The Study Of Anti-HBV Effects With A HBsAg And CTLA-4 ScFv Co-modified DC Vaccine

Hepatitis B virus(HBV)is the most common etiologic agent for infectious liver diseases.Following initial infection,T cellular immune response is the most important component of host defense mechanisms responsible for HBV clearance in patients,which is also associated with the liver pathological injury.Acutely infected patients characteristically produce a vigorous,polyclonal,and multispecific cytotoxic T lymphocytes(CTL)response that is usually sufficient to clear the HBV,while persistently infected patients produce a weak or undetectable HBV-specific CTL response.Thus,therapeutic enhancement of the T cell responses to HBV has the potential to terminate chronic HBV infection.Dendritic cells(DC)are the most potent antigen-presenting cells and play a central role in the induction of antiviral immune responses.The clearance of HBV dependents on the activation of HBV-specific T cellular responses,meanwhile,the establishment and extensity of HBV-specific T cellular responses are mostly based on the full function of DC.However,it has been shown that monocyte-derived DC (MoDC)from patients with chronic HBV infection is functionally impaired.On the other hand,during the process of the host DC presentated the HBV antigens and activated the viral specific T cells,the co-stimulation signal of B7 binding with CD28 was as important as the recognition signal of MHC/TCR and peptides.Meanwhile, CTLA-4 is commonly recognized as the co-inhibitory signal,which binds to B7 molecule that expressed on DC with 20- to 100-fold higher affinity than CD28.And many evidences demonstrated that the antigen specific T cell response was enhanced through the blockage of CTLA-4/B7 signaling.Therefore,by blocking the CTLA-4/B7 pathway with a single chain varable region(ScFv)of CTLA-4 antibody, combined with the improvement of DC function,would probably get the full enhancement of the anti-HBV T cellular responses,and thus clear the persistent HBV infection completely.HBV Transgenic mice(HBV-Tg)contain the complete HBV genome,express all HBV gene products,and replicate HBV in their hepatocytes similar to chronically infected patients,but they do not develop chronic hepatitis because they are immunologically tolerant to the viral Ags.The availability of these mice provides a model system to evaluate immunotherapeutic strategies to break tolerance and terminate persistent HBV infection.So far,it has been demonstrated that HBsAg- or peptide-pulsed DC can induce quantitatively normal HBsAg-specific CTL response, stronger than that induced by recombinant HBsAg vaccine or plasmid DNA immunization in HBV-Tg.However,they had no effect on viral replication and they did not cause hepatitis,suggesting that more efficient strategies must be developed to generate a functionally effective CTL response.Besides,our study group found at an early time that the HBsAg gene-modified DC more efficiently prime HBV-specific cytotoxic T lymphocytes than HBsAg-pulsed DC.But notably,the anti-HBs levels and the functional time of HBsAg gene-modified DC are still out of the expectation. So,more effective strategies that could induce much stronger and more persistent antiviraI T cellular responses,are need to be carried out.in this study,we constructed the ScFv of mouse CTLA-4,as well as the replication-defective recombinant adenovirus expressing HBsAg and ScFv.We studied the potential of DC transduced with this recombinant adenovirus both in vitro and in HBV mice,and investigated the possible mechanism of its high immunoreaction.The results we obtained may provide more information about specific gene-modified DC vaccine and lay ground for exploring more effective HBV immunotherapeutic ways in future.This study includes three parts. Partâ… Construction,purification and affinity examination of mouse CTLA-4 ScFv and fused HBsAg-ScFvObjectivesTo clone the ScFv of mouse CTLA-4,and to construct the eukaryotic expression vectors that expressing ScFv or fused HBsAg-ScFv(S-ScFv).After purification of the expressed ScFv and S-ScFv,the affinity activities of both were examined.MethodsFrom the mouse hybridoma cell line that subcloned from 4F 10,the VL and VH genes were amplified by reverse transcriptase polymerase chain reaction(RT-PCR)with family specific primer pairs,respectively.The PCR products were cloned into pGEM(?)