Vaccination With A Fusion DNA Vaccine Encoding Hepatitis B Surface Antigen Fused To The Extracellular Domain Of CTLA-4 Enhances HBV-specific Immune Responses In Mice

HBV infection is one of the most common health problems in the world,which can further cause liver cirrhosis and hepatocellular carcinoma in the chronic HBV carriers.Up to date,the treatment for chronic hepatitis B is limited,basically including type-1 interferons and nucleosides analogues,which exhibit limited efficacy in clearane of HBV cccDNA and considerable side effects in human.Therefore it is necessary to develop an alternative,effective therapeutic approach for the chronic infection patients.Persistent HBV infection is associated with HBV-specific immune hyporesponsiveness or tolerance,although the precise mechanism for this phenomenon is not entirely clear.Inductions of vigorous,polyclonal,and multispecific immune responses are crucial to the treatment of chronic HBV infection.To date,the studies on therapeutic vaccines have shown DNA vaccine has great potential in the prevention and treatment of hepatitis B virus(HBV)infection,but its potency is limited, especially in large animals and mankind,because only a limited fraction injectied DNA are taken up by antigen-presenting cells(APC).Targeting the specific antigen to the surfaces of APCs,which is able to broad enhancement of antigen-specific CD4+ helper,CD8+ cytotoxic T-cell,and B-cell responses,is a very effective way to enhance the immunogenicity of DNA vaccines.CTLA-4 is mainly expressed on the surface of activated T cells,which binds to B7 molecule with 20- to 50-fold higher affinity than CD28.Fusion of specific antigens to extracellular domain of cytotoxic T-lymphocyte associated antigen 4 (CTLA-4)represents a promising approach to increase the immunogenicity of DNA vaccines.In this study,we constructed a fusion plasmid by linking the extracellular domain of CTLA-4 to HBsAg,and evaluated its enhancement on HBsAg-specific immune responses in both BALB/c and HBV transgenic mice.Furthermore,its antiviral effects in transgenic mice were also detected,and the possible mechanism of this genetic regimen for its high immunogenicity was investigated.The present study evaluated the potential of the CTLA4-based DNA vaccine as an immunotherapeutic vaccine and may provide a novel stratege in the prevention and treatment of HBV infection.This study includes four parts.Partâ… Construction and characterization of a recombinant fusion plamid encoding HBsAg fused to the extracellular domain of CTLA-4Objective:An eukaryotic expression fusion plasmid encoding HBsAg fused to the extracellular domain of CTLA4 was constructed and its target protein was examined in the transfected cells.Methods:DNA fragment encoding extracellular domain of mouse CTLA-4 and HBsAg was amplified by RT-PCR and PCR,respectively.The two PCR fragments were inserted into pSecTagB vector respectively and generated the recombinant pCTLA and pS plasmids.To construct the CTLA4-HBs fusion plasmid, DNA fragment of HBsAg was placed downstream of the extracellular domain of CTLA-4 gene in frame of pCTLA,named pCTLA-S.The recombinant plasmids were transfected to Cos-7 cells by lipofection.The transcription and expression levels of the inserted target genes were detected by RT-PCR and radioimmunological assay(RIA), respectively.Results:Sequence analyses demonstrated that the inserted DNA sequences in the recombinant plasmids(pCTLA,pS and pCTLA-S)are correct. The target mRNAs can be detected in the transfected Cos-7 by RT-PCR.The expressions of HBsAg were positive in both intracellular and extracellular portions of the transfected Cos-7 with pCTLA-S and pS.Conclusions:The eukaryotic expression fusion plasmid encoding HBsAg fused to the extracellular domain of CTLA4 (pCTLA-S)and the control recombinant plasmids(pCTLA and pS)were constructed successfully.The target antigen was expressed effectively in the pCTLA-S-transfected cells.Partâ…¡Enhancement of HBsAg-specific immune responses by vaccination with a fusion DNA vaccine encoding hepatitis B surface antigen fused to the extracellular domain of CTLA-4 in BALB/c miceObjective:The HBsAg-specific immune responses were investigated in BALB/c mice after vaccination with the fusion DNA vaccine encoding hepatitis B surface antigen fused to the extracellular domain of CTLA4.Methods:BALB/c