| | Fusion of hC3d3 to hCGβ enhances responsiveness in vitro of human peripheral immunocompetent cells upon the antigen primary challengeReceived 8 February 2007; received in revised form 17 January 2008; accepted 7 March 2008. published online 12 May 2008. Abstract Contraceptive vaccines based on hCGβ have not met clinical application because of poor immunogenicity. In the present study, the eukaryotic expression vectors pCI-gs-signal-6His-hCGβ and pCI-gs-signal-6His-hCGβ-hC3d3 were constructed, and transfected into CHO cells with aid of Lipofectaine 2000 reagent to gain the secretory recombinant protein. Isolated B cells from human peripheral blood, combined B cells with T cells, and PBMC were treated in vitro, respectively, with 1 nM, 10nM, 100nM hCGβ, hCGβ-hC3d3 or PWM for 12 days. Immunoglobulin (Ig) and anti-hCG antibody levels in the supernatant were measured by an indirect enzyme-linked immunosorbent assay (ELISA). The expressions of CD80/CD86 on B cells, and CD154/CD25 on T cells, were analyzed by flow cytometry (FCM), and IL-2 production was assayed by ELISA. It was found that the Ig levels in the B-cell supernatants, the combined B with T cells, and PBMC treated with 100nM hCGβ-C3d3 fusion protein were 4-fold, 10-fold and 10.9-fold more, respectively, than that of hCGβ. The anti-hCG antibody could be produced in the combined B cells with T cells, as well as PBMC challenged with 100nM hCGβ-C3d3, but no anti-hCG antibody was produced in the challenge with hCGβ. The hCGβ-hC3d3 fusion protein enhanced the expression of CD80 and CD86 on B cells, especially CD86 (P<0.05), and significantly increased the expression of CD154 and CD25 molecules on T cells compared to that of hCGβ (P<0.05). The hCGβ-hC3d3 promoted human PBMC producing more IL-2 than hCGβ. These findings indicate that the fusion of hC3d3 to hCGβ, as a means of harnessing the adjuvant potential of the innate immune system, may contribute to a more efficient humoral immune response, and might provide a potential application of protein vaccine strategies in humans in the future. 1. Introduction  Contraceptive vaccines based on hCGβ have undergone clinical trials. A vaccine supported by the World Health Organization and developed by Stevens and coworkers used a 37 aminoacid carboxy-terminal peptide (CTP) of the hCGβ-chain (Stevens, 1999). In phase I clinical trials, the vaccine appeared to be safe and did not provoke any autoimmune-mediated side effects (Rose, 2000). However, the local reactions at the injection site were observed in some volunteers in phase II trials. Furthermore, the avidity of specific antibodies in women immunized with CTP was lower than in women immunized with intact hCGβ (Cekan and Aedo, 1998). To date, a separate heterospecies vaccine consisting of ovine α-chain noncovalently associated with hCG β-chain conjugated to either tetanus toxoid (TT) or diphtheria toxoid (DT), is the only antifertility vaccine to have completed phase II clinical trials to establish efficacy in humans (Talwar et al., 1994, Talwar, 1999). The heterospecies dimer was successful in preventing pregnancy in moderate to high responders to the vaccine, with only one pregnancy recorded out of 1224 cycles in those immunized females who developed a level of circulating neutralizing antibody >50ng/ml. However, although all women in the trial produced hCG-reactive antibodies in response to the vaccination, 20% of the 148 females given the initial three primary injections were low responders and failed to develop protective levels of the antibody. The technology underpinning hCGβ vaccine development requires innovations such as the use of cytokines or novel adjuvants to induce selectively specific classes of the immune response. In previous work, we have successfully constructed eukaryotic vectors of pcDNA3-hCGβ, pcDNA3-hCGβ-mC3d3, phCMV1-hCGβ and phCMV1-hCGβ-mC3d3, and found the humoral immune response of pcDNA3-hCGβ-mC3d3 to be enhanced 243-fold compared with that of pcDNA3-hCGβ following DNA immunization in BALB/c mice (Li et al., 2003). However, the antibody titer of hCGβ-mC3d3 immunization was 1995-fold higher than that of hCGβ alone in protein immunization 2 weeks following the booster immunization in BALB/c mice, which suggests that the protein vaccine can significantly increase the immunogenicity compared