Preparation Of A.baumannii Ghosts And Its Characteristics As A Vaccine And Adjuvant

In recent years,Acinetobacter baumannii(A.baumannii)has been one of the main pathogens causing nosocomial infections.With the continuous increase in the clinical isolation rate and drug resistance of A.baumannii and the lack of effective vaccines,it has brought great challenges to its anti-infective treatment and prevention.In addition,the large-scale persistent infection caused by SARS-CoV-2 increases the risk of ventilator-associated pneumonia caused by Acinetobacter baumannii in the hospital.Therefore,the development of a safe and efficient vaccine is of great significance for the prevention and control of A.baumannii infection.Bacterial ghosts(BGs)is a kind of bacterial shell,which only contain the outer membrane by removing the cytoplasmic contents of bacteria after certain treatment.The methods commonly used to prepare bacterial ghosts include:the minimum inhibitory concentration method using chemical reagents and the bacteriophage PhiX174 lytic gene E induction method.The advantages of BGs are:firstly,they contain bacterial surface antigen structure and strong immunogenicity,which can be used directly as a vaccine antigen;secondly,they can be used as natural immune adjuvants in combination with other antigen to improve the immune response level of the antigen.This article aims to use chemical and genetic methods to prepare A.baumannii ghosts(ABG),evaluate its immune protection as a vaccine,and investigate its immunoprotective effect when the bacterial virulence becomes stronger.On this basis,ABG was used as an adjuvant in combination with OVA antigen to evaluate its immune enhancement effect on antigen-presenting cells.Simultaneously the role of ABG as an adjuvant in stimulating the body’s cellular immune response and humoral immune response were evaluated by immunizing mice.The research content includes the following three parts.Part Ⅰ:Preparation of Acinetobacter baumannii ghostsA.baumannii ghosts was prepared by chemical method and genetic method respectively.The chemical method by using the minimum inhibitory concentration of NaOH include:the A.baumannii was incubated with different concentrations of NaOH solution,and the OD600 values before and after the incubation were detected to determine the minimum inhibitory concentration of NaOH against the Acinetobacter baumannii,and then use this concentration to prepare the ABG.The genetic method by using the pBBR-EBOX lysis plasmid include:First,the lysis plasmid is electrotransformed into A.baumannii,and A.baumannii containing the lysis plasmid was induced at 42℃ to prepare the A.baumannii ghosts.The morphology of A.baumannii ghosts was identified by light microscope and electron microscope.The results showed that the structure of the A.baumannii ghosts was relatively complete,with holes on the surface,and the contents flowed out in the form of "empty pockets".Part Ⅱ:Research on the characteristics of Acinetobacter baumannii ghosts as a vaccineBefore A baumannii ghosts was used as a vaccine to immunize mice,the AB43 strain Csy2 deletion mutant was constructed by one-step knockout method,and its growth curve,drug resistance,biofilm formation ability,serum resistance and virulence in mice were detected The results found that its drug resistance and virulence were significantly enhanced.The construction of the deleted strain is aimed at later evaluating whether the Acinetobacter baumannii ghosts can stimulate the body to resist drug resistance and virulence to enhance the immunity of the strain infection after being used as a vaccine to immunize mice.C57BL/6 mice were randomly divided into four groups:ABG group prepared by chemical method,ABG group prepared by lysis method,formalin inactivated,A.baumannii vaccine group and PBS group.The mice were subcutaneously immunized three times with an interval of two weeks.Two weeks after the third immunization,the survival of mice was recorded by intraperitoneal injection of AB43 strain.The results showed that the ABG group,ABF group and PBS control group mice prepared by chemical method all died within 48 hours.But compared with the PBS control group,the ABG group prepared by chemical method,the ABF group and the ABG prepared by lysis method can prolong the survival time of mice.Among them,ABG prepared by lysis method provides higher protection to mice after immunization.C57BL/6 mice were randomly divided into 3 groups,namely ABG group,formalin inactivated A.baumannii(ABF)group and PBS control group.The mice were subcutaneously immunized three times at two weeks interval.Indirect ELISA was used to detect the level of serum antibodies from immunized mice.Two weeks after the third immunization,each group of mice was divided into two groups,and AB43Δcsy2::kanr and AB43 were injected intraperitoneally and survival of the mice were recorded.The results showed that after infection with A.baumannii AB43 strain or AB43Δcsy2::kanr strain,compared with the control group,both the ABG group and the ABF group can provide a certain degree of immune protection.Among them,the mice in the ABG group have a late onset and have higher immune protection.The indirect ELISA results showed that IgG,IgGl and IgG2a antibody levels can be detected in the sera of ABG group and ABF group after the first immunization.After the second and third immunity,compared with the PBS control group,the serum levels of IgG,IgG1 and IgG2a in the ABG group and ABF group were significantly increased.And IgG levels in ABG group is higher than those in ABF group.Part Ⅲ:Study on the characteristics of Acinetobacter baumannii ghosts as an adjuvantUsing genetically prepared ABG as an adjuvant,it was used in combination with OVA(ABG+OVA).After incubating with BMDC,the cell surface costimulatory molecules CD40,CD80,CD86 and MHC-Ⅱ were detected by flow cytometry.The secretion of cytokines IL-1β,IL-6,IL-10,IL-12,and TNF-α in the supernatant of BMDC were detected by ELISA.The results showed that BMDC,stimulated by ABG+OVA,highly expressed surface mature molecules CD40,CD80,CD86 and MHC-Ⅱ,and highly secreted cytokines IL-1β,IL-6,IL-10,IL-12 and TNF-α.At the same time,24 6-week-old C57BL/6 mice were randomly divided into 4 groups,namely OVA group,ABG group,ABG+OVA group and PBS control group.The mice were subcutaneously immunized three times,with two weeks apart each time..Indirect ELISA was used to detect the levels of IgG,IgG1,and IgG2a antibodies in the sera of immunized mice.Flow cytometry was used to detect the frequency changes of CD3+CD4+T cells and CD3+CD8+T cells in the spleen.At the same time,the MTT method was used to detect the in vitro proliferation of splenic lymphocytes in immunized mice.The results showed that after three immunizations,compared with the PBS control group and the ABG group,specific IgG,IgGl and IgG2a antibodies against OVA in the OVA group and the ABG+OVA group could detected,and IgG,IgG1 and IgG2a antibody level in the ABG+OVA group were significantly higher than those in the OVA group.The frequency of CD3+CD4+T cells and CD3+CD8+T cells in the spleen of mice in the ABG+OVA group increased significantly.At the same time,mouse spleen cells proliferated significantly.