Antigen analysis of Mycobacterium tuberculosis H37Rv culture filtrate proteins

Culture filtrates of Mycobacterium tuberculosis H37Rv highly enriched with secreted proteins were used to identify antigens recognized by a serum pool from tuberculosis patients. Two different approaches were used to separate the culture filtrate protein mixture: (i) proteins were fractionated according to their hydrophobicity using an HPLC-C18 chromatography column followed by separation based on their molecular mass by SDS-PAGE and subsequent immunoblotting or (ii) proteins were separated by two-dimensional gel electrophoresis, based on their isoelectric point and their molecular mass. Twenty serologically reactive proteins were ultimately identified by both methods, including four novel antigens. Further, to estimate the immunogenicity of the identified culture filtrate proteins, the relative antibody quantities were measured using I mage master software. Our results show that the antibodies against proteins belonging to the antigen 85 complex were the most abundant in the serum of patients with active tuberculosis. The most immunogenic proteins in terms of high antibody-to-protein-ratio were Rv3881c and three lipoproteins Rv0934 (the 38 kDa antigen), Rv0932c (pstS2), and Rv3006 (LppZ). Rv3881c is located in the region of difference 1 (RD1) which is deleted from Mycobacterium bovis BCG, and is therefore a particularly promising candidate for development of serodiagnostic assays to detect active tuberculosis. The proteins from the M. tuberculosis H37Rv culture filtrate are strong candidates to be evaluated for improvement of the serodiagnostic tests of tuberculosis.     

Secreted Proteins of Mycobacterium leprae

In mycobacteria, secreted proteins represent a distinct group, probably of particular importance for development of immune responses following infection. Quantification of individual proteins in culture fluid and corresponding disrupted bacilli permits determination of a localization index for identification of secreted proteins. This procedure cannot be applied to Mycobacterium leprae because secreted proteins are lost during isolation of bacilli from tissues. The DNA sequences of secreted proteins of Mycobacterium tuberculosis were compared with sequences of M. leprae . Genes for homologues of the 85a, 85b, 85c , mpt32 ( apa ), mpt51 , erp, mtc28, Rv2376c, Rv3354 and Rv0526 genes were identified. All of these contain signal sequences typical for secretion in M. leprae . In several instances the local distance between marker genes and occurrence on the same or the complementary DNA strand was similar in these two species. The genomic organisation of genes for secreted proteins is thus very similar in M. leprae and M. tuberculosis , the homology being higher for the mature polypeptide chains than for the corresponding signal peptides.     

Evaluation of T-cell responses to novel RD1- and RD2-encoded Mycobacterium tuberculosis gene products for specific detection of human tuberculosis infection

The tuberculin skin test for diagnosing Mycobacterium tuberculosis infection suffers from antigenic cross-reactivity of purified protein derivative with BCG, resulting in poor specificity in BCG-vaccinated populations. Comparative genomics has identified several genetic regions in M. tuberculosis and M. bovis that are deleted in M. bovis BCG. Proteins encoded in these regions will form the basis of new specific T-cell-based blood tests that do not cross-react with BCG, but only two, early secretory antigen target 6 and culture filtrate protein 10, have been studied in detail in humans. We investigated four novel gene products, encoded by RD2 (Rv1989c) and RD1 (Rv3873, Rv3878, and Rv3879c), that are absent from most or all of the vaccine strains of BCG, respectively. Sixty-seven overlapping peptides were tested in ex vivo gamma interferon enzyme-linked immunospot assays in 49 patients with culture-confirmed tuberculosis and 38 healthy BCG-vaccinated donors. Forty-five percent (95% confidence interval [CI], 31 to 57%) and 53% (95% CI, 39 to 67%) of the tuberculosis patients responded to Rv3879c and Rv3873, respectively, identifying these proteins as major M. tuberculosis T-cell antigens in humans, while 35 and 25% of the patients responded to Rv3878 and Rv1989c, respectively. Of the 38 BCG-vaccinated donors, 1 (2.6%) responded to peptides from Rv3878 and Rv3879c, 3 (7.9%) responded to Rv3873, and none responded to Rv1989c. Exclusion of cross-reactive peptides encoded in conserved motifs of Rv3873, a PPE family member, increased its specificity to 97.4%. The high specificity of Rv3879c peptides and nonconserved Rv3873 sequences, together with their moderate sensitivity in tuberculosis patients, identifies these peptides as candidates for inclusion in new T-cell-based tests for M. tuberculosis infection.     

