Antibody reactivity to Mycobacterium tuberculosis-specific regions of differences 1 and regions of differences 9 proteins and peptides in rabbits, mice, and humans.

Background: The major antigens encoded by Mycobacterium tuberculosis-specific genomic regions of differences (RDs) could be useful in the development of new vaccines and/or diagnostic reagents using T-cell and/or antibody assays. In particular, RD1 proteins PE35, PPE68, ESXA, ESXB, and RD9 protein ESXV and their peptides have been identified as major T-cell antigens. However, little is known about their antibody reactivities in different mammalian species. This study aims to determine the antigen-specific antibody reactivities to the above antigens and their peptides in three different mammalian species, i.e., rabbits, mice, and humans. Methods: Sera were obtained from (i) rabbits immunized with purified recombinant proteins PE35, PPE68, ESXA, ESXB, and ESXV; (ii) mice immunized with recombinant DNA vaccine constructs of pUMVC6 and pUMVC7 containing RD1 and RD9 genes; and (iii) tuberculosis (TB) patients and healthy humans. Enzyme-linked immunosorbent assays (ELISAs) were performed with the sera to determine the antibody reactivity to purified recombinant proteins, peptide pools, and individual peptides of RD1 and RD9 proteins. Results: The ELISA results with sera from rabbits immunized with pure recombinant proteins showed positive antibody reactivity with all of the immunizing proteins and their synthetic peptide pools. Testing of the sera with individual peptides showed positive antibody reactivity with PE35 peptides P1 (aa 1-25), P2 (aa 16-40), P5 (aa 61-85), and P6 (aa 76-99); PPE68 peptides P9 (aa 121-145), P11 (aa 151-175), P14 (aa 196-220), P22 (aa 316-340), P23 (aa 331-355), and P24 (aa 346-371); all peptides (P1 to P6) of ESXA and ESXB; and ESXV peptides P1 (aa 1-25), P2 (aa 16-40), P3 (aa 31-55), P5 (aa 61-85), and P6 (aa 76-94). The sera from mice immunized with DNA vaccine constructs showed antibody reactivity to all proteins and the peptide P6 (aa 76-99) of PE35 and peptides P19 (aa 271-295) and P24 (aa 346-371) of PPE68. In humans, the peptides P11 (aa 151-175), P14 (aa 196-220), P22 (aa 316-340), P23 (aa 331-355), and P24 (aa 346-371) of PPE68 and the peptides P4 (aa 46-70), P5 (aa 61-85), and P6 (aa 76-94) of ESXV showed positive reactivity with sera from TB patients and healthy controls. Conclusion: The results demonstrate the presence of several antibody epitopes in each protein, but variations in the epitopes recognized were observed among mice, rabbits, and humans, which could be due to mammalian species differences and/or mode of antigen delivery.

Synthesis, Antioxidant and Antiproliferative Evaluation, Molecular Docking and DFT Studies of Some Novel Coumarin and Fused Coumarin Derivatives.

N'-[(4-Chloro-2-oxo-2H-chromen-3-yl)methylene]-2-cyanoacetohydrazide (3) was synthesized in excellent yield from the condensation of 4-Chloro-2-oxo-2H-chromene-3-carbaldehyde with cyanoacetohydrazide. Compound 3 was utilized as a building block to synthesize novel coumarin and heterocycle-fused coumarin derivatives. The chemical structures of all the new coumarin compounds were identified by spectral analyses. Some of the new coumarins compounds were screened in human cancer cell lines (HEPG-2, MCF-7, HCT-116 and PC-3) to learn about their cytotoxic effects in addition to the study of their DNA damage and antioxidant activity. Three of these compounds exhibited remarkable antioxidant and anti-proliferative activities. Moreover, they have the capability to protect DNA from damage induced by bleomycin. Molecular docking, DFT and molecular electrostatic potential studies were performed on the compounds in vitro.

An Overview of the Development of New Vaccines for Tuberculosis.

