Abstract
The immunological diagnosis and development of new antituberculosis vaccines require the characterization of Mycobacterium tuberculosis antigens inducing cell-mediated immune responses. In this study, we have tested peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients (n = 43) and Bacille Calmette–Guérin (BCG)-vaccinated healthy subjects (n = 24) for in vitro cellular immune responses, as indicated by antigen-induced proliferation and interferon (IFN)-γ secretion, in response to a panel of complex (culture filtrate and cell wall preparations) and single recombinant antigens (Mtb8·4, Mtb9·8, Mtb9·9, Mtb32A, Mtb39A, Mtb40, Mtb41 and Ag85B) of M. tuberculosis. The results of cellular responses showed that the majority (ranging from 70 to 98%) of TB patients and healthy donors responded to the complex antigens in antigen-induced proliferation and IFN-γ secretion assays. However, when PBMC from the same groups of patients and healthy donors were tested with the recombinant antigens, TB patients showed strong recognition (>50% responders) of Mtb9·8 and Mtb39A in proliferation assays (median SI = 6·2 and 6·4, respectively) and of Mtb9·8, Mtb39A, Mtb40 and Ag85B in IFN-γ assays (median delta IFN-γ = 15·5, 10·8, 7·8 and 8·1 U/ml, respectively). BCG-vaccinated healthy donors showed weak (<30% responders) to moderate (31–50% responders) responses to all of the recombinant antigens in both assays. When PBMC of a subset of TB patients (n = 11) were tested for secretion of protective Th1 cytokines [IFN-γ, tumour necrosis factor (TNF)-α and interleukin (IL)-12] and the immunosuppressive cytokine IL-10, the complex CF and CW antigens as well as the recombinant Mtb9·8, Mtb9·9, Mtb40 and Ag85B induced the secretion of both types of cytokines. On the other hand, Mtb41 induced only IL-10, while Mtb8·4, Mtb32Aand Mtb39A induced the secretion of one or more of Th1 cytokines, but not IL-10. In conclusion, the recombinant antigens inducing the secretion of Th1 cytokines could be useful as subunit vaccine candidates against TB.
Keywords: IFN-γ secretion, newly defined recombinant antigens, peripheral blood mononuclear cell, proliferation, tuberculosis
INTRODUCTION
Tuberculosis (TB) is a disease of great antiquity that has caused almost more suffering and death than any other infection [1]. It remains a major health problem of global concern. The recent estimates suggest that about 2 billion people are infected with Mycobacterium tuberculosis, of which 8–10 million develop active disease with 2 million deaths annually [2]. Although M. bovis Bacille Calmette–Guérin (BCG) is used widely as a vaccine against TB, the protection offered has been highly variable [3]. In addition, being live mycobacteria, BCG may cause disease in immunocompromized subjects [4]. For diagnostic purposes purified protein derivative (PPD) of M. tuberculosis is used to detect delayed type hypersensitivity (DTH) reaction to M. tuberculosis antigens. However, a positive PPD skin test may be detected in patients with active disease in healthy donors following BCG vaccination and in healthy subjects exposed to cross-sensitization by other mycobacterial species [5]. Therefore, there is a need to identify and evaluate M. tuberculosis antigens for the specific diagnosis of TB and to develop effective and safer vaccines to replace BCG.
