Construction And Immunological Activity Of Three Tandem Repeats Of Minigene DNA Vaccine Derived From Carcinoembryonic Antigen (CEA) Gene

Carcinoembryonic antigen (CEA) is an ideal TAA (tumor associated antigen) for the development of immunotherapy because it is produced by most colorectal, gastric, and pancreatic carcinomas and it is expressed to a far lesser extent on normal epithelium and also on some fetal tissues. At present, CEA-based immuno-biological cancer therapy includes active immunity and passive immunity. Active immunity mostly indicates DNA vaccine, while passive immunity includes peptide vaccine and antibody. Thereinto, DNA vaccine has been paid close attention to for its security, long action time, simple design, hign-production and economy. To produce DNA vaccine, we can use cDNA encoding full - length protein and also can use partial cDNA encoding specific epitope. We introduced the term minigene to describe such isolated epitope sequences. The use of minigene vaccines is an attractive approach because of the ease of synthesis and manipulation of these vaccines. Moreover, in contrast to whole-gene vaccines, minigene vaccines can induce immune responses against specific Ag epitopes while avoiding the interference of nonrelevant Ag epitopes. Consequently, such vaccines lend themselves to in-depth studies of immunological mechanisms far more readily than do DNA vaccines encoding entire genes.Much work on different links of gene immunity has been done to enhance the immune effect of DNA vaccines. In antigen modifying, many research groups are interested in selecting and combining the predominant epitope and in removing the suppressed epitope. We can analyse the sequence of Ag epitope and recognize its immunologic function exactly along with the development of gene technology and immunology. Because MHC class I-restricted CTLs have a direct lytic effect on tumor cells, most efforts have focused on the identification of peptide epitopes capable of stimulating these types of responses, while HTLs play an important role both in the induction and maintenance of CTL responses, vaccines that activate both CTLs and HTLs should be more effective than vaccines that only target CTL responses. In view of this, one obvious way to improve vaccines designed to induce antitumor CTLs is to include in these vaccines MHC class II-restricted epitopes that would trigger HTL responses to TAAs. In agreement with this assessment, we selected a minigene which contained two high-affinity HTL epitopes (CEA625-639 and CEA653-667). In addition to potentially being a promiscuous HTL epitope, we deliberately selected the CEA625–667 minigene sequence because it was absent in BGP and NCA. This decision served two purposes: (i) we were concerned that peptides corresponding to CEA sequences that are identical (or similar) to BGP or NCA would not be immunogenic because immune tolerance is likely to exist to widely expressed proteins such as BGP and NCA; and (ii) most importantly, we wanted to diminish the chances of inducing serious autoimmune pathology if a vaccine containing a cross-reactive T-cell epitope would be effective in overcoming immune tolerance because BGP and NCA are widely expressed in various tissues.The main deficiency in minigene vaccine appears to be in placing sufficient copies of the correct peptide-MHC complex on the cell surface which results in a low CTL or HTL precursor frequency. We showed here that this could be rectified by being tandemly repeated. We applied gene technology to construct three tandem repeats of minigene DNA vaccine to broaden the immunogenicity of a minigene vaccine and its immunological activity was studied in mice. Furthermore, DNA immunization ensures access of the antigen to the endogenous antigen presentation pathway which avoids low-specificity, low-affinity or immunological tolerance.First, we obtained the DNA fragment encoding CEA625-667 from human genome by PCR. The sequenced minigene was subcloned into PQE30 expression vector, then purified from transformed E.coli DH5αusing Ni-NTA affinity chromatography column and assessed by Western blotting with anti-his monoclonal antibody. Lymphocytes from mice were tested in vitro for specific proliferation responses to the CEA625-667 peptide by 3H-TdR incorporation. The result showed that A 6×his-CEA625-667 minigene fusion protein with relative molecular mass of 6 700 was expressed in E.coli DH5αand mainly located in inclusion bodies. Western blotting with anti-His monoclonal antibody identified the fusion protein. In the present of various concentration of CEA625-667 peptide, splenocytes from mice exhibited at least 10-fold increase of proliferation response after 7-9 days. Concluded that the CEA625-667 minigene peptide was successfully expressed in E.coli DH5αand high pure protein could promote antigen-specific proliferation of lymphocytes from mice in vitro. Our research made a helpful exploration into expression and purification of short peptide and suggested that CEA625-667 minigene peptide could act as a lymphocyte epitope processing strong immunogenicity to na?ve mice.Our observations confirmed that CEA625-667 minigene peptide processed antigenicity in mice. According as this conclusion, by using the Ecor I in T-easy and the matching relationship between the cutting site of target gene and multiple cloning sites of pcDNA3.0, the target DNA was cloned into the vector pCDNA3.0 directionally by repeated excision and ligation successively. The tandem recombinant vector pCDNA3.0- triCEA625-667 was thus constructed and confirmed by automated DNA sequencing, PCR and restriction enzymes analysis. At same time, our work led us to an in-depth study of methodology of connecting short sequence in series.The immunoreaction was induced by intramuscular injection with pc-DNA3.0, pcDNA-CEA625-667 and pcDNA-triCEA625-667 in BALB/c. Four weeks after injection, the spleen cells and serum were separated respectively from the mice for the in vitro assessment. Changes of the T lymphocytes subset was analyzed by flow cytometry. Lymphproliferation responses were tested by 3H-TdR incorporation, IFN-γ,IL-4 and GM-CSF in their cultural supernatants were detected with ELISA and seral IgG antibody against CEA were detected with western blotting and ELISA. The result showed that the difference of the ratio of CD4+/CD8+ of the mice immuned by pc-DNA3.0, pcDNA-CEA625-667 or pcDNA-triCEA625-667 was not significant. Lymphproliferation responses were more significant in the mice immuned by pcDNA-CEA625-667 and pcDNA-triCEA625-667 in a shorter time by contrast with na?ve mice. We detected low tilter IgG antibody against CEA in the antiserum of the mice immuned by repeats of minigene DNA vaccine, which suggested the activation of Helper T-cell. ELISA showed that the level of IFNγin the 3 days culture of the splenocytes was relatively higher in the groups of minigene DNA vaccination than in the control groups, while IL-4 expression was absent in all groups. Our observations suggested that the higher-affinity HTL epitope we selected skewed T cells toward a Th1 response.In addition, the immune response level elicited by three tandem repeats of minigene DNA vaccine pcDNA-triCEA625-667 was superior to that elicited by pcDNA-CEA625-667, which showed that any immunogenic inadequacies in minigene presentation can be rectified by linking itself in a string-of-beads vaccine.On the whole, to enhance the immunogenicity of a minigene vaccine, this research approached the methods about minigene molecule design and short sequence series connection according new route, did an in-depth study of the function of DNA vaccine based HTL epitope and established foundation for developing potent CEA recombinant vaccines and its pre-clinical study.