| For resident infectious pathogenic microorganisms,the formation of herd immunity through vaccination to produce an immune barrier is the only way to restore normal learning,working and living conditions.Nasal mucosal immunity has the characteristics of no injection,convenient administration and high immune efficacy.It can not only induce systemic and mucosal immune responses,but also stimulate cellular immune response,which has become an increasingly popular new vaccination approach.However,due to the weak immunogenicity of recombinant protein and peptide antigen and the special environment and structural characteristics of the nasal cavity,the immune activity of nasal vaccines is low,so it is necessary to develop new nasal mucosal immune adjuvants to improve the immune response level of nasal vaccines.In this study,surfactants with enhanced nasal administration effect or potential immunomodulatory activity were selected to form mixed systems with Tween 80(T80),a chemical chaperone promoting protein folding.The interactions between T80 mixed system and model antigen ovalbumin(OVA)were analyzed,and the compositions of adjuvant were optimized according to the physicochemical properties that affected the immune performance of the nasal mucosa of the composite system and the structural stability of OVA in the mixed system.The hemolytic activities of T80 mixed system and the nasal mucosal immune activities of OVA model vaccine were studied.The synergistic immune effects of adjuvants were achieved by combining T80 mixed system with selenium-doped carbon nanoparticles(Se/C)which had immunomodulatory activity or dibenzyldiselenide(DDSe)which mimicsed the activity of glutathione peroxidase(GSH-Px).The research results can be used to guide the development of safe,high-efficiency,low-cost and easy-to-produce nasal mucosal immune adjuvants.1.Through the optimization of adjuvant composition,a 2 wt%T80/B30(4:6)vesicle system composed of T80 and nonionic surfactant Brij 30(B30)was obtained.When the total concentration of T80/B30(4:6)was lower than cac,the alkyl chain of T80/B30 monomer interacted with the hydrophobic micro region formed by the non-polar side chains and non-polar side chains of OVA residues to make the tertiary structure changes slightly.When the total concentration of T80/B30(4:6)was between cac-cmc*,the adsorption of T80/B30 on OVA had little effect on the tertiary structure of the protein.When the total concentration of T80/B30(4:6)was higher than that of cmc*,the mixed micelle of T80/B30 interacted with OVA and had slight effect on the tertiary structure of OVA.The particle size of the 2 wt%T80/B30(4:6)vesicle system was about 30 nm,which was the ideal size of vaccine adjuvant.It had low surface tension and small contact angle,which was conducive to the wetting and spreading of vaccine on nasal mucosa.The secondary and tertiary structure of OVA in this vesicle system maintained relative integrity.2 wt%T80/B30(4:6)system had good hemolytic safety,and the titer of OVA-specific IgG antibody was significantly higher than that of control OVA(p<0.001).The type of immune response was Th2,which mediated humoral immune response.2.The polyoxyethylene-9-lauryl ether(P9LE),which was the same hydrophobic tail chain as B30 in Chapter 2,but had a significantly longer polyoxyethylene chain,was compounded with T80.When the total concentration of T80/P9LE(4:6)was between cac-cmc*,the fluorescence intensity of OVA changed little with the increase of T80/P9LE(4:6)concentration.When the total concentration of T80/P9LE(4:6)was greater than cmc*,T80/P9LE mixed micelle interacted weakly with OVA,causing minor changes in the tertiary structure of OVA.The 2 wt%T80/P9LE(4:6)mixed micelle system was selected through the optimization of the adjuvant composition.The secondary structure and tertiary structure of OVA in this system were stable,and the hemolytic activity was relatively low.The titer of OVA-specific IgG antibody induced by this mixed micelle system was significantly higher than that of control OVA(p<0.001),and it stimulated Th2 immune response.Compared with the 2 wt%T80/B30(4:6)in Chapter 2,2 wt%T80/P9LE(4:6)had a lower nasal mucosal