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Infection and Immunity, November 2004, p. 6255-6261, Vol. 72, No. 11
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.11.6255-6261.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Recognition of Mycobacterial Antigens Delivered by Genetically Detoxified Bordetella pertussis Adenylate Cyclase by T Cells from Cattle with Bovine Tuberculosis

H. Martin Vordermeier,1* Marcela Simsova,2 Katalin A. Wilkinson,3 Robert J. Wilkinson,3 R. Glyn Hewinson,1 Peter Sebo,2 and Claude Leclerc4

TB Research Group, Veterinary Laboratories Agency, Weybridge, New Haw, Addlestone,1 Wellcome Centre for Research in Clinical Tropical Medicine, Division of Medicine, Faculty of Medicine, Imperial College London, Wright Fleming Institute, Paddington, London, United Kingdom,3 Cell and Molecular Biology Division, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic,2 Unite de Biologie des Regulations Immunitaires, INSERM E 352, Institut Pasteur, Paris, France4

Received 3 June 2004/ Returned for modification 15 July 2004/ Accepted 3 August 2004


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ABSTRACT
 
The exponential increase in the incidence of tuberculosis in cattle over the last two decades in the British national herd constitutes a significant economic problem. Therefore, research efforts are under way to develop cattle tuberculosis vaccines and specific diagnostic reagents to allow the distinction of vaccinated from infected animals. Mycobacterial antigens like ESAT-6 and CFP10 allow this distinction. This study investigates whether fusion protein of ESAT-6 or CFP10 with genetically detoxified Bordetella pertussis adenylate cyclase (CyaA) are recognized by Mycobacterium bovis-infected cattle more effectively than conventional recombinant proteins are, thus enhancing sensitivity or reducing the amount of antigens required. By measuring the frequencies of gamma interferon (IFN-{gamma})-producing cells, we were able to show that the presentation of CFP10 as a CyaA fusion protein enhanced the molecular efficiency of its recognition 20-fold, while the recognition of ESAT-6 was not improved by CyaA delivery. Furthermore, in the whole-blood IFN-{gamma} test currently used in the field, the delivery of CFP10 and ESAT-6 by fusion to CyaA increased the amount of IFN-{gamma} produced and hence the proportion of infected animals responding to CFP10. The improved T-cell recognition of CyaA336/CFP10 was found to be dependent upon interaction with CD11b. In addition, presentation of CyaA336/CFP10 to CD4+ T cells was chloroquine sensitive, and CFP10 delivery by CyaA resulted in its accelerated presentation to T cells. In conclusion, the use of CyaA fusion proteins with ESAT-6 and CFP10 has the potential to improve the sensitivity of immunodiagnostic tests detecting bovine tuberculosis in cattle.


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INTRODUCTION
 
Bovine tuberculosis (BTB), caused by Mycobacterium bovis, is a zoonotic disease and was the cause of approximately 6% of total human deaths due to BTB in the 1930s and 1940s (5, 6). The introduction of pasteurization of milk in developed countries in the 1930s dramatically reduced the transmission from cattle to humans, although BTB is still a major human health problem in developing countries. Compulsory BTB eradication programs were introduced in many countries based on the slaughter of infected cattle detected by the single intradermal comparative tuberculin skin test. The implementation of this control strategy resulted in the dramatic reduction of BTB in Great Britain. However, possibly due to a wildlife reservoir, the incidence of TB in cattle caused by M. bovis has exponentially increased over the last two decades in the British national herd. This increase constitutes a significant economic and potential public health problem (17). To control this zoonotic disease, better and more specific diagnostic reagents, as well as effective vaccines for cattle, are urgently needed.

