The emergence of the Omicron variant of SARS-CoV-2 marked a significant turning point in the COVID-19 pandemic. Its rapid global spread and ability to displace earlier variants, such as Delta, highlighted its superior transmissibility. While immune evasion through vaccine-elicited adaptive immunity was recognized as a contributing factor, the underlying mechanisms responsible for Omicron's enhanced infectivity remained unclear. This research aimed to investigate the specific mechanisms driving Omicron's increased infectivity and its ability to overcome the innate immune defenses of the human nasal epithelium, the primary site of SARS-CoV-2 infection. The study sought to address the lack of precise characterization of Omicron's entry pathways in physiologically relevant primary cells and to determine the sensitivity of these pathways to antiviral inhibitors. Understanding the interaction between Omicron and the nasal epithelium is critical because this interaction initiates infection and the initial protective innate immune response. Previous research has indicated that Omicron's proteolytic activation might be less dependent on TMPRSS2, suggesting alternative entry pathways. The study specifically focused on comparing Omicron's ability to infect human nasal epithelial cells relative to ancestral SARS-CoV-2 and the Delta variant. The researchers hypothesized that Omicron's enhanced transmissibility in humans could be attributed to both its evasion of vaccine-elicited adaptive immunity and its superior invasion of nasal epithelial cells, coupled with resistance to the cell-intrinsic barriers found therein. This nuanced understanding of Omicron's infectivity mechanism is paramount in developing effective strategies for combating future variants.

The efficient infection of human cells by SARS-CoV-2 depends on the interaction of the viral Spike protein with its receptor, angiotensin-converting enzyme 2 (ACE2). Spike protein also interacts with various cellular factors to promote virus attachment, including glucose-regulated protein 78 kD, neuropeptides, scavenger receptors, CD209, and heparan sulfate. These factors aid virus attachment, enabling subsequent ACE2 binding. ACE2 engagement alters Spike conformation, facilitating processing by cellular proteases such as TMPRSS2, matrix metalloproteinases (MMPs), and ADAMs. Protease cleavage triggers fusion between viral and cellular membranes, completing cell entry. Omicron's emergence in late 2021, characterized by numerous unique Spike mutations, led to its rapid replacement of Delta as the dominant circulating variant. Functional characterization revealed that these mutations facilitated evasion of neutralizing antibodies induced by COVID-19 vaccination and, in some studies, increased affinity for ACE2. However, the role of antibody evasion and ACE2 affinity in Omicron's enhanced transmissibility remained uncertain. Previous research indicated that Omicron may utilize a distinct cellular entry pathway, less reliant on TMPRSS2 but possibly more dependent on cathepsin activity in endosomes or metalloproteinase activity at the plasma membrane. However, a detailed characterization of Omicron's entry pathways in primary cells, and their sensitivity to antiviral inhibitors, was lacking. Some studies suggested that Omicron BA.1 showed a decreased propensity for lower respiratory tract infection, potentially explaining reduced pathogenicity. This provided the impetus for the current study, which focused on the upper respiratory tract and, specifically, the nasal epithelium.

The study used primary human nasal epithelial cells (hNAEC) obtained from multiple donors. Cells were cultured as submerged monolayers to maintain an undifferentiated state, allowing for viral propagation in the basal state. Confocal immunofluorescence microscopy was used to confirm ACE2 expression. In parallel, cells were cultured at the air-liquid interface (ALI) to mimic the natural physiological conditions of the nasal epithelium, which is critical for proper ciliary function and accurate assessment of viral infection. Researchers used RT-qPCR to measure viral RNA levels (ORF1ab) and infectious virus titers in both submerged and ALI cultures. Replication-competent SARS-CoV-2 strains, including WA1 (ancestral), Delta, Omicron BA.1, and BA.2, were used to infect the cells at various multiplicities of infection (MOI). To determine the role of Omicron Spike protein in enhanced infectivity, recombinant mCherry-expressing SARS-CoV-2 viruses were engineered with Spike proteins from WA1, Delta, or BA.1. The effects of various inhibitors were tested, including camostat (serine protease inhibitor), bafilomycin A1 (vacuolar-ATPase inhibitor), and metalloproteinase inhibitors (apstatin and batimastat), to elucidate the entry pathways. The role of neuraminidase and neurophilin-1 in BA.1 entry were also investigated through specific inhibitor assays. The sensitivity of viral entry to interferon (IFN)-induced antiviral states was evaluated by pretreating cells with IFN-β or IFN-λ before infection. Infectious virion titers were measured using focus-forming assays. The expression of IFITM proteins, which have antiviral activity, was assessed by immunofluorescence, and the effect of amphotericin B (an inhibitor of IFITM's antiviral activity) on viral entry was evaluated. RT-qPCR was used to measure the expression of viral RNA (ORF1a), IFN-β, and IFITM3; these were normalized to cellular ACTB. Focus-forming assays were conducted on Vero E6-ACE2-TMPRSS2 cells. Statistical analyses were performed using GraphPad Prism. The generation and use of recombinant SARS-CoV-2 adhered to Biosafety Level 3 practices.

