Construction And Immunogenicity Of HPV 16 Therapeutic MRNA Nano Vaccine

In this study,a therapeutic mRNA nanovaccine(mRNA nanovaccine)against human papillomavirus type 16(HPV)was prepared.The HPV16 oncogene E6E7 was first synthesized and inserted into the eukaryotic expression vector PVAX1 to construct a PVAX1-E6E7 recombinant plasmids.The plasmid was transformed into E.coli TOP 10,and the plasmid was extracted.The linearized DNA template was digested with XbaI and transcribed into E6E7 mRNA in vitro.The PVAX1-GFP recombinant plasmid was constructed using the same technical route to prepare GFP mRNA.Secondly,due to the ubiquitous RNase,this study constructed a cationic lipid nanoparticle(LNPs)designed to protect the mRNA vaccine from degradation,enter the cell smoothly,and reach the ribosome after lysosome escape.Translation of antigenic proteins.First,GFP mRNA was used as a reporter gene to explore the conditions of the mRNA-LNPs protective delivery system.The fluorescence expression of GFP mRNA-LNPs in 293 cells was significantly increased by inverting fluorescence microscopy compared to chemical transfection.E6E7 mRNA and LNPs were then self-assembled into HPV mRNA-LNPs nano vaccine.The ELISA showed that the antigen expression level of HPV mRNA-LNPs nano vaccine in 293 cells was 1.8 times higher than that of chemically transfected HPV mRNA.In this study,nano-vaccines were investigated by dynamic light scattering,potential and encapsulation efficiency.The particle size of HPV mRNA-LNPs nano-vaccine was about 97 nm,PDI was 0.137,potential was 21 mV,and entrapment efficiency was 95.9%.Subsequently,the E6E7 oncogene was cloned into the Pet-28a expression vector by BamhI and XhoI restriction sites at both ends,and Pet28a-E6E7 was constructed and transformed into BL21 strain for expression.The E6E7 fusion protein was induced by different concentrations of IPTG.The optimal induction conditions were determined to be:16 ℃,120 rpm,0.1 mM IPTG induction for 20 h.The induced inclusion body protein was denatured and renatured to obtain E6E7 soluble protein,and the target band and binding activity were identified by Western Blot.The results showed that the purified E6E7 fusion protein had good binding activity and laid the foundation for subsequent animal experiments.Finally,a therapeutic mouse model was constructed to compare the tumor growth curves of LNPs,HPV mRNA-LNPs,HPV DNA-LNPs,mouse serum antigen-specific IgG antibody titers,Th1 type cytokines TNF-α,IFN-γ,and Th2.The expression of the cytokine IL-4 and the changes in the T cell subset of the spleen cells of each group of mice.The results showed that compared with the LNPs control group,the tumor size of the HPV mRNA-LNPs nano-vaccine group was significantly inhibited(p<0.05),the antibody titer was increased,and the expression levels of cytokines TNF-α,IFN-γ and IL-4 were increased.Both were significantly improved(p<0.01).The CD4+T cells and CD8+T cells of the spleen cells of the HPV mRNA-LNPs nano-vaccine group were increased compared with the LNPs control group(p<0.05).In summary,the HPV mRNA-LNPs nano-vaccine constructed in this study produced significant tumor therapeutic effects in mice,inducing strong humoral immunity and cellular immune responses.