The diagnosis of BTB in cattle is at present almost exclusively based on the use of tuberculin purified protein derivative (PPD) in skin tests. In addition, a blood-based test measuring tuberculin-induced production of gamma interferon (IFN-{gamma}) is now also in limited field use (34). The specificity of these tuberculin-based tests is limited due to the undefined and cross-reactive nature of PPD. Furthermore, the specificity of tuberculin-based reagents is also compromised following vaccination with the human TB vaccine M. bovis BCG (16). Diagnostic reagents allowing the differential diagnosis of M. bovis-infected and -vaccinated animals are therefore a precondition for the development of novel TB vaccines in cattle (17). The specificity of diagnostic reagents can be improved by using antigens that are highly expressed by M. bovis yet whose genes have been deleted from the genome of BCG. The antigens ESAT-6 and CFP10, which are encoded in the RD1 region of M. bovis-Mycobacterium tuberculosis—which is deleted in all strains of BCG (2, 19)—have shown particular promise as such improved diagnostic reagents when used as recombinant proteins or synthetic peptides in the IFN-{gamma} test (3, 4, 28, 31). Such antigens not only allowed the differential diagnosis of infected and BCG-vaccinated cattle but also improved the specificity of the test per se compared to tuberculin PPD in the absence of vaccination (22, 23, 31).

An attractive recent approach to effectively delivering proteins to the immune system is through nonreplicating protein vectors such as bacterial toxins (20). The Bordetella pertussis adenylate cyclase (CyaA) is such a vector system that has shown promise in mouse models (21, 25, 26). Indeed, we have recently demonstrated that CD11b/CD18, a member of the ß2-integrin family, is a specific cell receptor for this toxin (14). CD11b/CD18, also known as complement type 3 receptor or MAC-1, is expressed on macrophages, neutrophils, dendritic cells, and NK cells. Thus, the cellular specificity of CyaA allows its selective targeting to CD11c+ CD11bhigh dendritic cells in vivo (13). Peptide and small proteins can be inserted into this protein and expressed as fusion proteins (11, 12, 21, 25, 27). CyaA facilitates direct translocation of these inserted antigens across the plasma membrane of target cells (15). Importantly, it has been shown that CyaA vaccination can not only induce major histocompatibility complex (MHC) class I-restricted CD8+-T-cell responses (8, 10, 12, 21, 25, 27) but also result in the presentation of CD4+-T-cell epitopes restricted by MHC class II (8, 18). Thus, CyaA fusion proteins that contain mycobacterial antigens could constitute not only candidates for subunit vaccines but also diagnostic antigens, particularly if they will be recognized in cattle more effectively than conventional recombinant proteins, thereby enhancing sensitivity, or are recognized at lower protein concentrations. The latter consideration could have major cost benefits because this could significantly reduce the amount of antigen that would have to be produced to implement testing. Consequently, the experiments conducted in this study have been performed to determine whether CyaA-based recombinant proteins fused with either ESAT-6 or CFP10 are recognized by bovine T cells more efficiently than the corresponding nonfusion proteins and to establish the mechanisms of this improved recognition.


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MATERIALS AND METHODS
 
Antigens. Bovine (PPD-B) and avian (PPD-A) tuberculin were obtained from the Tuberculin Production Unit at the Veterinary Laboratories Agency-Weybridge (VLA Weybridge) and used in culture at 10 µg/ml. Recombinant ESAT-6 was supplied by A. Whelan (VLA Weybridge), and recombinant CFP10 was obtained from M. Singh, Gesellschaft für BioTechnologische Forschung, Braunschweig, Germany.

Escherichia coli XL1-Blue (Stratagene) was used throughout this work for recombinant DNA construction and for expression of antigens inserted into CyaA. Bacteria transformed with appropriate plasmids derived from pT7CACT1 were grown at 37°C in Luria-Bertani medium supplemented with 150 µg of ampicillin/ml. The open reading frames of M. tuberculosis H37Rv genes esat-6 and cfp-10 were amplified by PCR from the pYUB412 cosmid clone of the RD1 region with the following primers: Esat6-I, 5'-GATGTGTACACATGACAGAGCAGCAGTGG-3'; Esat6-II, 5'-GATGTGTACACTGAGCGAACATCCCAGTGACG-3'; CFP-10-I, 5'-CATGTGTACACATGGCAGAGATGAAGACC-3'; CFP-10-II, 5'-CATGTGTACACTGAAGCCCATTTGCGAGGA-3'.

