Presentation Of SARS-CoV-2 RBD Mediated By ClyA On Bacterial Vesicles And Polymerized Porin Nanoparticles And Their Immune Characteristics

Background:The disease caused by SARS-CoV-2 infection threatens human health.The development of vaccines that are simple to prepare and inexpensive can reduce the economic burden of vaccination in low-and middle-income countries,and at the same time facilitate the rapid development of vaccines against mutant strains.The S protein of SARS-CoV-2 consists of two subunits,S1 and S2.S1 can combine with angiotensin-converting enzyme Ⅱ(ACE2)to promote virus invasion of target cells,and the receptor-binding domain(RBD)on S1 is the direct recognition region for the virus to bind to its receptor and is therefore a key target for vaccine development.The use of bacterial,insect or mammalian cells to express immunogenic viral proteins can often make effective and safe subunit vaccines.Adjuvants can be used to enhance the immune response,while multimeric antigen display and conjugation to nanoparticles can also be used to improve the immunogenicity of vaccines.Due to lack of protein glycosylation and possible formation of denatured protein inclusion bodies in prokaryotic expression systems,most vaccine development against SARS-CoV-2 has focused on the expensive and less efficient mammalian expression system rather than the highly efficient Escherichia coli system.However,a large number of clinical and preclinical studies have shown successful cases and beneficial explorations for the development of antiviral vaccines using E.coli expression systems,such as the recombinant virus-like particle(VLP)of human papillomavirus(HPV)and hepatitis E virus(HEV)has been applied clinically,and the use of bacterial outer-membrane vesicles(OMVs)to deliver viral antigens from dengue,influenza,and Middle East respiratory syndrome coronaviruses(MERS-CoV)has proven effective in animal models.The RBD of SARS-CoV-2 exhibits a trimeric structure in its natural conformation,and the host immune response elicited by this trimeric structure may be different from that elicited by the monomeric structure.Many studies have shown that the dimer and multimeric forms of RBD have better immune protection ability than RBD monomer.Therefore,multimerization of RBD may be a critical step in designing a SARS-CoV-2 vaccine.Objective:In the E.coli expression system,realize the correct structural folding and multimerization nano-assembly of SARS-CoV-2 RBD recombinant protein,and explore the effective subunit nano-vaccine delivery strategy,for the preparation of highly immunogenic,high immune protection and inexpensive recombinant vaccines such as SARS-CoV-2 provide a platform.Methods:In the first part of the study "Presentation of the SARS-CoV-2 RBD protein based on the novel bacterial biomimetic vesicle platform".The porin cytolysin A(ClyA)is used to carry the full-length RBD to form the fusion protein ClyA-RBD.Based on the membrane localization and self-assembly properties of ClyA,the highly regular polymerization presentation of RBD on bacterial membranes is realized.E.coli cells overexpressing ClyA-RBD form a new type of bacterial biomimetic vesicle(BBV)after the bacterial membrane passes through a narrow gap driven by high-pressure homogenization technology.First,the physicochemical properties of the vesicles were evaluated,the protein composition of RBD-BBV was analyzed by proteomics,and the conformational folding of RBD was investigated through binding experiments with hACE2 and neutralizing antibodies.Using in vitro cell experiments to analyze the effect of RBD-BBV on DC uptake,lysosome escape and DC phenotype maturation.Then,in the in vivo experiment,the distribution of RBD-BBV at the injection site and lymph nodes was analyzed by in vivo and tissue ex vivo fluorescence imaging,the cell types of antigen uptake in lymph nodes were analyzed by flow cytometry,and the gene expression of lymph nodes was analyzed by microarray.Finally,the mice were immunized,and the antibody response level and its blocking effect on the combination of SARS-CoV-2 S1 and hACE2 were analyzed by ELISA.Spleen immune cells and their cytokine levels were analyzed by flow cytometry and ELISA,and antigen-specific effector cell responses were analyzed by ELISpot.In the second part of the study " Presentation of the SARS-CoV-2 RBD protein based on the polymeric pore nanoparticle platform",the ClyA-RBD was