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Infection and Immunity, June 2002, p. 3026-3032, Vol. 70, No. 6
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.6.3026-3032.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Correlation of ESAT-6-Specific Gamma Interferon Production with Pathology in Cattle following Mycobacterium bovis BCG Vaccination against Experimental Bovine Tuberculosis

H. Martin Vordermeier,* Mark A. Chambers, Paul J. Cockle, Adam O. Whelan, Jennifer Simmons, and R. Glyn Hewinson

Veterinary Laboratories Agency Weybridge, TB Research Group, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom

Received 27 November 2001/ Returned for modification 8 February 2002/ Accepted 15 March 2002


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ABSTRACT
 
Vaccine development and the understanding of the pathology of bovine tuberculosis in cattle would be greatly facilitated by the definition of immunological correlates of protection and/or pathology. To address these questions, cattle were vaccinated with Mycobacterium bovis bacillus Calmette-Guérin (BCG) and were then challenged with virulent M. bovis. Applying a semiquantitative pathology-scoring system, we were able to demonstrate that BCG vaccination imparted significant protection by reducing the disease severity on average by 75%. Analysis of cellular immune responses following M. bovis challenge demonstrated that proliferative T-cell and gamma interferon (IFN-{gamma}) responses towards the M. bovis-specific antigen ESAT-6, whose gene is absent from BCG, were generally low in vaccinated animals but were high in all nonvaccinated calves. Importantly, the amount of ESAT-6-specific IFN-{gamma} measured by enzyme-linked immunosorbent assay after M. bovis challenge, but not the frequency of responding cells, correlated positively with the degree of pathology found 18 weeks after infection. Diagnostic reagents based on antigens not present in BCG, like ESAT-6 and CFP-10, were still able to distinguish BCG-vaccinated, diseased animals from BCG-vaccinated animals without signs of disease. In summary, our results suggest that the determination of ESAT-6-specific IFN-{gamma}, while not a direct correlate of protection, constitutes nevertheless a useful prognostic immunological marker predicting both vaccine efficacy and disease severity.


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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 (13, 26). Although the introduction of pasteurization of milk in developed countries in the 1930s dramatically reduced the transmission from cattle to humans, compulsory 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. 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 (32). 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 (32). The introduction of a vaccine for cattle has the potential to reduce their risk of infection and hence result in lower tuberculin test frequencies and significant cost savings. Recently, an independent scientific review panel concluded that the development of a vaccine for cattle holds the best long-term prospect for BTB control in British herds (32). Moreover, a bovine vaccine would be particularly useful in developing countries that cannot afford to implement costly test and slaughter control strategies.

M. bovis bacillus Calmette-Guérin (BCG) is an attenuated strain of M. bovis and is presently the only available vaccine against BTB. Since its introduction as a vaccine against tuberculosis in humans in the early 1920s and 1930s, a large number of BCG vaccination experiments and trials have been conducted with cattle in a number of countries ranging from the United Kingdom and the United States to Malawi and New Zealand. Early results in the 1930s were encouraging, and significant degrees of protection were reported (24, 27). However, the reported protective efficacies of BCG vaccination in cattle over the last 70 years have been highly variable, ranging from none to about 70% protection (4, 5, 17, 18, 27, 30, 52). This variable outcome is similar to that observed for BCG vaccination in human populations (20). Interestingly, trials of BCG vaccination against tuberculosis in humans and cattle conducted in the United Kingdom in the 1940s and 1950s documented high levels of protection (28, 53), which encouraged us to undertake the present study.

It is generally accepted that cellular responses characterized by CD4+-T-cell-derived gamma interferon (IFN-{gamma}) are instrumental in containing tubercle bacilli, although a role for major histocompatibility complex class I-restricted T cells has also been postulated, particularly at later, chronic stages of disease (recently reviewed in references 21 and 23). Nevertheless, it is also evident that the mechanisms of antituberculous immunity are more complex as, e.g., humans and cattle mount potent IFN-{gamma} responses following Mycobacterium tuberculosis or M. bovis infection and development of active disease. In particular, precise correlates of protection still await definition. Identification of such correlates in cattle would greatly facilitate the development of more effective BTB vaccines, because at present cattle require lengthy experimentation periods post-M. bovis challenge to determine vaccine efficacy through postmortem examinations. This not only limits the number of vaccines that can be evaluated but has also serious cost implications due to the necessity of housing infected cattle at high levels of biosafety.

