As the dominant antigens, early secreted antigenic target 6 (ESAT6, E6) and culture filtrate protein 10 (CFP10, C10) had once been the focus of tuberculosis (TB) vaccine due to their capability of inducing strong cell immune response in the host. They are also endowed with promising future of prevention against and diagnosis of TB. In this review, we systematically introduce recent research progress of E6 and C10, especially in structure-function, biological characteristics, protein expression and secretion, host immunity and vaccine development, and the prospects of their application are also discussed.
Antigens, Bacterial ; chemistry ; genetics ; immunology ; Bacterial Proteins ; chemistry ; genetics ; immunology ; Humans ; Immunodominant Epitopes ; immunology ; Molecular Biology ; Peptide Fragments ; chemistry ; genetics ; immunology ; Tuberculosis Vaccines ; genetics ; immunology ; Vaccines, DNA ; immunology

The levels of Streptococcus (S.) mutans infections in saliva were evaluated and a comparison for specific antibody levels among children with different levels of S. mutans infection was made. The promising epitopic regions of antigen AgI/II (PAc) and glucosyltransferase (GTF) for potential vaccine targets related to S. mutans adherence were screened. A total of 94 children aged 3-4 years were randomly selected, including 53 caries-negative and 41 caries-positive children. The values of S. mutans and those of salivary total secretory immunoglobulin A (sIgA), anti-PAc and anti-Glucan binding domain (anti-GLU) were compared to determine the correlation among them. It was found the level of s-IgA against specific antigens did not increase with increasing severity of S. mutans infection, and the complete amino acid sequence of PAc and GTFB was analyzed using the DNAStar Protean system for developing specific anti-caries vaccines related to S. mutans adherence. A significantly positive correlation between the amount of S. mutans and children decayed, missing, and filled teeth index was observed. No significant difference was detected in specific sIgA against PAc or GLU between any two groups. No significant correlation was found between such specific sIgA and caries index. A total of 16 peptides from PAc as well as 13 peptides from GTFB were chosen for further investigation. S. mutans colonization contributed to early children caries as an important etiological factor. The level of sIgA against specific antigens did not increase with increasing severity of S. mutans infection in children. The epitopes of PAc and GTF have been screened to develop the peptide-based or protein-based anti-caries vaccines.
Antibodies, Bacterial ; biosynthesis ; Antigens, Bacterial ; chemistry ; immunology ; Bacterial Proteins ; chemistry ; immunology ; Case-Control Studies ; Child, Preschool ; Dental Caries ; immunology ; pathology ; prevention & control ; Epitopes ; chemistry ; immunology ; Female ; Glucosyltransferases ; chemistry ; immunology ; Humans ; Immunoglobulin A, Secretory ; biosynthesis ; Male ; Peptides ; chemistry ; immunology ; Saliva ; chemistry ; microbiology ; Severity of Illness Index ; Streptococcal Vaccines ; biosynthesis ; chemistry ; immunology ; Streptococcus mutans ; chemistry ; immunology ; pathogenicity ; Vaccines, Subunit ; Virulence Factors ; chemistry ; immunology

OBJECTIVETo compare the substrate specificity of three murine GDP fucose: beta-galactoside alpha1,2-fucosyltransferases (alpha1,2-FT).

METHODSThree members of MFUT- I, -II and -III, coding for a alpha1,2-FT, a GDP-fucose, were cloned from a cDNA of murine small intestine by reverse transcription-polymerase chain reaction. The coding regions were ligated into mammalian expression vector pcDNA 3.1 (pcDNA3.1-MFUT-I, pcDNA3.1-MFUT- II , and pcDNA3.1-MFUT- III) and were transiently transfected into COS-7 cells using a cellphect transfection kit. Then the cells were analyzed for expression and function of alpha1,2-FT and the substrate specificity of three alpha1,2-FT was compared.

RESULTSMFUT- I, -II, and -III exhibited sequence homology with human H (77%), Se (79%), and Sec1 (75%) genes, respectively. COS-7 cells transfected with pcDNA3.1-MFUT- I and pcDNA3.1-MFUT- II showed alpha1,2-FT activity, but no activity was detected in COS-7 cells transfected with pcDNA3.1- MFUT-III. MFUT- II showed alpha1,2-FT activity with both asialo-monosialoteterahexosyl ganglioside (GA1) and monosialoteterahexosyl ganglioside (GM1) as substrates to produce fucosyl GA1(FGA1) and fucosyl GM1(FGM1), respectively, but MFUT- I only showed alpha1,2-FT activity with GA1. The relative activity of MFUT- II with GA1 was 80-90-folds higher compared with MFUT- I, and the relative activity of MFUT- II with GA1 was 10-20-folds higher than that of GM1. The fucosyltransferase encoded by the MFUT- II gene showed the enzyme activity not only responsible for the synthesis of type 4-H antigens FGA1 and FGM1, but also responsible for the synthesis of type 1-H and 2-H antigens with lactotetraosylceramide and neolactotetraosylceramide as substrates.