extracted from the bacterial membrane using surfactants and then self-assembled into polymeric pore nanoparticles.In vitro experiments were carried out to investigate the morphology,folding and DC-promoting effect of the particles.In vivo experiments were used to investigate the distribution of major tissues and organs,the activation of immune cells in lymph node germinal centers,and further immunization of mice to investigate humoral and cellular immune responses.Results:In the first part of the study,a bacterial biomimetic vesicle RBD-BBV that presents RBD mediated by ClyA was successfully prepared.Compared with the OMV naturally secreted by bacteria,the yield of BBV has been greatly improved,and the loading of exogenous proteins on the vesicle surface has been significantly increased,which solves two important problems that limit the application and development of bacterial vesicles.Based on the membrane pore assembly properties of ClyA,a large number of ring structures formed by RBD polymerization were observed on the surface of BBV.Compared with the monomer,the RBD displayed on the surface of the vesicle in the form of nanopores has a higher affinity with ACE2,and the affinity is comparable to that of S1-Fc expressed in the eukaryotic system.In addition,RBDBBV can bind to a variety of neutralizing monoclonal antibodies,suggesting that RBD-BBV is correctly folded and has a similar spatial conformation to S1 expressed in eukaryotic systems,suggesting that RBD can provide correct immunogenicity.Further studies have found that BBV promotes the antigen uptake and maturation of DCs,and at the same time,lysosomal escape that is conducive to the induction of cellular immunity can occur.In addition,the characteristics of nano-proteolipid vesicles were demonstrated in terms of sustained release at the injection site,lymph node targeting,and activation of immune responses.In the mouse immune model evaluation,RBD-BBV induced specific humoral immunity,and the antiserum showed neutralizing protection in the wild-type SARS-CoV-2 virus neutralization experiment.At the same time,RBD-BBV immunization stimulated antigen-specific cellular immune responses,significantly stimulating the production of effector and memory CD4+and CD8+T cells.In order to explore a more simplified form of vaccine,based on the characteristics that the surfactant can simulate the bacterial membrane triggering the conformational change of ClyA and promote the self-assembly of the pore structure,the polymeric porin nanoparticle(RBD-PP)composed of recombinant ClyA-RBD was futher prepared.Compared with ClyA-RBD,RBD-PP showed a stronger ability to recognize neutralizing antibodies and ACE2,suggesting that it was folded in a more correct spatial conformation.Compared with ClyA-RBD,RBD-PP showed a stronger ability to recognize neutralizing antibodies and ACE2,suggesting that it is folded in a more correct spatial conformation,presumably because RBD is highly exposed to PP in a regular repeat arrangement and the steric conformational folding that occurs during concomitant ClyA polymerization.RBD-PP efficiently targets lymph nodes and promotes efficient uptake and maturation of antigen-presenting cells,as well as the generation of T follicular helper(Tfh)cells and germinal center(GC)B cells.In mouse immunization,RBD-PP induced more significant neutralizing antibody responses,effector and memory CD4+and CD8+T cell responses than RBD monomers,and exhibited balanced Thl and Th2 response characteristics.Conclusion:Using ClyA as a carrier to carry RBD achieved high-efficiency expression of RBD in Escherichia coli,and developed a bacterial biomimetic vesicle system and a polymeric porin nanoparticle as the novel vaccine antigen delivery platform.The polymerized nano-delivery of RBD with the correct conformation has been successfully realized.The ability to promote antigen processing and presentation and stimulate the body to produce an effective immune response has been demonstrated at the cellular level in vitro and in mouse immune models.The paper lays an important foundation for the development of SARS-CoV-2 subunit vaccines,especially in response to the challenges posed by constantly mutating viruses.At the same time,it also provides new ideas for the research and development of other virus vaccines,and can be extended to research and application fields such as bacterial vaccines,tumor vaccines,and drug delivery.