Mycobacterium Tuberculosis Heat Shock Protein 70 Enhances Immunization Effectiveness Of The Gene Vaccine Against Hepatocellular Carcinoma-associated Antigens 661

Genetic vaccine, also named as DNA vaccine, is recombinant eukaryotic expressionplasmid, which carries exogenous genes under the control of eukaryotic promoters, can beused to immunize animals. The direct injection of a naked plasmid DNA vaccineencoding a foreign antigen results in plasmid uptake and protein expression leading to theinduction of antigen-specific cellular and humoral immune responses. The ability of DNAvaccine-elicited immune responses to prevent beforehand or treat viral and bacterialinfections, parasites, cancers, and autoimmune diseases has been well documented innumerous animal models effectively. Phase I human clinical trials have shown thatexperimental DNA vaccines are safe and well tolerated, however, its immunogenicity islow. Furthermore, the immunogenicity of DNA vaccines in human is lower than that inmice. These preliminary studies indicate that measures must be taken to improve geneticvaccine immunogenicity.One strategy of genetic vaccines is fusion gene vaccine consisting of target gene andmycobacterium tuberculosis heat protein 70 (mtHSP70), in which mtHSP70 acts aspolypeptide carrier and genetic adjuvant to improve the immunogenicity of target gene.As a CD40 ligand, mtHSP70 stimulates macrophage or dendritic cells secreting variouscytokine, expressing numerous MHCI moleculars quickly and directly through binding toCD40, and enhances antigen presentation function. mtHSP70 helps cross-antigenpresenting cells to capture antigens from cell spaces or cell surface, then the CTLs andCD4+ Th cells are elicited through MHCI and MHCII molecular. On the other hand,mtHSP70 also promote B cells to proliferate by binding with CD40 expressed on thesurface of B cells together with CD4+ Th cells. At last, B cells differentiate into plasmacells secreting antibodies and memory B cells. Polypeptide binding domain at carboxylterminus of mtHSP70, the mini combining site with CD40, owns complete adjuvantfunction and reduce the immune response to mtHSP70 in body preferably. Only themtHSP70 cross-linking with polypeptide or fusion with target gene in molecular level isable to work as immunologic adjuvant. Because the fused big protein fragment or totallength protein is splited into numerous short peptides in MHCI presentation pathway,mtHSP70-polypeptide complex or mtHSP70-polypeptide fusion gene is the promisingvaccine to elicit antigenic specificity immune responds in individualities with differentMHC.In this study, the antigen was huaman hepatocellular carcinoma-associated antigens661 (HCA661), which was found in 2002 firstly, belongs to cancer/testis (CT) antigen andowns the following characters: ①It is expressed in hepatocellular carcinoma andminority melanoma selectively and not in the normal tissues except the spermatogenouscells in testis. ②It was identified by serological identification of antigens by recombinantexpression cloning (SEREX) and CTL reaction doubly, possesses the immunogenicity toelicit the humoral immunity and cell immunity. ③The gene locates in X chromosome.Since the tunica albuginea around the testis tissue acts as immunologic barrier, the hightumor restricted CT antigen is expected to develop antigenic specificity tumor vaccine asthe best candidate in tumor immunotherapy.Several molecular biology techniques were used for construction of recombinanteukaryotic expression plasmids pCI-HCA661 and pCI-HCA661-mtHSP70C. Sequencingresult showed that a point mutation in 831bp (G→A) between ATPase domain inNH2-terminal and polypeptide binding domain in C-terminal was found in the mtHSP70gene from bovine mycobacterium tuberculosis genome amplified by PCR, then an EcoRIsite was formed. HCA661 gene without stop codon and C-terminal of mtHSP70 wasfused together through the EcoRI site. The sequencing result showed that there was notreading frame displacement at EcoRI site. Concluded