key: cord-1046578-fvjh3g7v authors: Xiong, Hua-Long; Wu, Yang-Tao; Cao, Jia-Li; Yang, Ren; Ma, Jian; Qiao, Xiao-Yang; Yao, Xiang-Yang; Zhang, Bao-Hui; Zhang, Ya-Li; Hou, Wang-Heng; Yang-Shi,; Xu, Jing-Jing; Liang-Zhang,; Wang, Shao-Juan; Fu, Bao-Rong; Yang, Ting; Ge, Sheng-Xiang; Zhang, Jun; Yuan, Quan; Huang, Bao-Ying; Li, Zhi-Yong; Zhang, Tian-Ying; Xia, Ning-Shao title: Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells date: 2020-04-13 journal: bioRxiv DOI: 10.1101/2020.04.08.026948 sha: bda5dbec80bd7051a00c53767a833556e96fa1ab doc_id: 1046578 cord_uid: fvjh3g7v The global pandemic of Coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable in vitro neutralization assay is very important for the development of neutralizing antibodies, vaccines and other inhibitors. In this study, G protein-deficient vesicular stomatitis virus (VSVdG) bearing full-length and truncated spike (S) protein of SARS-CoV-2 were evaluated. The virus packaging efficiency of VSV-SARS-CoV-2-Sdel18 (S with C-terminal 18 amino acid truncation) is much higher than VSV-SARS-CoV-2-S. A neutralization assay for antibody screening and serum neutralizing titer quantification was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and human angiotensin-converting enzyme 2 (ACE2) overexpressed BHK21 cell (BHK21-hACE2). The experimental results can be obtained by automatically counting EGFP positive cell number at 12 hours after infection, making the assay convenient and high-throughput. The serum neutralizing titer of COVID-19 convalescent patients measured by VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with live SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting receptor binding domain (RBD) of SARS-CoV-2-S were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines. As of April 5, 2020, over 1.2 million cases of coronavirus disease 2019 (COVID-19) have been reported, including 68,000 deaths. The spread of the SARS-CoV-2 virus is difficult to control due to the highly contagious and asymptomatic infection. There is an urgent need to develop vaccines or therapeutics against SARS-CoV-2 infection. However, SARS-CoV-2 culture and assays need to be carried out in biosafety level-3 laboratory, which limits the efficiency of research and development (R & D). Widely available and convenient functional assays, such as cell-based assays for neutralizing quantification, are essential for vaccine and therapeutic antibody R & D [1] [2] [3] . The SARS-CoV-2 belonging to the genus of betacoronavirus with envelope [4] . The glycosylated spike (S) protein is the major surface protein and is responsible for receptor binding and fusion of the viral membrane with cellular membranes [5, 6] . Studies has showed that SARS-CoV-2 S and SARS-CoV S bind with the same receptor, human angiotensin-converting enzyme 2 (hACE2), with similar affinities [7] [8] [9] . Therefore, the studies of SARS-CoV neutralization assays, which based on pseudovirus bearing the spike protein of SARS-CoV, would provide valuable experience and reference for the SARS-CoV-2 assays. In the past years, pseudotyping system based on vesicular stomatitis virus (VSV) was reported to produce pseudotypes incorporating the envelope protein of heterologous risk group- coronavirus et al., in which the VSV G gene is deleted (VSVdG) and the gene encoding GFP, luciferase or other reporter genes was integrated [10] [11] [12] . Pseudotyped viruses provide a safe viral entry model because of their inability to produce infectious progeny virus. VSV assembly occurs at the plasma membrane and involves budding of virions from the cell surface. During budding, VSV acquires an envelope consisting of a lipid bilayer derived from the plasma membrane and spike proteins consisting of trimers of the VSV glycoprotein (VSV-G). When the VSV-G is absence and the glycoprotein from heterologous virus is complacently expressed in cells infected with rVSV-dG, the glycoprotein of heterologous virus could be assembled into the VSV membrane. Recently, Michael Letko et al. used VSVdG-luc bearing SARS-spike chimeras to study the cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses [13] . Jianhui Nie et al. have successfully constructed a pseudovirus neutralization assay for SARS-CoV-2, which consist of pseudotyped VSV bearing the full-length spike protein of SARS-CoV-2 and Huh7 cell [12] . Lentiviral pseudotype bearing the truncated spike protein of SARS-CoV-2 was also constructed and used to study the virus entry and its immune cross-reactivity with SARS-CoV [6] . Noteworthy, the correlation between pseudovirus system and live virus system hasn't been verified. And the virus package and infection efficiency is also one of the limiting factors for high-throughput in vitro neutralization assay. It was reported that a pseudotyped VSV and retrovirus bearing a SARS-CoV-S protein variant with a truncation in the cytoplasmic tail had much higher infection efficiency than that with full-length S protein [11, 14] . In this study, the VSVdG pseudotyped with full-length SARS-CoV-2-S protein or truncated SARS-CoV-2-Sdel18 protein with C-terminal 18 aa truncation were compared, we found that the infection efficiency of VSV-SARS-CoV-2-Sdel18 was much higher than