immune activity than 2 wt%T80/B30(4:6),but its hemolysis was significantly better than 2 wt%T80/B30(4:6),and this system had little effect on the structure of the antigen protein.3.According to the characteristics of nasal mucosa with negative charge under physiological pH condition,cationic surfactant cetylpyridinium chloride(CPC)was introduced,and 0.5 wt%T80/CPC(8:2)mixed micelle system was obtained by optimizing the composition.When the total concentration of T80/CPC(8:2)was lower than cac,the T80/CPC monomer was mainly combined with OVA through electrostatic interaction,and the fluorescence intensity of OVA decreased slightly.After the total concentration of T80/CPC(8:2)was greater than cmc*,the T80/CPC mixed micelle interacted with OVA to slightly change the tertiary structure of OVA.The 0.5 wt%T80/CPC(8:2)system had lower hemolytic activity.After intranasal immunization,the titer of OVA-specific IgG antibody induced by this system was significantly higher than that of control OVA(p<0.001),which was close to that of positive control Cholera toxin B subunit(CTB).The results showed that the mixed micelle system significantly enhanced cellular immunity,stimulated a mixed type immune response with a slight bias to Th2,and induced high levels of OVA-specific sIgA antibody in nasal mucosa and lung.The titer of OVA-specific IgG antibody induced by 0.5 wt%T80/CPC(8:2)was similar to the 2 wt%T80/B30(4:6)in Chapter 2,but the sIgA antibody level was significantly higher than 2 wt%T80/B30(4:6)(p<0.001).4.The 1 wt%T80/PN320(3:5)mixed micelle system was prepared by mixing T80 with PN320,a new polymer cationic surfactant with high cationic charge density and good biocompatibility.After this system was compounded with OVA,the zeta potential was about 20.31 mV,which could be adsorbed well in the nasal mucosa,but it would not cause cytotoxicity due to the excessively high cationic charge density.The α-helix structure of OVA in this system was 97.8%of that of pure OVA,and the fluorescence intensity was 85.1%of that of pure OVA(λex=295 nm),which maintained the integrity of protein structure.1 wt%T80/PN320(3:5)mixed micelle system had low hemolytic activity,and the titer of IgG antibody induced by 1 wt%T80/PN320(3:5)was slightly higher than that induced by 2 wt%T80/B30(4:6)in Chapter 2,significantly higher than that induced by 2 wt%T80/P9LE(4:6)in Chapter 3(p<0.01),similar to that induced by 0.5 wt%T80/CPC(8:2)and CTB in Chapter 4,and the immune response type is a mixed immune response slightly biased to Th2.The level of sIgA antibody produced by 1 wt%T80/PN320(3:5)in the nasal mucosa and lung was significantly higher than that of 2 wt%T80/B30(4:6)(p<0.001),indicating that it was a potential adjuvant for nasal vaccine.5.The T80 mixed system with excellent nasal mucosal immune activity in Chapters 2 to 5 of the thesis was compounded with Se/C that had immunomodulatory activity or DDSe that mimicsed the activity of GSH-Px to achieve the synergistic immune effect of adjuvant.After mixing with Se/C,the immune performances of the 2 wt%T80/B30(4:6)system and the 1 wt%T80/PN320(3:5)system were significantly enhanced.The nasal mucosal immune activity of 1 wt%T80/PN320(3:5)+Se/C was significantly stronger than 2 wt%T80/B30(4:6)+ Se/C,which was close to CTB.The addition of Se/C changed the type of immune response of 1 wt%T80/PN320(3:5)from a mixed immune response that was slightly biased towards Th2 to a mixed immune response that was slightly biased towards Th1,which effectively enhanced the cellular immune response.Moreover,the new synthetic Se/C material had strong antibacterial properties,further expanding the use of adjuvants,such as vaccine antisepsis and drug resistance.After combined with DDSe,the levels of OVA-specific IgG and sIgA antibodies induced by the 0.5%T80/CPC(8:2)system were significantly increased,which were similar to those of CTB,and the type of immune response changed from a mixed immune response that was slightly biased towards Th2 to a mixed immune response that was slightly biased towards Th1.It obviously enhanced cellular immunity,humoral immunity and mucosal immunity. |
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