| Subunit vaccines are the research hotspot and development direction of new animal vaccines.Subunit vaccine antigens are usually pathogen-protective functional proteins expressed recombinantly in vitro,which are safe but induce limited levels of antibodies that are insufficient to provide complete protection to the organism.Improving the immunogenicity of subunit vaccine antigens is a key issue that needs to be addressed in the development of novel animal vaccines.Multivalent presentation of antigens on the platform of nanoparticles(NPs)is an effective strategy to enhance the immunogenicity of subunit vaccines and thereby induce high levels of organismal immune response by increasing the affinity and particle size to improve the uptake of antigen-presenting cells(APCs),lymph nodes(LNs)transport,and B-cell activation,and it has been successfully utilized in both clinical research and commercial applications.Porcine circovirus type 2(PCV2)is one of the most important swine-origin viruses,causing severe immunosuppression and often mixed or secondary infections with other viruses,resulting in significant losses to the world pig industry.Its capsid(Cap)protein is capable of self-assembling into virus-like particles(VLPs),making it an ideal antigenic platform for the development of subunit vaccines for pigs.This study aimed to develop a versatile modular nano-antigen display platform based on PCV2 VLPs.It is proposed to investigate the potential of such a platform to display important porcine-derived viral protective peptides,monomeric proteins,dimeric proteins,and trimeric proteins and to study their induced immune responses in the mouse model.The main points of this study and the results obtained are briefly described below:1.The Cap-Cat VLPs nano-antigen display platform was constructedTo realize a convenient assembly method and expand more applications of PCV2 VLPs,a Cap-Spy Catcher003(referred to as Cap-Cat)VLPs nano-antigen display platform was developed by combining PCV2 VLPs with the modular linker element Spy Tag003/Spy Catcher003 system.Firstly,the Spy Catcher003 gene sequence was fused to the C-terminus of the PCV2 Cap protein gene sequence,and the prokaryotic expression vector of PCV2 Cap and Spy Catcher003 fusion protein was constructed.Then the fusion protein was prepared by expression using the Escherichia coli expression system,and a combination purified the protein of ammonium sulfate precipitation and size exclusion chromatography.The self-assembly of Cap-Cat VLPs into particles with a diameter of 20 nm was confirmed by dynamic light scattering(DLS)and transmission electron microscope(TEM).Stability and safety evaluations showed that Cap-Cat VLPs had a suitable temperature,freeze-thaw,and storage stability: they retained solubility after 1 h of incubation at 75°C,resisted 10 freezethaw cycles,and could be stored at 4°C for more than 30 days.In addition,Cap-Cat VLPs showed no significant cytotoxicity and good biocompatibility.2.The Cap-Cat VLPs platform is capable of efficiently demonstrating porcine-derived viral protective antigens of different sizes and multimeric structuresTo evaluate the antigenic display potential of the Cap-Cat VLPs platform,four Spy Tag003 fusion antigens with different sizes and multimeric structures were prepared,including the B-cell epitopes of porcine reproductive and respiratory syndrome virus(PRRSV)in a peptide form,the core neutralizing epitope(COE)protein of porcine epidemic diarrhea virus(PEDV)in monomeric form,the E2 protein of classical swine fever virus(CSFV)in dimeric form,and the hemagglutinin(HA)protein of swine influenza virus(SIV)in trimeric form.Firstly,to facilitate the purification of multiple Spy Tag003 fusion antigens,a highaffinity monoclonal antibody against the Spy Tag003 protein was successfully prepared after immunizing mice by also displaying the Spy Tag003 protein through NPs.The above Spy Tag003 fusion antigens were successfully purified by affinity chromatography using this antibody as the ligand.SDS-PAGE results showed that the four Spy Tag003 fusion antigens could be well demonstrated on the Cap-Cat VLPs platform,their assembly efficiencies could reach about 59% to 91%,and their solubility was well maintained.DLS and TEM analysis showed that the particle sizes of the four fusion antigens were about 22 to 28 nm after binding to VLPs.3.The Cap-Cat VLPs platform demonstrates antigens that trigger a robust organismal immune responseTo investigate the immunogenicity of four antigen-modified Cap-Cat VLPs,mice were immunized to analyze the humoral and cellular immune responses as well as the protective efficacy after the attack.The results showed that the four antigen-modified Cap-Cat VLPs induced a high level of immune