Immunogenicity of the Mycobacterium tuberculosis PPE55 (Rv3347c) protein during incipient and clinical tuberculosis

Clinical tuberculosis (TB), whether noncavitary or cavitary, is the late stage of a chronic disease process, since Mycobacterium tuberculosis is a slowly growing organism. Our studies have shown that the profiles of antigenic proteins expressed by the in vivo bacteria that elicit antibodies differ in cavitary and noncavitary TB. To gain insight into antigenic proteins expressed during incipient, subclinical TB, an expression library of M. tuberculosis genomic DNA was screened with sera obtained during subclinical TB from guinea pigs infected with aerosols of M. tuberculosis H37Rv. One of the proteins recognized by antibodies elicited during subclinical TB infection of guinea pigs is the 309-kDa PPE55 (Rv3347c) protein. Genomic hybridization studies suggest that the PPE55 gene is specific to the M. tuberculosis complex and is present in a majority of clinical isolates tested. Antibodies to the C-terminal, approximately 100-kDa fragment of PPE55 (PPE-C) were detectable in sera from 29/30 (97%) human immunodeficiency virus-negative/TB-positive (HIV(-) TB(+)) patients and 17/24 (71%) HIV(+) TB(+) patients tested but not in sera from purified-protein derivative-positive healthy controls, suggesting that the in vivo expression of PPE55 protein correlates with active M. tuberculosis infection. Anti-PPE-C antibodies were also detected in retrospective sera obtained months prior to manifestation of clinical TB from 17/21 (81%) HIV(+) TB(+) individuals tested, providing evidence that the protein is expressed during incipient, subclinical TB in HIV-infected humans. Thus, PPE55 is a highly immunogenic protein that may be useful for differentiating between latent TB and incipient, subclinical TB.     

Strong Antibody Responses to Mycobacterium tuberculosis PE-PGRS62 Protein Are Associated with Latent and Active Tuberculosis

Mycobacterium tuberculosis has a unique family of PE-PGRS proteins with conserved N-terminal domains (PE) containing site-specific proline-glutamine residues and polymorphic GC-rich repetitive sequences (PGRS). Tuberculosis (TB) patients produce antibodies against some such proteins, but it is not clear whether these responses correlate with disease. Clinical groups with different mycobacterium exposure were studied for their seroreactivity to PE-PGRS17 and PE-PGRS62 proteins and their respective PE domains. There were minimal antibody responses against both PE domains and full-length PE-PGRS17, even in patients with active TB. However, patients with active and latent TB showed significantly higher PE-PGRS62-specific immunoglobulin G antibody responses than treated TB patients and mycobacterium-reactive TB contacts without latent infection. Latently infected persons had high anti-PE-PGRS62 responses but low responses to the 38-kDa antigen commonly used for TB serology, while treated TB cases showed the opposite response. Thus, patterns of seroreactivity to PE-PGRS62 correlate with clinical status and are associated with latent TB infection.The genome sequence of the Mycobacterium tuberculosis H37Rv strain reveals a family of highly homologous PE-PGRS genes that encode a highly conserved N-terminal domain with Pro-Glu (PE) residues at specific sites and a polymorphic GC-rich repetitive-sequence (PGRS) domain at the C terminus (7). The abundance of these unique genes suggests that this gene family may have important roles, but many members have unknown functions. The PGRS domain of PE-PGRS proteins is generally rich in glycine-alanine (Gly-Ala) repetitive sequences (7). Comparison of some of the PE-PGRS gene sequences between an M. tuberculosis laboratory strain (H37Rv), an M. tuberculosis clinical isolate (CDC1551), and Mycobacterium bovis (2, 14) reveals that the repetitive regions of PGRS domains are prone to insertion-deletion mutations. Moreover, M. tuberculosis clinical isolates exhibit variations in restriction fragment length polymorphism patterns in their PE-PGRS genes (11). Hence, it has been speculated that the PGRS domain has a functional role in generating antigenic diversity to avoid host recognition.Antibodies against the PGRS domains of some such proteins, e.g., wag22 (Rv1759c^PE-PGRS) and PE-PGRS51, have been detected in sera of tuberculosis (TB) patients (10, 21). Murine immunization with a DNA vaccine encoding PE-PGRS33 also elicits substantial antibody responses (9). Thus, at least some of the M. tuberculosis PE-PGRS proteins are expressed in vivo and are recognized by host immunity. However, it is not known whether latent versus active infection, the stage and extent of disease, or duration of treatment may influence immune reactivity to PE-PGRS proteins in human TB. The relevance of such reactivity to TB diagnosis and prognosis is also unknown.In this study, the association of seroreactivity against PE-PGRS proteins with disease status was evaluated to uncover any differences in immune recognition of these proteins between healthy persons and persons with TB. We aimed to determine whether latent infection or prior mycobacterium exposure results in responses to these proteins similar to those in persons with active infection and whether such antibodies persist after treatment. As prior studies suggest that there are differential immune responses to the two domains (PE versus PGRS) and that the Gly-Ala repeats may inhibit immune recognition of the whole protein (3), we also wanted to know if these repetitive sequences influence the induction of antibodies against specific PE-PGRS proteins. Therefore, we studied two contrasting PE-PGRS proteins with only 20% identity—one rich in these repeats (PE-PGRS17) and one with a relative paucity of these repeats (PE-PGRS62). We also chose these two proteins because of evidence based on expression or knockout studies (19, 23) suggesting that they have relevance to host-pathogen interactions in vivo, which increases the likelihood that they may interact with host immunity in infected persons. Our study reveals markedly different antibody responses to PE-PGRS62 and to PE-PGRS17. We also demonstrate that strong responses to the former are particularly associated with latent and active TB infection but not treated TB, suggesting that immune recognition of this protein may be a feature of the acute response to TB infection.     