MTC28, a novel 28-kilodalton proline-rich secreted antigen specific for the Mycobacterium tuberculosis complex.

Proteins that are actively secreted by Mycobacterium tuberculosis serve as major targets of immune responses in the infected host. To identify and purify novel proteins in the filtrates of M. tuberculosis cultures, a bacteriophage lambda library of M. tuberculosis H37Rv DNA was immunoscreened by using an anti-culture filtrate rabbit antiserum. Of 20 positive clones isolated, 6 were analyzed and found to express the genes for two known components of the early culture filtrate, the secreted 45/47-kDa antigen complex and the KatG protein, and two novel genes. Here we report the molecular cloning and nucleotide sequence of one of the new genes encoding a culture filtrate protein of 310 amino acid (aa) residues. We called this gene mtc28. The deduced polypeptide sequence contained an NH2-terminal, highly hydrophobic 32-aa region having properties of a secretion signal peptide. The putative 278-aa mature MTC28 protein was characterized at its NH2 and COOH termini by a high content of proline and alanine residues organized in an (AP)n motif. Thus, MTC28 is a new member of a group of proline-rich antigens found in M. tuberculosis and Mycobacterium leprae. As shown by DNA hybridization experiments, the mtc28 gene was present only in species of the M. tuberculosis complex. Purified recombinant MTC28 antigen evoked strong delayed-type hypersensitivity and antibody responses in guinea pigs immunized with Mycobacterium bovis BCG, but not in guinea pigs immunized with Mycobacterium avium. The strong immunological activity of MTC28 and the absence of B- and T-cell epitopes cross-reactive with a common environmental mycobacterial species, such as M. avium, make this novel antigen an attractive reagent for immunodiagnosis of tuberculosis.     

Molecular and immunological characterization of Mycobacterium tuberculosis CFP-10, an immunodiagnostic antigen missing in Mycobacterium bovis BCG

In order to identify antigens that may be used in the serodiagnosis of active tuberculosis (TB), we screened a Mycobacterium tuberculosis genomic expression library with a pool of sera from patients diagnosed with active pulmonary TB. The sera used lacked reactivity with a recombinant form of the M. tuberculosis 38-kDa antigen (r38kDa), and the goal was to identify antigens that might complement r38kDa in a serodiagnostic assay. Utilizing this strategy, we identified a gene, previously designated lhp, which encodes a 100-amino-acid protein referred to as culture filtrate protein 10 (CFP-10). The lhp gene is located directly upstream of esat-6, within a region missing in M. bovis BCG. Immunoblot analysis demonstrated that CFP-10 is present in M. tuberculosis CFP, indicating that it is likely a secreted or shed antigen. Purified recombinant CFP-10 (rCFP-10) was shown to be capable of detecting specific antibody in a percentage of TB patients that lack reactivity with r38kDa, most notably in smear-negative cases, where sensitivity was increased from 21% for r38kDa alone to 40% with the inclusion of rCFP-10. In smear-positive patient sera, sensitivity was increased from 49% for r38kDa alone to 58% with the inclusion of rCFP-10. In addition, rCFP-10 was shown to be a potent T-cell antigen, eliciting proliferative responses and gamma interferon production from peripheral blood mononuclear cells in 70% of purified protein derivative-positive individuals without evident disease. The responses to this antigen argue for the inclusion of rCFP-10 in a polyvalent serodiagnostic test for detection of active TB infection. rCFP-10 could also contribute to the development of a recombinant T-cell diagnostic test capable of detecting exposure to M. tuberculosis.     

Comparative immune response to PE and PE_PGRS antigens of Mycobacterium tuberculosis

Sequencing of the entire genome of Mycobacterium tuberculosis identified a novel multigene family composed of two closely related subfamilies designated PE and PE_PGRS. The major difference between these two families is the presence of a domain containing numerous Gly-Ala repeats extending to the C terminus of the PE_PGRS genes. We have used a representative PE_PGRS gene from M. tuberculosis, Rv1818c (1818PE_PGRS), and its amino-terminal PE region (1818PE), to investigate the immunological response to these proteins during experimental tuberculosis and following immunization with DNA constructs. During infection of mice with M. tuberculosis, a significant humoral immune response was observed against recombinant 1818PE_PGRS but not toward the 1818PE protein. Similarly, immunization with a 1818PE_PGRS DNA construct induced antibodies directed against 1818PE_PGRS but not against 1818PE proteins, and no humoral response was induced by 1818PE DNA. These results suggest that certain PE_PGRS genes are expressed during infection of the host with M. tuberculosis and that an antibody response is directed solely against the Gly-Ala-rich PGRS domain. Conversely, splenocytes from 1818PE-vaccinated mice but not mice immunized with 1818PE_PGRS secreted gamma interferon following in vitro restimulation and demonstrated protection in the mouse tuberculosis challenge model. These results suggest that the PE vaccine can elicit an effective cellular immune response and that immune recognition of the PE antigen is influenced by the Gly-Ala-rich PGRS domain.     

