Evaluation Of Immune Protection And Mechanism Study Of Klebsiella Pneumoniae Bivalent Subunit Fusion Vaccine

ObjectiveKlebsiella pneumoniae(KP)is an opportunistic pathogen that mainly causes serious infections in critically patinets and immunocompromised people.It usually colonizes the human mucosa,including the gastrointestinal tract,genitourinary,oropharynx and so on.Klebsiella pneumoniae can invade tissues through mucous membranes to expand infection,resulting in pneumonia,urinary tract infection,meningitis,bacteremia and other serious diseases.In recent years,due to the abuse of antibiotics and the limitations of new antibiotic research,multidrug-resistant Klebsiella pneumoniae has emerged in clinical and community environments,especially multiple strains of extended-spectrum β-lactamase and carbapenemase,which brought pressure to clinical treatment.Therefore,it’s critical to develop vaccine to prevent the spread of drug resistance and outbreaks of Klebsiella pneumoniae.The purpose of this study was to screen,construct,express,and purify candidate antigens of GlnH(glutamine ABC transporter periplasmic protein)and Fim A(major type 1subunit fimbrin),and co-immunized with the mucosal adjuvant curdlan.To evaluate the protective effect of bivalent fusion subunit vaccines.And,to further study the mechanism of the bivalent subunit vaccine exerting immune protection.The aim is to provide effective candidate antigen proteins and serotype-independent vaccine strategies for the development of Klebsiella pneumoniae vaccines.Method1.Using p GEX-6P-2 vector,construct GST-GlnH and GST-Fim A recombinant plasmids.Transformed into BL21,and then expansion culture,low temperature induction with IPTG,collection supernatant by breaking bacteria,binding of GST-beads,crude PP enzyme,remove endotoxin,and concentration to obtain the target candidate antigen protein.2.C57BL/6 mice were immunized with two candidate antigen proteins and the mucosal adjuvant curdlan.On the 7th day after the last immunization,the tracheal intubation was injected with YBQ strain for infection,evaluate the protective effect by survival rate,colonization,lung tissue pathological sections and cytokine levels.3.Flow cytometry was used to analyze the frequency of TRM cells.Use GK1.5 to block CD4 T cells,detecting the colonization and lung tissue pathological sections.Use FTY720 to block peripheral lymphocytes,analyzing the colonization,lung tissue pathological sections,the cytokine levels and the frequency of TRM cells.Result1.Successfully constructed a soluble vaccine GlnH and Fim A that can be efficiently expressed and purified by prokaryotic expression system.The antigen purity can reach 95%,and the molecular weight of the antigen is consistent with the target protein.The molecular weight of GlnH protein is 25.3 k Da,and the molecular weight of Fim A protein is 17.3 k Da.2.After lethal dose challenge infection,the survival rate of immunization group was60%,and the control group was 0%,with statistical difference.After infection with sublethal dose,the mean lung colonization of mice in the immunized group was 7.62 Log10CFU/Lung,while the control group was 4.88 Log10 CFU/Lung(P=0.0002);compared with the control group,the pathological sections of the immunized group showed more complete alveolar structure,less damage and less inflammatory cell infiltration,and the pathological score showed that the immunized group was significantly lower than the control group,with significant statistical differences;the detection results of chemokines CXCL1 and CXCL2 showed that the immunized group was significantly higher.3.Compared with immunized group,immunized+GK1.5 showed increased colonization amount,more serious pathological inflammatory damage,and decreased chemokine levels,all of which were statistically different.Flow cytometry analysis showed that the average positive rate of CD4+CD69+CD62-TRM cells in the immune group was 54%,while that in the control group was only 21%(P=0.0006),with a statistically significant difference.The positive rate of IL-17 A in the immune group was 20%,the control group was 5.8%,there was a statistical difference(P=0.0194).Compared with the control group,Immunized+FTY720 had no statistical difference in colonization amount,lung histopathological score and cytokine level.The average positive rate of CD4+CD69+CD62-TRM cells in the immunized+FTY720 group was 73%,and that in the unimmunized+FTY720 group was 31%,with a statistically significant difference(P<0.0001).Conclusion1.The combined preparation of GlnH and Fim A candidate antigen proteins and mucosal adjuvant curdlan can resist the infection of mice by Klebsiella pneumoniae.2.The immune mechanism of this bivalent subunit vaccine is mainly cellular immunity,which may be related to the resident memory T cells in lung tissue.