In recent years, several defined antigens of M. tuberculosis have become available either through the purification of natural antigens using biochemical techniques or by producing large quantities of recombinant antigens using molecular biology procedures [6]. This has led to the identification of several major antigens of M. tuberculosis such as heat shock proteins (hsp), e.g. hsp60, hsp70, etc. and secreted antigens, e.g. Ag85, MPT64, MPB70, LPPX, CFP10 and ESAT6, etc. [6]. Some of these antigens have been shown to be useful as new vaccine candidates against TB [7,8]. However, most of these antigens were found to be shared between the virulent strains of M. tuberculosis, the vaccine strains of BCG and other environmental mycobacteria [6,7], and therefore cannot be used for the specific diagnosis of TB. More recently, a set of mycobacterial antigens were identified at Corixa Corporation using PBMC of healthy donors or infected TB patients [9–13]. The healthy donors were PPD+ subjects with no history of TB. This suggested that these donors were infected with M. tuberculosis and were able to control the infection and thus antigens recognized by such subjects could be useful to develop new vaccines against TB. Further selection was based on the robustness of the immune response and the percentage of donors responding to any individual antigen. These antigens were found primarily in the cell lysate with low levels detected in the CF, e.g. Mtb9·9 [12] and Mtb39A [10]; others were mainly present in the CF, e.g. Mtb8·4 [9] and Mtb32A [11]. The previous work also reported a significant antigen-induced proliferation and interferon (IFN)-γ production by PPD+ healthy donors but not by PPD– donors to these recombinant antigens. In addition, immunization with the newly defined antigens such as Mtb9·9, Mtb39A and Mtb41 was protective against M. tuberculosis challenge in animal models [10,12,13].
With respect to diagnostic potential, the above antigens have not been evaluated for discrimination between active TB patients and M. bovis BCG-vaccinated healthy subjects. In this study, we have tested these antigens for their ability to differentiate between TB patients and M. bovis BCG-vaccinated healthy subjects by using peripheral blood mononuclear cells (PBMC) in cellular immune responses, as indicated by antigen-induced proliferation and IFN-γ secretion. The results showed that some of the recombinant antigens such as Mtb9·8, Mtb39A and Mtb40 induced strong antigen-specific proliferation and/or IFN-γ secretion by PBMC of TB patients but not BCG-vaccinated healthy donors.
The protective immunity in TB is mediated by Th1 cytokines [IFN-γ, interleukin (IL)-12 and tumour necrosis factor (TNF)-α], whereas the immunosuppressive cytokine IL-10 appears to play a role in the activation of latent TB [14,15]. An ideal vaccine candidate against TB should therefore induce predominantly the production of protective cytokines with little or no IL-10. In addition to IFN-γ, we have evaluated the ability of the recombinant antigens to induce secretion of IL-12, TNF-α and IL-10 by PBMC of TB patients. The results showed that among the tested antigens, Mtb8·4, Mtb32A and Mtb39A induced the secretion of one or more of Th1 cytokines but not IL-10. Thus, these antigens could be prime candidates to develop a new anti-TB vaccine.
MATERIALS AND METHODS
Donor groups
Heparinized venous blood was collected from 43 newly diagnosed and culture-confirmed cases of pulmonary TB patients attending the Chest Diseases Hospital, Kuwait. Buffy coats were obtained from 24 BCG-vaccinated healthy subjects donating blood at the Central Blood Bank, Kuwait. The groups of healthy donors and TB patient included Kuwaiti and non-Kuwaiti nationals. Informed consent was obtained from all the subjects and the study was approved by the Ethical Committee of the Faculty of Medicine, Kuwait University, Kuwait.
Complex mycobacterial antigens
The complex antigens used in this study include the M. tuberculosis culture filtrate (CF), highly enriched for the secreted antigens with only trace amounts of intracellular soluble antigens, and purified M. tuberculosis cell wall (CW). Both of these complex antigenic preparations were kindly provided by Professor P. J. Brennan (Colorado State University, CO, USA) through the repository of tuberculosis research materials, NIAID, NIH, contact no: AI-25147, USA.
Newly defined recombinant mycobacterial antigens
Table 1 lists the recombinant antigens used, their Rv designation and predicted molecular weights. The production of these recombinant antigens has been described previously [9–13]. In brief, the full-length open reading frames of the cloned genes or the mature form (if a secreted antigen with a putative signal sequence) were polymerase chain reaction (PCR) amplified using sequence specific oligonucleotides followed by subcloning into pET17b expression vector. The recombinant plasmids were used to transform Escherichia coli and the transformants with the correct insert and orientation were identified by restriction digestion and verified by DNA sequencing. For expression of the recombinant antigens, plasmids were transformed into the E. coli expression host BL-21 (pLysE). The recombinant antigens were purified from induced batch cultures by affinity chromatography.
Table 1.
