Aluminum hydroxide (alum) is the most widely used adjuvant in human vaccines. Nevertheless, it is virtually unknown whether alum acts on B cells. In the present study, we explored the direct effect of alum on Ig expression by murine B cells in vitro. LPS-activated mouse spleen B cells were cultured with alum, and the level of isotype-specific Ig secretion, IgG1 secreting cell numbers, and Ig germ-line transcripts (GLT) were measured using ELISA, ELISPOT, and RT-PCR, respectively. Alum consistently enhanced total IgG1 production, numbers of IgG1 secreting cells, and GLTgamma1 expression. These results demonstrate that alum can directly cause IgG1 isotype switching leading to IgG1 production.
Alum Compounds ; Aluminum Hydroxide ; Animals ; B-Lymphocytes ; Cell Count ; Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Humans ; Hydroxides ; Immunoglobulin Class Switching ; Immunoglobulin G ; Mice ; Spleen ; Vaccines

BACKGROUND: Foot-and-mouth disease virus (FMDV) is a small single-stranded RNA virus which belongs to the family Picornaviridae, genus Apthovirus. It is a principal cause of FMD which is highly contagious in livestock. In a wild type virus infection, infected animals usually elicit antibodies against structural and non-structural protein of FMDV. A structural protein, VP1, is involved in neutralization of virus particle, and has both B and T cell epitopes. A RNA-dependent RNA polymerase, 3D, is highly conserved among other serotypes and strongly immunogenic, therefore, we selected VP1 and 3D as vaccine targets. METHODS: VP1 and 3D genes were codon-optimized to enhance protein expression level and cloned into mammalian expression vector. To produce recombinant protein, VP1 and 3D genes were also cloned into pET vector. The VP1 and 3D DNA or proteins were co-immunized into 5 weeks old BALB/C mice. RESULTS: Antigen-specific serum antibody (Ab) responses were detected by Ab ELISA. Cellular immune response against VP1 and 3D was confirmed by ELISpot assay. CONCLUSION: The results showed that all DNA- and protein-immunized groups induced cellular immune responses, suggesting that both DNA and recombinant protein vaccine administration efficiently induced Ag-specific humoral and cellular immune responses.
Animals ; Antibodies ; Clone Cells ; DNA ; DNA, Recombinant ; Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Epitopes, T-Lymphocyte ; Foot-and-Mouth Disease ; Foot-and-Mouth Disease Virus ; Humans ; Immunity, Cellular ; Livestock ; Mice ; Picornaviridae ; Proteins ; RNA Replicase ; RNA Viruses ; Vaccines ; Virion ; Viruses

Varicella vaccine has been included in the national immunization program for children since 2005 and zoster vaccine has been released since 2012 in Korea. Even though both varicella and zoster are caused by varicella-zoster virus (VZV), pathogeneses are different. In varicella, neutralizing antibody is very important to protect disease because VZV spreads via blood or lymph. In contrast, cell-mediated immunity is more important in zoster because of the neuronal spread of VZV. Therefore, the measurement methods of the immunogenicity against varicella and zoster vaccines are different. Fluorescent antibody to membrane antigen (FAMA) assay is the gold standard method to detect the protective antibody against VZV. It is still used as a reference test for the other methods. However, the fastidious nature required to perform the FAMA assay limits its use as a routine assay for the evaluation of vaccine immunogenicity. Nowadays, glycoprotein ELISA (gpEIA) is used as an alternative method for FAMA assay. However, there is no agreement over the protective level of gpEIA antibody titer with WHO standard international unit. The immunogenicity of zoster vaccine has been evaluated by responder cell frequency assay and IFN-gamma ELISpot assay. Nevertheless, skin test is considered to be a more accurate biomarker for cell-mediated immunity against zoster. For the evaluation of varicella vaccine, it is necessary to standardize the FAMA assay and to set the cut-off value for the gpEIA antibody titer through long-term follow-up study. For zoster vaccine, the evaluation of cell-mediated immunity in Korean adults is urgently needed.
Adult ; Antibodies, Neutralizing ; Chickenpox Vaccine ; Chickenpox* ; Child ; Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Follow-Up Studies ; Glycoproteins ; Herpes Zoster Vaccine ; Herpes Zoster* ; Herpesvirus 3, Human ; Humans ; Immunity, Cellular ; Immunization Programs ; Korea ; Membranes ; Methods* ; Neurons ; Skin Tests ; Vaccines*

