Cytomegalovirus (CMV) infection in healthy individuals is usually asymptomatic and results in latent infection. CMV reactivation occasionally occurs in healthy individuals according to their immune status over time. T cell responses to CMV are restricted to a limited number of immunodominant epitopes, as compared to responses to other chronic or persistent viruses. This response results in progressive, prolonged expansion of CMV-specific CD8+ T cells, termed 'memory inflation'. The expanded CMV-specific CD8+ T cell population is extraordinarily large and is more prominent in the elderly. CMV-specific CD8+ T cells possess rather similar phenotypic and functional features to those of replicative senescent T cells. In this review, we discuss the general features of CMV-specific inflationary memory T cells and the factors involved in memory inflation.
Aged ; Cytomegalovirus Infections* ; Cytomegalovirus* ; Humans ; Immunodominant Epitopes ; Inflation, Economic* ; Memory* ; T-Lymphocytes

BACKGROUND: In recent years, tuberculosis has re-emerged as a major health problem in both industrialized and developing countries. Recent advances in identifying and purifying antigens secreted in active tuberculosis infection have lead to the development of serological assays based on a number of immunodominant antigens. To date, the most sensitive and specific of these antigens has been the 38-kDa antigen. METHOD: Two rapid membrane-based serologic assays using antigen(38-kDa) from mycobacterium tuberculosis for the diagnosis of tuberculosis were evaluated in 22 patients with smear-positive pulmonary tuberculosis, 14 patients with inactive pulmonary tuberculosis, and 9 patients with non-tuberculous lung disease. RESULT: The evaluation of validity(sensitivity, specificity, positive predictive value, negative predictive value, false positivity and false negativity) of STAT-PAK ULTRA FAST(R) were 77.3%, 28.6%, 63.0%, 44.4%, 71.4%, and 22.7% for differential diagnosis of active pulmonary tuberculosis and inactive pulmonary tuberculosis, respectively. The evaluation of validity of STAT-PAK ULTRA FAST(R) were 77.3%, 33.3%, 73.9%, 37.5%, 66.7%, and 22.7% for differential diagnosis of active pulmonary tuberculosis and non-tuberculosis. The evaluation of validity of ICT Tuberculosis were 54.5%, 57%, 66.7%, 44.4%, 42.9%, and 45.5% for differential diagnosis of active pulmonary tuberculosis and inactive pulmonary tuberculosis. The evaluation of validity of ICT Tuberculosis were 54.5%, 100%, 100%, 47.4%, 0%, and 45.4% for differential diagnosis of active pulmonary tuberculosis and non-tuberculosis. CONCLUSION: We concluded no effectiveness of STAT-PAK ULTRA FAST(R) and ICT tuberculosis on serologic diagnosis of pulmonary tuberculosis. In the future, further large-scale study should be needed for serologic diagnosis of pulmonary tuberculosis.
Developing Countries ; Diagnosis* ; Diagnosis, Differential ; Humans ; Immunodominant Epitopes ; Lung Diseases ; Mycobacterium tuberculosis ; Tuberculosis* ; Tuberculosis, Pulmonary

A chimeric antigen designated B103 containing six immunodominant regions derived from three structural proteins of Rubella virus (RV) was designed and its utility in serological diagnosis was assessed. Protein B103 is comprised of aa 1-30 & aa 96-123 of C protein, aa 31-105 of E2 protein, as well as aa 11-39, aa 154-277 & aa 389-412 of E1 protein. In addition, it contains thioredoxin (TRX) at the N-terminal and His tag at the C-terminal. B103 was expressed in Escherichia coli BL21(DE3) and purified by Streamline Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B103 as verified by Western blotting analysis, we constructed and evaluated a novel capture ELISA for RV-IgM detection. B103 was expressed in a soluble form, accounting for 18.57% of the total bacterial proteins. After purification, the concentration and purity of protein B103 were 3.026 mg/mL and 95.35%, respectively. Western blotting analysis demonstrated that protein B103 could react with acute-phase serum of RV. By ELISA, 40 negative sera and 40 RV-acute phase sera were detected. The sensitivity, specificity, positive predictive value, negative predictive value and coincidence rate of the ELISA were 92.50%, 95.00%, 94.87%, 92.68% and 93.75%, respectively. The McNemer analysis suggested that there was no statistical difference between the 'Gold standard' and the novel ELISA with a kappa coefficient of 0.900, indicating excellent consistency. B103 chimeric protein with excellent antigenicity obtained from prokaryotic expression followed by chromatography purification could prove useful for early diagnosis of RV infection.
Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Immunodominant Epitopes ; Immunoglobulin M ; Rubella virus

