In order to research immunogenicity of the recombinant rVP2-IL-2 fusion protein, we obtained the rVP2-IL-2 fusion protein using Pichia pastoris expression system, and then evaluated its potential to induce immune responses in chicken. The effect was determined in the form of protective anti-IBDV VP2 titers, antibodies (IgG1 and IgG2a), lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and challenge experiment. Antibody titers and proliferation lymphocyte level suggested that the fusion protein could elicit specific humoral immune and cellular immune responses, antibody sub-type results indicated that the rVP2-IL-2 fusion protein induced secretion both of IgG1 and IgG2a. The seem result elicited from cytokines ELISA test, secretion of both of Th1 (gamma-IFN) and Th2 (IL-4) were induced by the rVP2-IL-2 fusion protein. Challenge experiment result shown that chicken immunized the rVP2-IL-2 fusion protein obtained 85% protection. These results confirm that the fusion protein enhances the protection against IBDV through both humoral and cell-mediated immunity, and thus could serve as a candidate for the development of IBDV subunit vaccine.
Animals ; Antibodies, Viral ; biosynthesis ; blood ; Chickens ; immunology ; Immunoglobulin G ; blood ; Interleukin-2 ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Vaccines, Subunit ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

To investigate the value of apoptosis of the allo-antigen specific T cells induced by Fas/FasL pathway in preventing graft-versus-host disease (GVHD), the CD34+ cells transfected with FasL or not, used as stimulus cells, were mixed with allo-antigen specific T lymphocytes in presence or absence of IFN-gamma and IL-2. After 5 days, apoptosis of T cells was detected by TdT nick end mediated dUTP labeling (TUNEL) and flow cytometry (FCM). The affects of these two cytokines on CD34+ cells in the graft were also compared. The ratio of apoptosis of T cells was 12.1+/-1.5% when CD34+ cells transfected with FasL was used as stimulus cells, much higher than that of CD34+ cells non-transfected (3.2+/-1.1%, P<0.01). And in presence of IFN-gamma or IL-2, the ratio reached 20.1+/-2.3%, 17.6+/-1.3% respectively (P<0.01). However, IFN-gamma up-regulated Fas expression of CD34+ cells and increased the sensibility of CD34+ cells to soluble FasL (sFasL); IL-2 showed no such effect. It is possible to induce apoptosis of the allo-antigen specific T cells of grafts activated by allo-antigen by exogenous Fas ligand expressed on recipient cells and this might provide a new approach for preventing GVHD and IL-2 may be more suitable for clinical application.
Antigens, CD34 ; biosynthesis ; immunology ; Apoptosis ; Cytotoxicity, Immunologic ; DNA, Complementary ; genetics ; Fas Ligand Protein ; Graft vs Host Disease ; prevention & control ; Interferon-gamma ; biosynthesis ; immunology ; Interleukin-2 ; biosynthesis ; immunology ; Membrane Glycoproteins ; biosynthesis ; immunology ; T-Lymphocytes ; cytology ; physiology ; fas Receptor ; biosynthesis ; immunology

The purpose of this study was to construct the IgHV and IL-2 coexpressed vector. The IgHV gene fragments were obtained from the peripheral blood of patients with lymphoma, and were cloned into eukaryotic expression vector. Meanwhile, the gene fragments of IgHV linked with gene of IL-2 were inserted into pcDNA3.0 to form a fusion gene of IgHV-IL-2. Then fusion genes were transfected into COS cells by Lipofectin and the expression of IL-2 was detected by ELISA. The results showed that the IgHV/pcDNA3.0 expression vector was successfully constructed. The 3' end of IgHV was linked to IL-2 gene, and IL-2 could be correctly expressed. In conclusion, the expression vector of IgHV-IL-2 can express IL-2 correctly in COS cells.
Cancer Vaccines ; immunology ; Eukaryotic Cells ; metabolism ; Genes, Immunoglobulin ; Genetic Vectors ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Interleukin-2 ; biosynthesis ; genetics ; Lymphoma ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, DNA ; biosynthesis ; genetics ; immunology

