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Clinical and Diagnostic Laboratory Immunology, May 2001, p. 571-578, Vol. 8, No. 3
TB Research Group1 and
TB Diagnostic Section,3 Department of
Bacterial Diseases, Veterinary Laboratories Agency-Weybridge, New
Haw, Addlestone KT15 3NB, and Ministry of Agriculture,
Fisheries and Food, State Veterinary Service, Exeter EX5
1DY,2 United Kingdom
Received 24 July 2000/Returned for modification 6 December
2000/Accepted 9 February 2001
In Great Britain an independent scientific review for the
government has concluded that the development of a cattle vaccine against Mycobacterium bovis infection holds the best
long-term prospect for tuberculosis control in British herds. A
precondition for vaccination is the development of a complementary
diagnostic test to differentiate between vaccinated animals and those
infected with M. bovis so that testing and
slaughter-based control strategies can continue alongside vaccination.
To date bacillus Calmette-Guérin (BCG), an attenuated strain of
M. bovis, is the only available vaccine for the
prevention of tuberculosis. However, tests based on tuberculin purified
protein derivative cannot distinguish between M. bovis
infection and BCG vaccination. Therefore, specific antigens expressed
by M. bovis but absent from BCG constitute prime
candidates for differential diagnostic reagents. Recently, two such
antigens, ESAT-6 and CFP-10, have been reported to be promising
candidates as diagnostic reagents for the detection of M.
bovis infection in cattle. Here we report the identification of
promiscuous peptides of CFP-10 that were recognized by M.
bovis-infected cattle. Five of these peptides were formulated
into a peptide cocktail together with five peptides derived from
ESAT-6. Using this peptide cocktail in T-cell assays, M.
bovis-infected animals were detected, while BCG-vaccinated or
Mycobacterium avium-sensitized animals did not respond.
The sensitivity of the peptide cocktail as an antigen in a whole-blood
gamma interferon assay was determined using naturally infected field
reactor cattle, and the specificity was determined using blood from
BCG-vaccinated and noninfected, nonvaccinated animals. The sensitivity
of the assay in cattle with confirmed tuberculosis was found to be
77.9%, with a specificity of 100% in BCG-vaccinated or nonvaccinated
animals. This compares favorably with the specificity of tuberculin
when tested in noninfected or vaccinated animals. In summary, our
results demonstrate that this peptide cocktail can discriminate between
M. bovis infection and BCG vaccination with a high
degree of sensitivity and specificity.
Bovine tuberculosis (BTB) is caused
by Mycobacterium bovis. It is a zoonotic disease and was the
cause of approximately 6% of total human deaths due to BTB in the
1930s and 1940s and of more than 50% of all cervical lymphadenitis
cases in children (13, 23). The introduction of
pasteurization of milk in the 1930s dramatically reduced the
transmission from cattle to humans, and in 1995 only 1% of 3,200 isolates from patients with TB in Great Britain were identified as
M. bovis (23). However, human TB caused by
M. bovis is still a major health issue in many developing countries (12, 14, 15).
BTB has severe implications for animal welfare in both developed
and developing countries, since it causes reduced productivity and
premature death in cattle and since affected farms also suffer severe
economic losses. A compulsory eradication program based on the
slaughter of infected animals detected by the single intradermal comparative cervical tuberculin test (SICCT) began in Great
Britain in 1950, and by 1960 it had been implemented in all of Great
Britain. These measures resulted in a dramatic reduction of BTB in
Great Britain. In 1934 approximately 40% of all cattle were infected with M. bovis, whereas in 1996 the annual incidence of
confirmed herd breakdowns had been reduced to 0.41% in Great Britain
(reviewed in reference 29). However, despite continued
implementation of these control measures, the incidence of BTB in
cattle has been steadily rising since 1988, possibly due to a wildlife
reservoir of BTB. A cattle vaccine would reduce the risk of cattle
infection and hence result in lower tuberculin test frequencies and
significant cost savings. Recently, a panel of scientists was
commissioned by the British government to conduct an independent review
of this problem, and they concluded that the development of a cattle vaccine holds the best long-term prospect for BTB control in British herds (29). It was also recommended that a complementary
diagnostic test to differentiate between vaccinated animals and those
infected with M. bovis (differential diagnosis) should be
developed in parallel with the vaccine to ensure continuation of the
testing and slaughter-based control strategies (29). These
recommendations have been accepted and are now being implemented by the
British government (1).