BCG vaccination can be considered an attractive model to evaluate potential correlates of protection, and we therefore performed a BCG vaccination experiment where we challenged vaccinated calves with a virulent British field strain of M. bovis. We were able to demonstrate a significant degree of protection against M. bovis challenge in the vaccinated animals. In addition, we were able to show that the extent of peripheral blood IFN-{gamma} responses induced by the M. bovis-specific antigen ESAT-6 (25, 43) in vitro positively correlated with the severity of pathology. While this does not constitute a direct correlate of protection, it can nevertheless be a useful prognostic immunological marker predicting both vaccine efficacy and disease severity.


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MATERIALS AND METHODS
 
Cattle. Approximately 6-month-old calves (Friesian or Friesian crosses, castrated males) were obtained from herds free of BTB and were kept in the Animal Services Unit in category 3 biosafety accommodation.

Experimental schedule. Six calves were vaccinated with M. bovis BCG Pasteur by subcutaneous injection of 106 CFU into the side of the neck followed 6 weeks later by a booster injection using the same route and dose (9, 50). A group of six unvaccinated calves served as controls. Seven weeks after the second BCG vaccination, both vaccinated and unvaccinated animals were infected with an M. bovis field strain from Great Britain (AF 2122/97) by endobronchial instillation of 4 x 104 CFU as described previously (9, 50). Blood samples were collected at regular intervals throughout the vaccination and challenge period. Animals were skin tested with the single intradermal comparative cervical tuberculin test 16 weeks after M. bovis infection. The skin tests were performed as specified in the European Economic Community Directive 80/219EEC, amending directive 64/422/EEC, annex B (19), and the animals were slaughtered 2 weeks later and postmortem examinations were performed to assess the protective efficacy of vaccination (see below).

Postmortem examination. At the end of the experimental period (18 weeks postinfection), the calves were euthanatized by intravenous injection of sodium pentabarbitone and a postmortem was performed (7). The personnel performing the postmortems were unaware of the vaccination status of the animals examined. Lungs were examined externally for the occurrence of lesions, followed by slicing of the lung into 0.5- to 1-cm-thick slices that were then individually examined for lesions. In addition, lymph nodes of the head and pulmonary regions were removed and weighed. They were sliced into thin section (1 to 2 mm thick) and examined for the presence of visible lesions. Tissue samples, removed from the central parts of the lymph nodes, were taken for M. bovis culture and for histopathological examination (Ziehl-Neelsen to stain for acid-fast bacilli and hematoxylin and eosin staining). The samples taken for culture were weighed to allow an estimation of the bacterial burdens per lymph node. These samples were homogenized and plated on 7H10 agar (see below). The severity of the gross pathological changes was scored applying the following semiquantitative system.

Lungs. Lung lobes (left apical, left cardiac, left diaphragmatic, right apical, right cardiac, right diaphragmatic, and right accessory lobes) were examined individually, and the following scoring system was applied: 0 = no visible lesions; 1 = no gross lesions but lesions apparent on slicing; 2 = < 5 gross lesions of <10 mm in diameter; 3 = >6 gross lesions of <10 mm in diameter or a single distinct gross lesion of >10 mm in diameter; 4 = >1 distinct gross lesion of >10 mm in diameter; 5 = gross coalescing lesions. The scores of the individual lobes were added up to calculate the lung score.

Lymph nodes. The severity of the observed gross pathology in individual lymph nodes was scored applying the following scoring system: 0 = no necrosis or visible lesions; 1 = small focus (1 to 2 mm in diameter); 2 = several small foci or necrotic area of at least 5 by 5 mm; 3 = extensive necrosis. Individual lymph node scores were added up to calculate the lymph node score. Both lymph node and pathology scores were added to determine the total pathology score per animal. All scoring was performed by the same operator for all animals to ensure scoring consistency.

