| Background and SignificanceStaphylococcus aureus is a familiar commensal organism in humans and it can cause the bacteremia and the hospital infection.According to the spread of strains resistant to the antibiotics,particularly the emergence of methicillin-resistant S.aureus(MRSA),these infections are becoming more and more difficult to cure.Therefore,seeking the anti-staphylococcal vaccine is very important for us.Vaccines have always playing an extremely important role in the prevention and treatment of major infectious diseases.However,Existing immune adjuvant due to its composition and effect of the immune response is relatively single,greatly limits the vaccine immunoprotective,give full play to the development of safe and effective new immune adjuvant is an important developing direction of the future vaccine development.This topic in the comprehensive analysis of the advantages and disadvantages of the traditional immune adjuvant and reference to the research and development experience and development trend of modern immune adjuvant,the new nano emulsion adjuvant research work on the basis of the organic integration of the active ingredients of adjuvant,people now use reasonable optimized proportion of adjuvant components,design and preparation of a new type of nanoemulsion with independent intellectual property rights of adjuvant,the model of systemic infection of staphylococcus aureus as experimental models,in to optimize the prescription,preparation technology and quality standard,on the basis of further through the serology level,cell level,the overall level of the animal acquired immune response to its excitation spectroscopy characteristics and mechanism of action of system research.MethodsSection 1: The preparation of nanoemulsion adjuvant for S.aureus vaccine and the surface characters identification(1)Three kinds of nanoemulsion adjuvantformulations vaccine were prepared by low energy emulsion method,namely,encapsulation formulation,mixture formulation,and combination formulation.(2)The morphological characters of the nanoemulsion adjuvant vaccine were evaluated through high speed centrifugation and particle size analyzer,transmission electron microscope and high resolution scanning electron microscope.Section 2 Immunological evaluation of the nanoemulsion adjuvant for S.aureus vaccine(1)The immunization of mice and the collection of serum and splenocytes: Three kinds of nanoemulsion adjuvant vaccine were used to immunize the 6-8 week old female BALB / c mice by intramuscular injection,and then respectively on the 6th day,the 13 th day blood was taken from the tail vein,collected,and antiseptically preserved in – 80?.After 10 days of the last immunization,the blood of the experimental group and control group mice were collected,centrifugated.As a result,serum was collected and antiseptically preserved in – 80?.At the same time,the spleens of mice were separated byoperation,prepared single cell suspension,and antiseptically kept in constant temperature cell culture box.(2)The Serological evaluation of immune mice:the serum specific IgG,IgG1,IgG1,IgG2 a,Ig G2 b antibody level were detected after immunization by ELISA method.(3)Cytological evaluation of experiment mice: the proliferation ability of isolated spleen cells was detected by cell proliferation assay.The number of spleen lymphocytes which secreted IFN-?,IL-4 were detected by ELISPOT assay.The cytokine secreted by isolated spleen cells after being stimulated by HI antigen was detected by ELISA method.The memory T cellsamong CD4 + T cells were detected by flow cytometry.(4)Systematic evaluation of experiment mice:the bacterial colonization of main organs was detected after the immune mice were infected.Kidney sections were stained with hematoxylin and eosin(HE)staining and pathological analysis after the mice were infected.The survival rate and protective rate of vaccine was analyzed when the immune mice were infected the lethal dose.Section3Thepreliminary exploration of the immunoprotective mechanism of the nanoemulsion adjuvant for S.aureus vaccine(1)6-8 weeks old female BALB/c mice were immunized by intramuscular injection.At the eighth days,the axillary lymph nodes of mice were grinded and centrifuged.As a result,the concentration of cells was adjusted to 105 cells/100? L/ well and cultured at 37 ? and 5% CO2.Besides,MHC molecules and CD86 molecules on the surface of DC cells in axillary lymph nodes were detected.(2)Culturing mouse primary bone marrow derived DC cells and detecting phagocytosis efficiency that the DC cell swallowed the nanoemulsion vaccine by the laser confocal scanning technology.