Induction of Humoral and Cellular Immune Responses in Mice by Multiepitope Vaccines Composing of Both T and B Lymphocyte Epitopes of MAGE-A3 which are Recombined into HBcAg.

BACKGROUND

Melanoma-associated antigen-A3 (MAGE-A3) is a tumor specific antigen and a potential candidate for cancer immunotherapy. We had screened three immunodominant multiepitopes of MAGE-A3, and identified these multiepitope peptides had significantly higher reactivity to serum samples from gastric cancer patients. However, the immune responses of three multiepitope peptides carried by HBcAg in mice have not been investigated.

OBJECTIVES

The main objective of this study was to analyze the humoral and cellular immune responses in mice induced by these three multiepitope vaccines of MAGE-A3.

METHODS

Three multiepitopes of MAGE-A3 (MAGE-A3(EPI-1, or -2, or -3)) were respectively inserted at HBcAg major immunodominant region (HBcAg(MIR)) of the pET21a(+)/HBcAg(MIR) recombinant plasmid. These recombinant chimeras were identified by PCR, and transfected respectively into E. Coli Ressotta strain. The expression products of rHBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) were purified respectively by Ni2+ chelated affinity column, and then confirmed by SDS-PAGE and Western-blot analysis.Purified three rHBcAg(MIR)/MAGE-A3 multiepitopes were administrated respectively into BALB/c (H-2Kd) mice by intradermal injection. The production of rHBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) specific IgG in serum from immunized mice were measured by ELISA. Spleen cells from all immunized mice were harvested after one week of last immunization for lymphocyte proliferation assay and cytotoxic T-lymphocyte assay.

RESULTS

PCR and Sequencing analysis showed the presence of the required gene fragment in pET21a(+)/ HBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) recombinant plasmid. Purified rHBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) could be probed specifically by McAb of 6×his-tag. ELISA analysis indicated that serum from immunized mice with rHBcAg(MIR)/MAGE-A3(EPI-1, -2, or -3) proteins could be discerned specifically by complete MAGE-A3 protein, and high level of antibodies in immune serum were obtained, and all antibody titers could reach above 1:1600. The splenocytes from groups of rHBcAg(MIR)/MAGE-A3(EPI-1,-2, or -3), stimulated respectively with corresponding peptides showed the higher proliferative responses comparing with control groups of HBcAg(MIR) or PBS (p<0.05, respectively). Splenocytes from mice immunized with rHBcAg(MIR)/MAGE-A3 (EPI-1, or -2, or -3) could killed target cells effectively, and there were significant difference of CTL activities compared with control groups of HBcAg(MIR), or PBS (p<0.05, respectively) at any ratio of effector : target.

CONCLUSION

Our results indicated MIR in HBcAg presenting platform could present MAGE-A3 multiepitopes efficiently and induced significant humoral or cellular immunity. The immune strategy based on multiepitopeimmunization could have potential for preventing or controlling MAGE-A3 associated malignant disease.