Omicron Has Shifted To Using Cathepsins For Cell Entry Via Endosomal Fusion


We are no longer dealing with the respiratory disease-causing pathogen that debuted in late 2019 in Wuhan but rather the SARS-CoV-2 coronavirus has evolved into a new pathogen which we are referring to as Omicron variant that has done a complete biological about-face of shifting from gaining entry via cell surface fusion following proteolysis by TMPRSS2 to entry via endosomal fusion after activation by the endosomal proteases Cathepsin B or L.

This shift will render a lot of things that we come to know through past studies about the SARS-CoV-2 virus as absolute as the shift to using cathepsins instead of TMPRSS2 will cause a total change in cellular tropism and also pathogenesis.

Scientists from the University of Glasgow Centre for Virus Research along with experts from the NHS Greater Glasgow & Clyde, University of Cambridge, Public Health Scotland. University of St Andrews and the London School of Hygiene and Tropical Medicine in a new study found that the Omicron variant has made a major switch in the cell entry mechanism.
The study findings were published as a preprint format and is currently being peer reviewed for publication into a well-known journal.

Omicron spike has switched entry route preference

Entry of SARS-CoV-2, and related coronaviruses, can proceed via two routes55. Cell surface fusion following proteolysis by TMPRSS2, as described above (Route 1; Fig. 6A), or fusion from the endosome after endocytosis and activation by the endosomal proteases Cathepsin B or L (Route 2; Fig. 6A).

The ability of SARS-CoV-2 to achieve cell surface fusion is dependent on its S1/S2 polybasic cleavage site; this is absent from most closely related sarbecoviruses, which are confined to endosomal fusion56–58. Given the reduced fusogenicity and replication kinetics of Omicron, we used HIV pseudotypes to evaluate entry route preference.

We evaluated Wuhan D614G, Alpha, Delta and Omicron spike and as a control we included Pangolin CoV (Guangdong isolate) spike, which exhibits high affinity interactions with human ACE2 but lacks a polybasic cleavage site and, therefore, enters via the endosome only59–62.

Calu-3 cells predominantly support cell surface (Route 1) fusion, owing to their high endogenous expression of TMPRSS257,63; in these cells, Delta yielded the highest infection, being ~4 fold higher than Omicron (Fig. 6B). Pangolin CoV infection was low, indicating that Calu-3 cells do not support robust endosomal entry.

On the contrary, HEK only support endosomal entry and in these cells Pangolin CoV had high infection. Notably, Omicron also achieved high infection in HEK cells, producing ~10 fold greater signal than Delta. This suggests that Omicron, like Pangolin CoV, is optimised for endosomal entry. All pseudotypes exhibited robust infection in A549 ACE2 TMPRSS2, where both entry routes are available64,65.

Entry pathway preference was further investigated using protease inhibitors targeting either TMPRSS2 (Camostat) or cathepsins (E64d)58. In Calu-3 cells, all SARS-CoV-2 pseudotypes were inhibited by Camostat, whereas only Omicron exhibited E64d sensitivity, indicating that a component of infection occurs via endosomal entry (Fig. 6C).

In HEK cells all pseudotypes were inhibited by E64d, whereas Camostat was non-inhibitory; this confirms that only endosomal entry is available in these cells. Inhibitor treatment in A549 ACE2 TMPRSS2 provided the clearest evidence of altered entry by Omicron. D614G, Alpha and Delta were potently inhibited by Camostat, but not E64d.

For Omicron, and Pangolin CoV, this pattern was completely reversed, suggesting a binary switch from cell surface to endosomal fusion; this conclusion was supported by titration of either inhibitor in A549 ACE2 TMPRSS2 cells (Fig. 6D).

These data indicate that, whilst Delta is optimised for fusion at the cell surface, Omicron preferentially achieves entry through endosomal fusion; this biological about-face may impact transmission, cellular tropism and pathogenesis. Moreover, this switch away from TMPRSS2-mediated activation offers a mechanistic explanation for reduced syncytia formation by Omicron infected cells.

