| The research of immuno-contraceptive vaccine is mainly focused on three target antigens, namely zona pellucida, sperm antigens, and implantation-associated antigens. Human chorionic gonadotropin (hCG) is a member of the glycoprotein hormone family. Like the other members of this family, hCG is a heterodimeric molecule consisting of an a-subunit (hCGα) common to all other members of the glycoprotein hormone family, non-covalently associated with a hormone specific β-subunit (hCGβ). The hCG is initially expressed by the syncytiotrophoblast in the early blastocyst following implantation, and an early signal for conception. The most well characterised biological function of hCG is its ability to maintain life of the corpus luteum and the corresponding progesterone production via receptors present on the luteinised cells. Because of the physiological and temporal specificity, the hCGβ has been as a target antigen to develop immuno-contraceptive vaccine for several decades. Although the hCGβ vaccine based on synthetic peptide have undergone clinical trials of II phase, none is fully applicable for manufacture and widespread application for birth control so far, because of the poor immunogenicity, the weak Th2 humoral immunity, and the antibody response diversity from recipient to recipient. Hence, it is imperative to study a new hCGβ contraceptive vaccine that can improve the antibody response and diminish the individual differences in population.DNA vaccination, namely genetic vaccination, induces protective immunity against a variety of pathogens DNA inoculation. Previous studies have demonstrated that DNA vaccination effectively induces immune responses to different infectious immunogens. Although hCGβ gene vaccine has high specificity and no carrier-mediated inhibition, there are some challenges for its practical application,such as poor immunogenicity and the Thl bias of immune response. Molecular adjuvant C3d3 should be a valuable breakthrough for the hCGp gene contraceptive vaccine.C3d is one of the final degradation products of the third complement protein, C3. The effect of C3d3 on selectively enhancing humoral immune response was demonstrated by a series of studies. The C3d receptor, CD21 or CR2, is located on B cells, follicular dendritic cells (FDC), and possibly some epithelial tissues. A consequence of complement activation is the covalent attachment of the C3d to antigen. On B lymphocytes, C3d in turn binds to CD21 and amplify B lymphocyte activation. Thus, Gene fusion of molecular adjuvant C3d3 to hCGp would achieve the goal of hCGp gene contraceptive vaccine that enhancing the humoral immune response and diminishing the individual difference.1. The preparation and expression in vivo of eukaryotic vectors pCMV4-hCGp-C3d3, pCMV4-hCGp and pCMV4-C3d3The eukaryotic expression vectors pCMV4-hCGp-C3d3, pCMV4-hCGp and pCMV4-C3d3 were large-scale prepared. BALB/c and C57BL/6J mice were inoculated with pCMV4-hCGp-C3d3 and pCMV4-hCGp respectively, followed by a boost in 3 week interval. The transcriptions in vivo were analyzed using RT-PCR at the third or the sixth week after the primary inoculation. The results showed that the pCMV4-hCGp-C3d3, PCMV4-hCGp and PCMV4-C3d3 appeared to the correct by restriction endonucleases analysis, and both pCMV4-hCGp-C3d3 and pCMV4-hCGP had been expressed in the muscles of the inoculated sites in the two strains mice. It was suggested that the hCGp gene vaccines were expressed continually in the different strains mice.2. Molecular adjuvant C3d3 fused to hCGp enhanced Th2-biased anti-hCGp* humoral response in gene immunization in different strains miceThe BALB/c and C57BL/6J mice were inoculated intramuscularly with pCMV4-hCGp-C3d3, pCMV4-hCG(3 alone or pCMV4-hCGp plus pCMV4-C3d3respectively, followed by a boost in the same dosage and immunizing pathway in 3 week interval. The titers and subclasses of anti-hCGP IgG antibody were determined by indirect enzyme-linked immunosorbent assay (ELISA). Avidity of anti-hCGP antibody was analyzed using Sodium thiocyanate (NaSCN)-displacement ELISA. The findings revealed that both in BAL B/c and C57BL/6J mice, the hCGp-C3d3 DNA immunization achieved an earlier and stronger antibody response than hCGp alone or hCGp plus C3d3, and C3d3 showed the strongest adjuvant efficiency at the dose of 50pmol. The IgGl / IgG2a ratio of the hCGP-C3d3 immunizing group was apparently higher than that of the hCGP immunizing group or the hCGp plus C3d3 immunizing group, and the avidity maturation of the antibody was accelerated in inoculation with hCGP-C3d3 than hCGp alone or hCGp plus C3d3. It demonstrated that the molecular adjuvant C3d3 could significantly increase the antigen-specific antibody response, and promote antibody IgG subclass turnover in hCGp DNA vaccine in mice of different strains.3. The molecular adjuvant C3d3 selectively promoted clonal expansion of B cell on response to hCG antigen recharge in vitro in BAL B/c miceThe BALB/c mice were inoculated with 50pmol pCMV4-hCGp-C3d3 or pCMV4-hCGp respectively, followed by a boost in 3 week interval. The proliferation of B cell and T cell in each group was analyzed using MTT assay, while the specific anti-hCGP IgG antibody secreting cells in hCGP-C3d3 and hCGp immunization were evaluated with ELISPOT assay. It was showed that in 1 or 2 weeks after the boost immunization, the proliferation of B cell in hCGP-C3d3 immunization is significantly higher than that of hCGP and the control, while no significant difference was found in T cell proliferation among the three groups. Compared to hCGP immunization, both secretion of the anti-hCGP IgG antibody and number of the antibody secreting cells were obviously increased in hCGp-C3d3 immunization. The molecular adjuvant C3d3 appeared to promote selectively a clonal expansion of B cell.4. The molecular adjuvant C3d3 up-regulated both B7-1 and B7-2 expression on Raji cells...
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