Listeria monocytogenes (LM), a Gram-positive facultative intracellular bacterium, can be used as an effective exogenous antigen expression vector in tumor-target therapy. But for successful clinical application, it is necessary to construct attenuated LM stain that is safe yet retains the potency of LM based on the full virulent pathogen. In this study, attenuated LM and recombinants of LM expressing melanoma inhibitory activity (MIA) were constructed successfully. The median lethal dose (LD(50)) and invasion efficiency of attenuated LM strains were detected. The recombinants were utilized for immunotherapy of animal model of B16F10 melanoma. The level of MIA mRNA expression in tumor tissue was detected by using real-time polymerase chain reaction (PCR) with specific sequence, meanwhile the anti-tumor immune response was assayed by flow cytometric analysis and enzyme-linked immunosorbent spot (ELISPOT) assay. The results showed the toxicity and invasiveness of attenuated LM were decreased as compared with LM, and attenuated LM expressing MIA, especially the double-genes attenuated LM recombinant, could significantly induce anti-tumor immune response and inhibit tumor growth. This study implicates attenuated LM may be a safer and more effective vector for immunotherapy of melanoma.
Animals ; Cancer Vaccines ; genetics ; immunology ; Extracellular Matrix Proteins ; genetics ; immunology ; Listeria monocytogenes ; immunology ; Male ; Melanoma ; genetics ; immunology ; Mice ; Mice, Inbred C57BL ; Vaccines, Attenuated ; genetics ; immunology

OBJECTIVETo study the feasibility of preparing a therapeutic lung cancer vaccine by transfecting dendritic cells (DCs) with adeno-associated virus vector carrying carcino-embryonic antigen gene (rAAV/CEA).

METHODSAdherent cells (monocytes) isolated from the peripheral blood of a healthy donor were infected with rAAV/CEA virus stock or pulsed with CEA peptide (control). The monocytes in both groups were induced into mature DCs with recombinant human GM-CSF, IL-4 and TNF-α. At day 7 of induction, the mature DCs were harvested and mixed with T lymphocytes. T cell proliferation stimulated by the DCs was assessed with (3)H-thymidine uptake, and the expression of IL-4, IFN-γ, CD8, CD4, CD25 and CD69 in cytotoxic T lymphocytes (CTL) was analyzed with flow cytometry. The cytotoxicity of the CTL against the target CEA-positive lung cancer A549 cells was tested by (51)Cr releasing assay.

RESULTSThe DCs transfected with rAAV/CEA strongly stimulated the proliferation of the T cell populations, and the induced CTL showed high expressions of CD8, CD69 and IFN-γ. The transfected DCs exhibited a high killing ability of CEA-positive lung cancer cells, and the killing showed a CEA antigen specificity and was limited by MHC I. These results suggested the ability of rAAV/CEA-transfected DCs in generating specific cellular immunity in vitro.

CONCLUSIONIt is feasible to prepare therapeutic lung cancer vaccines by transfecting DCs with rAAV/CEA.

Cancer Vaccines ; Carcinoembryonic Antigen ; genetics ; Cell Line ; Dendritic Cells ; immunology ; Dependovirus ; genetics ; Genetic Vectors ; Humans ; Monocytes ; immunology ; Transfection

OBJECTIVETo construct a novel immunogene therapeutic plasmid that expresses human interleukin-12 (IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF) and B7.1 and observe its expression in vivo and in vitro.

METHODSHuman IL-12 gene fragment was cloned into the upper stream of IRES gene in the previously constructed plasmid pVAX-IRES-GM-CSF-B7.1, and the positive recombinant plasmid pVAX-IL-12-GB was transfected into 293T cells via Lipofectamine 2000. The expressions of IL-12 and GM-CSF-B7.1 mRNA and proteins in the transfected cells were assayed by RT-PCR and ELISA, and B7.1 expression was tested by fluorescence-activated cell sorting and immunofluorescence assay. The plasmid pVAX-IL-12-GB was delivered into mouse muscle by electroporation, and the expression of IL-12 in the muscle tissue was identified by immunohistochemistry.