Figure 6. Omicron spike has switched entry route preference. A. SARS CoV-2 entry can occur via two routes. Route 1 permits rapid fusion at the cell surface following proteolytic processing by TMPRSS2. In Route 2 fusion occurs following endocytosis after processing by cathepsin B or L. Route 1 and 2 can be specifically inhibited using the protease inhibitors Camostat and E64d, respectively. B. SARS-CoV-2 pseudotype infection of the stated cell lines, data represent mean luciferase values from one representative experiment. In Calu-3 cells Route 1 entry predominates whereas HEK exclusively support Route 2, A549 ACE2 TMPRSS2 cells permit both routes. Pangolin CoV spike is included as a control; it can only achieve entry via Route 2. Pseudotypes without viral glycoproteins (No) are included as a negative control. C. Relative SARS-CoV-2 pseudotype infection (compared to untreated control) of cells treated with 10µM protease inhibitors. Data represent mean of four replicates, error bars indicate standard error of the mean, asterisks indicate statistical significance (ANOVA). D. Titration of Camostat and E64d against Delta, Omicron and Pangolin CoV in A549 ACE2 TMPRSS2 cells, data points represent mean relative infection, compared to untreated control.


The Omicron variant represents a major change in biological function and antigenicity of SARS-CoV-2 virus. In this study, we demonstrate substantial immune escape of this variant with clear evidence of vaccine failure in dual vaccinated individuals and partial restoration of immunity following a third booster dose of mRNA vaccine.

In addition, we demonstrate a shift in the SARS-CoV-2 entry pathway from cell surface fusion, triggered by TMPRSS2, to cathepsin-dependent fusion within the endosome. This fundamental biological shift may affect the pathogenesis and severity of disease and requires further evaluation in population-based studies.

Using sera from double vaccine recipients, we found that Omicron is associated with a drop in neutralisation greater in magnitude than that reported in all other variants of concern (including Beta and Delta).

Boosting enhanced neutralising responses to both Wu-Hu-1 and Omicron, particularly in recipients of ChAdOx1, but did not completely overcome the inherent immune escape properties of Omicron.

Importantly, we did not assess the impact of vaccination on clinical severity of disease which is likely to be much higher than detection of infection. Protection against severe disease is longer lasting than prevention of infection. We also did not measure the impact of vaccination on T cell immunity which may be better preserved as only 14% of CD8+ and 28% of CD4+ epitopes are predicted to be affected by key Omicron mutations17.

In order to evaluate the impact of reduced neutralisation responses in vaccine recipients, we next assessed vaccine effectiveness. The probability of infection with Omicron versus the preceding Delta variant was significantly higher in double vaccine recipients, in keeping with the neutralisation data. A third dose of mRNA vaccine substantially reduced the probability of infection but did not restore immunity fully.

The observation of a highly transmissible variant that is associated with escape from vaccine-induced immune responses means that over time, Omicron-specific vaccines would be required if disease severity was high, either directed at the general population or vulnerable groups. Early indications in young people are that Omicron is 40-70% less severe than Delta66,67 – similar calculations in the most vulnerable part of the population over the age of 40 years are awaited.

Genotypic change in new variants have previously been shown to alter viral phenotype by modulating innate immune responses as well as evasion of the adaptive immune response15,68. Additionally, mutations can alter spike functionality to impact transmission and pathogenesis. For example, a polybasic insertion at the S1/S2 spike junction that facilitates cleavage of the spike glycoprotein by furin during virus assembly13. This may have provided a selective advantage in lung cells and primary

human airway epithelial cells for the original emergent SARS-CoV-2, and previous VOCs by permitting spike activation by the plasma membrane protease TMPRSS2, enabling rapid cell surface fusion55. In this study, we found that the Omicron variant has switched entry pathway to preferentially use endosomal fusion that is independent of TMPRSS2; a major change in the biological behaviour of the virus.

This switching in the mechanism of fusion activation also manifests in reduced syncytia formation in infected cells, likely to reduce the cell-to-cell transmission characteristic of other variants. These properties have the potential to substantially change cellular tropism and pathogenesis of disease.

Nonetheless, even a variant that is less virulent with a very high transmission rate may still present a substantial risk to older people and those with co-morbidities, especially those with immunosuppression. Moreover, our work demonstrates that SARS-CoV-2 exhibits high antigenic and functional plasticity; further fundamental shifts in transmission and disease should be anticipated.


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