The PCR product was digested by bsrG1 at the sites incorporated into the PCR primers, and the purified fragments encoding the antigens were inserted in-frame between codons 335 and 336 of CyaA on the pT7CACT-336-BsrGI expression vector (21). The exact sequence of the cloned inserts was verified by DNA sequencing. The control detoxified mock CyaA and the recombinant CyaA proteins carrying the ESAT-6 and CFP10 antigens, respectively, were produced in E. coli, purified from inclusion bodies in 8 M urea-50 mM Tris-Cl (pH 8)-2 mM EDTA, and characterized as previously described. The resulting proteins were free of any detectable CyaA enzymatic activity.

M. bovis-infected cattle and BCG vaccination. Calves were infected with an M. bovis field strain from Great Britain (AF 2122/97) by intratracheal instillation of between 5 x 103 and 5 x 104 CFU (28). Infection was confirmed by the presence of tuberculous lesions in the lungs and lymph nodes of these animals as well as by the culture of M. bovis from tissue collected at the postmortems performed 20 weeks after the infection. Heparinized blood samples were obtained at least 6 weeks after infection when strong and sustained in vitro tuberculin responses were observed. Another group of 11 calves was vaccinated with BCG (Pasteur) by subcutaneous injection of 106 CFU. Blood was taken 3 weeks postvaccination when peak responses were observed.

IFN-{gamma} ELISPOT assay. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by Histopaque 1077 (Sigma) gradient centrifugation and cultured in tissue culture medium (RPMI 1640; Life Technologies, Paisley, Scotland, United Kingdom) supplemented with 5% controlled process serum replacement type 1 (Sigma Aldrich, Poole, United Kingdom), nonessential amino acids (Sigma Aldrich), 5 x 10–5 M 2-mercaptoethanol, 100 U of penicillin/ml, and 100 µg of streptomycin sulfate/ml. Direct ELISPOTs were enumerated, as described previously (28). Briefly, ELISPOT plates (Immunobilon-P polyvinylidene difluoride membranes; Millipore, Molsheim, France) were coated overnight at 4°C with the bovine IFN-{gamma}-specific monoclonal antibody (MAb) 2.2.1. Unbound antibody was removed by washing, and the wells were blocked with 10% fetal calf serum in RPMI 1640 medium. PBMC (2 x 105 to 5 x 105/well) suspended in tissue culture medium (RPMI 1640 supplemented with 5% controlled process serum replacement type 1) were then added and cultured at 37°C and 5% CO2 in a humidified incubator for 24 h. Spots were developed with rabbit serum specific for IFN-{gamma} followed by incubation with an alkaline phosphatase-conjugated MAb specific for rabbit immunoglobulin G (IgG; Sigma Aldrich). The MAb 2.2.1 was kindly supplied by D. Godson (Veterinary Infectious Disease Organization, Saskatoon, Saskatchewan, Canada). The spots were visualized with 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium substrate (Sigma Aldrich).

The involvement of CD11b was determined by addition (50 µl of ELISPOT plate/well) of the mouse MAbs CC94 and ILA15 (both IgG1; kindly provided by C. Howard, Institute for Animal Health, Compton, United Kingdom) to 2 x 105 PBMC dispensed in 100 µl. After 30 min of preincubation at 37°C, serial dilutions of CyaA336/CFP10 were added and the cultures were incubated for 24 h as described above, after which time ELISPOT analysis was performed.

CD4+- and CD8+-T-cell subpopulations were depleted by magnetic negative selection with the anti-bovine CD4- or CD8-specific MAbs CC30 and CC58 (C. Howard, Institute for Animal Health) in conjunction with the MACS system (goat anti-mouse IgG-coated beads, LS separation columns; Miltenyi Biotec Ltd., Bergisch-Gladbach, Germany) as described previously (30).

IFN-{gamma} assay. Whole-blood cultures were performed in 96-well plates in 0.2-ml/well aliquots by mixing 0.1 ml of heparinized blood with an equal volume of antigen-containing solution. Supernatants were harvested after 24 h of culture, and the levels of IFN-{gamma} present were determined using the Bovigam enzyme immunoassay (EIA) kit (CSL, Melbourne, Australia) (29, 34). The data are expressed as units of optical density at 450 nm ({Delta}OD450) (OD450 x 1,000). Background levels obtained after stimulation with control CyaA were subtracted from values obtained after stimulation with CyaA336/ESAT-6 and CyaA336/CFP10. The cutoff for positivity was determined by assessing responses of BCG-vaccinated animals (cutoff, 125 {Delta}OD450 units).