Bacterial enumeration. Tissue sections collected from lymph nodes postmortem were individually homogenized in 5 ml of sterile distilled water using a rotating-blade macerator system. Viable counts were performed on serial dilutions of the macerate in water containing 0.05% (vol/vol) Tween 80 to maintain dispersion. Suspensions were plated on 7H10 agar containing sodium pyruvate (4.16 mg/ml) and 10% (vol/vol) Middlebrook oleic acid-albumin-dextrose-catalase enrichment.

Antigens and peptides. Bovine (PPD-B) and avian (PPD-A) tuberculins were obtained from the Tuberculin Production Unit at the Veterinary Laboratories Agency Weybridge and were used in culture at 10 µg/ml. Recombinant ESAT-6 was expressed as a histidine-tagged fusion protein cloned into pET21d and was purified with Ni-affinity chromatography as described by the manufacturer (Novagen, Cambridge, United Kingdom) (50). Recombinant ESAT-6 was used at 5 µg/ml in T-cell assays. Five ESAT-6 and five CFP-10 peptides were formulated into a peptide cocktail. The details of this cocktail have been described previously in reference 51. Peptides were synthesized; their quality was assessed as described earlier, and they were used at 5 µg/ml each (51).

Lymphocyte transformation assay and production of cytokine-containing supernatants. T-cell proliferative responses were determined as described earlier (51). Briefly, peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by Histopaque-1077 (Sigma) gradient centrifugation and were cultured in tissue culture medium (RPMI 1640 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). PBMC (2 x 105/well in 0.2-ml aliquots) were cultured in triplicate for 6 days in flat-bottomed 96-well microtiter plates in the presence of antigen and radiolabeled during the last 16 to 20 h of culture with 37 kBq of [3H]thymidine/well (Amersham, Amersham, United Kingdom), harvested onto glass fiber filters, and counted in a scintillation counter (TopCount; Packard, Pangborne, United Kingdom).

IFN-{gamma} assay. Whole blood cultures were performed in 96-well plates in 0.20-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 IFN-{gamma} was determined using the BOVIGAM enzyme immunoassay (EIA) kit (CSL, Melbourne, Australia) (51). Results obtained with recombinant ESAT-6 protein and the ESAT-6-CFP-10 synthetic peptide pool and diagnostic cocktails were deemed positive when the optical densities at 450 nm with antigens divided by the optical densities at 450 nm without antigens (IFN-{gamma} stimulation index) were >= 3.0 (51).

IFN-{gamma} ELISPOT assay. Direct enzyme-linked immunospots (ELISPOTs) were enumerated as described earlier (51), by modifying the protocol for indirect ELISPOTs by van Drunen Littel-van den Hurk et al. (46). Briefly, ELISPOT plates (Immobilon-P polyvinylidene difluoride membranes; Millipore, Molsheim, France) were coated overnight at 4°C with the bovine IFN-{gamma} 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 x 105/well suspended in AIM-V-2% fetal calf serum or tissue culture medium [RPMI 1640 supplemented with 5% controlled process serum replacement type 1; Sigma Aldrich; nonessential amino acids; Sigma Aldrich; 5 x 10-5 M 2-mercaptoethanol, 100 U of penicillin/ml, and 100 µg of streptomycin sulfate/ml; Sigma Aldrich]) 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 monoclonal antibody specific for rabbit immunoglobulin G (Sigma Aldrich). The monoclonal antibody 2.2.1 was kindly supplied by D. Godson (VIDO, Saskatoon, Saskatchewan, Canada). The spots were visualized with 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium substrate (Sigma Aldrich).

Statistical analysis. Differences in cellular and humoral immune responses as well as differences in the degree of pathology and bacterial burdens between BCG-vaccinated calves and the unvaccinated control group were compared by nonparametrical statistical analysis employing the Mann-Whitney test. Correlations between immune responses and the degree of pathology were assessed by nonparametrical analysis applying the Spearman rank correlation.