Currently, there is only one licensed vaccine against tuberculosis (TB), the Bacillus Calmette-Guérin (BCG). Despite its protective efficacy against TB in children, BCG has failed to protect adults against pulmonary TB, lacks therapeutic value, and causes complications in immunocompromised individuals. Furthermore, it compromises the use of antigens present in the purified protein derivate of Mycobacterium tuberculosis in the diagnosis of TB. Many approaches, e.g., whole-cell organisms, subunit, and recombinant vaccines are currently being explored for safer and more efficacious TB vaccines than BCG. These approaches have been successful in developing a large number of vaccine candidates included in the TB vaccine pipeline and are at different stages of clinical trials in humans. This paper discusses current vaccination strategies, provides directions for the possible routes towards the development of new TB vaccines and highlights recent findings. The efforts for improved TB vaccines may lead to new licensed vaccines capable of replacing/supplementing BCG and conferring therapeutic value in patients with active/latent TB.

Humoral immune responses in mice immunized with region of difference DNA vaccine constructs of pUMVC6 and pUMVC7.

BACKGROUND: We aimed to study the antigen-specific antibody responses in mice immunized with recombinant DNA vaccines constructs of pUMVC6 and pUMVC7, containing RD1 and RD9 genes of Mycobacterium tuberculosis. METHODS: We immunized mice with the parent and recombinant plasmids and sera were collected and tested for antibodies against pure recombinant proteins of RD1 (PE35, PPE68, EsxA, EsxB) and RD9 (EsxV), peptide mixtures of each protein and their individual peptides using enzyme-linked immunosorbent assays. The optical density (OD) values were measured at 405 nm. E/C (OD in antigen-coated wells/OD in antigen uncoated wells) were calculated, and the values of E/C>2 were considered positive. RESULTS: RD1 and RD9 antigen-specific antibodies were detected in sera of mice immunized with the recombinant DNA vaccine constructs (E/C >2.0). With respect to peptide mixtures and single peptides, only PE35mixand P6 of PE35; PPE68mixand P19, P24 of PPE68 showed antibody reactivity with sera of mice immunized with the corresponding recombinant pUMVC6 and/or pUMVC7 DNA vaccine constructs. CONCLUSIONS: The results confirm in vivo expression and immunogenicity of all the five RD1 and RD9 genes cloned in both of the DNA vaccine vectors.

Comparative analysis of spontaneous and mycobacterial antigen-induced secretion of Th1, Th2 and pro-inflammatory cytokines by peripheral blood mononuclear cells of tuberculosis patients.

The cytokines produced by T helper (Th)1 cells (IFN-γ, IL-2 and TNF-ß) correlate with protection, whereas the cytokines released by Th2 cells (IL-4, IL-5) and the anti-inflammatory cytokine IL-10 correlate with pathogenesis of tuberculosis (TB). However, the pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, TNF-α and IL-12p70) are responsible for both protection and pathogenesis of TB. The aim of this work was to carry out a comparative analysis of cytokines present in early (day 2) and late (day 6) cultures of peripheral blood mononuclear cells (PBMCs) obtained from pulmonary tuberculosis patients. PBMCs were cultured in vitro in the absence and presence of exogenously added complex mycobacterial antigens and RD1 peptide pool. The supernatants were collected on day 2 and day 6 of culture and assayed for secreted cytokines using the flow cytomix assay. All of the cytokines, except for IL-12p70, were spontaneously secreted by PBMCs of 27-100% TB patients, but only TNF-α concentration was significantly higher on day 2 than day 6 (P < 0.05). Two days following antigenic stimulation, only IL-1ß, IL-6, TNF-α and IL-10 were secreted in response to some mycobacterial antigens. However, 6 days later, all of the cytokines, except for IL-2, IL-4, IL-5 and IL-8, were secreted significantly in response to all complex antigens and RD1 peptides, compared with the non-stimulated cultures (P < 0.05). In conclusion, the study shows that the longer in vitro stimulation time (6 days) was necessary for the optimal induction of IFN-γ and TNF-ß, and practically convenient for the detection of IL-10, IL-1 ß, TNF-α and IL-6.

