[C3d enhances the immune response against HBV-preS2/S following gene immunization in mice].

OBJECTIVE: To investigate whether C3d can enhance the immune response to HBV-preS2/S induced by direct injecting naked plasmid containing three copies of C3d and HBV-preS2/S in fusion form. METHODS: HBV-preS2/S coding sequence was introduced into the eukaryotic expression vectors TR421 and TR421-C3d3 containing 3 copies of C3d respectively. PCR was used to identify the direction of insertion, DNA sequencing analysis was used on the positive clones. The recombinant plasmids TR421-preS2/S and TR421-preS2/S-C3d3 were transfected into the human breast cancer cells of the line 4T1 as a transient expression system. Semi-quantitative RT-PCR was used to detect the expression of HBV-preS2/S protein. TR421-preS2/S and TR421-preS2/S-C3d3 were injected into the anterior tibial muscles of female BALB/c mice, 6 in each group, 3 times at an interval of 3 weeks. Six mice were injected with blank plasmid TR421 as controls. The total blood was collected from the mice. ELISA was used to detect the level of specific anti-HBs antibody. The splenocytes of the immunized mice were collected and stimulated with HbsAg protein and then harvested to analyze the specific lympho-proliferative response by (3)H-thymidine incorporation assay. CR2 positive Raji cells were added with plasmid transfected and mouse anti-HBs serum. Flow cytometry was used to detect the integration of preS2/S-C3d3 fusion protein. RESULTS: The 4T1 cells transfected with TR421-preS2/S and TR421-preS2/S-C3d3 effectively expressed HBV-preS2/S, with the expression level of TR421-preS2/S-C3d3 lower by 15.25% than that of TR421-preS2/S. Twelve weeks after immunization, the specific anti-HBs-IgG level (A(490nm)) was 0.81 +/- 0.09 in the TR421-preS2/S primed mice, significantly higher than that in the blank plasmid injected mice (0.49 +/- 0.02, P < 0.05); and the specific anti-HBs-IgG level (A(490nm)) was 1.24 +/- 0.29 in the TR421-preS2/S-C3d3 immunized mice, significantly higher than that of the TR421-preS2/S primed mice (P < 0.001). The lympho-proliferative response of the TR421-preS2/S primed mice was significantly stronger than that of the blank plasmid injected mice (P < 0.05) and the lympho-proliferative response of the TR421-preS2/S-C3d3 primed mice was significantly stronger than that of the TR421-preS2/S primed mice (P < 0.05). Surface binding of protein could be detected only in the TR421-preS2/S-C3d3 transfected supernatant. About 43.19% of the CR2 positive Raji cells integrated C3d fusion protein. TR421-preS2/S transfected supernatant and negative control did not show obvious integration. CONCLUSION: C3d enhances the humoral and cell-mediated immune responses against HBV induced by gene immunization.

Contribution of C3d-P28 repeats to enhancement of immune responses against HBV-preS2/S induced by gene immunization.

AIM: To investigate whether P28 derived from C3d can enhance the immune response to HBV-preS2/S induced by directly injection of naked plasmids containing variable repeats of P28 and HBV-preS2/S in fusion form. METHODS: One to four copies of C3d-P28 coding gene, amplified by PCR and modified by restriction endonucleases digestion, were subcloned into a eukaryotic expression vector pVAON33 to construct pVAON33-P28, pVAON33-P28.2, pVAON33-P28.3 and pVAON33-P28.4 (pVAON33-P28.[1-4]). HBV-preS2/S coding sequence was then introduced into the pVAON33-P28.[1-4] and identified by both PCR and DNA sequencing. BALB/c mice were primed by intramuscular gene immunization with 100 microg different recombinant plasmids on day 0 and were boosted by subcutaneous inoculation with HBsAg protein (1 microg) 12 wk post-priming. The levels and avidity of specific IgG in sera collected at the indicated times from each group were determined by ELISA and NaSCN-displacement ELISA, respectively. RESULTS: HBsAg specific antibody response was elicited in groups primed with plasmids pVAON33-S2/S-P28.[1-4] and pVAON33-S2/S. However, the response against HBsAg in the groups primed with pVAON33-S2/S-P28.[1-4] was significantly higher than that in pVAON33-S2/S group, the highest level of the specific antibody response was observed in the groups primed with pVAON33-S2/S-P28.4 (P<0.01). After secondary immunization with specific antigen, the acceleration of antibody levels was significantly higher and faster in the mice primed with DNA expressing preS2/S-P28 fusions than that with DNA expressing preS2/S only (P<0.05). Interestingly, mice primed with DNA expressing preS2/S-P28.4 fusions maintained the highest levels of anti-HBs antibodies in all animals. The avidity assay showed that the avidity index (AI) collected at 18 wk from mice primed with pVAON33-S2/S-P28.3 and pVAON33-S2/S-P28.4 were significantly higher than that from preS2/S-DNA vaccinated mice (P<0.01). CONCLUSION: Different repeats of C3d-P28 can enhance both humoral immune response and avidity maturation of specific antibodies induced by gene immunization, in which four copies of C3d-P28 may be necessary to achieve the most modest antibody response.

[Specifics anti-tumor immunity induced by gene immunization with ectopic hCGbeta encoding gene].

