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Clinical and Diagnostic Laboratory Immunology, May 2001, p. 604-611, Vol. 8, No. 3
Immunology Unit, Department of Infectious and
Tropical Diseases, London School of Hygiene & Tropical Medicine, London
WC1E 7HT, United Kingdom1; Unidad de
Investigacion Medica en Dermatologia y Micologia "Dr Ernesto
Macotela," Department of Dermatology and Medical Mycology, Hospital
de Especialidades "Dr Bernardo Sepulveda," Centro Medico Nacional
Siglo XXI,2 and Centro Dermatologico Dr
Ladislao de la Pascua,4 Mexico City DF, and
Hospital Amigo del Nino y la Mujer, Instituto Mexicano del
Seguro Social, Celaya GTO,5 Mexico;
and Department of Immunohematology and Blood Bank, Leiden
University Hospital Medical Center, 2300 RC Leiden, The
Netherlands3
Received 14 August 2000/Returned for modification 16 November
2000/Accepted 15 February 2001
The ability of the 45-kDa serine-rich Mycobacterium
leprae antigen to stimulate peripheral blood mononuclear cell
(PBMC) proliferation and gamma interferon (IFN- Leprosy is a chronic infectious
disease of the skin and peripheral nerves caused by infection with
Mycobacterium leprae. Leprosy patients present with a
spectrum of clinical disease that is closely correlated with the
ability of the host to make a cellular immune response to the organism
(19). Tuberculoid leprosy patients have strong
cell-mediated immunity to M. leprae both in vitro and in
vivo, and few bacilli are present in skin lesions. At the other end of
the spectrum, T cells from lepromatous leprosy patients are
nonresponsive to M. leprae and there is uncontrolled growth of the organism in skin lesions, often leading to disability and social
isolation. The widespread implementation of multidrug therapy during
the past 10 years has resulted in a dramatic reduction in the numbers
of registered leprosy patients (25). Despite these
achievements, it is presently unclear whether the implementation of
multidrug therapy has had any effect on M. leprae
transmission or reduced the incidence of disease (34), as
the number of new cases being reported each year has remained the same
at over 500,000 worldwide (25). Therefore, a major
priority in leprosy research is the identification of new specific
M. leprae antigens for use as skin test reagents to identify
those who have been exposed to the organism and also to help detect
individuals with early subclinical infection (21).
A number of antigens have been reported to induce T-cell responses from
tuberculoid leprosy patients and leprosy patient contacts in
vitro, including the 70-, 65-, 18-, and 10-kDa heat shock proteins (2, 6, 9, 15, 16, 22, 26), the 30/31-kDa secretory proteins (13-15), and the M. leprae 35-kDa
antigen (28). However, all these M. leprae
antigens have been shown to be cross-reactive, with homologous genes
identified in other mycobacterial species (27, 36), and
thus would not be useful as diagnostic reagents.
The importance of gamma interferon (IFN- Vega-Lopez et al. (29) used pooled sera from lepromatous
leprosy patients to screen an M. leprae lambda gt11
expression library (37) and isolated and sequenced a gene
encoding a serine-rich protein with a predicted molecular mass of 45 kDa; (25L or Sra) (27). A high proportion of leprosy
patient sera (78% of multibacillary and 68% of paucibacillary leprosy
patient sera) contained immunoglobulin G (IgG) antibodies to a
Patient population and controls.
Leprosy patients were
recruited from the Centro Dermatologiico Dr Ladislao de la Pascua,
Mexico City, Mexico, the Hospital del Especialidades de Centro Medico
Nacional Siglo XXI (IMSS), Mexico City, Mexico, the Centro Medico La
Raza, Mexico City, Mexico, and the Hospital Amigo del Nino y la Mujer,
Celaya, Mexico. Patients were categorized according to clinical
diagnosis, histopathology, and bacterial index of skin slit smears. A
total of 48 leprosy patients were used for the study. The tuberculoid
leprosy patient group consisted of 9 polar tuberculoid (TT) and 5 borderline tuberculoid (BT) patients, and the lepromatous
leprosy patient group was made up of 34 individuals with the diffuse or
nodular forms of lepromatous leprosy. All the leprosy patients
recruited were receiving chemotherapy at the time of the study. The
contact group consisted of 17 healthy individuals living in the same
house as a leprosy patient. Of these, 14 were contacts of lepromatous
leprosy patients, 2 were contacts of patients with the indeterminate
form of disease, and one was a contact of a borderline case. A further
20 healthy blood donors with no known exposure to the disease were
recruited from the Hospital Amigo del Nino y la Mujer, in the
leprosy-endemic area of Guanajuato, and Centro Dermatologico Dr
Ladislao de la Pascua. Eighteen patients with pulmonary tuberculosis
were also recruited from the Instituto Nacional de Enfermedades
Respiratorias, Mexico City, Mexico.
