Splenocytes Of Immunized Mice Research Articles

Strong antibody reaction against glycosphingolipids injected in liposome-embedded forms in beta3GN-T5 knockout mice.

It is known that mutant mice of the beta-1,3-N-acetylglucosaminyltransferase gene (beta3Gn-T5) respond well to T-cell dependent and independent antigens. Here, we examined the effectiveness of anti-ganglioside antibody generation by immunization of beta3Gn-T5 mutant mice with liposome-embedded glycosphingolipids such as GD1a and GT1b. Consequently, the mutant mice showed a more efficient generation of anti-GD1a or anti-GT1b antibodies than wild-type mice in an enzyme-linked immunosorbent assay using sera during immunization. Thus, the beta3Gn-T5 deficient mutant mice proved more responsive than wild-type mice to not only protein antigens, but also to carbohydrates in glycolipids. Furthermore, about 50% of monoclonal antibodies generated using splenocytes of the immunized mutant mice were of the IgG class. Besides general high responsiveness to proteins and glycolipids, it could be expected that the mutant mice of beta3Gn-T5 would be useful in the generation of monoclonal antibodies towards lacto-/neolacto-series glycolipids, since these mutants lack lacto-/neolacto-series glycolipids. In fact, they showed a good serum response in immuno-fluorescence assay with cultured living cells when immunized by glycolipids extracted from ovarian cancer cell lines. These results suggested that beta3Gn-T5 mutant mice are useful for the generation of anti-glycolipid antigens with lacto-/neolacto-core structures expressed in cancer cells.

Strong immune responses induced by a DNA vaccine containing HPV16 truncated E7 C-terminal linked to HSP70 gene.

Vaccines capable of controlling tumor virus based infections are found difficult to develop due to the consistence latent infection in the host. DNA vaccines are attractive tools for the development of HPV vaccines and inducing antigen-specific immunity owing to the stability, simplicity of delivery, safety and cost effectiveness. However, there is a need to increase their potency by procedures such as using HSP70 gene as an adjuvant. To evaluate a DNA vaccine containing HPV16 truncated E7 C-terminal cytotoxic T-lymphocyte epitopes linked to HSP70 gene (HSP70-tE7) in an animal model. Mice were immunized with the plasmid DNA after pre-treatment with cardiotoxin. The splenocytes of immunized mice were then tested for CTL activity by detecting the apoptosis and necrosis in target cells, cytokine production by ELISA, CD4 and CD8 frequencies by flow cytometry, and lymphocyte stimulation by MTT assay. The recombinant expression vector was able to elicit immune responses close to that of full length E7 complete gene. Although the use of a small part of a target antigen can induce immune responses equivalent to the full length antigen, it fails to elicit statistically significant stronger immune responses when fused with HSP70 compared to the complete E7 gene alone. The potent immunogenicity of HPV16 E7 was preserved in the HSP70-tE7 vaccine and may represent a target of choice for the therapeutic vaccination strategies. However, to improve the immunogenicity polytope DNA vaccines which elicit multiple effector and memory CTL responses should be considered in future studies of DNA-based cancer vaccines.

Direct Construction of an Open-Sandwich Enzyme Immunoassay for One-Step Noncompetitive Detection of Thyroid Hormone T4

To establish a sensitive noncompetitive immunoassay for thyroxine (T4), we attempted to isolate anti-T4 antibodies from a phage display library based on a phagemid pDong1 ( Dong et al. Anal. Biochem.2009, 36, 386 ), which was designed to enable open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) after selection on immobilized antigen. After the Fab-displaying phage library made from the splenocytes of T4-KLH immunized mice was subjected to biopanning on T4-BSA, two T4-specific clones were obtained. When they were assayed by indirect competitive ELISA, both clones showed low IC(50) (5-13 ng/mL), indicating their high affinity to T4. When they were used for OS-ELISA that detects antigen-dependency of the interaction between variable domains V(H) and V(L), a clone successfully detected 1 ng/mL of T4 with a working range superior to that of competitive IA. OS-ELISA was also performed with maltose binding protein (MBP)-fused V(H)/V(L) of this clone, which showed a detection limit less than 0.1 ng/mL T4. Moreover, the assay showed cross-reactivity with T3 similar to that of competitive ELISA, and also gave a reasonable total serum T4 concentration (90 ng/mL) from ethanol-extracted sample serum using the recombinant proteins. This is the first direct construction of an OS-ELISA system bypassing hybridoma, which will be applicable to the detection of many other small molecule antigens.

