The RNA demethylase FTO controls m6A marking on SARS-CoV-2 and classifies COVID-19 severity in patients (preprint)

The RNA modification N6-methyladenosine (m6A) plays a key role in the life cycles of several RNA viruses. Whether this applies to SARS-CoV-2 and whether m6A affects the outcome of COVID-19 disease is still poorly explored. Here we report that the RNA demethylase FTO strongly affects both m6A marking of SARS-CoV-2 and COVID-19 severity. By m6A profiling of SARS-CoV-2, we confirmed in infected cultured cells and showed for the first time in vivo in hamsters that the regions encoding TRS_L and the nucleocapsid protein are multiply marked by m6A, preferentially within RRACH motifs that are specific to {beta}-coronaviruses and well conserved across SARS-CoV-2 variants. In cells, downregulation of the m6A demethylase FTO, occurring upon SARS-CoV-2 infection, increased m6A marking of SARS-CoV-2 RNA and slightly promoted viral replication. In COVID-19 patients, a negative correlation was found between FTO expression and both SARS-CoV-2 expression and disease severity. FTO emerged as a classifier of disease severity and hence a potential stratifier of COVID-19 patients.

SARS-CoV-2 variant B.1.617 is resistant to Bamlanivimab and evades antibodies induced by infection and vaccination (preprint)

The emergence of SARS-CoV-2 variants threatens efforts to contain the COVID-19 pandemic. The number of COVID-19 cases and deaths in India has risen steeply in recent weeks and a novel SARS-CoV-2 variant, B.1.617, is believed to be responsible for many of these cases. The spike protein of B.1.617 harbors two mutations in the receptor binding domain, which interacts with the ACE2 receptor and constitutes the main target of neutralizing antibodies. Therefore, we analyzed whether B.1.617 is more adept in entering cells and/or evades antibody responses. B.1.617 entered two out of eight cell lines tested with slightly increased efficiency and was blocked by entry inhibitors. In contrast, B.1.617 was resistant against Bamlanivimab, an antibody used for COVID-19 treatment. Finally, B.1.617 evaded antibodies induced by infection or vaccination, although with moderate efficiency. Collectively, our study reveals that antibody evasion of B.1.617 may contribute to the rapid spread of this variant.

Humoral and cellular immune responses against SARS-CoV-2 variants and human coronaviruses after single BNT162b2 vaccination. (preprint)

Vaccine-induced neutralizing antibodies are key in combating the COVID-19 pandemic. However, delays of boost immunization due to limited availability of vaccines may leave individuals vulnerable to infection and disease for prolonged periods. The emergence of SARS-CoV-2 variants of concern (VOC), B.1.1.7 (United Kingdom), B.1.351 (South Africa) and P.1 (Brazil), may reinforce this issue with the latter two being able to evade control by antibodies. We assessed humoral and T cell responses against SARS-CoV-2 WT and VOC and endemic human coronaviruses (hCoV) that were induced after single and double vaccination with BNT162b2. Despite readily detectable IgG against the receptor-binding domain (RBD) of the SARS-CoV-2 S protein at day 14 after a single vaccination, inhibition of SARS-CoV-2 S-driven host cell entry was weak and particularly low for the B.1.351 variant. Frequencies of SARS-CoV-2 specific T cells were low in many vaccinees after application of a single dose and influenced by immunity against endemic hCoV. The second vaccination significantly boosted T cell frequencies reactive for WT, B.1.1.7 and B.1.351 variants. These results call into question whether neutralizing antibodies significantly contribute to protection against COVID-19 upon single vaccination and suggest that cellular immunity is central for the early defenses against COVID-19.

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

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 variants B.1.351 and B.1.1.248: Escape from therapeutic antibodies and antibodies induced by infection and vaccination (preprint)

SUMMARY The global spread of SARS-CoV-2/COVID-19 is devastating health systems and economies worldwide. Recombinant or vaccine-induced neutralizing antibodies are used to combat the COVID-19 pandemic. However, recently emerged SARS-CoV-2 variants B.1.1.7 (UK), B.1.351 (South Africa) and B.1.1.248 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies. Here, using pseudoparticles, we show that entry of UK, South Africa and Brazil variant into human cells is susceptible to blockade by entry inhibitors. In contrast, entry of the South Africa and Brazil variant was partially (Casirivimab) or fully (Bamlanivimab) resistant to antibodies used for COVID-19 treatment and was less efficiently inhibited by serum/plasma from convalescent or BNT162b2 vaccinated individuals. These results suggest that SARS-CoV-2 may escape antibody responses, which has important implications for efforts to contain the pandemic.

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity (preprint)

Antiviral therapy is urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The protease inhibitor camostat mesylate inhibits SARS-CoV-2 infection of lung cells by blocking the virus-activating host cell protease TMPRSS2. Camostat mesylate has been approved for treatment of pancreatitis in Japan and is currently being repurposed for COVID-19 treatment. However, potential mechanisms of viral resistance as well as camostat mesylate metabolization and antiviral activity of metabolites are unclear. Here, we show that SARS-CoV-2 can employ TMPRSS2-related host cell proteases for activation and that several of them are expressed in viral target cells. However, entry mediated by these proteases was blocked by camostat mesylate. The camostat metabolite GBPA inhibited the activity of recombinant TMPRSS2 with reduced efficiency as compared to camostat mesylate and was rapidly generated in the presence of serum. Importantly, the infection experiments in which camostat mesylate was identified as a SARS-CoV-2 inhibitor involved preincubation of target cells with camostat mesylate in the presence of serum for 2 h and thus allowed conversion of camostat mesylate into GBPA. Indeed, when the antiviral activities of GBPA and camostat mesylate were compared in this setting, no major differences were identified. Our results indicate that use of TMPRSS2-related proteases for entry into target cells will not render SARS-CoV-2 camostat mesylate resistant. Moreover, the present and previous findings suggest that the peak concentrations of GBPA established after the clinically approved camostat mesylate dose (600 mg/day) will result in antiviral activity.