Novel HBsAg Vaccine Adjuvanted With H. Polymorpha

Hepatitis B virus(HBV) infection has been a major public health problem.There are approximately 350 million chronic Hepatitis B virus infected patients worldwide.These patients have a great propensity to develop live cirrhosis and hepatocellular carcinoma. Interferons(IFN) and nucleosides analogues such as lamivudine and adefovir are the limited methods available for the control of chronic HBV infection.However,their efficacy is unsatisfied.Vaccination remains effective approach for preventing infectious diseases.HBsAg alone is poor in immunogenicity,and adjuvants are required to enhance its efficacy.Although several adjuvant candidates have been extensively evaluated over years,alum is the only human vaccine adjuvant approved for use.Unfortunately,alum can not induce Th1 type immune response which plays a vital role in elimination of HBV infection.It is generally believed that host's immune tolerance to HBV accounts for the disabilities to clear HBV efficiently.Therefore,it is important to break the immune tolerance to HBV by induction of HBsAg-specific humoral and cell immune response for controlling chronic HBV infection.Studies have showed that Th1 responses are a major contributor to controlling intracellular infections,while humoral immune responses are essential for controlling extracellular infections.Currently,an ideal therapeutic vaccine development is strongly recommended to focus on the induction of Th1 immune responses.Toll-like receptors(TLRs) are pattern recognition receptors that can recognize pathogens via pathogen-specific molecular patters(PAMPs).TLRs play a crucial role in both innate and adaptive immunity.So far 11 different human TLRs have been identified,and the natural ligands for most of these TLRs have also been recognized.Many TLR ligands have been studied as vaccine adjuvants,such as R.848,CpG.Immature dendritic cells(DCs) residing in peripheral tissues recognize these invading pathogens via numerous TLRs present on them.The direct activation of macrophages and DCs result in cluster designation(CD) for independent maturation and subsequent release of chemokines and pro-inflammatory cytokines including IL-12,upregulation of MHC classⅡ, CD80,CD86,CD40 expression.This leads to the activation,maturation and trafficking of the DCs to local lymph nodes and presentation of microbial antigens to n(a|¨)ive T cells,leading to the induction of adaptive immunity against the invading pathogen.Furthermore,DCs can also regulate the T cell differentiation(Th1 versus Th2) based on the proinflammatory cytokines produced by them.The proinflammatory cytokine IL-12 can bias the immune response towards Th1.Th1 response is characterized by potent cell immune and proper humoral immune response.Recent studies have demonstrated that yeast cell wall components possess multiple adjuvant properties.Interactions between yeast and DCs result in DC maturation.Whole recombinant yeast internalized by DCs can deliver heterologous antigens to both MHC classⅠand classⅡpathways and induce potent cell-mediated immunity.Vaccination with heat-killed S.cerevisiae expressing tumor-associated antigens could induce antigen-specific T cell responses and protect against tumor challenge.Furthermore,S.cerevisiae is inherently nonpathogenic and heat-killed recombinant S cerevisiae shows no toxicity in clinical studies.Yeast can be easily engineered to express multiple antigens.Additionally,the inherent adjuvant properties on S.cerevisiae avoid the need for additional adjuvants.These characteristics make S.cerevisiae a potential vaccine vehicle for cancer and infectious diseases.Antigen-specific T cell immune-enhancing effects of yeast have been reported for a number of antigens.However,there are some limitations and drawback in S.cerevisiae expression systems.For example,S.cerevisiae has a tendency to hyperglycosylate recombinant proteins,and N-linked carbohydrate chains are terminated with alpha-1,3-linked mannose residues which is considered to be allergenic.Other restriction is that the variety of carbon sources that can be utilized by this species are limited. H.polymorpha can potentially overcome the described limitations of the traditional S. cerevisiae.A non-allergenic terminal alpha-1,2-linked mannose residues is observed in N-linked chains of glycoproteins produced by H.polymorpha.Furthermore,H.polymorpha can grow on a broad range of carbon sources,and the productivity of protein is higher than that of traditional S.cerevisiae.Based on the potential application of recombinant S.cerevisiae in cancer therapy and the special characteristics of H.polymorpha,we sought to determine whether H.polymorpha could induce Th1 immune responses,and have the potential of being used for developing novel vaccine for HBV infection.In this study,we sought to determine whether heat-killed recombinant H.polymorpha expressing HBsAg(Yeast-HBsAg) could induce Th1 immune responses.In order to improve the production of HBsAg in H.polymorpha,the HBsAg gene was synthesized according to the codon usage bias of H.polymorpha,subsequently subcloned into the multi-copy expression vectors pDGXHP1.0(MOX promotor),and engineered to form a 4-copy expression plasmid.The recombinant plasmid was transformed into H.polymorpha ATCC26012(Ura3-) by electroporation,and HBsAg expression was induced.We detected HBsAg in supernatant of H.polymorpha cell.expressing HBsAg.by ELISA,and HBsAg in H. polymorpha cell.expressing HBsAg by ELISA and western blot.The results demonstrated that HBsAg was mainly expressed in H.polymorpha cell.In this study,mice were divided into 5 