Preparation And Evaluation Of Two Bacterial Nano-polysaccharide Conjugate Vaccines

Pathogenic bacterial infections pose a serious threat to human health,and immunization is the most cost-effective means of preventing infectious diseases.Bacterial exopolysaccharides(mainly CPS and O-PS)are important virulence factors that cause bacterial invasive infections,and are also the main antigenic components of vaccines to prevent and control these bacterial infections.Since most of these polysaccharides belong to the thymus non-dependent antigen(T-independent antigen,TI),can not form immune memory B cells,especially for infants under 2 years old,the elderly,some immunodeficiency patients and other groups can not provide sufficient protection,so the polysaccharide conjugate vaccine strategy can be adopted: polysaccharide units with variable structure and number and non-sugar units(proteins or short peptides,lipids)are covalently linked to form vaccines,mainly protein-polysaccharide combination.Non-sugar units act like adjuvants,activating helper T cells and then promoting the maturation and differentiation of B cells to form memories,thereby providing long-term immune protection.Therefore,polysaccharide conjugate vaccines were prepared for gram-positive and gram-negative bacteria respectively in this study.Objective: In order to prepare gram-positive bacterial Streptococcus pneumoniae and gram-negative bacteria Shigella spp.polysaccharide conjugate vaccines respectively and explore new vaccine immunization strategies,a safer and more efficient candidate polysaccharide conjugate vaccine variety was developed.Methods: W3110Δwaa L Δwbb H-L,which was constructed by our laboratory with high heteroglycoprotein expression efficiency,was selected as the expression host.By introducing the p BAC-CPSSp K5 expression vector of Streptococcus pneumoniae polysaccharide serotype K5,Meanwhile,the plasmids p ET28a-pgl L-CTB and p ET28a-pgl L-CTBTri were introduced respectively,and Protein glycan coupling technologies(PGCT)were used in Escherichia coli directly synthesized the polysaccharide conjugate vaccine of K5 serotype using CTB and CTBTri nanoparticles(NPs)as the carrier,and evaluated the particle size,serum antibody titer,protection ratio and in vitro bactericidal efficiency of the two vaccines.The mature and high-yield Shigella spp.polysaccharide conjugate vaccine NP-OPSS.flexneri 2a combined with microneedle preparation technology prepared in the laboratory was used to prepare microneedle tablets for animal immunization through percutaneous immunization,and its immune effect was comprehensively evaluated.Meanwhile,combined with PGCT and O-glycosylation system,Shigella spp.glycoprotein SC-OPSS.flexneri 2a with Spy Catcher as substrate protein was prepared,which was connected with Spy Tag-HBC-VLPs in vitro,and Shigella spp.polysaccharide was displayed on its surface.The particle size and immune effect of the polysaccharide conjugate vaccine were evaluated.Results: Using the expression vector of polysaccharide from Streptococcus pneumoniae serotype K5,the polysaccharide conjugate vaccine of serotype K5 was synthesized directly by glycosyltransferase Pgl L in Escherichia coli for the first time.Both of them can stimulate the body to produce specific immune response against Streptococcus pneumoniae serotype K5 CPS.Among them,the size of nano-vaccines is detected by transmission electron microscopy at about 25 nm,which can induce the production of more efficient Ig G antibodies,and the protection rate in fatal infections can reach 100%.To assess the neutralizing activity of the antibodies excited by the conjugate vaccine,the Opsono-phagocytic killing(OPK)assay was performed in vitro.In the presence of complement and HL60 cells,the post-serum of C-CPS+Al group and CPS+Al group showed opsonization activity against Streptococcus pneumoniae,and under pro-double serum,the NP-OPS group killed 75% of Streptococcus pneumoniae,which was significantly higher than that of C-OPS+Al group and CPS+Al group(p<0.0001).The above results showed that the specific antibodies produced by nano-conjugate vaccines mediated efficient killing ability against Streptococcus pneumoniae under the synergy of complement and phagocytes.The effect of animal immunization by percutaneous immunization of Shigella spp.nano-polysaccharide conjugate vaccine by microneedle technology showed that both experimental groups produced high levels of Ig G antibodies compared with the control group.In the same case without the addition of adjuvants,the microneedle experimental group was about 8 times higher than the conventional subcutaneous injection group-specific antibody titer Ig G,and there was a difference in serum titer between the two groups(p<0.0001).HBC-VLPs with Spy Tag joint in the MIR region of particle surface were successfully constructed.Meanwhile,the combination of Spy Catcher/Spy Tag system and PGCT technology was adopted to successfully display Shigella spp.polysaccharide on the surface of HBC-VLPs,and its particle size was detected,which was 33~45nm.Conclusion: The conjugate vaccine of serotype K5 with CTB and nanoconjugate vaccine with CTBTri nanoparticles(NPs)were prepared by biological method,which proved that both can stimulate the body to produce a specific immune response against Streptococcus pneumoniae K5 serotype CPS,and Streptococcus pneumoniae nano-conjugate vaccine can still produce an efficient immune response in the absence of adjuvants,producing a significantly better protective effect than non-nano-conjugate vaccines and monosaccharide vaccines,providing a new option for the vaccine development of Streptococcus pneumoniae.In this study,the use of microneedle technology to use Shigella spp.nano-polysaccharide conjugate vaccine to perform animal immunization by percutaneous immunization produces a more efficient immune response than conventional subcutaneous injection,which provides a new idea for the development of Shigella spp.vaccine.In addition,a Shigella spp.nano-polysaccharide conjugate vaccine with HBC VLPs as the carrier was prepared,and polysaccharide antigens were successfully displayed in the main immunodominant region(MIR)of HBC particles,forming densely arranged and repeated epitopes to induce immune response in the body,which provided a new nano-carrier for the preparation of Shigella spp.nano-polysaccharide vaccine.