Preparation And Immune Response Evaluation Of An OMV-Based SARS-CoV-2 Nanoparticle Vaccine

BackgroundSevere acute respiratory syndrome coronavirus 2(SARS-Co V-2)has swept through the world since 2019,infecting more than 760 million people cumulatively,posing a significant threat to global governance.With the emergence of multiple mutant strains,including Alpha(B.1.1.7),Beta(B.1.352),Delta(B.1.617.2),and Omicron(B.1.1.529),the protective effect of existing vaccines on human beings are gradually declining,and it is urgent to develop new strategies for SARS-Co V-2 vaccine research.Several vaccines developed with the antigen of SARS-Co V-2 receptor-binding domain(RBD)have entered clinical trials.However,the low immunogenicity of RBD calls for a combination of adjuvants,polymers-designing,and polyvalent displaying.The outer membrane vesicles(OMV)are nano-spherical vesicles secreted by Gram-negative bacteria.On the one hand,OMV can be used as a vaccine carrier to present and deliver antigens.On the other hand,a large amount of pathogen-associated molecular patterns(PAMP)in OMV also make it an adjuvant.Therefore,applying OMV as the carrier to design and realize the multivalent display of RBD and developing a "plug-and-play" vaccine platform may be a new route for SARSCo V-2 vaccine research and development.ObjectivesThe Spy Tag(ST)/ Spy Catcher(SC)biological conjugating system is applied in order to develop a "plug-and-display" SARS-Co V-2 nanoparticle vaccine based on OMV vector,make full use of the adjuvant activity and antigen delivery capability of OMV,and realize the purpose of multivalent displaying RBD with OMV.Escherichia coli was used to express OMV-SC,and HEK293 F cells were used to express RBD-ST.After purification,OMV and RBD were conjugated in vitro and characterized preliminarily.The effect of the OMV-RBD vaccine on DCs activation was evaluated.To evaluate the humoral and cellular immune response,analyzing the types and characteristics of immune responses induced by the OMV-RBD vaccine,mice were immunized intranasally or intramuscularly,and the protective effects of OMV-RBD vaccine were preliminarily evaluated by h ACE2 competitive inhibition experiment and pseudovirus neutralization test.Methods1.Preparation and characterization of OMV-RBDThe p Thio-His A-Cly A-GFP plasmid was constructed and transformed into Escherichia coli BL21(DE3).The induction conditions were screened by SDS-PAGE,and the OMV-SC was purified by methods like ultrafiltration and ultracentrifugation.HEK293 F cells were transfected with constructed pc DNA3.1-RBD-ST plasmid,and RBD-ST was expressed and purified by cell fermentation,affinity chromatography,and ultrafiltration.The reaction ratio between OMV-SC and RBD-ST and the content of RBD was determined by Western blot.Use transmission electron microscope(TEM)and dynamic light scattering(DLS)to characterize the morphology,average particle size,and size distribution of OMV-RBD.2.The effect of DCs activation by OMV-RBD in vitroDC2.4 was used as the model to evaluate the safe concentration of OMV-RBD in vitro by hemolysis test,MMT colorimetry,and CCK-8 reagent.The phagocytosis of DCs on OMV-RBD was observed by confocal microscopy after protein fluorescence labeling.Mouse bone marrowderived monocytes were obtained and differentiated into bone marrow-derived dendritic cells(BMDCs)in vitro.Flow cytometry was used to detect the expression levels of CD40,CD80,and CD86 in BMDCs stimulated by OMV-RBD.The concentrations of IL-1β,IL-6,IL-10,IL-12p70,and TNF-α in BMDCs culture supernatant were determined by ELISA to evaluate the DCs activation stimulated by OMV-RBD in vitro.3.Evaluation of the immune response of OMV-RBDMice were immunized with OMV-RBD intranasally or intramuscularly on day 0,day 14,and day 28 and sacrificed on day 42.Samples were also collected.Use ELISA to determine specific Ig G and Ig A levels in serum.The Ig G and s Ig A levels in