| Background:Infection is one of the main causes of neonatal death and severe sequelae,and neonatal bacterial meningitis has a high mortality and become a serious burden to the family and society.Escherichia coli K1(E.coli K1)is the main pathogen of neonatal bacterial meningitis,which causes more than 80% of the patients.Outer membrane protein A(OmpA)is the key pathogenic factor of E.coli K1.Its extracellular Loops plays a decisive role in the process of E.coli K1 resisting immune system damage,causing bacteremia and invading the blood-brain barrier(BBB)leading to intracranial infection.Neonatal infectious pneumonia is the most common infectious disease and an important cause of death in the neonatal period.even after infection control,irreversible damage is caused,which seriously affects the development of the brain and body.Pseudomonas aeruginosa(PA)is oneof the three main pathogens causing neonatal infectious pneumonia.There are many pathogenic factors of PA,among which exotoxin A(ETA)is the earliest,most virulent and classical virulence factor of PA.It plays an important role in neonatal infectious pneumonia caused by PA,mainly through cell recognition,transmembrane translocation and catalytic activity.With the drug resistance of E.coli K1 and PA becoming more and more serious,the conventional anti-infection therapy has been stretched.Therefore,the selection of appropriate antibodies is an effective alternative therapy for the treatment of PA,especially for drug-resistant PA infection.Historical experience has proved that vaccine is the best scientific means to prevent and control the prevalence,infection and pathogenesis of pathogenic bacteria,and antibody is a special drug to neutralize pathogenic bacteria toxin and treat infectious diseases.If specific vaccines or antibody drugs against E.coli K1 OmpA and PA ETA can be successfully developed,the transmission and pathogenicity of these bacteria will be effectively controlled and reduced.Therefore,in view of the severe prevention and control situation of common neonatal pathogenic bacteria E.coli.K1 and PA,this study studied the immune intervention strategies and drugs with new vaccines and antibodies as the core.In order to prevent the occurrence of neonatal infection and improve the level of treatment lay the foundation.Part I Development of nano-vaccine based on extracellular functional fragment of extracellular membrane protein A of meningitis Escherichia coli K1Objectives:In this study,in view of the severe prevention and control situation of E.coli.K1,a common pathogen of neonatal bacterial meningitis and a new vaccine was developed for its key virulence factor OmpA,in order to lay a foundation for preventing the occurrence of neonatal E.coli.K1 meningitis and improving its treatment level.Methods:1.On the basis of finding that Vo antigen has good immune protection effect in the early stage,polylactic acid-glycolic acid copolymer(PLGA)and chitosan(CS)were used as excipients to construct nano-vaccine coated with Vo.The blank nanoparticles were prepared by double emulsion solvent volatilization method and direct adsorption method.the effects of PLGA,acetone,dichloromethane and different modification methods on the nanoparticles were evaluated by measuring the morphology,particle size and dispersion index of the nanoparticles.The optimal formulation of blank nanoparticles was screened out,and the optimal formulation of Vo nanoparticles(VoNP)with maximum drug loading was obtained by measuring the encapsulation efficiency of Vo coated with nanoparticlesunder different conditions.By detecting the changes of appearance,morphology,particle size,potential,physical and chemical stability of VoNP before and after freeze-drying,the effect of freeze-drying process on the quality of VoNP was evaluated,and the optimum preparation process of VoNP was obtained.VoNP,was prepared according to the optimum process.the basic physical and chemical characteristics and stability of nanoparticle vaccine were investigated by transmission electron microscope,scanning electron microscope and atomic microscope,particle size and potential analyzer,mass spectrometer and so on.2.The cytotoxicity of VoNP in vitro was evaluated by using L929 national standard cytotoxic cells as model,and the pathological changes of heart,liver,spleen,lung and kidney were observed after intramuscular injection of VoNP in BALB/c mice,and the toxicity in vivo was evaluated.Mice were immunized with VoNP three times on 0,7 and 14 days,the level and type of anti-Vo specific antibody in serum were detected,the immunogenicity of VoNP was evaluated,and the mice were immunized with lethal dose of VoNP.The survival rate and body weight of mice were observed to evaluate the protective effect of VoNP.The mice were immunized with sublethal dose of VoNP,the amount of bacterial colonization in spleen and blood was observed,and the immune protection mechanism of VoNP was preliminarily evaluated.The immunogenicity andimmune protective effect of the prepared VoNP were evaluated according to the above methods after being stored at 4 ℃ for 180 days.compared with the Vo vaccine adsorbed by the same treatment of Al(OH)3 adjuvant,the long-term stability of VoNP was evaluated.Results:1.The optimum formulation of nanoparticles was as follows: PLGA nanoparticles modified by chitosan with acetone as solvent,the maximum drug loading of VoNP was 1 mg,and the entrapment efficiency was 59%.The freeze-drying process(freeze-drying for 3 h,drying for 22 h)had no significant effect on the morphology,particle size and dispersion of VoNP.The average particle size of VoNP prepared by the optimum process was250.8 ±2.13 nm,the dispersion index was 0.09 ±0.01,the potential was0.6110 ±0.0267 mVVoN,P,the surface was spherical and smooth,there was no obvious aggregation phenomenon,and the dispersion was good.MALDI-TOF MS results showed that Vo protein existed stably in nanoparticle vaccine,and the particle size,dispersion and Zeta potential of VoNP placed at 4 ℃ for 180 days did not change significantly,and the stability was good.2.VoNP had no obvious cytotoxic effect on L929 cells,BALB/c mice injected