Preparation Of K.pneumoniae Ghosts And Characteristic Of K.pneumoniae Ghosts Enhancing Immune Responses Of A.baumannii Subunit Vaccine In Mice

Acinetobacter baumannii(A.baumannii,Ab)is an extremely important opportunistic pathogen and one of the main bacteria causing nosocomial infections.A.baumannii is susceptible to induction of drug-resistant phenotypes.Some patients with autoimmune dysfunction,such as cancer and HIV,are vulnerable to A.baumannii infection,which leads to multiple complications such as pneumonia,meningitis,bacteremia,wound and urinary tract infection.Antibiotics are currently the main treatments for A.baumannii infection.Preventive vaccination is also an important strategy to control A.baumannii infection with the gradual increase of drug resistance.There is currently no clinical vaccine against Acinetobacter baumannii.As one of the most abundant surface proteins in A.baumannii,Outer membrane protein A(OmpA)plays an important role in regulating the permeability of antibiotics and small molecules while maintaining structural integrity of bacteria.It also plays a key role in the pathogenic mechanism of bacteria.OmpA-targeted subunit vaccine is an ideal candidate due to their advantages of single composition,highly conserved and cross-protection.The bacterial ghosts(BGs)is a bacterial shell which is safe and effective.The induced expression of phage lysate genes in bacteria can form transmembrane channels on the surface of bacteria,allowing nucleic acid and protein to spill out.BGs still retaining the same antigenic structure as living bacteria.In addition,BGs has strong immunogenicity,antigen presentation ability and bacterial components which have adjuvant properties,such as lipopolysaccharide,peptidoglycan,and lipid A.BGs can be used as an adjuvant in the development of novel vaccines.In this study,clinical strain T952 was used to prepare K.pneumoniae ghost(KPG)by inducing phiX174 cracking gene to explore its influence on the maturation and activation of bone marrow dendritic cells(BMDCs).The OmpA protein was expressed in E.coli.The purified OmpA protein combined with KPG was used as the candidate vaccine.The immune characteristics and immune effects of the candidate vaccine were evaluated through in vivo and in vitro tests,and the immune response of spleen lymphocytes to antigen restimulation was explored.The objective of this study was to elucidate the mechanism of KPG enhancing the immune response to OmpA in mice and lay a foundation for the development of new A.baumannii vaccines.Part 1.Study on the characteristics of K.pneumoniae ghost and its stimulation of immune function on BMDCsK.pneumoniae T952 competent cells were prepared,and the recombinant T952-EBOX strain was constructed by transforming pBBR-EBOX plasmid into bacteria.After induction at 42℃,the growth and lysis state of T952-EBOX strain(OD600 and colony for ming unit)were detected,and the biological characteristics of KPG in the preparation process were studied.Scanning electron microscopy(SEM)was used to observe the morphology of KPG.The results showed that the induced KPG produce holes at both ends of the bacteria cell,forming a bag structure with openings,and a large amount of contents spilled over.Mouse bone marrow derived dendritic cells(BMDCs)were isolated and stimulated with different doses of KPG.The surface molecules(MHC-Ⅱ,CD40,CD86,CD80),early activation marker CD69,migration marker CCR7,endocytosis of Dextran of BMDCs and the ability of BMDCs to promote CD4+T cell proliferation were detected by flow cytometry.The results showed that when BMDCs:KPG=1:10,the effect of KPG on the activation of BMDCs reached the best.After stimulation with KPG,the endocytosis of BMDCs was weakened,while the ability of promoting T cell proliferation was enhanced.The secretion of cytokines in the supernatant of BMDCs after KPG stimulation showed that the levels of IL-1β,IL-12 and TNF-α were significantly increased.These results indicate that KPG can promote the maturation and activation of BMDCs.Part 2.Expression of OmpA and effect of combined vaccine on immune function of BMDCs in vitroUsing the genome of A.Baumannii AB43 strain as template,ompA gene was amplified by PCR and cloned into prokaryotic expression vector pET-30a(+).The recombinant plasmid pET-30a-ompA was constructed and transformed into BL21(DE3)receptive cells.It was found that OmpA was mainly expressed in the inclusion bodies.The expressed OmpA protein was collected by purification,renaturation and other steps.The results of SDS-PAGE and Western blot showed that OmpA protein was successfully expressed.BMDCs were isolated from mice and stimulated with KPG combined with OmpA.OmpA,KPG and PBS were used as controls.Flow cytometry was used to detect the expression of surface molecules(MHC-Ⅱ,CD40,CD86,CD80)of BMDCs and the ability of BMDCs to promote the proliferation of CD4+T cells.The results showed that the expression of surface molecules and the ability to promote CD4+T cell proliferation were significantly increased in OmpA+KPG group compared with OmpA alone.The results also showed that KPG can enhance the expression of cytokines(IL-1β,IL-12,TNF-α)as an adjuvant.Part 3.Protective study of OmpA and KPG combined vaccine against A.baumannii infectionThe 6 weeks C57BL/6 mice were randomly divided into 4 groups:KPG group,OmpA group,OmpA+KPG group,and PBS group.Mice were immunized by subcutaneous injection with KPG at dose of 1 × 108 CFU/mouse and OmpA at dose of 50 μg/mouse.The mice were vaccined at week 0,week 2,and week 4.At week 6 of immunization,IgG,IgGl,and IgG2a antibody levels in mouse sera were measured by indirect ELISA.The results showed that IgG,IgGl and IgG2a antibody levels in sera of OmpA+KPG immunized mice were significantly increased compared with OmpA alone.At the same time,the immunized mice were challenged with different doses of A.baumannii AB43 according to the purpose of the experiment:the infection dose in survival experiment was 2 × 109 CFU/mouse,and the infection dose in bacteria loading experiment was 5 × 108 CFU/mouse.The results showed that the survival rate of OmpA+KPG combined vaccine group was significantly higher than that of OmpA alone,and the bacteria loading level was significantly lower than that of OmpA alone.HE staining showed the least pathological damage in the OmpA+KPG combined group.Central memory T cells(CD44hiCD62hi)and effector memory T cells(CD44hiCD621o)in spleen and lymph nodes were detected by flow cytometry.The results showed that the frequency of CD4+CD44hiCD62hi cells in the spleen and lymph nodes in OmpA+KPG group was significantly higher than those in OmpA alone and other controls,while the frequency of CD8+TCD44hiCD62hi cells was not significantly changed.Spleen cells were collected and the proliferation of spleen cells was detected by CCK-8 and flow cytometry.The results showed that the proliferation of spleen cell was significantly increased in the OmpA+KPG combined group compared with the OmpA alone.To further evaluate the activation of T cells,the percentage of central memory T cells(CD44hiCD62hi)stimulated with OmpA in vitro was measured by flow cytometry.The results showed that the frequencies of CD4+CD44hiCD62hi T cells and CD8+CD44hiCD62hi T cells were significantly increased in OmpA+KPG group.Flow cytometry was used to detect the early activation marker CD69.Compared with OmpA alone,T cells from mice immunized with OmpA+KPG were significantly activated.The intracellular cytokines were detected by flow cytometry.The results showed that the spleen of OmpA or OmpA+KPG immunized mice mainly secreted IFN-y after being stimulated,which indicated that the immune type favored Thl,and the level of IFN-γ secreted by OmpA+KPG group was significantly higher.These results indicated that KPG effectively enhanced the immune response to OmpA subunit vaccine.