Monoclonal Antibodies to Ki-67 Protein Suitable for Immunohistochemical Analysis

Detection of cell proliferation index is widely used in experimental and clinical research. Earlier it was shown that nuclear Ki-67 protein expression is strictly related to cell proliferation. It was revealed during all active phases of the cell cycle in mammals but was absent in G0 phase, so Ki-67 presence in cell nuclei reflects a potential growth fraction of whole cell population. The main area of Ki-67 antibody application is in immunocytochemical and immunohistochemical analyses. The aim of our work was to generate mouse monoclonal antibodies for Ki-67 antigen detection in mammalian tissues and in cultured cells. His-tagged fragment of Ki-67 expressed in bacteria was used as an antigen. Antibody-producing hybridoma cells were generated by standard procedure by fusing SP2/0 myeloma cells with splenocytes of immunized mice. Monoclonal antibodies were analyzed using paraffin-embedded human melanoma tissue samples and breast cancer cell line MCF-7. It was shown that generated anti-Ki-67 antibodies revealed proliferating cells in MCF-7 culture and after heat-induced epitope retrieval on paraffin sections of human melanoma tissue. In summary, generated antibodies could be useful for detection of proliferating cells in immunohistochemical and immunofluorescence studies of mammalian cells and tissues.

Antigen dose‐dependent suppression of murine IgE responses is mediated by CD4−CD8− double‐negative T cells

The IgE response against protein antigens is profoundly influenced by the dose used for sensitization. The aim of the study was to identify immune cells that are involved in antigen dose-dependent regulation of IgE formation. Wild-type mice as well as T helper (Th)1-deficient IL-12p40(-/-) and IFN-gamma(-/-) mice were immunized by repeated intraperitoneal injection of either low doses (K01 mice) or high doses (K100 mice) of keyhole limpet haemocyanin adsorbed to aluminium hydroxide. Splenocytes of immunized mice were restimulated in vitro and antigen-dependent T cell proliferation and cytokine production were measured. The frequency of regulatory T cell subsets among splenocytes from K01 and K100 mice was compared using fluorocytometry and RT-PCR analysis. Splenocytes or T cell subpopulations were transferred into naïve mice and the effect of lymphocyte transfer on IgE production after priming of recipients with low antigen doses was determined. Specific IgE production was considerably impaired in K100 mice. Antigenic restimulation revealed hypoproliferation of K100 splenocytes and reduced production of Th2 cytokines IL-4, IL-5 and IL-13, but no induction of IFN-gamma production. Moreover, lymphocytes from K01 and K100 mice did not show significant differences in the expression of molecules associated with the phenotype or activity of conventional regulatory T cells. Transfer of splenocytes or purified T cells from K100 mice substantially suppressed the induction of IgE production in the recipients in an antigen- and isotype-specific manner. Neither CD4(+) nor CD8(+) T cells from K100 mice were able to inhibit IgE formation; instead, we identified CD4(-)CD8(-) double-negative T cells (dnT cells) as the principal T cell population, which potently suppressed IgE production. Our data demonstrate that CD4(-)CD8(-) dnT cells play a major role in the regulation of IgE responses induced by high antigen doses.

