Omicron Spike confers enhanced infectivity and interferon resistance to SARS-CoV-2 in human nasal tissue

SARS-CoV-2 entry requires Spike engagement of ACE2 and proteolytic activation, historically via serine proteases such as TMPRSS2 or endosomal cathepsins. Omicron (BA.1 and subsequent sublineages) rapidly displaced Delta, with Spike mutations known to mediate antibody escape and possibly altered ACE2 affinity. It has been suggested that Omicron uses a distinct, less TMPRSS2-dependent entry route, potentially shifting tropism toward the upper respiratory tract. As the nasal epithelium is the initial site of infection and innate defense, the study set out to compare Omicron versus ancestral (WA1) and Delta variants for infectivity in primary human nasal epithelial cells, to determine whether Omicron Spike alone confers enhanced nasal entry, to define the protease pathway used in physiologically relevant cells, and to test whether this entry is resistant to constitutive and interferon-induced antiviral mechanisms.

Prior work established ACE2 as the primary receptor and implicated multiple attachment factors (e.g., neuropilin-1, heparan sulfate, sialic acids) in facilitating SARS-CoV-2 entry. TMPRSS2 usage and endosomal cathepsins govern entry route choice, impacting cell tropism and pathogenicity. Omicron Spike accumulates numerous mutations, enabling broad neutralizing antibody escape; reports on increased ACE2 affinity are mixed. Several studies indicate Omicron’s reduced TMPRSS2 dependence and potential reliance on cathepsins or metalloproteinases, with implications for altered tropism and reduced lower respiratory tract infection. Increasing interferon resistance across variants has been reported, with Omicron often showing the greatest resistance, but the contribution of Spike-mediated entry to interferon resistance in primary nasal cells had not been clearly defined.

- Primary human nasal epithelial cells (hNAEC) from adult donors were used in two formats: submerged undifferentiated monolayers (pooled from three donors) and differentiated air–liquid interface (ALI) cultures (pooled from up to 14 donors). Human small airway epithelial cells (lung) were also tested in submerged culture for comparison. - Virus infections included authentic replication-competent SARS-CoV-2 strains: ancestral WA1, Delta, Omicron BA.1, BA.2, and XBB, and recombinant WA1 engineered to encode WA1, Delta, or BA.1 Spike and express mCherry (designated WA1 (WA1 Spike), WA1 (Delta Spike), WA1 (BA.1 Spike)). Inoculations were typically performed at MOI 0.05 or with 10,000 PFU on ALI cultures. Apical infection on ALI was carried out with 50 µL inoculum for 2 h at 37°C, followed by wash. - Viral replication and entry were quantified by RT-qPCR of viral ORF1a RNA using 2^-ΔCt or 2^-ΔΔCt relative to ACTB, by infectious titers (focus-forming units) on Vero E6–ACE2–TMPRSS2 cells with anti-N immunostaining and high-content imaging, and by mCherry fluorescence for recombinant viruses at defined time points (e.g., 24 hpi). Confocal immunofluorescence was used to assess cellular markers (e.g., ACE2, acetylated tubulin) and IFITM2/3 expression. - Interferon sensitivity: ALI cultures were pretreated for 18 h with type I (IFN-β) or type III (IFN-λ) interferons at graded doses prior to infection. Effects were assessed by ORF1a RT-qPCR and infectious titers. - Entry pathway inhibitors: compounds targeting serine proteases (camostat), cathepsins/endosomal acidification (E64d, bafilomycin A1), and metalloproteinases/ADAMs (indinavir/“incyclidine”, apratastat, batimastat) were applied to probe entry routes in ALI cultures infected with clinical and recombinant viruses. A neuropilin-1 inhibitor (E620002) was tested for attachment factor contribution. - IFITM restriction: Amphotericin B pretreatment was used to counteract IFITM2/3-mediated membrane effects and assess impact on infection by WA1 (WA1 Spike), WA1 (Delta Spike), and WA1 (BA.1 Spike) at multiple time points. - Statistics: Student’s paired two-sided t-test and one-way ANOVA with multiple-comparisons adjustments (GraphPad Prism 10.1.0) were used; exact p-values provided for select comparisons.

