key: cord-0261088-4bvxj7u3 authors: Bojkova, Denisa; Widera, Marek; Ciesek, Sandra; Wass, Mark N.; Michaelis, Martin; Cinatl, Jindrich title: Reduced interferon antagonism but similar drug sensitivity in Omicron variant compared to Delta variant SARS-CoV-2 isolates date: 2022-01-04 journal: bioRxiv DOI: 10.1101/2022.01.03.474773 sha: ec951a26a434ac5b3e9510e5edd663558692e138 doc_id: 261088 cord_uid: 4bvxj7u3 The SARS-CoV-2 Omicron variant is currently causing a large number of infections in many countries. A number of antiviral agents are approved or in clinical testing for the treatment of COVID-19. Despite the high number of mutations in the Omicron variant, we here show that Omicron isolates display similar sensitivity to eight of the most important anti-SARS-CoV-2 drugs and drug candidates (including remdesivir, molnupiravir, and PF-07321332, the active compound in paxlovid), which is of timely relevance for the treatment of the increasing number of Omicron patients. Most importantly, we also found that the Omicron variant displays a reduced capability of antagonising the host cell interferon response. This provides a potential mechanistic explanation for the clinically observed reduced pathogenicity of Omicron variant viruses compared to Delta variant viruses. Omicron (B.1.1.529), is a heavily mutated and highly contagious SARS-CoV-2 variant, which was first detected in southern Africa. It has already replaced the previously dominating Delta variant (B.1.617.2) in some places and is expected to become the dominant SARS-CoV-2 variant in most parts of the world. Protection provided by the current vaccines is substantially reduced against Omicron [1] [2] [3] . Moreover, there are many immunocompromised individuals who cannot be effectively protected by vaccines [4] . Hence, antiviral therapies will be essential to protect the most vulnerable individuals from severe COVID-19. A number of antibody therapies have been approved for use in individuals at a high risk from COVID-19 [5] . Moreover, a range of antiviral small molecule drugs are under investigation or already approved for the treatment of COVID-19. Remdesivir, an intravenous inhibitor of the viral RNA-dependent RNA polymerase (nsp12), was the first antiviral drug to be approved for the treatment of COVID-19 [5, 6] . Molnupiravir and PF-07321332 are oral antiviral drugs that are hoped to be able to overcome the issues associated with an intravenous agent [5] . Molnupiravir, a derivative of the broad-spectrum antiviral drug ribavirin, is metabolised into the active compound EIDD-1931, which is incorporated into the complementary RNA strand that is used as a template for the synthesis of viral genomic RNA during replication of the SARS-CoV-2 RNA genome. The incorporation of EIDD-1931 into the template strand causes excessive mutations in the newly produced viral genomes, which affect their functionality in a process called 'error catastrophe' or 'lethal mutagenesis' [7] . In the UK, molnupiravir is approved and treatment of vulnerable SARS-CoV-2-infected individuals early after diagnosis has been started. The combination of PF-07321332 (nirmatrelvir) and ritonavir (which reduces PF-07321332 metabolism), also known as paxlovid, has been reported to reduce hospitalisation of SARS-CoV-2-infected individuals in clinical trials [5] . Other antiviral drug candidates for SARS-CoV-2 include the protease inhibitors, camostat, nafamostat, and aprotinin, which inhibit cleavage and activation of the viral spike (S) protein by host cell proteases and, in turn, SARS-CoV-2 entry into host cells [8] . Initial findings suggested that antibody therapies display reduced activity Methods) [10] in Caco-2 and Calu-3 cells as previously described [11] [12] [13] [14] . The Omicron isolates infected fewer cells in Calu-3 and Caco-2 cell cultures when compared with the Delta isolate ( Figure 1A , Figure 1B ), which is in agreement with previous findings in Calu-3 cells [15] and in the hamster upper respiratory tract [16] . However, all three isolates displayed comparable infection patterns in Vero cells ( Figure 1A , Figure 1B ). In contrast to Caco-2 and Calu-3 cells, Vero cells display a defective interferon response and represent an established model for studying virus replication in an interferon-deficient host cell background [17, 18] . Hence, the antagonising cellular interferon signalling than Delta viruses. In agreement, the Delta isolate displayed superior infection patterns in A549 cells transduced with ACE2 (cellular receptor for the SARS-CoV-2 S protein) and TMPRSS2 (cleaves and activates S) [19] , but not in the same cell model with defective interferon signalling due to MDA5 knock-out [20] ( Figure 1C) . Moreover, the Omicron isolates, but not the Delta isolate, activated interferon signalling as indicated by activation of