Sandra M. Arend

Detection of Active Tuberculosis Infection by T Cell Responses to Early-Secreted Antigenic Target 6-kDa Protein and Culture Filtrate Protein 10

The purified protein derivative (PPD) skin test has no predictive value for tuberculosis (TB) in Mycobacterium bovis bacillus Calmette-Guérin (BCG)-vaccinated individuals because of cross-reactive responses to nonspecific constituents of PPD. T cell responses to early-secreted antigenic target 6-kDa protein (ESAT-6) and the newly identified culture filtrate protein 10 (CFP-10), 2 proteins specifically expressed by M. tuberculosis (MTB) but not by BCG strains, were evaluated. Most TB patients responded to ESAT-6 (92%) or CFP-10 (89%). A minority of BCG-vaccinated individuals responded to both ESAT-6 and CFP-10, their history being consistent with latent infection with MTB in the presence of protective immunity. No responses were found in PPD-negative controls. The sensitivity and specificity of the assay were 84% and 100%, respectively, at a cutoff of 300 pg of interferon-gamma/mL. These data indicate that ESAT-6 and CFP-10 are promising antigens for highly specific immunodiagnosis of TB, even in BCG-vaccinated individuals.

Recognition of Stage-Specific Mycobacterial Antigens Differentiates between Acute and Latent Infections with Mycobacterium tuberculosis

ABSTRACT Mycobacterium tuberculosis is estimated to infect 80 to 100 million people annually, the majority of whom do not develop clinical tuberculosis (TB) but instead maintain the infection in a latent state. These individuals generally become positive in response to a tuberculin skin test and may develop clinical TB at a later date, particularly if their immune systems are compromised. Latently infected individuals are interesting for two reasons. First, they are an important reservoir of M. tuberculosis, which needs to be considered for TB control. Second, if detected prior to recrudescence of the disease, they represent a human population that is making a protective immune response to M. tuberculosis, which is very important for defining correlates of protective immunity. In this study, we show that while responsiveness to early secretory antigenic target 6 is a good marker for M. tuberculosis infection, a strong response to the 16-kDa Rv2031c antigen (HspX or α-crystallin) is largely restricted to latently infected individuals, offering the possibility of differential immunodiagnosis of, or therapeutic vaccination against, TB.

Antigenic equivalence of human T-cell responses to Mycobacterium tuberculosis-specific RD1-encoded protein antigens ESAT-6 and culture filtrate protein 10 and to mixtures of synthetic peptides.

ABSTRACT The early secreted antigenic target 6-kDa protein (ESAT-6) and culture filtrate protein 10 (CFP-10) are promising antigens for reliable immunodiagnosis of tuberculosis. Both antigens are encoded by RD1, a genomic region present in all strains of Mycobacterium tuberculosis and M. bovis but lacking in all M. bovis bacillus Calmette-Guérin vaccine strains. Production and purification of recombinant antigens are laborious and costly, precluding rapid and large-scale testing. Aiming to develop alternative diagnostic reagents, we have investigated whether recombinant ESAT-6 (rESAT-6) and recombinant CFP-10 (rCFP-10) can be replaced with corresponding mixtures of overlapping peptides spanning the complete amino acid sequence of each antigen. Proliferation of M. tuberculosis-specific human T-cell lines in response to rESAT-6 and rCFP-10 and that in response to the corresponding peptide mixtures were almost completely correlated (r = 0.96,P < 0.0001 for ESAT-6; r = 0.98,P < 0.0001 for CFP-10). More importantly, the same was found when gamma interferon production by peripheral blood mononuclear cells in response to these stimuli was analyzed (r = 0.89, P < 0.0001 for ESAT-6;r = 0.89, P < 0.0001 for CFP-10). Whole protein antigens and the peptide mixtures resulted in identical sensitivity and specificity for detection of infection with M. tuberculosis. The peptides in each mixture contributing to the overall response varied between individuals with different HLA-DR types. Interestingly, responses to CFP-10 were significantly higher in the presence of HLA-DR15, which is the major subtype of DR2. These results show that mixtures of synthetic overlapping peptides have potency equivalent to that of whole ESAT-6 and CFP-10 for sensitive and specific detection of infection with M. tuberculosis, and peptides have the advantage of faster production at lower cost.

Ag85B-ESAT-6 adjuvanted with IC31® promotes strong and long-lived Mycobacterium tuberculosis specific T cell responses in naïve human volunteers.

