The novel coronavirus can reach deep tissues and organs of the human host that do not have ACE2 receptors

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Researchers from Ohio State University have found that the novel coronavirus and its variants are adopting some stealth and covert moves to stay alive and kicking, and one secret to its success and survival is hiding from the human host’s immune system by spreading through cell-to-cell transmission.

The study team provided evidence that SARS-CoV-2 spreads through cell–cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell–cell fusion activity.
 
Importantly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism.
 
Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients.
 
Although angiotensin-converting enzyme 2 enhances cell-to-cell transmission, the study findings found that it is not absolutely required. (This has vast implications as it shows that the novel coronavirus can reach deep tissues and organs of the human host that do not have ACE2 receptors!)
 
Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission.
 
The study findings revealed critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.
 
The study findings were published in the peer reviewed journal: Proceedings of the National Academy of Sciences. https://www.pnas.org/content/119/1/e2111400119

SARS-CoV-2 is a novel beta-coronavirus that is closely related to two other highly pathogenic human coronaviruses, SARS-CoV and MERS-CoV (1). The spike (S) proteins of SARS-CoV-2 and SARS-CoV mediate entry into target cells, and both use angiotensin-converting enzyme 2 (ACE2) as the primary receptor (2⇓⇓⇓–6).

The spike protein of SARS-CoV-2 is also responsible for induction of neutralizing antibodies, thus playing a critical role in host immunity to viral infection (7⇓⇓–10).

Similar to HIV and other class I viral fusion proteins, SARS-CoV-2 spike is synthesized as a precursor that is subsequently cleaved and highly glycosylated; these properties are critical for regulating viral fusion activation, native spike structure, and evasion of host immunity (11⇓⇓⇓–15).

However, distinct from SARS-CoV, yet similar to MERS-CoV, the spike protein of SARS-CoV-2 is cleaved by furin into S1 and S2 subunits during the maturation process in producer cells (6, 16, 17).

S1 is responsible for binding to the ACE2 receptor, whereas S2 mediates viral membrane fusion (18, 19). SARS-CoV-2 spike can also be cleaved by additional host proteases, including transmembrane serine protease 2 (TMPRSS2) on the plasma membrane and several cathepsins in the endosome, which facilitate viral membrane fusion and entry into host cells (20⇓–22).

Enveloped viruses spread in cultured cells and tissues via two routes: by cell-free particles and through cell–cell contact (23⇓⇓–26). The latter mode of viral transmission normally involves tight cell–cell contacts, sometimes forming virological synapses, where local viral particle density increases (27), resulting in efficient transfer of virus to neighboring cells (24).

Additionally, cell-to-cell transmission has the ability to evade antibody neutralization, accounting for efficient virus spread and pathogenesis, as has been shown for HIV and hepatitis C virus (HCV) (28⇓⇓⇓–32). Low levels of neutralizing antibodies, as well as a deficiency in type I IFNs, have been reported for SARS-CoV-2 (18, 33⇓⇓⇓–37) and may have contributed to the COVID-19 pandemic and disease progression (38⇓⇓⇓⇓–43).

In this work, we evaluated cell-to-cell transmission of SARS-CoV-2 in the context of cell-free infection and in comparison with SARS-CoV. Results from this in vitro study reveal the heretofore unrecognized role of cell-to-cell transmission that potentially impacts SARS-CoV-2 spread, pathogenesis, and shielding from antibodies in vivo.