mice were vaccinated with the recombinant plasmids(pCTLA-S and several control plasmids pCTLA,pS and pSecTagB)intramuscularly.At different time points before and after vaccination,blood was collected from mice by retrobulbar puncture. Anti-HBs titers were quantitated by ELISA.The HBsAg-specific cytotoxic activity of splenocytes was measured with LDH release assay,HBsAg-specific T cell proliferation were detected by MTT assay,the percentage of HBsAg-specific CD⁸⁺ IFN-γ⁺ T cells were detected by flow cytometry.Both the HBsAg-specific IgG subclasses and the concentrations of IFN-γand IL-4 in the culture supematant of splenocytes were determined by ELISA.Results:pCTLA-S plasmids elicited significantly titers of anti-HBs at the beginning of 2 weeks and reach their highest levels at 8 weeks after immunization.The higest antibody titer in pCTLA-S vaccinated mice is 7123mIU/ml,while 261mIU/ml in pS group.Thus,compared with mice vaccinated with pS,those receiving the fusion plasmid pCTLA-S resulted in much higher level of anti-HBs antibody with about several hundreds-fold enhancement(P＜0.001).Both HBsAg-specific T cell proliferation and CTL response were enhanced significantly in pCTLA-S vaccinated mice as compared with the pS group(CTL activity,E/T=80:38.1±5.4%vs 25.4±3.7%,P＜0.05). Much more proportions of HBsAg-specific CD8⁺ IFN-γ⁺ T cells were induced in the mice vaccinated with pCTLA-S than those vaccinated with pS(7.27±1.29%vs 2.76±0.6%,P＜0.01).Moreover,compared with pS,pCTLA-S enhanced both HBsAg-specific IgG2a(Th1)and IgG1(Th2)antibody responses,and induced both HBsAg-specific IFN-γand IL-4 releases.(pCTLA-S vs pS,IFN-γ:369.7±117.8pg/ml vs 101.7±31.9pg/ml,IL-4:94.2±15.6pg/ml vs 42.0±10.2pg/ml,both P＜0.01).Conclusion:Vaccination with pCTLA-S plasmid induced not only HBsAg-specific antibody response but also specifc cellular response,including the enhancements of CTL activity,Tcl response,and Th immune response,indicating this fusion DNA vaccine might be used as a therapeutic vaccine in the control of HBV infection. Partâ…¢Induction of HBsAg-specific immune responses by vaccination with a fusion DNA vaccine encoding hepatitis B surface antigen fused to the extracellular domain of CTLA-4 in HBV transgenic mice and its anti-HBV effectsObjective:The induction of HBsAg-specific immune responses and antiviral effects were investigated in HBV transgenic mice after vaccination with the fusion plasmid pCTLA-S,and its potential use as a therapeutic vaccine in persistent HBV infection was also evaluated.Methods:HBV-Tg mice were vaccinated with the recombinant plasmids(pCTLA-S and several control plasmids pS and pSecTagB) intramuscularly.At different time points before and after vaccination,blood was collected from mice by retrobulbar puncture.HBsAg and anti-HBs titers were quantitated by RIA and ELISA,respectively.HBV DNA in the sera of Tg mice was detected by a real-time fluorescent quantitative PCR.HBsAg-specific cytotoxic activity of splenocytes was measured with LDH release assay,the percentage of HBsAg-specific CD8⁺IFN-γ⁺ T cells were detected by flow cytometry. HBsAg-specific T cell proliferation was detected by MTT assay,and the concentrations of IFN-γand IL-4 in the culture supematant of splenocytes were determined by ELISA.Hepatocellular injury was monitored at different time points after immunization by measuring serum alanine aminotransferase(ALT)activity. Tissue samples of the liver were stained with hematoxylin and eosin for histological analysis.The expression of HBsAg in tissue samples was assessed by immunohistochemical analysis.Results:pCTLA-S vaccination induced more powerful inhibition of HBsAg expression and HBV DNA replication than vaccination with pS,especially at 8,12 and 16 weeks after immunization for both HBV DNA and HBsAg.The inhibition began 2 weeks after immunization and peaked at 8 weeks,then maintained at relatively low level within 16 weeks[at 16 weeks,the titers of HBVDNA(log copies/ml)in pCTLA-S vs pS:3.58±0.09 vs 4.46±0.18,P＜0.05; the inhibition percentages of HBsAg in pCTLA-S vs pS:33.2±4.6%and 17.04±2.76%,P＜0.01].pCTLA-S vaccination was able to induce significant HBsAg-specific antibody response as compared with pS(P＜0.001).When the E/T ratio reached to 80:1,pCTLA-S vaccination was also able to elicit higher CTL response than pS(26.1±4.6%vs 16.1±3.8%,P＜0.05).More proportions