Production and Identification of Monoclonal Antibody Against Flumequine and Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay

The hapten of Flumequine (FLU) with four carbon atoms spacer arm (FLUABA) was synthesized and coupled to bovine serum albumin (BSA) for immunogen by the activated ester method. BALB/C mice were immunized with the artificial immunogen and the splenocytes of immunized mice were fused with SP2/0 cells to obtain the monoclonal antibody (McAb). A hybridoma cell line (named DB6-E7) secreting anti-FLU McAbs was obtained by limited dilution method and screened by heterologous enzyme-linked immunosorbent assay (ELISA). The results showed that the subtype of the obtained McAb was IgG1, and the affinity was about 8.19 × 108 M−1. The haptens of FLU, FLUABA, and FLUACA with different space arms were linked to ovalbumin (OVA) for heterologous or homologous coating antigens. The results of indirect ELISA and indirect competitive ELISA indicated that the heterologous coating antigen could improve the sensitivity of ELISA significantly. Using FLU-OVA as coating antigen, the ELISA showed an IC50 value of 26.33 μg L−1, a limit of detection (LOD) of 4.0 μg L−1, and the workable range of 8–114 μg L−1 (IC20–IC80). Cross-reactivity studies showed that the McAbs were quiet specific for FLU, no cross-reactivity (< 0.1%) was detected between the obtained McAbs and the several major quinolones compounds or other structural analogs. The developed ELISA can satisfy the detection criteria of FLU in animal food-products.

Oral vaccination with Ts87 DNA vaccine delivered by attenuated Salmonella typhimurium elicits a protective immune response against Trichinella spiralis larval challenge

We have previously reported that Ts87 is an immunodominant antigen that induces protective immunity against Trichinella spiralis larval challenge. In this study, the Ts87 gene was cloned into an expression plasmid, pVAX1, and the recombinant Ts87 DNA was transformed into attenuated Salmonella typhimurium strain SL7207. Oral immunization of mice with Ts87 DNA delivered in S. typhimurium elicited a significant local mucosal IgA response and a systemic Th1/Th2 immune response. Cytokine profiling also showed a significant increase in the Th1 (IFN-γ) and Th2 (IL-5, 6, 10) responses in splenocytes of immunized mice upon stimulation with Ts87 antigen. An immunofluorescence assay performed with antisera revealed that the recombinant Ts87 protein was expressed in mesenteric lymph nodes of immunized mice. The mice immunized with Salmonella-delivered Ts87 DNA displayed a statistically significant 29.8% reduction in adult worm burden and a 34.2% reduction in muscle larvae following challenge with T. spiralis larvae, compared with mice immunized with empty Salmonella or a PBS control. Our results demonstrate that Ts87 DNA delivered by attenuated live S. typhimurium elicits a local IgA response and a balanced Th1/Th2 immune response and produces partial protection against T. spiralis infection in mice.

Impact of the Underlying Mutation and the Route of Vector Administration on Immune Responses to Factor IX in Gene Therapy for Hemophilia B

Immune responses to factor IX (F.IX), a major concern in gene therapy for hemophilia, were analyzed for adeno-associated viral (AAV-2) gene transfer to skeletal muscle and liver as a function of the F9 underlying mutation. Vectors identical to those recently used in clinical trials were administered to four lines of hemophilia B mice on a defined genetic background [C3H/HeJ with deletion of endogenous F9 and transgenic for a range of nonfunctional human F.IX (hF.IX) variants]. The strength of the immune response to AAV-encoded F.IX inversely correlated with the degree of conservation of endogenous coding information and levels of endogenous antigen. Null mutation animals developed T- and B-cell responses in both protocols. However, inhibitor titers were considerably higher upon muscle gene transfer (or protein therapy). Transduced muscles of Null mice had strong infiltrates with CD8+ cells, which were much more limited in the liver and not seen for the other mutations. Sustained expression was achieved with liver transduction in mice with crm(-) nonsense and missense mutations, although they still formed antibodies upon muscle gene transfer. Therefore, endogenous expression prevented T-cell responses more effectively than antibody formation, and immune responses varied substantially depending on the protocol and the underlying mutation.

