| Background:Staphylococcus aureus is one of the most common pathogenic bacteria in ICU,burn unit and orthopedics department.Due to its characteristics of high infection rate,serious complications and strong drug resistance,it has been listed as "extremely harmful super drug-resistant bacteria" by WHO.The abuse of antibiotics leads to the rapid spread of Methicillin-resistant Staphylococcus aureus(MRSA),a short latency window after infection and difficult treatment.New vaccine strategies have emerged as the most cost-effective way to prevent and control MRSA infections and the spread of drug resistance.Based on reverse vaccinology strategy and genetic engineering recombination technology,our research group developed 1.1 original MRSA recombinant subunit vaccines in the early stage,and the results of phase I and II clinical trials showed that they had good safety and immunogenicity.In previous clinical studies,we found that in order to meet the characteristics of rapid onset,severe severity and short vaccination window period of high-risk patients with MRSA clinical infection,the MRSA vaccine needed to be developed could closely follow the clinical objective reality and quickly and efficiently stimulate a highly specific and protective immune response in a very limited vaccination window period.At present,the global market adjuvants include aluminum salt,AS series and MF59 and a few other adjuvants,due to their composition and caused by a relatively single immune response type,limited the vaccine immune response spectrum and protective effect of the full play,and patented technology and products are monopolized by foreign countries,become one of the key technical bottlenecks in the development of new vaccines in China.The combined use of two or more single adjuvants can not only overcome the deficiency of single adjuvant components,but also synergistic effect,play a role in enriching and enhancing immune response types,which has become a new trend and hot spot in the research and development of new adjuvants.With the development of vaccine engineering technology,nano immune adjuvant,the fusion product of nano and immunology technology,has become a hotspot of new vaccine research.Compared with traditional adjuvants,nano-sized immune adjuvants modified by engineering technology have the characteristics of small particle size and large specific surface area,which can adsorb vaccine antigens more efficiently and reach the immune response site quickly,giving full play to the small size effect of nano-sized adjuvants,and further reduce the immune side effects of vaccines while improving vaccine bioavailability and enhancing the intensity of immune response.Among them,nanoemulsion adjuvants have good stability and uniformity,and can more effectively encapsulate or adsorb antigens than traditional emulsions.In addition,as a traditional immune adjuvant,aluminum hydroxide has long-term proven safety and efficacy,but its slow-release effect and immune response intensity still need to be strengthened.Nano-sized aluminum salt adjuvant modified by engineering can improve the efficiency and stability of antigen adsorption,and further enhance the immune response aging and intensity of vaccine antigen.Therefore,it will be a beneficial exploration and research idea for the development of vaccine adjuvants to develop new composite nano adjuvants by using the nano adjuvants mentioned above.Objectives:This project has developed novel Nanoemulsion adjuvants(NEA)with independent intellectual property rights in the early stage.And on the basis of cooperating with the School of Chemical Engineering of Dalian University of Technology to develop four engineered nano-alumina hydroxide adjuvants(Rod1,Rod2,Nanoplates and Nanopolyhedron),the subunit HI with relatively weak immunogenicity in MRSA vaccine was used as the target antigen.Through the research and development strategies of different adjuvant components,prescription optimization and positive and negative charge mutual attraction,a novel composite nanoadjuvant(NEA/Rod1)was successfully developed.While completing its physicochemical characterization,its enhanced immune response and protective effect of MRSA vaccine were studied.It lays the experimental foundation and provides scientific basis for the research and development of novel composite nanoadjuvant.Methods:1.Combined adjuvant component formulation optimization and prescription process research.NEA was prepared on the basis of the patented formula,and its physicochemical properties were characterized and morphological detection was carried out.Two nanoemulsion adjuvants(NEA and MF59)and two commercial aluminum salt adjuvants[Al(OH)3 and Al PO4] were paired together,combined with HI antigen,and specific Ig G antibody titer was used as evaluation index to optimize the composition of composite adjuvant.According to the principle of comprehensive design of pharmaceutical science,different proportions of combination adjuvants were prepared,and the adsorption