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Infection and Immunity, December 1999, p. 6663-6669, Vol. 67, No. 12
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Differential Decline in Leishmania Membrane
Antigen-Specific Immunoglobulin G (IgG), IgM, IgE, and IgG Subclass
Antibodies in Indian Kala-Azar Patients after
Chemotherapy
Khairul
Anam,1
Farhat
Afrin,1
Dwijadas
Banerjee,2
Netai
Pramanik,2
Subhasis K.
Guha,2
Rama P.
Goswami,2
Shiben K.
Saha,2 and
Nahid
Ali1,*
Leishmania Group, Indian Institute of
Chemical Biology,1 and Department of
Tropical Medicine, School of Tropical Medicine,2
Calcutta 700032, India
Received 22 July 1999/Accepted 31 August 1999
 |
ABSTRACT |
Pathogenesis in kala-azar is associated with depressed cellular
immunity and significant elevation of antileishmanial antibodies. Since
these antibodies are present even after cure, analysis of the
parasite-specific isotypes and immunoglobulin G (IgG) subclasses in
kala-azar patients may shed new light on the immune responses during
progression and resolution of infection. Using leishmanial membrane
antigenic extracts, we investigated the relative levels of specific
IgG, IgM, IgA, IgE, and IgG subclasses in Indian kala-azar patient sera
during disease, drug resistance, and cure. Acute-phase sera showed
strong stimulation of IgG, followed by IgE and IgM and lastly by IgA
antibodies. IgG subclass analysis revealed expression of all of the
subclasses, with a predominance of IgG1 during disease. Following
sodium stibogluconate (SAG) resistance, the levels of IgG, IgM, IgE,
and IgG4 remained constant, while there was a decrease in the titers of
IgG2 and IgG3. In contrast, a significant (2.2-fold) increase in IgG1
was observed in these individuals. Cure, in both SAG-responsive and
unresponsive patients, correlated with a decline in the levels of IgG,
IgM, IgE, and all of the IgG subclasses. The stimulation of IgG1 and
the persistence, most importantly, of IgE and IgG4 following drug
resistance, along with a decline in IgE, IgG4, and IgG1 with cure,
demonstrate the potential of these isotypes as possible markers for
monitoring effective treatment in kala-azar.
| |
INTRODUCTION |
Human visceral leishmaniasis (VL),
or kala-azar, a systemic fatal disease, is caused by Leishmania
donovani, an intracellular protozoan parasite that infects and
multiplies in the macrophages of the spleen, liver, bone marrow, and
lymph nodes. The disease is associated with severe immunosuppression as
evidenced by the failure to respond to L. donovani antigens
in terms of delayed-type hypersensitivity, lymphoproliferation, and
interleukin-2 (IL-2) and gamma interferon (IFN-
) production in vitro
(13, 15, 37, 40). Enhanced induction of IL-10 and/or IL-4
mRNA in tissues and elevated levels of IL-4, IL-10, and IgE over
IFN- in serum (20, 26, 28, 46, 48) suggest that a
dominant Th2 response suppresses the activity of Th1 during disease.
With successful drug therapy, T-cell proliferation and IL-2 and IFN-
production in response to Leishmania antigen are restored
(13, 40). Cured individuals, however, show
Leishmania-reactive T cells with Th1- and Th2-type
lymphokines coexisting after infection (7, 26, 27). Thus,
the heterogeneous set of cytokine responses provoked by kala-azar
during disease and resolution of infection reflects a complex Th1-Th2
cell picture that is difficult to delineate for indicators of clinical improvement.
VL is also marked by high levels of Leishmania-specific
antibodies (10, 34) which appear soon after infection and
before the development of cellular immunologic abnormalities. While the antibody titers in kala-azar have been exploited for specific diagnosis, their role in resolution of disease and protective immunity
is largely unknown. It is, however, evident that resistance in a large
population of individuals residing in areas of endemicity is detectable
only by the development of specific antibodies and/or T-cell response
to leishmanial antigens (17, 29, 40). In our attempts to
induce protection against L. donovani infection in BALB/c
mice, we have demonstrated the involvement of cell-mediated and humoral
immune responses in resistance against the disease (2, 4).
Analysis of the immunoglobulin G (IgG) subclasses revealed preferential
stimulation of IgG1 in infected mice and of IgG2a and IgG2b in
protected mice (2, 3). A study of Leishmania
membrane antigen (LAg)-specific Ig isotypes in Indian kala-azar
patients revealed the elevation of IgG, IgM, IgE, and IgG subclass
antibodies, with IgG3 being specifically associated with this disease
(5). In this study, we report the LAg-specific Ig isotypes
and IgG subclasses in the sera of kala-azar patients during active
disease, drug unresponsiveness, and successful cure, to establish a
correlation of these isotypes with progression and resolution of
infection. These observations may have important implications for
vaccine development as well as noninvasive assessment of the success of
treatment of visceral leishmaniasis. Since resistance to
pentavalent antimonials, the mainstay of VL chemotherapy, is on
the rise, especially in India (25, 46), the prediction of
clinical relapse would be desired.
| |
MATERIALS AND METHODS |
Study subjects.