of HBsAg-specific CD8⁺ IFN-γ⁺ T cells were induced in the mice vaccinated with pCTLA-S than those vaccinated with pS with about three-fold increase(P＜0.01).Both HBsAg-specific T cell proliferation and IFN-γ/IL-4 releases from splenocytes were enhanced in pCTLA-S vaccinated mice compared with pS(both P＜0.05).The serum ALT activity was slightly elevated in a few of pCTLA-S- and pS-vaccinated mice,but they reduced to normal level quickly.The difference of ALT between the groups was not significant(P＞0.05).The immunohistochemical analysis showed that HBsAg was strongly expressed in the tissue samples of pSecTagB-vaccinated mice,while slightly positive was shown in 3 of 8 tissue samples of pCTLA-S-vaccinated mice and 1 of 8 tissue samples of pS-vaccinated mice.Conclusion:In HBV-Tg mice,the fusion recombinant plasmid pCTLA-S was capable of inducing not only HBsAg-specific antibody response but also cellular immune response including enhancement of CTL activity,induction of HBsAg-specific CD8⁺ T cell,and improvement of T cell proliferation as well as Th response,which let to the inhibition on circulating HBsAg expression and HBV DNA replication in HBV-Tg mice. Partâ…£Preliminary study on the mechanism of high immunogenicity of the fusion DNA vaccine encoding HBsAg fused to the extracellular domain of CTLA-4Objective:To study the mechanism of high immunogenicity of the fusion DNA vaccine pCTLA-S,a mutant fusion plasmid named pmCTLA-S was constructed,in which contains a point mutation at residue 104(Tyr→Ala)of CTLA4 in the MYPPPY motif.Then,BALB/c and HBV-Tg mice were vaccinated with pmCTLA-S,pCTLA-S and pS.After vaccination,HBsAg-specific immune responses and anti-viral effects induced by pmCTLA-S vaccination were monitored and compared with pCTLA-S and pS vaccinations.Methods:The mutant DNA fragment of extracellular domain of CTLA4 was amplified by PCR.The extracellular domain of CTLA-4 DNA fragment in pCTLA-S was replaced by the mutant CTLA-4 PCR fragment,thus a mutant fusion plasmid named pmCTLA-S was generated.The mutant construct was transiently transfected into COS-7.The culture supematants of transfected cells were collected. The binding activity of CTLA-4 to B7-expressing cells of the mutant fusion protein was detected by flow cytometry.The BALB/c and HBV-Tg mice were vaccinated by pmCTLA-S,pCTLA-S and pS intramuscularly.After various time points of vaccination,serum anti-HBs in BALB/c mice were detected by ELISA. HBsAg-specific T cell proliferation and CTL response were also determined by MTT and LDH release assay in BALB/c mice,repectively.Serum levels of HBsAg and HBV DNA were quantitied in Tg mice by RIA and a real-time fluorescent quantitative PCR,respectively.Results:Sequence analysis demonstrated that the inserted DNA sequence in the mutant fusion plasmid pmCTLA-S is correct.By RIA,the expressions of HBsAg were positive in both intracellular and extracellular portions of pmCTLA-S-transfected Cos-7.The binding activity of CTLA-4 in the supernatant of pmCTLA-S-transfected cells to B7-expressing A20 decreased dramatically,when compared with that from pCTLA4-S(MIF:3.06 vs 12.3).Vaccination with pmCTLA-S resulted in a significant decrease in HBsAg-specific antibody response in BALB/c mice(pmCTLA-S vs pCTLA-S,P＜0.001).HBsAg-specific T cell proliferation and CTL response in BALB/c mice were also downregulated,when compared with pmCTLA-S vaccination(pmCTLA-S vs pCTLA-S,both P＜0.05). There is no significant difference in either antibody or CTL response between pmCTLA-S and pS vaccination(pmCTLA-S vs pS,both P＞0.05).Furthermore, vaccination with the mutant plasmid pmCTLA-S resulted in relatively low suppression of HBsAg and HBV-DNA,while the pCTLA-S immunization showed higher suppresssion in the expression of HBsAg and replication of HBV-DNA in transgenic mice(pmCTLA-S vs pCTLA-S,the suppression of HBsAg:17.05±5.75% vs 30.5±4.38%,p＜0.01).The suppression of HBsAg and HBV DNA induced by pmCTLA-S vaccination was quite similar to that of pS immunization(both P＞0.05, compared to pS group).Conclusion:These results clearly demonstrated the binding activity of CTLA4 to B7 on APCs was required for the enhanced antiviral immunity of pCTLA-S vaccination.Increasing the binding activity of CTLA4 to B7 on APCs by genetic engineering might be an effective way to enhance the immunogenicity of CTLA4-fused DNA vaccine.