestingly, there are naturally occurring SARS-CoV2 variants in which the full length ORF3b is restored; these are even more strongly inhibitory than is the truncated version.
The above presents a complicated picture that nonetheless points to the importance of type 1 INF in the outcome of Covid-19. A wide variety of data point to defects in the host IFN response that are correlative with severe disease. According to in vitro studies of the viral proteins (with the caveats inherent from the use of overexpression systems), the virus mounts multifactorial defenses against IFN antiviral activities, with ORF6 and PLpro (and perhaps ORF3b) being prime candidates. Hopefully, future in vivo studies will bring more clarity. Overall these studies point to type 1 IFN-signaling as a potential important therapeutic target.
- A. Park, A. Iwasaki, Type I and Type III Interferons – Induction, Signaling, Evasion, and Application to Combat COVID-19. Cell Host Microbe 27, 870-878 (2020).
- J. Hadjadj et al., Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients. Science 369, 718-724 (2020).
- C. I. van der Made et al., Presence of Genetic Variants Among Young Men With Severe COVID-19. JAMA 324, 663-673 (2020).
- Q. Zhang et al., Inborn errors of type I IFN immunity in patients with life-threatening COVID-19. Science, (2020).
- P. Bastard et al., Auto-antibodies against type I IFNs in patients with life-threatening COVID-19. Science, (2020).
- Y. Konno et al., SARS-CoV-2 ORF3b Is a Potent Interferon Antagonist Whose Activity Is Increased by a Naturally Occurring Elongation Variant. Cell Rep 32, 108185 (2020).
- X. Lei et al., Activation and evasion of type I interferon responses by SARS-CoV-2. Nat Commun 11, 3810 (2020).
- J. Y. Li et al., The ORF6, ORF8 and nucleocapsid proteins of SARS-CoV-2 inhibit type I interferon signaling pathway. Virus Res 286, 198074 (2020).
- G. Liu et al., ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease. bioRxiv, (2020).
- C. K. Yuen et al., SARS-CoV-2 nsp13, nsp14, nsp15 and orf6 function as potent interferon antagonists. Emerg Microbes Infect 9, 1418-1428 (2020).
Since the beginning of the COVID-19 pandemic, extensive research has focused on therapeutics. A combinational approach of traditional drug development and drug repurposing is necessary to make progress in treating and preventing COVID-19 in a timely manner. While developing new drugs is critical, repurposing existing molecules has been a major strategy. In the early stages of the COVID-19 pandemic, Remdesivir has been identified as a promising repurposing drug to treat COVID-19 patients (1). Indeed, it became the first treatment for COVID-19 to receive the U.S. Food and Drug Administration (FDA)’s Emergency Use Authorization (EUA) (2). Recently, the FDA issued an EUA for three more treatments. In this perspective, we will review these available treatments based on the findings of clinical trials. In addition, we are updating other COVID-19 therapeutics according to recent publications.
Remdesivir is an intravenous nucleotide prodrug of an adenosine analog and is recognized as an antiviral drug against various RNA viruses (3). It binds to the viral RNA-dependent RNA polymerase and incorporates into nascent viral RNA chains. This results in pre-mature termination of RNA transcription and inhibiting viral replication. The FDA approved remdesivir (Veklury) for use in adult and pediatric patients 12 years of age and older and weighing at least 40 kilograms (about 88 pounds) for the treatment of COVID-19 requiring hospitalization (2). In a recently published report, a double-blind, randomized, placebo-controlled trial of intravenous remdesivir was conducted with hospitalized adults having lower respiratory tract infection (4). A total of 1062 patients underwent randomization (with 541 assigned to remdesivir and 521 to placebo). The reported clinical effect of intravenous remdesivir was very modest. The recovery time was significantly shorter among patients who received remdesivir than among those who received placebo (10 days vs. 15 days). A trend toward lower mortality was observed among patients who received remdesivir than among those who received placebo, both at day 15 (6.7% vs. 11.9%) and at day 29 (11.4% vs. 15.2%), but the differences were not statistically significant. Therefore, the primary outcome was the time to recovery. In fact, WHO has issued a conditional recommendation against the use of remdesivir in hospitalized patients, regardless of disease severity (5). Consistent with this, in a huge WHO sponsored clinical study, remdesivir treatment did not improve mortality for hospitalized COVID-19 patients (6). This trial was conducted in 405 hospitals in 30 countries. A total of 11,266 adults were randomized, with 2750 allocated remdesivir, 954 hydroxychloroquine, 1411 lopinavir, 651 interferon plus lopinavir, 1412 interferon only, and 4088 no study drug. Interestingly, this study concluded that remdesivir, hydroxychloroquine, lopinavir and interferon regimens appeared to have little or no effect on hospitalized COVID-19, as indicated by overall mortality, initiation of ventilation and duration of hospital stay.