Computer-assisted prediction of HLA-DR binding and experimental analysis for human promiscuous Th1-cell peptides in the 24 kDa secreted lipoprotein (LppX) of Mycobacterium tuberculosis

The secreted 24 kDa lipoprotein (LppX) is an antigen that is specific for Mycobacterium tuberculosis complex and M. leprae. The present study was carried out to identify the promiscuous T helper 1 (Th1)-cell epitopes of the M. tuberculosis LppX (MT24, Rv2945c) antigen by using 15 overlapping synthetic peptides (25 mers overlapping by 10 residues) covering the sequence of the complete protein. The analysis of Rv2945c sequence for binding to 51 alleles of nine serologically defined HLA-DR molecules, by using a virtual matrix-based prediction program (propred), showed that eight of the 15 peptides of Rv2945c were predicted to bind promiscuously to >/=10 alleles from more than or equal to three serologically defined HLA-DR molecules. The Th1-cell reactivity of all the peptides was assessed in antigen-induced proliferation and interferon-gamma (IFN-gamma)-secretion assays with peripheral blood mononuclear cells (PBMCs) from 37 bacille Calmette-Guérin (BCG)-vaccinated healthy subjects. The results showed that 17 of the 37 donors, which represented an HLA-DR-heterogeneous group, responded to one or more peptides of Rv2945c in the Th1-cell assays. Although each peptide stimulated PBMCs from one or more donors in the above assays, the best positive responses (12/17 (71%) responders) were observed with the peptide p14 (aa 196-220). This suggested a highly promiscuous presentation of p14 to Th1 cells. In addition, the sequence of p14 is completely identical among the LppX of M. tuberculosis, M. bovis and M. leprae, which further supports the usefulness of Rv2945c and p14 in the subunit vaccine design against both tuberculosis and leprosy.     

Comparison of the proteome of isoniazid-resistant and -susceptible strains of Mycobacterium tuberculosis

In an effort to determine whether there is a major difference in protein level between isoniazid resistant and susceptible Mycobacterium tuberculosis strains, the proteins extracted from nine isoniazid (INH) monoresistant M. tuberculosis strains, seven isoniazid susceptible M. tuberculosis strains, and M. tuberculosis H37Rv, were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In all protein spot differences, five protein spots that are upregulated in INH-resistant strains were identified as Rv1446c, Rv3028c, Rv0491, Rv2971, and Rv2145 by matrix-assisted laser desorption/ionization-time of fight-mass spectrometry (MALDI-TOF-MS). Most of these differential proteins are membrane proteins, which are known to be rich in potential diagnostic and therapeutic protein targets. These results suggest that the differentially expressed proteins from INH-resistant strains might be used as potential immunodiagnosis antigens and candidate novel drug targets against drug-resistant tuberculosis.     

Human humoral responses to antigens of Mycobacterium tuberculosis: immunodominance of high-molecular-mass antigens.

The selection of antigens of Mycobacterium tuberculosis for most studies of humoral responses in tuberculosis patients has been restricted to molecules that were either immunodominant in immunized animals or amenable to biochemical purification rather than those that were reactive with the human immune system. Delineation of antigens that elicit humoral responses during the natural course of disease progression in humans has been hindered by the presence of cross-reactive antibodies to conserved regions on ubiquitous prokaryotic antigens in sera from healthy individuals and tuberculosis patients. The levels of cross-reactive antibodies in the sera were reduced by preadsorption with Escherichia coli lysates, prior to studying their reactivity against a large panel of M. tuberculosis antigens to which the human immune system may be exposed during natural infection and disease. Thus, reactivity against pools of secreted, cellular, and cell wall-associated antigens of M. tuberculosis was assessed by an enzyme-linked immunosorbent assay (ELISA). Initial results suggested that the secreted protein preparation contained antigens most frequently recognized by the humoral responses of pulmonary tuberculosis patients. The culture filtrate proteins were subsequently size fractionated by preparative polyacrylamide gel electrophoresis, characterized by reaction with murine monoclonal antibodies to known antigens of M. tuberculosis by an ELISA, and assessed for reactivity with tuberculous and nontuberculous sera. Results show that a secreted antigen of 88 kDa elicits a strong antibody response in a high percentage of patients with pulmonary tuberculosis. This and other antigens identified on the basis of their reactivity with patient sera may prove useful for developing serodiagnosis for tuberculosis.     