RESULTSEnzyme digestion, PCR and sequence analysis all confirmed successful construction of the recombinant plasmid pVAX-IL-12-GB. IL-12, GM-CSF and B7.1 expressions were all detected in transfected 293T cells, and the expression of IL-12 was also detected in the transfected mouse muscular tissues.

CONCLUSIONA novel anti-tumor immunogene vaccine constructed can be expressed both in vivo and in vitro, which facilitates further studies of tumor immunogene therapy.

Animals ; B7-1 Antigen ; genetics ; immunology ; Cancer Vaccines ; genetics ; immunology ; Electroporation ; Genetic Therapy ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; immunology ; Humans ; Interleukin-12 ; genetics ; immunology ; Mice ; Plasmids ; Transfection

NY-ESO-1 is an important member of cancer-testis antigen family and is widely distributed among many cancer types. As a tumor-specific antigen with the strongest immunogenicity so far identified, it can induce spontaneous antibody and T-cell responses in patients with NY-ESO-1-positive tumors. Therefore, it has been a good vaccine candidate in the immunotherapy against many malignancies. This article reviews the recent research advances in NY-ESO-1 and its relevant vaccines.
Antigens, Neoplasm ; genetics ; immunology ; therapeutic use ; Cancer Vaccines ; immunology ; therapeutic use ; Clinical Trials as Topic ; Humans ; Immunotherapy ; Membrane Proteins ; genetics ; immunology ; therapeutic use ; Neoplasms ; genetics ; immunology ; therapy

OBJECTIVETo construct a replicative anti-tumor DNA vaccine PSCK-2PFcGB based on Semliki Forest Virus (SFV) replicon vector and observe its expression in vivo and in vitro.

METHODSThe plasmid pVAX1-2PFcGB was digested with Nhe I, and the digestion product was blunted prior to further digestion with BssH II to obtain the fragment 2PFcGB, a fusion gene containing the multitarget complex antigen 2PAG encoding both the most cytotoxic T lymphocyte epitopes of human survivin and chorionic gonadotropin β chain-CTP37 of human and monkey. The 2PFcGB fragment was inserted into the PSCK vector digested by Sma I. The products with the expected size were extracted and ligated, and the positive clones were screened by kanamycin and amplified. The recombinant PSCK-2PFcGB, following identification by colony PCR and restriction endonuclease Nde I, was transfected into 293T cells via lipofectamine 2000 and its expression was detected. The recombinant plasmid was also transfected into mouse quadriceps femoris muscle to observe its expression in vivo by immunohistochemistry.

RESULTSNde I digestion resulted in a fragment of the expected size. Transfection with the recombinant plasmid PSCK-2PFcGB resulted in successful expression of the antigen and adjuvant molecular protein in 293T cells, with the positivity rates of 5.70% and 19.75%, respectively. The fusion tumor antigen survivin and hCGβ-CTP37 were also detected in the muscular tissues of the mice.

CONCLUSIONA novel replicative anti-tumor DNA vaccine PSCK-2PFcGB has been successfully constructed and can be expressed in 293T cells and in the muscular tissues of immunized mice, which provide a basis for further studies of the antitumor activity and immunological mechanism of the DNA vaccine.

Animals ; Antibodies, Antinuclear ; immunology ; Cancer Vaccines ; biosynthesis ; immunology ; Gene Expression ; Genetic Vectors ; HEK293 Cells ; Humans ; Mice ; Muscle, Skeletal ; metabolism ; Plasmids ; Semliki forest virus ; genetics ; Vaccines, DNA ; biosynthesis ; immunology

OBJECTIVETo establish a human lung cancer cell line expressing human interferon-gamma.

