IL-2/IFN-γ Fusion Protein Gene Plasmid And In Vivo Electroporation Enhanced Potency Of Therapeutic HBV DNA Vaccine

Strategies to boost or to broaden the weak virus-specific T-cell response of patients with chronic hepatitis B have been proposed as a means of curing this persistent infection.HBV envelope-and nucleocapsid-based vaccines,new formulations for recombinant vaccines and DNA-based vaccines are currently being assessed in clinical trials,among which DNA vaccine represents a promising immunotherapeutic approach that can induce T-cell mediated antigen specific immunity,owing to its de novo intracellular antigenic protein expression and synthesis.In clinical trials,although HBV DNA vaccination developed protective antibody responses and antigen-specific CD8 T cells in healthy hepatitis-naive human volunteers,the detectable HBV-specific IFN-γsecreting T cells and decreased serum HBV DNA levels only in some chronic HBV carriers vaccinated with HBV PreS2/S DNA vaccine were limited.One resolution for the main obstacles of the new technique development is to enhance the transfection efficiency of plasmids into host cellls;the other is to improve the immunogenicity of DNA vaccine by driving the na(i|¨)ve T cell responses towards the Th1 profile.To tackle the first problem of low transfection rate of DNA vaccine in this investigation,we have applied the in vivo electroporation(EP) for potency enhancement of HBV DNA vaccine,which dramatically improved the host cell transfection of the plasmids and enabled the DNA vaccine we prepared to elicit both humoral and cellular immune responses in the large body weight animals like rabbit and nonhuman primates.In order to achieve the second goal of immunogenicity improvement of HBV DNA vaccine for its therapeutic usage,we had designed and constructed the Th1 type cytokines (interleukin-2 and interferon-γ) fusion protein expression gene plasmids(pFP),in attempt to direct Th1 bias in favor of cellular immunity augment when being used in combination with HBV DNA vaccine.Therefore,in this study we had carried out the in vitro assay of the plasmid cytokine fusion protein and in vivo investigation of different HBV DNA vaccination regimens with pFP and EP either in combination or separately in various animal models of healthy mice,Newzealand rabbit,nonhuman primates and HBV transgenic (Tg) mice,tending to get relatively complete adjuvant efficacy evaluation of the plasmid(pFP) and EP for therapeutic HBV DNA vaccine.Chapter 1 Preparation and identification of dual plasmids HBV DNA vaccine and in vivo electroporation apparatusDNA vaccine seemed to be a promising anti-viral strategy for treatment of chronic hepatitis B virus(HBV) infection,due to its capacity of eliciting both humoral and cellular immunity.As it being used as a therapeutic vaccine however,the arm of cellular immunity of DNA vaccine should be stressed,which prompted us to construct an eukaryotic expression plasmid encoding IL-2 and IFN-γfusion protein (pFP) to form a dual plasmids therapeutic DNA vaccine in addition to HBV envelope middle protein encoding gene plasmid(pS2.S).The molecular modeling based upon the gene sequence of the IL-2/IFN-γfusion protein demonstrated the correct construct of pFP releasing each one of the cytokines or their bioactive domain separately,which had been confirmed by ELISA and bioassay for detection of the presence and biological activity of either IL-2 or IFN-γin pFP transfected cell culture supernatant.In BALB/c mice experiment(8 mice in each group),the luciferase activity (16136.3±9588.7RLU) elicited by in vivo electroporation(EP)-mediated report firefly lucferase gene plasmid(pLUC) transfection into mouse tibialis anterior(TA) muscle, was 4 digits higher than that(8.0±8.0RLU) of the non-EP control,which difference possessing very significance(P＜0.005).After EP-mediated green fluorescent protein gene(pGFP) transfection,extensive GFP expression revealed by strong fluorescence was observed in intact pre-existing muscle fibers within 3-5 days.But they were mostly seen