the interferon response factor (IRF) promotor in A549 cells, which was prevented by MDA5 knock-out ( Figure 1D ). Taken together, these data show that Omicron viruses are less effective than Delta viruses in antagonising the interferon response in human cells, which may contribute to the lower pathogenicity of the Omicron variant observed in patients [21, 22] . Notably, SARS-CoV-2 proteins known to inhibit the host cell interferon response including S, NSP3, NSP6, NSP14, nucleocapsid (N), and membrane (M) are mutated in the Omicron variant [23, 24] . EIDD-1931, PF-07321332, remdesivir, favipravir, ribavirin, nafamostat, camostat, and aprotinin and, hence, to a range of drugs representing different mechanisms of action ( Figure 1E ). This shows that the mutations in the Omicron variant do not cause substantial changes in the drug sensitivity profiles of the viruses. With regard to drugs targeting the RNA-dependent RNA polymerase and the replication of the viral genome, this may not come too much as a surprise. Across the replicase-transcriptase complex (nsp7, nsp8, nsp9, nsp10, nsp12, nsp14), only two missense mutations were present in the investigated Omicron isolates, both of which are part of the set of mutations that define the Omicron variant. The RNA-dependent RNA polymerase Nsp12 contains a single change, P323L, which was also present in the Alpha, Beta, and Gamma variants. P323L is far removed from the RNA binding site (Suppl. Figure 1) , and would not be expected to impact on RNA implication based on a structural analysis. One further variant-defining mutation was present in the exonuclease (nsp14), resulting in an I42V change, which is present near the interface site with nsp10. This is a conservative substitution of two small hydrophobic side chains. Structural analysis shows the I42 side chain contacting V40 and N41, which directly contact nsp10 (Suppl. Figure 2 ). However, this is a minor change that seems unlikely to have a significant impact on the interaction with nsp10 or on antiviral drug activity. In contrast to our study, which did not detect differences between the sensitivity of Omicron and Delta isolates to TMPRSS2 inhibitors, one previous study had found an Omicron isolate to be less sensitive to camostat than a Delta isolate [15] . Given that this study compared two isolates in one cell line, it is possible that genomic differences between these isolates, which are independent of those defining the Delta and Omicron variant, were responsible for the observed differences. Notably, we detected in Caco-2 cells a 16.3-fold difference between the camostat IC50 for our Delta isolate (0.49µM) compared to the Omicron 2 isolate (0.03µM) ( Figure 1E ). However, the Omicron 1 isolate displayed a camostat IC50 (0.40µM) very close to that obtained for the Delta isolate, and we did not observe a similar difference in Calu-3 cells ( Figure 1E ). Moreover, Omicron mutations are only detected in close vicinity to one of the S cleavage sites. H655Y, N679K, and P681H are close to the 685 furin cleavage site. Among these mutations, only N679K is specific for Omicron (numbering of residues based on the original virus protein sequence). There is no structure for this region of S, because it is a disordered, flexible region. N679K (and P681H) increases the positive charge, but there is no obvious indication that these mutations might affect S cleavage. In conclusion, our comparison of Omicron and Delta isolates in different cellular models shows that Omicron viruses remain sensitive to a broad range of anti-SARS-CoV-2 drugs and drug candidates with a broad range of mechanisms of action. Moreover, Omicron viruses are less effective in antagonising the host cell interferon response, which may explain why they cause less severe disease [21, 22] . Effectiveness of BNT162b2 Vaccine against Omicron Variant in South Africa Third BNT162b2 Vaccination Neutralization of SARS-CoV-2 Omicron Infection Considerable escape of SARS-CoV-2 Omicron to antibody neutralization Effectiveness of 2-Dose Vaccination with mRNA COVID-19 Vaccines Against COVID-19-Associated Hospitalizations Among Immunocompromised Adults -Nine States Pharmacological treatment of COVID-19: an opinion paper (PINETREE) Investigators. 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Cell Rep Decreased severity of disease during the first global omicron variant covid-19 outbreak in a large hospital in tshwane, south africa Characteristics and Outcomes of Hospitalized Patients in South Africa During the COVID-19 Omicron Wave Compared With Previous Waves Review Devil's tools: SARS-CoV-2 antagonists against innate immunity Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Membrane (M) and Spike This work was supported by the Frankfurter Stiftung für krebskranke Kinder. The authors declare no competing interests.