CFP10-specific bovine CD4+-T-cell line. A long-term T-cell line was established from PBMC collected from a calf experimentally infected with M. bovis (32). Briefly, PBMC (2 x 106/well of 24-well plates in a 1-ml aliquot) were cultured in the presence of recombinant CFP10 (2 µg/ml) for 7 days. Viable cells were then isolated by Histopaque 1077 (Sigma) gradient centrifugation, and cells were rested for 1 week in the presence of mitomycin C (Sigma)-treated freshly prepared autologous PBMC. Cells (2 x 105/well) were then restimulated in the presence of CFP10 protein (2 µg/ml) and mitomycin C-treated PBMC (106/ml) as a source of antigen-presenting cells (APC) for 5 days, after which cells were collected and rested as described above. After three such stimulation and rest cycles, the specificity of this T-cell line was established by incubating 2 x 104 T cells with 105 mitomycin C-treated PBMC in 0.2-ml portions in the presence of antigen. IFN-{gamma} production was determined in culture supernatants harvested 2 days later as described above by using the Bovigam EIA kit. The line was composed of CD4+ T cells because magnetic depletion of CD4+ cells reduced antigen-specific IFN-{gamma} production by 99.7%, whereas depletion of CD8+ T cells or WC1+ {gamma}{delta} T cells did not have any effects (data not shown).

Inhibition of T-cell recognition by chloroquine. PBMC were depleted of CD4+ T cells by using the bovine CD4-specific MAb CC30 (kindly donated by C. Howard) in conjunction with the Miltenyi magnetic sorting system (goat anti-mouse IgG-coated beads, LS separation columns) and mitomycin C treatment. These APC were plated at 105/well in 96-well microtiter plates and incubated with CFP10 or CyaA336/CFP10 (at 3.7 nM) for 0, 60, 120, and 240 min before the addition of chloroquine (Sigma) to a final concentration of 5 mM (33) and 2 x 104 cells of the CFP10-specific T-cell line described above per well. Supernatants were harvested after 2 days of culture, and their IFN-{gamma} contents were determined by IFN-{gamma} EIA as described above.

Statistical analysis. Statistical analysis was performed using Instat v3.0a (GraphPad, San Diego, Calif.) on an iMac personal computer. Data were analyzed using the one- or two-tailed Wilcoxon signed rank matched pairs test. See the figure legends for further details.


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RESULTS
 
IFN-{gamma} responses of experimentally infected cattle. PBMC were prepared from experimentally infected cattle and incubated with serial dilutions of antigens (recombinant ESAT-6, CFP10, CyaA336/ESAT-6, CyaA336/CFP10, and CyaA control), and the antigen-induced IFN-{gamma} responses were determined after 24 h of culture by ELISPOT assay. To illustrate how the data are subsequently expressed, representative results for CFP10 and CyaA336/CFP10 tested in one calf are given in Fig. 1. Responses to CyaA alone in this cow were ≤14 spot-forming cells (SFC)/2 x 105 cells at all concentrations tested (data not shown), and these values were subtracted from the CyaA336/CFP10 responses. CyaA336/CFP10 both induced a higher peak response than did recombinant CFP10 (as shown by comparison of values indicated by horizontal lines a and b in Fig. 1) and was recognized more effectively as indicated by the vertical lines d and e, representing the concentrations required for half-maximum (50% of peak responses) responses induced with the recombinant protein (line c).



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  		FIG. 1. Dose-response relationship of in vitro IFN-{gamma} production after stimulation with CFP10 (squares) and CyaA336/CFP10 (triangles). The readout system was the IFN-{gamma} ELISPOT assay. SFC numbers from cultures with medium alone were subtracted from all values. In addition, the numbers of spots after incubation with CyaA alone were subtracted from the SFC induced by CyaA336/CFP10 stimulation. Tests were performed in duplicate with 2 x 105 PBMC/well isolated from an M. bovis-infected calf. Horizontal lines indicate the maximum SFC numbers (peak values) induced after stimulation with CyaA336/CFP10 (a) and CFP10 (b); line c indicates the half-maximum SFC induced after CFP10 stimulation (50% maximum values). Vertical lines indicate the CyaA336/CFP10 (d) and CFP10 (e) concentrations required to induce 50% of CFP10-induced peak responses (50% maximum concentration).