VSV-SARS-CoV-2-S. Among the cell lines used in this study, BHK21-hACE2 stably expressing human ACE2 was most sensitive for infection, while Vero-E6 was most suitable for virus package. A neutralization assay for antibody screening and validation was established based on VSV-SARS-CoV-2-Sdel18 and BHK21-hACE2 cell, and 7 strains of neutralizing monoclonal antibodies targeting receptor binding domain (RBD) of SARS-CoV-2-S were obtained. Most importantly, the correlation between pseudovirus system and live virus system was verified. To construct VSV pseudovirus carrying the spike protein of SARS-CoV-2, the spike gene of Wuhan-Hu-1 strain (GenBank: MN908947) was codon-optimized for expression in human cells and cloned into the eukaryotic expression plasmid pCAG to generate recombinant plasmid pCAG-nCoVS. The spike gene mutant of SARS-CoV-2 that with C-terminal 18 aa truncation was also cloned into plasmid pCAG and generate pCAG-nCoVSde18. Plasmid pCAG-nCoVS and pCAG-nCoVSde18 were transfected into Vero-E6 respectively. 48 hours post transfection, the VSVdG-EGFP-G (Addgene, 31842) [15] virus were inoculated to the cell expressing SARS-CoV-2 spike protein or SARS-CoV-2 Sde18 truncated protein and incubated for one To compare the infection efficiency of pseudotypes bearing different glycosylated protein, single cycle infectious recombinant VSVdG-EGFP-G was prepared and used to infect Vero-E6 cells transiently expressing full-length SARS-CoV-2-S protein and truncated SARS-CoV-2-Sdel18 protein that with C-terminal 18 aa truncation respectively, pseudotyped VSV-SARS-CoV-2-S and VSV-SARS-CoV-2-Sdel18 was obtained following the procedures described in methods ( Figure 1 ). Vero-E6, BHK21, BHK21-hACE2 and 293T cells were seeded to compare the infection efficiency of pseudotypes. The infection efficiency of VSV pseudotypes bearing truncated Sdel18 was much higher than that with full-length S protein. The results suggested that the combination of VSV-SARS-CoV-2-Sdel18 and BHK21-hACE2 cells could be a better alternation for establishing pseudotyped SARS-CoV-2 entry and neutralization assays. Viral packaging efficiency is one of the limiting factors for high-throughput in vitro neutralization assay. To select a cell line that is most appropriate for pseudotypes production, we compared the viral titers of VSVdG-SARS-CoV-2-Sdel18 packaged by Vero-E6, BHK21 and 293T cells. The results showed that the Vero-E6 formed more syncytia than the other two cell lines during the packaging, and also produced highest viral titer (Figure 3) , which was about 3×10 5 infectious particles per miniliter. To determine the optimal incubation period for detection of VSVdG-SARS- [16] . It also means this pseudoviruse system can mimic the entry of live virus. This system can be used for high-throughput screening of neutralizing antibodies. As shown in Figure 5A and 5B, 35 strains of mouse monoclonal antibody hybridoma culture supernatants were evaluated using the pseudovirus system, and 7 of them showed significant neutralization. The IC50 of 7 selected neutralizing antibodies for antiviral activity were also analyzed ( Figure 5C ) using this pseudovirus system. VSVdG-SARS-CoV-2-Sdel18 based system was used to analyze the neutralizing antibodies in the serum of 18 convalescent patients. The serum was diluted 60-fold as the first concentration, and then prepared doubling dilutions (60, 120, 240, etc.). BHK21-hACE2 cell was seeded and incubated with the mixture of gradient diluted serum and VSVdG-SARS-CoV-2-Sdel18 virus (MOI=0.05). The GFP positive cell number was counted to analysed the neutralizing activity of serum ( Figure 6A ). Among the 18 tested sera, 5 of them show high neutralizing activity (ID 50 >1000). Similar assay was also performed using live SARS-CoV-2 system. 7 sera show high neutralizing activity (ID 50 >1000) according to live virus assay ( Figure 6B ). The analysis result of the pseudovirus system correlates well with live virus system (R 2 =0.639, P<0.0001, Figure 6C ). The COVID-19 epidemic is spreading rapidly around the world, there is an urgent need for vaccines or therapeutic drugs. The isolation and culture of SARS-CoV-2 have to be handled in biosafety level 3 (BSL-3) facilities, which limits the research and development of drugs. A safer and more efficient assay for drugs evaluation would accelerate the development of drugs. The pseudovirus system provides a convenient tool for the research of RG-3 viruses, it makes the evaluation of inhibitors for viral entry, neutralizing antibodies and immunized-serum much easier. Retrovirus and VSV are both common used pseudovirus system, but the pseudotype virus titer obtained with VSVdG system is generally higher than that of the retrovirus system [10] . Furthermore, infection of [12] . Noteworthy, the truncation of the cytoplasmic tail may increase fusion activity, which is consistent with the study of SIV-MuLV chimeric envelope protein [17] . Enhanced syncytia arising from the fusion may contribute to the improved packaging efficiency. The pseudotyped VSV with C- Xia had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis Drafting of the manuscript: Tian-Ying Zhang, Jia-li Cao, Hua-Long Xiong. 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