In this study, we tested four dominant proteins found in PPD for their potential to induce a DTH response in M. tuberculosis
-infected or BCG-vaccinated guinea pigs. A baseline DTH response was established for two of these proteins, DnaK and GroEL2. Products in PPD that interacted with these two chaperones were identified by pull-down assays and mass spectrometry, produced as recombinant antigens, and tested alone or in combination with DnaK and GroEL2 for the capacity to induce a DTH response. Three cocktails, each containing three proteins, were as potent as PPD in the guinea pig model of tuberculosis. The primary molecular mechanisms responsible for inducing a DTH response were indistinguishable when differences between these cocktails and PPD were evaluated. Thus, the biological potency of PPD can be reproduced using a simplified, three-protein cocktail mixture of DnaK/GroEL2/Rv0009, DnaK/GroEL2/Rv0569, or DnaK/GroEL2/Rv0685. These well-defined cocktails can be used to further define the molecular mechanisms of the DTH response in tuberculosis, including the kinetics of the response and the role of sensitization. PPD contains hundreds of antigens that cumulatively induce the DTH response (3
; Cho et al., unpublished). Prior to this study, it was unknown which, or which combination, of these products, were responsible for the specificity or sensitivity of the DTH response. This is further complicated by the abundance of denatured, aggregated, and lysed proteinaceous products in PPD, complicating analysis of the PPD proteome, such that only two publications describe the PPD proteome in detail (3
; Cho et al., unpublished). Both the work by McFadden and our own identified the chaperones DnaK, GroEL2, and GroES as dominant products in PPD. In this study, these three chaperones and an additional chaperone, HspX, were tested using the guinea pig model of M. tuberculosis
infection. DTH responses similar to that induced by PPD were observed when DnaK and GroEL2 were used separately or in combination in M. tuberculosis
-infected or BCG-vaccinated guinea pigs. HspX and GroES, on the other hand, were negative or gave a poor response, whether injected separately or as a cocktail, in the guinea pig model of tuberculosis. Based on the relative abundance of DnaK and GroEL2 in PPD, we hypothesize that these two proteins significantly contribute to both the potency and lack of specificity of PPD in the DTH response.
Because both DnaK and GroEL2 are chaperone proteins, it was proposed that other proteins in PPD may be interacting with DnaK and GroEL2, and addition of these may enhance and engender a more specific DTH response than the use of these two proteins alone. To test this hypothesis, a pull-down experiment was performed to identify these proteins in PPD. Six novel proteins in PPD were identified by this method (Rv0009, Rv0475, Rv0569, Rv0685, Rv2626c, Rv2632c). None of these proteins induced a DTH response when tested individually. However, the addition of some of these proteins to DnaK and GroEL2 elicited a potent DTH response. Specifically, the protein cocktails DnaK/GroEL2/Rv0009 and DnaK/GroEL2/Rv0685 induced stronger DTH responses in H37Rv-infected and BCG-vaccinated guinea pigs than did DnaK/GroEL2 only, while the addition of other proteins to the DnaK/ GroEL2 mixture resulted in either an equally active or a weakened DTH response. This illustrates that protein-protein interactions either enhance or abrogate the DTH response. In addition, it is possible that the proteins contributing to a weakened DTH response may be inducing an alternative type of immune response. An anti-inflammatory or regulatory T-cell-type response may be occurring, leading to an overall weakening of the DTH-mediated immune response. Combined, these factors explain, in part, the variability seen in DTH responses, further demonstrating the challenges faced when defining the DTH response in M. tuberculosis infection or after BCG vaccination. The specific cellular attributes responsible for the DTH response in this model of tuberculosis are currently being studied using these defined protein cocktails. Discovery of these essential components during infection with M. tuberculosis or protection by BCG vaccination may lead to the development of rapid, inexpensive second-generation skin test antigens for tuberculosis.
Many protein candidates have been described as tuberculosis-specific skin test antigens over the past 2 decades (5
); however, none have reached an optimal diagnostic sensitivity. In this study, the protein mixtures described elicited a response that was equal to or greater than the response to PPD and was indistinguishable from PPD at the molecular level. While these cocktails did not demonstrate significantly different responses between infected and vaccinated animals, they do provide a tool to define the DTH response during the course of infection. Once defined, additional protein formulations that induce a strong and tuberculosis-specific DTH response can be rapidly tested.
To elucidate the mechanism of the DTH response in M. tuberculosis
-infected guinea pigs, histological examination and cellular analyses were performed at PPD or protein formulation injection sites on the skin of infected animals. The analysis of the flow cytometry data demonstrated that almost the same number of activated CD4+
T cells are involved in the DTH responses. Previous studies identified more CD4+
T cells than CD8+
T cells in DTH sites (14
). There are two probable reasons for the discrepancy. One is the differences between the human experience and the guinea pig model of tuberculosis. The other is that, in this study, activated CD4+
T-cell populations were measured directly at the site of the DTH response. To the best of our knowledge, this study is the first in which flow cytometry was used to look at DTH responses in the guinea pig model and may more accurately reflect the essential mechanisms of a productive DTH response.
DTH is classically defined as the recruitment of T cells to be activated by antigen-presenting cells, mainly Langerhans cells in skin, to produce cytokines that mediate local inflammation. Although the role of CD8+
T cells was previously poorly recognized, there is now considerable evidence that during the process of DTH, the antigen is processed and presented to both CD4+
T cells (2
). It is well known that phagocytosed antigens enter the exogenous pathway and are processed for presentation on major histocompatibility complex (MHC) class II molecules to CD4+
T cells, while cytoplasmic antigens are processed by the endogenous pathway for presentation on MHC class I molecules to CD8+
T cells. However, exogenous antigen can also enter the endogenous pathway to be presented to CD8+
T cells (18
). Our data demonstrate the activation of CD8+
T cells in DTH responses using PPD and different formulations, indicating that after proteins are digested in vivo
, the specific peptides are processed and presented on both MHC class I and II molecules. The expression of cytokines during the DTH response was determined by measuring specific mRNA expression levels at the sites of the DTH response in infected guinea pigs. Our data demonstrated that IFN-γ and TNF-α are actively expressed at the DTH site, and the same immune response pattern is induced by defined protein cocktails and PPD. Further, the T-cell response appears to be biased toward a Th1 T-cell response, based on the increases in the Th1 cytokines IFN-γ and TNF-α and the absence of IL-10 at the DTH sites. Injection of PPD into the footpads of H37Rv-infected mice is also characterized by high expression of Th1 cytokines like IL-2 and IFN-γ and the absence of Th2 cytokines such as IL-4 detected by RT-PCR (26
). Similarly, the expression of IFN-γ, the major cytokine produced by Th1 cells at sites of DTH responses, has been confirmed by others (4
) and is consistent with our results. Real-time RT-PCR can thus be used for validation of candidate PPD alternatives.
In summary, in our study, DnaK/GroEL2/Rv0009 and DnaK/GroEL2/Rv0685 were found to induce stronger responses than single protein or DnaK/GroEL2 protein cocktail injections and DTH responses indistinguishable from that elicited by PPD in the guinea pig model of tuberculosis. This is the first time that mimicking the PPD response has been shown at the molecular level with defined protein cocktails. The use of defined formulations will not only help provide a more thorough understanding of the DTH response but also provide a platform for standardized, defined second-generation skin test reagents for the diagnosis of M. tuberculosis infection and possibly novel skin test reagents for the evaluation of vaccine efficacy.