Effects Of Flt3L Molecular Adjuvant's On The Specific Immune Response Elicited By A LCMV Peptide Vaccine

Aim:The tumors and chronic virus infections are currently the big challenges faced by humans. While immunotherapy is the promising stretagy against tumors and chronic virus infections,it is evident from clinical studies that the current immunotherapy including therapeutic vaccines show weak effect.Thus seeking for new stretagies to enhance the effect of immunotherapy has become the hot research.In the present study,we aim to investigate the influence of Flt3L molecular adjuvant on the immune reponses induced by KAV peptide vaccine and the underlying mechanism.This should provide data of Flt3L molecular adjuvant for clinical study.Methods:The primers were designed according to the known Flt3L gene,and the cDNA of Flt3L was cloned by RT-PCR.Then the recombinant plasmids for the extracellular domain of Flt3L,for the soluble and membrane-bound Flt3L were constrcted,respectively.After confirmed by DNA sequencing,the prokaryotic expression vector was transformed into BL21(DE3) strain while the eukaryotic plasmids were transfected into HeLa cells repectively,by using FuGENE HD reagent. SDS-PAGE and/or Western blotting were used to analyze the expression of these recombinant proteins.The biological activity of soluble Flt3L in the supernatant of transfected HeLa cells was determined.The recombinant plasmid of membrane-bound Flt3L was tested as a molecular adjuvant in mice to evaluate its effect on the immune responses induced by the LCMV peptide vaccine.The intervention on the frequency and phenotypes of KAV-specific CD8~+ T cells was analyzed by tetramer technology.Results:1.The full encoding sequence of mouse Flt3L was cloned by RT-PCR.The sequence was identical to the previously reported Flt3L cDNA and submitted to GenBank with an accession number of EU274583.The expression vector for the recombinant Flt3L extracellular domain was then constructed and verified by DNA sequencing.SDS-PAGE and Western blotting analysis revealed that the fusion protein was highly expressed in E.coli BL21(DE3).2.The eukaryotic recombinant plasmid for soluble Flt3L was then constructed and verified by DNA sequencing.The eukaryotic expression vector was transfected into HeLa cells and the culture supernatant was shown to have synergistic effect with GM-CSF on bone marrow cells proliferation and colony formation.3.Furthermore,eukaryotic expression plasmid for membrane-bound Flt3L was constructed and used as the molecular adjuvant.Western blotting showed that this membrane-bound protein was expressed in HeLa cells.The adjuvant effect of this Flt3L was tested in C57B1/6 mice.The results showed that Flt3L molecular adjuvant in combination with KAV peptide could increase the frequency of the vaccine-specific CD8~+ T cells as compared with KAV peptide alone.It also slightly changed the phenotype of these specific CD8~+ T cells.Conclusion:On the basis of successful cloning of mouse Flt3L's cDNA,the prokaryotic expression plasmid for Flt3L extracellular domain and the eukaryotic expression plasmids for both soluble and membrane-bound Flt3L were constructed.These plasmids are proved to be expressed either in E coli or in HeLa cells.The soluble Flt3L expressed in HeLa cells has biological activity. Furthermore,the membrane-bound Flt3L molecular adjuvant can significantly increased the frequency of LCMV-specific CDD8~+ T cells and slightly influence the phenotype of these cells, suggesting that this molecular adjuvant could enhance the cellular immune reponse of peptide vaccines.