The recombinant antigens used, their Rv designation and predicted molecular weights
The yield of purified recombinant proteins varied from 10 to 75 mg/l of induced bacterial culture with greater than 98% purity. Endotoxin levels were typically < 10 EU/mg protein [i.e <1 ng lipopolysaccharide (LPS)/mg].
Antigen-induced proliferation of PBMC
PBMC were isolated from buffy coats of healthy subjects and blood of TB patients by flotation on Lymphoprep gradients using standard procedures. The cells were finally suspended in complete tissue culture medium [RPMI-1640 + 10% human antibody serum + penicillin (100 U/ml) + streptomycin (100 µg/ml) + gentamycin (40 µg/ml) + fungizone (2·5 µg/ml)] and counted in a Coulter counter (Coulter Electronics Ltd, Luton, Beds, UK).
Antigen-induced proliferation of PBMC was performed according to standard procedures [16,17]. In brief, 2 × 105 PBMC suspended in 50 µl were seeded into the wells of 96-well tissue culture plates (Nunc, Denmark). CF was used at a final concentration of 5 µg/ml while the CW was used at 1 µg/ml. Recombinant antigens were used at a concentration of 5 µg/ml. The plates were incubated at 37°C in an atmosphere of 5% CO2. The cultures were pulsed on day 6 with 1 µCi [3H]-thymidine (Amersham Life Sciences, Amersham, UK), harvested on filter mats with a Skatron harvester (Skatron Instruments AS, Oslo, Norway) and radioactivity incorporated was measured by liquid scintillation counting. The radioactivity incorporated was obtained as counts per minute (cpm). The results were presented as stimulation index (SI), which is defined as: SI = cpm in antigen-stimulated cultures/cpm in cultures lacking antigen. A proliferative response with SI ≥ 5 was considered positive [18].
Cytokine assays
The supernatants (100 µl) from each well of the 96-well plates were collected from the cultures of PBMC on day 6, immediately before [3H]-thymidine pulsing. The supernatants were kept frozen at −20°C until assayed for cytokine level. The amount of IFN-γ in the supernatants was measured using PREDICTA immunoassay kits (Coulter/Immunotech, SA, Marseille, France) as specified by the manufacturer. The detection limit of the IFN-γ assay kit was 0·08 U/ml. Secretion of IFN-γ in response to a given antigen was considered positive when delta IFN-γ (the IFN-γ concentration in cultures stimulated with antigen minus the IFN-γ concentration cultures without antigen) was ≥5 U/ml [18]. Such values are shown in bold type in the tables.
In addition to IFN-γ, IL-10, TNF and IL-12 concentrations were measured in the culture supernatants of PBMC from TB patients (n = 11) using commercial kits (Coulter/Immunotech, SA, Marseille, France) according to the manufacturer's instructions. The minimum detectable concentration of these cytokines was 5 pg/ml. For practical reasons, we used the culture supernatants collected on day 6 to measure the cytokines secreted by monocytes such as TNF-α and IL-12, although day 6 is not the optimal time point to measure their secretion.
Statistical analysis
The Mann–Whitney U-test was used for comparisons between the two groups tested, the TB patients and healthy subjects. The P-values of <0·05 were considered positive.
RESULTS
Antigen-induced proliferation and IFN-γ responses of PBMC from TB patients and BCG-vaccinated healthy subjects to the complex antigens of M. tuberculosis
PBMC from TB patients (n = 43) and BCG-vaccinated healthy subjects (n = 24) were screened for antigen-induced proliferation and IFN-γ secretion in response to the complex M. tuberculosis antigens, CF and CW. The results showed that 79 and 90% of the TB patients and 70% and 78% of the healthy subjects responded to CF and CW antigens, respectively, in the proliferation assays (Table 2). Similarly, both CF and CW complex antigens induced secretion of significant concentrations (delta IFN-γ ≥ 5 IU/ml) of IFN-γ by PBMC from 98% and 80% of the TB patients and 83% and 71% of the healthy subjects, respectively (Table 2). The statistical analysis showed that, in both assay systems, the responses of PBMC from TB patients and healthy subjects to the complex antigens of M. tuberculosis were comparable (P > 0·05).