The use of a mutant TNF-α as a vaccine adjuvant for the induction of mucosal immune responses

Safe and potent adjuvants are required in order to establish effective mucosal vaccines. Cytokines are promising adjuvants because they are human-derived safe biomaterial and display immune-modulating functions. We have created a mutant tumor necrosis factor-α (TNF-α), mTNF-K90R, that exhibits high bioactivity and resistance to proteases. Here, we examined the potential of mTNF-K90R as a mucosal adjuvant. Initially, we showed that intranasal co-administration of mTNF-K90R with ovalbumin (OVA) potently produced OVA-specific Immunoglobulin (Ig) G antibodies (Abs) in serum and IgA Abs both at local and distal mucosal sites compared to co-administration with wild-type TNF-α. The OVA-specific immune response was characterized by high levels of serum IgG1 and increased production of interleukin-4 (IL-4), IL-5 and IL-10 from splenocytes of immunized mice, suggesting a Th2 response. Furthermore, intranasal immunization with an antigen from influenza virus plus mTNF-K90R exhibited mucosal adjuvant activity for induction of both systemic and mucosal immune responses. Importantly, histopathological examination of the nasal tissue of mTNF-K90R treated mice detected no signs of toxicity. These findings suggest that mTNF-K90R is safe and effective mucosal adjuvant and this system may have potential application as a universal mucosal adjuvant system for mucosal vaccines improving the immune response to a variety of viral antigens.

Effective screening method of agonistic diabodies based on autocrine growth

Agonistic diabodies that mimic the function of natural ligands are expected to increase the value of therapeutic antibodies. We have developed a method that detects agonistic diabodies based on their ability to transduce growth signals through receptors, thereby permitting cytokine-independent growth of BaF/3-derived cytokine-dependent cells. Retrovirus-mediated expression of the diabody in cytokine-dependent cells was followed by selection of clones for growth in the absence of cytokine. A diabody library derived from splenocytes of human Mpl immunized mice was constructed. Infection of cells with viral particles led to the isolation of over 500 autonomously growing clones whose cultured supernatants showed agonistic activities against Mpl. Genome-integrated diabodies were cloned; representative clone AB317 showed agonistic activities as potent as a natural ligand and cross-reactive against mouse Mpl.

TNF superfamily member, TL1A, is a potential mucosal vaccine adjuvant

The identification of cytokine adjuvants capable of inducing an efficient mucosal immune response against viral pathogens has been long anticipated. Here, we attempted to identify the potential of tumor necrosis factor superfamily (TNFS) cytokines to function as mucosal vaccine adjuvants. Sixteen different TNFS cytokines were used to screen mucosal vaccine adjuvants, after which their immune responses were compared. Among the TNFS cytokines, intranasal immunization with OVA plus APRIL, TL1A, and TNF-α exhibited stronger immune response than those immunized with OVA alone. TL1A induced the strongest immune response and augmented OVA-specific IgG and IgA responses in serum and mucosal compartments, respectively. The OVA-specific immune response of TL1A was characterized by high levels of serum IgG1 and increased production of IL-4 and IL-5 from splenocytes of immunized mice, suggesting that TL1A might induce Th2-type responses. These findings indicate that TL1A has the most potential as a mucosal adjuvant among the TNFS cytokines.

