B7-1 Antigen ; genetics ; immunology ; Carcinoma, Hepatocellular ; genetics ; immunology ; Enterotoxins ; genetics ; immunology ; Genetic Vectors ; Humans ; Liver Neoplasms ; genetics ; immunology ; T-Lymphocytes ; immunology ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo prepare and identify monoclonal antibodies against staphylococcal enterotoxin I (SEI).

METHODSSpleen cells obtained from mice immunized with the SEI protein were fused with the myeloma cells (SP2/0). Hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) and the stable monoclonal hybridomas were isolated by limiting dilution at least three times. The characters of purified monoclonal antibodies were identified by indirect ELISA and Western blotting.

RESULTThe monoclonal antibodies secreted by two hybridomas 8F7 and D8 belonged to IgG(2b) and IgG(1) subtypes. Both had high titer and specificity with no cross reaction to SEG, SEE and SEC.

CONCLUSIONThe monoclonal antibodies against SEI has been successfully prepared and identified in this study.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Enterotoxins ; immunology ; Hybridomas ; secretion ; Immunoglobulin G ; biosynthesis ; immunology ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; Staphylococcus aureus ; immunology

To investigate the adjuvant effect of plasmid DNA encoding superantigen SEA (D227A) (pmSEA) on immune responses induced by HBV DNA vaccine containing HBV preS2 and S antigen in BABL/c (H-2d). BALB/c mice were immunized intramuscular injection with HBV DNA vaccine (pHBVS2S) mixed with or without pmSEA plasmid. Antibodies againat HBV PreS2 and S antigen in the sera were accessed by Anti-HBs ELISA, and the HBsAg specific cytotoxic T lymphocytes (CTLs) activity was determined by 5 Chromium Release Assay. The HBs peptide-specific IFN-gamma secreting T cells were detected by ELISPOT. Anti-HBs antibody titers and CTLs activity in mice immunized with pmSEA + pHBVS2S group were significant higher (P < 0.05) than pHBVS2S DNA vaccine group. The ratio of IgG1/IgG2a (0.282) was apparently different from the group immunized with peptide (10). Mice immunized with HBV DNA vaccine plus adjuvant produce higher titer of IgG1 and IgG2a antibodies against HBV S antigen 1.36 and 1.73 time higher than that without adjuvant respectively. HBs peptide--specific IFN-gamma secreting T cells increased 2 - 3 times by the pmSEA adjuvant, compared to DNA vaccine group. HBV DNA vaccine (pHBVS2S) induces humoral and cellular immuno-responses in BALB/c mice, and the responses could be significantly boasted by the plasmid encoding mSEA. Therefore the pmSEA was a potential adjuvant for DNA vaccines.
Adjuvants, Immunologic ; Animals ; Enterotoxins ; immunology ; Hepatitis B ; immunology ; prevention & control ; therapy ; Hepatitis B Antibodies ; blood ; Hepatitis B Vaccines ; immunology ; Interferon-gamma ; secretion ; Mice ; Mice, Inbred BALB C ; Staphylococcus aureus ; immunology ; Superantigens ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccination ; Vaccines, DNA ; immunology

Clinical application of staphylococcal enterotoxin C2 (SEC2) was restricted during the cure of malignant tumor due to its side-effects. The aim of this study was to obtain SEC2 mutant, preserving the important functional sites responsible for the T-cell stimulatory activities but removing the sites responsible for emetic activity, through truncation of SEC2. It would efficiently solve the question of SEC2 side-effect. According to the results of methyl thiazol tetrazolium (MTT) assay in vitro, novel truncated SEC2 mutant (NSM) efficiently stimulated T-cell proliferation and inhibited the growth of such tumor cells as human colorectal cancer cells (Cx-1) and human breast cancer cells (MCF-7) in vitro. Activities of T cell stimulating and anti-tumor of NSM were similar to those of SEC2. According to results of animal experiments, the mutant no longer induced emetic response even if the dose was a 10-fold excess of the amount of SEC2 required. And also, NSM obviously inhibited the tumor growth in tumor-bearing mice. Therefore, we obtained novel truncated staphylococcal enterotoxin C2 mutant, which could efficiently inhibit the growth of tumor cells. It will become novel anti-tumor agents with the lowest side-effects and best treatment effects in clinic.
Animals ; Antineoplastic Agents ; adverse effects ; pharmacology ; Breast Neoplasms ; immunology ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colorectal Neoplasms ; immunology ; pathology ; Enterotoxins ; genetics ; immunology ; Humans ; Mice ; Mutant Proteins ; immunology ; Staphylococcus aureus ; immunology ; Superantigens ; immunology ; T-Lymphocytes ; immunology ; Vomiting ; prevention & control