METHODSThe full-length gene of human interferon-gamma (IFN-gamma) was introduced into the human lung adenocarcinoma cell line A549 through retroviral vector pLXSN. The established cell line A549-IFN-gamma was tested for expression of MHC class I and class II by flow cytometer (FCM) and tested for expression of IFN-gamma by enzyme-lined immunoadsorbent assay (ELISA ). The tumorigenesis of cell line A549-IFN-gamma was tested on nude mice.

RESULTSA high level of IFN-gamma protein was detected in the culture supernatants of cell line A549-IFN-gamma. The expressions of MHC class I and class II on A549-IFN-gamma cells increased significantly (P<0.01), when compared with parental cell line A549. However, there was no significant difference (P<0.05) between the growth of cell line A549-IFN-gamma and A549. Finally, the tumorigenesis test showed that A549-IFN-gamma had lower tumorigenetic effects than A549.

CONCLUSIONThe results indicate that introduction of human IFN-gamma gene into cell line A549 could increase its immunogenicity and decrease its tumorigenesis. With the established cell line A549-IFN-gamma, a tumor vaccine for human lung cancer may be developed.

Animals ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Genetic Therapy ; Humans ; Interferon-gamma ; genetics ; Lung Neoplasms ; immunology ; therapy ; Male ; Mice ; Transfection

OBJECTIVETo observe the in vivo antitumor activity of murine liver tumor vaccine expressing MIP-1alpha mediated by recombinant adenoviral vector.

METHODSThe infection efficacy was measured by GFP expression 48 hours after infection of Hepa1-6, and the number of cells was counted daily for 14 days. 5 x 10(6) modified Hepa1-6 cells were inoculated subcutaneously to C57BL/6 mice and the tumor-free animals were rechallenged by 2 x 10(6) wild-type Hepa1-6 cells or syngenic EL4 cells four weeks later. The tumor volume was measured twice a week.

RESULTSAdenoviral vectors could efficiently infect Hepa1-6 cells in vitro, and the in vitro growth rate of AdmMIP-1alpha modified Hepa1-6 cells was not affected; however the in vivo tumorigenicity was significantly decreased, compared with that of control vector modified Hepa1-6. Rechallenge of the tumor-free mice four weeks after administration of AdmMIP-1alpha with the parental Hepa1-6 cells resulted in significant inhibition of tumor growth, but there was no significant difference when rechallenged with EL4.

CONCLUSIONSThe liver cancer cells expressing mMIP-1alpha mediated by recombinant adenoviral vector decrease tumorigenicity and elicit specific immunological protection, and could be used as an effective liver tumor vaccine.

Adenoviridae ; genetics ; Animals ; Cancer Vaccines ; immunology ; Chemokine CCL3 ; Chemokine CCL4 ; Genetic Therapy ; Liver Neoplasms, Experimental ; therapy ; Macrophage Inflammatory Proteins ; genetics ; Mice ; Mice, Inbred C57BL ; Vaccines, Synthetic ; immunology

OBJECTIVESTo construct a novel virus-like particulate peptide-nucleic acid vaccine (VPNV) of human telomerase reverse transcriptase (hTERT), and to study its anti-liver cancer immunity.

METHODSA cationic antigenic peptide was synthesized and purified, and then human granulocyte macrophage colony stimulating factor (hGM-CSF) and TERT gene were cloned into the eukaryotic expression vector pTCAE. The peptide was combined with the nucleic acid vaccine to make a VPNV, which was transfected into eukaryotic cell COS-7. The immunogenicity of hGM-CSF and hTERT were detected using ELISA and Western blot. The efficacy of VPNV for inducing antigen specific CTL response was determined using the lactate dehydrogenase release method.

RESULTSVPNV was verified capable to trigger specific CTL responses and has shown a specific cytolytic activity to liver cancer cell HepG2.

CONCLUSIONA VPNV which can stimulate antigen specific CTL response was successfully constructed. This paves the way for our further investigation of anti-liver cancer immunity in mice.

