In this study, we investigated the potential of a tuberculosis subunit vaccine based on fusion proteins of the immunodominant antigens ESAT-6 and antigen 85B. When the fusion proteins were administered to mice in the adjuvant combination dimethyl dioctadecylammonium bromide-monophosphoryl lipid A, a strong dosedependent immune response was induced to both single components as well as to the fusion proteins. The immune response induced was accompanied by high levels of protective immunity and reached the level of Mycobacterium bovis BCG-induced protection over a broad dose range. The vaccine induced efficient immunological memory, which remained stable 30 weeks postvaccination.Tuberculosis (TB) is the leading infectious disease in the developing world, and the World Health Organization estimates 80 million new cases of tuberculosis in this decade (8). The current vaccine against Mycobacterium tuberculosis, M. bovis Bacillus Calmette-Gúerin (BCG), has been extensively evaluated and demonstrated variable protective efficacies ranging from 0 to 85% in different field trials (13). An improved second-generation vaccine is therefore urgently needed. Alternative strategies in TB vaccine development such as subunit vaccines (2, 16, 23), genetic immunization (17, 27), and attenuated strains of M. tuberculosis (14) are currently being explored in many laboratories. Due to the complexity of the host immune response against tuberculosis and the genetic restriction imposed by major histocompatibility complex molecules, it has become clear that an effective subunit vaccine containing multiple epitopes may be required to ensure a broad coverage of a genetically heterogeneous population. We and others have previously demonstrated that vaccines based on a mixture of culture filtrate antigens can induce levels of protection similar to BCG in mice (2, 16, 23), but so far only a few experimental vaccines based on a single antigen have proved successful in animal models (6,17,27).The strategy being explored in our laboratory is the molecular engineering of recombinant fusion proteins. Compared to mixtures of proteins extracted from cultures or cell lysates, the fusion protein approach offers at least two substantial advantages: (i) it is a more defined product and (ii) it reduces the number of recombinant expression and purification steps. The purpose of our study was to evaluate the potential of a subunit vaccine based on a fusion protein between two immunodominant antigens, Ag85B and the 6-kDa early secretory antigenic target (ESAT-6). In this study, we show that this approach is very promising and promotes an efficient immune response which is highly protective against TB in the mouse model. MATERIALS AND METHODSAnimals. Specific-pathogen-free female C57BL/6J (H-2 b ) and B6CBAF1 (H-2 b,k ) mice were purchased from Bomholtgaard (Ry, Denmark). All mice used were 6-to 12 weeks of age and were housed in cages contained within a BL-3 laminar flow safety enclosure. Animals were allowed free access to water and standard mouse chow. Bact...

A fusion protein of antigen 85B (Ag85B) and ESAT-6 administered in cationic lipid vesicles conferred a highly significant level of protection against Mycobacterium tuberculosis in the guinea pig aerosol model of infection. The protection was manifested as delayed clinical illness and prolonged survival. Neither Ag85B nor ESAT-6 (independently or as a cocktail) induced significant protection in this model.

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.The Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has undergone a number of genetic and biochemical changes during its propagation over the last 70 years (18). Although the current vaccine is effective for protecting against childhood forms of tuberculosis (TB), it has failed to prevent adult pulmonary manifestations of the disease in countries where TB is highly endemic (8,17). The question of whether the contemporary BCG strains have been attenuated to impotence has therefore been posed (5).In attempts to understand the limitations of BCG, workers have exploited modern molecular techniques to dissect the molecular differences between BCG, M. bovis, and Mycobacterium tuberculosis. Subtraction hybridization revealed three DNA segments (RD1, RD2, and RD3) that have been lost from BCG strains (15). More recently, DNA microarrays and bacterial artificial chromosome arrays have provided information about an additional 13 regions (representing 129 open reading frames [ORFs]) of the genome of M. tuberculosis that are lacking in various BCG strains (6,12). In addition to the importance of these findings for our understanding of the genetic makeup of the current vaccines, the antigens encoded by these ORFs have attracted the attention of researchers in the TB field because they could feasibly be used to both supplement the BCG vaccine or allow the development of specific diagnostic reagents. Several lines of evidence suggest that the proteins encoded by one such DNA segment, the RD1 region, might be of particular importance since this ...

