Emulsion Particulate Strategy For Enhanced Vaccine Adjuvant

With the rapid development of vaccine industry,accumulating research interest has been concentrated into the design of safe and efficient adjuvant.However,the limitations on the functionalization,multi-component loading,and recapitalizing the dynamic remodeling of natural pathogens hampered the clinical applications of these adjuvants.To address this,this work developed emulsion particulate strategy to combine the advantages of emulsion and particulate adjuvant.More importantly,inspired by synthetic biology,the strategy was further exploited to offer "chassis" for vaccine design,which develops stable,effective and adaptable particulate vaccine,by loading with "components"(such as antigens,target or immune-potentiate ligands).Through intrinsic emulsion particulate strategy,hierarchical structures were constructed with oily core and solid shell to co-encapsulate antigens and immune potentiator(retinoic acid,RA),which simultaneously provoke systemic and mucosal immunity to broaden the breadth of the immune responses.In addition,through extrinsic emulsion particulate strategy,particle-stabilized emulsion(Pickering emulsion)was developed to mimic the pliability and lateral mobility of the natural pathogens,which would increase the contact area and dynamically activate the immune recognition to deepen the depth of the immune responses.The thesis was unfolded as follow:1.By intrinsic emulsion particulate strategy,solid emulsion capsules were prepared to facilitate the spatial-temporal delivery of antigen and RA,and induced evident endosomal escape and cross-presentation of antigens.The core-shell structure was constructed with RA-loaded squalene on the inside,covering with the positive-charged PLGA/DDAB shells(253.3 ± 35.2 nm),which harbored high RA encapsulation efficiency(83.6 ± 4.3%),and antigen adsorption(88.5 ± 4.2%).Within the lysosomes,the capsules experienced "intracellular ripening",that aggregated into larger sizes with smaller specific surface area,and manifested enhanced RA control-release behavior.Meanwhile,the positive charged surfaces demonstrated "proton sponge effect",which caused the rupture of lysosomes for cytosolic delivery of antigens.Accordingly,the capsules elicited long-lasting release of RA and the cross-presentation of antigens.2.Through solid emulsion capsules,"immunoticket" strategy was developed to simultaneously activate systemic and mucosal immune responses via intramuscular injections.The solid emulsion capsules stimulated the recruitment,antigen uptake and cross-presentation of dendritic cells(DCs).Through spatial-temporal delivery of RA and antigen,RA/RAR signal was activated to stimulate the expression of gut-homing receptor,CCR9.In addition,the enzyme activity of RALDH2 was boosted to secret RA,which caused the gut-tropism of other immunocytes to home to the intestines along the concentration gradient of chemokine CCL25,and provoked the mucosal immunity.Meanwhile,RA-involved regulatory T responses were attenuated.In OVA and EV71 vaccinations,the capsules elicited potent antigen-specific IgG titer,cytotoxic T lymphocyte engagement and antigen-specific mucosal antibody IgA secretion,which proved to stimulate both systemic and mucosal immune responses.3.Through extrinsic emulsion particulate strategy,PLGA nanoparticle-stabilized emulsion(Pickering emulsion,PPAS)was developed to mimic the elasticity and lateral mobility of the natural pathogens.With the particle-adsorbed surfaces,PPAS manifested large specific surface area and increased antigen loading capability to recapitalize the condense array of surface antigens on the pathogen.On the cellular membrane,PPAS demonstrated force-dependent deformation to increase the contact area.Within the contact zone,antigens laterally moving to dynamically activate the recognition and internalization of DCs for enhanced immune responses.And,PPAS also harbored high storage and freeze-thaw stability for large-scale production and clinical applications.4.PPAS stimulated the internalization and cross-presentation of antigens,and boosted the recruitment and activation of APCs,which facilitated the mature of lymph nodes for enhanced immune responses.Under the acidic environment,the carboxyl groups on the surface was protonated to demonstrate positive charges,which caused the rupture of lysosomes for cytosolic delivery,and subsequently cross-presentation of the antigens.In addition,PPAS formed antigen depot at the injection sites to potently recruit and activate antigen presenting cells(APCs),and provoke the homing of APCs to the draining lymph nodes.The primed APCs maintained antigen persistence and germinal center engagement within the lymph nodes for enhanced adaptive immune activations.5.In OVA,H1NM and MUC1 vaccinations,PPAS demonstrated robust prophylactic and therapeutic adjuvant activity.Compared with commercial adjuvants,PPAS increased antigen-specific IgG titer and cytotoxic T lymphocytes activation.In prophylactic vaccinations,PPAS induced highest survival rates in H1N1 virus challenge model and E.G7/OVA tumor challenge model.In therapeutic evaluations,PPAS stimulated the actrivation and proliferation of IFN-? secreting cells for tumor lysis,and delayed the tumor growth and onset of E.G7/OVA tumor and MUC1/B16 melanoma.