In a recent study in Nature Microbiology, researchers compared severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern (VOC) replication in human cell lines and primary airway cultures and measured host responses to infection.
Background
SARS-CoV-2 evolution has led to VOC development, with Omicron being the first to evolve globally dominant sub-VOCs. These sub-VOCs, either being replaced or circulating concomitantly, indicate a shift from host adaptation to immunological escape from infection- and vaccine-induced memory responses. The BA.1 sub-VOC and the BA.2 sub-VOC of Omicron emerged with SARS-CoV-2 spike (S) protein mutations, threatening the efficacy of vaccines. However, Omicron sub-VOCs develop mutations beyond spike, indicating S-independent adaptations potentially crucial for SARS-CoV-2 Omicron dominance.
About the study
In the present study, researchers proposed a model in which the earliest host innate immune responses significantly contribute to SARS-CoV-2 transmission by influencing whether interactions with the first few cells in the airway establish a productive infection.
The team compared BA.1 to BA.5 replication with the SARS-CoV-2 Delta VOC in the Calu-3 cultured human airway epithelium cells (HAEs) to assess phenotypic differences between Omicron sub-VOCs and the selective forces driving their evolution. They equalized the input dosage of every VOC by copies of the SARS-CoV-2 envelope (E) gene using quantitative-type reverse transcription-polymerase chain reaction (RT-qPCR). The analysis ensured cellular exposure to equivalent initial quantities of SARS-CoV-2 ribonucleic acid (RNA), the primary viral pathogen-associated molecular pattern molecule (PAMP), stimulating defensive-type host innate immunological responses.
The team compared host innate immunological responses to Omicron sub-VOC infections in human airway Calu-3 cells, comparing replication at 32°C in the cells. They investigated the mechanism underlying differential innate immune activation by Omicron sub-VOCs and whether Omicron sub-VOCs have independently evolved enhanced intrinsic immunosuppression through similar mechanisms during human adaptation. To probe open-reading frame 6 (ORF6) mechanisms and its role in increasing innate antagonism by the VOCs, the team used reverse genetics, inserting two stop codons into ORF6 sequences of the Alpha VOC (Alpha ΔORF6) and the BA.5 VOC (BA.5 ΔORF6).
Reverse genetics confirmed the elimination of ORF6 expression during infection. They determined viral titers by 50% tissue culture infectious dose (TCID50) in Henrietta Lacks (Hela) cells expressing angiotensin-converting enzyme (ACE2). The team compared intracellular SARS-CoV-2 E RNA expression with that of ORF1a and non-structural protein 12 (NSP12), uniquely coded within genomic RNA (gRNA), using SARS-CoV-2 E gene measurement during infection. They performed flow cytometry to measure interferon (IFN)-β, λ1, λ3, and C-X-C motif chemokine ligand 10 (CXCL10) levels and enzyme-linked immunosorbent assays (ELISA) to detect mediators secreted in cell supernatants. They also performed immunofluorescence and transcription factor translocation analyses. Western blot analysis detected SARS-CoV-2 nucleocapsid (N), ORF6, ORF9b, S, and β-actin expression.
Results
The study examined the evolution of SARS-CoV-2 variants, specifically the BA.4 sub-VOC and the BA.5 sub-VOC, which suppress innate immunity more than earlier subvariants. BA.2.75 and XBB lineages also reduced innate immune activation, correlated with increased expression of viral innate antagonists ORF6 and nucleocapsid. Increased ORF6 levels suppressed host innate responses to infection by decreasing IFN regulatory factor 3 (IRF3) and signal transducer and activator of transcription 1 (STAT1) signaling. Omicron, specifically the BA.5 sub-VOC, has a more significant entry into transmembrane serine protease 2 (TMPRSS2)-lacking cells than prior VOCs like Delta. However, Calu-3 cell entry was TMPRSS2-dependent, resulting in cell-specific discrepancies in VOC titers.
Calu-3 human airway epithelium cell infection with Omicron BA.4 and Omicron BA.5 significantly lowered innate immunological activation than the BA.1 and BA.2 sub-VOCs, with lower IFN-beta and IFN-stimulated gene (ISG) induction, reducing host immunological responses to Omicron BA.4 and Omicron BA.5 infections. The slower proliferation of Omicron BA.4 likely contributed to lower innate immunological activation during Calu-3 infections. IFN-mediated Janus kinase (JAK)-STAT signaling inhibition with ruxolitinib rescued Omicron BA.1 and BA.2 infections in Calu-3 human airway epithelium cells to a greater extent than Omicron BA.4 and BA.2, reflecting differences in interferon induction following Calu-3 infections.
The lack of differences in sub-genomic RNAs (sgRNAs) in S and ORF3a indicated no general enhancement of sgRNA synthesis. Omicron sub-VOCs have synonymous and non-synonymous mutations in ORF6 and N. Yet, no mutations distinguish the BA.4 sub-VOC and BA.5 sub-VOC from BA.1 sub-VOC and BA.2 sub-VOC, indicating they have evolved independent mechanisms to increase ORF6 and N protein levels or by changes elsewhere in the genome. ORF6 expression was the primary determinant of enhanced innate immune antagonism in emerging VOCs. All Omicron subvariants triggered significantly less IFNB and CXCL10 expression than BA.2 at 24 hours post-infection.
Conclusion
Overall, the study findings showed that Omicron variants of SARS-CoV-2 increase innate immune evasion by increasing viral protein expression, indicating that the earliest host innate immune responses are crucial in SARS-CoV-2 transmission. Viruses with enhanced ability to evade or antagonize innate immunity, such as increased ORF6 and N expression, transmit more frequently due to their ability to avoid inducing or shutting down host responses that suppress this earliest replication. The team hypothesizes that upon infection establishment, innate immunosuppression may lead to increased disease due to higher viral burden and inflammatory responses.
