Inger Brock

Comparative Evaluation of Low-Molecular-Mass Proteins from Mycobacterium tuberculosis Identifies Members of the ESAT-6 Family as Immunodominant T-Cell Antigens

ABSTRACT Culture filtrate from Mycobacterium tuberculosiscontains protective antigens of relevance for the generation of a new antituberculosis vaccine. We have identified two previously uncharacterized M. tuberculosis proteins (TB7.3 and TB10.4) from the highly active low-mass fraction of culture filtrate. The molecules were characterized, mapped in a two-dimensional electrophoresis reference map of short-term culture filtrate, and compared with another recently identified low-mass protein, CFP10 (F. X. Berthet, P. B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel. Microbiology 144:3195–3203, 1998), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested in cultures of peripheral blood mononuclear cells from human tuberculosis (TB) patients, Mycobacterium bovisBCG-vaccinated donors, and nonvaccinated donors. The two ESAT-6 family members, TB10.4 and CFP10, were very strongly recognized and induced gamma interferon release at the same level (CFP10) as or at an even higher level (TB10.4) than ESAT-6. The non-ESAT-6 family member, TB7.3, for comparison, was recognized at a much lower level. CFP10 was found to distinguish TB patients from BCG-vaccinated donors and is, together with ESAT-6, an interesting candidate for the diagnosis of TB. The striking immunodominance of antigens within the ESAT-6 family is discussed, and hypotheses are presented to explain this targeting of the immune response during TB infection.

Latent Tuberculosis in HIV positive, diagnosed by the M. Tuberculosis Specific Interferon-γ test

BackgroundAlthough tuberculosis (TB) is a minor problem in Denmark, severe and complicated cases occur in HIV positive. Since the new M. tuberculosis specific test for latent TB, the QuantiFERON-TB In-Tube test (QFT-IT) became available the patients in our clinic have been screened for the presence of latent TB using the QFT-IT test. We here report the results from the first patients screened.MethodsOn a routine basis the QFT-IT test was performed and the results from 590 HIV positive individuals consecutively tested are presented here. CD4 cell count and TB risk-factors were recorded from patient files.Main findings27/590(4.6%) of the individuals were QFT-IT test positive, indicating the presence of latent TB infection. Among QFT-IT positive patients, 78% had risk factors such as long-term residency in a TB high endemic area (OR:5.7), known TB exposure (OR:4.9) or previous TB disease (OR:4.9). The prevalence of latent TB in these groups were 13%, 16% and 19% respectively. There was a strong correlation between low CD4 T-cell count and a low mitogen response (P < 0.001;Spearman) and more patients with low CD4 cell count had indeterminate results.ConclusionWe found an overall prevalence of latent TB infection of 4.6% among the HIV positive individuals and a much higher prevalence of latent infection among those with a history of exposure (16%) and long term residency in a high endemic country (13%). The QFT-IT test may indeed be a useful test for HIV positive individuals, but in severely immunocompromised, the test may be impaired by T-cell anergy.

Healthy Individuals That Control a Latent Infection with Mycobacterium tuberculosis Express High Levels of Th1 Cytokines and the IL-4 Antagonist IL-4δ2

The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop disease and identifying what constitutes “protective immunity” is one of the holy grails of M. tuberculosis immunology. It is known that IFN-γ is essential for protection, but it is also apparent that IFN-γ levels alone do not explain the immunity/susceptibility dichotomy. The controversy regarding correlates of immunity persists because identifying infected but healthy individuals (those who are immune) has been problematic. We have therefore used recognition of the M. tuberculosis virulence factor early secretory antigenic target 6 to identify healthy, but infected individuals from tuberculosis (TB)-endemic and nonendemic regions (Ethiopia and Denmark) and have compared signals for cytokines expressed directly ex vivo with the pattern found in TB patients. We find that TB patients are characterized by decreased levels of Th1 cytokines and increased levels of IL-10 compared with the healthy infected and noninfected community controls. Interestingly, the healthy infected subjects exhibited a selective increase of message for the IL-4 antagonist, IL-4δ2, compared with both TB patients or noninfected individuals. These data suggest that long-term control of M. tuberculosis infection is associated not just with elevated Th1 responses but also with inhibition of the Th2 response.

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.

Risk for tuberculosis among children

Risk among children is underestimated in countries with a high incidence of this disease.

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.