to DNA vaccination, and three copies of C3d (C3d3) can be used to improve the immunogenicity of weakly immunogenic antigens (Wang et al., 2004). In order to apply the immunocontraception vaccine in humans, we constructed the eukaryotic vectors pCI-gs-signal-6His-hCGβ and pCI-gs-signal-6His-hCGβ-hC3d3. In the present study, we have first compared the expression efficiency of these eukaryotic vectors with the previous constructed pCMV1 and pcDNA3 for the hCGβ and hCGβ-C3d3 fusion protein, and found the expression efficiency of pCI is significantly higher than pCMV1 and pcDNA3. Thereafter, isolated B cells, combined B cells with T cells, PBMC from normal blood donors, or Raji cells were treated in vitro, respectively, with 1nM, 10nM, 100nM hCGβ, hCGβ-hC3d3 or PWM for 12 days. Immunoglobulin (Ig) and anti-hCG antibody levels were measured by an indirect ELISA. The expressions of CD80/CD86 and CD154/CD25 were analyzed by flow cytometry, and the production of IL-2 assayed by ELISA so as to evaluate the potential application of the molecular adjuvant in a human contraceptive vaccine. 2. Materials and methods 2.1. Construction of the eukaryotic expression vectors of pCI-gs-signal-6His-hCGβ and pCI-gs-signal-6His-hCGβ-hC3d3 The eukaryotic expression vector pCI-gs (provided by Fudan-Zhangjiang Biopharmaceutical Co. Ltd.) contains resistant gs gene at the downstream of the polyclone sites. Human C3d gene fragment was cloned from human hepatic cDNA library by PCR techniques, and cloned into pMD18-T simple vector. The junction of three copies of the human homologue of C3d, C3d3, were made by flexibility adaptor (G4S)2, and the hCGβ and hCGβ-C3d3 segments as a EcoRI–EcoRI fragment, respectively, from pMD18-T-signal-6His-hCGβ and pMD18-T-signal-6His-hCGβ-hC3d3 and cloned into pCI-gs digested with EcoRI (Fig. 1). The 6His tag was cloned at the C-terminal of the signal peptide and the N-terminal of the protein of interest, which ensured that all recombinant proteins secreted into the supernatant contained the 6His tag for purification. All plasmids were purified with an endotoxin-free purification kit (tip-2500 kit, Qiagen, Hilden, Germany) prior to transfection. 2.2. Expression of hCGβ and hCGβ-C3d3 fusion protein in CHO cells and characterization by Western blotting and Raji cell immunocytochemistry CHO cells were transfected with pCI-gs-signal-6His-hCGβ and pCI-gs-signal-6His-hCGβ-hC3d3, respectively, with aid of Lipofectamine™ (Invitrogen Life Technology, Carlsbad, USA) according to the manufacturer's guidelines. Resistant clones secreting the protein of interest were screened on gradient concentrations of MSX. Clones were selected by resistance to MSX, and supernatants were harvested 24h after transfection and stored at −20°C. The concentration of hCGβ in the culture supernatant was determined by a chemiluminescent assay. For Western blot analysis, the supernatant was concentrated to one-fiftieth of the volume, and then dialyzed against phosphate-buffered saline (PBS) at 4°C for 24h. Then, the products were loaded on a 10% polyacrylamide-sodium dodecyl sulfate (SDS) gel. The resolved proteins were transferred onto a nitrocellulose membrane (Schleicher & Schuell, Whatman, UK) and incubated with a 1:1000 dilution of mouse anti-hCG monoclonal antibody (NeoMarkers, Fremont, CA) in PBS containing 0.05% Tween 20 and 5% fetal calf serum (Invitrogen Life Technology). After washing, bound primary antibodies were detected using a 1:2000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Kirkegaard & Perry Laboratories, UK) and a chemiluminescent detection system. Raji cells were used in immunocytochemistry to identify the hCGβ and hCGβ-hC3d3 fusion protein, respectively. Raji cells, a B-cell line which expresses the receptor for the C3d molecule, CR2, spread on poly-l-lysine glass slides, were incubated with culture supernatants from transfected CHO cells and then incubated with a 1:200 dilution of murine anti-hCGβ antibody. The bound primary antibody was subsequently detected using biotin-anti-mouse IgG, and 50μl S-A/HRP were added to each well, and developed by DAB substrate. 