Escherichia coli heat-labile enterotoxin (LT), which causes a characteristic diarrhea in humans and animals, is a strong mucosal immunogen and has powerful mucosal adjuvant activity towards coadministered unrelated antigens. Here we report the different mucosal adjuvanticity of nontoxic LT derivatives, LTS63Y and LTdelta110/112, generated by immunizing through two different mucosal routes. Intragastric (IG) immunization with Helicobacter pylori urease alone resulted in poor systemic IgG and IgA responses and no detectable local secretory IgA, but IG co-immunization with urease and LTdelta110/112 induced high titers of urease-specific local secretory IgA and systemic IgG and IgA, comparable to those induced by wild-type LT. LTS63Y showed far lower adjuvant activity towards urease than LTdelta110/112 in IG immunization, but was more active than LTdelta110/112 in inducing immune responses to urease by intranasal (IN) immunization. LTdelta110/112 predominantly enhanced the induction of urease-specific IgG1 levels following IG immunization, whereas LTS63Y induced high levels of IgG1, IgG2a and IgG2b following IN immunization. In addition, quantitative H. pylori culture of stomach tissue following challenge with H. pylori demonstrated a 90-95% reduction (p < 0.0002) in bacterial burden in mice immunized intranasally with urease using either mutant LT as an adjuvant. These results indicate that the mechanism(s) underlying the adjuvant activities of mutant LTs towards coadmnistered H. pylori urease may differ between the IN and IG mucosal immunization routes.
Adjuvants, Immunologic/administration & dosage* ; Administration, Intranasal ; Animal ; Bacterial Toxins/immunology* ; Bacterial Toxins/genetics ; Bacterial Toxins/administration & dosage ; Enterotoxins/immunology* ; Enterotoxins/genetics ; Enterotoxins/administration & dosage ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli* ; Feces ; Female ; Gastric Mucosa/microbiology ; Gastric Mucosa/immunology* ; Helicobacter pylori ; Human ; IgA, Secretory/immunology* ; IgG/immunology ; Mice ; Mice, Inbred BALB C ; Mutagenesis, Site-Directed ; NAD+ ADP-Ribosyltransferase/immunology ; NAD+ ADP-Ribosyltransferase/genetics ; Nasal Mucosa/immunology* ; Point Mutation ; Urease/immunology* ; Urease/administration & dosage ; Vaccination*

The anti-tumor effect and mechanism of the staphylococcal enterotoxin A (SEA) were studied. The mouse gastric tumor model was produced by subcutaneously inoculating gastric tumor cells (MGC80-3). The experimental group was treated with SEA, and the control group was treated with normal saline. The percentage of tumor generation and tumor mass was measured. The results showed that the percentage of the tumor generation in the SEA-treated mice was lower than in the control group, but there was no significant difference (P > 0.05). However, the tumor mass in the experimental group was significantly lighter than in the control group, with the difference being very significant (P < 0.001). There were more CD4+ T cells and CD8+ T cells in the tumor of the mice treated with SEA than those of the control group. SEA has an obvious anti-tumor effect on mice gastric tumor. The mechanism might be that SEA induces the effect of superantigen-dependent cell mediated cytotoxicity to the tumor cells.
Adjuvants, Immunologic ; pharmacology ; Animals ; Antineoplastic Agents ; pharmacology ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Enterotoxins ; immunology ; pharmacology ; Female ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Random Allocation ; Staphylococcus aureus ; immunology ; Stomach Neoplasms ; immunology ; pathology ; Superantigens ; immunology ; pharmacology

BACKGROUND AND OBJECTIVEImmunocompromised patients with malignant tumor always lack of strong anti-tumor immune response, because the antigenicity of tumor cells is weak, and antigen-presenting cell function is low, so that can not be effectively presenting tumor antigens to the lymphocytes. Therefore, how to effectively induce anti-tumor immune response is the key issue. Through the study on establishing a method to culture dendritic cells (DC) in vitro and to observe the anti-lung cancer immunological effect induced by DC, we provided definite experiment basis for the clinic application of vaccine based on DC.

METHODSThrough the experiment we get the soluble antigen polypeptide from lung cancer cells GLC-82 by 3 mol/L potassium chloride. DCs are cultured and obtained from peripheral blood mononuclear cell by GM-CSF, IL-4 and TNF-a. DCs are identified by flow cytometer (FCM) and immunostaining. DCs modified by lung cancer tumor soluble antigen (TSA) and staphylococcal enterotox in A (SEA), DCs modified by TSA or DCs modified by SEA or DCs modified by nothing were cultivated together with T lymphocyte, and the obtained cells are named TSA-SEA-DCL or TSA-DCL or SEA-DCL or DCL as effector cells. The anti-tumor activity of every effector cells against target cells was assayed with MT method. Shape of DCs and effector cells, and the process of killing target cells were observed in microscope.