Bacillus Calmette-Guérin (BCG), an attenuated strain of M. bovis, is presently the only available vaccine for the prevention of BTB. Encouraging results with BCG have been reported from
experiments in New Zealand, where a significant level of protection in
BCG-vaccinated cattle against experimental M. bovis
infection has been demonstrated recently (7, 8). However,
vaccination with BCG compromises tuberculin purified protein derivative
(PPD) specificity (see, e.g., references 4, 25, and
27). Thus, cattle BCG vaccination constitutes an
appropriate model to develop strategies for differential diagnosis
associated with vaccines based on attenuated M. bovis strains. Theoretically, diagnostic reagents which distinguish between
vaccinated and infected cattle could be developed using specific,
defined antigens that are present in virulent M. bovis but
absent from the vaccine strain. Genetic analysis of BCG has revealed
that the genes encoding the M. bovis antigens ESAT-6, CFP-10, and MPB64 have been deleted from the Pasteur strain of BCG
(5, 21, 22, 55). Phenotypic analysis of M. bovis and BCG Pasteur has also demonstrated that the M. bovis antigens MPB70 and MPB83 are highly expressed in M. bovis but expressed only at low levels in BCG Pasteur (26,
55). Using this information, it has recently been demonstrated
that protein cocktails composed of ESAT-6, MPB70, and MPB59
(6) or of ESAT-6, MPB64, and MPB83 (50,
51) can be used to distinguish between BCG-vaccinated and
M. bovis-infected cattle.
An alternative approach to using recombinant proteins is the
application of synthetic peptides derived from antigens such as those
described above. Synthetic peptides have the advantages of lower
production costs and easier standardization and quality control, and
they carry no risk of infection since they are fully chemically
synthesized. Encouragingly, we have recently provided proof of
principle that the development of peptide-based reagents is a feasible
approach by demonstrating that a pool of seven peptides derived from
ESAT-6, MPB70, MPB83, and MPB64 differentiates between infected and
BCG-vaccinated cattle. However, further improvement to the sensitivity
of this peptide-based reagent was required before the levels of
sensitivity would be acceptable for the implementation of testing
and slaughter-based strategies based on such reagents (50, 51).
The objective of the present study was to develop an improved
peptide-based diagnostic reagent. Since MPB70 and MPB83 have shown
promise as candidates for DNA-based subunit vaccination (9, 34,
52), we decided to concentrate our efforts on developing diagnostic reagents based on the antigens ESAT-6 and CFP-10. ESAT-6 is
a well-studied antigen found in short-term culture filtrates of
pathogenic mycobacteria of the M. tuberculosis complex. It has been shown to be able to discriminate between infected and BCG-vaccinated guinea pigs and humans (16, 28, 45, 47) and
also to discriminate between M. bovis-infected cattle and cattle sensitized with environmental mycobacteria (39).
CFP-10 has been also identified in the low-molecular-mass fraction of culture filtrate. The genes encoding CFP-10 and ESAT-6 are adjacent on
the genome and are transcribed together. Both encode small exported
proteins and share some degree of homology at the DNA level
(5). Both are therefore members of the so-called ESAT-6 family of small mycobacterial proteins (5, 11, 46). Like ESAT-6, CFP-10 is recognized by M. tuberculosis-infected
guinea pigs and humans but not by individuals vaccinated with BCG
(2, 45, 49). In addition, it is also recognized by
lymphocytes from cattle with BTB (49). In humans, the
combination of recombinant CFP-10 and ESAT-6 demonstrated a high
sensitivity in detecting TB patients, with a higher specificity than
PPD (49).
