Research on the Construction of a SARS-CoV-2 Pseudovirus Library Based on the VSV Vector and the Screening of Immune Escape Strains

CAO Gao-Jie; XU Chen-Hui; YU Lin-Qian; CI Ke-Ke; WU Bei-Bei

Journal of Microbes and Infections ›› 2025, Vol. 20 ›› Issue (3) : 139-149.

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Abstract

This study aims to construct a pseudovirus mutation library of the SARS-CoV-2 spike (S) protein based on the vesicular stomatitis virus (VSV) vector system, in order to screen potential immune escape variants and identify critical mutation sites, thereby providing scientific evidence for vaccine development and epidemic prevention and control. A random mutagenesis kit was employed to introduce random mutations into the receptor-binding domain (RBD) of the Wuhan strain S protein, followed by the construction of a pseudovirus plasmid library and subsequent virus rescue using the VSV vector system. Immune escape variants were screened through serum pressure and limited dilution methods, with their mutation sites validated by sequencing. Results demonstrated the successful construction of the SARS-CoV-2 pseudovirus plasmid library, achieving a positive rate of 98%. Immune escape variants carrying single mutations such as E484K, F486I, G339S, and S477R, as well as double mutations including E484K+K444Q and E484K+K462R, were identified. Some escape variants exhibited significant resistance to neutralizing sera, with a marked reduction in neutralizing titers observed for the E484K single mutant and E484K-related double mutants. This study confirms the feasibility of screening immune escape variants through the construction of a SARS-CoV-2 pseudovirus mutation library and elucidates their potential escape mechanisms and epidemic characteristics. These findings provide important insights into understanding the evolutionary trends of SARS-CoV-2 and offer scientific support for optimizing vaccine development and epidemic prevention strategies.

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SARS-CoV-2 / Immune escape / ?Random mutation library / Mutation sites / Vaccine development

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Research on the Construction of a SARS-CoV-2 Pseudovirus Library Based on the VSV Vector and the Screening of Immune Escape Strains[J]. Journal of Microbes and Infections. 2025, 20(3): 139-149

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