A further six experimentally M. bovis-infected calves were then tested. Responses to control CyaA in these calves were below 10% of those determined with the fusion proteins and were subtracted from the responses of CyaA336/CFP10 and CyaA/ESAT-6. As demonstrated by the comparison of peak responses induced by CFP10 and by CyaA336/CFP10 and the reduced concentration needed for 50% maximal responses, the CyaA336/CFP10 fusion protein was again superior to its nonfusion counterpart (Fig. 2). CyaA336/CFP10 peak responses were about twice as high as those observed with CFP10 (median responses: CyaA336/CFP10, 157 SFC; CFP10, 75 SFC; P = 0.03), and CyaA336/CFP10 was recognized about 20 times more efficiently than was CFP10 (50% maximum concentrations: CyaA336/CFP10, 0.3 nM; CFP10, 6.25 nM; P = 0. 017). The IFN-{gamma} responses induced by ESAT-6 and by the CyaA336/ESAT-6 fusion proteins were not significantly different (Fig. 2). Recombinant ESAT-6 was about 70 times more efficiently recognized than CFP10 was (median of 50% maximum concentrations: 0.09 with ESAT-6 compared to 6.25 with CFP10).



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  		FIG. 2. Comparison of abilities of CyaA fusion proteins and recombinant proteins to stimulate in vitro IFN-{gamma} production by PBMC from M. bovis-infected cattle. Left panel: 50% maximum concentrations determined as illustrated in Fig. 1. Right panel: peak values determined as illustrated in Fig. 1. The readout system was the IFN-{gamma} ELISPOT assay. SFC numbers from cultures with medium alone were subtracted from all values. In addition, the numbers of spots after incubation with CyaA alone were subtracted from the SFC induced by CyaA336/CFP10 or CyaA336/ESAT-6 stimulation. Tests were performed in duplicate with 2 x 105 PBMC/well. *, P < 0.05 (two-tailed Wilcoxon signed rank matched pairs test).

Performance of CyaA336/ESAT-6 and CyaA336/CFP10 in whole-blood IFN-{gamma} tests (Bovigam assay). A bovine IFN-{gamma} test is applied as a diagnostic assay in the field in the format of a whole-blood assay (Bovigam test). In this format heparinized blood is incubated in the presence of antigens for 24 h and the amount of antigen-induced IFN-{gamma} in plasma supernatants is determined by enzyme-linked immunosorbent assay (ELISA). To determine the performance of the CyaA fusion proteins with ESAT-6 and CFP10 in the Bovigam assay, blood was obtained from 11 BCG-vaccinated animals and eight calves experimentally infected with M. bovis. These blood samples were stimulated with bovine tuberculin PPD (PPD-B), as well as ESAT-6, CFP10, CyaA336/ESAT-6, and CyaA336/CFP10. Strong IFN-{gamma} responses were demonstrated in 11 of 11 vaccinated calves after stimulation with PPD-B. Stimulation with ESAT-6 and CFP10 resulted in no or only marginal IFN-{gamma} production (Table 1), nor did stimulation with CyaA336/ESAT-6 or CyaA336/CFP10 result in IFN-{gamma} production (Table 1). After assessment of the data by receiver operating characteristic analysis (including the data from the infected animals discussed below), a cutoff for positivity for this assay was set at 125 {Delta}OD450 units. Applying this cutoff, all 11 animals tested were classified positive after PPD-B stimulation, while none of the BCG-vaccinated animals tested positive after stimulation with ESAT-6, CFP10 at a 20 nM antigen concentration, and CyaA336/ESAT-6 or with CyaA336/CFP10 when used at 4 nM (Table 1). Only 1 of 11 animals tested positive after stimulation with CyaA336/ESAT-6 and CyaA336/CFP10 at 20 nM (Table 1). Taken together, these results confirmed the specific nature of these antigens.