Peptide epitopes within the sequence of CFP-10 were identified using a
set of highly overlapping peptides that were tested with lymphocytes
from cattle experimentally or naturally infected with M. bovis. This approach identified five promiscuously recognized peptides that were formulated together with ESAT-6-derived peptides previously identified by us (50, 51) into a peptide
cocktail composed of 10 peptides. Our results demonstrated that this
diagnostic cocktail composed of synthetic peptides from ESAT-6 and
CFP-10 can be employed to differentiate M. bovis-infected
cattle from those vaccinated with BCG with a high degree of sensitivity
and specificity.
Cattle.
For BCG vaccination and experimental M. bovis infection experiments, ca. 6-month-old calves (Friesian or
Friesian crosses) were obtained from herds free of BTB and kept in the
Animal Services Unit. The following groups of cattle were used in this study.
(i) M. bovis infection.
Calves were infected
with an M. bovis field strain from Great Britain (AF
2122/97) by intratracheal instillation of 2 × 104 CFU as described previously (7, 8,
44). 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 postmortems
performed ca. 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. Data from a
total of 23 experimentally infected cattle are presented in this study.
(ii) BCG vaccination.
Calves were vaccinated with BCG
Pasteur by subcutaneous injection of 106 CFU into
the side of the neck (7, 8) followed 8 weeks later by a
booster injection using the same route and dose. Heparinized blood
samples were taken 4 to 8 weeks after the booster vaccination. Data
from 16 calves are presented in this study.
(iii) Field samples with confirmed BTB.
Heparinized blood
was obtained from 76 cattle of eight different breeds and cross-breeds,
from different parts of Great Britain, that had been designated
tuberculin test reactors following skin testing with the SICCT
(field reactors). The skin tests were performed as specified in
reference 17. Blood samples were taken 10 to 20 days after
the disclosing skin test, and all samples were transported by courier
to our laboratory within 24 h of sampling and processed immediately upon arrival. M. bovis infection was confirmed
by bacterial culture from pooled head lymph nodes and/or by
histopathology on visible lesions found at necroscopy.
(iv) Negative controls.
Heparinized blood from
SICCT-negative animals from herds free of BTB (30 animals) was also obtained.
Antigens and peptides. (i) Antigens.
Bovine (PPD-B) and
avian (PPD-A) tuberculins were obtained from the Tuberculin Production
Unit at the Veterinary Laboratories Agency-Weybridge and used in
culture at 10 µg/ml.
(ii) Peptides.
An overlapping set of 44 synthetic peptides
spanning the entire sequence of CFP-10 (14 residues long with a
12-residue overlap) was prepared by multi-rod peptide synthesis
(pin-synthesized peptides) (35) (for sequence information,
see reference 5) and used in mapping experiments at 25 µg/ml. The peptides were purchased from Chiron Mimotopes (Clayton,
Australia). ESAT-6 and CFP-10 peptides, when part of the peptide
cocktail, were synthesized by solid-phase peptide synthesis as
described earlier (54). The purity and sequence fidelity
of these peptides were confirmed by analytical reverse-phase
high-pressure liquid chromatography and electron spray mass
spectrometry, respectively. Peptides were used at 25 µg/ml when used
individually and at 10 µg/ml each when used as components of the
peptide cocktail (see Table 1 for
sequences of the peptides included in this cocktail).
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.3.571-578.2001
Use of Synthetic Peptides Derived from the
Antigens ESAT-6 and CFP-10 for Differential Diagnosis
of Bovine Tuberculosis in Cattle
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Composition of ESAT-6-CFP-10 peptide cocktail
Lymphocyte transformation assay.
Peripheral blood
mononuclear cells (PBMC) were isolated from heparinized blood by
Histopaque-1077 (Sigma) gradient centrifugation and cultured in RPMI
1640 supplemented with 5% Controlled Process Serum Replacement type 1 (Sigma Aldrich, Poole, United Kingdom), nonessential amino acids (Sigma
Aldrich), 5 × 10
5 M 2-mercaptoethanol,
100 U of penicillin per ml, and 100 µg of streptomycin sulfate per
ml. PBMC (2 × 105/well in 0.2-ml aliquots)
were cultured in triplicate for 6 days in flat-bottom 96-well
microtiter plates in the presence of antigen, radiolabeled during the
last 16 to 20 h of culture with 37 kBq of
[3H]thymidine (Amersham, Little Chalfont,
United Kingdom) per well, and harvested onto glass fiber filters, and
radioactivity was counted in a scintillation counter (TopCount;
Packard, Pangborne, United Kingdom). Positive responses are defined by
a stimulation index (counts per minute with antigen/counts per minute
without antigen) of 
3 together with a signal strength of 1,000 cpm
(24, 51, 53, 56).
