Construction Of Virus-Like Particles Of SARS-CoV-2 Variants And Study On Its Immunogenicity

COVID-19,caused by the SARS-CoV-2 of worldwide pandemic in end 2019,has brought unprecedented challenges to mankinds.Vaccine research is one of the most active and effective measures to deal with the epidemic.However,the continuous emergence of SARS-CoV-2 variants had significantly weakened the protective effect of existing vaccines.The novel vaccines continued to be developed to deal with the mutation of the virus.Virus-like particle（VLP）was a special subunit vaccine.It had been successfully developed in several virus vaccines,and also was a better choice in the development of SARS-CoV-2 vaccine.Our team used the baculovirus-insect expression system to construct the VLP-EMS-pro that expresses the E,M,and S proteins of Prototype strain of SARS-CoV-2 in early 2020.With the prevalence of Delta and Omicron variants of SARS-CoV-2 in the world,we modified the S protein gene sequence to construct VLP based on mutated spike protein（S）in the SARS-CoV-2 of Omicron（ο）and Delta（δ）strains.In this study,the immunogenicity and neutralizing antibody levels stimulated by VLP were explored.We constitute a promising platform in SARS-CoV-2 vaccine development.Objective:The baculovirus-insect expression system was used to construct and generate VLP based on the mutation of spike protein（S）in the Omicron（ο）and Delta（δ）strains of severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）.Evaluation of the immunogenicity of VLP by using different immunization strategies in mouse models.This study can serve as a basis for the development of a SARS-CoV-2 VLP vaccineMethods:1.Construction of shuttle recombinant bacmids Bacmid-EMS-οand Bacmid-EMS-δ.The expression plasmid pFastBac-EM was obtained on the basis of our team’s previous study.For construction of the recombinant plasmid pFastBac-EMS-ο,the codon-optimized S genes of the Omicron strain（B.1.1529）was cloned into the expression vector pFastBac-EM and S gene identified by sequencing.The pFastBac-EMS-οwas transformed into DH10 Bac^TM E.coli competent cells to perform the gene transposition.After blue-white spot screening and PCR analysis,the recombinant bacmid Bacmid-EMS-οwas isolated from the successfully transposed colonies.The Bacmid-EMS-δbased on the Delta（B.1.617.2）strain was produced in the same protocol.2.Expression,purification,and characterization of VLP-EMS-οand VLP-EMS-δ.Bacmid-EMS-οwas transfected into ExpiSf9^TM insect cells to obtain P0 generation baculovirus and the virus continued to be used to infect ExpiSf9^TM cells to produce P1generation baculovirus.The VLP-EMS-o was purified and harvested by sucrose density gradient hypervelocity centrifugation.The morphology of VLP-EMS-o and the display of S protein on VLP were analyzed by immunoelectron microscopy,and the expression of S protein was detected by Western blotting.The VLP-EMS（δ）was produced in the same protocol.3.We evaluated the immunogenicity of VLP-EMS-pro,VLP-EMS-οand VLP-EMS-δ.The BALB/c female mice were immunized thrice with the three immunization schedules at 0,2,and 4 weeks,respectively.We had been immunized twice with the vaccine against the Prototype strain of SARS-CoV-2,and the vaccine against the Prototype strain may be inoculated a third dose during the Delta and Omicron strains epidemics.In order to mimic to SARS-CoV-2 vaccine inoculated in human,the immunization schedules we designed were the prototype antigen booster group and Omicron antigen booster group.The mice were immunized thrice with VLP-EMS-pro in the pro+pro+pro group（the prototype antigen booster group）,respectively.The mice that had been primarily immunized and secondarily boosted with VLP-EMS-pro were third boosted with VLP-EMS-οin the pro+pro+o group（the Omicron antigen booster group）.Because we thought those who had been possibly infected with three variant strains of SARS-CoV-2 that included the Prototype,Delta,and Omicron strains during the COVID-19 pandemic,we named the third immunization schedule to the heterologous antigen booster group.The mice that had been primarily immunized were boosted with VLP-EMS-pro were secondarily and third boosted respectively with VLP-EMS-δand VLP-EMS-οin the pro+δ+οgroup（the heterologous antigen booster group）.The three immune strategies were divided into three dose groups,namely,a mouse was immunized with 5μg/200μL,10μg/200μL,and 50μg/200μL of VLP-EMS（weight/volume,W/V）,respectively.The mice that were injected with the same volume of buffer solution of VLP were used as the control group.The splenocytes and serum were isolated from mice at 2 weeks after the last immunization.The proportions of CD4⁺T cells and CD8⁺T cells in the mouse splenocytes were analyzed by flow cytometry（FCM）.The mouse splenocytes were stimulated with VLP-EMS-pro in the prototype antigen booster