groups,with 10 mice in each group.Mice were immunized intramuscularly in the left tibialis anterior muscle 3 times at 4-week intervals with PBS,HBsAg alone,HBsAg+alum,H.polymorpha(Yeast vector),Yeast-HBsAg respectively. Mice were bled at weeks 2,4,6,8,10,12 after the first immunization.Murine immunoglobulin G(IgG) and IgG isotypes(IgG1 and IgG2a) specific for HBsAg were detected by enzyme-linked immunosorbent assay(ELISA) kits.Part of mice were sacrificed and cells were isolated from spleens at days 7 after the third immunization.Splenocytes were collected from mice and treated with lysis buffer to eliminate red cells.The surface expression of CD11c,co-expression of CD11c with CD80,CD86,and MHC classⅡmolecules respectively;CD3,co-expression of CD3 with CD4,CD8 on spleen cell were determined by flow cytometry.The results were analyzed by the CellQuest software package.Splenocytes were adjusted to a final concentration of 4×10~6 cells/ml and stimulated with recombinant HBsAg.The supernatants of cultured cells were collected and IFN-γ,IL-4 were detected by ELISA.Splenocytes were further seeded into a 96-well flat-bottom microtiter plate and stimulated with recombinant HBsAg.Splenocyte proliferation was examined and CTL activity was determined by calcein release assay.Results suggested that there was a 5.6-fold increase in total number of spleen cells in mice immunized with Yeast-HBsAg compared to mice immunized with HBsAg,while 2.2 fold increase was observed in mice immunized with HBsAg+alum.There were a 13.6-fold in DCs and a 4.4-fold increase in T cells in Yeast-HBsAg immunized mice compared to mice immunized with HBsAg.Our results suggested that both the percentages and the absolute number of DCs expressing CD80,CD86,and the MHC classⅡmolecules within total spleen cells in mice receiving Yeast-HBsAg were significantly higher than that in mice receiving HBsAg,HBsAg+alum or PBS.Similar results were obtained in Yeast vector immunized mice. However,HBsAg had little effect on the recruitment of mature DCs in the spleen.Splenocytes from animals immunized with yeast-HBsAg produced significantly higher levels of IFN-γand IL-4 than those from mice immunized with HBsAg alone or with HBsAg+alum.Significantly higher percentages of HBsAg-specific CD4~+ IFN-γ~+ and CD8~+ IFN-γ~+ T cells were observed in mice immunized with Yeast-HBsAg.Splenocyte proliferation in the mice immunized with Yeast-HBsAg was significantly higher than that in HBsAg or HBsAg+alum group.CTL assay was performed on day 7 after the third immunization.Result showed that the stimulated spleen cells from Yeast-HBsAg immunized mice killed P815-S target cells in a dose-dependent manner(between 5.4%and 24.3%lysis).In contrast,HBsAg+alum could not induce T cell immune response.This result suggested that Yeast-HBsAg could induce antigen-specific CTL activities in immunized mice,which was consistent with the high level of IFN-γexpressed in CD8~+ T cells.Results suggested that mice immunized with Yeast-HBsAg produced much higher HBsAg-specific serum IgG,IgG1,IgG2a antibodies than that with HBsAg or HBsAg+alum. Yeast-HBsAg stimulated the secretion of HBsAg-specific IFN-γand IL-4 from spleen cells. Yeast-HBsAg promoted the percentages of both CD4~+ and CD8~+ IFN-γ-producing T cells in spleen cells.Yeast-HBsAg induced potent HBsAg-specific lymphocyte proliferation.Studies have demonstrated that yeast cell wall components possess multiple adjuvant properties. In this study,HBsAg was mixed with heat-inactivated whole H.polymorpha(HBsAg+yeast) to immunize mice,in order to determine whether H.polymorpha could enhance HBsAg-specificTh1 immune responses.In this study,mice were divided into 5 groups,with 10 mice in each group.Mice were immunized intramuscularly in the left tibialis anterior muscle 3 times at 4-week intervals with PBS,HBsAg alone,HBsAg+alum,H.polymorpha(Yeast vector),HBsAg+yeast respectively. Mice were bled at weeks 2,4,6,8,10,12 after the first immunization.Murine immunoglobulin G(IgG) and IgG isotypes(IgG1 and IgG2a) specific for HBsAg were detected by enzyme-linked immunosorbent assay(ELISA) kits.Some mice were sacrificed and cells were isolated from spleens at days 7 after the third immunization,splenocytes were collected from mice,treated with lysis buffer to eliminate red cells.Splenocytes were adjusted to a final concentration of 4×10~6 cells/ml and stimulated with recombinant HBsAg,the supernatants of cultured cells were collected and IFN-γ,IL-4 were detected by ELISA.Splenocytes were seeded into a 96-well flat-bottom microtiter plate and stimulated with recombinant HBsAg,splenocyte proliferation was examined, Results suggested that mice immunized with HBsAg+yeast produced much higher HBsAg-specific serum IgG,IgG1,IgG2a antibodies than that with HBsAg or HBsAg+alum. HBsAg+yeast stimulated the secretion of IFN-γand IL-4 from spleen cells and the production of IFN-γin CD4~+ T and CD8~+ T cells in spleen cells.The results suggested that HBsAg+yeast could not only induce Th1 immune responses but also have remarkable effects on enhancement of Th2 immune responses.In summary,our results demonstrated that vaccination with Yeast-HBsAg resulted in effective recruitment of immune-mediated cells to mouse spleen.The immune responses in mice elicited by Yeast-HBsAg is similar to that by HBsAg+yeast.In addition,Yeast-HBsAg could not only induce Th1 immune responses,but also have remarkable effects on enhancement of Th2 immune responses.In contrast,HBsAg+alum could only enhance Th2 immune responses. Therefore,HBsAg vaccine adjuvanted with H.polymorpha may play an important role in prevention and control of HBV infection.