Table 2.
Antigen-induced proliferation and IFN-γ secretion by PBMC from healthy subjects and TB patients in response to complex M. tuberculosis antigens
| Response of PBMC [no. positivea/no. tested (%)] from: | ||||
|---|---|---|---|---|
| Healthy donors | Tuberculosis patients | |||
| Antigen | Proliferation | IFN-γ | Proliferation | IFN-γ |
| CF | 16/23 (70) | 20/24 (83) | 33/42 (79) | 42/43 (98) |
| CW | 18/23 (78) | 17/24 (71) | 17/19 (90) | 16/20 (80) |
Antigen-induced proliferation with an SI of ≥5 and IFN-γ secretion with a delta of ≥5 U/ml were considered positive responses.
Antigen-induced proliferation and IFN-γsecretion by PBMC from TB patients and BCG-vaccinated healthy subjects to the recombinant antigens of M. tuberculosis
To determine the immunological reactivity of the newly defined recombinant M. tuberculosis antigens, the proteins were tested for antigen-induced proliferation and IFN-γ secretion by PBMC from the groups of TB patients and BCG-vaccinated healthy subjects described above. The results demonstrated that among the eight different recombinant antigens tested, only Mtb9·8 and Mtb39A showed frequent recognition by PBMC from TB patients (>50% responders) in the proliferation assays (Table 3). These two antigens were recognized by PBMC from 59% of the TB patients with significant median SI values of 6·2 and 6·4 to Mtb9·8 and Mtb39A, respectively (Table 3). The positive responders in TB patients to the other recombinant antigens ranged from 16% to 49%, with non-significant median SI values ranging from 1·7 to 4·1 (Table 3). PBMC from healthy subjects showed weak to moderate responses to all the recombinant antigens (0–39% responders) with non-significant median SI values ranging from 1·3 to 3·2 (Table 3). The statistical analysis showed that only Mtb39A induced significantly better responses in TB patients than BCG-vaccinated healthy subjects (P < 0·03).
Table 3.
Antigen-induced proliferation of PBMC from healthy subjects and TB patients in response to recombinant antigens of M. tuberculosis
| Healthy donors | Tuberculosis patients | |||
|---|---|---|---|---|
| Antigen | SI1 | P/T2 (%) | SI | P/T (%) |
| Mtb8·4 | 1·6 (0–261) | 4/23 (17) | 2·4 (0–63) | 9/43 (21) |
| Mtb9·8 | 1·8 (1–93) | 9/23 (39) | 6·2* (0–113) | 23/39 (59) |
| Mtb9·9 | 1·9 (0–26) | 5/23 (22) | 3·2 (0–40) | 13/41 (32) |
| Mtb32A | 1·3 (1–4) | 0/23 (0) | 1·7 (0–18) | 7/43 (16) |
| Mtb39A | 3·2 (0–38) | 8/23 (35) | 6·4* (0–140) | 24/41 (59) |
| Mtb40 | 1·9 (0–35) | 6/23 (26) | 3·4 (0–143) | 19/39 (49) |
| Mtb41 | 2·9 (0–44) | 7/23 (30) | 2·7 (0–29) | 44/41 (26) |
| Ag85B | 2·8 (1–44) | 8/22 (36) | 4·1 (0–106) | 19/40 (48) |
SI is presented as median (± range) and is significant if ≥5 (bold type).
P/T = positive responders per total tested (% of positive responders).
Indicates P < 0·05.