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RESULTS
 
BCG vaccination protects cattle against M. bovis infection. The protective efficacy of BCG vaccination was determined by postmortem examinations. Results in relation to gross pathology are shown in Table 1. Where visible lesions were found, they were confirmed to be tuberculous in nature by histopathology (data not shown). Although only two of six vaccinated animals presented with no visible or histopathological lesions and were M. bovis culture negative, BCG vaccination significantly reduced the pathology associated with M. bovis infection in a further three animals. This was most evident in the lungs (Table 1). Only one of the six calves presented with a disease pattern in the lung comparable to that in the unvaccinated animals. Substantial, M. bovis-induced lung pathology characteristic of BTB was observed in this animal. In addition, reduction in the severity of disease in the lymph nodes of the head and pulmonary regions was also observed in the vaccinated animals (Table 1). These observations are reflected by the significantly reduced number of lung lobes infected, reduced lung scores, reduced lymph node scores, and reduced total pathogenesis scores in the vaccinated group of calves compared to the scores in the nonvaccinated controls (4 versus 13; 9 versus 50; 17 versus 59; and 27 versus 111, all P < 0.05, Table 1). The median severity of disease in lung lobes and lymph nodes with visible lesions was also significantly reduced (P < 0.5, Table 1). In addition, the bacterial burdens in the lymph nodes of vaccinated animals were significantly lower than in the control animals (median log CFU in controls: 5.17 versus 3.30 in the BCG-vaccinated group, P < 0.05, Table 1). Interestingly, the bacterial burdens in the lymph nodes correlated positively with the extent of pathology as described by the total pathology score (Spearman r = 0.7944; P = 0.002).


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  		TABLE 1. Protective efficacy of BCG: gross pathology and M. bovis culture results

Immune responses pre- and postchallenge in vaccinated and control animals. Cellular and humoral immune responses were determined at regular intervals after BCG vaccination and post-M. bovis challenge. After primary BCG vaccination, we observed a small but noticeable increase in both PPD-B-specific T-cell proliferation and IFN-{gamma} production as measured by ELISPOT and BOVIGAM EIA (Fig. 1A to C). Following the BCG booster vaccination, we observed a marked increase in PPD-B-specific T-cell proliferation that remained at stable levels until the animals were infected with M. bovis. While we also observed an anamnestic effect of BCG vaccination on the amount of IFN-{gamma} produced and the frequencies of IFN-{gamma}-secreting cells, this was less pronounced than the increase in proliferative responses (Fig. 1A to C). As expected, prior to M. bovis challenge, vaccinated animals did not respond to the M. bovis-specific antigen ESAT-6, whose gene is deleted in BCG. Similarly, unvaccinated animals did not respond to either PPD-B or ESAT-6 prior to M. bovis infection (Fig. 1A to C). The outcome of infection in relation to disease severity could not be correlated to any prechallenge immune parameter studied. For instance, the two animals that were free of any signs of productive infection (BCG-5 and -6) did not have elevated (or decreased) prechallenge proliferative or IFN-{gamma} responses compared to those in the other four vaccinated animals (data not shown).



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  		FIG. 1. Kinetics of cellular immune responses in BCG-vaccinated and control cattle. Proliferative responses (A), as well as the number of IFN-{gamma}-secreting cells measured by ELISPOT (B) and the amount of IFN-{gamma} produced measured by EIA (C), were determined after in vitro stimulation with PPD-B (10 µg/ml, circles) or recombinant ESAT-6 protein (5 µg/ml, triangles). OD450, optical density at 450 nm; kcpm, thousands of counts per minute. Open symbols, unvaccinated control animals; closed symbols, BCG-vaccinated animals. Results are expressed as mean responses with standard error. *, Mann-Whitney test, P < 0.05. SFC, spot-forming cells.

After M. bovis challenge, immune responses of unvaccinated animals were characterized by rapid and strong in vitro T-cell proliferation, both against PPD-B and ESAT-6, which developed within 3 weeks postinfection and remained strong throughout the 18-week postchallenge experimental period (Fig. 1). In contrast, proliferative responses of PBMC isolated from BCG-vaccinated calves towards PPD-B did not increase over prechallenge values and responses against ESAT-6 were significantly lower at all time points postinfection, with the exception of one time point at week 22 (Fig. 1A). IFN-{gamma} responses were, as in the prechallenge period, measured both by ELISPOT (Fig. 1B) and EIA (Fig. 1C). On stimulation with PPD-B or ESAT-6, unvaccinated animals also developed IFN-{gamma} responses within 2 to 4 weeks of infection. These responses remained relatively stable throughout the experimental period. In contrast, the responses to ESAT-6 in BCG-vaccinated animals were significantly lower at most time points measured. The PPD-B-specific IFN-{gamma} responses in these animals increased slowly and steadily throughout the postinfection period, although they never reached the levels observed in the unvaccinated animals (Fig. 1B and C). Interestingly, minor activity peaks of both ESAT-6-specific proliferative and IFN-{gamma} responses (and also tuberculin-specific responses) were observed in the vaccinated calves between weeks 22 and 25 (Fig. 1A to C). This was mainly due to transiently higher responses of animals BCG-5 and -6, which at later stages did not give rise to positive IFN-{gamma} or proliferative responses. As described above, both of these animals presented with no grossly visible or microscopic signs of disease at postmortem; i.e., they appeared to be fully protected.