Use of ESAT-6 and CFP-10 Antigens for Diagnosis of Extrapulmonary Tuberculosis

1. Doukhan L, Delwart EL. Use of tissue culture–amplified human immunodeficiency virus type 1 to study evolutionary changes in vivo. J Infect Dis 2001;183:173 (in this issue). 2. Dykes C, Mootsikapun P, Dexter A, et al. Analysis of env sequence evolution in human immunodeficiency virus–infected patients receiving therapy with nonnucleoside reverse-transcriptase inhibitors. J Infect Dis 2000;182: 316–20. 3. Wei X, Ghosh SK, Taylor ME, et al. Viral dynamics in human immunodeficiency virus type 1 infection. Nature 1995;373:117–22. 4. Chun TW, Carruth L, Finzi D, et al. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 1997;387:183–8. 5. Finzi D, Hermankova M, Pierson T, et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 1997; 278:1295–300. 6. Spira AI, Ho DD. Effect of different donor cells on human immunodeficiency virus type 1 replication and selection in vitro. J Virol 1995;69:422–9. 7. Sabino E, Pan LZ, Cheng-Mayer C, Mayer A. Comparison of in vivo plasma and peripheral blood mononuclear cell HIV-1 quasi-species to short-term tissue culture isolates: an analysis of tat and C2-V3 env regions. AIDS 1994;8:901–9. 8. Delwart EL, Pan H, Sheppard HW, et al. Slower evolution of human immunodeficiency virus type 1 quasispecies during progression to AIDS. J Virol 1997;71:7498–508. 9. Delwart EL, Pan H, Neumann A, Markowitz M. Rapid, transient changes at the env locus of plasma human immunodeficiency virus type 1 populations during the emergence of protease inhibitor resistance. J Virol 1998;72:2416–21. 10. Sheehy N, Desselberger U, Whitwell H, Ball JK. Concurrent evolution of regions of the envelope and polymerase genes of human immunodeficiency virus type 1 during zidovudine (AZT) therapy. J Gen Virol 1996;77:1071–81. 11. Gunthard HF, Frost SD, Leigh-Brown AJ, et al. Evolution of envelope sequences of human immunodeficiency virus type 1 in cellular reservoirs in the setting of potent antiviral therapy. J Virol 1999;73:9404–12. 12. Holmes EC, Zhang LQ, Simmonds P, Ludlam CA, Brown AJ. Convergent and divergent sequence evolution in the surface envelope glycoprotein of human immunodeficiency virus type 1 within a single infected patient. Proc Natl Acad Sci USA 1992;89:4835–9.

Mapping Immune Reactivity toward Rv2653 and Rv2654: Two Novel Low-Molecular-Mass Antigens Found Specifically in the Mycobacterium tuberculosis Complex

New tools are urgently needed for the detection of latent tuberculosis (TB). We evaluated the diagnostic potential of 2 novel Mycobacterium tuberculosis complex-specific candidate antigens (Rv2653 and Rv2654) and investigated T cell recognition during natural infection in humans and experimental infection in guinea pigs. Peripheral blood mononuclear cells stimulated with peptide pools covering the full length of Rv2654 induced interferon- gamma release in 10 of 19 patients with TB. Neither Rv2654 single peptides nor Rv2654 pools were recognized by bacille Calmette-Guerin-vaccinated donors. However, peptides from Rv2653 were recognized by both patients group. The cross-reactive epitope(s) in Rv2653 were located in a 36-amino acid stretch in the center of the molecule. Rv2654 also induced M. tuberculosis-specific skin-test responses in 3 of 4 aerosol-infected guinea pigs. Rv2654 is a strongly recognized T cell antigen that is highly specific for TB and has potential as a novel cell-mediated immunity-based TB diagnostic agent.

Human T-cell responses to the RD1-encoded protein TB27.4 (Rv3878) from Mycobacterium tuberculosis.

In recent years, there has been considerable focus on the discovery and characterization of proteins derived from Mycobacterium tuberculosis leading to the identification of a number of candidate antigens for use in vaccine development or for diagnostic purposes. Previous experiments have demonstrated an important immunological role for proteins encoded by the RD1 region, which is absent from all strains of bacillus Calmette–Guérin (BCG) but present in the genomes of virulent M. bovis and M. tuberculosis. Herein, we have studied human T‐cell responses to the antigen encoded by the putative open reading frame (rv3878) of the RD1 region. Immunoblot analysis revealed that rv3878 was expressed and the native protein was designated TB27.4. Immunological evaluations demonstrate that TB27.4 elicits a prominent immune response in human tuberculosis patients with a dominant region in the C‐terminal part of the molecule. In contrast, very limited responses were seen in M. bovis BCG‐vaccinated donors. This study therefore emphasizes the diagnostic potential of proteins encoded by the RD1 region.

Clinical features of Clostridium difficile infection and molecular characterization of the isolated strains in a cohort of Danish hospitalized patients

The purpose of this study was to compare clinical features of Clostridium difficile infection (CDI) to toxin gene profiles of the strains isolated from Danish hospitalized patients. C. difficile isolates were characterized by PCR based molecular typing methods including toxin gene profiling and analysis of deletions and truncating mutations in the toxin regulating gene tcdC. Clinical features were obtained by questionnaire. Thirty percent of the CDI cases were classified as community-acquired. Infection by C. difficile with genes encoding both toxin A, toxin B and the binary toxin was significantly associated with hospital-acquired/healthcare-associated CDI compared to community-acquired CDI. Significantly higher leukocyte counts and more severe clinical manifestations were observed in patients infected by C. difficile containing genes also encoding the binary toxin together with toxin A and B compared to patients infected by C. difficile harbouring only toxin A and B. In conclusion, infection by C. difficile harbouring genes encoding both toxin A, toxin B and the binary toxin were associated with hospital acquisition, higher leukocyte counts and severe clinical disease.