Cellular immune responses in mice induced by M. tuberculosis PE35-DNA vaccine construct.

The PE35 (Rv3872) gene of Mycobacterium tuberculosis is present in the region of difference (RD) one that is deleted in all vaccine strains of Mycobacterium bovis bacillus Calmette Guerin. The aim of this study was to clone PE35 DNA into a DNA vaccine plasmid with CMV promoter and interleukin-2 secretory signal and evaluate the recombinant plasmid for induction of antigen-specific cellular responses in mice. DNA corresponding to PE35 was PCR amplified from the genomic DNA of M. tuberculosis H(37) Rv, cloned into pGEMT-Easy vector and sub-cloned into the DNA vaccine vector pUMVC6. BALB/c mice were immunized with recombinant pUMVC6/PE35 and spleen cells were tested for T-helper (Th)1-type (antigen-induced proliferation and secretion of IFN-γ) and Th2-type (IL-5), and anti-inflammatory (IL-10) cytokine responses to pure recombinant PE35 protein and its synthetic peptides. Mice immunized with the recombinant plasmid DNA (pUMVC6/PE35) showed positive Th1-type cellular responses to pure PE35, but not to an irrelevant antigen, i.e. PPE68 (Rv3873). However, the vaccine construct did not induce antigen-specific Th2-type (IL-5) or anti-inflammatory (IL-10) reactivity to PE35. Testing with synthetic peptides showed that Th1-type cells recognizing various epitopes of PE35 were induced in mice immunized with pUMVC6/PE35 DNA. These results suggest that pUMVC6/PE35 may be useful as a safer vaccine candidate against TB.

DNA vaccine constructs expressing Mycobacterium tuberculosis-specific genes induce immune responses.

RD1 PE35, PPE68, EsxA, EsxB and RD9 EsxV genes are present in Mycobacterium tuberculosis genome but deleted in Mycobacterium bovis BCG. The aim of this study was to clone these genes into DNA vaccine vectors capable of expressing them in eukaryotic cells as fusion proteins, fused with immunostimulatory signal peptides of human interleukin-2 (hIL-2) and tissue plasminogen activator (tPA), and evaluate the recombinant DNA vaccine constructs for induction of antigen-specific cellular immune responses in mice. DNA corresponding to the aforementioned RD1 and RD9 genes was cloned into DNA vaccine plasmid vectors pUMVC6 and pUMVC7 (with hIL-2 and tPA signal peptides, respectively), and a total of 10 recombinant DNA vaccine constructs were obtained. BALB/c mice were immunized with the parent and recombinant plasmids and their spleen cells were tested for antigen-induced proliferation with antigens of M. tuberculosis and pure proteins corresponding to the cloned genes. The results showed that antigen-specific proliferation responses were observed for a given antigen only with spleen cells of mice immunized with the homologous recombinant DNA vaccine construct. The mice immunized with the parent plasmids did not show positive immune responses to any of the antigens of the cloned genes. The ability of the DNA vaccine constructs to elicit cellular immune responses makes them an attractive weapon as a safer vaccine candidate for preventive and therapeutic applications against tuberculosis.

Molecular cloning, expression, purification and immunological characterization of three low-molecular weight proteins encoded by genes in genomic regions of difference of mycobacterium tuberculosis.