-T easy vectors and sequenced.Primers were synthesized according to the sequence of VH_4F10,VLaFI0,four tandem glysines and one serine(G₄S)₃ linker and multiple cloning site(MCS)of pSectag2B vectors.ScFv_4F10gene was amplified through splicing by overlap extension(SOE),and the ScFV_4F10was then cloned into pSectag2B and pSectag2B-S vectors,respectively.The pSect2/ScFv_4F10and pSect2/S-ScFv_4F10were expressed in Chinese hamster ovary(CHO)cells,and the expressed protein could be observed through SDS-PAGE and Western blotting.After purification,the biological affinity of the expressed ScFv_4F10and S-ScFv_4F10were examined through the competitive ELISA and SPR assay.Results1.The ScFv_4F10of mouse CTLA-4 was successfully constructed,and the VL consists of 339 bps encoding a peptide of 113 amino acid residues,and VH contains of 345 bps encoding a peptide of 115 amino acid residues.According to online analysis by IMGT/V-QUEST,the VL_4F10and VH_4F10genes belong to mouse IG_κV and IGHV subgroups,respectively.On the position 23/88 of the light chain and 22/96 of the heavy chain genes,there are cysteine pairs,which play the key role in forming disulfo-bond between the two chains.Both VL_4F10and VH_4F10chain have 4 definite frame regions(FR)and 3 complementary determinant regions(CDR). 2.The eukaryotic expression vectors of pSect2/ScFv_4F10and pSect2/S-ScFv_4F10were successfully constructed,and the expressed protein were about 28 kDa and 52 kDa that verified by Western blotting.3.Though ultrafiltration concentration and affinity chromatography,the purified ScFv_4F10and S-ScFv_4F10could block the binding of 4F10-mAb with recombinant CTLA-4 antigen.The affinity constant of ScFv_4F10and S-ScFv_4F10with CTLA-4 antigen were 9.52×10⁶ M^-1and 2.04×10⁶ M^-1,respectively.Conclusions1.The ScFv_4F10of mouse CTLA-4 was successfully cloned.2.The eukaryotic expression vectors of pSect2/ScFv_4F10and pSect2/S-ScFv_4F10were successfully constructed.3.The expressed proteins were well purified and proved to have fairly high affinty with CTLA-4 antigen when tested in vitro.Partâ…¡Construction and examination of recombinant adenovirus vectors that expressing ScFv and S-ScFvObjectivesTo construct recombinant adenovirus vector expressing ScFv or S-ScFv,and examine the efficiency of transfection and the expression of target antigen.To explore the phenotypic characteristic and stimulation ability of these two adenovirus vectors transduced DC in vitro.MethodsFor preparation of the recombinant adenoviruses expressing ScFv_4F10and S-ScFv_4F10, the AdEasy^TMXL Adenovirus Vector System was used.These purified Ad plasmids (Ad-ScFv and Ad-S-ScFv)were further amplificated by infection in Ad-293 cells,and the virus particle titre were determined by Reed-Muench assay.The expression efficiency of GFP by Ad-GFP-transduced DC was detected by flow cytometry,and the expression of ScFv and S-ScFv was determined by Western blotting.DC were generated from bone marrow cells from the BALB/c(H-2^d)mice by culturing with IL-4,GM-CSF and TNF-αfor 7 days.The bone marrow-derived DCs were transfected with rAd and used for in vitro analysis.The phenotypic makers,antigen phagocytosis,and IL-12 production of rAd-tranduced DC were analyzed.The stimulatory capacity of DC was checked in allogeneic and autologous mixed leukocyte reaction(MLR).Intracellular cytokines of proliferative T cells in autologous MLR were analyzed by flow cytometry.Results1.PCR and sequence analysis proved the Ad-ScFv and Ad-S-ScFv plasmid contained the intact ScFv_4F10,S-ScFv_4F10gene sequence.The target protein expression of ScFv_4F10,S-ScFv_4F10was testified by Western blotting.2.The virus particle titre of Ad-ScFv,Ad-S-ScFv were determined by Reed-Muench assay with resulting 50%tissue culture infective doses(TCID 50/mL)of 10⁸/ml.3.The expression of GFP by Ad-GFP-transduced DC was 95.05±3.05(%),which was detected by flow cytometry as the efficiency oftransfection of rAd into DC at an MOI of 100.4.The expression of