at the peripheral areas surrounding the electroporation center.The GFP fluorescence in the peripheral areas quickly diminished with time and become barely visible after 14 days.At this time,however,some cells in the central areas became fluorescent,and they could be identified as centrally nucleated regenerated muscle fibers in H&E stained serial section.Chapter 2 pFP enhanced potency of HBV DNA vaccinationThe objective of this study is to investigate the aduvant effects of pFP and plasmids for IL-2/IFN-γcytokines either separately or in combination on HBV DNA vaccine(pS2.S) induced humoral and cellular immune responses.Healthy BALB/c mice were recruited in 3 experimenatal series:(1) Intramuscular injection of pFP(10μg/mouse) was combined with pS2.S(10μg/mouse) in 4 different ways,i.e,co-injection with pS2.S simutaneously(pS2.S+pFP), pre-injection 3 days before pS2.S(pFP+3d+pS2.S),post-injection 3 days later (pS2.S+3d+pFP) and separate injection in left and right leg(pS2.S/TA+pFP/TA);(2) Different doses were co-injected with the fixed dose(10μg/mouse) of pS2.S.(3) While fixing the dose ratio of pS2.S/pFP at 1:1,the low(L,5/5μg/mouse),middle (M,10/10μg/mouse) and high(H,50/50μg/mouse) dosages were chosen for co-injection..The results showed that the best immune respose was achieved in the group of co-injection of pS2.S+pFP at dose ratio of 1:1.Based on this method,the positive rate of serum anti-HBs as 100%was found at the 4th week post-prime immunization in 3 dosage groups,while serum anti-HBs level in group L was significantly lower than that of group M or H respectively(P＜0.05).The cell count results showed that the number of monocytes or macrophages(MPs) and its percentage in local draining lymphnode in the group of pS2.S+pFP was remarkably higher than that of the control(pS2.S+pcDNA3.1).The results suggest that co-injection of pFP with pS2.S at dose ratio of 1:1,can achieve the best effect of humoral immunity,as pFP may enhance proliferation and activity of MPs infiltrating the site of injection,thus facilitating antigen presentation.The adjuvant efficacy of plasmids encoding murine IL-2 or IFN-γ(pmIL-2 or pmIFN-γrespectively) had been investigated individually or in combination for HBV DNA vaccine potency enhancement.A strong synergistic adjuvant efficacy for DNA vaccination,similar to that achieved in present study by pFP of human origin, was reached by taking the two plasmid cytokines together(pmIL-2+pmIFN-γ) rather than each of them separately.However,we failed to achieve any synergism of the antigen-specific immune response by co-injection of murine IL-2/IFN-γfusion protein expression plasmid(pmFP) with HBV DNA vaccine,suggesting that the same peptide linker(8 lysine peptide) for the human cytokines of IL-2 and IFN-γin the form of fusion protein that keeps each bioactive domain of the two molecules apart,is inappropriate for the plasmid construct encoding the IL-2/IFN-γfusion protein of murine origin.Chapter 3 EP enhanced potency of HBV DNA vaccinationIn this study,we apply in vivo electroporation(EP) for HBV DNA vaccine administration to improve the cell transfection rate of plasmid DNA and enhance the immune response.In New Zealand rabbit and nonhuman primates(monkeys) experiment,none of the animals in the groups of HBV DNA vaccination without EP appeared to be positive for serum anti-HBs immune response,even at a very high dose(1000+1000μg/rabbit or monkey) of the plasmids used during the whole observation period.In New Zealand rabbit experiment with EP,the number of rabbits with positive serum anti-HBs in the large dose(50+50μg/rabbit) group at the 2nd,4th week after immunization,were 3 out of 5 rabbits in thegroup;at the 13th week,the serum anti-HBs positive number in the large,middle(25+25μg/rabbit) and small dose(5+5μg/rabbit) group is 5,4 and 4 respectively.In Rhesus monkey experiment(3 monkeys in each group),there are 3 and 2 monkeys whose serum anti-HBs became positive at the 8th week after immunization in both the large(1000+1000μg/monkey) and middle(500+500μg/monkey) dose