The Effect Of CpG ODN On Assisting HCC Associate Antigen To Induce Immune Response Against Liver Cancer In Mice

It is well established that hepatocellular carcinoma (HCC) is associated with thepersistent infection of hepatitis B virus (HBV). Approximately, one third of HCCpatients are identified with HBsAg expressed on their hepatocytes and HCC cells. Thepatients with chronic HBV infection has been recognized as a high risk factor for thedevelopment of HCC, implying that HBsAg could serve as a tumor-associated antigen(TAA) in HBsAg expressing liver cancer. Presumably, HBsAg, included incommercialized vaccines for preventing HBV infection, could be formulated into avaccine for the treatment of HBsAg expressing liver cancer.Currently, in commercialized vaccines for HBV infection prevention,recombinant HBsAg produced in yeast or mammalian cells is formulated in aluminumsalt (Alum).Though safe and well tolerated, the alum is relatively weak adjuvant forthe vaccines. About10percent of those people received3doses of the vaccine failedto produce protective level of anti-HBsAg antibody (HBsAb). Moreover, the Alumadjuvanted HBsAg is unable to induce Th1-biased immune responses with elevationof IFN-γ, TNF-α, IL-12and IgG2a antibodies and instead induce a Th2-biasedimmune responses characterized by enhancing production of IL-4, IL-10and IgG1antibodies. As reported, Th1-biased cytokines are significantly correlated with theclearance of tumor cells by providing a favorable milieu for a TAA to induce specificCTLs. Ideally, the adjuvant formulated with HBsAg should be capable of promoting aTh1-biased immune response with a vigorous generation of specific CTLs againstHBsAg expressing liver cancer cells. Apparently, it is required to have a potent Th1biasing adjuvant to develop therapeutic vaccines for the treatment of HBsAgexpressing liver cancer.In recent years, multiple studies have shown that the synthetic unmethylatedCpG containing ODN (CpG ODN) could be used as a Th1biasing adjuvant. Afterrecognizing toll like receptor9(TLR9), CpG ODN acts as a immune-stimulator toinduce Th1biasing cytokines in favor of the generation of specific CTLs. The properties make the CpG ODNs promising adjuvants for tumor vaccines. In mousemodels, co-administration of tumor vaccine with CpG ODN was capable of inducingrejection of glioblastoma, B16melanoma, prostate cancer and gastric cancer. Inclinical trials, CpG ODN adjuvanted cancer vaccines showed therapeutic potential inpatients with melanoma, breast cancer, sarcoma, ovarian cancer and glioblastoma.Although there are no approving researches on CpG ODN adjuvanted HBsAg astherapeutic cancer vaccines against HBsAg expressing liver cancer, the CpG ODNhas been shown to be a promising adjuvant for developing vaccines capable ofinducing HBsAg specific cellular immune responses in mice, baboons and human.When used in combination with Alum adjuvanted HBsAg, CpG ODN facilitated thevaccine to induce a Th1biased immune response to HBsAg. These data imply thatCpG ODN could be of value in assisting HBsAg to induce immune responses againstHBsAg-expressing liver cancer.In our previous study, BW006, a B-type CpG ODN, was found to be potent instimulating the proliferation of human PBMCs and mouse spleen cells. In currentstudy, we investigated whether BW006could assist HBsAg to induce therapeuticimmunity against HBsAg expressing liver cancer in mice. We found that therapeuticapplication of BW006adjuvanted HBsAg induced Th1biased immune responses thatinhibited the growth of HBsAg expressing liver cancer cells and prolonged thesurvival of mice bearing HBsAg expressing liver cancer.Immunostimulatory properties of BW006To find whether CpG ODNs could be used as an adjuvant for HBsAg to induceimmune response against HBsAg-expressing liver cancer, we