With respect to the IFN-γ responses of PBMC from TB patients to the recombinant antigens, Mtb9·8, Mtb39A, Mtb40 and Ag85B induced positive responses in 76%, 59%, 66% and 54% patients, respectively, with the significant median IFN-γ concentration of 15·5, 10·8, 7·8 and 8·1 U/ml, respectively (Table 4). The responder frequency of TB patients to the remaining antigens ranged from 19% to 45%, with non-significant median values ranging from 0·6 U/ml to 3·6 U/ml (Table 4). The recognition of the recombinant antigens by the PBMC of healthy donors in IFN-γ secretion assays was found to be low both with respect to the percentage of responders (13–42%) and the magnitude of the cytokine produced (0–1·35 U/ml) (Table 4). The statistical analysis showed that, compared to BCG-vaccinated healthy subjects, IFN-γ secretion by PBMC of TB patients was significantly higher in response to Mtb9·8 (P < 0·001), Mtb39A (P < 0·0001), Mtb40 (P < 0·002) and Ag85B (P < 0·005) (Table 4). However, the best discrimination between TB patients and BCG-vaccinated healthy subjects was seen with Mtb39A with respect to both percentage of positive responders (59% and 13%, respectively) and median IFN-γ-values (10·8 and < 0·05 U/ml, respectively).
Table 4.
IFN-γ secretion by PBMC from healthy subjects and TB patients in response to recombinant antigens of M. tuberculosis
| Healthy donors | Tuberculosis patients | |||
|---|---|---|---|---|
| Antigen | SI1 | P/T2 (%) | SI | P/T (%) |
| Mtb8·4 | 0 (0–35) | 3/24 (13) | 0·6 (0–56) | 13/43 (30) |
| Mtb9·8 | 1·35 (0–43) | 10/24 (42) | 15·5* (0–89) | 29/38 (76) |
| Mtb9·9 | 0 (0–100) | 6/24 (25) | 2·7 (0–57) | 18/40 (45) |
| Mtb32A | 0 (0–32) | 5/24 (21) | 0·8 (0–26) | 8/43 (19) |
| Mtb39A | 0 (0–35) | 3/24 (13) | 10·8* (0–75) | 24/41 (59) |
| Mtb40 | 0 (0–74) | 6/24 (25) | 7·75* (0–82) | 25/38 (66) |
| Mtb41 | 0·55 (0–135) | 7/24 (29) | 3·6 (0–73) | 18/41 (44) |
| Ag85B | 0·19 (0–18) | 8/24 (33) | 8·1* (0–56) | 21/39 (54) |
Delta IFN-γ is presented as median (± range) and is significant if ≥ 5 (bold type), it is in U/ml.
P/T = Positive responders per total tested (% of positive responders).
Indicates P < 0·05.
The secretion of IFN-γ, TNF-α, IL-12 and IL-10 by PBMC of TB patients in response to the complex and recombinant antigens of M. tuberculosis
In addition to IFN-γ, the secretion by PBMC of two other protective Th1 cytokines, i.e. TNF-α and IL-12, and an immunosuppressive cytokine, IL-10, was studied in 11 TB patients. PBMC were stimulated with the complex CF and CW antigens as well as with the battery of eight recombinant antigens of M. tuberculosis in separate culture wells. Following 6 days of incubation, the supernatants were collected and used to determine the concentrations of the above cytokines. The complex CF and CW antigens induced detectable levels of IFN-γ (median = 35 and 34 U/ml, respectively), TNF-α (median = 233 and 127 pg/ml, respectively), IL-12 (median = 7 and 8 pg/ml, respectively) and IL-10 (median = 210 and 865 pg/ml, respectively) (Table 5). In response to the recombinant antigens, significant levels of IFN-γ (delta IFN-γ > 5 U/ml) were secreted in response to Mtb9·8, Mtb9·9, Mtb39A, Mtb40 and Ag85B (Table 5). Detectable levels of TNF-α (median values ranging from 5 pg/ml to 65 pg/ml) were secreted in response to all the recombinant antigens, whereas IL-12 was secreted by PBMC (median values ranging from 5 pg/ml to 50 pg/ml) in response to Mtb9·8, Mtb9·9, Mtb32A, Mtb39A, Mtb40 and Ag85B but not in response to Mtb8·4 and Mtb41 (median ≤ 5 pg/ml) (Table 5). Large quantities of IL-10, comparable to the amounts secreted in response to the complex antigens CF and CW, were secreted by PBMC in response to Mtb9·8, Mtb9·9, Mtb40, Mtb41 and Ag85B (median values = 1032, 466, 151, 222 and 390 pg/ml, respectively) (Table 5). However, PBMC of TB patients did not secrete detectable levels of IL-10 (median ≤ 5 pg/ml) in response to Mtb8·4, Mtb32A and Mtb39A (Table 5).
