Mechanism Of Inducing Th2 Type Immune Responses By Gene-gun-mediated HBs DNA Immunization And Strategies On Shifting Antigen Specific Th2 Type Immune Responses To Th1 Type

DNA vaccine was regarded as one of the most promising approaches for future vaccine developmentin light of its capability of overcoming the disadvantages in conventional vaccines (such ascapability of inducing both humoral and cellular immunity) and of its stability as well as simplicityof production. Gene-gun-mediated DNA vaccination, which developed in the last decade, requiresonly minute quantities of DNA to induce a strong immune response, so as to have the great potentialof being applied to humans. However, compared to a more Th1 type biased response induced byintramuscular (i.m.) or intradermal (i.d.) injections of plasmid DNA with syringe and needle, apredominant Th2 type response has been observed after gene-gun-mediated DNA immunization. Theexact mechanism for this difference is yet to be completely understood. The tendency topreferentially prime Th2 response by gene-gun-mediated DNA immunization makes it less attractiveas the Th1 immunity, which was characterized by strong cellular immunity and moderate humoralimmunity, plays a vital role in host defense against viral, intracellular bacterial infections and tumors.Considerable efforts are now being focused on understanding the molecular mechanism of inducingdifferent type of immune response by gene-gun-mediated DNA immunization orsyringe-needle-injection DNA vaccination, and on developing ways of shifting the Th2 type immuneresponse induced by gene-gun-mediated DNA immunization to Th1 type. In this study, mechanismof the tendency of inducing Th2 immune response by gene-gun-mediated DNA immunization wasinvestigated and the adjuvant strategies to switch its Th2 type immune response to Th1 type werefuther explored by using HBsAg as target antigen.It is proposed that the possible differences between gene-gun-mediated andneedle-injection-mediated DNA immunization include the dosage of plasmid DNA, the mechanismof DNA transfection, the type of local antigen presenting cells (APCs) and the antigen presentingprocess etc. As the activation of APCs (especially the dendritic cells) has a great impact on both theinduction and the qualities of acquired immune response, it is hypothesized that gene-gun-mediateddelivery and needle-injection-mediated inoculation of DNA vaccine will result in different "dangersignals" to organism APCs ( while the danger signals of injection DNA in saline with syringe and 4博士学位论文 英文摘要needle are from the CpG motifs built in the plasmid construct, those of gene-gun mediated DNAimmunization come from the gold-particle bombardment of the skin) and this may account for theirdifferences in immune-response type. To test this hypothesis, local tissues of gene-gun-bombardedskin or needle-injected muscle were sectioned and analyzed by H&E staining. Results showed thatremarkable inflammatory infiltration could be seen in both gold-particle bombarded skin andCpG-motif injected muscle, which suggests that bombardment of gold-particle and needle-injectionof CpG motif are both "danger stimuli" to na?ve immune system of body. The possible impacts ofthose two "danger stimuli" on the polarity (Th1/Th2) of adaptive immune response were furtherstudied by ananlyzing the cytokine profile from lymph-node-derived mono-nuclear cells. Themono-nuclear cells of draining lymph nodes in inoculated mice were separated and stimulated invitro after inoculated by gene-gun bombardment of plasmid DNA coated on gold particle, goldparticle alone or CpG-ODN coated on gold particle, versus inoculated by saline needle-injection ofplasmid DNA, CpG-ODN or GC-ODN. Reverse transcription (RT) and real-time PCR were appliedto ananlyzed the cytokine production profile of IFN-γ, IL-12, IL-10 and IL-4. Results showed that:gene-gun bombardment of 1μg plasmid DNA coated on gold particle to skin resu...