IFN-
assays.
Whole-blood cultures were performed in
96-well plates in aliquots of 0.20 ml/well 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 gamma
interferon (IFN-) was determined using the BOVIGAM enzyme-linked
immunosorbent assay (ELISA) kit (CSL, Melbourne, Australia)
(57). Results obtained with individual peptides and
diagnostic cocktails were deemed positive when the ratios of optical
density at 450 nm (OD450) with antigens to
OD450 without antigens (IFN- stimulation
index) (31, 32) were 3.0. For comparative analysis of
PPD-B versus PPD-A responses, a positive result was defined as a value
for the OD450 with PPD-B minus the
OD450 with PPD-A of 0.1 and a value for the
OD450 with PPD-B minus the
OD450 without stimulation of 0.1.
IFN- ELISPOT assay.
Direct enzyme-linked immunospots
(ELISPOTs) were enumerated by modifying the protocol described
for indirect ELISPOTs by van Drunen Littel-van den Hurk et al.
(48). Briefly, ELISPOT plates (Immobilon-P polyvinylidene
difluoride membranes; Millipore, Molsheim, France) were coated
overnight at 4°C with the bovine IFN--specific monoclonal antibody
2.2.1. Unbound antibody was removed by washing, and the wells were
blocked with 10% fetal calf serum in AIM-V medium (Life Technologies,
Paisley, Scotland, United Kingdom). PBMC (2 × 105/well suspended in AIM-V-2% FCS) 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- followed by incubation with an alkaline
phosphatase-conjugated monoclonal antibody specific for rabbit
immunoglobulin G (Sigma Aldrich). Both the monoclonal antibody 2.2.1 and the rabbit anti-bovine IFN- serum were kindly supplied by D. Godson (VIDO, Saskatoon, Saskatchewan, Canada). The spots were
visualized with BCIP (5-bromo-4-chloro-3-indolylphosphate)-nitroblue tetrazolium substrate (Sigma Aldrich). Cutoff values for positive responses in Fig. 2 (>5 spot-forming cells [SFC]/200,000
PBMC) were calculated using the formula [mean SFC from cultures
without antigen + (3.2498 × standard error)], to
achieve a confidence interval of 99% (10, 36).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Identification of regions within CFP-10 recognized by bovine T
cells.
PBMC isolated from eight naturally infected, SICCT-positive
field reactor cattle were incubated in the presence of a set of 44 highly overlapping pin-synthesized peptides derived from the sequence of CFP-10 (14-mer peptides overlapping by 12 amino acid residues). Peptides inducing proliferative responses were identified, and thereby regions containing T-cell-stimulating determinants were
defined. The aim was to identify regions of the protein which were
recognized by at least 50% of the cattle tested to identify promiscuous epitopes. Responses to CFP-10-derived peptides were detected in all eight field reactors tested, and regions containing epitopes could be identified between amino acid residues 1 and 18, 13 and 28, 21 and 36, 27 and 44, 33 and 52, 41 and 62, 49 and 64, 55 and
76, 65 and 84, 75 and 92, and 85 and 100. Epitopes within the regions
defined by amino acids 1 to 18, 13 to 28, 55 to 76, 65 to 84, and 75 to
92 were recognized by lymphocytes from at least 50% of the animals
tested (Fig. 1).
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production in vitro (data not shown).
Encouragingly, none of these five peptides were recognized by T cells
isolated from BCG-vaccinated cattle, confirming the specificity of
these T-cell determinants (data not shown). The peptides covering
residues 59 to 84 and 65 to 84 were recognized by one and two animals, respectively; none of the tested animals recognized the peptide covering residues 21 to 44 (data not shown).
The combination of CFP-10 and ESAT-6 improves sensitivity.