结核分枝杆菌Rv1626的原核表达及其免疫功能研究

结核分枝杆菌;Rv1626;原核表达;免疫原性;结核病     

Identification of human T-cell responses to Mycobacterium tuberculosis resuscitation-promoting factors in long-term latently infected individuals

The Mycobacterium bovis BCG vaccine is the only tuberculosis (TB) vaccine available, yet it provides limited protection against pulmonary TB in adults and fails to protect against TB reactivation. We hypothesized that immunity against Mycobacterium tuberculosis "resuscitation-promoting factors" (Rpfs), which are small bacterial proteins that promote proliferation of dormant mycobacteria, may be relevant in the human immune response to M. tuberculosis. In previous unpublished work, we found that Rpfs Rv0867c and Rv2389c induced gamma interferon (IFN-γ) production in the blood of TB patients' healthy household contacts in several different African populations. Here we examine these two dominant Rpf antigens in more detail and define the nature of the responding T-cell subsets. Multiparameter cytokine profiling showed that Rv2389c and, to a lesser extent, Rv0867c were recognized by mycobacterium-responsive healthy Dutch individuals; peptide-scanning revealed several epitopes, including a single immunodominant epitope in Rv2389c. Rv0867c and, to a lesser extent, Rv2389c Rpf-specific T-cell responses were maintained for decades in long-term M. tuberculosis nonprogressors. Prominent Rv0867c-specific double- and single-cytokine-producing CD8(+) T-cell subset responses were found, including a large population of CD8(+) effector memory and effector T-cell subsets. We conclude that M. tuberculosis Rpf antigens are important targets in the human immune response to M. tuberculosis and represent interesting TB vaccine candidate antigens.     

A mycobacterial lipoarabinomannan specific monoclonal antibody and its F(ab') fragment prolong survival of mice infected with Mycobacterium tuberculosis

Lipoarabinomannan (LAM) is a major structural carbohydrate antigen of the outer surface of Mycobacterium tuberculosis. High antibody titres against LAM are often seen in active tuberculosis (TB). The role of such LAM-specific antibodies in the immune response against TB is unknown. Here we have investigated a monoclonal antibody (MoAb) SMITB14 of IgG1 subclass and its corresponding F(ab')(2) fragment directed against LAM from M. tuberculosis strain H37Rv. MoAb SMITB14 was shown by immunofluorescence to bind to whole cells of the clinical isolate M. tuberculosis strain Harlingen as well as to M. tuberculosis H37Rv. The binding of MoAb SMITB14 to LAM was inhibited by arabinomannan (AM) and oligosaccharides (5.2 kDa) derived from LAM, showing that the MoAb binds specifically to the AM carbohydrate portion of LAM. In passive protection experiments BALB/c mice were infected intravenously with M. tuberculosis Harlingen. MoAb SMITB14 was added intravenously either prior to, or together with, the bacteria. The antibody proved to be protective against the M. tuberculosis infection in terms of a dose-dependent reduction in bacterial load in spleens and lungs, reduced weight loss and, most importantly, increased long-term survival.     

Immunogenicity of eight dormancy regulon-encoded proteins of Mycobacterium tuberculosis in DNA-vaccinated and tuberculosis-infected mice

Hypoxia and low concentrations of nitric oxide have been reported to upregulate in vitro gene expression of 48 proteins of the dormancy (DosR) regulon of Mycobacterium tuberculosis. These proteins are thought to be essential for the survival of bacteria during persistence in vivo and are targeted by the immune system during latent infection in humans. Here we have analyzed the immunogenicity of eight DosR regulon-encoded antigens by plasmid DNA vaccination of BALB/c and C57BL/6 mice, i.e., Rv1733c, Rv1738, Rv2029c (pfkB), Rv2031c/hspX (acr), Rv2032 (acg), Rv2626c, Rv2627c, and Rv2628. Strong humoral and/or cellular Th1-type (interleukin-2 and gamma interferon) immune responses could be induced against all but one (Rv1738) of these antigens. The strongest Th1 responses were measured following vaccination with DNA encoding Rv2031c and Rv2626c. Using synthetic 20-mer overlapping peptides, 11 immunodominant, predicted major histocompatibility complex class II-restricted epitopes and one K(d)-restricted T-cell epitope could be identified. BALB/c and (B6D2)F(1) mice persistently infected with M. tuberculosis developed immune responses against Rv1733c, Rv2031c, and Rv2626c. These findings have implications for proof-of-concept studies in mice mimicking tuberculosis (TB) latency models and their extrapolation to humans for potential new vaccination strategies against TB.     

