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Abstract Spike‐protein‐based pseudotyped viruses were used to evaluate vaccines during the COVID‐19 pandemic. However, they cannot be used to evaluate the envelope (E), membrane (M), and nucleocapsid (N) proteins. The first generation of virus‐like particle (VLP) pseudotyped viruses contains these four structural proteins, but their titers for wild‐type severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are relatively low, even lower for the omicron variant, rendering them unsuitable for neutralizing antibody detection. By optimizing the spike glycoprotein signal peptide, substituting the complexed M and E proteins with SARS‐COV‐1, optimizing the N protein with specific mutations (P199L, S202R, and R203M), and truncating the packaging signal, PS9, we increased the titer of the wild‐type VLP pseudotyped virus over 100‐fold, and successfully packaged the omicron VLP pseudotyped virus. The SARS‐CoV‐2 VLP pseudotyped viruses maintained stable titers, even through 10 freeze–thaw cycles. The key neutralization assay parameters were optimized, including cell type, cell number, and viral inoculum. The assay demonstrated minimal variation in both intra‐ and interassay results, at 11.5% and 11.1%, respectively. The correlation between the VLP pseudotyped virus and the authentic virus was strong (r = 0.9). Suitable for high‐throughput detection of various mutant strains in clinical serum. In summary, we have developed a reliable neutralization assay for SARS‐CoV‐2 based on VLP pseudotyped virus.

Abstract Regarding the extensive global attention to severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) that constitutes an international public health emergency, pseudovirus neutralization assays have been widely applied due to their advantages of being able to be conducted in biosafety level 2 laboratories and having a high safety factor. In this study, by adding a blue fluorescent protein (AmCyan) gene to the HIV system pSG3‐△env backbone plasmid HpaI and truncating the C‐terminal 21 amino acids of the SARS‐CoV‐2 spike protein (S), high‐titer SARS‐CoV‐2‐Sdel21‐AmCyan fluorescent pseudovirus was successfully packaged. The fluorescent pseudovirus was used to establish a neutralization assay in a 96‐well plate using 293T cells stably transfected with the AF cells. Then, parameters such as the ratio of backbone and membrane plasmid, sensitive cells, inoculation of cells and virus, as well as incubation and detection time were optimized. The pseudovirus neutralization assay demonstrated high accuracy, sensitivity, repeatability, and a strong correlation with the luminescent pseudovirus neutralization assay. Additionally, we scaled up the neutralizing antibody determination method by increasing the plate size from 96 wells to 384 wells. We have established a robust fluorescent pseudotyped virus neutralization assay for SARS‐CoV‐2 using the HIV system, providing a foundation for serum neutralization antibody detection, monoclonal antibody screening, and vaccine development.

Abstract The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) variants, particularly those with multiple mutations in receptor‐binding domain (RBD), pose a critical challenge to the efficacy of coronavirus disease 2019 (COVID‐19) vaccines and therapeutic neutralizing monoclonal antibodies (mAbs). Omicron sublineages BA.1, BA.2, BA.3, as well as the recent emergence of C.1.2, B.1.630, B.1.640.1, and B.1.640.2, have multiple mutations in RBD and may lead to severe neutralizing antibody evasion. It is urgent to evaluate the antigenic change of the above seven variants against mAbs and sera from guinea pigs immunized with variants of concern (VOCs) (Alpha, Beta, Gamma, Delta, Omicron) and variants of interest (VOIs) (Lambda, Mu) immunogens. Only seven out of the 24 mAbs showed no reduction in neutralizing activity against BA.1, BA.2, and BA.3. However, among these seven mAbs, the neutralization activity of XGv337 and XGv338 against C.1.2, B.1.630, B.1.640.1, and B.1.640.2 were decreased. Therefore, only five neutralizing mAbs showed no significant change against these seven variants. Using VOCs and VOIs as immunogens, we found that the antigenicity of variants could be divided into three clusters, and each cluster showed similar antigenicity to different immunogens. Among them, D614G, B.1.640.1, and B.1.630 formed a cluster, C.1.2 and B.1.640.2 formed a cluster, and BA.1, BA.2, and BA.3 formed a cluster.