Vaccine-induced Th17 cells are established as resident memory cells in the lung and promote local IgA responses.

The ability to mount accelerated and efficient mucosal immune responses is critically important to prevent the establishment of many infections. Secretion of immunoglobulin A (IgA) is a key component in this first line of defense, but the underlying cellular mechanisms are still not completely understood. We have evaluated different routes of immunization and examined the requirements for IgA induction in the airway mucosa. We demonstrate that subcutaneous priming with a recombinant antigen in a T helper (Th)17-inducing adjuvant followed by airway boosting promotes high and sustained levels of IgA in the lungs. This response is associated with germinal center formation in the lung-draining lymph nodes. The lung IgA response is dependent on Th17 cells and absent if interleukin (IL)-17 is depleted or when priming with vaccines inducing only Th1 or Th2 responses. We used intravascular staining to demonstrate that IgA+ B cells and chemokine receptor 6 (CCR6)+Th17 cells are recruited to the lung parenchyma after the airway booster immunization. Once recruited to the lung parenchyma, the Th17 cells transform into resident lymphocytes that persist in the lung tissue for at least 10 weeks. Here, they facilitate the accelerated recruitment of T and B cells resulting in an accelerated IgA recall response to a second airway booster immunization.

Serpins from wheat grain.

Wheat serpin genes have been identified by Southern blot hybridization with three distinct barley protein Z probes. Immunoblot analysis with a monoclonal antibody towards barley protein Z confirmed expression of related M(r) approximately 40 kDa proteins in wheat grain. The wheat serpins were extracted under reducing conditions and separated from beta-amylase and other seed proteins by thiophilic adsorption and anion-exchange chromatography. One molecular form possessing chymotrypsin inhibitory activity was isolated in a reactive site cleaved form on a chymotrypsin affinity column. N-terminal amino acid sequences of a CNBr fragment and of the C-terminal peptide from the cleaved inhibitor (M(r) 4574 +/- 4 Da) verified homology with barley protein Z and mammalian serpins. The native inhibitory serpin was demonstrated to form an SDS-stable complex with alpha-chymotrypsin.

Preparation of Culture Filtrate Proteins from Mycobacterium tuberculosis.

Culture filtrates obtained by in vitro cultivation of Mycobacterium tuberculosis have been studied for more than 20 years to identify and characterize proteins of immunological relevance. Culture filtrate preparations have been shown to induce a protective immune response in mice and guinea pigs by several groups (1-4). Stimulated by these findings, the isolation and evaluation of culture filtrate antigens have been the subjects of intensified effort in several laboratories in recent years. This work has resulted in an increasing number of novel antigens such as MPT59 (5-7), ESAT-6 (8), MPT64 (9), MTB12 (10), MTB8.4 (11), CFP29 (12), TB10.4 (13), among several others.

Towards the proteome of Mycobacterium tuberculosis.

Human tuberculosis is caused by the intracellular pathogen Mycobacterium tuberculosis. Sequencing of the genome of M. tuberculosis strain H37Rv has predicted 3924 open reading frames, and enabled identification of proteins from this bacterium by peptide mass fingerprinting. Extracellular proteins from the culture medium and proteins in cellular extracts were examined by two-dimensional gel electrophoresis using immobilized pH gradient technology. By mass spectrometry and immunodetection, 49 culture filtrate proteins and 118 lysate proteins were identified, 83 of which were novel. To date, 288 proteins have been identified in M. tuberculosis proteome studies, and a list is presented which includes all identified proteins (available at http://www.ssi.dk/publichealth/tbimmun). The information obtained from the M. tuberculosis proteome so far is discussed in relation to the information obtained from the complete genome sequence.

Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG.

ESAT-6 is a secreted protein present in the short-term culture filtrate of Mycobacterium tuberculosis after growth on a synthetic Sauton medium. ESAT-6 has recently been demonstrated to induce strong T-cell responses in a mouse model of memory immunity after infection with M. tuberculosis. In Western blotting (immunoblotting), the monoclonal antibody HYB76-8, reacting with ESAT-6, gave a 6-kDa region was observed in filtrates from four of eight substrains of M. bovis BCG that produced high levels of MPB64, while no band occurred in the 6-kDa region with any of these BCG substrains. Southern blotting and PCR experiments with genomic mycobacterial DNA showed the presence of the esat-6 gene in reference strains and clinical isolates of M. tuberculosis as well as in virulent M. bovis. The esat-6 gene could not be demonstrated in any of the eight substrains of M. bovis BCG tested by these techniques. Two gene deletions that distinguish M. bovis BCG from virulent M. bovis have thus now been demonstrated. Deletion of mpb64 affects four of the eight substrains tested; deletion of esat-6 affects all of them. The reaction of HYB76-8 AT 26 kDa with four of the BCG substrains was demonstrated to result from cross-reactivity with MPB64. HYB76-8 was also shown to cross-react with the A, B, and C components of the antigen 85 complex and MPT51.

