| Background:Many microorganisms infect the human body through mucosal,such as respiratory tract,digestive tract and reproductive tract.Compared with the systemic immune response,the mucosal immune response,the first line of defense,is of great significance.Most of the vaccines can not induce strong mucosal immune response through injection,while mucosal vaccines can effectively induce antigen-specific mucosal immune response.At the same time,mucosal vaccines can also induce systemic immune response as vaccines that vaccinate by injection.There are many ways of mucosal vaccination,including oral vaccination,nasal vaccination,oral cavity vaccination and so on.Among them,nasal vaccination has many advantages for vaccine delivery,such as less degradation of antigen;nasal mucosa has rich capillaries and lymphatic vessels;nasal mucosa surface area is large,which is conducive to antigen uptake;nasal vaccination is very easy,and can be self-administered.Even though the nasal vaccine has many advantages,the development of the nasal vaccine is still difficult.The human body’s nasal mucosal surface has multiple natural defense mechanisms,which make it very difficult for the vaccine to pass through this barrier,so the nasal vaccine often needs the appropriate delivery system to increase the delivery efficiency of the antigen,reduce the degradation and removal of the antigen in the nasal mucosa,so as to enhance the immune protection effect of the vaccine.Nanoemulsion（NE）is a thermodynamically stable oil-water dispersion system,which can be loaded with antigens and delivered directly to the mucosal surface.Depending on the preparation and material,NE can be between 20 and 200nm in size,close to the size of pathogens,and can be rapidly absorbed by M cells on the mucosal surface and further delivered to antigen presenting cells（APCs）.Our previous studies have found that intranasal immunization with NE at 20-50 nm as a delivery system of protein antigen can significantly enhance the mucosal immune response of antigen and enhance the immune protection of host.For different antigens,NE can increase the level of antigen-specific antibodies,but also enhance the Th1 and Th17 responses.T cell epitopes identified by T cell receptor（TCR）are mainly peptide segments of antigens that processed and presented by APCs.CD4+T cells can be activated by their epitope peptides and further differentiated into different helper T cells（Th）.CD8+T cells can be activated by their epitope peptides and further differentiated into cytotoxic T cells（CTL）.At present,most vaccines play a role in prevention of infection mainly by inducing specific antibodies.However,many studies on viruses,bacteria and tumors have shown that T cells play a more important role in the adaptive immunity.Our previous studies also found that the CD4+T cell epitope peptide HpaA154-171（P22）of Helicobacter pylori HpaA protein has a good protective effect on mice,it can effectively reduce the bacteria load in the stomach.This suggests that focusing on protective T cell epitopes may be the key to prevent or treat infections and tumors.However,the applications of T cell epitope peptides are limited by their low molecular weight,weak immunogenicity and easy to clearance by the body.This research project focuses on the characteristics of intranasal vaccines and T cell epitopes,carries out the design and research of intranasal vaccines delivery system for T cell epitopes.At the same time,based on the previous research of our group,NE is selected as delivery system to promote intranasal delivery of T cell epitope peptides,and to enhance specific immune response intensity and improve the immune protection effect of T cell epitope peptides.Objectives:In order to overcome the shortcomings of small molecular weight,weak immunogenicity and susceptibility to elimination of T cell epitopes,we prepared T cell epitope nanoemulsion vaccine using Helicobacter pylori HpaA protein CD4+T cell epitope peptide（HpaA154-171,P22）and ovalbumin CD8+T cell epitope peptide（OVA257-264,OEP）as immunogen and nanoemulsion as carrier,and carried out intranasal inoculation.The study of immunobiological effects and mechanisms will provide technical and theoretical support for the development of a new T cell epitope peptide mucosal