Table 5.
Secretion of IFN-γ, TNF-α, IL-12 and IL-10 by PBMC of TB patients in response to the complex and recombinant antigens of M. tuberculosis
| Median values* (range) of the cytokines secreted by PBMC | ||||
|---|---|---|---|---|
| Antigen | IFN-γ U/ml | TNF-α pg/ml | IL-12 pg/ml | IL-10 pg/ml |
| CF | 35 (5–60) | 233 (0–362) | 7 (0–33) | 210 (0–2910) |
| CW | 34 (8–86) | 127 (0–443) | 8 (0–400) | 865 (0–5200) |
| Mtb8·4 | <5 (0–51) | 13 (0–109) | <5 (0–250) | <5 (0–2210) |
| Mtb 9·8 | 5·4 (0–89) | 5 (0–89) | 21 (0–200) | 1032 (0–3511) |
| Mtb 9·9 | 8 (0–57) | 6 (0–93) | 10 (0–467) | 466 (0–4010) |
| Mtb 32 A | <5 (0–14) | 22 (0–59) | 17 (0–450) | <5 (0–1352) |
| Mtb 39 A | 15 (0–75) | 39 (0–82) | 5 (0–330) | <5 (0–210) |
| Mtb 40 | 6 (1–82) | 65 (0–198) | 50 (0–200) | 151 (0–610) |
| Mtb 41 | <5 (0–73) | 21 (0–268) | <5 (0–130) | 222 (0–4110) |
| Ag85B | 11 (0–56) | 42 (0–97) | 8 (0–500) | 390 (0–2310) |
The values shown are the cytokine concentrations in antigen-stimulated wells minus the cytokine concentration in non-stimulated wells.
DISCUSSION
In order to identify new antigens of diagnostic and vaccine potential, we have characterized in this study a panel of seven recombinant M. tuberculosis antigens (Mtb8·4, Mtb9·8, Mtb9·9, Mtb32A, Mtb39A, Mtb40, Mtb41) identified recently at Corixa Corporation, and Ag85B for cellular immune responses in TB patients and BCG-vaccinated healthy subjects. Antigen-induced proliferation and IFN-γ secretion assays were employed as markers of cellular immune responses by using PBMC from the two donor groups. In addition, the responses of PBMC to the complex CF and CW preparations of M. tuberculosis were also assessed. The results showed that both of the complex antigenic preparations were equally effective in inducing proliferation and IFN-γ secretion by PBMC from the majority of TB patients and healthy subjects. These results suggest that the immune status of pulmonary TB patients included in this study was not compromised and BCG-vaccinated donors were reactive to the complex M. tuberculosis antigens Thus, both these donor groups were suitable to determine the reactivity and specificity of the cellular immune responses upon stimulation with the panel of recombinant antigens.
Recent advances in understanding immunity to mycobacterial infections suggest that PPD skin testing can be replaced with more specific in vitro tests based on collection of a single blood sample [19]. These tests include the cell proliferation assay and cytokine secretion by cultures stimulated with multiple and specific antigens of M. tuberculosis. The utilization of antigens specific to M. tuberculosis could allow the discrimination of cases infected or exposed to TB. The new tests also aim for improving the sensitivity, specificity and the speed by which TB is diagnosed.
Research work has been aiming to look for defined antigens that could afford protection against tuberculosis to a degree comparable to that obtained with M. bovis BCG. However, these new vaccines should be devoid of the variability and disadvantages associated with the live M. bovis BCG. In addition, the new vaccines should be able to induce protective Th1 cytokines including IFN-γ, TNF-α and IL-12 [3]. For a potential vaccine to be useful in a population, it is also essential that it should be recognized by T cells in association with either the most frequently expressed and less polymorphic HLA-DR molecules or multiple HLA-DR molecules in a promiscuous manner [6].