The aim of this study was to clone, express and purify three major antigenic proteins, i.e. Rv3874, Rv3875 and Rv3619c, encoded by genes located in regions of difference of Mycobacterium tuberculosis and characterize them for immunogenicity in rabbits. The respective genes were amplified using gene-specific primers and genomic DNA of M. tuberculosis by polymerase chain reaction. The amplified DNA were cloned into pGEM-T Easy and subcloned into pGES-TH-1 vector for high-level expression in Escherichia coli and efficient purification. The results showed that the three fusion proteins, i.e. glutathione-S-transferase (GST)-Rv3874, GST-Rv3875 and GST-Rv3619c, were expressed at high levels and were purified (free of the GST fusion partner) to homogeneity using glutathione-Sepharose and Ni-NTA agarose affinity matrix after cleavage of the column-bound fusion proteins by thrombin protease. The purified recombinant Rv3874, Rv3875 and Rv3619c proteins were immunogenic and induced antigen-specific antibodies in rabbits. Testing of the rabbit sera with overlapping synthetic peptides showed that the antibodies were induced to several epitopes that were scattered throughout the sequence of each protein. These results show immunogenicity of all the proteins for inducing antigen-specific antibodies in rabbits and demonstrate the usefulness of pGES-TH-1 vector for obtaining purified recombinant proteins of M. tuberculosis for immunological characterization.

Species-specific antigenic Mycobacterium tuberculosis proteins tested by delayed-type hypersensitivity response.

OBJECTIVE: To investigate the diagnostic potential of four Mycobacterium tuberculosis antigens encoded by M. tuberculosis-specific region of difference 1 (RD1) region genes (PE35, PPE68, culture filtrate protein 10 [CFP-10], early secreted antigenic target-6 [ESAT-6]) and RD9 region gene Rv3619c, for delayed-type hypersensitivity (DTH) responses in guinea pigs. DESIGN: Recombinant M. tuberculosis proteins were expressed in Escherichia coli and purified to homogeneity by affinity chromatography. Guinea pigs were injected with heat-killed M. tuberculosis and live bacille Calmette-Guérin (BCG), M. avium and M. vaccae. Two to four weeks later, the guinea pigs were challenged intradermally in the flank region with mycobacterial sonicates and purified recombinant proteins. The DTH responses were quantitated by measuring erythema at injection sites after 24 h. RESULTS: All mycobacterial sonicates induced positive DTH responses in guinea pigs injected with M. tuberculosis, M. bovis BCG, M. avium and M. vaccae. Purified proteins PE35, PPE68, CFP10 and ESAT-6 elicited positive DTH responses in the M. tuberculosis-injected group but not in BCG-, M. avium- and M. vaccae-injected guinea pigs, whereas Rv3619c elicited positive DTH responses in the M. tuberculosis- and BCG-injected groups, but not in the M. avium- and M. vaccae-injected guinea pigs. CONCLUSION: The recombinant RD1 antigens induced M. tuberculosis-specific DTH responses. These antigens may therefore be useful in the diagnosis of tuberculosis.

Mycobacterial antigen-induced T helper type 1 (Th1) and Th2 reactivity of peripheral blood mononuclear cells from diabetic and non-diabetic tuberculosis patients and Mycobacterium bovis bacilli Calmette-Guérin (BCG)-vaccinated healthy subjects.

Patients with diabetes mellitus are more susceptible to tuberculosis (TB), and the clinical conditions of diabetic TB patients deteriorate faster than non-diabetic TB patients, but the immunological basis for this phenomenon is not understood clearly. Given the role of cell-mediated immunity (CMI) in providing protection against TB, we investigated whether CMI responses in diabetic TB patients are compromised. Peripheral blood mononuclear cells (PBMC) obtained from diabetic TB patients, non-diabetic TB patients and Mycobacterium bovis bacilli Calmette-Guérin (BCG)-vaccinated healthy subjects were cultured in the presence of complex mycobacterial antigens and pools of M. tuberculosis regions of difference (RD)1, RD4, RD6 and RD10 peptides. The PBMC were assessed for antigen-induced cell proliferation and secretion of T helper 1 (Th1) [interferon (IFN)-gamma, interleukin (IL)-2, tumour necrosis factor (TNF)-beta], and Th2 (IL-4, IL-5, IL-10) cytokines as CMI parameters. All the complex mycobacterial antigens and RD1(pool) stimulated strong proliferation of PBMC of all groups, except moderate responses to RD1(pool) in healthy subjects. In response to complex mycobacterial antigens, both IFN-gamma and TNF-beta were secreted by PBMC of all groups whereas diabetic TB patients secreted IL-10 with concentrations higher than the other two groups. Furthermore, in response to RD peptides, IFN-gamma and IL-10 were secreted by PBMC of diabetic TB patients only. The analyses of data in relation to relative cytokine concentrations showed that diabetic TB patients had lower Th1 : Th2 cytokines ratios, and a higher Th2 bias. The results demonstrate a shift towards Th2 bias in diabetic TB patients which may explain, at least in part, a faster deterioration in their clinical conditions.

