Preparation And Immunological Evaluation Of Synthetic Vaccine Based On INKT Cell Agonist

Subunit vaccines exhibit a wide range of applications.Among them,the semi-synthetic vaccines based on carrier protein are faced the problems of complex structure and immune suppression caused by protein itself.iNKT cells are a specialized subset of T cells,and share the ftuntions of NK cell and T cell.Unlike conventional T cells,the receptors of iNKT cells can recognize glycolipid antigens presented by CD 1d molecules,instead of peptides presented by MHC molecules.In addition,iNKT cells can help B cells to induce antibody class switching from IgM to IgG after being activated,thus trigger immune response for T cell-independent antigens,and overcome the immune tolerance to small molecule antigens.We hypothesized that iNKT cell agonists introduced into synthetic vaccines can simplify the structure of vaccines and facilitate their preparation,and trigger immune responses against non-polypeptide antigens effectively.This thesis was divided into two parts.The first part was to develop a chemically synthetic vaccine against nicotine,an addictive small molecule,based on iNKT cell agonists.In the second part,chemical synthetic vaccines targeting carcinogen aflatoxin were studied based on iNKT cell agonists.The main contents were as follows:First,the history and current situation of chemically synthetic vaccines were introduced.The development of nicotine vaccines and aflatoxin vaccines were briefly summarized,and the application of iNKT cell agonists as adjuvants in synthetic vaccines was summarized.Second,we studied on whether the covalent linkage between the iNKT cell agonist aGalCer and nicotine antigen was conducive to enhance immune response.First of all,nicotine antigen was covalently coupled to the ?GalCer,which exhibited no immune activity,just to give an amphiphilic molecule Nic-?GalCer to present a nicotine antigen.Liposomes of Nic-?GalCer combined with aGalCer,Pam3CSK4 as common TLR agonists or Freund's adjuvant,respectively,were prepared to immunize mice.The immunological results of mice showed that aGalcer displayed better adjuvant activity than Pam3CSK4 and Freund's adjuvant.Based on the excellent results of non-covalent modality between nicotine and aGalCer,Nic-aGalCer was prepared by nicotine antigen covalently coupled to aGalCer.The immune results showed that antibody induced by Nic-aGalcer was dependent on the concentration,and showed excellent immune activity similar to that of non-covalent vaccine.Based on the above results,the iNKT cell agonist aGalCer was introduced into the nicotine vaccine,and a strong immune response could be induced without covalent between adjuvant and antigen.Besides,since aGalCer induces both Thl and Th2 cytokines equally,iNKT agonists which produced Thl(IFN-y)and Th2(IL-4)cycokines were introduced to further optimize the immune activity of the vaccine.We hypothesized that the introduction of different types of iNKT cell agonists into nicotine vaccines can induce stronger immune responses than aGalCer.In this thesis,liposomes of Nic-?GalCer combined with a-C-GalCer and C34 as Thl agonists,C20:2 and C10:0 as Th2 agonists,respectively,were prepared to immunize mice.Antibody titer and animal behavior test results showed that Thl and Th2 agonists exhibited no difference with aGalCer but both were superior to unvaccinated mice.Cytokines produced by Thl type and Th2 type iNKT cell agonists in liposome were all in favor of Thl type.Based on the above results,different types of iNKT cell agonists as adjuvants can induce anti-nicotine antibodies,and liposome preparation can enhance cytokine release and shift Thl/Th2 balance to Th1.Last,the above liposome vaccine model was applied to construct a simple,convenient and effective aflatoxin vaccine with definite structure.In this paper,the effects of iNKT cell agonists ?GralCer and aflatoxin B1 on immune activity were studied by covalent and non-covalent strategy.Aflatoxin B1 was oximated and then covalently coupled to aGalcer activated by selenate to obtain compound AFB1-aGalCer.At the same time,AFB1-?GalCer,which has no immune activity,was synthesized as a carrier molecule and mixed noncovalently with aGalCer.The results showed that the antibody titer and the ability of antibody produced by the non-covalent modality to recognize different types of aflatoxin were higher than the covalent.The iNKT cell agonist aGalCer was introduced into aflatoxin vaccine and could induce a strong immune response via non-covalent between aGalCer with AFB1-?GalCer.Therefore,based on the iNKT cell agonist as an adjuvant,T cell-inpendent antigen can induce immune response with peptide free,providing a new strategy for the construction of simple and effective vaccines.