RESULTSInduced DCs expressed more CD1a, CD80 and HLA-DR, which had typical cell traits such as tree branch. The killing ratio of the TSA-SEA-DCL in vitro to GLC-82 is larger than TSA-DCL, SEA-DCL and DCL, also larger than to K562. When the effector cells cultivate with target cells, we can observe the CTL approach and gather to the cancer cell, induce it necrosis and apoptosis.

CONCLUSIONRipe DCs that have typical characteristic and phenotype could be induced successfully. High potency and relatively specific antilung caner effect can be prepared in virtue ofDC Bacterin Induced by lung caner TSA and SEA.

Antigens, Neoplasm ; immunology ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; Enterotoxins ; immunology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Immunotherapy ; Interleukin-4 ; pharmacology ; Lung Neoplasms ; immunology ; therapy ; Superantigens ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

Escherichia coli heat-labile enterotoxin (LT) is composed of catalytic A and non-catalytic homo-pentameric B subunits and causes diarrheal disease in human and animals. In order to produce a nontoxic LT for vaccine and adjuvant development, two novel derivatives of LT were constructed by a site-directed mutagenesis of A subunit; Ser63 to Tyr63 in LTS63Y and Glu110, Glu112 were deleted in LT delta 110/112. The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB5 to that of wild LT. In contrast to wild-type LT, mLTs failed to induce either elongation activity, ADP-ribosyltransferase activity, cAMP synthesis in CHO cells or fluid accumulation in mouse small intestine in vivo. Mice immunized with mLTs either intragastrically or intranasally elicited high titers of LT-specific serum and mucosal antibodies comparable to those induced by wild-type LT. These results indicate that substitution of Ser63 to Tyr63 or deletion of Glu110 and Glu112 eliminate the toxicity of LT without a change of AB5 conformation, and both mutants are immunogenic to LT itself. Therefore, both mLTs may be used to develop novel anti-diarrheal vaccines against enterotoxigenic E. coli.
Amino Acid Substitution ; Animal ; Bacterial Toxins/toxicity* ; Bacterial Toxins/metabolism ; Bacterial Toxins/immunology* ; Bacterial Toxins/genetics ; CHO Cells ; Cyclic AMP/metabolism ; Enterotoxins/toxicity* ; Enterotoxins/metabolism ; Enterotoxins/immunology* ; Enterotoxins/genetics ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli*/metabolism ; Escherichia coli*/genetics ; Female ; Hamsters ; IgA, Secretory/blood ; Ileum/metabolism ; Immunity, Mucosal ; Mice ; Mice, Inbred BALB C ; Mutagenesis, Site-Directed ; NAD+ ADP-Ribosyltransferase/metabolism ; Recombinant Proteins/toxicity ; Recombinant Proteins/metabolism ; Recombinant Proteins/immunology ; Recombinant Proteins/chemistry

This paper is aimed to investigate the transcription and expression of BCR-ABL-pIRES-SEA fusion gene vaccines in vivo in mice. The reconstructed plasmids (BCR-ABL-pIRES-SEA) which were developed previously in our laboratory were injected into the skeletal muscles of BALB/c mice at 14d intervals for three cycles. The transcription and expression of BCR-ABL and staphylococcal enterotoxin A (SEA) in injection site were detected using RT-PCR and immunohistological methods. The BCR-ABL/SEA mRNA and protein could be identified in the injection site of BCR-ABL-pIRES-SEA vaccinated mice. The reconstructed BCR-ABL-pIRES-SEA plasmids can effectively express gene production in the skeletal muscles of mice and have the common features of DNA vaccine.
Animals ; Enterotoxins ; genetics ; immunology ; metabolism ; Fusion Proteins, bcr-abl ; genetics ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Muscle, Skeletal ; metabolism ; Plasmids ; immunology ; RNA, Messenger ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Vaccines, DNA ; administration & dosage ; immunology

Anti-HIV-1 gp120 single chain antibody(scFv) gene and staphylococcus extoxin A(SEA) gene were inserted into vector pPIC9K. The recombinant plasmid was integrated into Pichia pastoris by electroporation. High level expression was performed by determining the Muts phenotype and screening muti-copy integrants. The recombinant protein was about 57kD and the production was 50.1 mg/L. It was shown that the two kinds of protein affected the conformation of each other by antibody affinity assay, but the recombinant targeting toxins could highly mediate CTLs to kill HIV-1 target cells.
Enterotoxins ; genetics ; Genetic Vectors ; HIV Envelope Protein gp120 ; biosynthesis ; genetics ; immunology ; HIV-1 ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology