1 Japan BCG Central Laboratory, 3-1-5 Matsuyama, Kiyose-shi, Tokyo 204-0022, Japan
2 National Sanatorium Kumamotominami Hospital, 2338 Toyofuku Matsubase-machi, Shimomashiki-gun, Kumamoto 869-0593, Japan
Correspondence
Yukiko Fujita
y-fujita{at}bcg.gr.jp
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ABSTRACT |
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INTRODUCTION |
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In this study we examined humoral immune responses of patients with active TB against various mycobacterial lipid antigens by ELISA. The purpose of this investigation was to determine the humoral immune responses of TB patients to representative mycobacterial lipid antigens and to improve the sensitivity of IgG antibody detection system for simple and precise diagnosis of active TB and other mycobacterioses. We found that the IgG antibody responses of the individual TB patient sera were extremely heterogeneous depending on the structure of the antigens. However, the multiple combination of selected lipid antigens gave a high sensitivity, and the IgG antibody levels against lipid antigens varied greatly according to the progression of the disease. Therefore, this test is useful as a criterion for the completion of anti-TB chemotherapy.
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METHODS |
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Selection of healthy control subjects and determination of cut-off points for ELISA.
To select the healthy control subjects, we carefully chose individuals who had received a health examination including a chest X-ray. Among these, 70 % of individuals were BCG vaccinated when younger than 12 years old. However, the IgG antibody titres against mycobacterial lipid antigens were not elevated after BCG vaccination, although TST results were positive; hence a co-relationship between IgG antibody titre elevation and the TST results was not observed. Therefore, we simply determined that the normal range for IgG antibody titres was lower than the mean A value +2SD of the healthy control
A.
The control subjects consisted of 85 sera from HIV-negative healthy adult individuals with no history of TB in their families, and included both TST-positive and TST-negative individuals.
Antigens.
Lipid antigens were isolated from heat-killed M. tuberculosis Aoyama B, Mycobacterium bovis BCG and Mycobacterium avium complex (MAC) serotype 16 strain. Lipids were extracted from packed cells with chloroform/methanol (2 : 1, v/v) and the solvent was evaporated off with a rotary evaporator. The lipids were first partially separated by solvent fractionation and then the acetone-soluble or chloroform-soluble, or tetrahydrofuran-soluble or -insoluble fractions obtained were further separated by thin-layer chromatography on silica gel (Uniplate, Analtech) with the solvent system chloroform/methanol/water (65 : 25 : 4, by vol.), chloroform/methanol/acetone/acetic acid (90 : 10 : 6 : 1, by vol.) or chloroform/methanol/water (90 : 10 : 1, by vol.). Antigenic lipids were purified until a single spot was obtained by repeating thin-layer chromatography, and these were identified by mass spectrometric analysis. Each component such as carbohydrates or fatty acids was analysed by gas-chromatography/mass spectrometry (GC/MS) after hydrolysis or methanolysis of the original lipids, and the molecular mass of the intact lipids was determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.
In a preliminary study, we tested the reactivity of 40 active TB patient sera (IgG antibodies) against 12 lipid antigens [TDM from M. tuberculosis Aoyama B, M. bovis BCG Tokyo 172 and BCG Connaught and MAC serotype 16 strain, sulfolipid I, triacyl (PL-2) and tetraacyl (PL-1) phosphatidylinositol dimannosides from M. tuberculosis Aoyama B, mycoside B and cardiolipin from M. bovis BCG Tokyo 172, TMM from M. bovis BCG Tokyo 172 and BCG Connaught and MAC serotype 16 strain]. None of the single antigens gave a sufficiently high positive rate to diagnose active TB, although up to 70 % of the 40 active TB patient sera tested positive against a single lipid antigen (data not shown). However, if an overall positive was scored when any one or more tests among the 12 antigens were positive, the positive rate increased to a much higher level than that obtained with a single antigen. Based on the antigenic reactivity, the stability in ELISA plates, the species specificity in mycobacteria and the availability of lipid antigens, we selected the following six antigens as most appropriate: TDM-T, TDM-M, TMM-T, TMM-M, PL-1 and PL-2.
MALDI-TOF mass analysis showed that TMM-T derived from M. bovis BCG Tokyo was a trehalose monomycolate possessing one molecule of -, methoxy- or keto-mycolic acid ranging from C76 to C91 (the methoxy- and keto-mycolic acid subclasses predominated), while TDM-T from the same strain was a trehalose dimycolate possessing two molecules of mycolic acid from among the above subclasses or molecular species, thus showing a highly complex mixture of trehalose diesters with molecular masses ranging from 2660 to 2920 Da. TMM-M from MAC serotype 16 strain showed a different mass spectrometric pattern, showing the existence of C82 or longer wax ester-mycolic acids characteristic of the MAC, instead of the methoxy-mycolic acids of M. bovis BCG Tokyo. TDM-M from MAC serotype 16 strain also showed multiplex molecular ions in mass spectra, due to the combined
-, keto- and wax ester-mycolic acids. On the other hand, both PL-1 and PL-2 derived from M. tuberculosis Aoyama B were major components of membrane mannophospholipids possessing C16 : 0 and/or branched-chain C19 (tuberculostearic) fatty acids. Thus, TDM (-T and -M) and TMM (-T and -M) were species-specific mycobacterial cell wall antigens, although they shared part of their molecular structure and were therefore cross-reactive. PL-1 and PL-2 were essentially common antigens in the mycobacterial strains. By the combination of above six antigens, the multiple-antigen ELISA gave a highly positive rate, reliable for the diagnosis of active TB and other mycobacterioses.
Multiple-antigen ELISA microplate system.
TDM-T and TDM-M were dissolved in n-hexane (0·2 µg TDM in 50 µl n-hexane per well), while TMM-T, TMM-M, PL-1 and PL-2 were dissolved in ethanol (1·0 µg TMM, PL-1 and 2·0 µg PL-2 in 50 µl ethanol per well). One antigen was deposited in a polystyrene microplate well (Nunc-Immunoplate; Nalge Nunc International) and the plates were allowed to dry at room temperature overnight. Plates were either used immediately or sealed in aluminium foil and stored at 4 °C. We confirmed that no difference was observed in results between plates used immediately after preparation and those stored for a month (data not shown). All chemicals used were purchased from Wako Pure Chemical Industries. For multiple-antigen ELISA, non-specific binding was blocked with 150 µl per well 0·05 % Tween 20 in phosphate-buffered saline (PBS-T) adjusted to pH 7·4 with 5 M NaOH and incubated for 10 min at room temperature. The plates were then washed three times with 250 µl PBS-T per well. Serum samples tested for determination of the titre of IgG antibodies were diluted 1 : 201 with PBS-T. The diluted serum samples (50 µl per well) were added to each well, and the plate was incubated for 1 h. Horseradish-peroxidase-conjugated goat anti-human IgG (H+L) (Zymed) diluted 1 : 500 in PBS-T was used as a secondary antibody. After incubation for 1 h, the substrate, o-phenylenediamine (Sigma; 1 mg ml1) in citrate buffer containing 0·06 % H2O2 was added. The reaction was stopped with 0·5 M H2SO4, and absorbance was measured in a microplate reader (NPR-A4i; Tosoh) at 492600 nm. Each incubation was performed at room temperature, and after each step of the procedure the plates were washed three times with PBS-T.
Classification of types of pathological lesion based on chest X-ray examination.
The extent and severity of the pathological lesion, with or without cavity, were classified based on chest X-ray examination, according to the 1963 Gakken classification of pulmonary TB. Extent of disease or the basic lesion including exudative, caseous, fibrotic, disseminated or far-advanced mixed was classified as follows: I, largest (larger than one side of the lung lobe); II, middle (intermediate between I and III); and III, small (less than one third of one side of the lung lobe). Side was classified as r (right lobe only), l (left lobe only) or b (both lung lobes). Severity was classified as 1 (unstable lesion without cavity confined in one third of one side of the lobe), 2 (lesion limited to one side of the lobe with or without cavity) or 3 (larger lesion with cavity in more than one side of the lobe). For example, bI3 indicates a large caseous lesion with large cavities over both lung lobes, and rIII1 indicates a small but unstable caseous and fibrotic lesion without cavity confined to one third of right lobe.
Data analysis.
The titre of serum IgG antibodies against TDM-T, TDM-M, TMM-T, TMM-M, PL-1 and PL-2 was expressed as the absorption index (A), equal to the absorption value of the test serum sample minus that of the same serum sample in wells without coated antigen. Statistical analysis was done according to the MannWhitney U test.
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RESULTS |
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DISCUSSION |
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Previously, we reported that anti-cord-factor IgG antibody titres were elevated significantly in active TB patient sera (He et al., 1991) and demonstrated that the detection of anti-cord factor antibody was applicable for the early diagnosis of active pulmonary TB (Maekura et al., 1993
), colonic TB (Kashima et al., 1995
) and later tuberculous uveitis (Sakai et al., 2001
). Cord factor is one of the most characteristic and ubiquitous cell wall glycolipids and the structure of the mycoloyl moiety varies widely among mycobacterial species. It has also been reported that glycolipids are extremely stable physico-chemically on microplate ELISA (Julian et al., 2001
). A TBGL (tuberculous glycolipid) antibody detection system using mainly cord factor with minor glycolipid antigens derived from M. tuberculosis H37Rv has been developed (Kawamura et al., 1997
) and evaluated clinically to be useful for the diagnosis of TB at early stages of the disease by a multiple-centre study (Maekura et al., 2001
). Compared to protein antigens, ELISA using lipid antigens shows better stability and reproducibility, and low cross-reactivity, if we choose appropriate antigens and antibody detection system. Chan et al. (1990)
reported haemagglutination tests with three glycolipid antigens and ELISA with antigen-5 on pulmonary TB patient sera; tests with all four antigens were of similar efficiency, with 3052 % of smear-positive patients. More recently, it has also been reported that the detection of immune complexes and IgG antibodies against the three glycolipid antigens is useful for the serodiagnosis of children with a high probability of pulmonary TB (Simonney et al., 2000
). Furthermore, such immunecomplex multiple-antigen ELISA has been described to serve as a fast, cheap and easy method for the diagnosis of TB (Harrington et al., 2000
). Julian et al. (2004)
reported antibody responses against a combination of specific glycolipids and proteins for test sensitivity improvement in TB serodiagnosis.
In our present study, we carefully selected six antigens from among 12 lipid components obtained from mycobacterial cell walls and membranes, and evaluated the reactivities of IgG antibody in active-TB patients and healthy control subjects. A diverse patient-to-patient variation of IgG antibody responses against each of the six lipid antigens was observed; when we combined the six antigens and an overall positive was scored when any one of the six tests was positive, a markedly higher sensitivity was obtained. Some patients gave a highly positive result against all six antigens generally. However, some patients scored highly positive against only mycoloyl glycolipids derived from cell walls, whereas others were positive against only mannophospholipids from cell membranes. Some were positive to TMM, but not to TDM. Some were positive to TDM-T but not to TDM-M, or positive to TMM-T but not to TMM-M, indicating that the mycolyl moiety of the trehalose esters was strictly recognized by IgG antibody in TB patient sera, as we have already reported (Pan et al., 1999).
Recently, the lipid antigen presenting molecules group 1 CD1 (such as CD1a, CD1b, CD1c) and group 2 CD1 (CD1d and CD1e) in human dendritic cells or macrophages have been investigated (Beckman et al., 1994; Moody et al., 1997
; Porcelli et al., 1992
), and T-cell responses in humans infected with M. tuberculosis to CD-1 restricted lipid antigens were reported (Ulrichs et al., 2003
), in which the T cell response in TB patients was the most active against the glycolipid fraction by proliferation assay. However, the lipid antigen presentation to B cells in humoral immune responses has not yet been reported. Mycobacterial lipid antigen epitopes may be presented by novel mechanisms different from the classical MHC class I or class II restricted protein antigens. Although in the case of protein antigens the diversity of T cell responses has been reported to be related to HLA type exclusively, a diversity mechanism of humoral response in the human system against lipid antigens has not yet been reported. To date, serodiagnostics have been designed for early diagnosis of TB or other mycobacterial diseases, and therefore the changes or variation of antibody titres according to the progression or stage of the disease have not been focused on. To reveal the diversity mechanism of antibody responses, we examined the time-course changes in IgG antibody titres. We found that IgG antibody levels against lipid antigens in TB patients' sera varied greatly depending on the stages of the disease. This differs from TST results, which show long-lasting positive scores, once changed to positive from negative. In most cases after the initiation of anti-TB chemotherapy, the IgG antibody titres of active TB patient sera against mycobacterial lipid antigen decreased immediately or were first elevated for a few weeks, and then decreased distinctly near to the normal healthy control level after 34 months, when the excretion of bacilli had ceased. The above results and our previous reports (Maekura et al., 1993
, 2001
), strongly suggest that in parallel with the bacterial burden, IgG antibody levels changed dramatically. To demonstrate the relationship between positive rate and/or mean
A values and loading amount of bacteria, we carefully compared the IgG antibody responses of smear-negative (Gaffky 0), paucibacillary (Gaffky +1 to +3), moderate bacillary (Gaffky +4 to +7) and multibacillary (Gaffky +8 to +10) groups. Unexpectedly, the smear-negative group did not necessarily show the lowest positive rate and IgG antibody levels, and the paucibacillary group showed the lowest. This may reflect the incompleteness of the detection technique of the smear test for acid-fast bacteria. IgG antibody responses were also related to the severity or extent of the pathological lesions determined by chest X-ray examination. In general, patients with the larger and more severe pathological lesions showed a higher positive rate against all antigens, while patients with smaller or less severe lesions showed a lower rate. As the chemotherapy for TB proceeded, excretion of bacilli was eradicated and the pathological lesions were minimized, IgG antibody levels against the lipid antigens decreased distinctly. Therefore, the serodiagnostic test can be useful for the diagnosis of the progression of the disease or as a criterion to determine whether the patient should end or continue the anti-TB chemotherapy. Although the pathological role of the humoral antibody response in TB is not known, marked elevation of IgG antibody against cell-surface lipid antigens may promote the generation of granulomatous lesions or induction of cytokine production by the cellular immune responses, as reported previously (Fujiwara et al., 1999
).
Recently, the incidence of non-TB mycobacteriosis such as MAC infection has increased in TB patients being treated with anti-TB chemotherapy and in immunosuppressed hosts such as AIDS. Therefore, a differential diagnostic test is required. In the present study, we observed that TB patient sera were more reactive against TDM-T and TMM-T, while MAC patient sera were more reactive against TDM-M and TMM-M, indicating that IgG antibody recognized the mycoloyl residue of TDM and/or TMM, as reported previously (Enomoto et al., 1998; Pan et al., 1999
). However, in the present study, IgG antibody responses against species-specific TDM and TMM were cross-reactive, and 100 % sensitivity or specificity was not obtained. This is probably due to the fact that natural trehalose esters consist of a mixture of mycolic acid subclasses. In contrast, phosphoinositomannnosides are widely distributed in mycobacterial species; thus PL-1 and PL-2 are essentially mycobacterial common antigens. Therefore, with the present combination of six antigens we cannot strictly differentiate between TB and MAC disease. However, if we use species-specific antigens, more precise differential diagnosis of TB and MAC disease would be possible. Our recent papers have described highly sensitive discrimination between TB and MAC disease, using ELISA with serotype-specific glycopeptidolipid (GPL) cocktail antigens (Kitada et al., 2002
) or GPL core (Kitada et al., 2005
) from MAC. A more comprehensive serological analysis of MAC infection using mycobacterial multi-lipid antigens including MAC-specific GPL core will be reported in a later paper.
Another use of multiple-antigen ELISA is for serological testing of high-risk contacts who might have been infected with M. tuberculosis recently and may develop active disease if chemoprophylaxis is not applied. Silva et al. (2003), using multivariate analysis, reported that the humoral response to 38 kDa protein was associated with active disease, while those to 14 kDa and ESAT-6 antigens were associated with inactive TB, namely recent M. tuberculosis infection or latent TB. Since in our study also, there were probably two types of antigens early-responsive or infection-specific antigens such as PL-1, PL-2, TMM-T and TMM-M, and late-responsive or disease-specific antigens such as TDM-T and TDM-M it would be possible for serological screening to use serodiagnostics with appropriate antigens. In contrast to TST, which is an infection-associated criterion based on the cellular immune response, humoral IgG antibody responses well reflected the loading amount of bacilli and were more disease specific. Therefore, TST-negative active-TB patients who have been excreting bacilli and are immunocompromised due to steroid use, diabetes mellitus or anti-TNF-
antibody treatment, and old-aged or low-protein nutritional cases, may yet give a positive IgG antibody response (data not shown). To establish whether a serological tool using a particular single antigen or multiple antigens is useful for the screening of latent infection of M. tuberculosis, it is essential to perform clinical tests on a population from an area of high TB incidence. Overall, multiple-antigen ELISA, based on the humoral immune responses, may be the most useful for the rapid diagnosis of active disease due to mycobacterial infection, diagnosis of progression of the disease and differential diagnosis of the mycobacterial species responsible for the infection.
The future interest will be to investigate the possible immunological role of IgG antibodies against each lipid antigen and the relationship between the humoral and cellular immune responses.
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Received 26 November 2004;
revised 16 February 2005;
accepted 17 February 2005.
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