BACKGROUND: Heat shock proteins (HSPs), or stress proteins, are immunodominant antigens of many microorganisms. In this study, we have detected the anti-HSP 70 antibody and tried to explain the role of the antibody with respect to the pathogenesis of SLE. Furthermore, we have attempted to find out the possibility to link the presence of the autoantibody with the monitoring and diagnosis of systemic lupus erythematosus (SLE). METHODS: A total of 80 samples from 55 SLE patients were screened for the presence of anti-HSP 70 antibodies. Simultaneously 59 healthy people were tested as a control group. The anti-HSP 70 antibodies were measured by enzyme-linked immunosorbent assay (ELISA) and confirmed by western blot in anti-HSP 70 antibody ELISA positive samples. The activity of disease state was confirmed by the patients' medical record and systemic lupus activity measure (SLAM). RESULTS: The mean optical density (O.D.450) of ELISA in healthy controls and SLE patients were 0.15+/-0.18 (mean+/-S.D.) and 0.13+/-0.14. The correlation of SLAM Score and ELISA O.D. was r2=0.19, P=0.014. And, the mean O.D. value of ELISA was 0.18+/-0.02 and 0.11+/-0.01 before and after treatment (P <0.05). We compared samples with SLAM Score. The O.D. of anti-HSP 70 ELISA in these patients were 0.20+/-0.02 and 0.08+/-0.002 before and after treatment respectively (n=10, mean+/-S.D., P <0.01). CONCLUSIONS: Anti-HSP 70 antibody was not a clinically useful diagnostic marker in SLE patients. However, the titer of anti-HSP 70 antibody can be used for the monitoring of the therapeutic effectiveness in these patients.
Antibodies ; Blotting, Western ; Diagnosis ; Enzyme-Linked Immunosorbent Assay ; Heat-Shock Proteins ; Humans ; Immunodominant Epitopes ; Lupus Erythematosus, Systemic* ; Medical Records

CFP10, ESAT6, Antigen 85A (Ag85A) and antigen 85B (Ag85B) are the key immunodominant antigens of Mycobacterium tuberculosis. In order to construct a eukaryotic vector able to co-express the four genes in one vector, we amplified the target gene fragments encoding the CFP10, ESAT6, Ag85A and Ag85B antigens and inserted them into the multicloning site of the shuttle plasmid vector pcDNA3.1 (+), of which the CFP10 and ESAT6 encoding genes were in frame fused with a linker encoding (Gly4Ser)3 residue, before the fused gene was inserted downstream of CMV promoter with a bovine growth hormone poly A(BGH pA) sequence at the 3'-end; Ag85A and Ag85B encoding genes were fused with a separation of internal ribosome entry site (IRES) sequence before the fused gene cassette was inserted downstream of RSV promoter with a BGH pA sequence at the 3'-end. The final plasmid containing all four genes was confirmed by sequence analysis and designated as pcDNA-CFP10-ESAT6-Ag85A-Ag85B (pcDNA-CEAB). In order to verify the ability of this construct to express target proteins, we then transfected the recombinant plasmid into Human embryonic kidney (HEK) 293T cells and harvested the cell lysates, and the cell lysates were then separated by SDS-PAGE and subjected to Western blot analysis 48 h after transfection. All four of the target proteins were detected in the cell lysates against the respective specific antibodies, suggesting that we have successfully constructed a eukaryotic vector co-expressing the four immunodominant antigens of Mycobacterium tuberculosis, which lay a foundation for the further study of the immunogenicity and protective activity of the four antigens.
Acyltransferases ; biosynthesis ; Antigens, Bacterial ; biosynthesis ; Bacterial Proteins ; biosynthesis ; Genetic Vectors ; HEK293 Cells ; Humans ; Immunodominant Epitopes ; Mycobacterium tuberculosis ; Plasmids