Though widely used, the BCG vaccine has had little apparent effect on rates of adult pulmonary tuberculosis. Moreover, the risk of disseminated BCG disease in immunocompromised individuals means that improved TB vaccines ideally need to be able to efficiently prime mycobacterially-naïve individuals as well as boost individuals previously vaccinated with BCG. Protective immunity against Mycobacterium tuberculosis is thought to depend on the generation of a Th1-type cellular immune response characterized by interferon-gamma (IFN-gamma) production. In the present study, we monitored safety and IFN-gamma responses in healthy TB-naïve humans receiving an entirely novel vaccine, composed of the fusion protein Ag85B-ESAT-6, administered at 0 and 2 months either as recombinant protein alone or combined with two concentrations of the novel adjuvant IC31. Vaccination did not cause local or systemic adverse effects besides transient soreness at the injection site, but it elicited strong antigen-specific T cell responses against H1 and both the Ag85B and the ESAT-6 components. These strong responses persisted through 2.5 years of follow-up, indicating the induction of a substantial memory response in the vaccine recipients.

Epitope Mapping of the Immunodominant Antigen TB10.4 and the Two Homologous Proteins TB10.3 and TB12.9, Which Constitute a Subfamily of the esat-6 Gene Family

ABSTRACT The human T-cell recognition of the low-molecular-mass culture filtrate antigen TB10.4 was evaluated in detail. The molecule was strongly recognized by T cells isolated from tuberculosis (TB) patients and from BCG-vaccinated donors. The epitopes on TB10.4 were mapped with overlapping peptides and found to be distributed throughout the molecule. The broadest response was found in TB patients, whereas the response in BCG-vaccinated donors was focused mainly toward a dominant epitope located in the N terminus (amino acids 1 to 18). The gene encoding TB10.4 was found to belong to a subfamily within the esat-6 family that consists of the three highly homologous proteins TB10.4, TB10.3, and TB12.9 (Rv0288, Rv3019c, and Rv3017c, respectively). Southern blot analysis combined with database searches revealed that the three members of the TB10.4 family were present only in strains of the Mycobacterium tuberculosis complex, including BCG, and M. kansasii, whereas other atypical mycobacteria had either one (M. avium, M. intracellulare, and M. marinum) or none (M. scrofulaceum, M. fortuitum, and M. szulgai) of the genes. The fine specificity of the T-cell response to the three closely related esat-6 family members was markedly different, with only a few epitopes shared between the molecules. Minimal differences in the amino acid sequence translated into large differences in recognition by T cells and secretion of gamma interferon. In general, the peptides from TB10.4 stimulated the largest responses, but epitopes unique to both TB10.3 and TB12.9 were found. The relevance of the findings for TB vaccine development and as a potential mechanism for immune evasion is discussed.

Tuberculin Skin Testing and In Vitro T Cell Responses to ESAT-6 and Culture Filtrate Protein 10 after Infection with Mycobacterium marinum or M. kansasii

T cell responses to ESAT-6 and culture filtrate protein 10 (CFP-10), antigens expressed by Mycobacterium tuberculosis but not by M. bovis bacille Calmette-Guérin (BCG), were found to discriminate reliably between infection with M. tuberculosis and BCG vaccination. Because the esat-6 and cfp-10 genes occur in M. kansasii and M. marinum, T cell responses to ESAT-6 and CFP-10 were investigated in patients infected with M. kansasii or M. marinum, persons intensively exposed to environmental mycobacteria, and unexposed control subjects. Tuberculin skin tests were performed, and peripheral blood mononuclear cells were cocultured with ESAT-6, CFP-10, peptide mixtures of ESAT-6 and CFP-10, and control antigens. When enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT) were used to measure interferon-gamma production, most M. kansasii- or M. marinum-infected patients and several persons exposed to environmental mycobacteria were found to respond to ESAT-6 and/or CFP-10. ELISA and ELISPOT yielded comparable results, as did whole antigen and peptides (P<.0001). These results may be relevant for the development of novel assays for diagnosis of tuberculosis.

Predictive value for progression to tuberculosis by IGRA and TST in immigrant contacts

The authors determined the positive predictive value (PPV) for progression to tuberculosis (TB) of two interferon-γ release assays (IGRAs), QuantiFERON-TB® Gold In-tube (QFT-GIT) and T-SPOT.TB®, and the tuberculin skin test (TST) in immigrants contacts. Immigrant close contacts of sputum smear-positive TB patients were included when aged ≥16 yrs and their TST result was ≥5 mm 0 or 3 months after diagnosis of the index patient. Contacts were followed for the next 2 yrs for development of TB disease. Of 339 immigrant contacts with TST ≥5 mm, 324 and 299 had valid results of QFT-GIT and T-SPOT.TB®, respectively. Nine contacts developed active TB. One patient had not been tested with TST, while another patient had not been tested with QFT-GIT and T-SPOT.TB®. The PPV for progression to TB during this period was 9/288 = 3.1% (95% CI 1.3–5.0%) for TST ≥10 mm, 7/184 = 3.8% (95% CI 1.7–5.9%) for TST ≥15 mm, 5/178 = 2.8% (95% CI 1.0–4.6%) for QFT-GIT and 6/181 = 3.3% (95% CI 1.3–5.3%) for T-SPOT.TB®. Sensitivity was 100%, 88%, 63% and 75%, respectively. The predictive values of QFT-GIT, T-SPOT.TB® and TST for progression to TB disease among immigrant close contacts were comparable.