2.3. Indirect ELISA for determination of Ig and anti-hCG antibody The recombinant proteins of interest were purified using immobilized metal affinity chromatography under native conditions, and Sephadex G150 column gel filtration chromatography. B-cell and T cells were isolated with MicroBeads from peripheral blood (B/T Cell Kit Miltenyi Biotec Inc., Auburn, USA). The isolated B cells, the combined B cells with T cells and PBMC were treated in vitro, respectively, with 1nM, 10nM, 100nM hCGβ, hCGβ-hC3d3 or PWM for 10–12 days. Supernatants were harvested to detect the titers of Ig according to the biotinylated anti-human immunoglobulin kit. An indirect enzyme-linked immunosorbent assay (ELISA) was performed to assess the titers of anti-hCG antibody in the supernatants using purified hCG (0.5μg/ml; Sigma, St. Louis, USA) to coat plates, and nonspecific sites were saturated with 5% of FCS in PBS. After incubation with supernatants at 37°C for 2h, biotinylated anti-human IgG (Vector Laboratories Inc., Burlingame, USA) was added at 1:1000 dilution. After washing, bound antibody was incubated with HRP-avidin at 37°C for 1h. 3,3,5,5-Tetramethylbenzidine (TMB) was employed as substrate. The optical density (OD) values of each well were measured at 450nM after termination of the reaction by 2M H2SO4. 2.4. FCM for expression of CD80/CD86 and CD154/CD25 on human immune cells Isolated B cells, combined B cells with T cells, and PBMC were treated in vitro, respectively, with 1nM, 10nM, 100nM hCGβ, hCGβ-hC3d3 or PWM for 48h. The expressions of CD80/CD86 and CD154/CD25 on these cells were analyzed by flow cytometry. Briefly, cells were prepared by simple cell suspension at a concentration of 3×105ml−1, washed twice with PBS, and incubated at 4°C for 20min with FITC-conjugated mouse anti-human CD3, CD19, PE-conjugated mouse anti-human CD86, CD154, PE/Cy5-conjugated mouse anti-human CD80, CD25, and the relevant isotype control, respectively (Biolegend, San Diego, USA). All analyses were conducted on a BD Biosciences FACScan flow cytometer using CellQuest software. Three independent experiments were performed, each in triplicate. 3. Results 3.1. Expression efficiency of pCI eukaryotic vectors and characterization for hCGβ and hCGβ-C3d3 fusion protein Western blotting analysis revealed that CHO cells transfected by pCI-gs-signal-6His-hCGβ and pCI-gs-signal-6His-hCGβ-hC3d3 could express efficiently the corresponding recombinant protein with expected molecule sizes in a secreted form. The hCGβ was 24kDa, and the higher molecular weight band at 128kDa is consistent with the hCGβ-C3d3 fusion protein (Fig. 2). The successful fusion of hCGβ to hC3d3 was demonstrated by Raji cell immunocytochemistry for the hCGβ and hCGβ-hC3d3 fusion protein (Fig. 3). 3.2. Ig and anti-hCGβ antibody secretion by B cells challenged with hCGβ or hCGβ-hC3d3 fusion protein The isolated B cells, combined B cells with T cells, and PBMC were treated, respectively, with hCGβ, hCGβ-hC3d3 and PWM at serial of concentrations 0nM, 1nM, 10nM, 100nM, and the results in Fig. 4 show that the Ig level in culture supernatants at the dose of 100nM was highest, and thus 100nM was considered as the optimal dosage in this study. The level of Ig produced by hCGβ challenge was almost too low to detect in spite of the optimal concentration of 100nM. The Ig levels secreted by isolated B cells, combined B cells with T cells, and PBMC challenged with hCGβ at the dose of 100nM were 79.59±23.18pg/ml, 65.77±21.21pg/ml, 122.14±38.17pg/ml, respectively. The Ig levels secreted by isolated B cells, combined B cells with T cells, and PBMC challenged with hCGβ-C3d3 at the dose of 100nM were 316.91±87.10pg/ml, 643.73±101.72pg/ml, 1324±162.17pg/ml, respectively, and were respectively 4-, 10- and 10.9-fold more than that of hCGβ challenge. After the isolated B cells, combined B cells with T cells, and PBMC were treated, respectively, with hCGβ, hCGβ-hC3d3 or PWM at the dose of 100nM for 12 days, only the combined B cells with T cells and PBMC treated with hCGβ-C3d3 fusion protein produced specific anti-hCG antibody (Fig. 5). 