SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization

Transmission of SARS-CoV-2 from humans to farmed mink was observed in Europe and the US. In the infected animals viral variants arose that harbored mutations in the spike (S) protein, the target of neutralizing antibodies, and these variants were transmitted back to humans. This raised concerns that mink might become a constant source of human infection with SARS-CoV-2 variants associated with an increased threat to human health and resulted in mass culling of mink. Here, we report that mutations frequently found in the S proteins of SARS-CoV-2 from mink were mostly compatible with efficient entry into human cells and its inhibition by soluble ACE2. In contrast, mutation Y453F reduced neutralization by an antibody with emergency use authorization for COVID-19 therapy and by sera/plasma from COVID-19 patients. These results suggest that antibody responses induced upon infection or certain antibodies used for treatment might offer insufficient protection against SARS-CoV-2 variants from mink.

SARS-CoV-2 differs from SARS-CoV in the requirements for receptor expression and proteolytic activation to trigger cell-cell fusion and is not inhibited by Bromhexine

The severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with Angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and the sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S(SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by Batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector-target-cell fusion when ACE2 or TMPRSS2 were limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target-effector-cell fusion was unaltered compared to wt SARS2-S, but syncytia remained smaller. Mutation of the S2 site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor Bromhexine was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S as opposed to the inhibitor Camostat. Paradoxically, Bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite Ambroxol exhibited inhibitory activity in some conditions. On Calu-3 lung cells, Ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend towards weak inhibition of authentic SARS-CoV-2. IMPORTANCECell-cell fusion allows the virus to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2 cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently tested in clinical trials against coronavirus disease 2019. Our results indicate that Bromhexine enhances fusion in some conditions. We therefore caution against use of Bromhexine in higher dosage until its effects on SARS-CoV-2 spike activation are better understood. The related compound Ambroxol, which similarly to Bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for Ambroxol.

Novel surrogate virus neutralization test reveals low serum neutralizing anti-SARS-CoV-2-S antibodies levels in mildly affected COVID-19 convalescents

Neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells using surface-expressed angiotensin-converting enzyme 2 (ACE2). We developed a surrogate neutralization test (sVNT) to assess at what degree serum antibodies interfere with the binding of SARS-CoV-2-S-RBD to ACE2. The sVNT revealed neutralizing anti-SARS-CoV-2-S antibodies in the sera of 90% of mildly and 100% of severely affected coronavirus-disease-2019 (COVID-19) convalescent patients. Importantly, sVNT results correlated strongly to the results from pseudotyped-vesicular stomatitis virus-vector-based neutralization assay and to levels of anti-SARS-CoV-2-S1 IgG and IgA antibodies. Moreover, levels of neutralizing antibodies also correlated to duration and severity of clinical symptoms, but not patient age or gender. These findings together with the sVNT will not only be important for evaluating the prevalence of neutralizing antibodies in a population but also for identifying promising plasma donors for successful passive antibody therapy.

LY6E impairs coronavirus fusion and confers immune control of viral disease

Zoonotic coronaviruses (CoVs) are significant threats to global health, as exemplified by the recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. Host immune responses to CoV are complex and regulated in part through antiviral interferons. However, the interferon-stimulated gene products that inhibit CoV are not well characterized2. Here, we show that interferon-inducible lymphocyte antigen 6 complex, locus E (LY6E) potently restricts cellular infection by multiple CoVs, including SARS-CoV, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in hematopoietic cells were highly susceptible to murine CoV infection. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic and splenic immune cells and reduction in global antiviral gene pathways. Accordingly, we found that Ly6e directly protects primary B cells and dendritic cells from murine CoV infection. Our results demonstrate that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo, knowledge that could help inform strategies to combat infection by emerging CoV.

The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells

The emergence of a novel, highly pathogenic coronavirus, 2019-nCoV, in China, and its rapid national and international spread pose a global health emergency. Coronaviruses use their spike proteins to select and enter target cells and insights into nCoV-2019 spike (S)-driven entry might facilitate assessment of pandemic potential and reveal therapeutic targets. Here, we demonstrate that 2019-nCoV-S uses the SARS-coronavirus receptor, ACE2, for entry and the cellular protease TMPRSS2 for 2019-nCoV-S priming. A TMPRSS2 inhibitor blocked entry and might constitute a treatment option. Finally, we show that the serum form a convalescent SARS patient neutralized 2019-nCoV-S-driven entry. Our results reveal important commonalities between 2019-nCoV and SARS-coronavirus infection, which might translate into similar transmissibility and disease pathogenesis. Moreover, they identify a target for antiviral intervention. One sentence summaryThe novel 2019 coronavirus and the SARS-coronavirus share central biological properties which can guide risk assessment and intervention.