Characterization of spike glycoprotein of 2019-nCoV on virus entry and its immune cross-reactivity with spike glycoprotein of SARS-CoV

Abstract

Since beginning of this century, there have already been three zoonotic outbreaks caused by beta coronaviruses (CoV), SARS-CoV in 2002, MERS-CoV in 2012, and the newly identified 2019-nCoV in late 2019, Wuhan, China. As to Feb 10 th , 2020, there are over 40,000 confirmed cases and over 900 deaths. However, little is known about the biology of this newly emerged virus. Here we developed a lentiviral based pseudovirus system for S protein of 2019-nCoV to study virus entry in BSL2 settings. First, we confirmed that human angiotensin converting enzyme 2 (hACE2) is the main entry receptor for 2019-nCoV.Second, we found that 2019-nCoV S protein mediated entry on 293/hACE2 cells was mainly through endocytosis, and PIKfyve, TPC2, and cathepsin L are critical for virus entry. Third, 2019-nCoV S protein is less stable than SARS-CoV, and it could trigger proteaseindependent and receptor dependent cell-cell fusion, which might help virus rapidly spread from cell to cell. Finally and more importantly, polyclonal anti-SARS S1 antibodies T62 effectively inhibited entry of SARS-CoV S pseudovirions, but almost had no effect on entry of 2019-nCoV S pseudovirions. Further studies using sera from one recovered SARS-CoV patient and five 2019-nCoV patients showed that there was only limited crossneutralization activities between SARS-CoV and 2019-nCoV sera, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for 2019-nCoV.

Human monoclonal antibodies block the binding of SARS-CoV-2 spike protein to angiotensin converting enzyme 2 receptor

“…Furthermore, we determined the neutralization of these three mAbs using a SARS-CoV-2 S pseudotyped lentiviral particle. 22 In line with ELISAand flow cytometry-based blockade results, both 311mab-31B5 and 311mab-32D4 effectively neutralized pseudovirus entry to host cells ectopically expressing hACE2 (IC 50 = 0.0338, and 0.0698 μg/ml, respectively). As expected, 311mab-31B9 clone failed to show any neutralization activities (Fig.…”

Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19

“…The pseudovirus neutralization assay was previously described 15,32 .Briefly, HEK-293T cells were transfected with pLenti-luciferase, psPAX2, and 2019-nCov S plasmids by using Trans IT-293 Transfection reagent (Mirus, MIR 2700). After 12 hours, the culture media was changed to fresh media.…”

On the origin and continuing evolution of SARS-CoV-2

“…Both SARS-CoV and SARS-CoV-2 bind to ACE2 through the RBD of spike protein in order to initiate membrane fusion and enter human cells [1,2,[22][23][24][25][26]. Five out of the six critical amino acid (AA) residues in RBD were different between SARS-CoV-2 and SARS-CoV ( Fig.…”