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: Zoster vaccination is recommended for people with a history of herpes zoster (HZ), but the most effective timing of vaccine administration after zoster illness is unresolved. This prospective observational study compared the immunogenicity and safety of administering HZ vaccine at 6-12 months and 1-5 years after zoster illness. MATERIALS AND METHODS: Blood samples were collected before the administration of live zoster vaccine and 6 weeks after vaccination. Varicella-zoster virus (VZV) IgG concentrations and T-cell responses were assessed by glycoprotein enzyme-linked immunosorbent assay and interferon-γ enzyme-linked immunospot assay (ELISPOT), respectively. RESULTS: The baseline geometric mean value (GMV) of VZV IgG was higher in the 6-12 months group than in the 1-5 years group (245.5 IU/mL vs. 125.9 IU/mL; P = 0.021). However, the GMV increased significantly in both groups (P = 0.002 in the 6-12 months group; P <0.001 in the 1-5 years group). The results of the ELISPOT assay were not significant for differences of the GMV between baseline and 6-week post-vaccination groups, while the GMV increased significantly in both groups (P = 0.001 in the 6-12 months group; P <0.001 in the 1-5 years group). CONCLUSION: The immunogenicity of zoster vaccine may be similar whether administered 6-12 months, or >1 year after zoster illness. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02704572
Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Glycoproteins ; Herpes Zoster Vaccine* ; Herpes Zoster* ; Herpesvirus 3, Human ; Immunoglobulin G ; Observational Study ; Prospective Studies* ; T-Lymphocytes ; Vaccination*

BACKGROUND: Zoster vaccination is recommended for people with a history of herpes zoster (HZ), but the most effective timing of vaccine administration after zoster illness is unresolved. This prospective observational study compared the immunogenicity and safety of administering HZ vaccine at 6-12 months and 1-5 years after zoster illness. MATERIALS AND METHODS: Blood samples were collected before the administration of live zoster vaccine and 6 weeks after vaccination. Varicella-zoster virus (VZV) IgG concentrations and T-cell responses were assessed by glycoprotein enzyme-linked immunosorbent assay and interferon-γ enzyme-linked immunospot assay (ELISPOT), respectively. RESULTS: The baseline geometric mean value (GMV) of VZV IgG was higher in the 6-12 months group than in the 1-5 years group (245.5 IU/mL vs. 125.9 IU/mL; P = 0.021). However, the GMV increased significantly in both groups (P = 0.002 in the 6-12 months group; P <0.001 in the 1-5 years group). The results of the ELISPOT assay were not significant for differences of the GMV between baseline and 6-week post-vaccination groups, while the GMV increased significantly in both groups (P = 0.001 in the 6-12 months group; P <0.001 in the 1-5 years group). CONCLUSION: The immunogenicity of zoster vaccine may be similar whether administered 6-12 months, or >1 year after zoster illness. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02704572
Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Glycoproteins ; Herpes Zoster Vaccine* ; Herpes Zoster* ; Herpesvirus 3, Human ; Immunoglobulin G ; Observational Study ; Prospective Studies* ; T-Lymphocytes ; Vaccination*

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*

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

OBJECTIVETo express HPV6bL2deltaN360E7E6 fusion protein in E. coli and preliminarily evaluate its immune effect.

METHODSThree HPV6b gene fragments, which were L2(1-360 bp), E7 and E6, were fused by overlapping PCR, then were inserted into a prokaryotic expression vector and expressed in E. coli. C57BL/6 mice were immunized with purified fusion protein plus Al (OH)3 and/or CpG adjuvants through intramuscular route, the cellular and humoral immune responses were detected by IFN-gamma ELISPOT and ELISA respectively.

RESULTSProtein plus CpG adjuvant could induce the strongest cellular immune response to E7 and E6, high antibody titer against L2 could be detected in all immunized groups but there were no significant difference among these groups.

