Immunogenicity Analysis Of Recombinant Exosome Derived From Chicken Macrophage Cell Line HD11

As the extracellular vesicle structure secreted by cells,exosome plays an important role in molecular events related to substance and information exchange between cells,such as antigen presentation,and regulation of the cellular microenvironment,makes it become a biomarker for diseases diagnosis or a natural nanocarrier.Exosomes loaded with biomolecules have received extensive attention.There are various methods for loading proteins,nucleic acids or small molecule drugs to construct recombinant exosomes.Among this,proteins or peptides can be loaded into the exosome by fusion expression of signal sequences,such as vesicular stomatitis virus（VSV）G protein.Since the immunogenicity of most subunit vaccines are poor,they are usually not suitable for primary immunization.Exosomes secreted by antigen-presenting cells after antigen stimulation and activation can induce adaptive immune response.Thus,the immunogenicity of subunit antigen can be enhanced by preparing a recombinant antigen presenting cell-derived exosome loaded with the antigenic protein.However,the related functions of chicken antigen-presenting exosomes are still unknown.The recombinant chicken macrophage HD11 constructed previously was employed to generate engineered exosome loading virus antigen.The recombinant HD11 cell can stably express avian infectious bronchitis virus S1 protein which C-terminal fused with transmembrane region of VSV G.Firstly,the recombinant exosomes were extracted and purified from the culture supernatant of recombinant HD11 cells by ultracentrifugation.Then the purification of exosome was confirmed by analysis of exosome biomarkers,the transmission electron microscope and nano-flow cytometry.It was proved by Western Blot that S1^VSVprotein was successfully loaded into the exosome.The exosomes were then labeled with PKH67 dye and incubated with HD11 cells and chicken bone marrow-derived dendritic cells,respectively.The expressions of related cytokines were synchronously detected by quantitative PCR to analyze the effect of recombinant exosomes on antigen-presenting activity.Finally,7-day-old SPF chickens were further immunized with recombinant exosome by intramuscular injection to detect the IBV antibody level in serum and the distribution of T lymphocyte subsets in peripheral blood of the immunized chickens.The results showed that the recombinant HD11 exosomes could be uptaken by HD11cells and chicken bone marrow-derived dendritic cells.The exosomes induced M2-type polarization of HD11 cells as well as the activation of dendritic cells.The HD11-derived recombinant exosome can induce the production of anti-IBV antibodies and changes in the distribution of T lymphocyte subsets,indicating that the HD11-derived exosome carrying the S1 antigen has immunogenicity.However,the level of immune response produced in this study varied among individuals.It is possible that the ultra-centrifugal purification method could not obtain homogeneous exosomes,while exosomes with various particle sizes and generated from different synthetic pathways have different biological functions.Therefore,the large-scale purification of eukaryotic exosomes with high homogeneity is the key to the development of exosomal vaccines.