The pathogenesis of SARS-CoV-2 involves not only viral replication, but also immunomodulation and inflammation (7). Therefore, a combination therapy of remdesivir with other antivirals or antiinflammatory agents could enhance its efficacy. On November 19, 2020, the FDA authorized a combination of remdesivir (Veklury) and the JAK (Janus kinase) inhibitor, baricitinib (Olumiant) for the treatment of suspected or laboratory confirmed COVID-19 in hospitalized adults and pediatric patients 2 years of age or older requiring supplemental oxygen, invasive mechanical ventilation. This approval is based on data from the Adaptive COVID-19 Treatment Trial (ACTT-2), a randomized double-blind, placebo-controlled study to evaluate the efficacy and safety of baricitinib in combination with remdesivir versus placebo with remdesivir in hospitalized patients with or without oxygen requirements (8). The recommended dose for this approval is baricitinib 4-mg once daily for 14 days or until hospital discharge. Baricitinib has been already approved to treat moderate to severe rheumatoid arthritis. Baricitinib, taken orally, inhibits cytokine signaling in the body that play roles in causing inflammatory responses. Rational of this clinical study is that adding an anti-inflammatory agent to the remdesivir treatment can provide additional benefit for patients, including improving mortality outcomes (8). The putative benefit of baricitinib for COVID-19 has been described in a case series of critically ill patients who recovered from COVID-19. The data from this clinical study have not been published yet. It has been suggested that patients treated with baricitinib in combination with remdesivir had a significant reduction in median time to recovery from 8 to 7 days (12.5% improvement) compared to remdesivir (9). The proportion of patients who progressed to ventilation (non-invasive or invasive) or died by Day 29 was lower in baricitinib in combination with remdesivir (23%) compared to remdesivir (28%). The proportion of patients who died by Day 29 was 4.7% for baricitinib in combination with remdesivir vs. 7.1% for remdesivir. However, the safety of this treatment needs to be validated.
In November 9, 2020, the FDA approved investigational monoclonal antibody therapy bamlanivimab for the treatment of mild-to-moderate COVID-19 in adult and pediatric patients (10). Bamlanivimab is authorized for the treatment of nonhospitalized patients with mild to moderate COVID-19 who are at high risk for progressing to severe disease and/or hospitalization This includes those who are 65 years of age or older, or who have certain chronic medical conditions. The approval is based on data from BLAZE-1, a randomized, double-blind, placebo-controlled Phase 2 study in patients with recently diagnosed mild to moderate COVID-19 in the outpatient setting (11). It is not authorized for patients who are hospitalized due to COVID-19 or require oxygen therapy due to COVID-19. Bamlanivimab (Lily)also known as LY-CoV555 and LY3819253) is a neutralizing monoclonal antibody that targets the receptor-binding domain of the spike protein of SARS-CoV-2. Because this drug may block SARS-CoV-2 entry into host cells, it is being evaluated for the treatment of COVID-19. This trial randomly assigned 452 patients to receive a single intravenous infusion of neutralizing antibody LY-CoV555 in one of three doses (700 mg, 2800 mg, or 7000 mg) or placebo (12). After undergoing randomization, patients received an infusion of LY-CoV555 or placebo within a median of 4 days after the onset of symptoms. The viral load at day 11 (the primary outcome) was lower than that in the placebo group only among those who received the 2800-mg dose. However, the evaluation of the effect of LY-CoV555 therapy on patients’ symptoms at earlier time points during treatment (e.g., on day 3) showed a possible treatment effect, with no substantial differences observed among the three doses. While the safety and effectiveness of this investigational therapy continues to be evaluated, bamlanivimab was shown in this trial to reduce COVID-19-related hospitalization or emergency room visits in patients at high risk for disease progression within 28 days after treatment when compared to placebo. For patients at high risk for disease progression, hospitalizations and emergency room visits occurred in 3% of bamlanivimab-treated patients on average compared to 10% in placebo-treated patients. However, a benefit of bamlanivimab treatment has not been shown in patients hospitalized due to COVID-19. Monoclonal antibodies, such as bamlanivimab, may be associated with worse clinical outcomes when administered to hospitalized patients with COVID-19 requiring high flow oxygen or mechanical ventilation (10).