(3)Using imaging technique in vivo to detect the sustained release effect of the nano emulsion adjuvant vaccine in two kinds of administration modes of subcutaneous injection and intramuscular injection.ResultsSection 1 The preparation of nanoemulsion adjuvant for S.aureus vaccine and the surface characters identificationFound by using the method of high speed centrifugation,three batch of nano emulsion adjuvant vaccines did not occur stratified and demulsification phenomenon under the condition of 6000rpm(5min),6000rpm(15min),12000rpm(5min)and12000rpm(15min).Preparation of three kinds formulations of nanoemulsion adjuvant vaccine average particle size of about 30 nm,the potential stability were around 20 mV,dispersion coefficients is less than 0.2.By transmission electron microscope analysis,proving that the preparation of nanoemulsion adjuvant vaccine is stable nanometer spherical particle,and good dispersion.By high resolution scanning electron microscope analysis,more intuitive to prove that the preparation of nanoemulsion adjuvant vaccine morphology is characterized by particle size,uniform dispersion of good spherical particles.Section 2 Immunological evaluation of the nanoemulsion adjuvant for S.aureus vaccine(1)Serological analysis of the experiment miceWithin a week after immunization,MF59 adjuvant could not stimulate the body to produce IgG antibody response rapidly,but the encapsulation formulation of nano emulsion adjuvant vaccine could induce high level of antibody response and adjuvant vaccine had significant statistical difference(vs MF59 group,P< 0.05).The three formulation groups and the MF59 group could effectively improve Ig G1,IgG2 a,IgG2b antibody response levels.By analyzing Ig G1 level,the encapsulation formulation group induced significant statistical difference(vs HI group,P < 0.001);By analyzing IgG2 a level,the three formulation groups induced significant statistical difference(vs HI group,P < 0.001),and the encapsulation formulation induced the highest level of antibody response;By analyzing IgG2 b level,the three formulation groups induced significant statistical difference(vs HI group,P < 0.01)Tip:The encapsulation formulation is a kind of high efficient immune enhancement effect of the vaccine preparation form.(2)Cytological analysis of the experiment mice1)Three kinds of nanoemulsion adjuvant vaccine and MF59 adjuvant vaccine immuned mice,and secondary antigen stimulation after take the spleen lymphocytes,spleen lymphocytes were appeared different degree of proliferation,and compared with health spleen lymphocytes in mice,were statistically significant differences.Tip:The nanoemulsion adjuvant could can effectively stimulate the body to produce memory cells,at the second stimulus antigen,spleen lymphocytes can rapidly achieve proliferation differentiation2)The encapsulation formulation and combination formulation groups could induced more cells to produce the IFN-?compared with the HI group.The encapsulation formulation group induced significant statistical difference(vs HI group,P < 0.001)and it induced significant statistical difference(vs MF59 group,P < 0.05);The encapsulation formulation group induced more cells to produce the IL-4 and had the significant statistical difference(vs HI group,P < 0.01)and it also induced significant statistical difference(vs MF59 group,P < 0.05);Tip:The encapsulation formulationgroup could efficiently stimulate the secretion of IFN-?related to cellular immunity and IL – 4related to humoral immunity.3)After the 3 days antigen stimulation to the splenic lymphocytes,the three formulation groups and the MF59 group could induce the higher IFN-?production than HI group,and the encapsulation formulation group and MF59 group produced more IFN-?and had the significant statistical difference(vs HI group,P < 0.001);After the 6 days antigen stimulation,the MF59 group induced the highest IFN-?level,and had the significant statistical difference(vs HI group,P < 0.01).Tip:The encapsulation formulationgroup and MF59 group could efficiently stimulate the secretion of IFN-? to promote Th1 cells differentiation.4)After the 3 days antigen stimulation to the splenic lymphocytes,the mixture formulation group and the combination group produced more IL-4 and had the significant statistical difference(vs HI group,P < 0.01);After the 6 days antigen stimulation,the combination group induced the highest IL-4 level,and had the significant statistical difference(vs HI group,P < 0.05).Tip:The nanoemulsion adjuvant vaccine especially combination formulation could efficiently stimulate the secretion of IL-4 to promote Th2 cells differentiation.5)After the 3 days antigen stimulation to the splenic lymphocytes,the encapsulation formulation group and the MF59 group produced more IL-10 and had the significant statistical difference(vs HI group,P < 0.05);After the 6 days antigen stimulation,the encapsulation formulation group had the significant statistical difference(vs HI group,P < 0.001).and the MF59 group had decreased the production of IL-10.Tip:The encapsulation formulationgroup had a kind of high efficient immune enhancement effect to humoral immune.6)After the 3 days antigen stimulation to the splenic lymphocytes,the three formulation groups and the MF59 group could induce the higher IL-12 production than HI group,and the encapsulation formulation group and the combination group produced more IL-12 and had the significant statistical difference(vs HI group,P < 0.001),but the MF59 group had no significant change.After the 6 days antigen stimulation,the encapsulation formulation group had