that recombinant plasmidpCI-HCA661-mtHSP70 was constructed successfully.To evaluate whether the target gene subcloned into these recombinant plasmids couldtranscript and translate effectively, another two recombinant eukaryotic plasmidspCI-eGFP and pCI-HCA661-eGFP were constructed and transfected into φA cells aswell as pCI-neo. It showed that the cells transfected with pCI-neo displayed noautofluorescence, and green fluorescence was observed in the cells transfected withpCI-eGFP or pCI-HCA661-eGFP. It was certificated that pCI-neo expression systemexpressed eGFP and fusion gene HCA661-eGFP well in the eukaryocytes in vitro.Concluded from above data, pCI-HCA661 and pCI-HCA661-mtHSP70C could expressproportional protein in the eukaryocytes and could be use as genetic vaccine in vivo toverify its immunological activity.To detect the humoral immunity effect elicited by genetic vaccine in the animalbodies, recombinant prokaryotic expression plasmid with 6 continuous histidine residuetag pQE-HCA661 was constructed. The protein purified by Ni-NTA metal affinitychromatography techniques and assessed by Western blotting with His monoclonalantibody. The result showed that there was a positive staining at 39kD. Concluded that theinterest protein is HCA661 about 39kD with 6 continuous histidine residue.In viro, the function of DNA vaccine has been carried out. The spleen cells andserum were separated respectively from the mice immunized with pCI-neo, pCI-HCA661and pCI-HCA661-mtHSP70. Changes of the T lymphocytes subset was analyzed by flowcytometry. The production of polyclonal antibody against HCA661 was analyzed byWestern blotting, immunohistochemistry and flow cytometry. It shows that: ① Thenumber of CD3+, CD4+, CD8+ T lymphocytes and the ratio of CD4+/CD8+ of themice immuned by pCI-HCA661 or pCI-HCA661-mtHSP70C increased significantlycompared with control ( P < 0.05 ) . The ratio of CD4 + /CD8 + ofpCI-HCA661-mtHSP70C group was notably higher than that of pCI-HCA661 group (P<0.05).②The flow cytometry result showed that the binding ratio of the antiserumimmuned by pCI-HCA661 or pCI-HCA661-mtHSP70C was higher notably compairedwith control(P<0.05). The binding ratio of pCI-HCA661-mtHSP70 group were morethan that of pCI-HCA661 group at the same dilution(P<0.05). ③SABC stain showedthat HCA661 protein expressed in SMMC7721 cells. There are no positive staining cellswhen the antiserum of pCI-neo group was used as the first antibody, but there are positivestaining cells when the antiserum of pCI-HCA661 or pCI-HCA661-mtHSP70 group wasused as the first antibody. The number and staining intensity of positive staining cells inpCI-HCA661-mtHSP70 group were more than that of pCI-HCA661 group at the samedilution. ④The Western blotting result showed that only the serum from mice immunedby pCI-HCA661 or pCI-HCA661-mtHSP70C could bind with recombinant proteinHCA661. And the antiserum of pCI-HCA661-mtHSP70C group did not recognizerecombinant protein mtHSP70. ⑤Using the antiserum of pCI-HCA661-mtHSP70Cgroup as the first antibody, SABC stain showed that the cytoplasm of spermatogenouscells in seminoma of testis was positive staining, and about 40% hepatocellularcarcinomas were positive staining. ⑥ELISA showed that the highest dilution presentingpositive reaction of the antiserum of pCI-HCA661 group was 1:4000,and that ofpCI-HCA661-mtHSP70C group was 1:8000. Concluded from above data: ①The geneticvaccine pCI-HCA661 and pCI-HCA661-mtHSP70 elicited antigen specificity andnon-specificity immune reaction effectively in mice. ②The immune response levelelicited by fusion genetic vaccine pCI-HCA661-mtHSP70 was more superior than thatelicited by pCI-HCA661 alone. ③The polypeptide carrier and immunologic adjuvant asthe C-terminal of mtHSP70 was, it can decrease the immune response to mtHSP70 atmaximum extent, and enhanced the immune action to the antigen fused with mtHSP70 inthe immuned animals simultaneously. ④HCA661 protein was located in the body ofcells and expressed in about 40% HCC..