In TB patients, the recombinant antigens Mtb9·8, Mtb39A, Mtb40 and Ag85B were found to be recognized strongly by PBMC as reflected by antigen-induced proliferation and/or IFN-γ secretion by PBMC from >50% patients. HLA-DR typing of PBMC from 21 TB patients and 14 healthy subjects selected randomly from the tested groups showed that both groups were highly HLA-heterogeneous, as they were positive for most of the serologically defined HLA-DR specificities (data not shown). Therefore, this HLA-heterogeneity of the donors would suggest that these recombinant antigens were recognized promiscuously in cellular immune responses. The promiscuous recognition of single antigens is required for their usefulness as a vaccine candidate in the highly HLA-heterogeneous human populations [6]. When tested in BCG-vaccinated healthy subjects, all the recombinant antigens showed low to moderate responder frequency, i.e. <50% responders, in both antigen-induced proliferation and IFN-γ responses. The recognition of recombinant antigens by healthy donors may be due to either the prior vaccination with BCG [20] or previous exposure to M. tuberculosis, as shown by the reactivity of 30% of the healthy donors to M. tuberculosis-specific antigens, ESAT-6 and/or CFP10, in this study (data not shown) and a previous study [21]. The differences between TB patients and BCG-vaccinated healthy subjects with respect to median IFN-γ values in response to Mtb9·8 (P < 0·001), Mtb39A (P < 0·0001), Mtb40 (P < 0·002) and Ag85B (P < 0·005) were found highly significant. Despite the fact that these antigens showed more frequent and significantly stronger recognition in TB patients than BCG-vaccinated healthy individuals, their use in diagnosis of TB is questionable because of positive responses in 13–42% subjects in the BCG-vaccinated group.
A recent study carried out in The Gambia showed that neonatal BCG vaccination induced IFN-γ responses to Mtb8·4, Mtb9·9 A, Mtb32-N, Mtb32-C and Mtb39A [20]. However, TB patients with advanced disease showed low IFN-γ responses to these antigens as well as to PPD. The patients included in the present study showed strong IFN-γresponses to complex antigens of M. tuberculosis as well as to the defined antigens. The differences in the results obtained in these two studies could be due to several reasons, including differences in the patients' populations, exposure to environmental mycobacteria and differences in the assays used.
More recent data have indicated that protection induced by selected defined antigens in animal models of TB are comparable to that obtained with BCG vaccination. In this context, a recombinant vaccine consisting of Mtb32-C- Mtb39A-Mtb32-N (rMtb72F) has been shown to induce protection in mice and prolonged survival in guinea pigs against tuberculosis for periods lasting over 1 year [22]. This vaccine formulation is now in phase I clinical trials, which makes it the first recombinant protein tuberculosis vaccine tested in humans.
To identify the antigens most suitable for vaccine development against TB, we have also tested the recombinant antigens for cytokines that have been shown to play important roles either in protection against or susceptibility/reactivation of TB. Among the tested cytokines, IFN-γ, IL-12 and TNF-α have been associated with protection and IL-10 with susceptibility/reactivation [14,15] of the disease. Therefore, in the context of a protective vaccine, it will be desirable to choose those antigens that induce the biased production of protective Th1 cytokines. In this study, we show that detectable levels of both types of cytokines, i.e. protective Th1 cytokines and IL-10, were secreted by PBMC of TB patients in response to the complex CF and CW antigens of M. tuberculosis. Furthermore, the results show that several of the recombinant antigens also induced the secretion of the protective cytokines as well as IL-10. However, three of the recombinant antigens (Mtb8·4, Mtb32A and Mtb39A) induced the secretion of one or more Th1 cytokines associated with protection, but with no detectable levels of IL-10. Among the Th1 cytokines produced by monocytes, Mtb8·4 induced the secretion of TNF-α only, whereas Mtb32A and Mtb39A induced the secretion of TNF-α as well as IL-12. These results further support the use of Mtb32A and Mtb39A (rMtb72F) as a new anti-TB vaccine candidate.
Acknowledgments
This work was supported the Kuwait Foundation for the Advancement of Sciences (KFAS) grant no. 97-07-05 and Kuwait University Research Administration grant MI02/02. The supply of buffy coats from the Central Blood Bank, Kuwait is gratefully acknowledged.
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