Activity And Mechanism Study Of Cholera Toxin-like Chimeric Protein In Cross Presentation Of Exogenous Antigen

Sole or multiple exogenous tumor antigen epitopes are used in tumor subunit vaccines to stimulate immune cells（usually are antigen-presenting cells）in vivo and generate immune reaction.In these cells,exogenous tumor antigen epitopes can be processed and presented to major histocompatibility complex Ⅰ（MHC Ⅰ）to form MHC Ⅰ-antigen peptide complexes.Then,the complexes are transferred to cell surface and recognized by CD8⁺T cells.The CD8⁺T cells differentiate into cytotoxic T lymphocytes（CTLs）which exert direct cytotoxic effect on tumor.This process of exogenous antigen epitopes presented by MHC I is called cross presentation of exogenous antigen.Whole tumor antigen vaccines are replaced by tumor subunit vaccines to reduce side effects and realize precise treatment.However,tumor antigen epitope has low immunogenicity.Administration of exogenous tumor antigen epitopes results in weak cross presentation and antitumor immunity.Thus,tumor subunit vaccines are often administered with immune adjuvant.Immune adjuvant can nonspecifically enhance immune response to epitope in vivo or change the type of immune response to produce anti-tumor immune effect.Native cholera toxin（CT）is a kind of bio-adjuvant.We constructed a CT-like chimeric protein based on hexameric structure of CT as an anti-prostate cancer subunit vaccine in our previous studies.It was found that the chimera could enhance the immunogenicity of prostate cancer antigen epitope,induce antigen-specific CTL and inhibit tumor growth via cross presentation.CT-like chimeric protein is a molecule with AB5 structure and specific functional sequence.To study the cellular mechanism of CT-like chimeric protein in cross presentation,MHC I-restricted epitope 257-264 of ovalbumin（OVA257-264,OVAT）was used as a model antigen peptide in this study.Three different chimeric biomacromolecules were constructed according to CT molecule characteristic,the different fusion sites and whether the endoplasmic reticulum（ER）location sequence was included.The main contents of our study are as follows:（1）Obtaining three CT-like chimeric proteins containing OVAT epitope.Constructing plasmids and transforming into Escherichia coli expression system.The recombinant proteins m G-CTA2-OVAT,m G-CTA2（KO）and CTB-OVAT were all expressed as inclusion bodies.After affinity chromatography purification,the purified proteins assembled respectively into three CT-like chimeric proteins in vitro by citric acid-tris alkali denaturation and renaturation method.The chimeric proteins were determined by SDS-PAGE,Western blot,Native-PAGE,gel filtration and MALDI-TOF/MS.（2）Study of the in-vitro biological activity of CT-like chimeric proteins.The cells from C57BL/6J mice bone marrow proliferation assay and the ganglioside binding assay（GM1-ELISA）were used to examine whether the chimeric proteins retained both m GM-CSF and（CTB）5 activities.The results showed that CT-like chimeric proteins possessed m GM-CSF activity to stimulate myeloid cell proliferation and the ability of（CTB）5 to bind to GM1.（3）The effects of CT-like chimeric protein on cross presentation mediated by mouse bone marrow-derived dendritic cells（BMDCs）.BMDCs were co-cultured with CT-like chimeric proteins in vitro and the expressions of co-stimulatory molecules and MHC I/II molecules on cell surface were detected via flow cytometry.The results showed that CT-like chimeric proteins up-regulated CD80,CD86 and MHC II on BMDCs to induce activation and maturation of BMDCs and increased expression of MHC I-OVAT complex to improve cross presentation of exogenous antigen.（4）The mechanism study of improvement in cross presentation induced by CT-like chimeric protein.Effects of dose and inhibitor on BMDCs uptake of three chimeras were explored,and transportation pathways of three CT-like chimeric proteins in DCs were compared.The results showed that BMDCs uptake of CT-like chimeric proteins was in a dose-dependent manner.GM1-mediated endocytosis was a major participant which was affected by endosomal alkalization.A comparison of chimera uptake and transportation showed that A2 and B subunit of CT were both available for loading antigen peptide and did not affect cell uptake.Hexameric structure of CT-like chimeric proteins was beneficial to lysosomal escape to avoid excessive degeneration.KDEL sequence in hexameric CT-like chimeric proteins helped to reside in ER and promote cross presentation.Our study further clarified the pathway and mechanism of cross presentation promoted by the chimeric,deepened the understanding of cross presentation of exogenous antigen and provided a new theoretical basis for the adjuvant developments of tumor subunit vaccines based on CT-like chimeric protein.