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

Brian D Quinlan; Huihui Mou; Lizhou Zhang; Yan Gao; Wenhui He; Amrita Ojha; Mark S Parcells; Guangxiang Luo; Wenhui Li; Guocai Zhong; Hyeryun Choe; Michael Farzan

To further characterize the effects of the two successive sets of immunizations, we compared day-0, day-15, and day-40 sera by pooling these sera from all rats, and also compared it with pooled day-0 serum mixed with 10 and 100 µg/ml ACE2-Ig, diluted as indicated ( Figure 1C ). Again day-40 serum potently neutralized S-protein-mediated entry, with significantly greater efficiency than day-0 serum mixed with 100 µg/ml ACE2-Ig. At lower dilutions (1:20 and 1:60) day-15 serum neutralized more efficiently than day-0 serum, somewhere between 10 and 100 µg/ml ACE2 in preserum. However day-40 serum did not efficiently neutralize retroviruses pseudotyped with SARS-CoV-1 S protein (SARS1-PV), except at the lowest (1:20) dilution, excluding the possibility that immunization with a single RBD could protect against SARS-like viruses with divergent RBD. Finally, to confirm that sera from vaccinated rats neutralized SARS2-PV by recognizing the SARS-CoV-2 S protein, we used each pooled serum to displace an ACE2-Ig variant with a rabbit Fc (ACE2-rIg) domain from cells expressing the SARS-CoV-2 S protein ( Figure 2D ). The ability of each pooled serum to displace ACE2-rIg correlated with its ability to neutralize SARS2-PV. Specifically, day-40 sera displaced ACE2-rIg more efficiently than preimmune sera containing 100 µg/ml ACE2-Ig, and day-15 sera displaced ACE2-rIg more efficiently than preimmune sera containing 10 µg/ml ACE2-Ig. Thus immunization with SARS-CoV-2 RBD elicits antibodies that potently neutralize S-protein mediated entry by directly binding the S protein.