3.3. Expression of CD80/CD86 and CD154/CD25 on human immune cells after challenge by hCGβ or hCGβ-hC3d3 fusion protein Isolated B cells, the combined B cells with T cells, and PBMC were treated, respectively, with hCGβ, hCGβ-hC3d3, hCGβ-hC3d3 plus anti-CD21Ab or PWM at 100nM. The expressions of CD80/CD86 on B cells (labelled with mouse anti-human CD19-FITC) and CD154/CD25 on T cells (labelled with mouse anti-human CD3-FITC) in all groups were analyzed by flow cytometric assay. The expression of CD86 on isolated B cells, the combined B cells with T cells and PBMC, and CD154/CD25 on T cells in the combined B cells with T cells and PBMC challenged with hCGβ-hC3d3, were significantly higher than that of hCGβ challenge, and the increased expression could be blocked efficiently by anti-CD21Ab. Expression of CD80 on isolated B cells and CD25 on T cells in PBMC treated with PWM were significantly higher than the other groups (Fig. 6). 4. Discussion hCG, a hormone that indicates the presence of the embryo, is considered essential for both establishment and maintenance of pregnancy. In principle, induction of immunity against hCG should lead to a sequence of normal, or slightly extended, menstrual cycles during which pregnancy would be terminated around the blastocyst stage of embryo. The elegance of this approach is emphasized by its potential reversibility. A dimeric vaccine consisting of ovine α-chain noncovalently associated with hCGβ-chain conjugated to either tetanus toxoid (TT) or diphtheria toxoid (DT), was constructed by Talwar et al. (1994). The phase I clinical trials involving 101 women resulted in generation of high anti-hCG antibody titers, with high avidity and enhanced bioactivity. A phase II efficacy trial had been completed with only one pregnancy observed in 1224 cycles in females producing levels of neutralizing antibodies above 50ng/ml. However, the approach is far from becoming a reality because of its insufficient immunogenicity. To enhance the immunogenicity of the hCGβ contraceptive vaccine, we have chosen C3d3 as a molecular adjuvant of hCGβ DNA vaccine based on pcDNA3 (Li et al., 2003), phCMV1 (Wang et al., 2004) and pCMV4 (Wang et al., 2006) vector, and found that the humoral immune response of pcDNA3-hCGβ-C3d3, phCMV1-hCGβ-C3d3, and pCMV4-hCGβ-C3d3 has been enhanced compared to pcDNA3-hCGβ, phCMV1-hCGβ, and pCMV4-hCGβ following DNA immunization or protein inoculation in BALB/c mice. In order to apply the hCGβ contraceptive vaccine in humans, in the present study, we have conjugated a new hCGβ-hC3d3 fusion vaccine containing the molecular adjuvant based on the pCI vector. The expression efficiency of pCI-gs-signal-6His-hCGβ and pCI-gs-signal-6His-hCGβ- hC3d3 was higher than that of pcDNA3 and phCMV1 for hCGβ and hCGβ-C3d3 fusion protein in mammalian cells. The Ig level produced by human B cells challenged with hCGβ-C3d3 were 4-, 10-, and 10.9-fold, respectively, more than that of hCGβ; specific anti-hCG antibody could be found only in the supernatant of B cells treated with hCGβ-C3d3, but anti-hCG antibody was too low to detect in the supernatant of B cells treated with hCGβ. The cloned molecular adjuvant hC3d3 induced of more Ig and anti-hCGβ antibody synthesis by in vitro cultured B cells compared to hCGβ alone. C3d, a split product of C3, interacts with its receptor (CR2 or CD21) on B cells and follicular dendritic cells (FDCs) and is crucial for induction and maintenance of a normal humoral immune response (Carroll, 2000). It has been shown that DNA immunization with plasmids encoding influenza virus hemagglutinin (Ross et al., 2000), measles virus hemagglutinin (Green et al., 2002) or HIV envelope protein gp120 (Green et al., 2003) covalently linked to three C3d domains elicited a much higher level of antigen-specific antibodies of enhanced affinity than these antigens alone. Recently, Terrazzini et al. (2004) found also that C3d3 did not exert an adjuvant activity on hCGβ DNA vaccine. The differences in conclusions may be explained by differences in experimental approach. Dempsey et al. (1996) originally proposed that the adjuvant effect of C3d bound to antigens was mediated through coligation of the CD19/CD21 complex with a HEL (hen egg lysozyme)-specific B-cell receptor (BCR). C3d has been postulated to augment humoral responses by targeting Ag complexes to B cells and FDCs that express CD21/35. On B cells, coligation of the BCR and the CD19/CD21 complex by C3d–Ag complexes is proposed to lower the signaling threshold required for B cell activation and expansion (Carter et al., 1988, Carter and Fearon, 1992, Fingeroth et al., 1989). C3d3 is proposed to play an important role in follicular localization of antigen and generation of germinal centers (Banki et