Since we were encouraged by the results described above, the peptides
listed in Table 1 were selected and formulated into a peptide cocktail
for further evaluation together with five previously identified
peptides derived from the sequence of ESAT-6 (50).The amino acid sequences of these peptides are given in Table 1. We first
determined whether combining peptides from both antigens could improve
the sensitivity of detection of an immune response to M. bovis infection over that with the use of single antigens. The
IFN- responses of PBMC from 10 field reactors with confirmed BTB
against the five peptides from ESAT-6 or the five peptides from CFP-10
listed in Table 1, or against the combined ESAT-6-CFP-10 peptide
cocktail, were compared using the highly sensitive ELISPOT assay format
to detect IFN--secreting cells. As shown in Fig. 2, PBMC from 5 of 10 cattle produced
IFN- upon in vitro challenge with both ESAT-6- and CFP-10-derived
peptides, whereas T cells from one cow responded exclusively to the
ESAT-6-derived peptides and those from two cows responded exclusively
to the CFP-10-derived peptides. T cells from 8 of 10 animals responded
to the combined ESAT-6-CFP-10 peptide cocktail (Fig. 2).
Interestingly, when heparinized blood from the same animals was
stimulated with the same peptide preparations and the supernatants were
tested with the conventional BOVIGAM IFN- ELISA, only four and three
animals, respectively, responded to CFP-10 or ESAT-6 peptide
stimulation alone, whereas all eight identified by ELISPOT were also
identified by ELISA (data not shown). This suggests that the
combination of the two peptide pools increased the ELISA signal
strength, resulting in sensitivity comparable to that of the IFN-
ELISPOT.
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ELISPOT responses in five animals whereas seven
animals responded to the combined ESAT-6-CFP-10 peptide cocktail (data
not shown). Taking both experiments together, a total of 10 of 18 animals responded to ESAT-6 alone, whereas 15 of 18 responded to the
combined ESAT-6-CFP-10 peptide cocktail. Thus, we have provided proof
of principle for a strategy of combining several antigens to improve
the sensitivity of detection of the immune response to M. bovis infection in cattle over that with the use of single antigens.
The ESAT-6-CFP-10 peptide cocktail can distinguish between
infected and BCG-vaccinated cattle.
We next investigated
whether the ESAT-6-CFP-10 peptide cocktails described above
would be able to discriminate between M. bovis-infected
animals and either BCG-vaccinated or nonvaccinated control cattle. The
peptide cocktail was therefore tested in a first set of experiments
with PBMC from cattle infected experimentally with M. bovis
(10 animals), BCG-vaccinated cattle (9 animals), or noninfected
negative controls (5 animals). The results of this experiment are shown
in Fig. 3. Proliferative immune responses were induced by the peptide cocktail in all experimentally infected calves tested in this experiment. In contrast, none of the
BCG-vaccinated or negative control cattle responded to the peptide
cocktail. Significantly, T cells from all BCG-vaccinated animals
proliferated strongly to both avian and bovine tuberculin. This
confirmed the lack of specificity of tuberculin in the face of BCG
vaccination. In addition, four of the noninfected control animals
responded to avian tuberculin, indicating that they had been exposed to environmental mycobacteria (Fig. 3). Thus, we have demonstrated that a
cocktail composed of peptides from antigens not expressed in BCG can
differentiate between M. bovis-infected and BCG-vaccinated animals, whereas tuberculin cannot.
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), 9 BCG-vaccinated cattle (
), and
5 nonvaccinated controls (
) were incubated with either the
protein cocktail, PPD-B, or PPD-A. Results are expressed as mean
proliferative responses, and the black lines indicate response means.
responses in whole-blood cultures induced by the
peptide cocktail. Heparinized blood samples were obtained from
tuberculin-positive field reactors with confirmed BTB (68 animals),
from BCG-vaccinated cattle (16 animals), and from cattle from herds
free of BTB (25 animals) and were stimulated within 24 h of blood
sampling with tuberculins or the peptide cocktail. IFN- in these
supernatants was measured with the commercially available BOVIGAM
IFN- ELISA kit. In this assay the ESAT-6-CFP-10 cocktail detected
77.9% of the confirmed reactors (Table
2). This compared well with the sensitivity achieved by the comparison of PPD-B and PPD-A responses, which detected 88.2% of confirmed reactors (Table 2).