Specific T-Cell Epitopes for Immunoassay-Based Diagnosis of Mycobacterium tuberculosis Infection

ABSTRACT The currently used method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity. Antigens specific for Mycobacterium tuberculosis to replace purified protein derivative are therefore urgently needed. We have performed a rigorous assessment of the diagnostic potential of four recently identified antigens (Rv2653, Rv2654, Rv3873, and Rv3878) from genomic regions that are lacking from the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine strains as well as from the most common nontuberculous mycobacteria. The fine specificity of potential epitopes in these molecules was evaluated by sensitive testing of the T-cell responses of peripheral blood mononuclear cells derived from M. bovis BCG-vaccinated healthy individuals to synthesized overlapping peptides. Three of the four molecules contained regions with significant specificity problems (Rv2653, Rv3873, and Rv3878). We selected and combined the specific peptide stretches from the four proteins not recognized by M. bovis BCG-vaccinated individuals. These peptide stretches were tested with peripheral blood mononuclear cells obtained from patients with microscopy- or culture-confirmed tuberculosis and from healthy M. bovis BCG-vaccinated controls. The combination of the most promising stretches from this analysis showed a sensitivity level (57%) comparable to the level found with the two well-known M. tuberculosis-specific proteins ESAT-6 and CFP-10 (75 and 66%, respectively). The combination of ESAT-6, CFP-10, and the novel specific peptide stretches gave an overall sensitivity of 84% at a specificity of 97%. In a validation experiment with new experimental groups, the sensitivities obtained were 57% for the combination of peptides and 90% for the combination of the peptides, ESAT-6, and CFP-10. This combination gave a specificity of 95%.

PPE protein (Rv3873) from DNA segment RD1 of Mycobacterium tuberculosis: strong recognition of both specific T-cell epitopes and epitopes conserved within the PPE family.

ABSTRACT Proteins encoded by DNA segment RD1 of Mycobacterium tuberculosis have recently been demonstrated to play important roles in bacterial virulence, vaccine development, and diagnostic reagent design. Previously, we characterized two immunodominant T-cell antigens, the early secreted antigen target (ESAT-6) and the 10-kDa culture filtrate protein (CFP10), which are encoded by the esx-lhp operon in this region. In the present study we characterized a third putative open reading frame in this region, rv3873, which encodes a PPE protein. We found that the rv3873 gene is expressed in M. tuberculosis H37Rv and that the native protein, Rv3873, is predominantly associated with the mycobacterial cell or wall. When tested as a His-tagged recombinant protein, Rv3873 stimulated high levels of gamma interferon secretion in peripheral blood mononuclear cells isolated from tuberculosis (TB) patients, as well as from healthy tuberculin purified protein derivative-positive donors. In contrast to other RD1-encoded antigens, Rv3873 was also found to be recognized by a significant proportion of Mycobacterium bovis BCG-vaccinated donors. Epitope mapping performed with overlapping peptides revealed a broad pattern of T-cell recognition comprising both TB-specific epitopes and epitopes also recognized by BCG-vaccinated donors. The immunodominant epitope (residues 118 to 135) for both TB patients and BCG-vaccinated individuals was found to be highly conserved among a large number of PPE family members.

Nontuberculous mycobacterial infection in children: a 2-year prospective surveillance study in the Netherlands

We performed a prospective, 2-year nationwide study to assess incidence and disease characteristics of suspected infections with nontuberculous mycobacteria (NTM) in children, via the Netherlands Pediatric Surveillance Unit. Data for 61 children were reported (median age, 31 months; interquartile range, 22-50 months; female sex, 37 subjects); 2 subjects had an underlying disease. Most children (53 [87%] of 61) had cervical lymph node enlargement, with abscess in 25 (47%) and fistula in 11 (21%). The estimated annual incidence of NTM infection was 77 cases per 100,000 children. In 16 children, the diagnosis was based solely on the results of skin tests with mycobacterial antigens. Cultures were performed in 36 cases and yielded mycobacteria in 27 (75%); Mycobacterium avium was isolated from 18 cultures. Children with a culture positive for mycobacteria did not differ in presentation, complications, or treatment from those whose cultures showed no growth. Thirty children underwent surgery, and chemotherapy was the single treatment in 24 (39%) of the cases. The treatment of localized NTM infection in immunocompetent children by antimycobacterial drugs should be evaluated further.