Data from several laboratories have demonstrated the vaccine potential of antigens secreted by M. tuberculosis
, and high-resolution separation techniques have identified molecules in the low-molecular-mass region of culture filtrate (masses of <15 kDa) to be of particular relevance (2
). Antigen discovery efforts from several laboratories have in recent years resulted in the identification of a number of these low-molecular-mass antigens (Mtb8.4 [1
]; Mtb12 [20
]; GroES, CFP10 [4
]; TB10.4 [17
]; Mtb9.9 [1
]; alpha chrystallin, CFP6 [5
]; TB7.3 [17
]; and TB9.8, TB9.4, TB11.0, and TB11.2 [14
]). In analyses of these molecules, it is striking that a significant number of them were found to be located in one mycobacterial protein-gene family-the esat-6
family. This family consists of at least 14 low-mass proteins with some homology to ESAT-6 and a defined genomic organization. With the proteins described here, a total of 10 proteins (ESAT-6 [15
], CFP10 [17
], MTB9.9A-E [1
], TB10.4 [17
], TB10.3, and TB12.9) from this family have now been published as strongly recognized T-cell antigens.
In the present study we describe the identification and immunological evaluation of the TB10.4 family, consisting of TB10.3, TB10.4, and TB12.9, as a new subfamily of the esat-6
family. We used synthetic peptides to identify and compare the epitopes recognized in these proteins and found that especially TB10.4 contained a number of strongly recognized T-cell epitopes distributed throughout the protein sequence. The major epitopes recognized at the PBMC level were located in the N-terminal part of the molecule, but this region was less frequently recognized by T-cell lines, which may suggest that the skewing of responses occurs during in vitro propagation of a T-cell line. TB10.3 and TB12.9 were in general less antigenic than TB10.4 but contained T-cell epitopes, several of which were unique to these proteins. Sharing of the major T-cell epitopes by these proteins would be expected based on their highly homologous amino acid sequence, but the present study, together with recent observations from Alderson et al. (1
), clearly demonstrate that the highly homologous proteins from the esat-6
family (Mtb9.9 and TB10.4 subfamilies) contain predominantly protein-specific epitopes and that shared epitopes are an exception. This finding underlines the highly specific binding of peptides to HLA-class II molecules and demonstrates that a difference of just one amino acid at critical positions may have dramatic consequences for T-cell recognition.
Expression of TB10.4 has been verified by N-terminal sequencing of the corresponding protein spot in a two-dimensional gel of a M. tuberculosis
short-term culture filtrate fraction enriched in monoclonal antibody PV-2 (a monoclonal antibody that recognizes TB10.4) reactive protein (17
). Although not formally demonstrated, the recognition of a number of unique epitopes on TB10.3 and TB12.9 also strongly suggests that these proteins are expressed by the bacteria during TB infection.
Pathogenic organisms will often use sequence polymorphism of highly immunogenic proteins to escape the host's immune defenses (9
). This is not the case for M. tuberculosis
as recently demonstrated by Musser et al. (11
) and Streevatsan et al. (19
), who found negligible sequence diversity in a large number of M. tuberculosis
genes encoding important antigens recognized by the host immune system. This finding is in agreement with the recent finding of a lack of sequence diversity in the TB10.4 sequence originating from 13 clinical isolates of M. tuberculosis
obtained from different geographical locations (unpublished observations).
An interesting possibility is that M. tuberculosis
may compensate for this lack of sequence polymorphism by having duplicated the genes encoding major T-cell antigens, leading to several copies (homologues) of proteins that can substitute each other functionally but which differ in their immunodominant epitopes. Tightly controlled expression of these homologues may result in antigen variation and immune evasion. This hypothesis may be relevant for the whole esat-6
family, since the genomic organization of these proteins suggests that these proteins have similar functions. Apart from the esat-6
family, the immunodominant molecules within the antigen 85 complex in which the three molecules presumably have the same function in living bacteria could be examples of this phenomenon as well. In this regard, the TIGR database defines the three tb10.4
family genes plus esat-6
as one paralogous gene family (http://www.tigr.org
), meaning that these genes possibly have been duplicated in M. tuberculosis
during evolution. These duplication events may even involve whole clusters of genes, including regions surrounding the esat-6
family members such as PE and PPE genes as suggested by Cole et al. (7
The lack of sequence homology to proteins and genes from other organisms illustrates that esat-6
family members have Mycobacterium
-specific functions, and the need for several copies of the same gene may indicate that the genes encode function(s) of crucial importance for bacterial survival in the host. Using differential display for comparison of gene expression in M. tuberculosis
H37Rv and the avirulent H37Ra, Rindi et al. (13
) showed that TB10.4 was produced in the virulent strain but not in the avirulent strain, suggesting that this protein or gene may be involved in functions important for M. tuberculosis
virulence. The presence of this gene in BCG (which is deleted of the esat-6-cfp10
operon) illustrates that attenuation can be obtained through the inactivation of various combinations of these different genes.
The availability of the M. tuberculosis genome and the current efforts to sequence a large number of additional mycobacterial genomes has set the stage for postgenomic approaches to the identification of novel antigens. The identification and characterization of protein-gene families is a very important outcome of these initiatives. If the gene families contain components that can substitute for each other functionally and represent mechanisms for immune evasion, the finding of markedly different epitope recognition of the individual components, as observed in the present study, may have important consequences for vaccine design. The extreme consequence may be that a successful TB vaccine will have to promote an immune response not only to a selected target antigen such as TB10.4 but also to epitopes characteristic for the rest of the closely related gene family. We are currently conducting experiments to address this important question.