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Improved diagnostic reagents are needed for the detection of Mycobacterium tuberculosis infections, and the development of a serodiagnostic test would complement presently available diagnostic methods. The aim of the present study was to identify novel serological targets for use for the future serodiagnosis of tuberculosis (TB). We cloned and expressed 35 M. tuberculosis proteins as recombinant proteins in Escherichia coli and analyzed their serodiagnostic potentials. By a two-step selection process, four superior seroantigens, TB9.7, TB15.3, TB16.3, and TB51, were identified, none of which has been described before. The four novel antigens were tested with panels of sera from smear-positive and smear-negative TB patients from areas both where TB is endemic and where TB is not endemic, with recognition frequencies ranging from 31 to 93% and with a specificity of at least 97%. The single most potent antigen was TB16.3, which had a sensitivity of 48 to 55% with samples from Danish resident TB patients and a sensitivity of 88 to 98% with samples from African TB patients. Importantly, the TB16.3 and the TB9.7 antigens were recognized by more than 85% of the samples from TB patients coinfected with human immunodeficiency virus, a patient group for which it is in general difficult to detect M. tuberculosis-specific antibodies.Approximately one-third of the world's population is infected with Mycobacterium tuberculosis, and 7 million to 8 million new cases of tuberculosis (TB) occur each year (49). The majority of TB cases occur in developing countries with limited resources, and today, the diagnosis of TB largely depends upon clinical examination and radiographic findings, confirmed by sputum smear microscopy and bacterial culture. Culture is not useful as a first-line means of diagnosis due to the long cultivation period required for M. tuberculosis (6 to 8 weeks); therefore, confirmation of the diagnosis relies on sputum smear examinations. However, smear microscopy has a sensitivity of only 50 to 60%, and the detection rate in children and patients coinfected with human immunodeficiency virus (HIV) is even lower (for a review, see reference 41).To improve the diagnosis of TB, more rapid diagnostic techniques have been investigated in recent years. These methods include the detection of M. tuberculosis components in clinical specimens by PCR and immune reactions, based on the cellmediated immune (CMI) response or on humoral immune responses. The in vitro CMI-based tests allow the early detection of latently infected individuals and are of tremendous value for contact tracing and screening of high-risk groups in a setting of low endemicity (10, 16). However, this method is not suitable as an alternative to culture and microscopy in developing countries, as large proportions of the populations in these countries are likely to be latently infected with M. tuberculosis (24).A serological test is an attractive diagnostic method because it is rapid, easy to perform, and robust and can easily be implemented under the cond...

ESAT-6 (the 6 kDa early secreted antigenic target) protein species in short-term culture filtrate of Mycobacterium tuberculosis were separated in a 4-5 narrow range pI gradient two-dimensional gel electrophoresis (2-DE). Eight ESAT-6 protein species were analyzed in detail by peptide mass fingerprinting matrix-assisted laser desorption/ionization-mass spectrometry as well as by electrospray ionization-tandem mass spectrometry. An N-terminal Thr acetylation was identified in four species and a C-terminal truncation was identified in two species. In 2-DE blot overlay assays, the recombinant 10 kDa culture filtrate protein (CFP10) discriminated N-terminal acetylated and nonacetylated ESAT-6 by differential interaction, whereas removal of the C-terminal 11 residues of ESAT-6 had no effects thereon. This example shows that the access to the protein species level can be a prerequisite to understand regulation of protein-protein interaction.