vaccine.Methods:Part I:Design,preparation and characterization of Helicobacter pylori HpaA CD4+T cell epitope peptide nanoemulsion vaccine（NE-P22 nanoemulsion vaccine）（1）Formulation research methods of NE-P22 nanoemulsion vaccine preparationOn the basis of the previous work of our group,the oil-in-water nanoemulsion system with IPM as oil phase,EL-35 as surfactant and propylene glycol as cosurfactant was prepared.The determination criteria are particle size range of 20-50 nm,Zeta potential range of-30 to 30 mV,encapsulation efficiency>80%.The drug loading concentration of P22 and the ratio of surfactant to cosurfactant（Smix）were determined by different surfactants.Multivariate matching screening was used to determine the optimal formulation of the P22 epitope peptide vaccine（NE-P22）.（2）Characterization and identification of NE-P22 nano-emulsion vaccine preparationDynamic light scattering particle size potential analyzer,transmission electron microscopy（TEM）,atomic force microscopy（AFM）,MALDI-TOF MS and CCK8 were used to detect the particle size,Zeta potential,dispersion,morphology,stability of P22epitope peptide in delivery system,cytotoxicity to BEAS-2B cells and BMDC cells in mice.PartⅡ:Evaluation and mechanism of intranasal immune protection of NE-P22nanoemulsion vaccine（1）Evaluation method of immune protection effect of NE-P22 vaccine intranasal inoculationThe mice were inoculated with NE-P22 in 0,7,14 and 21 days.After 7 days of the last immunization,the mice were infected with Helicobacter pylori by gastric perfusion.After30 days,the colonization of Helicobacter pylori in stomach was detected.The pathological inflammation score of gastric tissue was made to evaluate the protective effect of NE-P22intranasal immunization.（2）Research methods of immune protection mechanism of NE-P22 vaccine intranasal inoculationConfocal microscopy,flow cytometry,IVIS in vivo imaging system,ELISA and ELISPOT were used to detect the uptake efficiency of BEAS-2B cells of NE-P22,the ability of inducing BMDC maturation,nasal retention time,uptake effect of nasal mucosa tissue,antibody level,cytokine level and the proportion of P22-specific IFN-gamma cells secreted from spleen cells.PartⅢ:Design,preparation and characterization of IKVAV-PA conjugated OVA257-264 CD8+T cell epitope peptide nanoemulsion vaccine（NE-IO/MPLA vaccine）（1）Formulation research methods of NE-IO/MPLA nano-emulsion vaccine preparationOn the basis of the previous work of our group,oil-in-water nanoemulsion system with squalene as oil phase and Tween-80 as surfactant was used.The drug-loading concentration of epitope peptide of IKVAV-PA conjugated OVA257-264（IO）was screened to determine the epitope peptide with the standard of particle size ranging from 20-50 nm,Zeta potential between-30 to 30 mV and entrapment efficiency>80%.Determine the best formulation for NE-IO/MPLA nanoemulsion vaccine.（2）Characterization and identification of NE-IO/MPLA nano-emulsion vaccine preparationDynamic light scattering particle size potential analyzer,transmission electron microscopy,atomic force microscopy and CCK8 were used to detect NE-P22 particle size,Zeta potential,dispersion,morphological characteristics,stability in the presence of mucin and toxicity to BEAS-2B cells.PartⅣ:Evaluation and mechanism of intranasal immune protection of NE-IO/MPLA nanoemulsion vaccine（1）Evaluation method of immune protection effect of NE-IO/MPLA vaccine intranasal inoculationThe subcutaneous E.G7 cell model of C57BL/6 mice was established.The mice were immunized with NE-IO/MPLA intranasally before and after implantation.The preventive and therapeutic protective effects were evaluated according to the tumor volume and survival rate of mice.