Th1 cell reactivity and HLA-DR binding prediction for promiscuous recognition of MPT63 (Rv1926c), a major secreted protein of Mycobacterium tuberculosis.

MPT63 (Rv1926c), a major secreted protein of Mycobacterium tuberculosis, is immunoreactive in antibody assays in humans and animals and provides protection as a combined DNA vaccine in mice. This study was undertaken to determine the reactivity of MPT63 in T helper 1 (Th1) cell assays, i.e. antigen-induced proliferation and interferon-gamma secretion, using peripheral blood mononuclear cells (PBMCs) obtained from 72 Mycobacterium bovis Bacille Calmette-Guérin vaccinated healthy subjects. PBMCs were tested with complex mycobacterial antigens and pools of synthetic peptides corresponding to MPT63, MPB70, MT24, PPE68, CFP10 and ESAT-6. The results showed that MPT63 induced moderate Th1 cell reactivity which was equivalent to the reactivity induced by other secreted antigens of M. tuberculosis, i.e. MT24 and MPB70. Furthermore, human leucocyte antigen (HLA) heterogeneity of the responding donors suggested that MPT63 was presented to Th1 cells promiscuously. Analysis of the MPT63 sequence and its peptides for binding to 51 alleles of 9 serologically defined HLA-DR molecules, using a virtual matrix-based prediction program (ProPred) showed that MPT63 sequence could bind to all the 51 alleles, whereas 9 of the 10 peptides of MPT63 were also predicted to bind promiscuously. When tested with PBMCs of HLA-DR heterogeneous donors that responded to MPT63 in interferon-gamma assays, at least 9 of the 10 peptides of MPT63 were recognized by PBMCs from HLA heterogeneous donors. These results suggested that promiscuous Th1 cell reactive epitopes are scattered throughout the sequence of MPT63, and further support the inclusion of this protein in an antigen cocktail to develop a new anti-tuberculosis vaccine.

Characterization of human cellular immune responses to novel Mycobacterium tuberculosis antigens encoded by genomic regions absent in Mycobacterium bovis BCG.

Comparative genomics has identified several regions of differences (RDs) between the infectious Mycobacterium tuberculosis and the vaccine strains of Mycobacterium bovis BCG. We aimed to evaluate the cellular immune responses induced by antigens encoded by genes predicted in 11 RDs. Synthetic peptides covering the sequences of RD1, RD4 to RD7, RD9 to RD13, and RD15 were tested for antigen-induced proliferation and secretion of Th1 cytokine, gamma interferon (IFN-gamma), by peripheral blood mononuclear cells (PBMC) obtained from culture-proven pulmonary tuberculosis (TB) patients and M. bovis BCG-vaccinated healthy subjects. Among the peptide pools, RD1 induced the best responses in both donor groups and in both assays. In addition, testing of TB patients' PBMC for secretion of proinflammatory cytokines (tumor necrosis factor alpha [TNF-alpha], interleukin 6 [IL-6], IL-8, and IL-1beta), Th1 cytokines (IFN-gamma, IL-2, and TNF-beta), and Th2 cytokines (IL-4, IL-5, and IL-10) showed differential effects of RD peptides in the secretion of IFN-gamma and IL-10, with high IFN-gamma/IL-10 ratios (32 to 5.0) in response to RD1, RD5, RD7, RD9, and RD10 and low IFN-gamma/IL-10 ratios (<1.0) in response to RD12, RD13, and RD15. Peptide-mixing experiments with PBMC from healthy subjects showed that secretion of large quantities of IL-10 in response to RD12 and RD13 correlated with inhibition of Th1 responses induced by RD1 peptides. In conclusion, our results suggest that M. tuberculosis RDs can be divided into two major groups--one group that activates PBMC to preferentially secrete IFN-gamma and another group that activates preferential secretion of IL-10--and that these two groups of RDs may have roles in protection against and pathogenesis of TB, respectively.