PURPOSE: Epitope spreading is a phenomenon in which distinct subdominant epitopes become major targets of the immune response. Heat shock protein (HSP) 60 from Porphyromonas gingivalis (PgHSP60) and peptide 19 from PgHSP60 (Pep19) are immunodominant epitopes in autoimmune disease patients, including those with periodontitis. It remains unclear whether Pep19 is a dominant epitope in subjects without periodontitis or autoimmune disease. The purpose of this study was to determine the epitope spreading pattern and verify Pep19 as an immunodominant epitope in healthy teenagers using dot immunoblot analysis. The patterns of epitope spreading in age-matched patients with type 1 diabetes mellitus (type 1 DM) and healthy 20- to 29-year old subjects were compared with those of healthy teenagers. METHODS: Peptide from PgHSP60, Mycobacterium tuberculosis HSP60 (MtHSP60), and Chlamydia pneumoniae HSP60 (CpHSP60) was synthesized for comparative recognition by sera from healthy subjects and patients with autoimmune disease (type 1 DM). Dot immunoblot analysis against a panel of peptides of PgHSP60 and human HSP60 (HuHSP60) was performed to identify epitope spreading, and a densitometric image analysis was conducted. RESULTS: Of the peptide from PgHSP60, MtHSP60, and CpHSP60, PgHSP60 was the predominant epitope and was most consistently recognized by the serum samples of healthy teenagers. Most sera from healthy subjects and patients with type 1 DM reacted more strongly with PgHSP60 and Pep19 than the other peptides. The relative intensity of antibody reactivity to Pep19 was higher in the type 1 DM group than in the healthy groups. CONCLUSIONS: Pep19 is an immunodominant epitope, not only in autoimmune disease patients, but also in healthy young subjects, as evidenced by their robust immunoreactivity. This result suggests that the Pep19-specific immune response may be an initiator that triggers autoimmune diseases.
Adolescent* ; Autoimmune Diseases ; Autoimmunity ; Chlamydophila pneumoniae ; Diabetes Mellitus, Type 1 ; Epitopes ; Healthy Volunteers ; Heat-Shock Proteins ; Humans ; Immunodominant Epitopes ; Mycobacterium tuberculosis ; Peptides ; Periodontitis ; Porphyromonas gingivalis* ; Porphyromonas*

This study was purposed to construct a fusion DNA vaccine containing WT1 multi-epitope and stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and to detect its expression and immunogenicity. On the basis of published data, a multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted CTL epitopes: one HLA*2402-restricted CTL epitope, two Th epitopes and one universal Th Pan-DR epitope (PADRE) was constructed. DNA-coding sequence was modified by Computer-Aided Design (CAD) to optimize proteasome-mediated epitope processing through the introduction of different amino acid spacer sequences. The synthetic nucleotide sequence was then inserted into an eukaryotic vector to construct the plasmid pcDNA3.1-WT1.For enhancing CTL activity, HSP70 fragment including stimulatory domain P407-426 was amplified by PCR from mycobacterial HSP70 gene and cloned into pcDNA3.1(+). Then Multi-WT1 was fused to the N-terminal of pcDNA3.1-mHSP70(407-426) to make the multi-epitope fusion gene vaccine pcDNA3.1-WT1-mHSP70(407-426). HEK-293T cells were transfected with this vaccine and the expressed product was identified by RT-PCR. Enzyme-linked immunospot assay (ELISPOT) was used to evaluate the immunological responses elicited by vaccine. The results showed that the most of WT1 epitopes could be correctly cleaved which was confirmed by software Net Chop 3.1 and PAPROCIanalysis. RT-PCR showed correct expression of target gene in HEK293T cells and ELISPOT showed specific T-cell responses. It is concluded that the eukaryotic expression vector PcDNA3.1-WT1-mHSP70(407-426) fusion gene has been successfully constructed and the immunity response is also elicited, which is a good candidate for further research of DNA vaccine.
Bacterial Proteins ; genetics ; immunology ; Epitopes ; genetics ; immunology ; Genetic Vectors ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Humans ; Immunodominant Epitopes ; Vaccines, DNA ; genetics ; immunology ; WT1 Proteins ; genetics ; immunology