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.     

Isolation of acid-inducible genes of Mycobacterium tuberculosis with the use of recombinase-based in vivo expression technology

A better understanding of mycobacterial gene regulation under certain stress conditions (e.g., low pH) may provide insight into mechanisms of adaptation during infection. To identify mycobacterial promoters induced at low pH, we adapted the recombinase-based in vivo expression technology (RIVET) promoter trap system for use with mycobacteria. Our results show that the TnpR recombinase of transposon gammadelta is active in Mycobacterium smegmatis and Mycobacterium tuberculosis. We developed a method to perform sequential double selection with mycobacteria by using RIVET, with a kanamycin preselection and a sucrose postselection. A library of M. tuberculosis DNA inserted upstream of tnpR was created, and using the double selection, we identified two promoters which are upregulated at low pH. The promoter regions drive the expression of a gene encoding a putative lipase, lipF (Rv3487c), as well as a PE-PGRS gene, Rv0834c, in a pH-dependent manner in both M. smegmatis and M. tuberculosis. The acid inducibility of lipF and Rv0834c was independent of the stress response sigma factor, SigF, as acid induction of the two genes in an M. tuberculosis sigF mutant strain was similar to that in the wild-type strain. No induction of lipF or Rv0834c was observed during infection of J774 murine macrophages, an observation which is in agreement with previous reports on the failure of phagosomes containing M. tuberculosis to acidify.     

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.     

LipC (Rv0220) is an immunogenic cell surface esterase of Mycobacterium tuberculosis

We have reported previously the identification of novel proteins of Mycobacterium tuberculosis by the immunoscreening of an expression library of M. tuberculosis genomic DNA with sera obtained from M. tuberculosis-infected rabbits at 5 weeks postinfection. In this study, we report the further characterization of one of these antigens, LipC (Rv0220). LipC is annotated as a member of the Lip family based on the presence of the consensus motif "GXSXG" characteristic of esterases. Although predicted to be a cytoplasmic enzyme, we provide evidence that LipC is a cell surface protein that is present in both the cell wall and the capsule of M. tuberculosis. Consistent with this localization, LipC elicits strong humoral immune responses in both HIV-negative (HIV-) and HIV-positive (HIV+) tuberculosis (TB) patients. The absence of anti-LipC antibodies in sera from purified protein derivative-positive (PPD+) healthy subjects confirms its expression only during active M. tuberculosis infection. Epitope mapping of LipC identified 6 immunodominant epitopes, 5 of which map to the exposed surface of the modeled LipC protein. The recombinant LipC (rLipC) protein also elicits proinflammatory cytokine and chemokine responses from macrophages and pulmonary epithelial cells. rLipC can hydrolyze short-chain esters with the carbon chain containing 2 to 10 carbon atoms. Together, these studies demonstrate that LipC is a novel cell surface-associated esterase of M. tuberculosis that is highly immunogenic and elicits both antibodies and cytokines/chemokines.     