ESAT-6 subunit vaccination against Mycobacterium tuberculosis.

The ESAT-6 antigen from Mycobacterium tuberculosis is a dominant target for cell-mediated immunity in the early phase of tuberculosis (TB) in TB patients as well as in various animal models. The purpose of our study was to evaluate the potential of ESAT-6 in an experimental TB vaccine. We started out using dimethyl dioctadecylammonium bromide (DDA), an adjuvant which has been demonstrated to be efficient for the induction of cellular immune responses and has been used successfully before as a delivery system for TB vaccines. Here we demonstrate that, whereas immune responses to both short-term-culture filtrate and Ag85B are efficiently induced with DDA, this adjuvant was inefficient for the induction of immune responses to ESAT-6. Therefore, we investigated the modulatory effect of monophosphoryl lipid A (MPL), an immunomodulator which in different combinations has demonstrated strong adjuvant activity for both cellular and humoral immune responses. We show in the present study that vaccination with ESAT-6 delivered in a combination of MPL and DDA elicited a strong ESAT-6-specific T-cell response and protective immunity comparable to that achieved with Mycobacterium bovis BCG.

Two-dimensional electrophoresis for analysis of Mycobacterium tuberculosis culture filtrate and purification and characterization of six novel proteins.

Culture filtrate from Mycobacterium tuberculosis contains molecules which promote high levels of protective immunity in animal models of subunit vaccination against tuberculosis. We have used two-dimensional electrophoresis for analysis and purification of six novel M. tuberculosis culture filtrate proteins (CFPs): CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28. The proteins were tested for recognition by M. tuberculosis-reactive memory cells from different strains of inbred mice and for their capacity to induce a skin test response in M. tuberculosis-infected guinea pigs. CFP17, CFP20, CFP21 and CFP25 induced both a high gamma interferon release and a strong delayed-type hypersensitivity response, and CFP21 was broadly recognized by different strains of inbred mice. N-terminal sequences were obtained for the six proteins, and the corresponding genes were identified in the Sanger M. tuberculosis genome database. In parallel we established a two-dimensional electrophoresis reference map of short-term culture filtrate components and mapped novel proteins as well as already-known CFP.

Identification and characterization of a 29-kilodalton protein from Mycobacterium tuberculosis culture filtrate recognized by mouse memory effector cells.

Culture filtrate proteins from Mycobacterium tuberculosis induce protective immunity in various animal models of tuberculosis. Two molecular mass regions (6 to 10 kDa and 24 to 36 kDa) of short-term culture filtrate are preferentially recognized by Th1 cells in animal models as well as by patients with minimal disease. In the present study, the 24- to 36-kDa region has been studied, and the T-cell reactivity has been mapped in detail. Monoclonal antibodies were generated, and one monoclonal antibody, HYB 71-2, with reactivity against a 29-kDa antigen located in the highly reactive region below the antigen 85 complex was selected. The 29-kDa antigen (CFP29) was purified from M. tuberculosis short-term culture filtrate by thiophilic adsorption chromatography, anion-exchange chromatography, and gel filtration. In its native form, CFP29 forms a polymer with a high molecular mass. CFP29 was mapped in two-dimensional electrophoresis gels as three distinct spots just below the antigen 85 complex component MPT59. CFP29 is present in both culture filtrate and the membrane fraction from M. tuberculosis, suggesting that this antigen is released from the envelope to culture filtrate during growth. Determination of the N-terminal amino acid sequence allowed cloning and sequencing of the cfp29 gene. The nucleotide sequence showed 62% identity to the bacteriocin Linocin from Brevibacterium linens. Purified recombinant histidine-tagged CFP29 and native CFP29 had similar T-cell stimulatory properties, and they both elicited the release of high levels of gamma interferon from mouse memory effector cells isolated during the recall of protective immunity to tuberculosis. Interspecies analysis by immunoblotting and PCR demonstrated that CFP29 is widely distributed in mycobacterial species.

Specific acquired resistance in mice immunized with killed mycobacteria.

Past attempts to raise resistance against Mycobacterium tuberculosis using various preparations of killed mycobacteria have questioned the specificity of the generated immune response. In the present study, we have focused on the protective efficacy of experimental vaccines based on killed mycobacteria. We demonstrate that killed mycobacteria can confer high levels of protection, which can be adoptively transferred to recipient T-cell-deficient mice. Moreover, protective antigens can be found in the cell wall, membrane and cytosol of the mycobacterial cell, and hence emphasize the importance of searching for protective antigens in various compartments of the mycobacterial cell.

Specific delayed-type hypersensitivity responses to ESAT-6 identify tuberculosis-infected cattle.

