Affiliation:
1. Changping Laboratory Beijing China
2. Chinese Academy of Medical Sciences & Peking Union Medical College Beijing China
3. Division of HIV/AIDS and Sex‐Transmitted Virus Vaccines National Institutes for Food and Drug Control (NIFDC) Beijing China
4. CAS Key Laboratory of Infection and Immunity, National Laboratory of Macromolecules, Institute of Biophysics Chinese Academy of Sciences Beijing China
5. University of Chinese Academy of Sciences Beijing China
6. National Kunming High‐level Biosafety Primate Research Center, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming Yunnan, China Kunming China
Abstract
AbstractSpike‐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.