Legionella in clinical specimens and hospital water supply facilities: molecular detection and genotyping of the isolates.

OBJECTIVE: To evaluate genus- and species-specific polymerase chain reactions (PCRs) for the detection of the genus Legionella and the species Legionella pneumophila in clinical specimens and hospital water supplies, and to establish a simple and reproducible random amplification of polymorphic DNA (RAPD)-PCR technique for genotyping of Legionella. MATERIALS AND METHODS: A total of 70 respiratory tract specimens(bronchoalveolar lavage: n = 46; endotracheal secretions: n = 9; sputum: n = 15) from patients with atypical pneumonia, and 283 environmental samples (water: 20; swabs: 263) collected from water storage and supply facilities of the Mubarak Al-Kabeer Hospital, Kuwait, were tested by culture and genus-specific PCR for the detection of Legionella. The L. pneumophila isolates were subsequently typed by serology and RAPD-PCR using serotype-specific sera and arbitrary primers, respectively. RESULTS: Of the 70 clinical samples, culture yielded 2 (2.9%) whereas genus-specific PCR detected Legionella in 20 (28.6%) samples. The 2 culture-positive specimens were also positive for L.-pneumophila-specific PCR. Testing of swab and water samples by culture and genus-specific PCR yielded 61 (21.6%) and 67 (23.7%) positive samples, respectively. All of the 61 culture-positive samples were also positive by genus-specific PCR and 45 of them were positive for L.-pneumophila-specific PCR. Serological typing of 43 L. pneumophila isolates showed that 8 of these belonged to serotype 1 and 35 to serotype 3; however, RAPD-PCR analyses demonstrated polymorphisms among the isolates of both serotypes. CONCLUSION: A higher association between PCR and culture was observed for the environmental samples than for the clinical samples. The application of genus- and species-specific PCRs and RAPD is useful in the detection and typing of Legionella in clinical and environmental samples.

Demonstration of in vivo expression of a hypothetical open reading frame (ORF-14) encoded by the RD1 region of Mycobacterium tuberculosis.

Previously we identified a novel antigenic open reading frame (ORF), designated as ORF-14, on the RD1 region of Mycobacterium tuberculosis that was not originally predicted by Mahairas or by annotation of the M. tuberculosis H37 Rv genome. Here we show that anti-ORF-14 antibodies either from mice immunized with recombinant ORF-14 protein or isolated from serum samples from tuberculosis patients, react with a protein in culture filtrate but not in cytoplasmic or cell wall fractions from M. tuberculosis. Our data indicate that the ORF-14 protein is expressed as a secreted protein, representing one more secreted protein antigen not previously identified by genomics.

Polymerase-chain-reaction-based detection of fetal rhesus D and Y-chromosome-specific DNA in the whole blood of pregnant women during different trimesters of pregnancy.