Antigens.
Phytohemagglutinin (PHA-P) was supplied by
Difco (Detroit, Mich.) and used at a 1:200 dilution. Purified protein
derivative (PPD) from M. tuberculosis (batch RT48) was
obtained from the Statens Serum Institute (Copenhagen, Denmark).
Armadillo-derived M. leprae sonicate (batch CD235) (prepared
as described in the report of the fifth meeting of the Scientific
Working Group on the Immunology of Leprosy, World Health Organization
[WHO] document TDR/IMM-LEP-SWG [5] 80.3, annex 4, p. 23, 1980) was kindly provided by R. J. W. Rees (National
Institute for Medical Research, Mill Hill, United Kingdom). The
30/31-kDa antigen (1) was purified from the culture
filtrate of M. tuberculosis H37Rv as outlined previously
(5). The M. leprae 65- and 10-kDa recombinant
antigens used in the study were kindly provided by J. van Embden and M. Singh through the WHO Immunology of Mycobacteria antigen bank.
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.3.604-611.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Presence of Human T-Cell Responses to the Mycobacterium
leprae 45-Kilodalton Antigen Reflects Infection with or
Exposure to M. leprae
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) production was
measured in leprosy patients, household contacts, and healthy controls
from areas of endemicity in Mexico. Almost all the tuberculoid leprosy
patients gave strong PBMC proliferation responses to the M.
leprae 45-kDa antigen (92.8%; n = 14).
Responses were lower in lepromatous leprosy patients (60.6%;
n = 34), but some responses to the 45-kDa antigen were detected in patients unresponsive to M. leprae
sonicate. The proportion of positive responses to the M.
leprae 45-kDa antigen was much higher in leprosy contacts
(88%; n = 17) than in controls from areas of
endemicity (10%; n = 20). None of 15 patients with pulmonary tuberculosis gave a positive proliferation response to the
45-kDa antigen. The 45-kDa antigen induced IFN- secretion similar to
that induced by the native Mycobacterium tuberculosis 30/31-kDa antigen in tuberculoid leprosy patients and higher
responses than those induced by the other recombinant antigens
(M. leprae 10- and 65-kDa antigens, thioredoxin, and
thioredoxin reductase); in patients with pulmonary tuberculosis
it induced lower IFN- secretion than the other recombinant antigens.
These results suggest that the M. leprae 45-kDa antigen
is a potent T-cell antigen which is M. leprae specific
in these Mexican donors. This antigen may therefore have diagnostic
potential as a new skin test reagent or as an antigen in a simple
whole-blood cytokine test.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) in reducing infection with
M. leprae is well documented in vitro and in vivo. Th1 T
cells specific for mycobacterial antigens have been isolated from the
lesions and peripheral blood of tuberculoid leprosy patients (8,
17, 23). The skin lesions from tuberculoid leprosy patients have
also been shown to contain abundant Th1 cytokine mRNA, which was rare
in lepromatous lesions (35). Furthermore, when skin
lesions of lepromatous leprosy patients were inoculated with
recombinant IFN-, marked reductions in bacterial load were observed
(11). The ability to induce secretion of IFN- is
therefore an important property of those leprosy antigens involved in
protective immunity.
-galactosidase 45-kDa fusion protein (29). Work by
others (20) suggested that this antigen may be specific to
M. leprae as DNA from Mycobacterium tuberculosis and several atypical mycobacteria failed to hybridize with a 45-kDa protein-encoding DNA probe in Southern blots. We have now
compared peripheral blood mononuclear cell (PBMC) proliferation and
IFN- production by leprosy patients in response to the 45-kDa
protein with those induced by other proteins including the 65-, 30/31-, and 10-kDa antigens, as well as the M. leprae thioredoxin
(Trx) and thioredoxin reductase (TR) proteins (31-32).
