SARS-CoV-2 Virus Is Evolving To Evade Innate Responses And Interferons

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A new study by scientist from University of Colorado Anschutz Medical Campus-USA has worryingly found that the SARS-CoV-2 coronavirus is actually evolving to evade innate immunity responses and interferons.

The study findings were published in the peer reviewed journal: Proceedings of the National Academy of Sciences-USA. https://www.pnas.org/doi/full/10.1073/pnas.2203760119

Numerous studies have shown that IFNs are important for host defense against SARS-CoV-2. This sarbecovirus is believed to have recently crossed the species barrier to humans, either directly from bats or via an intermediate mammalian host(s) (40). Here, we demonstrate that SARS-CoV-2 has in fact evolved after host switching to become more resistant to human IFNs.

Moreover, we establish an order of antiviral potency for the diverse type I and III IFNs. IFNλ initially showed promise as an antiviral that can reduce inflammation (41), but our data suggest that for SARS-CoV-2, higher doses of IFNλ may be needed to achieve a similar antiviral effect in vivo as the IFN-Is.

Nebulized IFNβ showed potential as a therapeutic against COVID-19 (11), and our data confirm that IFNβ is highly potent against SARS-CoV-2.

However, IFNβ was also linked to pathogenic outcomes in chronic mucosal HIV-1 (30), murine lymphocytic choriomeningitis virus (42) and, if administered late in mice, SARS-CoV-1 and Middle East respiratory syndrome-CoV (43, 44) infection. We previously reported that IFNβ up-regulated 2.4-fold more genes than individual IFNα subtypes, suggesting that IFNβ may induce more pleiotropic effects (30).

Among the IFNα subtypes, IFNα8 showed an anti-SARS-CoV-2 potency similar to that of IFNβ. IFNα8 also exhibited high antiviral activity against HIV-1 (3), raising its potential for treatment against both pandemic viruses. Notably, IFNα8 appeared to be an outlier in this regard, as the antiviral potencies of the IFNα subtypes against SARS-CoV-2 and HIV-1 did not correlate strongly.

IFNα6 potently restricted HIV-1 (3, 4) but was one of the weakest IFNα subtypes against SARS-CoV-2. Conversely, IFNα5 strongly inhibited SARS-CoV-2, but weakly inhibited HIV-1 (3). This lack of correlation is a key point for future studies. Of note, the high potency of IFNα5 and low potency of IFNα6 against an isolate of SARS-CoV-2 (not a VOC) were corroborated by another group (45).

Collectively, these data strengthen the theory that diverse IFNs may have evolved to restrict distinct virus families (2, 30). The mechanisms underlying these interesting qualitative differences remain unclear. While IFNΑR signaling contributes to antiviral potency (3, 4, 31), diverse IFNs may have distinct abilities to mobilize antiviral effectors in specific cell types.

Comparing the interferomes induced by distinct IFNs in lung epithelial cells (45) may be useful in prioritizing further studies on this point.

Most significantly, our data reveal the concerning trend for SARS-CoV-2 variants emerging later in the pandemic—in the setting of prolific replication of the virus in human populations—to resist the antiviral IFN response. Before the present work, the emergence and fixation of variants was linked to enhanced viral infectivity and/or neutralizing antibody evasion due to mutations in the Spike protein.

However, previous studies with HIV-1 suggested that IFNs also can shape the evolution of pandemic viruses (46, 47). In fact, SARS-CoV-2-infected individuals with either genetic defects in IFN signaling (48) or IFN-reactive autoantibodies (49) had an increased risk of developing severe COVID-19. As IFNs are critical in controlling early virus infection levels, IFN-resistant SARS-CoV-2 variants may produce higher viral loads that could in turn promote transmission and/or exacerbate pathogenesis.

Consistent with this hypothesis, the alpha VOC was associated with increased viral loads (50) and risk of death (51). Infection with Delta may yield even higher viral loads than that with Alpha (52), but to date, no discernible differences in viral loads were found between Delta and Omicron (53).