In November 21, 2020, the FDA approved monoclonal antibodies, casirivimab and imdevimab (Regeneron) to be administered together by intravenous infusion for the treatment of mild to moderate COVID-19 in adults and pediatric patients (12 years of age or older weighing at least 40 kilograms) with positive results of direct SARS-CoV-2 viral testing and who are at high risk for progressing to severe COVID-19 (13). This includes those who are 65 years of age or older or who have certain chronic medical conditions. However, this treatment is not authorized for patients who are hospitalized due to COVID-19 or require oxygen therapy due to COVID-19. These monoclonal antibodies are specifically directed against the spike protein of SARS-CoV-2, designed to block the virus’ attachment and entry into human cells. This approval is based on a randomized, double-blind, placebo-controlled clinical trial in 799 non-hospitalized adults with mild to moderate COVID-19 symptoms. Of these patients, 266 received a cocktail of casirivimab and imdevimab (1,200 mg each), 267 received a cocktail of casirivimab and imdevimab (4,000 mg each), and 266 received a placebo intravenously, within three days of obtaining a positive SARS-CoV-2 test. Viral load reduction in patients treated with casirivimab and imdevimab was larger than in patients treated with placebo at day seven. Further, for patients at high risk for disease progression, hospitalizations and emergency room visits occurred in 3% of casirivimab and imdevimab-treated patients on average compared to 9% in placebo-treated patients. The findings of this study have not been published in a peer-reviewed journal yet. The safety and effectiveness of this investigational therapy for use in the treatment of COVID-19 continues to be evaluated.
As an experimental monoclonal antibody treatment, two ultrapotent SARS-CoV-2 human neutralizing antibodies (S2E12 and S2M11) were isolated and characterized for their potential use as a prophylaxis or therapy (14). Cryo–electron microscopy structures show that S2E12 and S2M11 competitively block angiotensin-converting enzyme 2 (ACE2) attachment. Furthermore, S2M11 is a distinct class of potent neutralizers of SARS-CoV-2 by additionally locking the spike in a closed conformation by recognition of a quaternary epitope spanning two adjacent receptor-binding domains. Combinations of monoclonal antibodies leveraging multiple distinct mechanisms of action with additive or synergistic effects could provide additional benefits for clinical application. Indeed, using cocktail of these antibodies enhanced antibody responses to SARS-CoV-2 S and conferred significant protection in hamster models. The mAb cocktails are also expected to take advantage of both ultrapotent neutralization, different mechanisms of action, and Fc-mediated effector functions to protect from a broad spectrum of circulating SARSCoV-2 isolates. The findings of this study also has important implication in implementing antibody cocktails for circumventing or limiting the emergence of viral escape mutants.
Ivermectin is an FDA-approved broad-spectrum antiparasitic agent with demonstrated antiviral activity against a number of DNA and RNA viruses, including SARS-CoV-2 (15). The inhibition of importin α/β1-mediated nuclear import of viral proteins is suggested as the probable mechanism underlying its antiviral activity. In addition, ivermectin could ultimately induce an ionic imbalance that disrupts the potential of the viral membrane, thereby threatening its integrity and functionality. A recent study showed that a single dose of ivermectin was able to reduce the replication of an Australian isolate of SARS-CoV-2 in Vero/hSLAM cells by 5000-fold (16). In addition to the indication for antiviral therapy, anti-inflammatory intervention may also be necessary to prevent acute lung injury in SARS-CoV-2 infection. With regard to its anti-inflammatory properties, ivermectin have been shown to mitigate skin inflammation. A study evaluating the ability of ivermectin to inhibit lipopolysaccharide (LPS)-induced inflammation revealed significantly decreased production of TNF-alpha, IL-1ss and IL-6 in vivo and in vitro. Further studies may establish the role of ivermectin in inflammatory response caused by SARS-CoV-2. A number of clinical studies are being conducted in various countries. The data from these studies are not available yet. However, it has been suggested that the necessary inhibitory concentration may only be achieved via high dosage regimes in humans. Further, safety of using high-dose antiviral therapy needs to be evaluated. Currently, no commercially available injectable forms of ivermectin are available for human use. The development of ivermectin formulations presents challenges, primarily due to its property of poor water solubility. Novel delivery strategies are needed to optimize ivermectin bioavailability. Furthermore, efficacy of ivermectin for COVID-19 treatment needs to be defined with extensive in vivo study and clinical trials.
Lastly, antiparasitic drug nitazoxanide is widely available and exerts broad-spectrum antiviral activity in vitro. For its use for treatment of mild COVID-19 patients, randomized, double-blind, placebo-controlled was conducted in a multicenter (17). In patients with mild Covid-19, symptom resolution did not differ between the nitazoxanide and placebo groups after 5 days of therapy. However, at the 1-week follow-up, 78% in the nitazoxanide group and 57% in the placebo group reported complete resolution of symptoms. Further, viral load was also reduced after nitazoxanide treatment compared to placebo. This study showed only partially analyzed data from the clinical trials. Complete analysis of this study will be required to clearly assess the efficacy of nitazoxanide for the treatment.
