HSP65-HBV Multiple-epitope Fusion Protein As A Theraputic Vaccine Against Hepatitis B Virus

Heat shock proteins HSPs are molecular chaperones, which can helpother protein in folding and degradation. As a danger signal, HSPs may act as abridge between innate immunity and the acquired immunity. The researches haveshown that the HSPs play an important role in immune response. HSPs can helpthe antigenic peptide enter the dendritic cell (DC) through specific receptor andelicit signal transductions. After entering the APC, HSP assists antigens to beprocessed and presented in MHC class I pathways. Thus, antigenic peptideseither fused or associated to/with HSP can be co-expressed with MHC class Imolecules on DCs and consequently stimulate the generation of specific cytotoxicT lymphocyte (CTL).Maket opportunity for an HBV theraputic vaccine Hepatitis B is a kind ofinfectious diseases caused by hepatitis B virus (HBV). According to theinvestigation of WHO in 2004, about 400 millions people had infected with HBVworldwide and among them 120 millions people are Chinese. Among the carriers30 millions people are chronic hepatitis B patients. The treatment of hepatitis B isa big hard nut in the field of medical science. There are two important antiviralmedicines for Hepatitis B: IFN-α and Lamivudine. But both of them haveshortcomings. IFN-α can only be used to the patients with active HBV DNAduplication. However, IFN-α is only effective in 20–40% of patients. With long-termusing, it can stimulate the production of antibody to IFN and cause side effectssuch as anemia. Lamivudine must be used for long-term therapy, which caninduce tolerance to the medicine. Because there are so many questions with themedicine used now, it is urgent to develop new antiviral medicines.The foundation and the safty of HBV theraputic vaccineUnderstanding mechanisms behind pathogenesis is central to thedevelopment of successful immunotherapies. The recognition of infectedhepatocytes by HBV-specific CTLs has been assumed to be the centralmechanism causing liver damage.Acompained with the 2 new technology of HBV transgenic mice and Tetramermethod, the recent results show that In the presence of an effective HBV-specificCD8 response, inhibition of virus replication can be independent of liver damage.When the HBV-specific CD8 response is unable to control virus replication, it maycontribute to liver pathology not only directly but also by causing the recruitment ofnonvirus-specific T cells. Therefore, the effective and timely reconstitution of HBVspecific CTL is the key issue of the HBV therapeutic vaccine.A Theraputic HBV vaccine developed by us In this project, we expressedand purified a BCG HSP65-PADRE-HBV epitope fusion protein ( HSP65-HBV epi)in E.coli. In order to identify whether it can be used for Hepatitis B therapy, wetested if it can generate HBV specific CTL both in mice and in human. We alsotested if it can stimulate lymphcyte proliferation, produce antiviral cytokines,enhance the cytotoxicity of NK cell.The main results of the study are divided into 5 parts as follows:Part 1. gene cloning, gene expression and protein purification1.1 Obtaining the HSP-HBV epi fusion protein:PADRE-HBV Multiple-epitope gene (in short, HBV epi) was synthesized by 4round-PCR and sequenced. The HBV multiple-epitope gene was inserted to the Cterminal of the BCG HSP65 gene in the expression vector pET28a-HSP65 tocreate a heat shock protein-65-Multi-epitope-HBV fusion gene (HSP-HBV epigene). The pET28a-HSP65-HBV epi was transformed into BL21(DE3) bacteria.The transformed bacteria were cultured, induced by IPTG for 3 hours and thenharvested. The HSP65-HBV epi was purifyied from the bacteria and purified. Thepurified recombinant HSP-HBV epi showed a single band of 80KD in SDS-PAGE,and the LPS contamination is below 100EU/mg protein. That was suited for thefunctional tests.1.2 Obtaining Chaperonin10-HBV epi fusion proteinChaperonin 10 is a member of heat shock protein family. The HBVmultiple-epitope gene was inserted to the C terminal of the chaperonin 10 gene inthe expression vector pET28a-chaperonin 10 to produce a chaperonin 10-HBVMulti-epitope fusion gene, Chap10-HBV epi gene. The recombinant plasmid wastransformed into BL21 (DE3) bacteria. The recombinant chaperonin 10-HBV epifusion protein was purifyied from the transformed bacteria induced by IPTG for 3hours and showed a 25KD protein in SDS-PAGE.Part 2. Biological effects of HSP65-HBV epi in mice2.1 Effect of HSP-HBV epi on the induction of HBV specific CTL in mice.6-8 weeks old BAB/c female mice were immunized with recombinantHSP-HBV epi on day 0, 21, 42. For the control group, the same mocular of HSP65,or HBsAg or the same volume of saline was administered to the mice. On thetenth day after the last immunization, the mice were sacrificed. Single cellsuspensions were prepared from spleen of the mice to detect the HBV specificCTL. The results showed that HSP65-HBV epi stimulated the generation ofHBsAg specific CTL in mice.2.2 antibody subtype detection by ELISAIgG2a represents the IgG subtype of Th1 response, wherease