New baculovirus recombinants expressing Pseudorabies virus (PRV) glycoproteins protect mice against lethal challenge infection

The present study demonstrates the protective potential of novel baculovirus recombinants, which express the glycoproteins gB, gC, or gD of Pseudorabies virus (PRV; Alphaherpesvirus of swine) and additionally contain the glycoprotein G of Vesicular Stomatitis Virus (VSV-G) in the virion (Bac-G-PRV). To evaluate the protective capacity, mixtures of equal amounts of the PRV gB-, gC-, and gD-expressing baculoviruses were used for immunization. Three intramuscular immunizations with that Bac-G-PRV mixture could protect mice against a lethal PRV challenge infection. To achieve complete protection high titers of Bac-G-PRV and three immunizations were necessary. This immunization with Bac-G-PRV resulted in the induction of high titers of PRV-specific serum antibodies of the IgG2a subclass and of interferon (IFN)-gamma, indicating a Th1-type immune response. Moreover, splenocytes of immunized mice exhibited natural killer cell activity accompanied by the production of IFN-alpha and IFN-gamma. Collectively, the presented data demonstrate for the first time that co-expression of VSV-G in baculovirus recombinant vaccines can improve the induction of a protective immune response against foreign antigens.

Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries

These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks, resulting in a panel of scFvs specific for the target antigen.

Production and Identification of High Affinity Monoclonal Antibodies Against Pesticide Carbofuran

Abstract To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people's health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]-amino]-butanoic acid (BFNB) of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier (BFNB-bovine serum albumin, BFNB-BSA) conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay (ELISA), based on BFNB-ovoalbumin conjugates (BFNB-OVA). Purified monoclonal antibody (McAb) was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line (5D3) secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1:2.048 × 10 3 and 1:1.024 × 10 6, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 10 9 L mol −1, with an IC 50 value of 1.18 ng mL −1 and a detection limit of 0.01 ng mL −1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized (4.59 × 10 −4% for 2,3-dihydro-2,2-dimethyl-7-benzofuranol and less than 3.0 × 10 −4% for others). The prepared McAb had a very high affinity and specificity, and it could be used to develop ELISA for rapid determination of carbofuran.

The α-adrenoceptor mediation of the immunomodulatory effects of electroacupuncture in DNP-KLH immunized mice

Our previous study demonstrated that successive electroacupuncture (EA) at ST36 acupoint reduces IgE production in BALB/c mice immunized with 2,4-dinitrophenylated keyhole limpet protein (DNP-KLH) by suppression of the Th2 cell lineage development. Here, we report that pretreatment of phentolamine (alpha-adrenoceptor antagonist, 10mg/kg, i.p.) completely blocks the EA-induced suppression of antigen-specific and total IgE levels in serum and IL-4 production in anti-CD3 mAb-activated splenocytes in DNP-KLH immunized mice. The results suggest that alpha-adrenoceptor play an important role in mediating the suppressive effects of EA on IgE production and Th2 cell response in DNP-KLH immunized mice.

The neurotrans mechanism of electronic-acupuncture in DNP-KLH immunized mice. (B21)

Abstract Our previous study demonstrated that successive electroacupuncture (EA) at the ST36 acupoint reduces IgE production in BALB/c mice immunized with 2,4-dinitrophenylated keyhole limpet protein (DNP-KLH) by suppression of the Th2 cell lineage development. Here we report that pretreatment of phentolamine (¥á-adrenergic receptor antagonist, 10 mg/kg, i.p.) completely blocks the EA-induced suppression of antigen-specific and total IgE levels in serum, and IL-4 production in anti-CD3 mAb-activated splenocytes in DNP-KLH immunized mice. And pretreatment of Y-25130 (5HT3 receptor antagonist, 3 mg/kg, i.p.) partly blocks EA-induced suppression effect shown above. These results suggest that ¥á-adrenergic receptor play an important role in mediating the suppressive effects of EA on IgE production and Th2 cell response in DNP-KLH immunized mice, and 5HT3 receptor play as a minor factor. This work was supported by the Brain Korea 21 project in 2007

Novel anti-cholesterol monoclonal immunoglobulin G antibodies as probes and potential modulators of membrane raft-dependent immune functions

