SARS-CoV-2 Omicron variant virus isolates are highly sensitive to interferon treatment

The SARS-CoV-2 Omicron variant (B.1.1.529) causes less severe disease than previous SARS-CoV-2 variants, although immune protection provided by vaccinations and previous infections is reduced against Omicron compared to previous variants 1 . In agreement, evidence is emerging that Omicron is inherently less pathogenic than previous SARS-CoV-2 variants. Omicron variant viruses cause less severe disease in animal studies 2 and appear to display a lower capacity than other variants to replicate in the lower respiratory tract 2 . Additionally, initial clinical data indicated that the Omicron variant causes less severe disease than previous SARS-CoV-2 variants in unvacci-nated individuals 1 . We have most recently shown that Omicron variant viruses are less effective at antagonizing the host cell interferon response than Delta variant viruses 3 , which provides a mechanistic explanation for the reduced clinical severity of Omicron disease in individuals without pre-existing adaptive immunity 1 . Omicron virus replication was attenuated relative to Delta virus in interferon-competent Caco-2 and Calu-3 cells, but not in interferon-de ﬁ cient Vero cells, and Omicron viruses caused enhanced interferon promoter activity compared to Delta viruses 3 . Additionally, depletion of the pattern recognition receptor MDA5, which plays a critical role in SARS-CoV-2 detection and interferon response initiation 4 , resulted in increased Omicron


C O R R E S P O N D E N C E
O p e n A c c e s s SARS-CoV-2 Omicron variant virus isolates are highly sensitive to interferon treatment Denisa Bojkova 1 , Tamara Rothenburger 1 , Sandra Ciesek 1,2,3 , Mark N. Wass 4✉ , Martin Michaelis 4✉ and Jindrich CinatlJr 1,5 ✉ Dear Editor, The SARS-CoV-2 Omicron variant (B.1.1.529) causes less severe disease than previous SARS-CoV-2 variants, although immune protection provided by vaccinations and previous infections is reduced against Omicron compared to previous variants 1 . In agreement, evidence is emerging that Omicron is inherently less pathogenic than previous SARS-CoV-2 variants. Omicron variant viruses cause less severe disease in animal studies 2 and appear to display a lower capacity than other variants to replicate in the lower respiratory tract 2 . Additionally, initial clinical data indicated that the Omicron variant causes less severe disease than previous SARS-CoV-2 variants in unvaccinated individuals 1 .
We have most recently shown that Omicron variant viruses are less effective at antagonizing the host cell interferon response than Delta variant viruses 3 , which provides a mechanistic explanation for the reduced clinical severity of Omicron disease in individuals without pre-existing adaptive immunity 1 . Omicron virus replication was attenuated relative to Delta virus in interferoncompetent Caco-2 and Calu-3 cells, but not in interferondeficient Vero cells, and Omicron viruses caused enhanced interferon promoter activity compared to Delta viruses 3 . Additionally, depletion of the pattern recognition receptor MDA5, which plays a critical role in SARS-CoV-2 detection and interferon response initiation 4 , resulted in increased Omicron virus replication in interferoncompetent cells 3 .
The exact molecular reasons for the alleviated interferon response antagonism by Omicron viruses remain to be elucidated. Notably, the Omicron and Delta virus isolates that we investigated (see Supplementary Information) display sequence variants in the viral interferon antagonists nsp3, nsp12, nsp13, nsp14, the membrane (M) protein, the nucleocapsid protein, and ORF3a 5 (Supplementary Table S1), which may be of relevance.
In A549 cells transduced with ACE2 (SARS-CoV-2 receptor) and TMPRSS2 (mediates SARS-CoV-2 cell entry by cleaving and activating the viral spike protein), the Omicron viruses also displayed alleviated infection capacity compared to the Delta virus (Fig. 1c). This difference largely disappeared upon depletion of either of the pattern recognition receptors MDA5 and RIG-I, both of which mediate the host cell interferon response in virusinfected cells 6 . However, when we compared interferon activity in the supernatants of SARS-CoV-2-infected cells in a HEK-Blue IFNα/β reporter cell assay, the supernatants of Omicron virus-infected RIG-I-knock out cells induced higher interferon promoter activation than the supernatants of Omicron virus-infected MDA5-knock out cells (Fig. 1d). This is in agreement with previous data showing that MDA5 is primarily responsible for virus recognition and the induction of an interferon response in SARS-CoV-2-infected cells 4,6,7 .
Taken together, these findings further confirm that Omicron viruses are less effective than Delta viruses in antagonizing the host cell interferon response 3 and that MDA5 is a major player in SARS-CoV-2 recognition 4,6 .

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Open  Accordingly, elevated MDA5 levels were detected in the upper airways of SARS-CoV-2-infected individuals with mild or asymptomatic disease 8 . Since Delta has been found to display a similar level of interferon antagonism and sensitivity as previous SARS-CoV-2 variants 9,10 , the reduced interferon antagonism appears to be unique to Omicron.
Most notably, treatment with interferon-α, interferon-β, and interferon-γ revealed that the weaker interferon antagonism by Omicron virus isolates translates into a profoundly increased Omicron sensitivity to interferon treatment (Fig. 1e). Further experiments showed that antiviral interferon-β effects were further increased in combination with nirmatrelvir (the antivirally active agent in paxlovid), remdesivir, and EIDD-1931 (the active metabolite of molnupiravir) (Fig. 1f). Combination experiments using the Chou-Talalay approach 11 indicated moderate synergism of interferon-β with remdesivir, synergism with nirmatrelvir, and strong synergism with EIDD-1931 (Fig. 1g, h). So far, clinical studies reported mixed outcomes in COVID-19 patients treated with different interferons [12][13][14][15] . Given the newly discovered substantially increased interferon sensitivity of Omicron viruses, interferons represent a promising option for the treatment of Omicron patients.
In conclusion, we present further evidence that reduced interferon-antagonizing activity explains at least in part why Omicron variant infections are inherently less severe than infections with other SARS-CoV-2 variants. Sequence variations in the SARS-CoV-2 interferon antagonists nsp3, nsp12, nsp13, nsp14, M protein, the nucleocapsid protein, and/or ORF3a may contribute to these differences. Most importantly, this study shows that Omicron variant viruses display enhanced sensitivity to interferon treatment, which makes interferons promising therapeutic candidates for Omicron patients, in particular in combination with other antiviral agents.