Research On Therapeutic Vaccines Of Hepatitis B

Current antigen-based vaccines have been widely used by inducing effective humoral immunity. The major shortcoming of these vaccines is the disability to induce cellular immunity, which is essential for the prophylaxis and treatment of the disease. DNA vaccine has been well-documented to elicit durable humoral and cell-mediated immunity including CTL and cytokines in rodents and primates. It is a good candidate for immunization of non-responders of recombinant HBsAg vaccines and for therapeutic vaccination.The envelope gene of HBV was amplified by PCR from HBV genome (adr subtype of Chinese epidemic strain) and cloned into plasmid vector. Three copies of CpG motif, immunostimulatory sequence were also constructed into pVAX1. Optimal conditions for purification of supercoiled plasmid DNA in a large scale of semi-works production were identified. Several procedures involving alkaline lysis, ion exchange chromatography and gel chromatography were performed to separate plasmid isoforms from each other and from other host cell contaminants, including RNA, genomic DNA, protein, and endotoxins. Finally, the purified supercoiled plasmid DNA was suitable for use in DNA vaccine applications, according to Points to Consider on Plasmid DNA Vaccine set by FDA.The immunogecity of HBV DNA vaccine were tested in both normal mice and HBV-Tg mice. HBsAg was detected in serum and bilateral tibialis anterior muscle of normal mice post-immunization. HBsAg-specific humoral and cellular immune response was efficiently elicited. The therapeutic value of DNA vaccine was demonstrated in HBV-Tg mice injected with HBV DNA vaccine, resulting in the clearance of HBsAg from 2 out of 5 HBV-Tg mice and the HBsAg level decreased in the other mice. Anti-HBs was detected in all HBV-Tg mice at 4 weeks after vaccination. The results indicated that DNA-based vaccine was capable to break the immune tolerance of HBV-Tg and would be a promising therapeutic choice for the treatment of HBV carriers.However, some phase 1 clinical trials of HBV DNA vaccine and other DNA vaccines have demonstrated that the magnitude of immune responses induced in human is generally lower than that in small animals, and the amount of DNA required for effective immunization is much larger. Therefore, the potency of DNA vaccines must be increased to enable this technology for successful human application.Several reports have indicated that combinatorial regimens with DNA and protein vaccines canelicit both strong immune responses, to circumvent the limits of each vaccine. Surprisingly little was known on HBV vaccine. Here, we investigated the immunization effects of several regimens involving different components, different orders and different times of vaccination in BALB/c mice. The regimen involving twice priming pVAX(S) encoding HBsAg and once rHBsAg boosting, induced strong and homogenous antibody responses. The regimen induced significant stronger responses of IL-12 and IFN-y in splenocytes and elicited stronger CD8+ CTL responses than that elicited by immunization with rHBsAg or pVAX(S) alone. The efficacy of immunoprotection against the challenge of HBsAg-expressing tumor was also examined. A greater immunopretectional efficacy was found in group of mice immunized with rHBsAg after twice pVAX(S) priming. Our regimen may thus provide a strategy for developing an improved immunization against HBV and many other pathogens.Plasmid DNA encoding HBV envelope antigen was encapsulated into or absorbed onto PLGA and generated highly uniform microparticles. The immunogenicity of PLGA-DNA microparticles was demonstrated by significantly enhanced humoral and cellular immune responses in immunized mice, including higher levels of antibody, IFN^y and CTL activity, compared with that elicited by naked ' plasmid DNA. A greater efficacy of immunoprotection against the challenge of transplanted HBsAg-expressing tumor cells was also shown in the group of mice immunized with PLGA-DNA microparticles. Oral administration of PLGA-DNA microparticles induced a long-lasting and stable intestinal IgA and cellular immune responses in GALT of immunized mice. The feature of controlled release of PLGA-DNA microparticles was demonstrated in vitro and in vivo. Flow cytometry and confocal laser scanning microscopy were used to localize and quantitatively evaluate APCs involved in HBsAg expression. Much higher percentage of CDllc+ APCs involved in HBsAg expressing at 24 h and day 14 postinoculation, as well as prolonged transcription of plasmid DNA detected by RT-PCR, was demonstrated in the draining lymph nodes from mice immunized with PLGA-DNA microparticles, compared with that in mice vaccinated by naked DNA. The detection of controlled release in vitro and in vivo of PLGA-DNA microparticles provided direct evidence that the formulation not only directed the delivery of plasmid DNA to APCs, but also prolonged the existence of plasmid DNA expressing target antigen. The significant association between the feature of controlled release of microparticles and its augmented immunogenicity revealed some mechanism of immunopotentiation. The results may facilitate the development of a new type of protective HBV vaccine in humans.HBx is a multifunctional protein, participating in viral pathogenesis as a transactivator of viral...