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.