completeness of HI antigen was detected by SDS-PAGE.The influence weight of specific antibody response was evaluated by orthogonal experiment,and the optimal proportion of adjuvant compatibility and adjuvant dosage were obtained,and the protective effect of enhancing humoral and cellular immune response and sepsis animal infection model was evaluated.2.Drug delivery mode and activity evaluation of NEA and engineered nano-Al(OH)3adjuvantOn the basis of the above optimized composite adjuvant formulation and prescription technology of nanoemulsion adjuvant NEA and commercial aluminum hydroxide Al(OH)3,the optimal loading mode of NEA and HI antigen was optimized through different loading modes of NEA to HI antigen.The physicochemical properties and morphology of four kinds of engineered sodium(OH)3 were characterized.Using commercial Al(OH)3 adjuvant as control,combined with HI antigen and Ig G antibody titer as evaluation index,the immune response of four engineered nano Al(OH)3 adjuvants was compared and evaluated,and the appropriate components of engineered nano Al(OH)3 adjuvants were optimized.3.Preparation of a novel composite nano adjuvant(NEA/Rod1)and evaluation of its immune-enhancing effectThe above studies determined that the loading mode of Nanoemulsion adjuvants NEA and HI antigen was encapsulated HNE(HI Nanoemulsion)and the preferred engineered nano Al(OH)3 adjuvant was Rod1.Furthermore,HNE and Rod1 were used as the components of the novel composite nano adjuvant to optimize their proportions,characterize their physical and chemical properties and detect their morphology.SDS-PAGE,Circular Dichroism(CD)and Western-Blot were used to evaluate the integrity and specificity of the primary and secondary structures of HI antigen in HNE/Rod1.After 6 months ’investigation of particle size,Zeta potential and appearance,the preliminary stability was evaluated.The immune protective effect of HNE/Rod1 was evaluated by lethal model of sepsis.ELISpot was used to detect the secretion levels of IFN-γ,IL-4 and IL-17 A in spleen lymphocytes after immunization.Flow cytometry was used to detect the activation level of dendritic cells in the inguinal lymph nodes of immunized mice.Results:1.The combination adjuvant of NEA and commercial Al(OH)3 has the advantage of significantly enhancing specific Ig G antibody response compared with other combination adjuvants;When the mixture ratio of NEA and Al(OH)3 was 1:4(w/w),the adsorption completeness of HI antigen was the best.The matching parameters of compound adjuvant NEA/Al(OH)3 and antigen were determined by orthogonal experiment: antigen amount was60 μg,adjuvant amount was 200 μg[NEA: Al(OH)3)was 1:4(w/w)].The immunoprotective effect evaluation showed that NEA/Al(OH)3 had good immunogenicity and immunoprotective effect.2.The results showed that NEA encapsulation of HI antigen was the preferred delivery form of vaccine antigen.Among the four engineered nano-Al(OH)3,Rod1 has the smallest particle size,the best dispersion,and a long rod-like shape,showing a better immune effect in the immune evaluation.3.When the combination ratio of HNE/Rod1 with composite nano-adjuvant vaccine was1:4(w/w),it had better immune enhancement effect than other ratios;No HI antigen degradation was detected in HNE/Rod1,which showed good structural integrity and specific immune reactivity.Preliminary stability studies showed that NEA/Rod1 had good stability at room temperature at 1st,2nd,3rd and 6th months.HNE/Rod1 can significantly increase the Ig G antibody level,showing good bacterial clearance effect and immune protection effect.Compared with the single adjuvant group,it can enrich the types of immune response and effectively induce the high expression of costimulatory molecules CD80,CD86,CD40 in lymph nodes and MHC-II molecules on the surface of dendritic cells.Conclusions:1.Nanoemulsion NEA adjuvant and commercial Al(OH)3 adjuvant are the preferred emulsion and aluminum salt mixing components,the suitable prescription ratio of NEA/Al(OH)3 is 1:4(w/w),and confirmed that NEA encapsulation HI antigen delivery mode(HNE)is the preferred delivery form of vaccine antigen.2.Among the four engineered nano-Al(OH)3 adjuvant of different shapes and sizes,rod-like Rod1 has the smallest average particle size(269.2 nm)and the largest Zeta potential(45.9 m V),and has good dispersion,which can induce rapid and efficient specific Ig G antibody response.It showed better enhanced immune response than the other three forms.3.NEA can form a 98.4 nm combined nano adjuvant form with Rod1 through positive and negative charge attraction.HNE/Rod1,a novel combined nano adjuvant with a mass ratio of 1:4,has good structural stability within 6 months,and effectively enhances the humoral and cellular immune response aging of MRSA vaccine in animal infection and lethal models.It can be used as a novel type of combined nano adjuvant to further carry out systematic safety and efficacy evaluation research. |
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