Natural autoantibodies against cholesterol are present in the sera of all healthy individuals; their function, production, and regulation, however, are still unclear. Here, we managed to produce two monoclonal anti-cholesterol antibodies (ACHAs) by immunizing mice with cholesterol-rich liposomes. The new ACHAs were specific to cholesterol and to some structurally closely related 3beta-hydroxyl sterols, and they reacted with human lipoproteins VLDL, LDL, and HDL. They bound, usually with low avidity, to live human or murine lymphocyte and monocyte-macrophage cell lines, which was enhanced substantially by a moderate papain digestion of the cell surface, removing some protruding extracellular protein domains. Cell-bound ACHAs strongly colocalized with markers of cholesterol-rich lipid rafts and caveolae at the cell surface and intracellularly with markers of the endoplasmic reticulum and Golgi complex. These data suggest that these IgG ACHAs may serve as probes of clustered cholesterol (e.g., different lipid rafts) in live cells and thus may also have immunomodulatory potential.

Anti‐idiotype antibody induced cellular immunity in mice transgenic for human carcinoembryonic antigen

In the present study, we have analysed the detailed cellular immune mechanisms involved in tumour rejection in carcinoembryonic antigen (CEA) transgenic mice after immunization with dendritic cells (DC) pulsed with an anti-idiotype (Id) antibody, 3H1, which mimics CEA. 3H1-pulsed DC vaccinations resulted in induction of CEA specific cytotoxic T lymphocyte (CTL) responses in vitro and the rejection of CEA-transfected MC-38 murine colon carcinoma cells, C15, in vivo (Saha et al.,Cancer Res 2004; 64: 4995-5003). These CTL mediated major histocompatibility complex (MHC) class I-restricted tumour cell lysis, production of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), and expression of Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) in response to C15 cells. CTL used perforin-, FasL-, and TRAIL-mediated death pathways to lyse C15 cells, although perforin-mediated killing was the predominant lytic mechanism in vitro. The cytokines IFN-gamma and TNF-alpha synergistically enhanced surface expression of Fas, TRAIL receptor, MHC class I and class II on C15 cells that increased the sensitivity of tumour cells to CTL lysis. CTL activity generated in 3H1-pulsed DC immunized mice was directed against an epitope defined by the idio-peptide LCD-2, derived from 3H1. In vivo lymphocyte depletion experiments demonstrated that induction of CTL response and antitumour immunity was dependent on both CD4+ and CD8+ T cells. The analysis of splenocytes of immunized mice that had rejected C15 tumour growth revealed up-regulated surface expression of memory phenotype Ly-6C and CD44 on both CD4+ and CD8+ T cells. The adoptive transfer experiments also suggested the role of both CD4+ and CD8+ T cells in this model system. Furthermore, mice that had rejected C15 tumour growth, developed tumour-specific immunological memory.

Mucosal genetic immunization against four adhesins protects against Staphylococcus aureus-induced mastitis in mice

Staphylococcus aureus is the most common etiologic agent of mastitis in bovines and a major cause of economic losses in the dairy industry. Since adhesins, that anchor bacteria to the extracellular matrix (ECM), are among the most important S. aureus virulence factors, we used four adhesion factors (fibrinogen binding protein, fibronectin binding protein A, clumping factor A and collagen adhesin) as target in a DNA vaccine. Intranasal immunization with a pDNA mixture coding the four adhesins, triggered significant levels of specific serum and mucosal Ig that inhibited S. aureus adhesion to cow mammary gland epithelial cells in vitro. Splenocytes of immunized mice challenged in vitro with S. aureus extracts showed a strong proliferative response. Finally, immunized mice were significantly protected against intramammary challenge with S. aureus. Our data confirm the feasibility of a genetic mucosal vaccine targeting S. aureus adhesins and prompt further study to test the efficacy in preventing cow mastitis.

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

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. 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. 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. C3d enhances the humoral and cell-mediated immune responses against HBV induced by gene immunization.