These responses were compared with those of leprosy contacts, healthy
individuals living in the same house as a leprosy patient, patients
with pulmonary tuberculosis, and individuals living in a
leprosy-endemic area in order to evaluate whether the 45-kDa antigen
contains M. leprae-specific T-cell epitopes.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-D-thiogalactopyranoside) induction
step was performed at 22°C rather than 37°C for the Trx and TR
proteins. This was followed by sonication under nondenaturing conditions to avoid the formation of insoluble aggregates
(31-32). The purity of the 45-kDa antigen was confirmed
on Coomassie blue-stained sodium dodecyl sulfate-12% polyacrylamide
gel electrophoresis gels, and visible contamination with
E. coli proteins was excluded by immunoblotting using a
peroxidase-labeled rabbit anti-E. coli antiserum (Dakopatts,
Glostrup, Denmark). Single batches of each antigen were used throughout
the study. All the purified and recombinant mycobacterial antigens were
used at final concentrations of 10, 1, and 0.1 µg/ml; data shown are
for the responses to the antigens used at 10 µg/ml, which was shown
to be optimal (results not shown). The histidine tag control was used
as a negative control for both the histidine sequence and for any
contaminating E. coli proteins. This control was used at a
final dilution of 1 in 100, comparable to that of the test antigen.
PBMC separation. Heparinized blood from healthy contacts, controls from areas of endemicity, and patients was diluted with an equal volume of RPMI 1640 and then layered over Histopaque 1077 (Sigma Chemical Co., Poole, Dorset, United Kingdom), followed by centrifugation at 400 × g for 30 min. The PBMCs were isolated from the plasma-Histopaque interface, washed three times with Hanks' balanced salt solution (Gibco BRL, Paisley, United Kingdom), and suspended in growth medium consisting of RPMI 1640 (Gibco BRL) containing 10% autologous plasma, 100 U of penicillin/ml, 100 µg of streptomycin/ml, and 2 mM L-glutamine (Gibco BRL). PBMCs were counted using the trypan blue exclusion method.
T-cell proliferation assays. T-cell proliferation assays were performed as previously described (6). Aliquots of 2 × 105 PBMCs in 180 µl of growth medium were dispensed into each well of 96-well round-bottom microtiter plates (Gibco BRL) containing either mitogen or antigen in triplicate wells. All the mycobacterial antigens were used at a final concentration of 10 µg/ml, which had previously been shown to be optimal. Negative-control cultures contained PBMCs in growth medium alone. Plates were incubated for 6 days at 37°C in a humidified atmosphere of 5% CO2 and 95% air and then were pulsed with 1 µCi of methyl-[3H]thymidine (specific activity, 2 Ci/mmol; Amersham International, Little Chalfont, United Kingdom). Cells were harvested 16 h later onto glass fiber filter discs (Cambridge Technology, Cambridge, Mass.) using a semiautomatic cell harvester (PHD; Cambridge Technology). The discs were transferred into biovials (Beckman, High Wycombe, United Kingdom), and 1.5 ml of scintillation fluid (Cytoscint ES; ICN Biomedicals Ltd., Irvine, Calif.) was added. Incorporation of [3H]thymidine was determined using a Beckman LS 880 scintillation counter. Proliferative responses to the antigens were considered positive when the stimulation index (SI; counts per minute in stimulated cultures divided by counts per minute in unstimulated cultures) was greater than 3 and the increase in counts per minute in stimulated cultures minus that in unstimulated cultures was >2,500 cpm; this criterion was used to calculate the percent responders for each antigen.
Enzyme-linked immunosorbent assay (ELISA) for the detection of
IFN-.
Supernatants from antigen-stimulated PBMCs and controls
were stored at 
20°C before testing. Immulon IV microtiter plates (Dynatech, Billingshurst, United Kingdom) were coated with 100 µl of
monoclonal antibody to human IFN- (MD-1; Gibco BRL) at 2.5 µg/ml
in 0.1 M carbonate buffer, pH 9.6, and incubated overnight at 4°C.
The contents of the wells were discarded, and then the wells were
blocked with 2.5% bovine serum albumin (BSA) in PBS, followed
by incubation in a humid box for 1 h at 37°C. After three washes
with PBS-Tween 20 (0.05%), sample supernatants (100 µl) or the
IFN- reference standard (GIF86-04; kindly provided by Hoffman
la Roche, Basel, Switzerland) was added and the plate was incubated for
2 h at 37°C; this was followed by a further four washes with
PBS-Tween 20. Rabbit polyclonal antibody to human IFN- (100 µl/well; kindly provided by P. Kaye, London School of Hygiene & Tropical Medicine), diluted in PBS-Tween 20 containing 5% human
AB serum and 0.25% BSA, was added in order to detect bound IFN-,
and the plate was incubated for another hour at 37°C. The washing
stage was repeated as before, and 100 µl of peroxidase-labeled goat
anti-rabbit IgG (heavy plus light chains) (KPL; Dynatech, Billingshurst, United Kingdom) was added to all wells. After an incubation of 30 min at 37°C, the plate was washed and 100 µl of
tetramethylbenzidine substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) was added. After 15 min, the color development was
read at 490 nm on a Dynatech MR600 microplate reader. The assay was
sensitive down to a lower limit of 3 U/ml and had an upper limit of
detection of 400 U/ml. The quantity of IFN- present in the
supernatants was determined from a standard curve.