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  		TABLE 1. IFN-{gamma} responses in BCG-vaccinated calvesa

The results of the ELISA conducted in experimentally infected calves are shown in Fig. 3. As shown above for PBMC responses measured by ELISPOT, significantly stronger IFN-{gamma} responses were observed with CyaA336/CFP10 at both test concentrations than with recombinant CFP10 protein (P = 0.0078 at both concentrations). These increased responses were particularly evident when the blood was stimulated at 4 nM antigen concentrations. While the responses for CyaA336/ESAT-6 and ESAT-6 were not significantly different at 20 nM, significantly elevated responses were observed after stimulation with CyaA336/ESAT-6 at 4 nM (P = 0.015).



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  		FIG. 3. Performance of CyaA fusion proteins and recombinant ESAT-6 and CFP10 in the whole-blood Bovigam IFN-{gamma} assay. Heparinized blood from eight M. bovis-infected calves was incubated with the antigens at 4 and 20 nM test concentrations. IFN-{gamma} in plasma culture supernatants was determined by ELISA. The results are expressed as {Delta}OD450 units (OD450 x 1,000) with {Delta}OD450 values from cultures with medium alone subtracted from all values. In addition, the {Delta}OD450 values after incubation with CyaA alone were subtracted from wells with CyaA336/CFP10 or CyaA336/ESAT-6 stimulation. The horizontal line indicates cutoff for positivity ({Delta}125 OD450 units). Cultures were performed in duplicate in 96-well flat-bottomed plates. *, P < 0.05; **, P < 0.01 (two-tailed Wilcoxon signed rank matched pairs test).

When the diagnostic outcome was evaluated using the commonly applied cutoff of 125 {Delta}OD450 units for the Bovigam assay, six of eight tested animals were deemed positive for BTB by using ESAT-6 and CyaA336/ESAT-6 applied at both test concentrations (Fig. 3). In contrast, the use of CyaA336/CFP10 improved the sensitivity of CFP10, because, respectively, seven of eight and six of eight animals tested positive at 20 and 4 nM test concentrations with CyaA336/CFP10, whereas six of eight and three of eight, respectively, were classified as positive after stimulation with recombinant CFP10 at corresponding test concentrations (Fig. 3).

One of the eight animals was skin test negative and presented without tuberculous lesions at postmortem examinations carried out several months after this experiment was performed; in addition M. bovis could not be detected by culture from tissue samples taken at the postmortem examination. Taken together, this suggests that the experimental infection in this animal was contained and did not result in disease. Consistent with this diagnosis, no IFN-{gamma} was induced in the blood of this calf after stimulation with PPD-B, CyaA336/ESAT-6, CyaA336/CFP10, ESAT-6, or CFP10, thus highlighting the specificity of these reagents (Fig. 3).

Mechanisms of the enhanced responses. To determine whether the presentation of CyaA336/CFP10 to bovine T cells is mediated via a CD11b-dependent mechanism, PBMC from an infected calf were stimulated with CyaA336/CFP10 in the presence of two MAbs of the same isotype (IgG1) specific for bovine CD11b. One of these MAbs (ILA15) interfered with the interaction of CyaA336/CFP10 with CD11b, as the number of SFC was reduced significantly, whereas the nonblocking isotype control MAb (CC94) did not modify this response (Fig. 4, P < 0.02 for each concentration tested; one-tailed Wilcoxon matched pairs test). These results therefore provide evidence that interaction between CyaA and CD11b on APC is required in order to increase the CFP10-specific T-cell responses.



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  		FIG. 4. Involvement of CD11b in the recognition of CyaA336/CFP10. Cultures were performed in the presence of a blocking CD11b-specific IgG1 MAb (ILA15) and an isotype control (CC94). The readout system was the IFN-{gamma} ELISPOT assay. SFC numbers from cultures with medium alone were subtracted from all values. Tests were performed in duplicate with 2 x 105 PBMC/well isolated from one infected calf. Responses are significantly different (P < 0.02) for each concentration tested except lowest concentration (one-tailed Wilcoxon signed rank matched pairs test).