have synthesized aseries of CpG ODNs and tested their immunostimulatory capacities to stimulate theproliferation of human PBMCs and the activation of immune cells in the PBMCs. Inthe process, a CpG ODN, designated as BW006, stood out. In human PBMCproliferation assay, BW006could stimulate human PBMCs to proliferate vigorously.The efficacy was stronger than that induced by2216(P=0.001), a prototype of type-ACpG ODN for human,and similar to those induced by1018, a prototype of type-BCpG ODN for human, and C274(p>0.05vs.BW006), a prototype of type-C CpGODN. Meanwhile, BW007, a GC reversed control for BW006, failed to stimulate the proliferation of the human PBMCs (P>0.05vs. medium). To determine the cellsactivated by BW006, the human PBMCs cultured with BW006were analyzed fortheir up-regulation of CD69molecule. The flow cytometry analysis showed thatBW006up-regulated the expression of CD69molecule in CD19+, CD56+or CD8+cells, indicating that BW006could activate human B cells, NK cells and T cells.Furthermore, the human PBMCs activated by BW006were analyzed for their up-regulation of human leukocyte antigen class I (HLA-A2) molecules.The resultdemonstrated that BW006could up-regulate HLA-A2in human PBMC in a similarcapacity as1018. Taken together, BW006could stimulate the proliferation of humanPBMCs, activate human B cells, NK cells and T cells and up-regulate MHC Imolecules, showing the potentials as a promising adjuvant to assist HBsAg to induceimmunity against HBsAg expressing liver cancer.Because of the requirement for evaluating the adjuvant activity of BW006forHBsAg in mouse models, we next tested whether BW006could also stimulate theproliferation of murine splenocytes, activate murine immune cells and up-regulationof MHC class I molecule of the cells. In proliferation assay, splenocytes of BALB/cmice were stimulated by BW006and control CpG ODN including1585(a mouse A-type CpG ODN),1826(a mouse B-type CpG ODN) and C274and then tested fortheir incorporation of [3H] TdR. The result showed that BW006could stimulate theproliferation of the splenocytes to a level similar to that induced by C274and a levelmuch higher than that induced by1826(P<0.05) and1585(P<0.001). To test theability of BW006for activating mouse immune cells, mouse splenocytes were stainedby fluorescence-conjugated mAb after CpG ODN stimulation and analyzed by flowcytometry. The result demonstrated that BW006could up-regulate CD69expressionin CD19+, CD56+or CD8+cells in a similar capacity as that induced by1826(P>0.05),and stimulate the up-regulation of IAb1molecules in mouse splenocytes. The resultsreveal that adjuvanticity of BW006could be evaluated in mice.Effect of BW006on assisting HBsAg to induce Th1-biased immune responses inmiceTo investigate whether BW006could facilitate HBsAg to induce a Th1polarizedimmune response, mice were injected subcutaneously (s.c.) at inguinal lymph node areas in both sides with HBsAg alone or HBsAg+Alum or HBsAg+BW006orHBsAg+Alum+BW006and PBS, respectively, for four times at a7-day interval. Onday7after the last immunization, the mice were bled and HBsAg specific IgG orIgG2a, a subtype Th1biased antibody, or IgG1, a subtype Th2biased antibody, in thesera were detected by ELISA. The levels of IgG induced by HBsAg+BW006+Alumwere the highest than those induced by the HBsAg+BW006(P<0.05), HBsAg+Alum(P<0.001) and HBsAg alone (P<0.001), respectively. Whereas the IgG2a/IgG1ratioinduced by HBsAg+BW006(1.02) was significantly higher than those induced byHBsAg+BW006+Alum (0.68)(P<0.05), HBsAg+Alum (P<0.001) and HBsAg alone(P<0.001), respectively. The data indicated that the addition of BW006to HBsAgresulted in a shift towards a Th1biased immune response. Next, we measured IFN-γand IL-12production induced by BW006adjuvanted HBsAg. Mice were immunizedas