Statistical analysis. A nonparametric test (Mann-Whitney U test) was used to evaluate the statistical significance of the data.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Lymphocyte proliferation responses induced by the M. leprae 45-kDa antigen. The M. leprae 45-kDa antigen was expressed as a recombinant protein with a leader sequence of six histidine residues followed by a 24-amino-acid linker at the N terminus (31). It was therefore important to exclude the possibility that there was any human T-cell recognition of this leader sequence. PBMCs from a group of 13 Mexican tuberculoid leprosy patients and 20 controls from an area of endemicity were stimulated with the M. leprae 45-kDa antigen or the histidine control prepared as described above. The results confirmed that whereas the M. leprae 45-kDa antigen induced positive proliferative responses, the histidine control did not. The inability of the leader sequence to induce positive proliferation responses was also confirmed in a group of 11 United Kingdom donors and in a group of 13 patients with pulmonary tuberculosis (results not shown).
The PBMC proliferative responses of tuberculoid leprosy and lepromatous leprosy patients were compared with those of healthy household contacts and individuals from a leprosy-endemic area in standard 7-day proliferation assays using a panel of mycobacterial antigens including the 45-kDa antigen (Fig. 1). All leprosy patients, household contacts, and controls from areas of endemicity gave strong proliferative responses to the mitogen PHA (data not shown). The majority of tuberculoid leprosy patients had positive proliferative responses to M. leprae sonicate and to the 45-kDa antigen (Fig. 1a). When the proportion of responders was calculated using a SI of >3 and an increase in counts per minute of >2,500 cpm to define a positive response, the tuberculoid leprosy patients showed 92.8% positive responders to both M. leprae sonicate and the M. leprae 45-kDa antigen. A lower proportion responded to the 65-kDa and 10-kDa antigens (29 and 15%, respectively). Tuberculoid leprosy patients made significantly higher proliferative responses than lepromatous leprosy patients after stimulation with M. leprae sonicate, the M. leprae 45-kDa antigen, and the M. tuberculosis 30/31-kDa antigen (P < 0.05) (Fig. 1b). Both tuberculoid leprosy and lepromatous leprosy patients made equally good responses to PPD and equally poor responses to the 65-kDa and 10-kDa antigens. Only 35% of the lepromatous leprosy patients failed to make PBMC proliferative responses to M. leprae sonicate, and a proportion of these made PBMC proliferative responses to the recombinant M. leprae 45-kDa antigen (25%) and to the M. tuberculosis 30/31-kDa antigen (36%).
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3 with an increase in counts per minute
(counts per minute in cultures plus antigen IFN- production by PBMCs in response to leprosy antigens.
Supernatants were removed from PBMC cultures after 6 days and assayed
for IFN- by ELISA. There was no significant difference in IFN-
production among any of the groups tested in response to the mitogen
PHA (P > 0.05) (data not shown). The pattern of IFN- production in response to the antigens tested in all groups of
patients and controls paralleled the PBMC proliferative responses (Fig.
2). The tuberculoid leprosy patient group
produced significantly higher IFN- than lepromatous leprosy patients
in response to PPD, M. leprae sonicate, and the 45- and
30-kDa antigens (P < 0.05) (Fig. 2a and b). Patients
in the tuberculoid leprosy group produced significantly higher
quantities of IFN- than the contact group in response to PPD
(P < 0.01), M. leprae sonicate
(P < 0.02), or the 45- (P < 0.01),
30- (P < 0.02), and 65-kDa (P < 0.01)
antigens. Interestingly, IFN- production in response to the 45-kDa
antigen by tuberculoid leprosy patients was higher than those in
response to M. leprae sonicate and the 65- and 10-kDa
antigens and comparable to that in response to the 30/31-kDa antigen.
Compared with that by tuberculoid leprosy patients, IFN- production
by controls was significantly higher in response to PPD
(P < 0.01) and the 30- (P < 0.02) and
65-kDa (P < 0.01) antigens but similar to that in
response to M. leprae sonicate (P > 0.05).