OBJECTIVE: The aim of this study was to determine whether or not a noninvasive procedure utilizing maternal peripheral blood as the source of DNA and polymerase chain reaction (PCR) could be used to detect fetal rhesus D (RhD) status as well as fetal gender during different gestational stages of pregnancy. MATERIALS AND METHODS: Maternal blood samples were obtained from 54 RhD-negative pregnant women during the first trimester (6-13 weeks, n = 14), second trimester (14-26 weeks, n = 26) and third trimester (27-40 weeks, n = 14). Genomic DNA was extracted from the whole blood and analyzed by seminested and nested PCR for detection of DNA sequences corresponding to RhD (n = 54) and Y chromosome (n = 48) using RhD and Y-chromosome-specific oligonucleotide primers, respectively. The seminested/nested PCR results were compared with the RhD status and gender of the babies after delivery. RESULTS: The sensitivity and specificity of seminested PCR for detection of fetal RhD positivity in whole blood of pregnant women were 81 and 100%, respectively, while the sensitivity and specificity of nested PCR for detection of male fetuses, using Y-chromosome-specific DNA as a marker, were 96 and 91%, respectively. There were no significant differences in the PCR results with samples obtained from women at different gestational stages of pregnancy. CONCLUSION: Seminested and nested PCRs for detection of fetal RhD and gender status, respectively, by using the blood of pregnant women during different gestational stages of pregnancy, are reliable noninvasive procedures with high sensitivity and specificity.

Utility of three mammalian cell entry proteins of Mycobacterium tuberculosis in the serodiagnosis of tuberculosis.

SETTING: The mammalian cell entry (mce) proteins Mce3A, Mce3D and Mce3E, encoded by the mce3 operon of Mycobacterium tuberculosis, have recently been shown to be expressed during natural infection in humans. OBJECTIVE: To determine the potential of Mce3A, Mce3D and Mce3E proteins in the serodiagnosis of tuberculosis (TB). DESIGN: The quantitative detection of anti-Mce3A, -Mce3D and -Mce3E antibodies in serum samples from active TB patients (n = 58), healthy contacts of TB patients (n = 24) and bacilli Calmette-Guérin (BCG) vaccinated healthy subjects (n = 24) was performed using enzyme-linked immunosorbent assay (ELISA). RESULTS: Antibodies in serum from 98%, 86% and 90% of active TB patients and from 92%, 75% and 96% of healthy contacts of TB patients reacted with Mce3A, Mce3D and Mce3E proteins, respectively. However, none of the serum from BCG-vaccinated healthy subjects reacted with Mce3A and Mce3E proteins, and only 8% of serum samples reacted with Mce3D protein. Overall, serum from 98% active TB patients, 96% healthy contacts and 0% BCG-vaccinated healthy subjects were positive for anti-Mce3A and/or -Mce3E antibodies. CONCLUSIONS: Our results suggest that Mce3A and Mce3E proteins may be useful for the serodiagnosis of TB infection.

Assessment of in vitro immunity to Mycobacterium tuberculosis in a human peripheral blood infection model using a luciferase reporter construct of M. tuberculosis H37Rv.

Protective immune responses to tuberculosis in man are primarily cell-mediated and require the interaction of specific T cells, cytokines and activated macrophages. In the present study, Mycobacterium tuberculosis H37Rv labelled with luciferase reporter enzyme was used to analyse the anti-mycobacterial immunity in man using an in vitro whole blood infection model. Peripheral blood samples obtained from M. bovis bacille Calmette-Guérin (BCG)-vaccinated tuberculin-positive healthy volunteers (n = 23) were cultured with M. tuberculosis H37Rv reporter strain. The growth of bacteria in the whole blood cultures was monitored after 48 and 96 h of infection. The results showed that the growth of M. tuberculosis was significantly inhibited after 96 h (P < 0.029) of culture. Among the cytokines studied, interleukin (IL)-10 and IL-12 were not detected at all, whereas low levels of interferon (IFN)-gamma after 96 h (0.4 IU/ml) and tumour necrosis factor (TNF)-alpha after 48 (135 pg/ml) and 96 h (47 pg/ml) of culture were detected in the supernatants of whole blood infected with M. tuberculosis. The magnitude of bacterial growth correlated directly with the concentration of TNF-alpha detected after 48 h (r = 0.722) and 96 h (r = 0.747) of culture (P

Immunogenicity of Mycobacterium tuberculosis antigens in Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle.