Reinforcing the efficacy of drug repurposing can provide much shorter and less costly development process than developing a new drug. Yet, we have to recognize that beside the very modest activity of remdesivir nothing has so far emerged from these efforts. In fact, patients with respiratory symptoms due to COVID-19 are still best treated by combining the vintage steroids combined with oxygen and anticoagulants. On the other hand, the monoclonal antibody-based treatments have now become available for people in high-risk groups and seem to be effective early in the course of the disease; yet there is a limitation in the production capacity of such therapies and their cost must not be a barrier to get them widely available in low- and middle-income countries. In general, a combination of two to three different therapeutic agents for the treatment has become a common approach. Finally, treatment which might potentially prevent infection (such as Nitazoxanide, Romark LLC) are being evaluated. Overall, despite many difficulties, there is now hope that innovative and cost-effective therapeutic agents can be effectively developed.
- Wang M, Cao R, Zhang L, et al. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 2020;30(3):269-271. Available at: https://www.ncbi.nlm.nih.gov/pubmed/32020029.
- Food and Drug Administration. FDA Approves First Treatment for COVID-19. 2020. Available at: https://www.fda.gov/news-events/press-announcements/fda-approves-first-treatment-covid-19.
- Warren TK, et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature. 2016;531:381–385. doi: 10.1038/nature17180.
- Beigel JH, Tomashek KM, Dodd LE, et al. Remdesivir for the treatment of Covid-19 — final report. N Engl J Med 2020;383:1813-1826.
- World Health Organization. WHO recommends against the use of remdesivir in COVID-19 patients. Available at: https://www.who.int/news-room/feature-stories/detail/who-recommends-against-the-use-of-remdesivir-in-covid-19-patients.
- WHO Solidarity Trial Consortium. Repurposed antiviral drugs for covid-19—interim WHO Solidarity trial results. 15 Oct 2020. doi:10.1101/2020.10.15.20209817.
- Mehta P, McAuley DF, Brown M, Sanchez E, Tattersall RS, Manson JJ. COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet 2020;395:1033-1034.
- National Institute of Health. NIH clinical trial testing antiviral remdesivir plus anti-inflammatory drug baricitinib for COVID-19 begins. Available at: https://www.nih.gov/news-events/news-releases/nih-clinical-trial-testing-antiviral-remdesivir-plus-anti-inflammatory-drug-baricitinib-covid-19-begins.
- Lilly. Baricitinib Receives Emergency Use Authorization from the FDA for the Treatment of Hospitalized Patients with COVID-19. Available at: https://investor.lilly.com/news-releases/news-release-details/baricitinib-receives-emergency-use-authorization-fda-treatment
- Food and Drug Administration. Fact sheet for healthcare providers: emergency use authorization (EUA) of bamlanivimab. 2020. Available at: https://www.fda.gov/media/143603/download.
- Lilly. Lilly’s neutralizing antibody bamlanivimab (LY-CoV555) receives FDA emergency use authorization for the treatment of recently diagnosed COVID-19. Available at: https://investor.lilly.com/news-releases/news-release-details/lillys-neutralizing-antibody-bamlanivimab-ly-cov555-receives-fda.
- Chen P, Nirula A, Heller B, et al. SARS-CoV-2 neutralizing antibody LY-CoV555 in outpatients with COVID-19. N Engl J Med. 2020; Available at: https://www.ncbi.nlm.nih.gov/pubmed/33113295.
- Food and Drug Administration Coronavirus (COVID-19) Update: FDA Authorizes Monoclonal Antibodies for Treatment of COVID-19. Available at: https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-monoclonal-antibodies-treatment-covid-19.
- M. ALEJANDRA TORTORICI et al., A potent antibody cocktail blocks attachment of SARS-CoV-2 to the host receptor and activates a protective immune response. SCIENCE20 NOV 2020 : 950-957.
- Fabio Rocha Formiga, Roger Leblanc, Juliana de Souza Rebouças, Leonardo Paiva Farias, Ronaldo Nascimento de Oliveira, Lindomar Pena, Ivermectin: an award-winning drug with expected antiviral activity against COVID-19, Journal of Controlled Release, 2020, https://doi.org/10.1016/j.jconrel.2020.10.009.
- L. Caly, J.D. Druce, M.G. Catton, D.A. Jans, K.M. Wagstaff. The FDA-approved drug Ivermectin inhibits the replication of SARS-CoV-2 in vitro. Antivir. Res., 178 (2020), p. 104787, 10.1016/j.antiviral.2020.104787.
- Patricia R. M. Rocco et al. Early use of nitazoxanide in mild Covid-19 disease: randomized, placebocontrolled trial. medRxiv preprint doi: https://doi.org/10.1101/2020.10.21.20217208.