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assay compared to tuberculin was confirmed because it did not induce
IFN- responses in blood from BCG-vaccinated cattle or cattle from
TB-free herds tested (100% specificity). In contrast, 12 of 16 of the
BCG-vaccinated cattle and 2 of 25 of the TB-free cattle gave rise to
positive responses when the production of IFN- upon stimulation with
PPD-B was compared to that induced by PPD-A (25 and 92% specificity,
respectively) (Table 2). The differences in ODs between PPD-B and PPD-A
responses of BCG-vaccinated animals were smaller than those for cattle
with confirmed BTB (medians of 0.258 for BCG-vaccinated cattle and
0.735 for cattle with BTB) (Table 2). This was due to the induction of
high PPD-A responses in the BCG-vaccinated animals concomitant with the
development of PPD-B responses rather than to significantly lower PPD-B
responses per se in BCG-vaccinated animals (median ODs with PPD-B in
animals with BTB and in BCG-vaccinated cattle, 1.119 and 0.804, respectively).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The development of diagnostic reagents capable of differentiating between infection and vaccination is necessary for the development of a vaccine against TB in cattle so that existing test and slaughter control strategies can continue alongside vaccination. BCG compromises the specificity of the tuberculin skin test and is therefore not used as a control measure for BTB in cattle. Vaccination of cattle with BCG therefore provides a challenging model system of an attenuated live mycobacterial vaccine to develop strategies and reagents for differential diagnosis. The application of recombinant ESAT-6 as a diagnostic antigen to differentiate between infected and BCG-vaccinated humans has been widely tested, and responder frequencies of between 60 and 95% have been reported (see, e.g., references 28, 38, 43, and 49). Comparable data on the application of recombinant CFP-10 are available from several reports, which indicated responder frequencies of between ca. 50 and 90% of the TB patients (2, 45, 49). Studies in cattle indicated that about the same percentage of M. bovis-infected cattle as human TB patients recognized ESAT-6 (6, 39, 42, 49, 51) and that it could be applied to the differential diagnosis of infected and vaccinated cattle (6, 51). It has also been reported recently that CFP-10 is recognized by a large proportion of infected cattle (49).
Only a subset of infected animals will respond to a single antigen (18, 19, 42, 49, 51), which means that it is unrealistic to expect that any single antigen will be able to detect the proportion of infected animals required of a successful diagnostic reagent. Therefore, cocktails of antigens will be required to achieve broad population coverage. Such cocktails, composed of, e.g., ESAT-6, MPB64, MPB83, MPB70, and CFP-10 in different combinations, have been described recently by us and other groups as useful in the detection of TB in cattle or humans (6, 49-51). An additional advantage to this approach is that higher signal strengths are often obtained with reagent pools than with their individual components (51). Furthermore, antigen cocktails reduce the possibility of selecting escape mutants, i.e., strains of bacilli lacking the diagnostic antigen. Such natural mutants have been described. For example, strains of M. tuberculosis which do not express the 19-kDa lipoprotein have been identified (30).
The approach described in the present report exploits the use of synthetic peptides derived from the sequences of these proteins as diagnostic reagents to detect TB in cattle. For a long time this approach has been deemed impractical. It has been argued that due to the genetic diversity of the major histocompatibility complex (MHC) system in mammalian species, a large pool of epitopes would be required to achieve sufficient population coverage to be a practical alternative to recombinant protein antigens. However, it has now been widely accepted that a significant proportion of MHC class II-restricted peptides are recognized by T cells in the context of multiple MHC haplotypes (promiscuous epitopes). This has been demonstrated in the recognition of mycobacterial antigens by murine, human, and bovine CD4+ T cells (33, 40, 41). In the present study, cattle of eight different breeds and cross-breeds from herds in different parts of Great Britain were used to define and subsequently validate the peptide epitopes, and ca. 78% of Great Britain field reactors tested responded to the peptide cocktail. It is therefore likely that we have defined truly promiscuous peptides within the antigens mapped in this study. However, we have not formally demonstrated the association of these peptides with multiple bovine MHC (BoLA) class II haplotypes, since this was beyond the applied objectives of this study. We also cannot exclude the existence of more such epitopes within the sequences of CFP-10 and ESAT-6. In addition, for obvious reasons we have tested only cattle from the Great Britain national herd, and we appreciate that further studies will be necessary to determine if those peptides are recognized by cattle from other national herds, particularly in Africa or Asia, where different breeds of cattle are more common.