The development of novel vaccine strategies supplementing Mycobacterium bovis BCG (BCG) constitutes an urgent research challenge. To identify potential subunit vaccine candidates, we have tested a series of eight recently identified Mycobacterium tuberculosis antigens in M. bovis-infected and BCG-vaccinated cattle. These antigens were characterized on the basis of their ability to induce in vitro gamma interferon responses in infected or BCG-vaccinated calves. We were able to establish a hierarchy of these antigens based on how frequently they were recognized in both groups of animals. In particular, we were able to prioritize frequently recognized proteins like Rv0287, Rv1174, and Rv1196 for future evaluation as subunit vaccines to be used in BCG-protein heterologous prime-boost vaccination scenarios. In addition, the antigen most dominantly recognized in M. bovis-infected cattle in this study, Rv3616c, was significantly less frequently recognized by BCG vaccinees and could be a target to improve BCG, for example, by increasing its secretion, in a recombinant BCG vaccine.

The six mammalian cell entry proteins (Mce3A-F) encoded by the mce3 operon are expressed during in vitro growth of Mycobacterium tuberculosis.

The pathogenesis of Mycobacterium tuberculosis is largely due to its ability to enter and survive within human macrophages. The mammalian cell entry (mce)3 operon is one of four homologous mce operons that encodes six putative invasin-like exported proteins (Mce3A-F), possibly involved in entry and survival of M. tuberculosis inside macrophages. We have recently shown that Mce3A, Mce3D and Mce3E are expressed and elicit antibody responses in a majority of human subjects during natural infection with M. tuberculosis. In this study, we demonstrate the expression of Mce3A-F proteins and their mRNA during in vitro growth of M. tuberculosis. To demonstrate the expression of mce3A-F proteins, the antibodies were raised in rabbits against three pure proteins (Mce3A, Mce3D and Mce3E), and their specificity was checked by immunoblotting with recombinant Mce1A-F proteins encoded by mce1 operon. The antibodies were also generated against all the six Mce3 proteins, which were expressed and purified as fusion proteins with glutathione S-transferase (GST) as the fusion partner (GST-Mce3A-F). The antibodies reacted, in each case, with a protein of expected molecular mass (Mr) for the corresponding Mce3 protein in the cell wall fraction but not in the soluble fraction of in vitro-grown M. tuberculosis cells. The presence of mRNA for mce3A-F genes was also shown by using mce3A-F gene-specific primers, and total RNA isolated from in vitro-grown M. tuberculosis cells by reverse transcription-polymerase chain reaction (RT-PCR). Pretreatment of the RNA preparation with RNase A abolished amplification in RT-PCR confirming that mce3A-F mRNA rather than genomic DNA was being amplified. The data show that Mce3A-F encoded by the mce3 operon are expressed during in vitro growth of M. tuberculosis.

Mycobacterial gene cloning and expression, comparative genomics, bioinformatics and proteomics in relation to the development of new vaccines and diagnostic reagents.

Recent advances in molecular and genomic techniques have facilitated research on several aspects of mycobacteriology, such as diagnosis and the identification of new vaccines and therapeutic targets for various diseases, including tuberculosis. The aim of this review was to analyze the implications of advances in molecular and genomic techniques on the development of new vaccines for tuberculosis as well as immunological reagents to diagnose the disease. Gene cloning and expression, DNA and protein sequencing, polymerase chain reaction, comparative genomics, bioinformatics, proteomics and DNA and peptide synthesis coupled with the application of cellular immunology techniques have led to the identification of several antigens of Mycobacterium tuberculosis, which have potential for diagnosis and vaccine applications. For example, cross-reactive mycobacterial antigens like heat shock proteins, MTB32 and MTB39, have been identified as new vaccine candidates, and antigens encoded by M. tuberculosis-specific genomic regions as new reagents for diagnosis.