No abstract available.
Enzyme-Linked Immunosorbent Assay*

No abstract available.
Enzyme-Linked Immunosorbent Assay*

No abstract available.
Enzyme-Linked Immunosorbent Assay* ; Korea*

No abstract available.
Autopsy* ; Enzyme-Linked Immunosorbent Assay*

PURPOSE: The human carcinoembryonic antigen (CEA) is expressed in several tumor types, including colorectal cancer, and is a tumor-associated antigen used as a target for antigen-specific immunotheraphy. CEA is a self-antigen associated with development, expressed in fetal cells and rarely expressed in normal colorectal epithelial cells. The induction of immune response to CEA is very difficult. In this study, we attempted to increase the tumor immunity specific to CEA by using dendritic cells pulsed with fusion proteins of CEA and Tat (transactivator of transcription), which transduces extracellular proteins into cytoplasm and causes antigens to be presented with MHC class I pathway. METHODS: The Tat gene was amplified in the PNL4-3 HIV plasmid and then inserted into PCEP4 plasmid vector. The CEA gene was cloned from cDNA from LoVo human colorectal cell line and then amplified through polymerase chain reaction method. After cloning of PCEP4 plasmid vector, the dendritic cell was sensitized and internalized with CEA and Tat-CEA protein. Then the Western blot analysis of the expression of CEA in the gene-modified dendritic cell and the immunofluorescent staining of the expression of CEA in CEA or Tat-CEA-pulsed dendritic cell were performed. A detection of IFN-gamma-releasing CD8 cell and a cytotoxicity of T-cell were was assesed using ELISPOT assay. The Immunoglobulin (Ig) G isotypes were analyzed with enzyme-linked immunosorbent assay. The statistical significance was assessed using Students t-test. RESULTS: CEA pulsed in dendritic cells was distributed over the cell surface and TatCEA was observed in the cytoplasm. The cellular immune responses by immunization with dendritic cells pulsed with TatCEA (322/10(4) lymphocytes) were significantly increased compared with those with CEA (244/10(4) lymphocytes) by IFN-gamma ELISPOT assay (P<0.05). The cytotoxic T lymphocyte (CTL) activity using mouse T-cell, EL-4 pulsed peptide (EAQNTTYL) as target cells was 23.3+/-2.75% (E:T=1:100) in the CEA group and 22.9+/-2.23% (E:T=1:100) in the TatCEA group. In ELISA analysis of the IgG isotype, the titer of IgG2a and IgG3, representing Th1 immune response, was lower than that of IgG1, representing Th2 immune response, in both the CEA group and the TatCEA group. CONCLUSIONS: These results suggest that TatCEA could be used for the development of a tumor vaccine and cellular immunotherapy using CTLs induced in vitro.
Animals ; Blotting, Western ; Carcinoembryonic Antigen ; Cell Line ; Clone Cells ; Cloning, Organism ; Colorectal Neoplasms ; Cytoplasm ; Dendritic Cells ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Epithelial Cells ; Equidae ; Genes, tat ; HIV ; Humans ; Immunity, Cellular ; Immunization ; Immunoglobulin G ; Immunoglobulins ; Immunotherapy ; Lymphocytes ; Mice ; Plasmids ; Polymerase Chain Reaction ; T-Lymphocytes