IFN-{gamma} responses as correlate of pathology. As described above, this experiment resulted in cattle presenting with a wide range of disease severity, ranging from no detectable BTB to animals that were heavily diseased. This animal-to-animal variation in the severity of M. bovis-induced pathology is accurately reflected in the range of total pathology scores assigned to different animals (ranging from 0 to 36, Table 1). This wide range of pathological changes gave us the ideal opportunity to compare the immunological responses with the disease severity for each animal. We therefore compared the pathology scores with the proliferative and IFN-{gamma} responses observed at different times postchallenge (weeks 5, 9, 11, and 16 postchallenge), as well as with the humoral responses following the tuberculin skin test. Neither tuberculin- nor ESAT-6-specific proliferative responses correlated with the severity of disease (not shown). However, we observed a significant positive correlation between the pathology score and the amount of IFN-{gamma} produced after in vitro stimulation with ESAT-6 at all four time points tested (Spearman r between 0.59 and 0.73, P values from 0.041 to 0.07). Representative results obtained 11 weeks postinfection are shown in Fig. 2. In contrast, the amount of IFN-{gamma} produced after in vitro challenge with bovine tuberculin (PPD-B) did not correlate with the degree of disease (data not shown). Interestingly, the frequency of ESAT-6-specific IFN-{gamma}-secreting cells measured by ELISPOT analysis did not consistently correlate with the severity of BTB in these animals (data not shown).



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  		FIG. 2. Positive correlation between ESAT-6-specific IFN-{gamma} and disease severity. IFN-{gamma} concentration was determined by EIA in supernatants from ESAT-6-stimulated (5 µg/ml) whole blood cultures performed 11 weeks after M. bovis infection (week 24 of experiment). Responses of individual cattle are shown in relation to the severity of disease observed at the postmortem (pathology score). Closed symbols, BCG-vaccinated animals; open symbols, unvaccinated control animals. OD450, optical density at 450 nm. Statistical analysis was conducted with the Spearman rank test.

Differential diagnosis of BCG-vaccinated, diseased animals from BCG-vaccinated, protected animals. A necessary precondition for vaccination against BTB to become a viable option is the parallel development of reagents that differentiate between vaccinated animals that still develop disease and the ones that do not; i.e., those that can be considered fully protected. Tuberculin skin tests performed on the animals used in this study 15 weeks postchallenge using both PPD-A and PPD-B showed no significant differences between the responses of BCG-vaccinated and control group animals (median differences between the skin reactions induced by PPD-B and PPD-A in BCG-vaccinated animals, 13 mm; range, 6 to 17 mm; in controls, median, 13 mm; range, 10 to 21 mm). Significantly, the two vaccinated animals without signs of BTB (BCG-5 and -6) had positive tuberculin skin responses (12- and 14-mm differences between PPD-A and PPD-B), which would have clearly diagnosed them as having BTB. The same diagnosis was obtained when tuberculins were used in in vitro IFN-{gamma} tests that are employed in some countries as an ancillary test to skin testing. The results depicted in Fig. 3 demonstrate that all animals, including BCG-5 and -6, tested positive in the ELISPOT (A) and EIA (B) format of this test upon stimulation with PPD-B. However, when we applied the same in vitro assays but used antigens that are encoded by genes that are deleted in BCG Pasteur, namely, ESAT-6 and CFP-10, all animals that presented with BTB at postmortem gave positive EIA and ELISPOT results. In contrast, calves that showed no sign of disease (BCG-5 and BCG-6) did not respond in these assays. This outcome was observed when we used recombinant ESAT-6 or a cocktail of synthetic peptides composed of immunodominant epitopes from ESAT-6 and CFP-10 (Fig. 3), demonstrating that, by using such antigens, the differential diagnosis of diseased from nondiseased animals following BCG vaccination is possible.