CONCLUSIONMFUT- II is the main alpha1,2-FT in mouse and MFUT- II can product type 4-H antigen FGA1 and FGM1, but MFUT- I only synthesizes FGA1. MFUT-III has no alpha1,2-FT activity.

Animals ; Antigens, Bacterial ; biosynthesis ; COS Cells ; Cercopithecus aethiops ; Cloning, Molecular ; Fucosyltransferases ; chemistry ; genetics ; metabolism ; Gangliosides ; metabolism ; Mice ; Substrate Specificity ; Transfection

OBJECTIVETo clone tpn17 and tpn47 genes of Treponema pallidum and then construct their prokaryotic expression systems,to establish ELISAs based on rTpN17 and rTpN47 as antigens and to evaluate the sensitivity and specificity of the ELISAs for detection of serological diagnosis of syphilis.

METHODSThe whole length of tpn17 and tpn47 genes was amplified by PCR and then their prokaryotic expression systems were constructed. SDS-PAGE was used to measure the expression of the target recombinant proteins rTpN17 and rTpN47. Ni-NTA affinity chromatography was applied to extract rTpN17 and rTpN47, while Western blot was performed to determine the specific immunoreactivity of rTpN17 and rTpN47. By using rTpN17 and rTpN47 as the coated antigen, respectively, ELISAs (rTpN17-ELISA and rTpN47-ELISA) were established to detect serum samples from 200 healthy individuals, 25 RA patients, 17 SLE patients and 211 syphilis patients. The detection effects of the ELISAs were compared to those of TRUST and TPHA.

RESULTThe sequence similarity of the cloned tpn17 and tpn47 genes was 100 % compared with the corresponding sequences in GenBank. The expression outputs of rTpN17 and rTpN47 were approximately 37.2 % and 26.8 % of the total bacterial proteins, respectively. Both the extracted rTpN17 and rTpN47 could take place remarkable conjugation reactions to the sera with positive antibody against Treponema pallidum.The positive detection rate of TPHA (99.1%) was the highest (P<0.001). The positive detection rates of rTpN17-ELISA (85.3 %) and rTpN47-ELISA (84.3 %) were similar (P>0.05). The positive detection rates of TRUST (72.5 %) was lower than that of rTpN17-ELISA (P=0.001) but similar to that of rTpN47-ELISA (P=0.014). The detection results of all the serum samples from healthy individuals, RA patients and SLE patients were negative, whereas 7.1 % (3/42) of the samples from RA or SLE patients were positive.

CONCLUSIONrTpN17 and rTpN47 are still maintaining their original immunoreactivity. The ELISAs using rTpN17 or rTpN47 as the antigen are rapid, simple and convenient, higher sensitivity and specificity methods for serological screening and detection of syphilis.

Antibodies, Bacterial ; Antigens, Bacterial ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Humans ; Male ; Syphilis ; diagnosis ; Syphilis Serodiagnosis ; Treponema pallidum ; chemistry ; immunology ; isolation & purification

aboratory diagnosis of tuberculosis (TB) traditionally relies on smear microscopy and culture of Mycobacterium tuberculosis from clinical samples. With recent advances in technology, there have been numerous efforts to develop new diagnostic tests for TB that overcome the low sensitivity and specificity and long turnover time associated with current diagnostic tests. Molecular biological tests based on nucleic acid amplification have brought an unprecedented opportunity for the rapid and specific detection of M. tuberculosis from clinical specimens. With automated sequencing analysis, species identification of mycobacteria is now easier and more accurate than with conventional methods, and rapid detection of mutations in the genes associated with resistance to TB drugs provides early information on the potential drug resistance for each clinical isolate or for clinical samples. In addition, immunological tests for the detection of M. tuberculosis antigens and antibodies to the antigens have been explored to identify individuals at risk of developing TB or with latent TB infection (LTBI). The recent introduction of commercial IFN-gamma assay kits for the detection of LTBI provides a new approach for TB control even in areas with a high incidence of TB. However, these molecular and immunological tools still require further evaluation using large scale cohort studies before implementation in TB control programs.
Antigens, Bacterial/immunology ; DNA, Bacterial/chemistry/genetics ; Humans ; Immunologic Tests/*methods ; Interferon-gamma/analysis ; Mycobacterium tuberculosis/genetics/immunology ; Sequence Analysis, DNA ; Tuberculin Test ; Tuberculosis/*diagnosis/immunology/microbiology

