Generation Of Chimeric HBc-VLP With HCC Epitopes In E.coli And Augmented Induction Of Ag-specific Cytotoxic Immune Response And Antitumor Effect By DCs Pulsed With The Particle Protein

ObjectiveCancer is a serious disease endangers human health. Hepatocellular carcinoma (HCC) is among the most common malignant tumors especially in our country because of the high exposure to hepatitis viruses like B and C and to regional exposure to environmental pathogens. The incidence rate is high. However, the detection can be difficult as most of the patients who develop this tumor have no symptoms. The development of HCC is rapid, and more with cirrhosis and other complications, surgical resection and postoperative survival rates are lower. Therefore, the treatment efficacy and prognoses for HCC are not ideal. So it is urgently needed to find effective novel therapeutic interventions or"vaccine"to stimulate the body of the cellular and humoral immune responses to control such diseases.Dendritic cells (DCs) are the professional antigen presenting cells (APCs) which are active in the taking up and processing of antigens (Ags) to T cells and possess the ability to stimulate na?ve T cells. DCs also have the property of control primary and secondary immune responses to various exogenous antigens. In fact, DCs can be considered as ideal tools for the development of therapeutic vaccines against cancer and DC-based vaccination strategies designed to induce tumor-specific CD8+ CTL are being widely considered for cancer immunotherapy.At present, many researchers use tumor antigens, such as tumor-specific antigen, tumor-associated antigen or fully cell antigen pulsing DCs in vitro, then the modified DCs were transferred back to body and the effective anti-tumor immune response could be detected. But the general immune response induced by DCs could not be maintained. Recent studies showed that the antigen loaded DCs have a weaker antigen presenting capacity or the life span of DCs even was reduced, so it is very difficult to induce a long-term of T cell responses.Virus-like particles (VLPs) are formed by the self-assembly of envelope and/or capsid proteins from many viruses, but VLPs are non-infectious because they assemble without incorporating genetic material. In many cases such VLPs have structural characteristics and antigenicity similar to the parental virus, and some have already been proven successful as vaccines against the cognate virus infection. VLPs are commonly more immunogenic than subunit or recombinant protein immunogens, and are able to stimulate both the humoral and cellular arms of the immune system. The structural components of some VLPs have also been proven amenable to the insertion or fusion of foreign antigenic sequences, allowing the production of chimeric VLPs exposing the foreign antigen on their surface.MAGE genes are silent in healthy tissues with the exception of placenta and male germ-line cells. MAGE-1 and MAGE-3 are widely expressed in cancers such as melanomas, lung carcinomas, ovarian tumors, hepatocellular carcinomas and so on. The usefulness of AFP as a diagnostic marker for the detection and/or differentiation of a great number of infantile diseases are well established, especially for some forms of malignant tumors and liver disorders. Four epitopes MAGE-1(278– 286aa), MAGE-3(271– 279aa), AFP1 (158–166aa) or AFP2 (542–550aa) which experssed highly in HCC, were fused to the 3'terminus of the truncated HBV core gene(HBc1-144), respectively or conjunctively, and then ligated into prokaryotic expression vector pET28a.Recombinant plasmids led to expression of chimeric proteins in expression strain E. coli BL21 (DE3), and chimeric proteins which are expressed in inclusion bodies resulted in the formation of complete"mature"VLPs. DCs pulsed with the chimeric HBc-VLPs could induce stronger CTL activity and greater IFN-γsecretion by responding T cells compared with peptid-pulsed DCs. We used tansgenic mouse in the current study to demonstrate the principal possibility of using HBc-VLPs pulsed DCs as vaccine for creation of a new type of HCC-specific immunogen.Methods1 Designing and obtaining of single-epitope chimeric HBc/HCC