group.The mouse splenocytes were stimulated respectively with VLP-EMS-pro and VLP-EMS-οin the Omicron antigen booster group.The mouse splenocytes were stimulated respectively with VLP-EMS-pro,VLP-EMS-οand VLP-EMS-δin the heterologous antigen booster group.Cell culture supernatants were collected after 72 hours to detect the levels of IFN-γand IL-4 by ELISA.The level of antigen-specific antibodies Ig G in the mice of sera was detected with ELISA.Then we also test neutralizing antibodies in the sera of mice immunized,the RBD-ACE2interaction inhibition kits detected the neutralization of the sera against RBD proteins of SARS-CoV-2 strains about Prototype（pro）,Omicron（ο）,and Delta（δ）respectively.For the pilot to the molecular mechanism of antibody immunized using VLP,the BALB/c female mice were inoculated twice with VLP-EMS-pro at 0 and 2 weeks.Similarly,a mouse was immunized with 5μg/200μL,10μg/200μL,and 50μg/200μL of VLP-EMS（W/V）.The mice of lymph nodes were isolated for 10 days after the last immunization.The transcription factor of bcl-6 and blimp-1 in those lymph nodes was detected by quantitative real-time PCR（q PCR）.Result:1.The sequencing results showed that the recombinant plasmids pFastBac-EMS（ο）and pFastBac-EMS（δ）were constructed successfully.These recombinant bacmid Bacmid-EMS-οand Bacmid-EMS-δwere confirmed by PCR.2.The recombinant bacmids were transfected into ExpiSf9^TM insect cells to obtain the VLP-EMS.The VLP-EMS was purified and collected using sucrose density gradient hypervelocity centrifugation.Immunoelectron microscopy showed that the diameter of VLP-EMS-οand VLP-EMS-δwere 20-100nm and S protein was displayed on the surface of VLP,respectively.Furthermore,the expression of S protein in VLPs was also identified by Western blotting,respectively3.The mouse splenocytes were detected analyzed by FCM.We contrasted to the control group,the CD4⁺T cells in the percentage of the prototype antigen booster group,Omicron antigen booster group and heterologous antigen booster group significantly increased（P<0.05）.The CD8⁺T cells in the percentage of the prototype antigen booster group and heterologous antigen booster group were more than the control group,and the Omicron antigen booster group was more than the control group with10μg/200μL（W/V）（P<0.05）.The level of IFN-γand IL-4 whether the prototype antigen booster group or Omicron antigen booster group stimulated respectively with VLP-EMS-pro were higher than the control group in the dose difference（P<0.05）.And the heterologous antigen booster group stimulated respectively with VLP-EMS-pro,VLP-EMS-οand VLP-EMS-δwere higher than the control group in the dose diffrence（P<0.05）.Contrasted to the control group,the antigen-specific Ig G in the sera of mice immunized with the VLP-EMS was significantly increased in these strategies（P<0.05）.The neutralizing antibody response against RBD proteins of the Prototype and of the Omicron strains in the prototype antigen booster group was significantly stronger than the control group（P<0.01）.The neutralizing antibody response against RBD proteins of the Prototype,Delta and Omicron strains in the Omicron antigen booster group was stronger than the control group（P<0.05）.But in the 5μg/200μL（W/V）group,the antibody had no neutralizing response against RBD proteins of the Omicron strains in the Omicron antigen booster group.The neutralizing antibody response against RBD proteins of the Prototype,Delta and Omicron strains in the heterologous antigen booster group was significantly stronger than the control group（P<0.01）.Meanwhile,The neutralizing antibody response against RBD proteins of the Delta and Omicron strains in the heterologous antigen booster group was stronger than the prototype antigen booster group（P<0.05）.Conclusion:The baculovirus-insect cell expression system is used to produce the VLP-EMS-οand VLP-EMS-δof SARS-CoV-2 successfully.The mutated S protein based on the Omicron and Delta strains was assembled and expressed stably on VLPs.The VLP-EMS based on three variants of SARS-CoV-2 effectively induced antigen-specific cellular and humoral immunity after immunizing mice with three strategies,and the sera of the dose different groups showed significantly neutralizing antibody against RBD of Prototype strain.In addition,the Omicron antigen booster group and heterologous antigen booster group uniformly produced neutralizing antibody response against RBD of different SARS-CoV-2 strains.And the neutralizing antibody response against RBD of Omicron and Delta strain was stronger in the heterologous antigen booster group.The heterologous antigen booster group had a better extensive and cross-neutralizing antibody response.Our study also suggests that the vaccine should be adjusted in time and the heterologous antigen booster strategy should be adopted for about variation of SARS-CoV-2.