IFN- production by PBMC from lepromatous leprosy patients in
response to antigen was similar to that of the contact group for all
antigens (P > 0.05) except for the 65-kDa antigen,
which induced higher IFN- responses in the lepromatous leprosy
patient group (P < 0.05). Compared with those from the
lepromatous leprosy group, PBMCs from controls gave higher IFN-
production in response to all the antigens except the 45-kDa antigen
(P < 0.05).
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in response to
M. leprae sonicate, PPD, and the 30- and 65-kDa antigens than the contact group (P < 0.01) (Fig. 2c and d). In
contrast, IFN- production by PBMC in response to the 45-kDa antigen
was found to be higher in the contact group than in the controls, but
this difference was not statistically significant (P > 0.05).
T-cell proliferation responses to the M. leprae Trx
and TR antigens.
PBMCs from the same groups were also stimulated
with two other novel M. leprae antigens, prepared as
histidine fusion proteins and purified using the same procedure as for
the 45-kDa antigen. Neither the Trx nor the TR antigen induced any
positive proliferation responses in PBMC from controls from areas of
endemicity (Table 1). The Trx antigen
induced positive responses in 46% of the leprosy contacts (median,
2,715 cpm) and in 23% of the tuberculoid leprosy patients (median,
1,633 cpm). The TR antigen induced a similar pattern of responses,
although the numbers of responders were lower, with 27% responders in
the contact group and 15% responders in the tuberculoid leprosy group.
Both antigens induced low responses in the lepromatous leprosy
patients. Therefore although all three recombinant antigens seemed to
be potentially leprosy specific, the responses to the M. leprae 45-kDa antigen were higher than those to the other fusion
proteins. These results also confirmed that the responses to the 45-kDa
antigen were not directed to the histidine leader sequence.
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Lymphocyte proliferation and IFN- responses to the 45-kDa
antigen in patients with pulmonary tuberculosis.
The M. leprae 45-kDa antigen therefore appeared to induce stronger T-cell
responses than the other recombinant leprosy antigens tested and
appeared to be more strongly recognized by T cells from leprosy
patients and contacts than controls. As a further test of specificity,
a group of patients with active tuberculosis was also tested for
responses to the M. leprae 45-kDa antigen. All the patients
with pulmonary tuberculosis responded to PHA (data not shown) and PPD.
In contrast, none of the patients tested responded to the 45-kDa
antigen and only a small number responded to the other recombinant
antigens (Fig. 3a). The responses to M. leprae sonicate in the patients with tuberculosis were
lower than those in the control group. The PBMC proliferative responses to the other antigens tested were generally low and not significantly different from those in the control groups (P > 0.05 for all antigens).
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responses to PPD and to the M. tuberculosis 30/31-kDa
antigen (Fig. 3b). No significant difference in IFN- production by
PBMCs between tuberculosis patients and controls in response to either
PHA or PPD were found (P > 0.05). The highest
concentrations of IFN- were produced in response to PPD and to the
65- and 30/31-kDa antigens, although they were not significantly
different from those produced by the control group. As seen for the
proliferative responses, the M. leprae 45-kDa antigen
induced lower IFN- responses in the PBMC cultures from patients with
pulmonary tuberculosis than any of the other mycobacterial antigens.
A comparison of the percent responders in the lymphocyte proliferation
assay to both M. leprae sonicate and the M. leprae 45-kDa antigen between tuberculosis patients and leprosy
patients, contacts, and controls is shown in Fig.
4. This highlights the difference in
recognition of the 45-kDa antigen in subjects known to be infected or
exposed to M. leprae compared to the patients infected with
M. tuberculosis and suggests that the M. leprae 45-kDa antigen is being recognized as an M. leprae-specific
antigen.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Multidrug therapy has been highly successful in the treatment of leprosy. However, the identification of novel antigens which could be used as specific, diagnostic antigens for leprosy is still of major importance. The aims of the present study were to test T-cell reactivity to the 45-kDa antigen, previously described as an M. leprae-specific protein (20, 29), and to evaluate the potential of the 45-kDa protein to identify individuals exposed to M. leprae or with subclinical infection. A specific skin test reagent could be an important epidemiological tool in monitoring M. leprae transmission and disease distribution. The results demonstrate that the M. leprae 45-kDa protein is a potent T-cell antigen and that it may have diagnostic potential.
