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

Retroviruses pseudotyped with the SARS-CoV-2 or SARS-CoV-1 S proteins (SARS2-PV, SARS1-PV) were produced as previously described (Moore et al., 2004) with modest modifications as described. HEK293T cells were transfected by polyethylenimine (PEI) transfection at a ratio of 5:5:1 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-1 (GenBank AY271119) or SARS-CoV-2 (GenBank YP_009724390). Cells were washed 6 hours later, and the culture supernatant containing pseudoviruses was harvested at 48-72 hours post transfection. Zika virus virus-like particles (ZIKV-VLP) were produced by transfecting HEK293T cells by the calcium phosphate transfection method with a ZIKV replicon (strain FS13025, GenBank KU955593.1), whose expression is controlled by tetracyclin, a plasmid encoding ZIKV capsid, prM, and E proteins (strain FSS13025, GenBank KU955593.1), and the pTet-On plasmid expressing a reverse Tet-responsive transcriptional activator (rtTA) at a ratio of 2:1:1. Cells were washed 6 h later and replenished with fresh media containing 1 μg/ml doxycycline. The VLP-containing culture supernatant was harvested 48 h post transfection. ZIKV replicon was generated by replacing the region spanning 39 th through 763 rd amino acids of the polyprotein of a ZIKV molecular clone we previously generated (Zhang et al., 2018) with Renilla luciferase with the 2A self-cleaving peptide fused at . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.10.036418 doi: bioRxiv preprint its C-terminus. This construct contains the tetracyclin-responsive Ptight promoter that drives ZIKV RNA transcription. The pseudovirus-or VLP-containing culture supernatants were cleared by 0.45 µm filtration. SARS2-PV and ZIKV-VLP titers were assessed by RT-qPCR targeting the CMV promoter in the retroviral vector pQCXIX and ZIKV NS3 gene, respectively. In some cases, clarified pseudovirus and VLP stocks were stored at -80 o C for long-term storage and reuse.