Differential delayed-type hypersensitivity skin testing with tuberculin purified protein derivatives from Mycobacterium bovis and M. avium is the standard for diagnosing bovine tuberculosis. However, improved tests based on defined, specific antigens are urgently needed. In the present study, a combination of bioinformatics, molecular biology, and bovine models of infection were used to screen mycobacterial proteins for their potential as diagnostic reagents which could be used in a whole-blood assay for diagnosis of tuberculosis. Initial screening of 28 proteins selected in silico and expressed as recombinants in Escherichia coli indicated that CFP-10, ESAT-6, TB27.4, TB16.2, TB15.8, and TB10.4 induced strong gamma interferon responses in experimentally infected cattle. A more thorough investigation over time in two groups of animals infected with a high (10 6 CFU) and a low (10 4 CFU) dose of M. bovis revealed that, for both groups, the strength of the in vitro response to individual antigens varied greatly over time. However, combining the results for ESAT-6, CFP-10, and TB27.4, possibly supplemented with TB10.4, gave sensitivities at different infection stages close to those obtained with M. bovis purified protein derivative. Importantly, while responsiveness to ESAT-6 and CFP-10 correlated strongly for individual samples, the same was not the case for ESAT-6 and TB27.4 responsiveness. The results suggest that combinations of specific antigens such as these have great potential in development of optimized diagnostic systems for bovine tuberculosis.

In the present study, we demonstrate that, in analogy with the genes encoding ESAT-6 and CFP-10, the genes rv0287 and rv0288 from the ESAT-6 gene family are cotranscribed. Using Western-Western blotting and protein-print overlay methodologies, we demonstrate that ESAT-6 and CFP-10, as well as the protein pair Rv0288/Rv0287, interact pairwise in a highly specific way. Most notably, the ESAT-6 proteins interact directly with Rv3873, a possible cell envelope component of the ESAT-6 secretion pathway.The 6-kDa early secretory antigenic target (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10) from Mycobacterium tuberculosis are two dominant targets for T cells in the early phases of infection (1,25,27). Furthermore, ESAT-6 has recently been demonstrated to induce protective immunity when administered as either a subunit (5) or a DNA (12) vaccine. The genes encoding ESAT-6 and CFP-10 (esx and lhp, respectively) lie next to each other in an operon-like structure (4). A dual knockout of ESAT-6 and CFP-10 in M. bovis results in decreased virulence of the pathogen (29), indicating that the two molecules may play important roles in immunopathogenesis and virulence. Recent genomic and genetic studies have revealed that lhp and esx and their neighboring genes constitute a gene cluster (esx cluster 1), which encodes a cellular function that is fundamental for the virulence of M. tuberculosis (15,19,28).Both ESAT-6 and CFP-10 are low-molecular-mass proteins that belong to a large protein family, the ESAT-6 family, which has 23 members in M. tuberculosis. When M. tuberculosis is grown in broth culture, these proteins are released into the surroundings (2, 4, 23) through a Sec-independent pathway, since none of the proteins contain common signal peptides. The genes encoding ESAT-6 family proteins are arranged in tandem pairs at 11 loci in M. tuberculosis H37Rv and are often preceded by a pe-ppe gene pair. At five loci, the esx-like gene pairs are part of larger gene clusters that have genetic contexts similar to that of the esx cluster 1. These clusters characteristically include a core region, consisting of a pair of esx-like genes next to a pe-ppe gene pair, flanked by open reading frames (ORFs) encoding potential membrane proteins, serine proteinases, and putative ABC transporters. It has been suggested that these gene clusters (the esx clusters) encode novel transporter systems that are responsible for the secretion of ESAT-6-like proteins (9,18,28). Experimental evidence that supports this theory is now available (20).Two of the esx clusters are conserved in M. leprae (9); one is the esx cluster 1, while the other is the esx cluster 3 encoding ESAT-6 family members Rv0287 and Rv0288. As it has been suggested that M. leprae may contain the minimum gene set required by a pathogenic mycobacterium (7), the function represented by the esx cluster 3 may thus also be essential for the pathogenesis of M. tuberculosis. In fact, although large numbers of genetic deletions have been identified in clinical isolates of M. tuberc...