The study revealed that Omicron BA.1 exhibited significantly superior replicative fitness compared to WA1 and Delta in primary human nasal epithelial cells cultured as submerged monolayers. This enhanced replication was specific to nasal cells and was not observed in human small airway epithelial cells. Further analysis demonstrated that Omicron BA.1 displayed approximately a 10-fold greater replication capacity and a 30-fold superior invasion capacity compared to WA1 in nasal monolayers. Using recombinant viruses, the study demonstrated that the Omicron Spike protein was responsible for this enhanced infectivity. In ALI cultures, Omicron BA.1 and BA.2 exhibited significantly higher replication than WA1 and Delta, with Omicron XBB also showing significantly greater replication than D614G. Recombinant viruses carrying the BA.1 Spike showed a substantial gain in infectivity compared to those carrying WA1 or Delta Spikes. The study revealed that Omicron's entry pathway in nasal ALI cells is predominantly mediated by metalloproteinases, unlike earlier variants that rely on TMPRSS2. Omicron Spike-mediated entry was insensitive to inhibitors of cathepsins and TMPRSS2 but was highly sensitive to pan-metalloproteinase inhibitors. Importantly, Omicron exhibited significantly reduced sensitivity to type I and II interferons compared to WA1 and Delta, a finding confirmed both with full-length clinical isolates and recombinant viruses. Omicron's resistance to interferons was attributed to its Spike protein, which enabled infection through an IFN-resistant entry pathway. The study demonstrated that the constitutive expression of IFITM proteins in nasal ALI cells restricted WA1 and Delta infection, an effect that was partially relieved by treatment with amphotericin B. However, Omicron infection was much less sensitive to IFITM-mediated restriction. These findings indicate that Omicron's enhanced infectivity arises from its unique Spike protein, leading to an entry pathway resistant to both interferon-induced and constitutive antiviral factors in the nasal epithelium.

The study's findings provide a compelling mechanistic explanation for Omicron's remarkable transmissibility and ability to outcompete earlier SARS-CoV-2 variants. The enhanced infectivity conferred by the Omicron Spike protein, along with its ability to bypass innate immune defenses in nasal epithelial cells, represent key factors driving Omicron's pandemic success. The reliance on metalloproteinases for entry offers a potential therapeutic target. The decreased sensitivity to interferon also has significant implications for antiviral therapeutic strategies. While interferon lambda has shown promise in preclinical studies, conflicting clinical trial results highlight the need for further research into the effectiveness of interferon-based therapies against Omicron and its emerging sublineages, especially considering Omicron's resistance to the effects of interferons. The study's findings necessitate a reassessment of the role of IFITM proteins in SARS-CoV-2 infection. Future research should focus on identifying specific mutations within the Omicron Spike protein responsible for its enhanced infectivity and interferon resistance, which would greatly enhance our understanding of the selective pressures driving viral evolution.

This study demonstrates that Omicron's enhanced transmissibility stems from its Spike protein's ability to mediate efficient entry into nasal epithelial cells via a metalloproteinase-dependent pathway, bypassing the antiviral effects of interferons and IFITM proteins. These findings offer crucial insights into Omicron's evolutionary success and underscore the need for continued research into novel antiviral strategies targeting the unique characteristics of Omicron and future SARS-CoV-2 variants.

The study primarily focused on Omicron BA.1 and BA.2, limiting its generalizability to other Omicron subvariants. The use of pooled samples from multiple donors may mask individual variations in responses to infection. The in vitro nature of the study might not fully replicate the complex in vivo environment of the human respiratory tract. Further research using animal models is necessary to validate the findings in a more physiologically relevant context.