Strategies And Mechanism Of Enhancing Immunopotency Of DNA Vaccines

Nucleic acid vaccine(DNA vaccine) is a kind of DNA vector which can carry exogenous gene encoding immunogenic antigen. Generally, the plasmids or some viruses can be used as DNA vector. When the plasmid carrying target gene is inoculated into the body, the transfected muscular cells or antigen-presenting cells(APC) can express the immunogenic antigen in cytoplasm and present the antigen to the Th cells to induce immune response, which can protect the body from infection. More and more attentions are paid to the usage of DNA vaccine for its special immunogenicity that can induce strong cellular and humoral immune responses. Furthermore, some phase â… -â…¡ clinical trials of DNA vaccines are ongoing recently. However, the potency of DNA vaccines in humans is disappointing. In addition, a large amount of plasmid DNA was required to induce immune response on the larger animals, such as rhesus macaques, chimpanzee, and so on. Therefore, it is urgent to develop new strategies in order to increase the efficacy of DNA vaccine with decreased amount of plasmid DNA.Novel formulation is one of the strategies to enhance the immunopotency of DNA vaccines. A new vaccine delivery system was selected and two kind of DNA vaccines were used as candidates in our experiments. At first, the plasmid DNA encoding HBsAg or FMD multiple epitope antigen (SG) was encapsulated by PLGA in order to form vaccine microparticles. It was confirmed that microparticles could be phagocytosed by APC. The phagocytosis process was simulated by the Raw 264.7 cells in vitro. After the cells have been incubated with particles for one hour, Giemsa agent was used to dye the cells. Many microparticles with no dye could be found in the cytoplasm. For the further showing of the phagocytotic process, the fluorescent dye was added to the plasmid solution in order to prepare fluorescent microparticles, which were added to cover slides seeded with Raw264.7 cells. Phagocytosis was observed directly and photographed using a digital camera linked to a fluorescence microscope. The fluorescent dots became confluent and larger with the prolongationof incubation time. These two phagocytosis experiments confirmed that the plasmid DNA encapsulated by PLGA could be taked up by APC directly. Whether the vaccine DNA could be released suitably and whether the target antigen could be expressed in the cytoplasm were the key points to the subsequent immune responses. The expression of target antigen was verified in vitro and in vivo. It was also found that the capsulation of PLGA could prolong the persistence of antigen in local tissue or draining lymph nodes. By this mechanism, DNA vaccine's immunopotency was enhanced. Moreover, the single oral administration HBV DNA vaccine encapsulated by PLGA could induce not only the cellular and humoral immune response, but also the mucosal immunity. In the research of FMD multiple epitope DNA vaccine, the expression of multiple epitope antigen(SG) was verified by immunocytochemistry. The results add support to the conclusion that the PLGA capsulation helps the vaccine target APC and prolong the persistence of DNA in vivo and in vitro, which facilitates the antigen presentation to Thl cells in draining lymph nodes. The encapsulated FMD DNA vaccine produced a broad spectrum of immune response. Meanwhile, the new formulation elicited the desired immune response with less amount of plasmid DNA and less injection times compared with naked plasmid DNA. So this kind of immune method against FMDV is more convenient, safer and cheaper.Annexin Bl gene was found by immune screening from cDNA library of Cysticercus cellulosae. The annexin Bl protein can be used as an antigen to diagnose Cysticercus cellulosae disease, and is a protective antigen. The DNA vaccine encoding annexin Bl could protect the pigs against Taenia solium cysticercosis. As far as a vaccine antigen with high immunogenicity was concerned, analyzing the structure and learning the bioactivity are the basis of using vaccine antigen safely. According to the data from the homology modeling three-dimensional structure of annexin Bl, we constructed three domain-deleted mutants in order to study the bioactivity of annexin Bl and membrane binding properties of its domains. The phosphatidylserine(PS) binding properties of wild annexin Bl and three domain-deleted mutants were confirmed in vivo and in vitro. PS exposure at thecellular surface is one of the earliest detectable molecular events in apoptosis and the main event in blood coagulation initiation. So the PS binding bioactivities of annexin Bl made it be used as anticoagulant protein to facilitate the migration of Cysticercus cellulosae in human body. The physiological and pathological roles of annexin Bl were elucidated. In reality, annexin Bl can be developed to be anticoagulant and imaging agent for apoptotic cells.The illumination of PS binding property of annexin Bl contributed to the understanding of annexin Bl possessing the adjuvant function. It was suggested that annexin Bl binding to the PS molecular on the surface of early apoptotic APC could protect the transfected APC from the phagocytosis which depended the PS as the apoptotic singal. It was assumed that the annexin Bl played adjuvant role by prolonging the life span of tranfected APCs and the expression of antigen in local tissue and draining lymph nodes.On the whole, this study developed a series of experiments surrounding the topic how to enhance the immunopotency of DNA vaccines. A novel delivery system, PLGA microparticle system, was confirmed to be effective on improving the immunogenicity of encapsulated DNA vaccines. The PS binding property of annexin Bl was observed on active platelets, apoptotic cells and PS lipid beads. The mechanism of annexin Bl as a novel adjuvant could be explained by blocking the PS on surface of early apoptotic APC.