Tuberculoid leprosy patients showed the greatest T-cell responses to the M. leprae 45-kDa antigen, and, as 13 of 14 of the tuberculoid leprosy patients tested responded to the 45-kDa antigen, it may contain one or more genetically permissive or promiscuous T-cell epitopes. Some lepromatous leprosy patients also showed good T-cell responses to M. leprae and to the M. leprae 45-kDa antigen. Although lepromatous leprosy patients are generally anergic to M. leprae (10), they can show T-cell responses to individual M. leprae antigens (18, 26). The 45-kDa antigen was similar to the secreted M. tuberculosis Ag85 (30/31-kDa) proteins in its capacity to stimulate T-cell responses in leprosy patients and contacts. Secreted proteins often appear to be immunodominant in the T-cell response to M. tuberculosis (3), and, in leprosy, the 30/31-kDa Ag85 antigen has been shown to induce strong responses in leprosy contacts (15). The high response rates to the 45-kDa antigen reported here are higher than those reported in Ethiopia using the L1 or L4 fragment of the 45-kDa antigen (20). Another study in Indonesia (12) showed that the histidine-tagged M. leprae 45-kDa fusion protein only induced positive responses in 3 to 8% of the leprosy patients and contacts tested. Further work is required to assess if the 45-kDa antigen gives stronger T-cell responses in some ethnic groups than others.
T-cell responses to the M. leprae Trx and TR antigens were also assessed using recombinant antigens expressed and purified in the same way as the 45-kDa antigen. M. leprae is unusual in that these proteins are expressed as a single fusion protein (33). Both of these antigens were recognized by a proportion of leprosy patients and household contacts but not by controls, indicating the presence of M. leprae-specific epitopes. Trx and TR could therefore also have potential as diagnostic reagents, although overall the responses induced were less strong than those stimulated by the 45-kDa antigen.
An important aim of this study was to assess the potential of the
M. leprae 45-kDa antigen as a diagnostic reagent. PBMC
proliferative and cytokine responses of patients were compared with
those of household contacts of leprosy patients and individuals living in a leprosy-endemic area. The leprosy contact group, generally considered at increased risk of developing leprosy (7),
showed very high responses to the M. leprae 45-kDa antigen.
Follow-up studies would be required to determine whether those contacts making poor T-cell responses to the 45-kDa antigen are at greater risk
of developing leprosy, as proposed for poor IFN- responders to
M. leprae sonicate (24). Similarly high
proportions of contacts have been reported to respond to other leprosy
antigens such as the 35-, 18-, and 10-kDa antigens (6, 15,
28) and the Mycobacterium bovis BCG 30/31-kDa antigen
(15). The M. leprae 65-kDa antigen gave lower
responses, although the proportions of responders differed in other
studies (2, 9, 15).
One of the functions of a skin test reagent is to differentiate individuals exposed to M. leprae from those merely living in areas of endemicity. Only 10% of controls responded to the 45-kDa antigen compared with the 100% that responded to M. leprae sonicate and 55% that responded to the 30/31-kDa antigen, both of which contain cross-reactive T-cell epitopes. Southern hybridization with a DNA fragment of the 45-kDa protein gene failed to identify a similar gene in M. tuberculosis or eight other atypical mycobacteria (20), although several hypothetical protein homologues in M. tuberculosis have now been identified (e.g., Rv0442c and Rv2108 [4]).
In conclusion, the data presented here show that the 45-kDa protein is
a potent T-cell antigen capable of inducing PBMC proliferation and
IFN- production in leprosy patients. The 45-kDa antigen was strongly
recognized by T cells from leprosy patients and contacts but gave weak
or negative responses in controls and patients with tuberculosis. This
suggests that the M. leprae 45-kDa antigen may be of use as
a diagnostic reagent, either in a skin test or a simple whole-blood
IFN- assay (30).
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ACKNOWLEDGMENTS |
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This study was supported by a contract from the European Union (TS3*-CT94-0299) and by The Netherlands Leprosy Relief Association (NSL).
A.M. and R.M.-G. contributed equally to this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel St., London WC1E 7HT, United Kingdom. Phone: 44 20 7927 2466. Fax: 44 20 7637 4314. E-mail: Hazel.Dockrell{at}lshtm.ac.uk.
Present address: University College London Hospitals, Department of
Dermatology, London W1N 8AA, United Kingdom.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Clinical and Diagnostic Laboratory Immunology, May 2001, p. 604-611, Vol. 8, No. 3
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.3.604-611.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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