intramuscularly with VoNP had no death and adverse reactions within 7 days,and there were no obvious histopathological changes in heart,liver,spleen,lung and kidney organs.The results showed that VoNP had no obvious toxic effect on BALB/c mice,and VoNP immunized mice could induce strong humoral immune response,and the type of immune response was Th2 type.In the experiment of challenge and protection,the immunity of VoNP can resist the infection of E.coli K1 by reducing the bacterial colonization in peripheral blood and spleen,reducing the change of body weight,and improve the survival rate of mice.Animal experiments confirmed that long-term preservation(180 days)of VoNP not only had stable physical and chemical properties,but also its immunogenicity and immune protection effect did not change significantly.The results show that the safety of VoNP is good and the quality is stable.Conclusions:In this study,the preparation process of VoNP was successfully established and optimized,and a stable and safe VoNP vaccine was obtained.Animal experiments showed that the vaccine had long-term and stable immunogenicity and immune protection effect.These findings lay a foundation for the further successful development of E.coli K1 vaccine and play an important role in promoting the study of immune intervention strategies for neonatal meningitis E.coli K1 infection.Part II Study on the Preventive and Therapeutic effects ofAnti-exotoxin A Human Immunoglobulin in Pseudomonas aeruginosa pneumoniaObjectives:Aiming at the difficult problem of the treatment of neonatal Pseudomonas aeruginosa pneumonia,this study studied the intervention strategy and drugs with immunoglobulin as the core,in order to lay a foundation for preventing the occurrence of neonatal PA infection and improving its treatment level.Methods:1.An anti-ETA antibody concentration detection method based on Luminex technique was established to detect the concentration of ETA specific antibody in serum of 320 healthy volunteers.Immunoglobulin(IVIG)containing high titer ETA antibody was purified by Protein A affinity chromatography,and the neutralizing activity of anti-ETA antibody to ETA toxin was evaluated by L929 cell model.BALB/c mice were intraperitoneally injected with anti-ETA antibody,followed by lethal dose of ETA in mice.The survival rate,body temperature and body weight were observed to evaluate the preventive protective effect of anti-ETA antibody.at the same time,the changes of cytokines and pathological tissue of liver were observed by sublethal dose of ETA in mice,and the protective mechanism was explored.2.BALB/c mice were injected intraperitoneally with different concentrations of anti-ETA antibody.one hour later,the lethal dose of PA103 bacteria was injected into the trachea of challenged mice.the changes of survival rate,body temperature and body weight were observed,and the preventive protective effect of anti-ETA antibody was evaluated.at the same time,The lung bacterial colonization and inflammatory pathological changes were observed by sublethal dose of PA103 bacteria,and the levels of TNF-α and IL-1β in alveolar lavage fluid were measured to explore its protective mechanism.Three hours after the lethal dose of PA103 was injected into the trachea of BALB/c mice,Different concentrations of anti-ETA antibody were injected intraperitoneally to observe the survival rate,body temperature and body weight,and to evaluate the therapeutic effect of anti-ETA antibody.at the same time,the amount of bacterial colonization and inflammatory pathological changes in the lungs of mice were observed by sublethal dose of PA103 bacteria.The levels of TNF-α and IL-1β in alveolar lavage fluid were measured to explore their protective mechanism.The solid phase carrier combined with GST-ETA was incubated with the purified IVIG containing high titer anti-ETA antibody,and the ETA specific IgG,was removed and the neutralization activity of the toxin was evaluated by the above method.The protective effect of prevention and treatment was changed to verify thespecificity of the protective effect.Results:1.In this study,an engineering strain expressing ETA and its non-toxic mutant(mETA)was successfully constructed,and the recombinant ETA toxin was purified,its activity was similar to that of natural toxin,and an anti-ETA antibody concentration detection method based on Luminex technology was established.The level of anti-ETA antibody in serum of 320 healthy volunteers was detected.it was found that the average concentration of anti-ETA antibody was 98.7 ng/ml,and the highest was 918.24 ng/ml.The concentration of serum anti-ETA antibody was about 54% at 0≦99 ng/ml.Human immunoglobulin containing high concentration of anti-ETA antibody was successfully purified by Protein A affinity chromatography.the purity of SDS-PAGE was more than 90%.2.It was found by L929 cell model that the anti-ETA antibody could effectively inhibit the cytotoxicity of ETA in a concentration-dependent manner,and by ETA toxin challenge mouse model,it was found that the antibody could block the liver damage induced by ETA,so as to play a good protective effect.In the model of acute PA pulmonary infection,it was found that high titer anti-ETA immunoglobulin could significantly reduce the colonization and inflammatory response of bacteria in the lung,so as to play a preventive and therapeutic effect.In addition,this studyfound that the use of rabbit anti-ETA antibody or non-specific human IgGs alone can also have a limited protective effect.Conclusions:In this study,high titer anti-ETA antibody immunoglobulin was successfully purified from healthy blood donors,which has good toxin neutralization activity.Animal experiments showed that the immunoglobulin has a good preventive and therapeutic effect on ETA toxin and PA acute pulmonary infection.These results lay a foundation for the prevention and treatment of PA pulmonary infection without antibiotics,provide a new strategy and scheme for the prevention and control of PA,and also provide an example for the immune intervention of other drug-resistant bacteria. |
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