aboratory diagnosis of tuberculosis (TB) traditionally relies on smear microscopy and culture of Mycobacterium tuberculosis from clinical samples. With recent advances in technology, there have been numerous efforts to develop new diagnostic tests for TB that overcome the low sensitivity and specificity and long turnover time associated with current diagnostic tests. Molecular biological tests based on nucleic acid amplification have brought an unprecedented opportunity for the rapid and specific detection of M. tuberculosis from clinical specimens. With automated sequencing analysis, species identification of mycobacteria is now easier and more accurate than with conventional methods, and rapid detection of mutations in the genes associated with resistance to TB drugs provides early information on the potential drug resistance for each clinical isolate or for clinical samples. In addition, immunological tests for the detection of M. tuberculosis antigens and antibodies to the antigens have been explored to identify individuals at risk of developing TB or with latent TB infection (LTBI). The recent introduction of commercial IFN-gamma assay kits for the detection of LTBI provides a new approach for TB control even in areas with a high incidence of TB. However, these molecular and immunological tools still require further evaluation using large scale cohort studies before implementation in TB control programs.
Antigens, Bacterial/immunology ; DNA, Bacterial/chemistry/genetics ; Humans ; Immunologic Tests/*methods ; Interferon-gamma/analysis ; Mycobacterium tuberculosis/genetics/immunology ; Sequence Analysis, DNA ; Tuberculin Test ; Tuberculosis/*diagnosis/immunology/microbiology

Perivasculitis and endothelial cell abnormalities are prominent histopathologic features of syphilis. Various cutaneous lesions are the main clinical features of syphilis. We examined whether Treponema pallidum 47 kDa antigen regulates the expression of cell adhesion molecules on human dermal microvascular endothelial cells (HDMEC) and the regulation of T-lymphocytes binding to HDMEC. Using immunofluorescence flow cytometry and enzyme-linked immunosorbent assay (ELISA), we demonstrated that T. pallidum upregulated the expression of adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin. The 47 kDa antigen of T. pallidum also activated endothelium as measured by the upregulation of the expression of adhesion molecules on HDMEC, and it also promoted an increased adherence of T-lymphocytes to HDMEC. The expressions of ICAM-1 and VCAM-1 on HDMEC and the adherence of T-lymphocytes to HDMEC were inhibited by treatment with anti-TNF-alpha antibody or anti-IL-1alpha antibody. These results show that T. pallidum or T. pallidum-specific 47 kDa antigen are capable of stimulating HDMEC to increase the expression of ICAM-1, VCAM-1 and E-selectin and thereby, promote the adherence of T-lymphocytes. The whole process may play an important role in the immunopathogenesis of syphilis and it is likely that TNF-alpha and IL-1alpha are involved.
Antigens, Bacterial/physiology* ; Antigens, Bacterial/chemistry ; Cell Adhesion Molecules/metabolism* ; Cells, Cultured ; Endothelium, Vascular/pathology ; Endothelium, Vascular/metabolism* ; Human ; Microcirculation ; Molecular Weight ; Skin/blood supply* ; T-Lymphocytes/metabolism* ; Treponema pallidum/pathogenicity ; Treponema pallidum/immunology*

An expression plasmid carrying anthrax protective antigen (PA) gene was constructed, which has an OmpA signal sequence attached to the 5' end of PA gene. The plasmid was transformed into E. coli and induced to express recombinant PA (rPA) . The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange, hydrophobic interaction chromatography, and gel filtration, about 15 mg of 95 % pure rPA was obtained from 1-liter culture. The bioactivity of rPA was proved by in vitro cytotoxicity assay. The polyclonal antiserum from rabbits immunized with rPA could inhibit the action of anthrax lethal toxin in vitro, which suggests that antibodies against rPA can provide high passive protection against anthrax. The results reported here may be helpful to develop a safe and efficacious recombinant PA vaccine against anthrax.
Amino Acid Sequence ; Animals ; Anthrax Vaccines ; immunology ; Antigens, Bacterial ; chemistry ; genetics ; immunology ; toxicity ; Bacterial Toxins ; chemistry ; genetics ; immunology ; toxicity ; Base Sequence ; Mice ; Molecular Sequence Data ; Plasmids ; Rabbits ; Recombinant Proteins ; biosynthesis ; immunology ; Vaccines, Synthetic ; immunology

OBJECTIVETo explore the important role of the terminal residues containing sialic acid (SA) in Campylobacter jejuni (CJ) lipopolysaccharide (LPS) as the critical antigen to induce nerve damage, and also to identify immunopathological evidence for the hypothesis of molecular mimicry and cross-immunity between CJ LPS and gangliosides.