The study was purposed to investigate the effects of humanized recombined CD25 monoclonal antibody (rhCD25MAb) on activation and proliferation of T lymphocytes in vitro. Peripheral blood mononuclear cells (PBMNC) were incubated with phytohemagglutinin (PHA). Before or after T cell activation, the cells were cultured with or without rhCD25MAb or cyclosporine A (CsA) in vitro. After 72 hours incubation, the proliferation of lymphocytes was analyzed by MTT assay. The expression of CD3 and CD25 antigens on T lymphocytes were detected by flow cytometry. The levels of sIL-2R in the supernatants were determined with ELISA. The results showed that both rhCD25MAb and CsA could inhibit the proliferation of T lymphocytes significantly in concentration-dependent manner and CsA was more efficient than rhCD25MAb. Both rhCD25MAb and CsA could also decrease the levels of sIL-2R in the supernatant and inhibit the expression of CD25 antigen on T lymphocytes. The level of sIL-2R and the expression of CD25 on T lymphocytes decreases more profoundly in rhCD25MAb group. It is concluded that rhCD25MAb shows strong immunosuppressive activity both before and after T cell activation, suggesting that this agent may be useful in not only prophylaxis but also the treatment of acute graft-versus-host disease.
Antibodies, Monoclonal ; pharmacology ; CD3 Complex ; biosynthesis ; genetics ; Cell Proliferation ; Cells, Cultured ; Cyclosporine ; pharmacology ; Humans ; Immunosuppressive Agents ; pharmacology ; Interleukin-2 Receptor alpha Subunit ; biosynthesis ; genetics ; immunology ; Lymphocyte Activation ; immunology ; Receptors, Interleukin-2 ; analysis ; Recombinant Proteins ; immunology ; pharmacology ; T-Lymphocytes ; immunology

By combining interleukin2 (IL-2) with a tumor specific antibody, immunotherapy of tumors may become more effective in the future. Anti-GD2 single chain antibody directed to the extracellular domain of GD2 disialoganglioside can result in an antitumor response in some pateins with tumors expressing GD2. In this study, the fusion protein consisting of GD2 single chain antibody (ScFv) and IL-2(Ala125) was constructed. Anti-GD2 ScFv and IL-2 genes were obtained by PCR, then the ScFv-IL-2 gene was constructed by over lap PCR. The gene was inserted into the pMD18-T easy vector. Genes from pMD18-T -vector were inserted into expression vector pSE380. Recombinant expression vector was identified by restriction enzyme-cutting and then was transformed into BL21. SDS-PAGE and Western blot analysis confirmed that the transformed E. Coli BL21 could express ScFv-IL-2 fusion-proteins and the molecular weight is 43 kDa. The fusion protein was purified by affinity chromatograph and Sephacryl S-200HR then was identified through ELISA. The results show that the fusion protein retains the activities of both antigen binding and IL-2.
Antibodies ; genetics ; metabolism ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gangliosides ; immunology ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Interleukin-2 ; biosynthesis ; genetics ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate whether GM-CSF can enhance the antiviral effect of HBsAg DNA vaccine.

METHODSDivided animals into 8 groups. Group A: pcDNA3.1-S 100microg; Group B: pcDNA3.1-GM-CSF-S 100microg; Group C: pcDNA3.1-S-GM-CSF 100microg; Group D: pcDNA3.1-S 50microg + pcDNA3.1-GM-CSF 50microg; Group E: pcDNA3.1-GM-CSF 100microg; Group F: recombinant HBsAg vaccine 1microg; Group G: pcDNA3.1,100microg; Group H: PBS 100microl. Serum HBsAg level and concentration of IL-2, IL-4 and IFN-gamma were examined using commercial ELISA kit. The [3H] thymidine incorporation into DNA of Spleen cells was measured; HBsAg expression of hepatocytes from HBV-transgenic mice was assessed using immunohistochemical analysis.