Heterogenous Expression of the Related MBP70 and MPB83 Proteins Distinguish Various Substrains of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv

MPB70 and MPB83 are homologous cross-reactive secreted mycobacterial proteins with very limited species distribution. The expression of these two proteins was compared between several substrains of Mycobacterium bovis BCG, virulent M. bovis and Mycobacterium tuberculosis H37Rv. A polyclonal antibody specific for MPB70 in Western blotting, and a monoclonal antibody, MBS43, found to be specific for MPB83 in ELISA and Western blotting, were used for the comparison. The previously established pattern of high- and low-producing substrains of BCG for MPB70 is only partially applicable for MPB83. MPB70 low-producing strains are also MPB83 low-producing, but the expression of MPB83 is much more variable than the expression of MPB70 in the MPB70 high-producing strains. Purified MPB83 (23 kDa) was found to be glycosylated. A band in SDS-PAGE at 1–2 kDa lower than that of purified MPB83 may represent unglycosylated MPB83. Furthermore, it was confirmed that purified MPB70 (22 kDa) is unglycosylated. There is cross-reactive antigen at 26 kDa. The MPB83 related antigen at 26 kDa was found to be the most abundant. These findings indicate greater heterogeneity between different substrains of BCG than previously realized. Virulent M. bovis produce and secrete large amounts of MPB70 and MPB83 while both these proteins occur in a far lower concentration in M. tuberculosis     

Assessment of five antigens from Mycobacterium tuberculosis for serodiagnosis of tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a major public health issue, particularly in developing countries, and thus effective diagnostic methods for TB remain a central theme in basic and clinical research. To evaluate five antigens (38-kDa protein [38kDa], Rv3621c, Rv3618, 38kDa-ESAT-6 [38E6], and Ag85B-HBHA [AH]) in serological tests for TB patients, we recruited 288 patients and 201 healthy controls. The median IgG reactivity to 38kDa, 38E6, and AH was higher than that to Rv3618 and Rv3621c in pulmonary TB. 38kDa and 38E6 provided high sensitivities in pulmonary TB but low sensitivities in extrapulmonary TB (EPTB). The specificities achieved by 38kDa and 38E6 ranged from 82.0% to 93.9% in patients with non-TB respiratory disease (PD) and in controls. 38kDa and 38E6 exhibited lower sensitivities and higher specificities than their combinations with Rv3618. These findings provide useful information on the relative importance of the above five antigens and suggest that combinations of Rv3618 with 38kDa and 38E6 can increase their sensitivities, but their specificities need to be further increased.     

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.     

Proteins of the Rpf family: immune cell reactivity and vaccination efficacy against tuberculosis in mice

It was shown recently that Mycobacterium tuberculosis expresses five proteins that are homologous to Rpf (resuscitation promoting factor), which is secreted by growing cells of Micrococcus luteus. Rpf is required to resuscitate the growth of dormant Micrococcus luteus organisms, and its homologues may be involved in mycobacterial reactivation. Mycobacterial Rpf-like products are secreted proteins, which makes them candidates for recognition by the host immune system and anti-Rpf immune responses potentially protective against reactivated tuberculosis. Here we report that the Rpf protein itself and four out of five of its mycobacterial homologues, which were administered as subunit vaccines to C57BL/6 mice, are highly immunogenic. Rpf-like proteins elicit immunoglobulin G1 (IgG1) and IgG2a responses and T-cell proliferation and stimulate production of gamma interferon, interleukin-10 (IL-10), and IL-12 but not IL-4 or IL-5. Both humoral and T-cell responses against these antigens show a high degree of cross-reactivity. Vaccination of mice with Rpf-like proteins results in a significant level of protection against a subsequent high-dose challenge with virulent M. tuberculosis H37Rv, both in terms of survival times and mycobacterial multiplication in lungs and spleens.