PURPOSE: The human carcinoembryonic antigen (CEA) is expressed in several tumor types, including colorectal cancer, and is a tumor-associated antigen used as a target for antigen-specific immunotheraphy. CEA is a self-antigen associated with development, expressed in fetal cells and rarely expressed in normal colorectal epithelial cells. The induction of immune response to CEA is very difficult. In this study, we attempted to increase the tumor immunity specific to CEA by using dendritic cells pulsed with fusion proteins of CEA and Tat (transactivator of transcription), which transduces extracellular proteins into cytoplasm and causes antigens to be presented with MHC class I pathway. METHODS: The Tat gene was amplified in the PNL4-3 HIV plasmid and then inserted into PCEP4 plasmid vector. The CEA gene was cloned from cDNA from LoVo human colorectal cell line and then amplified through polymerase chain reaction method. After cloning of PCEP4 plasmid vector, the dendritic cell was sensitized and internalized with CEA and Tat-CEA protein. Then the Western blot analysis of the expression of CEA in the gene-modified dendritic cell and the immunofluorescent staining of the expression of CEA in CEA or Tat-CEA-pulsed dendritic cell were performed. A detection of IFN-gamma-releasing CD8 cell and a cytotoxicity of T-cell were was assesed using ELISPOT assay. The Immunoglobulin (Ig) G isotypes were analyzed with enzyme-linked immunosorbent assay. The statistical significance was assessed using Students t-test. RESULTS: CEA pulsed in dendritic cells was distributed over the cell surface and TatCEA was observed in the cytoplasm. The cellular immune responses by immunization with dendritic cells pulsed with TatCEA (322/10(4) lymphocytes) were significantly increased compared with those with CEA (244/10(4) lymphocytes) by IFN-gamma ELISPOT assay (P<0.05). The cytotoxic T lymphocyte (CTL) activity using mouse T-cell, EL-4 pulsed peptide (EAQNTTYL) as target cells was 23.3+/-2.75% (E:T=1:100) in the CEA group and 22.9+/-2.23% (E:T=1:100) in the TatCEA group. In ELISA analysis of the IgG isotype, the titer of IgG2a and IgG3, representing Th1 immune response, was lower than that of IgG1, representing Th2 immune response, in both the CEA group and the TatCEA group. CONCLUSIONS: These results suggest that TatCEA could be used for the development of a tumor vaccine and cellular immunotherapy using CTLs induced in vitro.
Animals ; Blotting, Western ; Carcinoembryonic Antigen ; Cell Line ; Clone Cells ; Cloning, Organism ; Colorectal Neoplasms ; Cytoplasm ; Dendritic Cells ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Epithelial Cells ; Equidae ; Genes, tat ; HIV ; Humans ; Immunity, Cellular ; Immunization ; Immunoglobulin G ; Immunoglobulins ; Immunotherapy ; Lymphocytes ; Mice ; Plasmids ; Polymerase Chain Reaction ; T-Lymphocytes

PURPOSE: The human carcinoembryonic antigen (CEA) is expressed in several tumor types, including colorectal cancer, and is a tumor-associated antigen used as a target for antigen-specific immunotheraphy. CEA is a self-antigen associated with development, expressed in fetal cells and rarely expressed in normal colorectal epithelial cells. The induction of immune response to CEA is very difficult. In this study, we attempted to increase the tumor immunity specific to CEA by using dendritic cells pulsed with fusion proteins of CEA and Tat (transactivator of transcription), which transduces extracellular proteins into cytoplasm and causes antigens to be presented with MHC class I pathway. METHODS: The Tat gene was amplified in the PNL4-3 HIV plasmid and then inserted into PCEP4 plasmid vector. The CEA gene was cloned from cDNA from LoVo human colorectal cell line and then amplified through polymerase chain reaction method. After cloning of PCEP4 plasmid vector, the dendritic cell was sensitized and internalized with CEA and Tat-CEA protein. Then the Western blot analysis of the expression of CEA in the gene-modified dendritic cell and the immunofluorescent staining of the expression of CEA in CEA or Tat-CEA-pulsed dendritic cell were performed. A detection of IFN-gamma-releasing CD8 cell and a cytotoxicity of T-cell were was assesed using ELISPOT assay. The Immunoglobulin (Ig) G isotypes were analyzed with enzyme-linked immunosorbent assay. The statistical significance was assessed using Students t-test. RESULTS: CEA pulsed in dendritic cells was distributed over the cell surface and TatCEA was observed in the cytoplasm. The cellular immune responses by immunization with dendritic cells pulsed with TatCEA (322/10(4) lymphocytes) were significantly increased compared with those with CEA (244/10(4) lymphocytes) by IFN-gamma ELISPOT assay (P<0.05). The cytotoxic T lymphocyte (CTL) activity using mouse T-cell, EL-4 pulsed peptide (EAQNTTYL) as target cells was 23.3+/-2.75% (E:T=1:100) in the CEA group and 22.9+/-2.23% (E:T=1:100) in the TatCEA group. In ELISA analysis of the IgG isotype, the titer of IgG2a and IgG3, representing Th1 immune response, was lower than that of IgG1, representing Th2 immune response, in both the CEA group and the TatCEA group. CONCLUSIONS: These results suggest that TatCEA could be used for the development of a tumor vaccine and cellular immunotherapy using CTLs induced in vitro.
Animals ; Blotting, Western ; Carcinoembryonic Antigen ; Cell Line ; Clone Cells ; Cloning, Organism ; Colorectal Neoplasms ; Cytoplasm ; Dendritic Cells ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Epithelial Cells ; Equidae ; Genes, tat ; HIV ; Humans ; Immunity, Cellular ; Immunization ; Immunoglobulin G ; Immunoglobulins ; Immunotherapy ; Lymphocytes ; Mice ; Plasmids ; Polymerase Chain Reaction ; T-Lymphocytes