METHODSA mutant of Pen O:19 CJ with neuB1 gene inactivated and LPS outer core terminal residues losing SA was to be constructed. PCR and RT-PCR were used to confirm the mutant. Capability of CJ LPS binding to cholera toxin B subunit (CTB) was tested. Guinea pigs were systematically immunized with LPS of the wild and the mutant strains, respectively. Titers of anti-LPS and anti-ganglioside GM(1) IgG antibodies in sera of immunized guinea pigs were detected by ELISA. Pathological study for sciatic nerves of both Guinea pigs either immunized systematically or perineural injection with their immunized serum was finished.

RESULTS(1) The mutant of CJ O:19 strain with inactivated neuB1 gene was successfully constructed and lost transcriptional activity of neuB1 gene in the mutant strain was confirmed by PCR and RT-PCR. SA was well demonstrated by both acidic ninhydrin reaction and periodate-resorcinol reaction in the LPS of wild strain but not in the mutant LPS; (2) Compared with the titers before immunization, the titers of anti-GM(1) IgG antibody increased in sera of guinea pigs immunized with LPS of the wild strain. However there were no detectable anti-GM(1) IgG antibody in sera of the animals immunized with mutant LPS and PBS. (3) The incidence of pathological fibers of sciatic nerves in wild CJ LPS group (17.3%) was significantly higher than the mutant CJ LPS group (chi(2) = 125, P < 0.01); the difference between the mutant CJ LPS group and control group was not statistically significant (chi(2) = 1.633, P > 0.05). (4) After perineural injection with immunized serum, the incidence of pathological fibers of sciatic nerves in wild strain group (67.8%) was also significantly higher than the incidence of mutant group (P < 0.01).

CONCLUSIONA mutant of CJ O:19 strain neuB1 gene inactivated and SA component of terminal structure of LPS lost was successfully constructed. And it no longer expressed SA component which is the normal terminal structure of LPS in wild strain. The capability of the wild strain to induce increased titers of anti-GM(1) antibody and immune-mediated nerve damage was simultaneously lost for the mutant strain. It could be a strong immunopathologic evidence to identify the molecular mimicry hypothesis between CJ LPS and ganglioside epitope in nerve on the pathogenesis of CJ related GBS. The terminal residues containing SA should be as the basic GM1-like structure in CJ LPS.

Animals ; Antibodies, Bacterial ; blood ; immunology ; Antigens, Bacterial ; genetics ; immunology ; Campylobacter jejuni ; genetics ; immunology ; G(M1) Ganglioside ; immunology ; Guinea Pigs ; Lipopolysaccharides ; chemistry ; immunology ; Molecular Mimicry ; Mutagenesis ; N-Acetylneuraminic Acid ; chemistry ; immunology ; Peripheral Nervous System Diseases ; immunology ; microbiology

Corn, one of the most important forage crops worldwide, has proven to be a useful expression vehicle due to the availability of established transformation procedures for this well-studied plant. The exotoxin Apx, a major virulence factor, is recognized as a common antigen of Actinobacillus (A.) pleuropneumoniae, the causative agent of porcine pleuropneumonia. In this study, a cholera toxin B (CTB)-ApxIIA#5 fusion protein and full-size ApxIIA expressed in corn seed, as a subunit vaccine candidate, were observed to induce Apx-specific immune responses in mice. These results suggest that transgenic corn-derived ApxIIA and CTB-ApxIIA#5 proteins are potential vaccine candidates against A. pleuropneumoniae infection.
Actinobacillus Infections/microbiology/*prevention & control ; Actinobacillus pleuropneumoniae ; Animals ; Antigens, Bacterial/immunology ; Bacterial Proteins/*immunology ; Bacterial Vaccines/*immunology ; Cholera Toxin/*chemistry ; Female ; Hemolysin Proteins/*immunology ; Immunization, Secondary ; Mice ; Mice, Inbred ICR ; Plants, Genetically Modified ; Zea mays/*genetics