The gag encoded p24 protein of human immunodeficiency virus-1 (HIV-1) is a major constitutent of the viral core, and is also known as one of the most immunodominant antigens in the host immune response against the HIV-1. Based on the neutralizing ability of anti-p24 antibodies as well as their rapid appearance in human serum after viral infection, the development of vaccines and diagnostic tools targeting the p24 protein and anti-p24 antibodies is of great interest. For the characterization of the immunological properties of the HIV-1 p24 protein, in a previous study, putative B-cell epitopes were identified by screening the reactivity of a goat anti-p24 antiserum to a large array of overlapping synthetic peptides covering the whole p24 sequence. Four peptides were identified for their abilities to elicit a strong B-cell response, which sequences comprises the regions p24 (164-182), (202-221), (217-236) and (232-256), respectively. In the present study, the immunogenicity and differential properties of each of these individual epitopes were further characterized. To evaluate the time course of the antibody response, BALB/c mice were immunized with the HIV-1 p24 protein and their serum titers against each of these peptides were determined. The earliest immune response was observed against the p24 (202-221) peptide, which also showed the highest antibody titer against the immunized antigen. Furthermore,. enzyme-linked immunosorbent assay with HIV-1 p24 protein coated microtiter plates revealed that anti-p24 (202-221) antiserum has the most pronounced reactivity against the native p24 protein. Since the p24 (202-221) epitope has also been reported to include a cytotoxic T-lymphocyte epitope, it is suggested that this region might represent a powerful antigenic site responsible for eliciting both T- and B-cell immune response. The possible application of this specific epitope in vaccine development or AIDS diagnosis is discussed.
Animals ; Antibodies ; Antibody Formation ; B-Lymphocytes ; Diagnosis ; Enzyme-Linked Immunosorbent Assay ; Epitopes* ; Epitopes, B-Lymphocyte ; Goats ; HIV* ; HIV-1 ; Humans ; Immunodominant Epitopes ; Mass Screening ; Mice ; Peptides* ; T-Lymphocytes, Cytotoxic ; Vaccines

The aim was to develop a single multiple-epitope fusion antigen which incorporates all of the major immunodominant epitopes from the six functional regions of the HCV genome. A nucleic acid sequence consisting of viral core, E1, E2, NS3, NS4, and NS5 regions was constructed and inserted into the Promega Pinpoint Xa-1 T vector for inducing expression. The protein was expressed in JM109 (DE3) as a fusion protein with a 13 kD biotinlated tag to be used for detection and affinity purification. Immunogenicity and biotinylated tag of the fusion protein were detected by Western blot analysis with positive anti-HCV serum and streptavidin alkaline phosphatase. After purified by Promega SoftLink Soft Release Avidin Resin, the protein was pre-coated on microwell and detected with anti-core, anti-NS3, anti-NS4 and anti-NS5 positive sera by EIA, respectively. The results indicated that the recombinant soluble protein was expressed and labelled with biotin successfully, it reacted with anti-HCV positive serum, and exposed all of the major immunogenic epitopes chosen. In conclusion, this recombinant antigen may be used to design an double antigen sandwich anti-HCV immunoassay.
Antigens, Viral ; genetics ; immunology ; Biotinylation ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Hepacivirus ; immunology ; Hepatitis C Antibodies ; blood ; Humans ; Immunodominant Epitopes ; Recombinant Fusion Proteins ; genetics ; immunology

OBJECTIVETo screen and identify dengue virus type 2 specific antigens and establish an enzyme-linked immunosorbent assay (ELISA) for detecting dengue virus type 2 antibody.

METHODSUsing the bioinformatic software DNAstar and ANTHEPROT, we analyzed the hydrophilicity, flexibility, surface probability and antigenicity of dengue virus type 1-4, Japanese encephalitis virus, and Yellow fever virus M and E protein amino acid sequences, and also evaluated the influence of secondary structure. The specific epitopes of dengue virus type 2 were predicted according to the epitope location and amino acid sequence similarity, and the epitope conservation was assessed using the sequence information of different dengue virus type 2 strains in GenBank. Based on the results of bioinformatic analysis, 5 specific epitopes were amplified and inserted into the prokaryotic expression vector pET32a, which were transferred into E. coli Rosetta (DE3) for expression of the proteins. SDS-PAGE and Western blotting were used to identify the expressed proteins and test their antigenicities. The antigen selected by Western blotting was used to establish the ELISA system for dengue virus type 2 antibody detection.

RESULTSBioinformatic analysis predicted 8 possible dengue virus type 2 specific epitopes, and 6 of them were efficiently expressed in E. coli. Western blotting confirmed 1 dengue virus type 2 specific antigen, the ELISA system for dengue virus antibody detection was successfully established using this specific antigen.

CONCLUSIONWe have obtained a dengue virus type 2 specific antigen and established an ELISA system for detection of dengue virus type 2 antibody.

Antibodies, Viral ; immunology ; Antigens, Viral ; immunology ; Computational Biology ; Dengue Virus ; classification ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Immunodominant Epitopes ; Software