CD11c,DEC-205,MHCâ… ,MHCâ…¡,CD80,CD86,CD40 and CD54 on cytokine-activated murine bone marrow-derived DC was up-regulated, and either the rAd-tranduced DC or untreated DC expressed similar levels of those markers.The ability of OVA phagocytosis and IL-12 production were also similar between different rAd-tranduced DC and untreated DC.5.The stimulatory capacity of rAd-tranduced DC had no significantly difference from untreated DC in either allogeneic MLR or autologous MLR,respectively. With the rAd-transduced DC stimulation,typeâ… immune responses comprising CD3⁺CD8^- Th1 and CD3⁺CD8⁺ Tc1 in autologous MLR could be activated.The Ad-S-ScFv-tranduced DC had the highest IFN-_γ⁺ T cells,however,no significant difference of this stimulatory activity was found between other DC groups.Conclusions1.The recombinant adenovirus vectors which expressed ScFv_4F10or S-ScFv_4F10were successfully constructed.The transfection efficiency of recombinant adenovirus in marrow DC was high and the target proteins were normally expressed.2.The expression of activation markers,OVA phagocytosis and IL-12 production for the rAd-transfected DC were slightly affected.3.The stimulatory capacity of Ad-S-ScFv transduced DC on T lymphocytes proliferation in allogeneic and autologous was similar with other DC groups, while the induction of typeâ… immune responses(especially IFN-_γ⁺ T-cell production)by DC/Ad-S-ScFv was higher than other DC groups.Partâ…¢Study on the immune responses against HBV induced by Ad-S-ScFv -modified DC in HBV transgenie miceObjectivesTo analysis the immune responses against HBV induced by DC/Ad-S-ScFv in HBV transgenic mice(HBV-Tg),and to compare its antiviral effects with that induced by DC/Ad-S,DC/Ad-ScFv,anti-PD-L1 mAb or plasmid DNA immunization.To investigate the mechanism and evaluate the therapeutic potential of this Ad-S-ScFv transduced DC in chronic HBV infection.MethodsHBV-Tg mice were randomly assigned to receive either Ad-S-ScFv-transduced DC(n =10)or Ad-ScFv-transduced DC(n=10)or Ad-S-transduced DC(n=10)or anti-PD-L1 mAb(n = 9)or plasmid pcDNA3.1(+)-S(n = 9).For immunization with DC,50μl PBS containing 1×10⁶ DC was injected into the tail vein of each mouse.For immunization with mAb,200μl anti-PD-L1(1mg/ml)was injected into peritoneum. For DNA vaccination,mice were injected in the leg muscle with 50μl sucrose in PBS containing 100μg of plasmid DNA.The same immunization schedule was repeated twice at 3-week intervals.The IFN-_γ⁺ CD8⁺ T proportion of splenic T cells after immunization was detected by flow cytometry,and the production of IFN-_γ,IL-2 and IL-10 in the culture supernatant of splenocytes were determined by ELISA.HBsAg-specific T cellular proliferation and cytotoxic activity of splenocytes were measured with CFSE incorporation assay,and the HBsAg and anti-HBs titers in sera of the HBV-Tg mice were tested by ELISA.Quantitative PCR was performed to detect HBV DNA in sera of HBV-Tg mice.Hepatocellular injury was monitored at various time points after immunization by measuring serum ALT levels.Some tissue samples of the liver,lung, kidney,heart,stomach and intestine were stained with hematoxylin and eosin for histological analysis,and the expression of Hepatitis B core Ag(HBcAg)and HBsAg in tissue samples of liver was also assessed by immunohistochemical analysis.The expression and phosphorlation level for the key components of the intracellular signal pathway of MAPK and PI3K/Akt were detected through Western blotting.Results1.At 2 week after immunization,DC/Ad-S-ScFv more efficiently stimulated splenic IFN-_γ⁺ CD8⁺T cells than DC/Ad-S,DC/Ad-ScFv or pcDNA3.1-S,anti-PD-L1 (p＜0.01 or p＜0.05).Meanwhile,DC/Ad-S-ScFv induced a stronger splenic CTLs than that induced by other groups(p all＜0.01 at different Eeffetor:Target ratio). While compared to other groups but not DC/Ad-S-ScFv,DC/Ad-S induced a higher splenic Tc1 response and HBsAg-specific CTLs(p all＜0.05).2.At 4 week after immunization,compared to the other groups,DC/Ad-S-ScFv still more efficiently stimulated splenic IFN-_γ⁺ CD8⁺T cells and HBsAg-specific CTLs(p＜0.01 or p＜0.05).Meanwhile,when compared DC/Ad-S to other groups but not DC/Ad-S-ScFv,there is no significant difference either for splenic IFN-_γ⁺ CD8⁺ T-cell proportion,or for splenic HBsAg-specific CTLs.3.The IFN-_γ⁺ CD8 T-cell and CTL response elicited by DC/Ad-S-ScFv persisted for at least 4 weeks,and then gradually decreased thereafter.Meanwhile,this stimulatory activity induced by DC/Ad-S was transient,being strongest at 2 weeks after immunization and decreased to a low level at 4 weeks.And at 8 weeks after immunization,the IFN-_γ⁺ CD8 T-cell and CTL response elicited by DC/Ad-S-ScFv were both very low,but still higher than that elicited by DC/Ad-S (p both＜0.05). 