constructsThe approach we used for construction of chimeric HBc-epitope genes was fusing the sequences of single-epitope MAGE-1(278-286aa), MAGE-3(271-279aa), AFP1(158-166aa) and AFP2(542- 550aa) to the C terminus of the truncated HBc protein via GA linker by PCR methods.2 Designing and obtaining of multi-epitopes chimeric HBc/ HCC constructsIn the same way, the approach we used for construction of multi-epitope chimeric HBc-epitope genes was fusing the sequences of multi-epitope S1 (AFP1-Th epitope-AFP2) and S2 (MAGE1- MAGE3-Th epitope) to the C terminus of the truncated HBc protein via GA linker by PCR methods.3 Construction of prokaryotic expression plasmid pET28a- HBc-epitopeFirst, the fusion genes were ligated into the plasmid of pET28a to construct recombinants. Then they were transfected into E. coli BL21 (DE3) and subjected to identification by restriction enzymes and sequencing.4 Expression and purification of fusion proteins in E. coliThe recombinant plasmids led to expression of chimeric proteins in expression strain E. coli BL21 (DE3). In order to obtain the optimized expression we changed the concentration of glucose and of IPTG as well as the time of induction. The chimeric proteins which are expressed in a form of inclusion bodies.The inclusion bodies were dissolved in 6 M urea, and the denatured fusion proteins were purified with the Ni–NTA affinity columns. We also showed that whether they could be internalized by DCs and their antigenicities were identified by ELISA and Western-blot.5 Investigation of the stronger T cell responses and an antitumor effect induced by HBc-VLPs-pulsed DCs in vivoWe investigated whether the immature mice BMDCs could uptake the HBc-VLPs efficiently and then process and present the antigen to syngeneic mice spleen T cells in vitro. We also showed that whether vaccination with VLPs-pulsed DCs could elicit stronger T cell responses, as measured by both intracellular production of IFN-γand killing assays by Ag-specific T cells.ResultsSix forms of HCC epitopes MAGE-1(278-286aa), MAGE -3(271-279aa), AFP1 (158-166aa), AFP2 (542-550aa), S1 (AFP1-Th epitope-AFP2) and S2 (MAGE1- MAGE3-Th epitope) were fused to the 3'terminus of the truncated HBV core gene respectively. Not all recombinant plasmids led to expression of chimeric proteins in expression strain E. coli BL21 (DE3), but chimeric proteins which are expressed in inclusion bodies resulted in the formation of complete"mature"VLPs. E. coli-derived HBc-epitope self-assembled into VLPs that both morphologically and physically are similar to the wild-type ones and they still remained activity after purification and refolding from 6M urea solution. Chimeric HBc-epitope VLPs with an N-terminal His-tag were purified by denatured Ni2+-chelate affinity chromatograph and then renatured by dialysis and their antigenicities were identified by ELISA and Western-blot.We also show immature mice BMDCs could capture HBc-VLPs efficiently and present the antigen to syngeneic mice spleen T cells in vitro. Comparing with peptide-pulsed DCs, immunization with VLPs-pulsed DCs could elicit stronger T cell responses in vivo, as measured by both intracellular productions of IFN-γand killing assays by Ag-specific T cells. In the B16-pIR-HH tumor therapy model, the growth of tumors was significantly inhibited by immunization of DCs pulsed with HBc-VLPs, resulting in significantly longer survival of immunized animals and strikingly, high frequencies of protective CTL could be induced and maintained.Conclusions1 This study established a series of technology platform with the chimeric HBc-VLPs: such as construction, prokaryotic expression and purification.2 The results of the current study have demonstrated that HBc-VLPs-pulsed DCs are a new, more effective DC vaccine which could induce stronger immune response and anti-tumor effect.3 Successfully established a tumor-bearing mouse model with B16-pIR-HH cell which could evaluat the HLA-A2- restricted CTL epitopes in HLA-A2 transgenic mice. 4 The results of this study lay a foundation for the further research into effective therapeutic HCC vaccine and provided a useful platform for optimizing and evaluating DC-based vaccine.