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

The ability of anti-SARS-CoV-2 RBD immune plasma to mediate antibody-dependent enhancement (ADE) was measured using HEK293T cells or HEK293T cells stably expressing human ACE2 (293T-ACE2 cells), transfected using the calcium phosphate transfection method to express the rat ortholog of FcγRI (CD64). The human monocytic cell line K562 (ATCC CCL-243), which endogenously expresses FcγRII, was also used for ADE assays. The RBD immune sera, collected from four different rats at day 40 after the first immunization, were mixed at an equal ratio, as was preimmune sera obtained at day 0 from the same rats. As a positive control, sera from ZIKV-infected rats (rat #13 and #15, distinct from the similarly numbered RBDinoculated rats), was also mixed at an equal ratio. Immune and pre-immune serum samples were heat inactivated for 30 min at 56°C and serially diluted in DMEM containing 10% heat-inactivated FBS. SARS2-PV or ZIKV-VLP in 50 µl was preincubated for 1 h at 37°C with 50 µl of diluted sera and added to the indicated cells plated on the 96 well plates. Two days later, infection levels were assessed using Luc-Pair Firefly Luciferase HS Assay Kit (Genocopia) for SARS-PV and Luc-Pair Renilla Luciferase HS Assay Kit (Genocopia) for ZIKV-VLP.