OBJECTIVE: To investigate the specific anti-tumor immunity induced by gene immunization with ectopic hCG encoding gene. METHODS: BALB/c mice were immunized with plasmid TR421-hCGbeta coding for hCGbeta and mock DNA for 3 times at 3 weekly intervals. The level of specific anti-hCGbeta IgG antibody in the serum was determined by ELISA at the indicated time in the two groups. The growth inhibitory activity of the sera against tumor cells was examined in vitro by [(3)H]-Thymidine incorporation assay. Specific lympho-proliferation versus hCGbeta was detected by [(3)H]-Thymidine incorporation assay with hCGbeta protein or inactivated SP2/0-hCGbeta cells as specific stimulating antigen. Cytotoxic T lymphocyte (CTL) activity of the splenocytes derived from the immunized mice was measured by [(3)H]-Thymidine release assay. Protective assay was performed by subcutaneous inoculation of SP2/0-hCGbeta cells into the immunized mice. The weight and formation rate of the tumor were evaluated after challenge. RESULTS: All mice immunized with plasmid TR421-hCGbeta developed high level of anti-hCGbeta antibodies, which could inhibit the growth of Hela cells and SP2/0-hCGbeta cells compared with the serum from animals immunized with mock DNA (P < 0.05). The high-level specific lympho-proliferation against hCGbeta protein or/and inactivated SP2/0-hCGbeta cells were shown in TR421-hCGbeta immunized mice, whereas no significant proliferative activity was found in mock DNA immunized animals (P < 0.01). A strong cytotoxic activity against SP2/0-hCGbeta in TR421-hCGbeta immunized mice was found. Inoculation of SP2/0-hCGbeta cells into the mice immunized with mock DNA developed large tumors within 25 days. But a marked reduction of tumor weight and formation rate was found after the tumor cells challenge in the mice immunized with TR421-hCGbeta plasmid DNA (P < 0.01). CONCLUSION: The gene immunization of ectopic hCGbeta encoding gene, eliciting high-level of specific humoral and cellular immune responses, could inhibit the growth of tumor cells harboring ectopic hCGbeta in vitro and in vivo.

The distinct effects of three tandem repeats of C3d in the immune responses against tumor-associated antigen hCGbeta by DNA immunization.

Several examples have shown that C3d3, when fused to a corresponding antigen, had a strong adjuvant effect on certain specific antibody production. In a previous study, we attempted to prove that this was the case of the human chorionic gonadotrophin beta chain (hCGbeta)-induced immunity following DNA vaccination. However, we found that C3d3 when fused to hCGbeta inhibited rather than enhanced the antigen-specific immune response. In the present study, using hCGbeta DNA vaccine preparations, we demonstrated that C3d3 inhibited the antigen-specific humoral antibody response and several other immune responses, such as the hCGbeta specific lymphoproliferation. Such inhibitory effects of C3d3 were not related to the expression level of the target protein, the gender of the test mice, or the vector used. Contrastingly, C3d3 fused with the envelope protein of hepatitis B virus (PreS2/S) used as a control system resulted in the enhancement of both humoral and cell-mediated antigen-specific immune responses against HBV-preS2/S, which was consistent with other groups' adjuvant-effect findings. We further showed that the mechanisms involved in the inhibitory effect of C3d3 might be possible due to impairing the function of antigen presenting B lymphocytes and reducing the expression of transcription factors (T-bet and GATA-3) and cytokine IL-4. Collectively, unlike its usual expected adjuvant function, the fusion of C3d3 with the tumor-associated antigen hCGbeta was found to inhibit both humoral and cell-mediated antigen-specific immune responses. These findings indicate that research concerning tumor immune escapes and vaccine designs require further extensive attention.

[Enhancement of HBV gene-induced specific cell-mediated immunoresponse by C3d-P28].

AIM: To investigate whether P28 derived from complement C3d can enhance the cell-mediated immunoresponse to HBV-preS2/S induced by direct injection of naked plasmid DNA containing four tandem repeats of C3d-P28 gene and HBV-preS2/S gene existed in fusion form. METHODS: Four copies of gene coding for C3d-P28, amplified by PCR and cut by restriction endonucleases digestion, were subcloned into a eukaryotic expression vector pVAON33 to construct pVAON33-P28.4. HBV-preS2/S gene was then introduced into the pVAON33 and pVAON33-P28.4 respectively to form pVAON33-S2/S and pVAON33-S2/S-P28.4. The recombinant plasmids were identified by PCR and restriction endonucleases digestion as well as DNA sequencing. BALB/c mice were immunized i.m. three times at 3 weeks' intervals with 100 microg of pVAON33-S2/S DNA, pVAON33-S2/S-P28.4 and mock DNA, respectively. Splenocytes from immunized mice were stimulated by HBsAg and then harvested to analyze the specific lymphocytic proliferative response and CTL cytotoxic activity by (3)H-TdR incorporation assay and isotopic release analysis, respectively. RESULTS: Specific lymphocytic proliferation and CTL cytotoxic activity against HBV-preS2/S were observed in mice immunized by both pVAON33-S2/S and pVAON33-S2/S-P28.4 in dose-dependent form. Specific lymphocytic proliferation and CTL response in mice immunized by pVAON33-S2/S-P28.4 were markedly stronger than those in mice immunized by pVAON33-S2/S. CONCLUSION: A C3d-P28 can enhance the cell-mediated immunoresponse induced by HBV-preS2/S gene immunization.