Study Of Construction And Mechanism Of CTL Epitope Peptide-based Microparticles Vaccine Against PHC

[Objective] To develope a effective epitope-based vaccination strategy against primary hepatocellular carcinoma(PHC),we try to identify immunodominant CTL epitopes from tumor associated antigens(TAA) expressed in PHC after determining whether CTL epiptope-based vaccine is suitable for its immunotherapy,then, we construct a epitope peptide-based vaccine with suitable carriers or adjuvants and examine its immunological activity and mechanism underlying.[Methods] The LSAB assay of immunohistochemistry were used for investigating the expression of target antigen human alpha-fetoprotein (hAFP) in human hepatocellular carcinoma cell lines and important molecules of MHC-I-restricted antigen presentation pathway expressed in human PHC tissues. The candidates of HLA-A2-restricted CTL epitope derived from hAFP were predicted and evaluated by a program named A2B developed by ourselve and tools for predicting CTL epitope available on the World Wide Web. The predicted candidates were synthesized, purified, and authenticated by peptide synthesizer, reverse-phase high pressure liquid chromatography, and mass spectrum respectively.The affinity between the peptide and HLA-A2molecule were measured through T2 cell binding assay. The cytotoxicity of the CTL induced in vitro by peptides were surveyed through MTT assay. Peptide-loading poly-L-lactide(PLA) microparticles were prepared by solvent evaporation and non-solvent precipitation methods, microparticle size distibution was determined by laser light scattering ,and surface morphology was examined by scanning electron microscopy. In vitro characteristics of peptide-loading microparticles including encapsulation efficiency, peptide loading and release of peptide, was examined by ultraviolet radiation. The ability of induction specific CTL by peptide-loading microparticles was surveyed by cytolytic assay. The interaction between monocyte-MΦ and peptide-loading microparticles was invetigated in phagocytosis, activation and cytotoxicity. In vitro MHC I-restricted presentation of peptide loaded by microparticles was studied through surveying effects of intracellular inhibitorsin its presentation.[Results] 1.human hepatocellular carcinoma cell lines Alexander and HepG-2 expressed hAFP;2.HLA-I antigens, TAP and B7 molecules were expressed strongly in the majority of PHC sepcimens;3.Two candidates of HLA-A2-restricted CTL epitope derived from hAFP were successfully predicted out by a program named A2B developed by ourselve and tools for predicting CTL epitope available on the World Wide Web;4. The two nonapeptides were successfully synthesized, purified, and authenticated with a purity near 100%;5.The candidate peptides both have high affinity to HLA-A2 molecule and can induce HLA-A2+ PBL from healthy volunteer to produce proliferation and peptide-specific cytolysis;6.Effectors induced by peptides can kill the HLA-A2+/AFP+HepG-2 and HLA-A27AFP+ Alexander cells efficiently;7.The size and morphology of peptide-loading PLA microparticles were disirable, and with high encapsulation efficiency, peptide loading;8.The cumulative release profile of peptide-loading microspheres and lamellae was quite different.The release of peptide from microspheres showed an initial burst(about 15% of load)in 24h,the remaining 85% of load was released slowly and smoothly, while peptide was released fastly from lamellae,approximately 80% of asorbed peptide was released within 3d;9.All of four kind microparticles can induce HLA-A2+ PBL from healthy volunteer to produce proliferation and high peptide-specific cytolysis, espicially two lamellae; lO.Peptide-loading microparticles showed no or little cytotoxicity on human monocyte-M $ ,they can activate human monocyte-M O strongly, and can be phagocytolized by them; 11.Peptides loded in microparticles can be presented by monocyte-M O and induce specific CTL;12.Blocking phagocytosis, proteolytic site of proteasome and hydrolysis in acidic vacuolarcompartments have no effects on MHC I-restricted presentation of peptide-loading lamellae,while inhibiting exocytosis of newly synthesized MHC-I molecules showed partly downregulation. But for peptide-loading microspheres, Blocking phagocytosis, hydrolysis in acidic vacuolarcompartments and exocytosis of newly synthesized MHC-I molecules showed some inhibiting on their MHC I-restricted presentation .[Conclusion] 1. human hepatocellular carcinoma cell lines Alexander and HepG-2 can be used in studies of CTL epitope-based vaccine against PHC targeting for hAFP; 2. CTL epitope-based vaccine therapy is suitable for treating PHC; 3. The majority of PHC tissues may possess intact MHC-I restricted presenting machinery, and it imply tumor immune escape may be associated with post evens of antigen presentation; 4.The program named A2B developed by ourselve and tools for predicting CTL epitope available onthe World Wide Web are potent tools for prediction of CTL epitope; 5. Two cadidates hAFPi56-i66 and hAFP2is-226both have high affinity to HLA-A2 molecule and potent capability to induce peptide-specific CTL, they are HLA-A2-restricted immunodominant CTL epitopes processed and presented naturally; 6. hAFP 156-166 and hAFP2is-226 maybe have supertype significance; 7.PLA can be used for preparing peptide-loading microparticle which we expect; 8.The release profiles of peptide-loading microspheres and lamellae are distinctively from each other; 9. All of four kind microparticles have potent capibility to induce high peptide-specific CTL, they are probably one kind of efficient CTL epitope vaccine against HPC; 10.peptide-loading microparticles can be efficiently phagocytosized and presented by monocyte-M ?; 11. peptides released from loading microparticles outcell can be presented by directly binding with MHC-I molecules expressed on surface of APC; 12.Peptides loaded in mcroparticles also can be presented through peptide regurgitation and phagolysosome-to-cytosol way.