Animals ; Cancer Vaccines ; immunology ; Cloning, Molecular ; Eukaryotic Cells ; metabolism ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; immunology ; Liver Neoplasms ; immunology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Peptide Nucleic Acids ; genetics ; immunology ; Telomerase ; genetics ; immunology ; Vaccines, DNA ; immunology

The idiotypic determinant of surface immunoglobulin of B-cell lymphoma, as a tumor-specific antigen, has proved to be able to induce immune responses. To analyze whether an idiotypic vaccine fused with cytokine can elicit more effectively protective antitumor immunity, an eukaryotic expression plasmid was constructed, which encoded the fusion gene of single-chain variable fragment as a tumor specific antigen against B-cell lymphoma with monocyte chemotactic protein-3 (MCP3) as immunogen to elicit T-cell-dependent protective antitumor immunity, and EGFP (Enhanced Green Fluorescent Protein) gene as a marker to trace the survival, growth, differentiation and expression of the former exogenetic genes. The cDNAs for immunoglobulin (Ig) VH and IgVL were amplified by RT-PCR and assembled into the single-chain variable fragment (scFv) connected with a (Gly(4)Ser)(3) linker by recombinant PCR method. Then, the fragments of scFv and MCP3 were ligated with a NDAQAPKS spacer by the same method. The results showed that the fusion genes of scFv and MCP3-scFv were inserted into an eukaryotic expression vector pTARGET, and EGFP was cloned into the downstream of scFv and MCP3-scFv respectively. Finally the constructed plasmids were confirmed by sequencing and restriction analysis. In conclusion, a tumor-derived idiotypic DNA vaccine, encoding the fusion gene of single-chain variable fragment and monocyte chemotactic protein-3 (MCP3) to elicit a T-cell dependent, antitumor immunity, and the EGFP gene was inserted correctly. The DNA vaccine could be used for further study of DNA vaccine against B cell lymphoma in vivo.
Animals ; Cancer Vaccines ; genetics ; immunology ; Cell Line, Tumor ; Chemokine CCL7 ; immunology ; Genetic Vectors ; Immunoglobulin Variable Region ; immunology ; Lymphoma, B-Cell ; genetics ; immunology ; prevention & control ; Mice ; Mice, Inbred BALB C ; Plasmids ; Single-Chain Antibodies ; immunology ; Vaccines, DNA ; genetics ; immunology

OBJECTIVETo obtain streptavidin-tagged human interleukin-15 (SA/hIL15) fusion protein and evaluate its bioactivity.

METHODSpET24a-6His-SA-hIL-15 and pET32a-hIL-15-SA-6His plasmids were constructed and expressed in BL 21(DE3) host bacteria to generate the fusion protein. The recombinant fusion protein IL-15/SA was purified using Ni-NTA affinity chromatography and refolded, and the efficiency of surface modification of the fusion protein on biotinylated cells was examined by fluorescence-activated cell sorting. CCK-8 method was used to evaluate the effect of IL-15/SA fusion protein in inducing the proliferation of human peripheral-blood lymphocyte (PBL) cells stimulated by PHA.

RESULTSThe recombinant SA-hIL-15 and hIL15-SA fusion proteins were highly expressed in BL21(DE3) at about 20% of the total bacterial proteins. The purified hIL15-SA fusion protein exhibited a bifunctionality by promoting the proliferation of PBL cells activated by PHA and high-affinity binding to biotinylated cell surface mediated by SA, with a cell surface modification efficiency exceeding 95%. SA-hIL-15 showed a 4-fold higher hIL15 bioactivity than hIL15-SA.

CONCLUSIONSA/hIL-15 bifunctional fusion protein has been successfully obtained to facilitate the future development of hIL-15-surface-modified cancer cell vaccine.

Cancer Vaccines ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Interleukin-15 ; biosynthesis ; genetics ; Lymphocyte Activation ; drug effects ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Streptavidin ; biosynthesis ; genetics