- Omicron BA.1 replicates substantially better than ancestral WA1 in primary nasal epithelial cells cultured as submerged monolayers, showing approximately 10-fold higher replication and ~30-fold greater cell surface invasion/adhesion capacity versus WA1. Delta showed only modest, non-significant improvement over WA1. - BA.1 failed to replicate in submerged small airway (lung) epithelial cells where WA1 replicated comparably to nasal cells, indicating an upper-airway-specific advantage for BA.1. - In ALI cultures, BA.1 and BA.2 achieved higher ORF1a RNA levels and infectious titers than WA1; BA.1 elicited more IFNB expression than other variants. Omicron XBB also replicated significantly more than D614G in ALI. - Spike determinant: Recombinant WA1 encoding BA.1 Spike exhibited a strong gain in nasal ALI infectivity (about 20-fold by mCherry fluorescence at 24 hpi and about 100-fold by ORF1a RT-qPCR) versus WA1 (WA1 Spike). WA1 (Delta Spike) did not confer this gain, demonstrating that BA.1 Spike specifically governs enhanced nasal entry. - Entry route: BA.1 entry in nasal ALI is largely TMPRSS2-independent and metalloproteinase-dependent. Pan-metalloproteinase inhibitors (e.g., apratastat, batimastat; also indinavir/“incyclidine”) reduced infection by WA1 (BA.1 Spike) by ~60% and/or markedly, while the same inhibitors did not inhibit and could modestly boost Delta Spike-mediated infection. Bafilomycin A1 enhanced BA.1 infection (~4-fold), consistent with a preference for non-endosomal entry. Cathepsin inhibitor E64d did not block BA.1 entry under conditions that inhibited other routes. Neuropilin-1 inhibition (E620002) most strongly affected WA1, had no effect on Delta, and only modestly affected BA.1, indicating NRP1 usage does not explain BA.1’s nasal advantage. - Interferon resistance: Pre-treatment with IFN-β or IFN-λ inhibited WA1 by up to two orders of magnitude in nasal ALI, whereas BA.1 and BA.2 were significantly less affected and maintained higher infectious titers. Recombinant WA1 (BA.1 Spike) recapitulated IFN resistance seen with full-length BA.1, while WA1 (Delta Spike) showed only a slight decrease in IFN sensitivity relative to WA1. Thus, BA.1 Spike is sufficient to confer IFN resistance at the entry step in nasal epithelium. - IFITM-related restriction: Nasal ALI constitutively expressed IFITM2/3. Amphotericin B pretreatment increased infection by WA1 (WA1 Spike) and WA1 (Delta Spike) by approximately 7-fold at 24 hpi, but had negligible impact on WA1 (BA.1 Spike), suggesting IFITM proteins restrict early variants at the entry step, whereas BA.1 Spike-mediated entry is resistant to this barrier.

The study shows that Omicron BA.1’s enhanced transmissibility can be partly explained by its Spike-mediated superiority at infecting human nasal epithelium. BA.1 Spike confers a distinct, TMPRSS2-independent, metalloproteinase-dependent entry pathway in primary nasal cells that enhances attachment/entry and circumvents constitutive and interferon-induced antiviral defenses. This Spike-driven IFN resistance operates at the entry stage, reducing the efficacy of innate barriers such as IFITM proteins that restrict earlier variants. These properties provide a mechanistic basis for Omicron’s efficient seeding of infection in the upper airway, facilitating person-to-person spread and displacement of prior variants, beyond its well-documented antibody escape. The findings refine assumptions that TMPRSS2 independence implies endosomal entry in primary cells, highlighting instead a prominent role for metalloproteinases at or near the plasma membrane. They also have implications for IFN-based therapeutics, as Omicron’s Spike-mediated entry shows relative insensitivity to type I and III IFNs in nasal epithelium.

Omicron BA.1 Spike is the principal determinant of enhanced infectivity in primary human nasal epithelial tissue, enabling metalloproteinase-dependent entry that bypasses TMPRSS2 and resists restriction by type I/III interferons and IFITM proteins. These Spike-driven features likely contributed to Omicron’s increased transmissibility and persistence in human populations. Future work should dissect the contributions of individual Spike mutations to nasal attachment, protease usage, and IFN resistance, and further evaluate the therapeutic potential and limitations of IFN-based interventions against contemporary and emerging Omicron-lineage variants.

- The study relies on in vitro primary human nasal epithelial cultures (submerged and ALI), which, while physiologically relevant, may not fully recapitulate in vivo conditions across diverse human populations. - Donor variability was mitigated by pooled donors, but individual differences in nasal epithelial composition and innate responses may influence outcomes. - Specific metalloproteinase(s) responsible for BA.1 Spike activation in primary nasal epithelium were not definitively identified; inhibition profiles suggest involvement but do not pinpoint single enzymes. - Some conclusions draw on pharmacologic inhibitors that can have off-target effects; while multiple inhibitors were used, definitive genetic validation was not presented in the text provided. - Discrepancies with other studies regarding relative nasal infectivity (e.g., Delta vs BA.1) underscore model-dependent differences (cell systems, culture conditions).