Research On The Establishment Of SARS-CoV-2 Pseudovirus System And Vaccine Preliminary Evaluation

Severe Acute Respiratory Syndrome Coronavirus 2（SARS-Co V-2）has spread globally,bringing great threats to human life,health and huge economic losses.Therefore,it is urgent to find effective specific drugs or vaccines against the virus.However,SARS-Co V-2 is highly infectious and pathogenic.More and more infectious mutants have appeared such as D614 G,N501Y,E484 K and so on.The research of the virus should be carried out only in the P3 laboratory or above,which greatly limits the research of most laboratories and scientific researchers and is not conducive to exploit effective treatment methods,drugs and vaccines.There is an urgent need for a safe,effective and suitable research technique for SARS-Co V-2 infection studies.SARS-Co V-2 pseudovirus particles can simulate well the way and dynamics of live virus strains entering cells,making it a very safe research method to solve the safety problem of this virus.In order to facilitate the study of SARS-Co V-2 infection in P2 biosafety laboratory,a production and infection system for SARS-Co V-2 pseudovirus particles（SARS2pp）was constructed.The SARS2 pp system introduced in this article is based on a lentiviral vector carrying dual reporter genes e GFP and Luc2 for better observation and quantitative analysis.In order to increase the pseudovirus titer,the following conditions were optimized: 1)Remove the last 19 amino acids of the Spike protein and replace the original signal peptide with the mouse Igk signal sequence;2)Use the CMV promoter instead of CAG to express the Spike protein;3)Screen the better Spike protein sequence for SARS2 pp production;4)Add 1% BSA to SARS2 pp production medium.These optimizated conditions has greatly increased the pseudovirus titer,and provided convenience for viral infection mechanism,the screening of infection inhibitors,and the neutralizing antibody experiments for vaccine evaluation.In the pre-experimental of SARS-Co V-2 vaccine,the SARS-Co V-2 candidate Spike antigen protein is used to replace the VEE virus structural protein to design a self-replicating vaccine.The designed Spike protein has two mutations: 1)C-terminal 19 amino acids were deleted to improve its cell membrane localization;2)Furin site was mutated to reduce Spike protein cleavage.A tandem RBD structure named RBDRBD was constructed for comparative study.The neutralizing activity of serum was verified after immunization of mice.The preliminary results showed that the designed antigen protein can better stimulate the body to produce effective antibodies with a strong neutralizing effect on the pseudovirus.Compared with other DNA vaccines,this vaccine has the advantages of high expression of candidate antigens,faster clearance,and less immunization dose.It is worthy of further study.