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.     

Homogeneity of antibody responses in tuberculosis patients

The goals of the present study were twofold: (i) to compare the repertoires of antigens in culture filtrates of in vitro-grown Mycobacterium tuberculosis that are recognized by antibodies from noncavitary and cavitary tuberculosis (TB) patients and (ii) to determine the extent of variation that exists between the antigen profiles recognized by individual TB patients. Lipoarabinomannan-free culture filtrate proteins of M. tuberculosis were fractionated by one-dimensional (1-D) and 2-D polyacrylamide gel electrophoresis, and the Western blots were probed with sera from non-human immunodeficiency virus (non-HIV)-infected cavitary and noncavitary TB patients and from HIV-infected, noncavitary TB patients. In contrast to earlier studies based on recombinant antigens of M. tuberculosis which suggested that antibody responses in TB patients were heterogeneous (K. Lyashchenko et al., 1998, Infect. Immun. 66:3936-3940, 1998), our studies with native culture filtrate proteins show that the antibody responses in TB patients show significant homogeneity in being directed against a well-defined subset of antigens. Thus, there is a well-defined subset of culture filtrate antigens that elicits antibodies during noncavitary and cavitary disease. In addition, another set of antigens is recognized primarily by cavitary TB patients. The mapping with individual patient sera presented here suggests that serodiagnostic tests based on the subset of antigens recognized during both noncavitary and cavitary TB will enhance the sensitivity of antibody detection in TB patients, especially in difficult-to-diagnose, smear-negative, noncavitary TB patients.     

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.     

Association of strong immune responses to PPE protein Rv1168c with active tuberculosis

Accurate diagnosis of tuberculosis (TB) infection is critical for the treatment, prevention, and control of TB. Conventional diagnostic tests based on purified protein derivative (PPD) do not achieve the required diagnostic sensitivity. Therefore, in this study, we have evaluated the immunogenic properties of Rv1168c, a member of the PPE family, in comparison with PPD, which is routinely used in the tuberculin test, and Hsp60 and ESAT-6, well-known immunodominant antigens of Mycobacterium tuberculosis. In a conventional enzyme immunoassay, the recombinant Rv1168c protein displayed stronger immunoreactivity against the sera obtained from patients with clinically active TB than did PPD, Hsp60, or ESAT-6 and could distinguish TB patients from Mycobacterium bovis BCG-vaccinated controls. Interestingly, Rv1168c antigen permits diagnosis of smear-negative pulmonary TB as well as extrapulmonary TB cases, which are often difficult to diagnose by conventional tests. The immunodominant nature of Rv1168c makes it a promising candidate to use in serodiagnosis of TB. In addition, our studies also show that Rv1168c is a potent T-cell antigen which elicits a strong gamma interferon response in sensitized peripheral blood mononuclear cells obtained from TB patients.     

Identification, diagnostic potential, and natural expression of immunodominant seroreactive peptides encoded by five Mycobacterium tuberculosis-specific genomic regions

Comparative genomic studies have identified several Mycobacterium tuberculosis-specific genomic regions of difference (RDs) which are absent in the vaccine strains of Mycobacterium bovis BCG and which may be useful in the specific diagnosis of tuberculosis (TB). In this study, a total of 775 synthetic peptides covering the sequences of 39 open reading frame (ORF) proteins encoded by genes predicted in five RDs of M. tuberculosis, i.e., RD1, RD4, RD5, RD6, and RD7, were tested by enzyme-linked immunosorbent assays for antibody reactivity with sera from HIV-negative pulmonary TB patients (n = 100) and M. bovis BCG-vaccinated healthy subjects (n = 100). The results identified three immunodominant peptides reactive with TB sera, i.e., amino acids (aa) 346 to 370 of RD1ORF Rv3876, aa 241 to 265 of RD6ORF Rv1508c, and aa 325 to 336 of RD6ORF Rv1516c. These peptides had significantly stronger antibody reactivity with sera from TB patients than with sera from healthy subjects (P < 0.05) and significantly higher rates of positivity with TB sera (positives = 66 to 93%) than sera from healthy subjects (positives = 10 to 28%). Antipeptide antibodies were raised in rabbits after immunization with pools of 11 peptides corresponding to each protein. Probing of culture filtrates and whole-cell lysates of M. tuberculosis with antipeptide antibodies suggested the natural expression of Rv1516c in whole-cell lysates of M. tuberculosis. The results suggest the potential of the identified immunodominant RD peptides in the serodiagnosis of TB.     