Human and bovine tuberculosis have long been detected by skin testing with purified protein derivative (PPD), a complex mix of partly denatured mycobacterial antigens with suboptimal specificity. In the present study, skin tests based on ESAT-6, a recombinantly produced antigen highly specific for tuberculosis infection, were investigated. Although ESAT-6 was strongly recognized in vitro and induced high levels of gamma interferon, initial investigations demonstrated that higher doses of ESAT-6 than of PPD were needed to induce substantial delayed-type hypersensitivity reactions. Also, the kinetics of the skin test response differed for the two reagents; PPD showed maximal response at 72 h, but the response to ESAT-6 often peaked later at 96 h. Tests based on an optimized strategy (400 micro g of ESAT-6 measured between 72 and 96 h), in cattle infected with Mycobacterium bovis (n = 22) and animals sensitized by exposure to environmental mycobacteria showed ESAT-6 to have a promising diagnostic potential (sensitivity, 82%; specificity, 100%; optimal cutoff, 3 mm), compared with PPD (sensitivity, 86%; specificity, 90%; optimal cutoff, 4 mm). Larger investigations are required to refine cutoff points for any new diagnostic test, but the present results indicate great potential for skin tests based on specific antigens for accurate in vivo diagnosis of tuberculosis.

Mapping and identification of Mycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection.

Mycobacterium tuberculosis is the infectious agent giving rise to human tuberculosis. The entire genome of M. tuberculosis, comprising approximately 4000 open reading frames, has been sequenced. The huge amount of information released from this project has facilitated proteome analysis of M. tuberculosis. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was applied to fractions derived from M. tuberculosis culture filtrate, cell wall, and cytosol, resulting in the resolution of 376, 413, and 395 spots, respectively, in silver-stained gels. By microsequencing and immunodetection, 38 culture filtrate proteins were identified and mapped, of which 12 were identified for the first time. In the same manner, 23 cell wall proteins and 19 cytosol proteins were identified and mapped, with 9 and 10, respectively, being novel proteins. One of the novel proteins was not predicted in the genome project, and for four of the identified proteins alternative start codons were suggested. Fourteen of the culture filtrate proteins were proposed to possess signal sequences. Seven of these proteins were microsequenced and the N-terminal sequences obtained confirmed the prediction. The data presented here are an important complement to the genetic information, and the established 2-D PAGE maps (also available at: www.ssi.dk/publichealth/tbimmun) provide a basis for comparative studies of protein expression.

Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens.

Culture filtrate from Mycobacterium tuberculosis contains 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 bovis BCG-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.

Differential T-cell recognition of native and recombinant Mycobacterium tuberculosis GroES.

Mycobacterium tuberculosis GroES was purified from culture filtrate, and its identity was confirmed by immunoblot analysis and N-terminal sequencing. Comparing the immunological recognition of native and recombinant GroES, we found that whereas native GroES elicited a strong proliferative response and release of gamma interferon-gamma by peripheral blood mononuclear cells from healthy tuberculin reactors, the recombinant protein failed to do so. The same difference in immunological recognition was observed in a mouse model of TB infection. Both the native and recombinant preparations were recognized by mice immunized with the recombinant protein. Biochemical characterization including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional electrophoresis, and mass spectrometry analysis of both proteins demonstrated no differences between the native and recombinant forms of GroES except for the eight additional N-terminal amino acids derived from the fusion partner in recombinant GroES. The recombinant fusion protein, still tagged with the maltose binding protein, was recognized by T cells isolated from TB-infected mice if mixed with culture filtrate before affinity purification on an amylose column. The maltose binding protein treated in the same manner as a control preparation was not recognized. Based on the data presented, we suggest that the association of biologically active molecules from culture filtrate with the chaperone GroES may be responsible for the observed T-cell recognition of the native preparation.

Human T cell responses to the ESAT-6 antigen from Mycobacterium tuberculosis.

Human T cell responses to ESAT-6 and eight synthetic overlapping peptides were investigated in tuberculosis (TB) patients and control subjects from regions of high and low endemicity for TB. ESAT-6 was recognized by 65% of all tuberculin purified protein derivative-responsive TB patients, whereas only 2 of 29 bacille Calmette-Guérin-vaccinated Danish healthy donors recognized this molecule. In Ethiopia, a high frequency (58%) of healthy contacts of TB patients recognized ESAT-6. All of the peptides were recognized by some donors, indicating that the molecule holds multiple epitopes. Danish and Ethiopian patients differed in the fine specificity of their peptide responses. Recognition of the C-terminal region (aa 72-95) was predominant in Danish patients, whereas recognition of aa 42-75 was predominant in Ethiopia. The relationship of these differences to the distribution of HLA types in the two populations is discussed. This study demonstrates that ESAT-6 is frequently recognized during early infection and holds potential as a component of a future TB-specific diagnostic reagent.