CONCLUSIONSHPV6bL2deltaN360E7E6 gene was successfully cloned into pQE30 vector and expressed in E. coli, the fusion protein was also purified and proved that could induce strong cellular and humoral immune responses with appropriate adjuvant in C57BL/ 6 mice and could be used for future research.

Adjuvants, Immunologic ; Animals ; Condylomata Acuminata ; genetics ; Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; methods ; Escherichia coli ; genetics ; Gene Expression ; Genetic Vectors ; Immunity, Humoral ; Immunization ; Mice ; Mice, Inbred C57BL ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacology ; Recombinant Proteins ; chemistry ; genetics

BACKGROUND: The immune responses mediated by CD8+T cells are known to be significant in controlling M. tuberculosis infections. In order to determine the role of cytotoxic CD8+T cells in the protective immune mechanism in latently infected subjects, this study examined whether or not the cytotoxic immune responses of CD8+T cells specific to the M. tuberculosis somatic antigens are induced in BCG vaccinated healthy subjects. METHODS: Cytotoxicity and IFN-gamma elispot assays were used to investigate the activities of CD8+T cells specific for the thyA30-38 peptide epitope in circulating peripheral blood mononuclear cells (PBMC) from BCG-vaccinated HLA-A*0201 and A*0206 subjects. RESULTS: The results indicate the cytotoxic and IFN-gamma immune responses of CD8+T cells specific for thyA30-38 were induced in BCG vaccinated healthy subjects. CONCLUSION: The cytotoxic and IFN-gamma responses by CD8+T cells specific for the M. tuberculosis somatic antigens are induced in BCG-vaccinated subjects, and appear to be involved in the protective immune mechanism in latently infected people against a M. tuberculosis infection.
Enzyme-Linked Immunospot Assay ; Mycobacterium bovis ; Tuberculosis*

Background: Mosquitoes and pigs play important roles in maintaining and increasing the Japanese Encephalitis (JE) virus in nature and which is then transmitted to humans. Thus, surveillance of the JE infection frequency in the pig population may predict the human JE cases. \r\n', u'Objectives: The study aimed to determine IgG antibody against the JE virus in the pig population in Hanam province \r\n', u'Subjects and methods: The study included 1791 pig serum samples collected from 3 districts of Hanam province from Apr 2006 to Mar 2007. GAC-ELISA technique was used to determine the JE virus infection in the swine population.\r\n', u'Results: The average positive rate in pig population was 34.9 % (626/1791); with the highest frequency occurring in the summer (37.7%- 84.0 %), co-incident with the JE season in Northern Vietnam. On the contrary, in winter JE case are rare, frequency of IgG antibody against JE virus in the swine population was low, ranging from 9.2% to 22.0.%. \r\n', u'Conclusions: These results have shown the ecologically close relationship between the amplification of the JE virus in the swine population, vector and JE cases in northern Vietnam. \r\n', u'
Japanese encephalitis ; pig population ; GAC-ELISA.

Background: Japanese Encephalitis (JE) is common in the plains and mountainous areas in Asia \u2013 Pacific. Japanese encephalitis vaccine shows effectiveness in protecting children from JE in some countries such as Japan and Korea. Objective: To evaluate the efficacy of Japanese Encephalitis (JE) vaccination in Thai Binh province during 2003-2007. Subject and Method: Prospective, retrospective and sero-epidemiological methods were carried out on 329 samples collected from viral encephalitis patients and tested by JE MAC-ELISA, the positive average was 41.6% (137/329). Result: It had dramatically dropped from 85.2% in 2003 to 8.5 % in 2007 related to the rate of JE vaccination for children from 1 to 5 years old increasing from 49 % in 2003 to 77 % in 2007. Most of JE confirmed cases were un-vaccinated. Conclusion: JE etiology cause viral encephalitis in children in Thai Binh province was reduced thanks to JE vaccination in EPI program for 1 to 5 year old children. But more than 96% (131/136) of viral encephalitis in 15 years old upward was unknown etiology, the need for further study of the etiology cause viral encephalitis in adults.
Japanese encephalitis ; virus encephalitis ; MAC-ELISA ; Vaccine