To determine whether CD11b-mediated uptake of CyaA fusion proteins also resulted in accelerated processing and presentation of the antigen, we investigated the effects of inhibiting the MHC class II processing pathway. Chloroquine was added between 0 and 240 min after the coculture of CyaA336/CFP10 or CFP10 and APC to stop further antigen processing. Then cells from a CFP10-specific CD4+-T-cell line were added and IFN-{gamma} production was determined. As expected, the recognition of both CFP10 and CyaA336/CFP10 by CD4+ T cells was chloroquine sensitive, as addition of chloroquine at the same time as the proteins completely inhibited IFN-{gamma} production (Fig. 5). However, CyaA336/CFP10 was presented at an accelerated rate compared to that of CFP10 because a significant IFN-{gamma} response to CyaA/CFP10 was observed when processing was inhibited after 120 to 240 min whereas processing of CFP10 was not sufficient at these time points to allow IFN-{gamma} production (Fig. 5).



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  		FIG. 5. Accelerated presentation kinetics of CyaA336/CFP10 to CD4+ T cells. CyaA336/CFP10 and CFP10 (both at 3.7 nM) were incubated with CD4-depleted PBMC as a source of APC. At the indicated time points, chloroquine was added to stop further processing. APC were then cultured for 48 h in the presence of 2 x 104 cells from a CFP10-specific CD4+-T-cell line added. IFN-{gamma} in culture supernatants was determined by ELISA. Triangles, CFP10; circles, CyaA336/CFP10; open symbols, no addition of chloroquine; closed symbols, chloroquine was added at indicated time points. Box, addition of chloroquine and antigens simultaneously. Results are expressed as means of triplicate determinations ± standard errors.


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DISCUSSION
 
Based on the experiments described above, we conclude that fusion of ESAT-6 and CFP10 with CyaA improved antigen recognition. These results extend previous observations made in the murine model (18) where 100-times-higher molar efficiency has been described for the CyaA fusion protein. This constitutes a novel approach to the generation of potential in vitro diagnostic reagents for the detection of BTB in cattle. In particular the observation that they can be used in a whole-blood format is encouraging, as this is the format of choice when contemplating in vitro diagnosis of BTB. Our results demonstrate that, compared to the recombinant protein, presentation in vitro of CFP10 in the form of a CyaA fusion protein resulted in a significant improvement of IFN-{gamma} responses. A recombinant CyaA carrying a human melanoma epitope was also shown to be 100-fold more efficient than the synthetic peptide in inducing the presentation of the epitope to specific human T cells by dendritic cells (7). In addition, a recent study testing human TB patients with CyaA336/ESAT-6 and CyaA336/CFP10 also showed 10-times-higher molar efficiencies of recognition compared to those of purified recombinant CFP10 and ESAT-6 (K. A. Wilkinson et al., unpublished data). It has to be highlighted that CyaA336/CFP10 when used in the whole-blood test format not only increased the strength of IFN-{gamma} responses but also resulted in increased test sensitivity by detecting more of the truly infected animals than recombinant CFP10 alone did. Again this result is in agreement with the results obtained with human TB patients (K. A. Wilkinson et al., unpublished). Interestingly, and in contrast to the results with human TB patients, CyaA336/ESAT-6 did not increase the molar efficiency of its recognition in cattle compared to the recombinant ESAT-6 protein. Recombinant ESAT-6 was about 70 times more efficiently recognized than CFP10 was, and this difference in the efficiency of recognition between those two proteins might explain why an additional benefit of presenting ESAT-6 as a CyaA fusion protein could not be realized in these experiments. Interestingly, preliminary results in our laboratory suggest that ESAT-6 does not need to be lysosomally processed to induce a response in vitro, as we observed strong responses (>50% of responses observed without addition of chloroquine) when we prevented processing by adding chloroquine prior to antigen addition (H. M. Vordermeier and A. O. Whelan, unpublished data). This observation, if confirmed by further studies, could therefore also account for the failure of CyaA336/ESAT-6 to improve in vitro recognition. Generation of MHC class II-restricted epitopes by extracellular processing has been described for epitopes of, e.g., hepatitis {delta} antigen (1).