described above. On day7after the last immunization, splenocytes were isolatedand cultured with HBsAg (50μg/ml) for24h. The levels of IFN-γ and IL-12in thesupernatants were determined by ELISA. Levels of IFN-γ and IL-12in spleen cellcultures from the mice immunized with HBsAg+BW006were not significantlydifferent from those induced by HBsAg+Alum+BW006(P>0.05) but much higherthan those induced by HBsAg+Alum, HBsAg or PBS (P<0.001). Thus, these datasuggest that BW006could induce a milieu in favor of a Th1polarized immuneresponse to HBsAg.Establisment of HBsAg expressing liver cancer cellsTo establish a HBsAg-expressing liver cancer cell line for assessing the efficacyof BW006adjuvanted HBsAg inducing an immune response against HBsAg-expressing liver cancer, pcDNA3plasmids carrying HBsAg encoding gene werestably transfected into murine H22liver cancer cells (H22cells). The transfected cells,selected in a medium containing G418(800μg/ml) and through cloning by limiteddilution, were identified by RT-PCR and Western blotting. A DNA fragment of703bp, representing HBsAg specific mRNA, was amplified from the transfected cells,and proteins in a28Kd band, representing the expressed HBsAg, were recognized byanti-HBsAg mAb in the cell lysate of the transfected cells. The identified cells,designated as HBsAg+H22cells, were used for tumor inoculation and in vitro assays. Therapeutic effect of HBsAg+BW006against HBsAg expressing liver cancer inmiceTo investigate whether BW006could promote HBsAg to induce therapeuticimmune response against HBsAg-expressing liver cancer, mice (n=8) were inoculatedwith HBsAg+H22cells (2×106/mouse) on day-1and then s.c injected with PBS orHBsAg or HBsAg+Alum or HBsAg+BW006or HBsAg+Alum+BW006at inguinallymph node areas in both sides on days0,7,14,21, respectively. The mice weremonitored for measuring tumor volumes every other day and recoding the survival.The result showed that HBsAg+BW006significantly inhibited the growth of HBsAg+H22cells compared with PBS (P=0.028), however, HBsAg+Alum orHBsAg+Alum+BW006failed to induce tumor growth inhibition (P>0.05)(Fig.5A).Interestingly, the established tumors were completely rejected in2out of8micetreated with HBsAg+BW006. By day90post tumor challenge, all of mice receivedwith PBS or HBsAg+Alum or HBsAg+Alum+BW006had succumbed to theimplanted HBsAg+H22cells and the average survival of mice of all groups wasapproximately61-64days. In contrast, by day64and90post tumor implantation,87.5%and37.5%of mice in HBsAg+BW006group were still alive. These resultsindicated that therapeutic administration of HBsAg+BW006induced effectiveimmune responses against HBsAg expressing liver cancer in mice.CTL induced by HBsAg+BW006in miceTo test whether HBsAg+BW006induced therapeutic effect on HBsAg-expressing liver cancer was associated with generation of HBsAg specific CTLresponse, mice (n=5) were immunized as described above. The immunized mice weresacrificed on day7after the last immunization and their spleen cells were isolated.The cells were incubated with HBsAg for2days, and then co-cultured with HBsAg+H22cells at effector/target ratio of12.5:1,25:1,50:1, respectively, for12h. The livingHBsAg+H22cells were stained by trypan blue and counted. It was found that at theE:T ratios25:1and50:1, the CTLs from mice immunized by HBsAg+BW006lysedHBsAg+H22and failed to lyse wild-type H22cells. The result showed thatHBsAg+BW006could induce the generation of CTL specific to HBsAg+H22cells. In addition to, we tested the effect of CpG ODN on assisting TCL from H22cellsto induce immune responses against orthotopic-transplantated liver cancer in mice.The result showed that therapeutic administration of TCL or TCL+0800, a C-typeCpG ODN, completely inhibited the growh of orthotopic transplantation liver cancercells in mice.