IgG1represents the IgG subtype of Th2 response. Th1 response is the desired immuneresponse elicits by HSP65-HBV epi.We detected the IgG subtype of the mice immunized by HSP65-HBV epi.Remakble IgG2a of anti-HSP65-HBV epi was detedcted on the forth week afterthe last immuneization. This suggests the HSP65-HBV epi can be presented byMHC I pathway. That is the fundation of HSP65-HBV epi to become a theraputicvaccine.2.3 Promotion of the proliferation of spleen cell by HSP65-HBV epiThe 3H-thymidine incorporation assay was used to test the proliferation.The results show: HSP65-HBV epi can stimulate the proliferation of spleencell in mice. The stimulatory effect of HSP65-HBV epi is similar to that of HSP65control.Part 3. Biological effects of HSP65-HBV epi on human PBMC3.1 Activation of PBMC by HSP-HBV epiThe activation of human PBMC by HSP65-HBV epi was studied firstly. CD69was expressed only on the surface of activated lymphcytes. The up-regulation ofthe CD69 indicated the acute activation of lymphcytes.The results showed that the HSP65-HBV epi could up-regulate theexpression of CD69 of Th cell, B cell and NK cell.3.2 Activation of HSP-HBV epi on NK cellThe NK cytotoxicity tests were used to examine the activation of HSP-HBVepi to NK cells. The results show: HSP65-HBV epi and HSP65 can activate NKcells with similar efficiency.Part 4. Biological effects of HSP65-HBV epi on human DC4.1 Stimulation of DC surface marker by HSP-HBV epiThe HLA-A2 positive PBMC were co-cultured with GM-CSF and IL-4 todifferentiate into immature dendritic cell (iDC). The HSP-HBV epi was used tostimulate the iDCs to mature. The surface markers of the DC were detected byFACS method. The results revealed that HSP-HBV epi could upregulate theexpression of CD40, CD80, CD83, CD86, HLA-A2, HLA-DR on the surface of DC.The cell displayed typical characteristics of mature DC after loaded by HSP-HBVepi.4.2 Stimulation of HSP-HBV epi to cytokine secretion by human DCMeasuring the content of IL-12 in the supernatant by ELISAThe results show: The content of IL-12 in the supernatant of the mediumgroup are similar to that in the plasma. The content of IL-12 in the supernatantfrom DC stimulated by HSP-HBV epi are 100～400 pg/ml. The stimulatory activityof HSP-HBV is very significant.Part 5. Biological effects of HSP65-HBV epi on human CTL stimulatedby DC.5.1 Promotion of the proliferation of purified CD8+ T cell by HSP65-HBV epiloaded DCThe 3H-thymidine incorporation assay was used to test the proliferation.The results show: HSP65-HBV epi loaded DC can stimulate the proliferationof purified CD8+ T cell The stimulatory effects of HSP65-HBV epi loaded DC areobviously stronger than that of HSP65 control.5.2 cytotoxity assey of human HBV specific CTL generated by HSP-HBVepi loded DCHuman HLA-A2+ CD8+T cells were stimulated in vitro with mature autologousDC loaded by HSP-HBV epi to generate the HBV specific CTL. The CTL wereused as effector cells. T2 cells loaded with 10μM of HBcAg18-27 peptide wereused as targeted cells. The CTL assays were conducted by the methord of 3H andthe method of PKH26 and CFSE staining. The results suggested that the maturemononuclear cell-derived DC loaded by HSP-HBV epi, could induce and generateHBV specific CTL. The CTL can lyse the T2 cell loaded with HBcAg18-27peptides.5.3 Measuring the frequency of IFN-γ secreating cell by IFN-γ ELISPOTThe frequency of IFN-γ secreating cell were measured using commerciallyavailable IFN-γ ELISPOT set.When using HepG2.2.15 or HBcAg 18-27 loaded T2 as target cell,thefrequency of IFN-γ+ cell is greatly increased in the group of DC P65-HBV activited Tcell. The results show: The frequency of IFN-γ secreating cell between groups hasobvious difference. The frequency of IFN-γ secreating cell stimulated byHSP65-HBV epi loaded DC are much higher than other groups. HSP65-HBV epiloded DC can obviously stimulate CD8+ T cell to secrete IFN-γ in the coculturesystem with target cells.5.4 Measuring the frequency of IFN-γ secreating CD8+ T cell by IFN-γ byintracelullar stainingSimilar to the result of IFN-γ ELISPOT, HSP65-HBV epi loded DC canobviously stimulate CD8+ T cell to secrete IFN-γ in the coculture system with targetcells.5.5 Dimer assay to detect the frequency of HBcAg18-27 specific CTLThe Dimer assay suggested that the mature mononuclear cell-derived DCloaded by HSP-HBV epi, could induce and generate HBcAg18-27 specific CTL.In summary, we first fused the BCG HSP65 gene to a multiple-epitope-HBVepi gene to form a fusion gene named as HSP65-HBV epi gene. The recombinantHSP65-HBV epi fusion protein was expressed in E.coli.The fusion protein was tested in BALB/c mice in vitro. It was demonstratedthat HSP65-HBV epi could elicit HBsAg specific CTL and produce IgG2a antibodyin mice.In human in vitro test, the HSP65-HBV epi can activate the HLA-A2+ PBMC,mature the DCs derived from mononuclear cells, and generate the HBV specificCTLs.Therefore, the data presented here strongly surported the development ofHSP65-PADRE-HBV epitope fusion protein as a therapeutic vaccine againstchronic HBV infection by induction of HBV specific CTL.