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  		FIG. 3. Differential diagnosis to differentiate vaccinated, diseased cattle from vaccinated, protected cattle. IFN-{gamma} responses were determined 16 weeks postchallenge using either ELISPOT (A) or EIA (B) as readout systems. Antigens were as follows: PPD-B (10 µg/ml), recombinant ESAT-6 (5 µg/ml), or a synthetic peptide cocktail (5 µg of each peptide/ml) (51). Filled symbols, BCG-vaccinated animals without signs of disease; shaded symbols, BCG-vaccinated animals with confirmed BTB; open symbols, unvaccinated controls; horizontal lines, cutoffs for positivity as determined by testing of uninfected animals. ISI, IFN-{gamma} stimulation index; ELISA, enzyme-linked immunosorbent assay. SFC, spot-forming cells.


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DISCUSSION
 
In this paper we demonstrate a high degree of protection of BCG vaccination against BTB in cattle. The degree of protection of around 75% is comparable to the best results reported over the years in studies where BCG was shown to be protective in cattle (e.g., references 8 and 9). In trials where significant protection has been reported in cattle, protection was characterized by significant reductions in disease-induced pathology and bacterial loads, rather than in sterilizing immunity (8, 9, 18, 52). Our findings confirm these earlier reports.

Although precise correlates of protection await definition, it is generally accepted that control of mycobacterial infections is characterized by the emergence of CD4+ cells producing type 1 cytokines and, in particular, IFN-{gamma}. For example, mice in which the gene for IFN-{gamma} was disrupted are unable to control disease and develop progressive and widespread tissue destruction (12, 22). Furthermore, the central role of IFN-{gamma} in controlling human mycobacterial diseases has been highlighted in patients who, due to their inability to produce or respond to IFN-{gamma}, display a heightened susceptibility to mycobacterial infections (reviewed in reference 37). IFN-{gamma} production is also a dominant feature of BTB in cattle (7, 38, 39). Paradoxically, our data show that the lack of production of ESAT-6-induced IFN-{gamma} in vitro in the peripheral blood after infection can be a reliable indicator of the protective efficacy of BCG vaccination. Results consistent with this observation have been reported recently in primate models of human tuberculosis where BCG-vaccinated cynomolgus monkeys that were almost completely protected produced relatively low levels of PPD-specific IFN-{gamma} in BCG- or DNA-vaccinated animals in vitro, whereas rhesus monkeys, which produced higher levels of IFN-{gamma}, were not protected (33).

To reconcile this apparent paradox in the role of IFN-{gamma} in antituberculous immunity with respect to protection, it is pertinent to consider results obtained in rodent models that highlight the importance of very early IFN-{gamma} responses for protection against tuberculosis in BCG, DNA-vaccinated, or tuberculosis-resistant mice (10, 31, 35). This is consistent with the transient early peak of tuberculin and ESAT-6-specific IFN-{gamma} responses that we observed several weeks postinfection in both vaccinated animals that were disease free at postmortem. Another feature to consider is that one might expect "protective" IFN-{gamma} responses to occur at sites of infection, i.e., the lungs (references 11 and 34-36), and that peripheral blood responses might not accurately reflect this (2, 3, 40).

It has been accepted for a long time that acquired immune responses against tuberculous infection promote not only protection but also contribute to the development of pathology (reviewed in reference 14). The data presented here are consistent with this model in that the amount of ESAT-6-specific IFN-{gamma} fed positively with disease severity. However, the animals in these experiments presented with no apparent signs of disease at the time of postmortem, i.e., were asymptomatic, and should therefore be considered to be at a relatively early stage of disease. It is therefore likely that the extent of disease in animals at more advanced stages of BTB will correlate with other immune parameters.

