Research On The Immunologic Mechanism Of DNA Vaccine

Deoxyribonucleic acid vaccine(DNA vaccine),which is also named nucleic acid vaccine or gene vaccine,is a novel vaccine developing with gene therapy technology in recent years.DNA vaccine not only has advantages of live attenuated vaccine,but also has safety of inactivated vaccine or subunit vaccine,and has both preventive effection and therapeutic effection.Therefore,DNA vaccine for therapy is the key point of research in recent years,and rapidly expands from the treatment of communicable disease to the treatment of the non-communicable disease.Recently,DNA vaccines have entered into a variety of human clinical trials for vaccines against various infectious diseases and for therapies against cancer.Despite their rapid and widespread development,however,the disappointing potency of the DNA vaccines in humans underscores the challenges encountered in the efforts to translate efficacy in preclinical models into clinical realities. In order to improve the efficacy of DNA vaccines,a lot of novel strategies have been performed.The definition of molecular mechanism of DNA vaccine must lead to gain more effective DNA vaccine approaches.However,the mechanisms of immune responses for DNA vaccines are still not entirely understood.In order to improve understanding of the immune response mechanisms,two diseases,Alzheimer's disease and HBV-related hepatocellular carcinoma,suggesting the development of DNA vaccine may be possible, are investigated in present study.Alzheimer disease(AD) is a neurodegenerative disorder characterized neuropathologically by neuritic plaques-amyloid deposits,neurofibrillary tangles,and selective neuronal loss.The various mouse models and clinical trial results demonstrated that the progressive deposition of Aβhad a central role in AD pathogenesis.Accordingly, Aβhas become a therapeutic target for the prevention and treatment of AD.In 1999, Schenk et al reported that vaccination of an amyloid precursor protein(APP) transgenic mouse model of AD with Aβpeptide resulted in a significant reduction in cerebral Aβdeposits.But this peptide vaccine was complicated by meningoencephalitis among 6%of subjects in phaseⅡa trial.Therefore,the advanced development of AβDNA vaccine having immunogenicity and no toxic,adverse effects is important strategy for AD immunotherapy.In previous study,we have constructed DNA vaccines containing Aβepitope sequences or full-length Aβgene.Although one of Aβepitope encoding plasmid and Aβfull-length encoding plasmid elicited higher Aβ-specific antibody titers than others, the humoral immune responses were not enough satisfied without any adjuvant.According to the positive correlation between antigenic expression levels and the extent of immune activation for DNA vaccine,the immunogenicity of gene would be enhanced by alteration of cellular location and leading product of gene to increased secretion.In the present study, to elevate the immunogenicity of Aβ,the tissue plasminogen activator signal sequence was fused to Aβgene.The results showed that humoral responses were enhanced in mice inoculated with DNA constructs expressing TPA-fused Aβantigen relative to those in mice that had been immunized with AβDNA vaccine.To investigate whether the enhanced immune activation of TPA-fused antigens compared to native protein is the result of increased secretion of protein,pEGFP-N1-Aβand pEGFP-N1-TPA-Aβwere transfected into 293T cells.The fluorescence of TPA-Aβ/EGFP was observed in both the nucleus and cytosol and a few was located at cell membrane at 72h after transfection.This result suggested that TPA- fused protein was secreting from intracellular into extracellular. Western blot analysis confirmed the results by detection of Aβ/EGFP from the cultured supernatant of 293-T-pEGFP-TPA-Aβcells.All of results of experiments in vitro or in vivo demonstrated the targeting antigen was successfully secreted into extracellular conducted by TPA signal sequence.Therefore,we concluded that TPA-fused Aβprotein in host cells should lead to increased secretion of this protein with elevated uptake by antigen-presenting cells,and thus,a more generalized activation of the immune system and the higher levels of specific antibody.In summary,this study provides a clue to apply TPA signal sequence in Aβepitope DNA vaccine and is in favor of elucidating the underlying mechanism of senile plaques clearance by DNA vaccine active immunotherapy in the future research.Chronic HBV infection is closely associated with the incidence of hepatocellular carcinoma.Among the four proteins that originate from the HBV genome,HBx has been reported to be associated with hepatocellular carcinogenesis.The molecular mechanisms of HBx in hepatocellular carcinoma(HCC) are not well understood,since the initiation and propagation of most tumors is a multistep process,and HBx has multiple effects on cellular transcription and the cytoplasmic modulation of signal transfection pathways.Further elucidation of the molecular events that lead to HCC holds promise for improved tumor prevention,and more effective therapies,involving DNA vaccine.HBx does not directly bind DNA but functions via protein-protein interaction.To reveal and identify proteins, interacting with HBx directly or indirectly,which are associated with HCC,the proteomic approach was applied.Eight spots were identified as SEC13L1,PA28a,STRAP,Nm23-H1, APRT,NDUFS3,PIMT and RXRbeta proteins.Among these proteins,one down-regulated pr-otein(PIMT),and two up-regulated proteins(STRAP and nm23-H1),were associated with the PI3K signaling pathway.Furthermore,our experiments showed that PIMT down-regulation by HBx was mediated by the up-regulation of STRAP and nm23-H1, which activated the PI3K/Akt signaling cascade.The altered expression of these proteins could provide more information about very complicated signaling molecular networks activated by HBx protein,and HBx-mediated PIMT down-regulation may provide clues to elucidate the molecular mechanisms of the development and progression of HCC and a target for therapy of HCC.