RESULTSSerum HBsAg level was lower and concentration of IL-2, IFN-gamma and SI was higher in mice immunized with pcDNA3.1-GM-CSF-S than those from mice immunized with pcDNA3.1-S and other groups (F=11.262, P<0.01, F=8.147, P<0.01, F=62.275, P<0.01, F=116.28, P<0.01. Less Hepatic HBsAg expression and decline of pcDNA3.1-GM-CSF-S of mice immunized with pcDNA3 were observed in comparison with control groups (F=41.439, P<0.01). Liver histological analysis showed no evidence of necrosis or inflammation in various groups.

CONCLUSIONThe plasmid co expressing GM-CSF and HBsAg could improve HBsAg-specific humoral and cellular immune responses induced by plasmid encoding HBsAg alone and enhance HBsAg DNA vaccine antivirus effect.

Animals ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B Vaccines ; immunology ; Interferon-gamma ; biosynthesis ; Interleukin-2 ; biosynthesis ; Interleukin-4 ; biosynthesis ; Lymphocyte Activation ; Male ; Mice ; Mice, Transgenic ; Plasmids ; Vaccines, DNA ; immunology

UNLABELLEDTo set up a new rapid and efficient way for the preparation of specific monoclonal antibodies (MAbs) with plasmid DNA immunization.

METHODSThe fusion gene of IL-2 signal peptide and profilin 1 by 'overlapping PCR' was obtained and inserted into the vector pVAX1 to construct the recombinant plasmid pVAX-IL2-profl. Another fusion gene of IgG kappa chain signal peptide and profilin 1 by 'no template PCR' was obtained and inserted into the vector pVAX1 to construct the recombinant plasmid pVAX-Igkappa-prof1. BALB/c mice were single intrasplenic immunized with plasmid DNA. Results After cell fusion and hybridomas screening by indirect ELISA, we charactered two mAbs (1D8A2B4 and 4B12A5E3) against profilin 1. The MAbs isotype were determined as IgM and IgG3.

CONCLUSIONA single intrasplenic plasmid DNA immunization is rapid and efficient and can be used as a helpful tool for the production of specific mAb.

Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; Antibody Specificity ; DNA ; genetics ; immunology ; Female ; Gene Fusion ; Immunization ; Interleukin-2 ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Profilins ; genetics ; immunology ; Spleen ; immunology ; metabolism

The mechanism underlying CD4CD25Foxp3regulatory T cells (Tregs) promoting the development of colorectal cancer (CRC) was elucidated in the present study. Forty-eight cases of colorectal carcinomas, 22 cases of colon polyps and 21 cases of normal colorectal tissues were collected. The correlation among Foxp3, IL-10 and Stat3, and the clinical relevance of these three indexes were analyzed. The results showed that the levels of Foxp3 expressed in infiltrating CD4CD25Foxp3Tregs, and IL-10 and Stat3 in CRC tissues were all significantly higher than those in polypus tissues and normal colon tissues (P< 0.01). Pearson correlation analysis indicated that the expression level of Foxp3 was positively correlated with Stat3 at mRNA level (r=0.526, P=0.036), and was positively correlated with IL-10 at protein level (r=0.314, P=0.030). The Foxp3 expressed in CD4CD25Foxp3Tregs was correlated with the histological grade, lymph node metastasis and TNM stage of CRC (P<0.05 for all). The IL-10 expression was correlated with the histological grade and TNM stage (both P<0.05). The Stat3 expression was correlated with the lymph node metastasis and TNM stage (both P<0.05). It was concluded that CD4CD25Foxp3Tregs can inhibit tumor immunity in combination with some other related inhibitory cytokines and that Foxp3 expression in CD4CD25Foxp3Tregs correlates with CRC progression.
Adult ; Aged ; CD4-Positive T-Lymphocytes ; immunology ; Colorectal Neoplasms ; genetics ; immunology ; pathology ; Female ; Forkhead Transcription Factors ; biosynthesis ; genetics ; immunology ; Gene Expression Regulation, Neoplastic ; immunology ; Humans ; Immunity ; genetics ; Interleukin-10 ; biosynthesis ; immunology ; Interleukin-2 Receptor alpha Subunit ; immunology ; Lymphatic Metastasis ; Male ; Middle Aged ; STAT3 Transcription Factor ; biosynthesis ; immunology ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo study the antitumor effect GPI-CD80 fusion protein and its mechanisms.

