ABSTRACT
Objective:To study the different effect of fused gene IL-2/Fc administrated at different time on immune responses induced by HBV preS_2S genetic vaccine.Methods:BALB/c mice were immunized with purified recombinant plasmids at 0, 2, 4 weeks. Two groups were designed. One group were injected with pcDNA3.1S_2S at first, then pcDNA3.1IL-2/Fc was administrated after three days. While the other group were injected two plasmids at the same time. We compared the immune responses through detecting the anti-HBs titers, CTL cytotoxicity level, proliferation of lymphocytes and cytokines levels secreted by cultural splenocytes.Results:The data demonstrated that the humoral and cellular immune responses induced by pcDNA3.1IL-2/Fc as adjuvant and administrated after HBV preS_2S DNA vaccine 3 days mice were dominant higher than other groups.Conclusion:Genetic adjuvant administrated after Ag priming can enhance its effect.
ABSTRACT
Objective:To develop an effective therapeutic double HBV DNA vaccine with two eukaryotic expressing motif,namely pS2.S and pIIF,a fusion ORF of hIL-2/hIFN-?.Methods:Two genes were amplified,which separately encoded HBV preS2.S middle envelope protein as a vaccine and human IL-2/IFN-? fusion protein as an adjuvant by PCR technique from plasmids pcDNA3.1/preS2.S and pcDNA3.1/hIL-2/IFN-?.Then the genes subcloned into eukaryotic vector pVAX1.The new plasmid was analysed by restriction endonuclease and DNA sequencing.The constructed plasmids were transfected into COS-7 cells in vitro with LipofectamineTM 2000.The expressing products in supernatants were quantified by ELISA.Space structure of fusion hIL-2/IFN-? expressed by the adjuvant plasmid was simulated by CE software.The double plasmid association were injected into BALB/c mice by in situ electroporation method,and specific humoral and cellular immune responses were examined.Results:The gene segments and inserting direction of pVAX1-S2.S(pS2.S)and pVAX1-IL-2IFN-?(pIIF)were both correct by genetic analysises.At 48 h after transfection,HBsAg and the cytokines of IL-2 and IFN-? were respectively 45.1 ng/ml,10.03 ng/ml and 11.5 ng/ml by ELISA.The activity domains of the two cycokines in fusion protein were entirely exposed on surface by CE software.Experiments were pefrormed in mice by injection of plasmid pS2.S.Protective antibodies of anti-HBs were produced in terms of dose-dependment pattern for the efficacy.Co-injection of plasmid pS2.S together with adjuvant pIIF resulted in more evident immune responses and dose of the plasmid pS2.S needed was diminished.There was strongly specific humoral and cell immunity by pS2.S and pIIF co-injection with EP technique.Conclusion:The double plasmids pS2.S and pIIF of anti-HBV are constructed succeedly and induce strongly specific immune activity in vitro and vivo.
ABSTRACT
In this paper, we reviewed the progress in the study of the therapeutic HBV DNA vaccine, particularly on the evidences for its therapeutic effect, its mechanism of action, and to compare it with the traditional vaccines, etc. We also presented our research results on the therapeutic HBV DNA vaccine, and evaluated its clinical effect and characteristics objectively.
ABSTRACT
To develop an effective therapeutic HBV DNA vaccine, two eukaryotic expressing plasmids, namely pcDNAS 2 ?S and pcDNAIIF , which respectively encoding HBV middle envelope protein preS 2 ?S and human IL 2/IFN ? fusion protein, were constructed by DNA recombinant technique. After identification by restriction analysis and DNA sequencing, the constructed recombinant plasmids were transfected into COS 7 cells in vitro with transfection reagent Lipofectamine. The expressed product in supernatant was quantified by ELISA , and the biologic activities of IL 2 and IFN ? were assayed by CTLL 2 cell line proliferation and cytopathogenic inhibition, respectively. The results showed that secretive expression of preS 2 ?S and hIL 2/hIFN ? fusion protein peaked at 48h after transfection, and the expression levels were HBsAg(P/N)=7 63, IL 2=10 35ng/ml, and IFN ?=7 90ng/ml. The IL 2 and IFN ? activities in the culture supernatant of COS 7 cells transfected with pcDNAIIF were 998U/ml and 249U/ml, respectively. These results suggested that the recombinant plasmids pcDNAS 2 ?S and pcDNAIIF were correctly constructed and the transfected cells could express secretive active protein.
ABSTRACT
The peptides from HBV cytotoxic T lymphocyte(CTL) epitope were used to either stimulate or impact the immune effector (E) or CTL target (T) cells respectively, in order to investigate the cellular immune response induced by DNA vaccination in both healthy and HBV transgenic (Tg) mice. It was found that HBV DNA based immunization could induce CTL activity in healthy BALB/c mice, with intensity correlated with the E/T ratio and IFN ? secretion level in its supernatants. The target cells hit by HBsAg CTL epitope peptide(pp20) could be lysed by the active CTL induced by HBV DNA vaccine encoding preS2 and HBsAg, while the cell lysis could not be observed in the target cells impacted by the HBcAg CTL epitope peptide (pp10). The supernatant IL 12 secretion level (211 3?39 8pg?ml -1 ) in the DNA vaccination group was significantly( P