bronchoalveolar lavage fluid(BALF),nasal lavage fluid(NLF),and vaginal wash were measured too.ELISpot was used to detect the number of IFN-γ-secreting cells in mice’s splenic and pulmonary lymphocytes.Cytokine concentrations of IFN-γ,IL-2,IL-4,and IL-17 in stimulated splenic lymphocyte supernatant were determined by ELISA.The ability of antibodies in serum,BALF,and NLF to competitively inhibit the combination of RBD and h ACE2 was detected by ELISA,serumneutralizing antibody levels are measured by pseudovirus neutralization test,evaluating the immune response of OMV-RBD comprehensively.Results1.OMV-SC and RBD-ST were expressed by E.coli prokaryotic expression system and HEK293 F eukaryotic expression system,then covalently conjugated with Spy Tag / Spy Catcher biological conjugating system.The optimal reaction ratio was 40:1(OMV-SC: RBD-ST).The average particle size of OMV-RBD was 152.6 nm,characterized by DLS and TEM.OMV-RBD was a regular vesicle,and the lipid bilayers were visible under transmission electron microscopy.The content of RBD was 1.63%(m/m),indicating that the OMV-RBD was successfully constructed.2.When the concentration of OMV-RBD is lower than 0.3 mg/m L,it does not cause hemolysis and has no apparent cytotoxicity.The phagocytic efficiency of OMV-RBD by DC2.4was significantly higher than that of the free RBD and OMV+RBD mixed group.OMV-RBD can promote the co-stimulatory molecules’ expression of CD40,CD80,and CD86 on the surface of BMDCs,and significantly increase the secretion levels of TNF-α,IL-1β,IL-12p70,and IL-6 and decrease the secretion of IL-10 in the supernatant of BMDCs culture,suggesting that OMV-RBD can significantly activate DCs.It also has the potential to activate innate immunity and activate T cells.3.OMV-RBD can significantly improve the level of s Ig A antibodies in NLF,BALF,and vaginal wash,as well as the level of specific Ig G antibodies in serum and mucosal.Mice immunized with OMV-RBD intramuscularly cannot produce specific s Ig A at the mucosal site.However,it can induce higher serum Ig G titer,suggesting that OMV-RBD can enhance mice’s systemic humoral immune response.The number of IFN-γ-secreting cells in the lung of intranasally immunized mice and the spleen of intramuscularly immunized mice were significantly increased.The secretion of IFN-γ,IL-2,IL-4,and IL-17 in lymphocytes was also significantly raised,suggesting that OMV-RBD can induce a specific cellular immune response in mice.Both methods of OMV-RBD immunization can significantly enhance the inhibitory effect of NLF,BALF,and serum antibodies on the binding of RBD to h ACE2,the serum antibodies induced by OMV-RBD can effectively neutralize the wild-type SARS-Co V-2pseudovirus,suggesting that functional antibodies induced by OMV-RBD may provide more robust protection for mice.Conclusion1.An OMV-based SARS-Co V-2 nanoparticle vaccine displaying multivalent RBD was successfully constructed in this study.2.OMV-RBD is relatively safe in vitro,can promote the antigen uptaking of DCs and the maturation of BMDCs,and significantly activates DCs.3.OMV-RBD can induce antigen-specific humoral and cellular immune responses by both intranasal and intramuscular immunization.The antibodies produced after immunization can significantly inhibit the binding of RBD to h ACE2 and neutralize the pseudovirus.All these results were significantly better than those of free RBD and physical mixed OMV+RBD.SignificanceIn this study,we designed a SARS-Co V-2 nanoparticle vaccine platform with OMV as the carrier,which can realize the purpose of "plug-and-display." We systematically evaluated the immune response in vivo and in vitro,providing a new idea for the research and development of the SARS-Co V-2 recombinant subunit vaccine.The vaccine platform can be used to rapidly develop SARS-Co V-2 mutant vaccines and demonstrate multiple mutant antigens to construct a SARS-Co V-2 "mosaic vaccine" in the future.