Use of multiepitope polyproteins in serodiagnosis of active tuberculosis

Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of approximately 98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of approximately 93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.     

Peripheral blood and pleural fluid mononuclear cell responses to low-molecular-mass secretory polypeptides of Mycobacterium tuberculosis in human models of immunity to tuberculosis

A total of 104 polypeptides were purified from the low-molecular-mass secretory proteome of Mycobacterium tuberculosis H(37)Rv using a combination of anion exchange column chromatography and high resolution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution. The goal of this study was to identify polypeptides from a low-molecular-mass secretory proteome recognized by human subjects infected with M. tuberculosis and to ascertain the differences in specificity of antigen recognition by the peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) of these individuals. The study identified CFP-8 (Rv0496), CFP-11 (Rv2433c), CFP-14.5 (Rv2445c), and CFP-31 (Rv0831c) as novel T-cell antigens apart from previously characterized ESAT-6, TB10.4, CFP10, GroES, MTSP14, MTSP17, CFP21, MPT64, Ag85A, and Ag85B on the basis of recognition by PBMCs of tuberculosis contacts and treated tuberculosis patients. Further, polypeptides prominently recognized by PFMCs of tuberculous pleurisy patients were the same as those recognized by PBMCs of healthy contacts and treated tuberculosis patients. The results of our study indicate the homogeneity of antigenic target recognition by lymphocytes at the site of infection and at the periphery in the human subjects studied and the need to evaluate these antigenic targets as components of future antituberculous vaccines.     

Mycobacterium tuberculosis HBHA protein reacts strongly with the serum immunoglobulin M of tuberculosis patients

Identification and characterization of serologically active mycobacterial antigens are prerequisites for the development of diagnostic reagents. We examined the humoral immune responses of active tuberculosis (TB) patients against Triton-soluble proteins extracted from Mycobacterium tuberculosis by immunoblotting. A 29-kDa protein reacted with immunoglobulin M (IgM) in the pooled sera of the patients, and its N-terminal amino acid sequence matched that of the heparin-binding hemagglutinin (HBHA). Recombinant full-length HBHA was expressed in Escherichia coli (rEC-HBHA) and M. smegmatis (rMS-HBHA). In immunoblot analysis, the IgM antibodies of the TB patients reacted strongly with rMS-HBHA but not with rEC-HBHA, whereas the IgG antibodies of these patients reacted weakly with both recombinant HBHA proteins. In enzyme-linked immunosorbent assay analysis using rMS-HBHA and 85B as antigens, the mean levels and sensitivities of the anti-HBHA IgM antibodies of the TB patients were significantly higher than those of the anti-antigen 85B IgM antibodies, while the IgG antibodies showed the opposite results. Of interest in this respect, the pooled sera from the TB patients that contained anti-HBHA IgM antibodies neutralized the entry of M. tuberculosis into epithelial cells. These findings suggest that IgM antibody to HBHA may play a role in protection against extrapulmonary dissemination.     

Glycolipids of Mycobacterium tuberculosis strain H37Rv are potential serological markers for diagnosis of active tuberculosis

A simple and cost-effective diagnostic tool (TB Screen Test) for the screening of patients with pulmonary and extrapulmonary tuberculosis and for differentiation of those individuals from individuals without tuberculosis, other common infections, and healthy controls has been developed. The serological responses of purified mycobacterial glycolipid antigens were examined by a liposome agglutination assay. The assay was able to detect very low antiglycolipid antibody concentrations in the infected individuals. The sera from the tuberculosis patient group had significantly higher concentrations of antiglycolipid antibody than the sera from uninfected control subjects, with 94% sensitivity and 98.3% specificity. Glycolipids of Mycobacterium tuberculosis H37Rv antigens were isolated, purified, and characterized. After interchelation with liposome particles, these purified antigens specifically bound to the antiglycolipid antibodies present in the sera of patients with tuberculosis, resulting in the formation of a blue agglutination. This protocol clearly differentiates healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. The resultant diagnostic tool, the TB Screen Test, is more economical and rapid (4 min) than other currently available products and can be used for the mass screening of a heavily afflicted population.     

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.     

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.