METHODSA tumor vaccine was prepared by culturing HepG2 cells in the presence of purified GPI-CD80 followed by inactivation with mitomycin, with mitomycin-inactivated HepG2 cells as the control group. The two preparations were co-cultured with nude mouse splenic lymphocytes, and the changes of lymphocyte proliferation and the production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) were detected by MTT assay. The cytotoxic T-lymphocyte (CTL) activity was evaluated by LDH-release assay, and the changes of gross tumor volume were measured in tumor-bearing nude mice after administration of different vaccines.

RESULTSThe application of GPI-CD80 tumor vaccine resulted in significantly increased optical density, IL-2 and IFN-gamma levels and CTL activity of the nude mouse splenic lymphocytes in comparison with the control groups. The average tumor volume in nude mice treated with GPI-CD80 tumor vaccine was significantly smaller than that in negative control and blank control groups.

CONCLUSIONGPI-CD80 fusion protein may inhibit the tumor growth velocity in nude mice, possibly by promoting lymphocyte proliferation, stimulating the production of the cytokines IL-2 and IFN-gamma, and enhancing of CTL activity.

Animals ; B7-1 Antigen ; biosynthesis ; genetics ; immunology ; isolation & purification ; CHO Cells ; Cancer Vaccines ; biosynthesis ; genetics ; immunology ; isolation & purification ; Cell Proliferation ; Cricetinae ; Cricetulus ; Glycosylphosphatidylinositols ; genetics ; metabolism ; Interferon-gamma ; biosynthesis ; Interleukin-2 ; biosynthesis ; Mice ; Mice, Nude ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification ; Spleen ; cytology ; immunology ; metabolism ; Tumor Burden ; immunology

OBJECTIVESTo investigate the improvement of specific immune responses induced by plasmid coexpressing hepatitis B surface antigen (HBsAg) and granulocyte-macrophage colony stimulating factor (GM-CSF).

METHODSAll Balb/c (H-2d) mice were immunized with pGM-CSF/S, pS/GM-CSF, pS or control plasmids. 4 weeks later, anti-HBs titer and the levels of IL-2, IL-4 and IFN-gamma in the supernatant of splenocytes were detected using enzyme- linked immunosorbent assay (ELISA), and HBsAg-specific cytotoxic T lymphocytes (CTL) activity was measured with a 51Cr release assay, using P815/S transfectants as target cells.

RESULTSThe anti-HBs antibody titers in the serum, the levels of IL-2 and IFN-gamma, and the CTL activity in pcDNA3.1-GM-CSF-S immuned mice were higher than those in PcDNA3.1-S immunized mice (F=4.176, P<0.01; F=31.188, P<0.01; F=31.796, P<0.01; F

CONCLUSIONIt will improve the specific immune responses induced by HBsAg DNA vaccine after it is binded to the gene of GM-CSF.

Animals ; Enzyme-Linked Immunosorbent Assay ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; immunology ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B Vaccines ; immunology ; Interferon-gamma ; biosynthesis ; Interleukin-2 ; biosynthesis ; Interleukin-4 ; biosynthesis ; Lymphocyte Activation ; Male ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; immunology