（2）Research methods of immune protection mechanism of NE-IO/MPLA vaccine intranasal inoculationConfocal microscopy,flow cytometry,ELISA,Bio-Plex liquid suspension chip,T-Select H-2Kb OVA tetramer and CTL assay in vitro were used to detect the uptake of BEAS-2B cells by NE-IO/MPLA,the effect of integrin blockade on the uptake rate of BEAS-2B cells,the level of serum antibodies,Th1 and Th2 cytokines,the proportion of OEP-specific CD8+T cells and the effect of CTL scavenging E.G7 in vitro.Results:Part Ⅰ:Design,preparation and characterization of Helicobacter pylori HpaA CD4+T cell epitope peptide nanoemulsion vaccine（NE-P22 nanoemulsion vaccine）The NE-P22 intranasal vaccine was successfully prepared with Smix=4:1 and drug loading concentration of 1000 ug/mL.The average particle size was 22.84±0.01 nm,Zeta potential was-5.168±1.687 mV,PDI was 0.1830±0.0026,encapsulation efficiency was96.13±0.81%,and the dispersion was good.TEM and AFM observation showed that the size of NE-P22 vaccine particles was about 20-30 nm,with smooth spherical surface and no obvious aggregation.MALDI-TOF MS assay confirmed that the P22 epitope peptide was stable in NE-P22.In addition,NE-P22 diluted 20 times had no significant toxic effect on BEAS-2B cells and mice BMDC cells.Part Ⅱ:Evaluation and mechanism of intranasal immune protection of NE-P22nanoemulsion vaccineNE-P22 intranasal inoculation significantly reduced gastric colonization（P<0.05）and inflammation score（P<0.01）after Helicobacter pylori infection in mice,and significantly enhanced the immune protection of P22 epitope peptide.NE-P22 increased the uptake of P22 epitope peptide by BEAS-2B cells（P<0.01）and promoted the maturation of DC cells（P<0.05）,prolonged the retention time of P22 epitope peptide in mouse nasal cavity（P<0.05）,and increased the uptake rate of P22 epitope peptide by DC cells in nasal epithelium and nasal mucosa（P<0.001）.After immunization with NE-P22,there was no specific Th2immune response,but it could significantly enhance the specific Th1 immune response of P22.At the same time,there was a synergistic effect between NE-P22 and CPG.PartⅢ:Design,preparation and characterization of IKVAV-PA conjugated OVA257-264 CD8+T cell epitope peptide nanoemulsion vaccine（NE-IO/MPLA vaccine）The results showed that the nano-emulsion vaccine prepared with IO loading concentration of 4 mg/mL had the smallest particle size,the average particle size was22.61±0.71 nm,the Zeta potential was-16.67±1.76 mV,the encapsulation rate was84.07±7.59%,the dispersion was good,and the particle size and Zeta potential did not change significantly in the presence of mucin.The results of transmission electron microscopy（TEM）and atomic force microscopy（AFM）showed that NE-IO/MPLA vaccine particles were smooth and spherical in surface,uniform in size,good dispersibility and no obvious aggregation.In addition,NE-IO/MPLA diluted 20 times had no significant toxic effect on BEAS-2B cells.PartⅣ:Evaluation and mechanism of intranasal immune protection of NE-IO/MPLA nanoemulsion vaccineFor E.G7 tumor bearing mice,NE-IO/MPLA immunization can play an effective preventive and therapeutic protective effect,can effectively inhibit the growth of tumors and prolong the survival time of mice.NE-IO/MPLA could promote the uptake of epitope peptides by BEAS-2B cells（P<0.001）,and the uptake rate decreased significantl y after integrin blockade（P<0.01）.There was no specific antibody in serum of mice immunized with NE-IO/MPLA.The level of Th2 cytokines in spleen cells stimulated by OEP did not change significantly,but the level of Th1 cytokines increased significantly,and the proportion of specific CD8+T cells increased significantly（P<0.01）.CTL test in vitro confirmed that CTL induced by immunization could kill and clear E.G7 cells effectively（P<0.001）.Conclusions:1.Two T cell epitope peptides nanoemulsion vaccines,NE-P22 and NE-IO/MPLA,were successfully prepared.Both of them have good quality characteristics.2.These two vaccines can effectively enhance the corresponding immune protection effect,which indicates that nanoemulsion is suitable for intranasal vaccine carrier as T cell epitope peptides,and is an effective delivery system design strategy.3.Nanoemulsion can effectively promote the uptake of antigens in nasal mucosa,prolong the retention time of antigens in mucosa,and promote the presentation of APCs to antigens.4.Both nanoemulsion carriers exhibit certain adjuvant activity,and their effects on enhancing specific immune response are synergistic with TLR agonists CpG and MPLA.Therefore,it is an ideal strategy to select suitable TLR agonists to combine in the design of nano-emulsion nasal vaccine.5.This study confirmed that T cell epitope peptide nano-emulsion nasal vaccine can effectively induce a high level of mucosal and systemic immune response,and has an effective immune protection effect on the body. |
