The SARS-CoV-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement

Abstract

The SARS-coronavirus 2 (SARS-CoV-2) spike (S) protein mediates entry of SARS-CoV-2 into cells expressing the angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino acid fragment of the 1273-amino acid S-protein protomer. Antibodies to the RBD domain of SARS-CoV (SARS-CoV-1), a closely related coronavirus which emerged in 2002-2003, have been shown to potently neutralize SARS-CoV-1 S-protein-mediated entry, and the presence of anti-RBD antibodies correlates with neutralization in SARS-CoV-2 convalescent sera. Here we show that immunization with the SARS-CoV-2 RBD elicits a robust neutralizing antibody response in rodents, comparable to 100 µg/ml of ACE2-Ig, a potent SARS-CoV-2 entry inhibitor. Importantly, anti-sera from immunized animals did not mediate antibodydependent enhancement (ADE) of S-protein-mediated entry under conditions in which Zika virus ADE was readily observed. These data suggest that an RBD-based vaccine for SARS-CoV-2 could be safe and effective.

Rapid isolation of potent SARS-CoV-2 neutralizing antibodies and protection in a small animal model

“…What do our results suggest for SARS-CoV-2 vaccine design? In the first instance, the results suggest a focus on the RBD and indeed strong nAb responses have been described by immunizing mice with a multivalent presentation of RBD [15]. The strong preponderance of non-neutralizing antibodies and very few nAbs to S protein that we isolated could arise for a number of reasons including: (1) the recombinant S protein that we used to select B cells is a poor representation of the native spike on virions.…”

Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays

“…We describe a detailed protocol for producing SARS-CoV-2 Spike-pseudotyped lentiviral particles and performing neutralization assays. Although this basic pseudotyping approach has been described previously [4,12,27,29,[37][38][39][40][41], we provide the first detailed protocol that makes all reagents available in a public repository (https://www.beiresources.org/). We hope this protocol and reagents will more easily enable others to assess the neutralizing activity of antibodies and sera reactive to SARS-CoV-2.…”

“…Spike from several coronaviruses can be "pseudotyped" onto safer non-replicative viral particles in place of their endogenous entry protein, thereby making entry of these particles into cells dependent on Spike [29][30][31][32][33][34][35][36]. For SARS-CoV-2, such pseudotyping has recently been reported using HIV-based lentiviral particles [4,27,37], MLV-based retroviral particles [12,38], and VSV [29,[39][40][41]. In the data reported to date, results from such pseudovirus neutralization assays correlate well with measurements made using live SARS-CoV-2 [1,12,27,39].…”

The role of high cholesterol in age-related COVID19 lethality

“…To test the cholesterol dependence of SARS-COV-2 on viral entry we loaded cholesterol into HEK293T cells with apolipoprotein E (apoE) and blood serum and treated the cells with retroviruse pseudotyped with the SARS-CoV-2 S protein (CoV-2-PV) 21,22 . A segment of the S protein binds to the ACE2 and recapitulates viral entry 23 .…”

“…Retroviruses pseudotyped with the SARS-CoV-2 S proteins (SARS2-PV) was produced as previously described 21,22 with modest modifications as described. HEK293T cells were transfected by X-tremeGENE™ 9 DNA Transfection Reagent (Millipore Sigma, #6365787001) at a ratio of 3:4:3 with a plasmid encoding murine leukemia virus (MLV) gag/pol proteins, a retroviral vector pQCXIX expressing firefly luciferase, and a plasmid expressing the spike protein of SARS-CoV-2 (GenBank YP_009724390).…”