4.The suppression of serum HBsAg induced by DC/Ad-S-ScFv was the strongest in all groups,which was persistent for at least 4 weeks(the inhibitory percentage for the 2^th,4^thand 8^thweek were 29.69%,38.35%and 17.76%,respectively).This effect of Ad-S-transduced DC weakened gradually from 3 to 4 weeks after immunization,and the suppression of serum HBsAg induced by plasmid DNA was weaker but more stable in 4 weeks(rang from 12.05%to 14.35%).5.The level of serum HBV DNA was the lowest in all groups,from 1 to 4 weeks alter DC/Ad-S-ScFv immunization(p＜0.05 or p＜0.01).Moreover,the levels of serum HBV DNA by all DC groups or anti-PD-L1,DNA immunization were rebounded a lot at 8 weeks after immunization.6.The anti-HBs response elicited by DC/Ad-S-ScFv and pcDNA3.1(+)-S arrived at 24.55±6.25(mIU/ml)and 38.46±7.88(mIU/ml)at 2 weeks after immunization, while this anti-HBs response was weak in other groups.Meanwhile,all groups not achieving 10 mIU/ml at 4 weeks except the pcDNA3.1(+)-S immunization.7.The serum ALT activity was slightly elevated at 1,2,or 4 weeks after immunization,and histological analysis of the liver displayed only focal inflammatory infiltration in the hepatic parenchyma from the Tg mice that immunized with DC/Ad-S-ScFv or DC/Ad-S.However,the liver inflammation of Tg mice in other groups was not marked.Meanwhile,DC/Ad-S-ScFv immunization did not induce the autoimmunological inflammation in the tissue of heart,stomach,intestine,kidney and lung.8.From 2 to 8 weeks after immunization,HBcAg was inferior positive in 8 of 10 tissue samples of DC/Ad-S-ScFv immunization,and HBcAg was inferior positive in 6 of 10 tissue samples of DC/Ad-S immunization.Meanwhile,inferior positive ratio of HBcAg in DC/Ad-ScFv,pcDNA3.1(+)-S,DC and anti-PD-L1 immunization were 3/10,3/9,1/3 and 1/9,respectively.9.From 2 to 8 weeks after immunization,the ratio of HBsAg inferior positive was 7/10 in DC/Ad-S-ScFv immunization,while the inferior positive ratio of HBsAg in DC/Ad-S,DC/Ad-ScFv,pcDNA3.1(+)-S and DC were 5/10,2/10,3/9 and 1/3, respectively. 10.Compared to other DC groups,the phosphorlation expression level of Erk1/2 in the liver of DC/Ad-S-ScFv immunized Tg mice was significantly increased.While there is no significant difference for the phosphorlation expression of the PI3K/Akt signal protein in all DC groups.Conclusions1.Compared with either DC/Ad-S or DNA immunization,DC/Ad-S-ScFv can induce much stronger typeâ… immune responses and HBV-specific CTLs,and more significantly reduce the titer of serum HBsAg and HBV DNA,and also reduced the expression of HBcAg and HBsAg in the liver of HBV-Tg mice. DC/Ad-S-ScFv did not induce the autoimmunological inflammation in the tissues of immunized Tg mice.2.The increased phosphorlation expression of Erk1/2 in the liver tissue of DC/Ad-S-ScFv immunized mice,suggested that probably enhanced the activation of TCR related intracellular signal transduction pathway.3.The humoral response to HBsAg induced by Ad-S-ScFv transduced DC was still weak,but was stronger than that induced by Ad-S-transduced DC at 2 weeks after immunization.4.The efficient antiviral effects triggered by Ad-S-ScFv transduced DC may persist at least 4 weeks,and be superior to that elicited by Ad-S transduced DC or plasmid DNA after immunization,though the effects were significantly deceased at 8 weeks after immunization.5.Thus,Ad-S-ScFv transduced DC may be a promising candidate for a CTL-based vaccine for chronic HBV infection,and the combination with the strategy of PD-1/PD-L1 pathway blocking,could probably enhance the antiviral effects of this DC vaccine more efficiently.