Studies in human tuberculosis have correlated disease severity with a range of, sometimes contrasting immunological parameters. For example, severe clinical disease in humans has been correlated with an increase in mRNA copy numbers for type 2 cytokines, like interleukin 4 (IL-4) and IL-13, although the IFN-{gamma} copy numbers still exceeded those of type 2 cytokines (41). Such balanced Th0 responses or gradual increases in IL-4 responses have been reported in other studies (16, 42, 45). In the present study, we also determined in vitro IL-4 responses using a bioassay (39). After challenge we observed a single IL-4 PPD-B-induced peak both in vaccinated and unvaccinated animals about 9 weeks postinfection, although the responses of BCG-vaccinated and control animals were largely identical (data not shown) and could not be correlated to disease severity. A more balanced cytokine profile in advanced, chronic stages of tuberculosis has been reported in rodent models (e.g., (29). In humans, some studies have described elevated IFN-{gamma} levels in sera (48) or in the lungs of patients with active pulmonary tuberculosis (44). In contrast, a recent study using culture filtrate antigens to induce proliferative and IFN-{gamma} responses in patients with minimal and severe disease revealed no significant differences in both parameters between the two groups (15).

However, human studies relating immune responses with disease severity might not be completely comparable to our data, because human patients studied presented with clinical symptoms of tuberculosis and were thus at more advanced stages of disease than the cattle in our study. Therefore, although the infection in these cattle has to be considered more advanced than in healthy human contacts, their immune responses could be more akin to those observed in such individuals. Interestingly, several reports comparing immune responses of patients with those of direct household contacts have reported increased responses to culture filtrate proteins or ESAT-6-specific IFN-{gamma} in contacts (15, 47). This has been interpreted as a sign of an early stage of infection, although the prognostic value of these observations on the eventual outcome of this infection, i.e., containment of the infection or progression to overt disease, remains to be elucidated.

BCG vaccination compromises the specificity of the tuberculin skin test and tuberculin-based blood assays (4, 6, 30), which was one of the main reasons that a World Health Organization-Food and Agriculture Organization panel in 1959 concluded that BCG vaccination had no role to play in the control of tuberculosis in cattle (1). Diagnostic tests distinguishing between infected and vaccinated animals are required for the continuation of test and slaughter strategies to remove animals, which are not protected from infection. However, over the last 5 years, a number of studies, including several from our laboratory, have reported that antigens whose genes were deleted from BCG yet expressed in M. bovis can distinguish BCG vaccinated from M. bovis-infected cattle when applied in blood-based assays (6, 49, 51). We extended these findings in the present paper by demonstrating that reagents based on genes located in the RD1 region of the M. bovis genome, which is deleted in all strains of BCG, can discriminate BCG-vaccinated but diseased cattle from those that were vaccinated yet remained free from disease.

In conclusion, based on the demonstration of a considerable degree of protection imparted by BCG vaccination of cattle against BTB, our results have demonstrated that the determination of ESAT-6-specific IFN-{gamma}, while not a direct correlate of protection, could constitute a useful prognostic immunological marker predicting both vaccine efficacy and disease severity. If these baseline data are confirmed in larger field studies, this parameter could be a useful marker to assist in the future development of novel tuberculosis vaccines.


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ACKNOWLEDGMENTS
 
This work was funded by the Department for Environment, Food & Rural Affairs, London, United Kingdom.

We express our appreciation to the staff of the Animal Services Unit at the Veterinary Laboratories Agency for their dedication to animal welfare. We thank S. Done, Pathology Department, Veterinary Laboratories Agency, for his histopathological analysis of tissue section and R. Sayers, Epidemiology Department, Veterinary Laboratories Agency, for advice on statistics. We are grateful to D. Godson, VIDO, Saskatoon, Saskatchewan, Canada, and to C. Howard, Institute for Animal Health, Compton, United Kingdom, for supplying monoclonal antibodies and reagents.


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FOOTNOTES
 
* Corresponding author. Mailing address: VLA Weybridge, TB Research Group, Woodham Ln., New Haw, Addlestone, Surrey KT15 3NB, United Kingdom. Phone: 44 1932 357 884. Fax: 44 1032 357 684. E-mail: mvordermeier.vla{at}gtnet.gov.uk. Back

Editor: S. H. E. Kaufmann


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Infection and Immunity, June 2002, p. 3026-3032, Vol. 70, No. 6
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.6.3026-3032.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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