response: serum antibody titers against the corresponding antigens were increased by approximately 6-to 8-fold compared with the immunized soluble antigens,and virus neutralization assays showed that serum neutralizing antibody titers were increased by approximately 6-to 24-fold.Antibody subtyping showed that antigen-modified Cap-Cat VLPs enhanced Th2 polarization and improved Th1 polarization.In addition,compared with mice immunized with the corresponding soluble antigens,the number of cells secreting the cytokines IFN-γ and IL-4 in splenic lymphocytes from mice immunized with several antigen-modified Cap-Cat VLPs was increased by approximately 3.8-to 15.6-fold,the splenic lymphocyte stimulation index was elevated by approximately 1.2-fold,the level of secretion of IFN-γ,IL-4,and IL-2 in the culture medium of splenic lymphocytes was elevated by approximately 3.5-to 4.6-fold,and the proportion of T helper cells(Th)and cytotoxic T lymphocytes(CTL)in splenic lymphocytes was increased by approximately 1.5-to 2-fold.The results of the takedown protection assay showed that Cap-Cat-HA VLPs significantly ameliorated the lung pathological changes in H1N1-infected mice and reduced the mouse lung virus titer by about 400-fold,and the secretion levels of inflammatory cytokines TNF-α and IL-6 in the mouse bronchoalveolar lavage fluid(BALF)by about 18-fold and 13-fold,respectively.In addition,analysis of the effect of in vivo preexisting immunization on immunization with Cap-Cat VLPs showed that the Cap-Cat VLPs platform increased the level of specific antibodies to the carried antigen by approximately 3.2-to 8.5-fold,although high levels of PCV2 antibodies or antibodies to the Cap-Cat scaffold already existed in vivo.4.Preliminary study of the mechanism of antigen-triggered robust organismal immune response demonstrated by the Cap-Cat VLPs platformIn this chapter,the Cap-Cat-HA was used as a representative to study the process of improving the uptake,transport,localization of LNs,and activation of immune cells by dendritic cells(DCs)by antigens demonstrated on the Cap-Cat VLPs platform,and initially analyzed the mechanism by which antigens demonstrated on the Cap-Cat VLPs platform enhanced the immune response of the body.Flow cytometry(FCM)and Confocal laser scanning microscope(CLSM)demonstrated that the antigens demonstrated by Cap-Cat VLPs were more readily taken up and internalized by DCs,and helped to stimulate the activation and maturation of DCs.Analysis of antigen transport kinetics in mice by in vivo imaging showed that antigens demonstrated by the Cap-Cat VLPs platform had a longer storage time in vivo than soluble antigens,which could be sustained at the injection site for more than 4 d,and at the same time were more helpful in delivering antigens to secondary immune organs.The accumulation and localization of antigens in LNs were analyzed by immunofluorescence imaging of frozen sections of LNs,and the results showed that the antigen carried by Cap-Cat VLPs platform could reach the LNs 2 h after immunization and could be gradually deposited in the areas of follicular dendritic cells(FDCs)and germinal centers(GCs),and the areas of FDCs and GCs in LNs at 14 days after immunization increased approximately 2.1-fold and3.3-fold compared with immunization with soluble antigens.Finally,FCM analysis showed that antigen demonstration using the Cap-Cat VLPs platform significantly activated immune cells,increasing the proportions of LNs GC B cells and follicular helper T(Tfh)cells by approximately 2-fold and 2.6-fold,respectively.In summary,in this study,a universal nano-antigen display platform based on PCV2 VLPs was constructed,and protective antigens of porcine-derived viruses with different sizes and multimeric structures were successfully demonstrated by modular assembly,and triggered strong humoral and cellular immune responses in mice.The potential mechanism that triggers this powerful organismal immune response was also preliminarily investigated: the appropriate size with repetitive,high-density antigenic presentation promotes the uptake of DCs and the transport,localization,and storage of antigens within LNs,and initiates the GCs response to promote the activation of GC B cells with the assistance of FDCs and Tfh cells,which then induces a highly efficient immune response.Overall,the development of a "plugand-display" universal nano-antigen display platform based on PCV2 VLPs can provide new ideas and technical support for the granular design of antigens for porcine subunit vaccines.It will be beneficial to the development of new porcine subunit vaccines that are more efficient and convenient,and will also facilitate the prevention,control,and differential diagnosis of porcine viral diseases. |
