The Study Of Pertussis Vaccine Based On Outer Membrane Vesicles

Pertussis,also named whooping cough,is an acute respiratory infectious disease caused by infection with B.pertussis.Since the middle of the last century,with the widespread inoculation of vaccines,the morbidity and mortality of whooping cough have been greatly reduced.But pertussis is still the main cause of death in vaccine-preventable diseases.According to WHO estimation,the number of whooping cough cases each year is about 20~40 million,and the number of deaths is about 400,000,of which more than 95% occur in developing countries.Vaccination is an effective way to prevent the spread of whooping cough.Whole-cell pertussis vaccine(wPV)and acellular pertussis vaccine(aPV)are two types of approved pertussis vaccines.Since wPV sometimes produces serious local or systemic side reactions,aPV has gradually replaced wPV since the 1980 s.The rate of pertussis vaccine in the target population has increased year by year,and in 2018 the rate was as high as 86%.However,the morbidity of whooping cough has not decreased significantly since the end of the last century,but has shown an upward trend in recent years.The duration of protection of aPV(4~7 years)is significantly lower than that of wPV(5~14 years),which may be one of the reasons for the above phenomena.Therefore,the development of new and more effective vaccines may be one of the solutions to the above problems.Outer membrane vesicle(OMV)is a membrane vesicle structure secreted by bacteria into the external environment.It contains a variety of substances such as outer membrane proteins,lipopolysaccharides and nucleic acids et al.Due to its unique structure and abundant antigen components,OMV has good immunogenicity.Group B Neisseria meningitidis vaccine based on OMV has been widely used in Cuba,Norway,New Zealand and other countries.The existing data show that OMV of B.pertussis also has good immunogenicity,which provides a scientific basis for the development of pertussis vaccine based on OMV.Therefore,this research intends to prepare OMV from B.pertussis fermentation broth on a large scale,through detailed study on its structure,composition,stability,immunogenicity and immune protection.The main findings are as follows:First,OMV were extracted from B.pertussis fermentation broth by ultracentrifugation or density gradient centrifugation.The OMV extraction method was established with yield and complexity of the preparation process as the main parameters.The structure and composition of OMV were characterized using transmission electron microscope,dynamic light scattering,the quantification of protein,Western blotting and proteomics.The results showed that the OMV prepared from the fermentation broth by ultracentrifugation were spherical,and the particle size distribution was between 20 and 300 nm(average particle size was 107.8 ± 1.7 nm)which were similar to the structure of OMV derived from the shake flasks.OMV contained 745 proteins such as pertussis toxin(PT),filamentous hemagglutinin(FHA),and pertactin(PRN),with a molecular weight between 10 and 368 kDa;its yield was 13.4 times that of shake flask culture.Second,we used Balb/c mice as animal models,using wPV,aPV(adjuvant-free)and commercial DTaP vaccine as controls,the immune protection and mechanism of pertussis OMV vaccines were explored.In addition,the adjuvant property of OMV was also studied.The results showed that:(1)Three types of OMV vaccines(OMV,OMV+alum,and aPV antigens+OMV),like wPV and the commercial DTaP vaccine,could induce mice to produce 100% immune protection.(2)The three types of OMV vaccines were similar to wPV,and the ratio of IgG2 a to IgG1 induced by mice is close to 1.(3)The three types of OMV vaccines were similar to wPV,and the levels of antigen-specific IgG produced by mice and IFN-γ,IL-2,IL-4,and IL-17 A produced by splenocytes were significantly higher than those of DTaP vaccine and aPV.It could be seen that,similar to wPV,the three types of OMV vaccines could produce a more balanced Th1 and Th2 immune response and a strong Th17 immune response and humoral immune response.In addition,OMV had the ability to make aPV antigen obtain immunity similar to wPV,indicating that OMV had good adjuvant property.Third,we investigated the effects of temperature and storage time on the stability of OMV and the changes in OMV structure and composition as indicators.OMV were resuspended in D-PBS at a protein concentration of 360μg/mL.The study found that:(1)The morphology,particle size and zeta potential of OMV did not change significantly when stored at-80 ℃ and-20 ℃ for 5 months;when stored at 4 ℃,the number of OMV decreased and most of them showed irregular shapes,but the average particle size and zeta potential did not change significantly;the structure of OMV was destroyed during storage at 37 ℃,and a large number of amorphous granular material appeared.The zeta potential gradually increased with storage time(from-21.77 mV to-16.70 mV);freeze-drying treatment,had a significant effect on the OMV structure.At 4 ℃,the particle size increased with time,the zeta potential gradually decreased(from-21.77 mV to-41.30 mV)and the polydispersity index significantly increased.(2)The samples stored at 4 ℃ and 37 ℃ showed an increase in total protein concentration(545.46 μg/mL and 746.70 μg/mL at 5 months,respectively),and the characteristics of SDS-PAGE protein band significantly changed.In summary,we established a method for preparing OMV from B.pertussis fermentation broth and obtained the parameters of temperature,times and lyophilization affecting the stability of OMV.We proved that OMV vaccine had the ability to induce mice to produce immune protection similar to wPV and clarified the mechanism of OMV vaccine.These results could provide technical methods,data references and theoretical basis for the further development of pertussis OMV vaccine.