Human chorionic gonadotropin (hCG) has been used as an anti-fertility vaccine and as a target for cancer immunotherapy. We have explored the use of three copies of C3d in DNA vaccine as molecular adjuvant to improve the immunogenicity of this hormone in previous work and found that the immune response induced by pcDNA3-hCGβ-C3d3 has been enhanced 243-fold compared with pcDNA3-hCGβ following DNA immunization in BALB/c mice. In the present study, a new functionally active DNA vaccine of hCGβ-C3d3 chimera based on pCMV4 vector has been described. We compared the expression efficiency of pCMV4 and pcDNA3 eukaryotic vectors for hCGβ and hCGβ-C3d3 fusion protein and the immune response of mice immunized with pcDNA3-hCGβ, pCMV4-hCGβ, pcDNA3-hCGβ-C3d3 and pCMV4-hCGβ-C3d3, respectively, at 25, 50 and 100 pmol dose, and further analyzed the levels of Th1 and Th2 cytokines produced by spleen lymphocytes of the immunized mice upon hCG restimulation in vitro. It was found that pCMV4 vector achieved 1.3-1.5-fold higher protein expression and raised 1.1-1.2 (primary) and 1.2-1.3 (booster) logs higher titer of anti-hCGβ IgG than pcDNA3. Mice vaccinated with 50 pmol of hCGβ-C3d3-DNAs elicited the highest titer of hCGβ-specific antibody among the serial doses and the immune response induced by pCMV4-hCGβ-C3d3 were, respectively, 1.3, 1.3 and 1.2 logs higher than that of pcDNA3-hCGβ-C3d3 and 2.2, 2.9 and 2.4 logs higher than that of pCMV4-hCGβ at week 2 following the booster immunization. Moreover, we observed that the production of IL-4 and IL-10 increased in mice vaccinated with hCGβ-C3d3-DNAs and the ratio of IL-4/IFN-γ showed a Th2 bias of immune response in the mice immunized with hCGβ-C3d3-DNAs. These findings indicated that gene fusion of C3d3 to hCGβ, as a means of harnessing the adjuvant potential of the innate immune system, may improve the antigen-specific Th2 humoral immune response of the hCGβ DNA vaccine and the pCMV4 vector is a more ideal eukaryotic vector for DNA vaccine than pcDNA3. Copyright Sons, Ltd.

The aim of this study was to investigate whether CXCL16/CXCR6, a newly identified chemokine pair, is expressed in first-trimester human placenta and whether they affect the trophoblast cell biology, since we have found CXCR6 highly transcribed in first-trimester human trophoblast cells previously. METHODS: We analysed the transcription and translation of CXCR6 and CXCL16 in purified first-trimester human trophoblast cells by real-time RT-PCR and immunochemical staining. We then examined the kinetic secretion of CXCL16 in the supernatant of primary-cultured trophoblast by enzyme-linked immunosorbent assay. We further investigated effects of CXCL16 on the proliferation and invasion of trophoblast cells in vitro. RESULTS: We found the chemokine pair CXCL16/CXCR6 was transcribed and translated in first-trimester trophoblast cells and JAR line. In addition, the primary-cultured trophoblasts secreted CXCL16 spontaneously and continuously in 100-h culture. Treating trophoblasts with CXCL16 induced marked proliferation and invasion in vitro. CONCLUSION: The findings from this study have demonstrated for the first time that CXCR6 and CXCL16 are co-expressed by first-trimester human trophoblast cells and stimulate their proliferation and invasion in an autocrine/paracrine manner. It suggests that CXCL16 plays important roles in human extravillous cytotrophoblast invasion and placentation.