the significant statistical difference(vs HI group,P < 0.001;vs MF59 group,P < 0.01).Tip:The encapsulation formulationgroup had a kind of high efficient immune enhancement effect to cellar immune.7)After the 3 days antigen stimulation to the splenic lymphocytes,the encapsulation formulation group and the mixture group could induce the higher IL-17 production than HI group,and had significant statistical difference(vs HI group,P < 0.01).After the 6 days antigen stimulation,the three formulation groups and the MF59 group could induce the higher IL-10 production than HI group(vs HI group,P < 0.001),and the MF59 group induced the highest level.Tip:The encapsulation formulationgroup and MF59 group could effectively stimulated thesecretion of IL-17,to promote the neutrophilic granulocyte playing a role removing bacteria.8)After the 3 days antigen stimulation to the splenic lymphocytes,the encapsulation formulation could induced the highest content of central memory CD4+ T cells,and had significant statistical difference(vs HI group,P < 0.001);The MF59 group could induced the highest content of effective memory CD4+ T cells and had significant statistical difference(vs HI group,P< 0.001).After the 6 days antigen stimulation to the splenic lymphocytes,the three formulation groups and the MF59 group could induced the production of central memory CD4+ T cells and effective memory CD4+ T cells.the combination formulation could induced the highest content of central memory CD4+ T cells,and had significant statistical difference(vs HI group,P < 0.01);The MF59 group could induced the highest content of effective memory CD4+ T cells and had significant statistical difference(vs HI group,P< 0.05).Tip:The encapsulation formulation group was easier to efficiently motivate center memory CD4 + T cells,while the MF59 adjuvant was more likely to stimulate the body to produce the effectivememory CD4 + T cells.(3)The Overall evaluation of the experiment mice1)Compared the 1day post infection results with the 3 day post infection results,we found the bacteria amount significant decreased in the blood and spleen at 3 dpi,but the bacteria amount significant increased in kidney at 3 dpi.The encapsulation formulation could effectively improve the body to produce bacterial removal effect,and proved to be a development potential formulation.2)Through comprehensive evaluation of the kidney biopsy pathological damage,The encapsulation formulation could produce the lightest pathological damage effect,and had the significant statistical difference(vs control group,P< 0.001).The prompt: the kidney biopsy pathology score results were corresponding to the result of bacteria amount,once again to verify the encapsulation formulation is expected to become a staphylococcus aureus vaccine adjuvant formulation.3)The three formulation groups and the MF59 group could produce the effective immunoprotective.The mice survival rate of the encapsulation and combination formulation groups were 90%;The mice survival rate of the mixture formulation group was 80%;The mice survival rate of the MF59 adjuvant group was 60%;The survival rate of HI group mice was 40%;The survival rate of the His control group mice was 20%.The vaccine protection ratio of the encapsulation and combination formulation groups were 77.78%;The vaccine protection ratio of the mixture formulation group was 75%;The vaccine protection ratio of the MF59 group was 66.67%;The vaccine protection ratio of the HI group was 66.67%Section3Thepreliminary exploration of the immunoprotective mechanismof the nanoemulsion adjuvant for S.aureus vaccine1)For the results,Theencapsulation formulationgroup induced the highest expression of MHC I in the lymph nodes DC(vs HI group,P < 0.001),the combined vaccine group followed.The encapsulation formulationgroup induced higher expression of MHC II(vs HI group,P < 0.001),Themixture formulationgroup followed.The MHC II expression in the mixture formulation group was higher than HI group,and showed statistically significant(P < 0.001),the encapsulation formulation group followed.2)From the results of laser confocal experiments,the encapsulation formulation groupwas more easier caught by DC,compared than the mixture formulation group and HI group.3)The living imaging experiments show that the injection site slow-release effect is better in the encapsulation formulation group,and the DC can constantly capture the release antigen,therefore resulting in efficient humoral immunity and cellular immune response.Conclusions1.The nanoemulsion adjuvantespecially the encapsulation formulation we designed significantly improves the levels of humoral and cellular immune response at same time,and improves organ bacterial clearance and animal survival,to resist the MRSA systemic infection.2.Based on the experimental Results of immunological differences in spectral characteristics analysis and systematic analysis of the experimental Results,the immune protective effect of the novel nanoemulsion adjuvant in particular the encapsulation formulation is multi-channel network. |
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