EP group respectively;at the 13th week,the serum anti-HBs of the whole 3 monkeys became positive in all the 3 dose groups with EP.In cancrivorous monkey experiment group with EP-mediated HBV DNA vaccination,the HBsAg specific IFN-γsecreting cells in large,middle and lower dose groups(range from 10 to 67 SFCs/3×10~5PBMCs) was much higher than its simultaneous non-HBsAg in vitro stimulation control wells(range from 0 to 9 SFCs /3×10~5pBMCs)(P＜0.05) and were much higher in comparison with that in the adjuvant and PBS groups(range from 0 to 12 SFCs/3×10~5PBMCs).To explore the potential of electroporation mediated HBV DNA vaccination for the treatment of chronic HBV infection,BALB/c mice were vaccinated with HBV DNA vaccine(pS2.S) mediated with or without EP prior to hydrodynamic injection of HBV surface antigen expression plasmid to mimic HBV infection.The results showed that the elicited immune responses by EP-mediated HBV DNA vaccine can indeed reduce the expression of HBsAg in hepatocytes of mice model with pS2.S hydrodynamic liver transfection to express HBsAg.The antigen clearance process didn't cause significant toxicity to liver tissue,suggesting a non-cytolytic mechanism is important in the anti-viral immune response elicited by DNA vaccination and that EP-aided DNA vaccination may have a therapeutic potential for chronic Hepatitis B patients.Chapter 4 Evaluation of pFP and EP enhanced potency of HBV DNA vaccination in both healthy and HBV transgenic mouse modelThe adjuvant efficacy of pFP for HBV DNA vaccination was evaluated in healthy mouse model.In both EP and pEP-mediated DNA vaccination,the DNA vaccine induced HBsAg-specific immune responses,both the humoral and cellular one by assessment of serum anti-HBs level or spleen IFN-γT cell counts and its corresponding positive response rate,in the groups co-injected with pFP were significantly stronger than that in the controls with pcDNA3.1.On the other hand,in both pFP and pcDNA3.1 assisted DNA vaccination,HBV DNA vaccine induced serum anti-HBs responses in the regimen groups mediated by EP were significantly stronger than those in the control groups by pEP,while the spleen IFN-γT cell responses were also improved,but with no statistic significance between the two regimens with or without EP.Although EP-mediated DNA vaccination had elicited much higher serum anti-HBs positivity in the group of pS2.S+pcDNA3.1+EP than that in the pFP co-injected group of pS2.S+pFP+pEP,the spleen IFN-γT cell response comparison between the two groups revealed no significant difference.By fixation the regimen of DNA vaccination(pS2.S+pFP+EP),the dose-effect evaluation demonstrated that pFP exerts its adjuvant efficacy in a dose-dependent manner,thus recommending us a more appropriate dose for therapeutic efficacy evaluation in HBV transgenic(Tg) mouse model.In HBV Tg mouse model,pFP could effectively enhance the therapeutic efficacy of HBV DNA vaccine by persistently suppressing the HBV DNA replication and expression till the endpoint of our observation when the more profound suppressive effects were accompanied by the favorable INF-γT cell responses and serum ALT elevation.In summary,the results from above investigation on pFP and EP enhanced potency of therapeutic HBV DNA vaccination suggest that the plasmid encoding IL-2/IFN-γfusion protein enhances the potency of HBV DNA vaccine and more favorably bias na(i|¨)ve T cells towards Th1 differentiation in comparison with EP.EP on the other hand strongly promotes the host cell transfection of the plasmids,thus probably providing one of the tactics to tackle the hurdle of very low potency of DNA vaccination in large body weight animal models encountered in currently investigation of therapeutic gene therapy.The results also elucidate that IL-2 and IFN-γ,as the Th1-type cytokines in the form of a fusion protein expression plasmid can enhance the potency of a HBV DNA vaccine,implicating the probability of this special formulation as a therapeutic vaccine for immunomodulatory treatment of chronic HBV infection.