Notably, our data on IFN resistance partially tracked with consecutive waves of the most dominant global VOCs Alpha, Delta, and Omicron. Alpha, as well as the Beta and Gamma VOCs that circulated concurrently, was more IFN resistant than ancestral isolates. Delta was more sensitive or had similar IFN sensitivity compared to Alpha in A549-ACE2 and Calu-3 cells, respectively.

This suggested that other factors, such as increased transmissibility and resistance to neutralizing antibodies, may have contributed to the shift from Alpha to Delta. Notably, the most infectious variant to date, Omicron, had the highest levels of residual virus replication at the highest doses of IFNβ, one of the most potent IFNs we determined in this study.

Alpha and Delta replicate efficiently in the lower respiratory tract; by contrast, Omicron appeared to replicate predominantly in the upper respiratory tract (53), which was proposed to be the main site of protective IFN action in vivo (32). Primary human bronchial airway epithelial cells are physiologically relevant models of the upper airway epithelial compartment (39). In these cells, Delta and Omicron were more resistant to IFNβ, IFNα2, and IFNλ1 than Alpha. In fact, our data in primary HBEC cultures suggested that SARS-CoV-2 resistance to IFNs may be increasing during the course of the COVID-19 pandemic.

In addition to Spike, emerging variants exhibit mutations in nucleocapsid, membrane, and nonstructural proteins NSP3, NSP6, and NSP12 (SI Appendix, Table S1). In the case of early pandemic viruses that predated the emergence of VOCs, these viral proteins were reported to antagonize IFN signaling in cells (36, 54).

To specifically map the virus mutations driving IFN-I resistance in emerging variants, panels of recombinant viruses can be generated to isolate specific mutations, singly or in combination against various IFNs, and candidate single viral protein antagonists can be individually tested as well.

This would help to confirm, for example, that the D3L mutation in the Alpha nucleocapsid may facilitate innate immune evasion by increasing the expression of an IFN antagonist, ORF9b (37). Of note, the nucleocapsid D3L mutation was not observed in the Beta, Gamma, Delta, and Omicron lineages (SI Appendix, Table S1), which exhibited IFN-I and IFN-III resistance in our experiments.

One of the Delta isolates we studied had a deletion in ORF7a, which may counteract IFN signaling (34, 35); this deletion was not a cell culture artifact as it was also observed in the clinical isolate. We tested another Delta isolate with an intact ORF7a and it exhibited similar, or even higher, IFN sensitivity. This would suggest that ORF7a may not be a dominant mechanism contributing to IFN antagonism in live virus.

In SI Appendix, Table S2, we highlight mutations in nucleocapsid, NSP12, and NSP6 in multiple VOCs, which may confer IFN resistance and will be prioritized in subsequent work. It is possible that mutations in multiple viral proteins may synergize to confer IFN resistance and that the combinatorial effect of these mutations may differ between the various VOCs.

Overall, the present study suggested a role for the innate immune response in driving the evolution of SARS-CoV-2 that could have practical implications for IFN-based therapies. Our findings reinforce the importance of continued full-genome surveillance of SARS-CoV-2, and assessments of emerging variants not only for resistance to vaccine-elicited neutralizing antibodies, but also for evasion of the host IFN response.

Selection of SARS-CoV-2 strains for IFN sensitivity studies. (A) Global distribution of SARS-CoV-2 clades. GISAID.org plotted the proportion of deposited sequences in designated clades against collection dates. The 10 isolates chosen are noted by colored dots. (B) SARS-CoV-2 strains selected for this study included representatives of lineages A, B, B.1, B.1.351, and B.1.1.7 (SI Appendix, Table S1). Lineage P.1 (which branched off from lineage B.1.1.28), B.1.617.2, and B.1.1.529 were added after the initial preprint submission and were evaluated for IFNβ and IFNλ1 sensitivity. Lineage B isolates encode the D614G mutation associated with increased transmissibility. *Amino acid mutations were relative to the reference hCOV-19/Wuhan/WIV04/2019 sequence.

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