al., 2005, Breukels et al., 2005). Moreover, C3d–CD21 interactions may allow antigen retention on FDC, increasing interactions between them and B cells within germinal centers, thus providing signals protecting B cells from apoptosis (Fischer et al., 1998). Cherukuri et al. (2001) demonstrated that binding of complement-tagged antigens stimulates translocation of both the BCR and CD19/CD21 complex into lipid rafts, resulting in prolonged residency in and signaling from the rafts compared to BCR cross-linking alone. This is in the agreement with results obtained later (Pierce, 2002). But, Haas et al. (2004) showed that C3d can function as a molecular adjuvant in the absence of CD21/35 expression. Fakher et al. (2001) and O’Rand and Lea (1997) suggested that T cells were not necessary for the adjuvant effect of C3d in the primary immune response, which indicates that the molecular adjuvant C3d can enhance humoral immunity without the restriction of MHC. This advantage will be very promising for constructing contraceptive vaccines to break through the current obstruction in immune contraception. In our previous research, we demonstrated that hCGβ-mC3d3 can up-regulate both B7-1 and B7-2 on Raji cells, and is blocked by anti-CD21mAb (Yu et al., 2006). Kozono et al. (1998) showed the same results. In the present study, isolated B cells, combined B cells with T cells and PBMC were treated, respectively, with hCGβ and hCGβ-hC3d3. hCGβ-hC3d3 enhances the expression of CD86 on B cells and CD154/CD25 on T cells compared to hCGβ, which can be blocked by anti-CD21mAb. Meanwhile, production of IL-2 by PBMC treated with hCGβ-hC3d3 was significantly higher than that of hCGβ, and the increase could be blocked by anti-CD21Ab. It is evidenced that the hCGβ-hC3d3 fusion protein involved in activation of B cells and T cells, and helpful for the interaction of B cells with T cells, results in secretion of antibody and the enhanced humoral immune response. The enhanced humoral immune response of the hCGβ-hC3d3 protein vaccine may be explained by the following mechanisms: first, after C3d binds to CD21, it cross-links with the signaling receptor, CD19, and results in the possible lowering of both the concentration and affinity threshold of C3d3-conjugated antigen for B-cell activation, and then B cells become activated and expression of B7-1/B7-2 increases, thereby enhance antigen presentation to T helper cells. IL-2 production by T cells further enhances the interaction of B cells with T cells, and lowers the signaling threshold required for B-cell activation and expansion, thus increasing the immunogenicity of hCGβ. Recently, Lyubchenko et al. (2005) demonstrated that CD21-enhanced BCR signaling may proceed not only through the previously described amplification of positive signaling pathways, but also by a lack of normal inhibitory/feedback signaling. Our study in vitro has demonstrated that C3d3 as a molecular adjuvant fused to the less immunogenic hCGβ increases antigen immunogenicity and responsiveness in vitro of human immunocompetent cells upon primary stimulation, which appears to be of significant potential for contraceptive vaccine from bench to bed. Acknowledgements We thank Prof. Liu Yan-Jun of Fudan-Zhangjiang Bio-Pharmaceutical Co. Ltd. for his kind technique support. This work was supported by National Basic Research Program of China 2006CB944009 (to D.-J. 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Yu et al., 2006. 28.Yu Min, Li DJ, Wang XL, Zhao XR, Yao XY. Molecular adjuvant C3d up-regulates both B7-1 and B7-2 expression on Raji cells. J. Mol. Cell. Biol. 2006;39:77–82. a Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics & Gynecology, Fudan University Shanghai Medical College, Shanghai 200011, China b Department of Obstetrics & Gynecology, The Affiliated Hospital, Hainan Medical College, Haikou 570102, China c Department of Obstetrics & Gynecology, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China d Shanghai Changning Maternity & Infant Health Institute, Shanghai 200052, China  Corresponding author at: Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics & Gynecology, Fudan University Shanghai Medical College, Shanghai 200011, China. Tel.: +86 21 63457331/55050x386; fax: +86 21 63457331.
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