To show that an anti-human chorionic gonadotrophin-β (hCGβ) antibody response can be induced by inoculating Lb. Expressing hCGβ through diVerent mucosal pathways in mice of two strains, female BALB/c and C57BL/6 mice were immunized via vaginal, oral or nasal routes with 108, 109, and 1010 Lb.hCGβ (a recombinant Lactobacillus expressing hCGβ). The mice were immunized twice with a booster in study week 3. An indirect ELISA was used to determine anti-hCGβ IgG and IgA antibodies in vaginal lavage and serum, obtained from the 2nd to 8th week after the primary immunization. Flow cytometry was used to analyze the lymphocyte proliferation from these tissues, 1 week after the primary immunization. The hCGβ antigen-speciWc antibody-secreting cells of spleen, uterus, and vagina were evaluated by enzyme-linked immunospot assay (ELISpot), 2 weeks after the booster. The analysis showed that 109 and 1010 Lb.hCGβ inoculations induced similar anti-hCGβ antibody responses, while the three mucosal pathways induced similar antibody responses. The antiserum obtained after boosters with 109 and 1010 Lb. hCGβ was able to neutralize more than 100 ng/ml hCGβantigen, both in BALB/c and C57BL/6 mice. The highest antibody titer induced by vaginal mucosal immunization was stronger than that obtained via the other mucosal pathways. The B cells in the vagina appeared to proliferate after vaginal immunization (P <0.05). The numbers of anti-hCGβ IgG and IgA antibody-secreting cells in the uterus and vagina were greater than in the spleen. Therefore, the vaginal mucosal route appears to be a better immunization pathway to induce higher anti-hCGβ antibody levels in the reproductive tract.

CD28/CTLA-4 interactions with their specific B7-ligands (CD80 and CD86) play a decisive role in antigenic and allogenic responses. Recently, experimental transplant studies demonstrated that donor-specific tolerance was achieved by blocking these interactions. However, the role of blockade of CD28/B7 costimulatory pathway in the maintenance of materno-fetal tolerance has received little attention. In the present study, abortion-prone CBA/J females mated with DBA/2 males were administered with anti-CD80 and anti-CD86 monoclonal antibodies (mAbs) on day 4 of gestation (time of murine implantation). We demonstrated that the combined use of anti-CD80 and anti-CD86 mAbs induced maternal tolerance to the fetus in the abortion-prone CBA/J mice, and displayed expansion of the maternal CD4+CD25+ regulatory T cell population and up-regulated expression of CTLA-4, suggesting an active mechanism of regulatory T cells in suppressing maternal rejection to the fetus. In addition, the anti-CD80/86 mAbs treatment enhanced Th2 and reduced Th1 cytokine production in mice, implying that the development of Th2 cells might contribute to maternal tolerance to her fetus. Together, these findings indicated that blocking CD80 and CD86 enhanced maternal tolerance to her fetus in mice by increasing regulatory T cell function and skewing toward a Th2 response. Our data might provide an enhanced understanding of the maternal-fetal immune relationship and be helpful in clinical trials for immunotherapy of recurrent spontaneous abortion.

Objectives: To test the possibility of vaccination with lactobacillus expressing hCGβ antigen administered by vaginal mucosal immunization. Methods: A plasmid pIlac-hCGβ was constructed and then transfected into Lactobacillus casei CECT5276, which stably expressed hCGβ protein. RIA was used to detect hCGβ in the culture supernatant and cell lysate.Western blotting was performed to evaluate the expressed protein of interest. Female BALB/c mice aged 6–8 weeks received inoculations in the vagina of the recombinant L. casei CECT5276. ELISA was used to determine the anti-hCGβ IgA antibody in vaginal lavage fluid from the BALB/c mice after vaginal mucosal immunization. Results: The pIlac alone appeared to have a higher efficiency than pIlac-hCGβ, and the highest transfection efficiency of both plasmids was at pulse voltages of 1200V and 1500V. About 78.5% of the hCGβ protein was excreted into the culture supernatant. Excretion of hCGβ was most efficient when the pH of the culture medium was adjusted to around 7.0 and the concentration of lactose was around 1%. The hCG protein in the vaginal lavage fluid of these BALB/c mice was positive on the third day after vaginal inoculation. Anti-hCGβ IgA antibody continued to be found in the vaginal lavage fluid for 2 weeks following a booster vaginal inoculation. The splenic lymphocytes of the mice immunized with hCGβthrough the vagina underwent a proliferative reaction to hCGβ antigen restimulation in vitro. Interferon gamma (IFN-γ) and interleukin (IL)-4 were secreted at higher levels after vaginal mucosal immunization of L. casei expressing hCG than after vaginal mucosal immunization of L. casei alone. Conclusions: Vaginal immunization of lactobacillus expressing hCG induced an antihCGβ antibody response in the murine vaginal mucosa. Induction of the antigen-specific antibodies in the reproductive tract following vaginal inoculation of recombinant lactobacillus will lead to the development of a safe, efficient, and easy-to-use form of immunocontraception.