A number of previous reports have conclusively shown that, in mice, CyaA fusion proteins facilitate the delivery of CD8+-T-cell epitopes directly into the cytosol of dendritic cells both in vitro and in vivo (13, 14). CyaA fusion proteins are therefore excellent vehicles to trigger class I-restricted CD8+ T cells in vitro and in vivo (7, 11, 12, 15, 21, 25, 27). In addition, murine studies have shown that MHC class II-restricted CD4+-T-cell epitopes could also be efficiently delivered by CyaA fusion proteins (8, 18). This was confirmed in the present study where we detected predominantly responses of CD4+ T cells (Fig. 5 and data not shown). We also demonstrated that this enhancement of T-cell responses by CyaA fusion proteins depends upon their interaction with CD11b in agreement with the results obtained in mice (9). Furthermore, we demonstrate that the processing and presentation of CyaA fusion proteins to CD4+ T cells via the MHC class II pathway are chloroquine sensitive and that CyaA fusion proteins are presented at accelerated kinetics compared to the nonfusion proteins (Fig. 5). Taken together, the CD11b-mediated uptake and improved presentation kinetics could account for the superior recognition of CyaA336/CFP10 compared to recombinant CFP10.

We also determined whether CyaA336/CFP10 was recognized by bovine CD8+ T cells. This was analyzed by depleting either subpopulation with magnetic beads. However, the cattle used in this study were at early stages of BTB, and it has been reported that such animals display only weak or undetectable CD8+-T-cell responses (24; H. M. Vordermeier, unpublished observation). Consequently, we observed significant PPD-B- and CFP10-specific CD8+-T-cell responses only in one of four cows tested. Nevertheless, the results obtained indicated that CyaA336/CFP10 induced higher in vitro CD8+-T-cell responses than did the recombinant protein (data not shown). These results are also in line with results obtained using the same CyaA constructs to test human TB patients (K. A. Wilkinson et al., unpublished).

One important aspect to determine when considering the practical diagnostic application of the CyaA fusion proteins described in the present study is whether background responses to the CyaA backbone protein can be found in cattle. We determined this by inclusion of the CyaA toxoid as vector control in our assays and by stratifying the data obtained with the fusion proteins by subtracting IFN-{gamma} responses obtained with this control. Interestingly, only one cow tested displayed significant responses to CyaA. Therefore, we conclude that background responses to CyaA are no obstacle to the practical application of CyaA fusion proteins in cattle, as they can be easily controlled and distinguished from ESAT-6- or CFP10-specific responses by the inclusion of CyaA alongside the fusion proteins.

In conclusion, the present study has demonstrated that the fusion of mycobacterial proteins to CyaA improved their recognition in vitro by increasing both signal strength and molecular efficacy of recognition. In addition, when applied to the whole-blood IFN-{gamma} test format used routinely to diagnose BTB in cattle, the CyaA336/CFP10 fusion protein increased test sensitivity compared to conventional CFP10, although precise cutoffs have to be defined to confirm these results in larger groups of naturally infected animals. Increased sensitivity and reduction in the amount of antigens needed to perform diagnostic tests are important attributes for immunodiagnostic reagents and prioritize these reagents for further evaluation to establish their performance in a larger field trial.


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ACKNOWLEDGMENTS
 
This study was funded by the Department for Environment, Food and Rural Affairs, United Kingdom, with additional support from the Wellcome Trust. P.S. was supported in part by grant no. S5020311 from the Academy of Sciences of the Czech Republic.

We express our appreciation to the staff of the Animal Services Unit at VLA, in particular Derek Clifford, for their dedication to the welfare of test animals. We also thank C. Howard, Institute for Animal Health, Compton, United Kingdom, for the supply of MAbs, and M. Singh, Lionex Ltd., Braunschweig, Germany, for the supply of recombinant CFP10.


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FOOTNOTES
 
* Corresponding author. Mailing address: VLA Weybridge, TB Research Group, Woodham Lane, New Haw, Addlestone KT15 3NB, United Kingdom. Phone: 44 1932 357 684. Fax: 44 1932 357 584. E-mail: mvordermeier.vla{at}gtnet.gov.uk. Back

Editor: J. T. Barbieri


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Infection and Immunity, November 2004, p. 6255-6261, Vol. 72, No. 11
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.11.6255-6261.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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