Construction And Evalution Of Immunogenicity Of Pha Nanocarrier Vaccine Against Streptococcus Suis And Glasser’s Disease

Streptococcus suis is a zoonotic pathogen that is commonly found in the upper respiratory tract of pigs as a conditional pathogen.It can infect pigs of all ages and cause a range of severe symptoms,including acute sepsis,meningitis,arthritis,endocarditis,and pneumonia.Glaesserella parasuis,on the other hand,is also a commensal bacterium in the upper respiratory tract of pigs,but it can invade the body and cause Gl?sser’s disease.This disease mainly affects pigs between the ages of 2 weeks and 4 months,and is characterized by fibrinous polyserositis,arthritis,and meningitis.Streptococcus suis and Glaesserella parasuis often co-infect pigs,posing a significant threat to both the pig industry and human public health security in our country.Therefore,the clinical prevention of these two pathogens is of utmost importance.Vaccination is widely considered as the most effective approach to prevent infections caused by Streptococcus suis and Glaesserella parasuis.Nevertheless,currently available inactivated vaccines for the prevention of these two bacterial diseases have various limitations such as poor cross-protection among different types,large immune doses and incomplete inactivation.Additionally,the attenuated vaccine also poses the risk of heightened toxicity.Despite these drawbacks,the first commercialized subunit vaccine for Streptococcus suis and Glaesserella parasuis globally has emerged as a promising alternative to address the limitations of the inactivated and attenuated vaccines.However,several challenges persist,including the requirement for high immune doses,complex production processes,relatively high manufacturing costs,and inadequate immunogenicity of individual protective antigens.Addressing these challenges is imperative for enhancing the effectiveness and feasibility of subunit vaccines.Polyhydroxyalkanoates(PHA)are natural polyesters that are self-assembled within bacteria.Due to their excellent biocompatibility,complete degradability,and appropriate particle size,PHA has emerged as a promising vaccine delivery system.Recent studies have confirmed that PHA particles,as a new type of vaccine,exhibit stronger immunological characteristics than the corresponding soluble subunit vaccines.Therefore,this study aimed to construct a universal PHA surface display antigen protein system in Clear Coli BL21(DE3)strain,and subsequently develop a PHA nanocarrier vaccine displaying the main antigens of Streptococcus suis and Glaesserella parasuis.To achieve this goal,the constructed PHA surface display antigen protein system was utilized to express and display the target antigens on the surface of PHA particles.The vaccine’s immunogenicity and efficacy were then evaluated through in vivo and in vitro experiments.The ultimate objective of this study is to develop a new,inexpensive,and efficient vaccine for the prevention of Streptococcus suis and Glaesserella parasuis infections.In conclusion,this study demonstrates the potential of PHA as a novel vaccine delivery system and the effectiveness of a PHA nanocarrier vaccine displaying the main antigens of Streptococcus suis and Glaesserella parasuis.The findings of this study contribute to the development of a safe,cost-effective,and efficient vaccine that can help prevent infections caused by these two important pathogens.The main results of this study are as follows :1.Construction of PHA surface display protein systemIn the present study,we aimed to synthesize Polyhydroxyalkanoates(PHA)polyester particles using the pha CAB biosynthesis operon from Cupriavidus necator.To achieve this goal,we first synthesized the Pha A,Pha B,and Pha C gene sequences via codon optimization in E.coli.Next,we constructed the p ACYC-Pha A-Pha B and p ET28a-Pha C plasmids according to the PHA synthesis process,and successfully expressed PHA protein.Under the transmission electron microscope,we observed that PHA spherical polyester particles were successfully formed with a diameter of approximately 100-500 nm.2.Construction of PHA nanocarrier vaccine of Streptococcus suis and Glaesserella parasuisBy taking into account the preliminary laboratory results and relevant literature at home and abroad,this study aimed to construct a polyhydroxyalkanoate(PHA)nanocarrier vaccine for Streptococcus suis and Glaesserella parasuis,using specific protective antigens as target antigens.In particular,the protective antigens HP0197,HP0272,and msly(P353L)of Streptococcus suis,and the protective antigens Afu A,06257,and Y167A-Tbp B of Glaesserella parasuis were chosen for this purpose.The p ET28a-Pha C plasmid was used as the basis for constructing the vaccine,and HP0197-PHA,HP0272-PHA,msly(P353L)-PHA,Afu A-PHA,06257-PHA,and Y167A-Tbp BPHA fusion proteins were successfully expressed.These PHA fusion proteins were observed in the form of spherical polyester precipitates,which were broken and centrifuged to obtain PHA particles.After washing the PHA particles three times with TBS,they were stored at 4℃.To prepare the nanoparticle vaccine of Streptococcus suis and Glaesserella parasuis,each PHA fusion protein was quantified based on an effective antigen dose of 15 μg / protein / mouse and 30 μg / protein / guinea pig.A total of 150 μL/ mouse and 300 μL / guinea pig of immune volume were used.The six PHA fusion proteins were then mixed in equal amounts with Montanide TM ISA206 VG(ISA206)adjuvant at a ratio of 1:1 to prepare the PHA nanoparticle vaccine of Streptococcus suis and Glaesserella parasuis.3.Establishment of antibody detection technology.The recombinant proteins,namely HP0197,HP0272,msly(P353L),Afu A,06257,and Y167A-Tbp B,were successfully expressed with a His tag at both ends.Each protein exhibited high expression levels in the supernatant.Following purification using a NiNTA affinity chromatography column,high-purity supernatant proteins were obtained,and the coating concentration of each protein was optimized to determine the serumspecific antibody titer of mice or guinea pigs immunized with the PHA nanocarrier vaccine.4.Evaluation of immunogenicity of PHA nanocarrier vaccine against Streptococcus suis and Glaesserella parasuisAfter immunizing mice with the PHA nanoparticle vaccine of Streptococcus suis and Glaesserella parasuis,significant levels of cellular and humoral immunity were induced.The induced antibodies had significant bactericidal effects against Streptococcus suis type2 and Glaesserella parasuis types 5 and 13,and had certain bactericidal effect on GPS13.Following challenge with type 2 Streptococcus suis SC19 strain,the vaccine was found to significantly reduce the bacterial load in various tissues,inflammatory response and tissue damage of mice,and it could 100% protect mice from infection caused by Type 2Streptococcus suis.Similarly,after immunization with the PHA nanoparticle vaccine of Streptococcus suis and Glaesserella parasuis,guinea pigs were found to produce significant levels of Ig G against each antigen protein.After challenge with H.parasuis type 5 H26 strain,the vaccine significantly reduced clinical symptoms and tissue damage in guinea pigs and provided 100% protection against H.parasuis type 5 infection in guinea pigs.In summary,this study successfully constructed a universal PHA surface display protein system and utilized it to construct a PHA nanocarrier vaccine for Streptococcus suis and Glaesserella parasuis.Immunization experiments in mice and guinea pigs demonstrated that the vaccine was capable of inducing high levels of cellular and humoral immunity,producing antibodies with bactericidal activity,and protecting against infection by Streptococcus suis type 2 and Glaesserella parasuis type 5.The vaccine is simple to prepare,cost-effective,easily obtainable,and has good protective efficacy.Therefore,this vaccine provides a potential vaccine candidate for Streptococcus suis and Glaesserella parasuis subunit vaccines.