Our strategy of using a large number of highly overlapping peptides prepared by pin synthesis to screen for regions of the sequence containing T-cell epitopes has been successful. A large number of peptides can be produced by this method at relatively low costs. However, the amount of each peptide produced is limited, as is the ability to perform quality assurance procedures. It is therefore important to confirm the results of such epitope scans by preparing peptide candidates employing conventional synthesis. Our chosen strategy was vindicated since we were able to confirm that T-cell epitopes were present within all of the regions identified by the pin-synthesized overlapping peptide set, with the exception of one peptide (residues 21 to 44), where we attempted to cover two adjacent sequence regions (residues 21 to 36 and 27 to 44) with a single peptide. This might have led to interference of MHC binding by the presence of secondary structures within this peptide linking at least two epitope cores and preventing MHC binding by steric hindrance (20, 37). Alternatively, the MHC haplotype(s) associated with residues 21 to 44 might not have been represented in the animals tested. The latter hypothesis is less likely, however, since two animals that had originally responded to peptides within the sequence regions 21 to 36 and 27 to 44 did not respond to the peptide containing residues 21 to 44.
The ESAT-6-CFP-10 peptide cocktail described in this study detected a
similar proportion of infected cattle (77.9%) as that described for
the respective recombinant proteins in the study by van Pinxteren and
coworkers (49), who detected 75% of infected cattle using
ESAT-6 and CFP-10 individually. This suggests that mixtures of
synthetic peptides can have potencies in cattle equivalent to those of
whole recombinant proteins. Supporting this notion, Arend and coworkers
(3) have recently used M. tuberculosis-specific human T-cell lines of different HLA-DR types as well as ex vivo PBMC
assays measuring IFN- production. They could demonstrate that
mixtures of synthetic overlapping peptides spanning the complete amino
acid sequence of each antigen have potencies equivalent to those of
whole ESAT-6 and CFP-10 for sensitive and specific detection of human
infection with M. tuberculosis. The ESAT-6-CFP-10 peptide
cocktail also constitutes an improvement on the peptide pool composed
of peptides from ESAT-6, MPB83, MPB64, and MPB70 that we have described
previously (50): 88.3% of all SICCT-positive reactors
that were identified as positive for BTB by comparison of the in vitro
IFN- production with PPD-B and PPD-A were also identified using the
ESAT-6-CFP-10 cocktail, which compares favorably to a 69.9%
congruence level between peptides and tuberculin responses observed
with the previously described peptide pool.
In conclusion, this study demonstrates that diagnostic cocktails based on synthetic peptides derived from antigens expressed in M. bovis but not in BCG can distinguish between vaccinated and infected cattle in blood-based assays. Such antigens could form the basis of next-generation diagnostic reagents whose sensitivity might reach or even surpass that of tuberculin PPD and which could be employed alongside vaccination.
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ACKNOWLEDGMENTS |
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This work performed was funded by the Ministry of Agriculture, Fisheries and Food, Great Britain.
We express our appreciation to the staff of the Animal Services Unit at the Veterinary Laboratories Agency for their dedication to animal welfare. We acknowledge the valuable assistance of Veterinary Field Service staff of the Ministry of Agriculture, Fisheries and Food in the collection of blood samples from field reactor cattle for use in this study.
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FOOTNOTES |
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* Corresponding author. Mailing address: TB Research Group, Department of Bacterial Diseases, Veterinary Laboratories Agency-Weybridge, New Haw, Addlestone KT15 3NB, United Kingdom. Phone: 44 1932 357 884. Fax: 44 1932 357 684. E-mail: mvordermeier.vla{at}gtnet.gov.uk.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Clinical and Diagnostic Laboratory Immunology, May 2001, p. 571-578, Vol. 8, No. 3
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.3.571-578.2001
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