key: cord-0931310-ntwvquqz authors: Yang, Ren; Huang, Baoying; Ruhan, A.; Li, Wenhui; Wang, Wenling; Deng, Yao; Tan, Wenjie title: Development and effectiveness of Pseudotyped SARS-CoV-2 system as determined by neutralizing efficiency and entry inhibition test in vitro date: 2020-08-21 journal: Biosaf Health DOI: 10.1016/j.bsheal.2020.08.004 sha: dd133875929c24ce0e91730307a41f80f439750c doc_id: 931310 cord_uid: ntwvquqz With the development of the COVID-19 epidemic, there is an urgent need to establish a system for determining the effectiveness and neutralizing activity of vaccine candidates in biosafety level 2 (BSL-2) facilities. Previously, researchers had developed a pseudotyped virus system for SARS-CoV and MERS-CoV, based on HIV-1 core, bearing virus spike protein. During the development of a pseudotyped SARS-CoV-2 system, a eukaryotic expression plasmid expressing SARS-CoV-2 spike (S) protein was constructed and then co-transfected with HIV-1 based plasmid which containing the firefly luciferase reporter gene, into HEK293T cells to prepare the pseudotyped SARS-CoV-2 virus (ppSARS-2). We have successfully established the pseudotyped SARS-CoV-2 system for neutralization and entry inhibition assays. Huh7.5 cell line was found to be the most susceptible to our pseudotyped virus model. Different levels of neutralizing antibodies were detected in convalescent serum samples of COVID-19 patients using ppSARS-2. The recombinant, soluble, angiotensin-converting enzyme 2 protein was found to inhibit the entry of ppSARS-2 in Huh7.5 cells effectively. Furthermore, the neutralization results for ppSARS-2 were consistent with those of live SARS-CoV-2 and determined using the serum samples from convalescent patients. In conclusion, we have developed an easily accessible and reliable tool for studying the neutralizing efficiency of antibodies against SARS-CoV-2 and the entry process of the virus in a BSL-2 laboratory. The causative agent of the unprecedented global pandemic of coronavirus disease 2019 (COVID-19) is a novel beta-coronavirus [1, 2, 3] , named as SARS-CoV-2 (also called as COVID-19 virus in China) [4] . The control of the COVID-19 pandemic is a great challenge because SARS-CoV-2 is a highly contagious virus, and COVID-19 has diverse clinical manifestations (such as asymptomatic infection, common cold, and pneumonia) [5] . Therefore, there is an urgent need to develop vaccines or therapeutics against COVID-19. However, currently, SARS-CoV-2 live virus-associated experiments can only be conducted in a biosafety level 3 (BSL- 3) facility, which limits the development of SARS-CoV-2 vaccines and drugs to several scientific teams and local departments of disease control and prevention. Hence, a reliable, rapid, and convenient neutralization assay, that can be handled in a BSL-2 laboratory is essential for screening and evaluation of antibodies and therapeutic agents against the SARS-CoV-2 infection. Previous studies have suggested that the glycosylated spike (S) protein is the major surface protein responsible for receptor binding and entry of the virus into the host cell [6, 7, 8] and that both SARS-CoV-2 and SARS-CoV have the same main receptor, which is angiotensin-converting enzyme 2 (ACE2) [9, 10] . Pseudotyped antibodies at 37°C for 2 h. Mouse anti-SARS-CoV-2 S pAb and convalescent serum from COVID-19 patients, diluted at 1:200 and 1:50, respectively, were used as primary antibodies. After the incubation period, the cells were washed with PBST 10 times and were incubated with goat anti-mouse IgG FITC or anti-human IgG FITC in 0.5% Evans blue PBS at RT for 1 h. The cells were then washed with PBST, and mounted on cover slips using mounting buffer. Fluorescence was observed using OLYMPUS IX50FL microscope and the fluorescent images were captured using the DP70 digital camera system. SARS-CoV-2 pseudotyped virus and mock virus were ultracentrifuged at 24,000 × g using 1 mL of 20% sucrose as a cushion. Then, the medium was discarded, and the precipitated pseudoviruses were suspended in 200 μL of SDS-PAGE loading buffer. The samples were heated for 10 min at 95°C and then subjected to SDS-PAGE for immunoblotting, as previously described [16] . SARS-CoV-2 S protein was J o u r n a l P r e -p r o o f Journal Pre-proof identified using the mouse anti-SARS-CoV-2 S pAb (Sino biological, China), while HIV p24, used as reference, was identified using the rabbit anti-p24 polyclonal Ab (Sino biological). Vero, Vero E6, Huh7, Huh7.5, and BHK21 cells were seeded into 96-well plates one day before infection. When infected, the cells were approximately 60-80% confluent. Following infection, the culture medium was removed, and 50 μL of serum-free DMEM medium was added. The cells were stabilized for 30 min, following which 50 μL of pseudotype virus culture suspension was added to the cells. The culture medium was replaced after 12-16 h with fresh DMEM supplemented with 2% FBS, and the culture was continued for 24 h. Bright-Glo Luciferase Assay System (Promega Co., US) was used to detect Fluc expression in the cells, and the titer of pseudotype virus in different cell lines was obtained. Thus, sensitive cell lines were identified. The sensitive cells were grown to 60-80% confluency for the experiment. The serum can be determined using the pseudotyped SARS-CoV-2 virus. Various levels of neutralizing antibodies were detected in the serum samples from COVID-19 convalescent patients, which exhibited 50% neutralization (ND 50 ) against ppSARS-2 from less than 1:50 dilution to more than 1:2048 dilution. Since hACE2 has been reported as the SARS-CoV-2 entry receptor, we have utilized soluble hACE2 as a binding competitor to inhibit the entry of pseudotyped SARS-CoV-2 [9] . ppSARS-CoV-2 entry was blocked using soluble ACE2, as indicated by the significant reduction in reporter gene expression level ( Figure 3B ). The IC50 of soluble hACE2 was identified as 0.032 μg/mL To verify the true reliability of the system, serum samples of the SARS-CoV-2 convalescent patients were analyzed by microneutralization using live SARS-CoV-2 virus ( Figure 4A) . Results revealed the similar neutralizing potency between the pseudotyped-SARS-CoV-2 system and live-SARS-CoV-2. We then compared the results of the analysis conducted using the ppNT and the live virus ( Figure 4B ) and found that the results from the ppNT were strongly correlated with those obtained using live virus (R 2 = 0.6931 and p < 0.005). 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