Fig. 1.The spike protein of SARS-CoV-2 and SARS-CoV mediates cell-to-cell transmission of HIV-1 lentiviral pseudotypes. (A and B) Schematic representations of cell-to-cell and cell-free infection assays (see details in Materials and Methods). Briefly, the inGluc-based lentiviral pseudotypes bearing spike were produced in 293T cells, which were cocultured with the target cells (293T/ACE2) for cell-to-cell transmission; the Gluc activity of cocultured cells was measured over time (A). Cell-free infection was performed by harvesting virus from the same number of producer cells, followed by infecting 293T/ACE2 target cells in the presence of the same number of untransfected 293T cells; alternatively, cell-free infection was carried out in Transwell plates, from which Gluc activity was measured (B). (C) Comparison of cell-to-cell transmission mediated by SARS-CoV-2 or SARS-CoV spike. Results shown were from six independent experiments, with cell-free infection measured at 48 and 72 h after coculture; the portion of cell-free infection was excluded (n = 6). (D) Comparison of cell-free infection mediated by SARS-CoV-2 or SARS-CoV spike. Results were from six independent experiments (n = 6). (E) The expression level of spike proteins on the plasma membrane of donor cells was measured by flow cytometry using a polycolonal antibody T62, which detects both SARS-CoV-2 and SARS-CoV. (F and G) The calculated ratios between cell-to-cell and cell-free infection mediated by SARS-CoV-2 or SARS-CoV-2 spike. Results from cell coculture are shown in F and from Transwell plates shown in G (n = ∼3 to 6). PV, pseudotyped virus. *P < 0.05, **P < 0.01 . ns, not significant.

Cell-to-Cell Transmission of SARS-CoV-2 Is Refractory to Neutralizing Antibody and Convalescent Plasma.

One important feature of the virus cell-to-cell transmission is evasion of host immunity, particularly neutralizing antibody-mediated response. We therefore examined the sensitivity of SARS-CoV-2 spike-mediated cell-to-cell transmission to neutralization by a monoclonal antibody against the receptor-binding domain of the spike, 2B04 (48), as well as convalescent plasma derived from COVID-19 patients (46, 59).

While 2B04 effectively inhibited cell-free infection of SARS-CoV-2 in 293T/ACE2 cells by more than 90%, its effect on cell-to-cell transmission between 293T and 293T/ACE2 was ∼50% (Fig. 6 A and B).

As would be expected, 2B04 had no effect on SARS-CoV, regardless of cell-to-cell transmission or cell-free infection (Fig. 6 A and B). We also performed cell–cell fusion in the presence of different concentrations of 2B04, and we found that the fusion activity of the SARS-CoV-2 spike was inhibited in a dose-dependent manner (Fig. 6C).

We then tested five serum samples of COVID-19 patients and observed that, although they potently inhibited the cell-free infection of SARS-CoV-2 (P < 0.001), they showed variable but no significant effect on cell-to-cell transmission of SARS-CoV-2; the effect of these sera on SARS-CoV infection, either cell-to-cell or cell-free, was minimal or modest (Fig. 6 D and E). Together, these results indicate that cell-to-cell transmission of SARS-CoV-2 lentiviral pseudotyped virus is mostly refractory to neutralization by neutralizing antibodies against spike relative to cell-free infection.

Fig. 6.Cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody and COVID-19 convalescent plasma. (AC) Effects of SARS-CoV-2 monoclonal antibody 2B04 on cell-to-cell transmission, cell-free infection, and cell–cell fusion mediated by SARS-CoV-2 or SARS-CoV-2 spike. The experiments were carried out as described in Fig. 1 C and D, except that 0.2 µg/mL or 2 µg/mL 2B04 were included during the infection period. Relative infections were plotted by setting the values of mock infection without 2B04 to100% for statistical analyses (A and B). The photos of syncytia formation were taken at 18 h after coculture and presented (C). (D and E) Effect of COVID-19 sera on cell-to-cell and cell-free infection of SARS-CoV-2 and SARS-CoV. Experiments were performed as described as above, except five diluted COVID-19 sera were included during the infection period. Effect on cell-to-cell (D) and cell-free (E) infection of SARS-CoV or SARS-CoV-2 were summarized and plotted by setting the values of mock infection control to 100% (n = ∼3 to 4). PV, pseudotyped virus. **P < 0.01, ***P < 0.001. ns, not significant.

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