The subjects of the present investigation
were 5- to 70-year-old VL patients (n = 15) living in areas
of eastern India, where kala-azar is endemic. The patients (5 females
and 10 males) were admitted to the School of Tropical Medicine,
Calcutta, India. Diagnosis of the disease and drug unresponsiveness
were confirmed parasitologically by the presence of
Leishmania amastigotes in spleen and/or bone marrow
aspirates. Blood was obtained after diagnosis, before the initiation of
chemotherapy, posttreatment, and after cure. Treatment with 20 injections of sodium stibogluconate (SAG), the first-line drug (20 mg/kg of body weight), led to successful cure in 10 patients, whereas
five failed to respond to SAG and were retreated with the second-line
drug, amphotericin B (seven injections; 1 mg/kg of body weight). Serum
samples were taken from each of the 15 patients at least twice: on day
0 (i.e., before initiation of therapy) and 50 days after successful
treatment or 45 days after unsuccessful treatment with SAG. Samples
from the latter five patients were taken again at 75 days following successful treatment with amphotericin B. A total of 35 different samples obtained were studied in two groups. All patients had given
informed consent to participate in this study.
Antigen preparation.
L. donovani AG83, originally
isolated from an Indian kala-azar patient, was cultured in vitro for
antigen preparation as described earlier (2). Briefly,
stationary-phase promastigotes, harvested after the third or fourth
passage, were washed four times in ice-cold 0.02 M phosphate-buffered
saline, pH 7.2 (PBS), and suspended at a concentration of 1.0 g of
cell pellet (ca. 5 × 1010 stationary-phase
promastigotes) in 50 ml of cold 5 mM Tris-HCl buffer, pH 7.6. The
suspension was vortexed six times for 2 min each on ice with 10-min
intervals in between and centrifuged at 2,310 × g for
10 min. The crude ghost membrane pellet thus obtained was resuspended
in 10 ml of the same Tris buffer and sonicated three times for 1 min
each on ice in an ultrasonicator. The suspension was finally
centrifuged at 4,390 × g for 30 min, and the
supernatant containing the LAg was harvested and stored in aliquots at
70°C until use. The amount of protein obtained from 1.0 g of
cell pellet, as assayed by the method of Lowry et al. (31),
was 16 mg.
ELISA for parasite-specific Igs.
Enzyme-linked immunosorbent
assay (ELISA) of IgG, IgM, IgA, IgE, and IgG subclass antibodies to LAg
was carried out on polystyrene round-bottom microtiter plates (Tarsons)
as described earlier (5). LAg extracted from L. donovani was applied to the plates at 20 µg/ml in 0.02 M
phosphate buffer (pH 7.5) and incubated at 4°C overnight. After the
plates were washed three times with PBS supplemented with 0.05% Tween
20, excess reactive sites were blocked with 1% bovine serum albumin
for 3 h at room temperature, washed as before, and subsequently
incubated overnight at 4°C with the kala-azar sera serially diluted
in PBS containing 1% bovine serum albumin. After washing,
peroxidase-conjugated goat polyclonal antibodies directed against human
IgG, IgM, IgA, and IgE (Sigma Immunochemicals, St. Louis, Mo.) were
applied at a 1:5,000 dilution in PBS for 3 h at room temperature.
After four washes, o-phenylenediamine dihydrochloride was
applied as an enzyme substrate for 45 min, and the optical density was
read at 492 nm in an ELISA reader.
For the determination of human IgG subclass antibodies, the LAg-washed
wells incubated with serially diluted kala-azar sera were treated with
mouse anti-human IgG subclass-restricted monoclonal antibodies (Sigma
Immunochemicals) at a 1:3,000 dilution for 3 h at room
temperature. After three washes, peroxidase-conjugated goat anti-mouse
IgG (Sigma Immunochemicals) was applied at a 1:5,000 dilution overnight
at 4°C. The color reaction was carried out as described above, and
the optical density was read at 492 nm. Titers were determined from the
extensive titration of each serum sample as the dilution of serum
required to reach half-maximal absorbance
(A492 = 1.0).
Western blot analysis.
After sodium dodecyl sulfate-10%
polyacrylamide gel electrophoresis, the LAg was electrophoretically
transferred to nitrocellulose by using a Transblot apparatus (Bio-Rad
Laboratories). Immunoblot assays were performed by the method of
Rolland-Burger et al. (38) with slight modifications. The
blot was blocked overnight in 100 mM Tris-buffered saline (pH 7.6)
containing 0.1% Tween 20 (T-20) (8), washed once with
0.05% T-20 in Tris-buffered saline (washing buffer), and incubated for
1 h with kala-azar acute-phase, SAG-resistant, or
convalescent-phase serum. Acute-phase and SAG-resistant sera were
diluted at 1:500, and convalescent-phase sera were diluted at 1:100, in
washing buffer. The blots were then washed three times for 20 min each
and incubated for 1 h with 1:500-diluted peroxidase-conjugated
goat anti-human IgG (Sigma Immunochemicals), followed by three washes
as described above. The last wash was done without T-20. Enzymatic
activity was revealed with 15 mg of 3,3'-diaminobenzidine
tetrahydrochloride (Sigma Immunochemicals) in 30 µl of TBS containing
15 µl of 30% H2O2.
Statistical analysis.
All data comparisons were analyzed for
statistical significance with the two-tailed paired Student
t test. Differences were considered significant at
P < 0.05.
| |
RESULTS |
Specific antibody responses of antimony-resistant Indian kala-azar
patients.
Five symptomatic kala-azar patients who failed to
respond to the conventional treatment with pentavalent antimonials were retreated successfully with amphotericin B. The serological profile of
the Ig isotypes and IgG subclasses in this group were studied at 0, 45, and 75 days posttreatment. Major laboratory findings of this
longitudinal study are shown in Fig. 1 and 2 and Table 1. High titers
of anti-LAg IgG antibodies before the initiation of therapy were
reduced insignificantly (Fig. 1A; Table
1) after unsuccessful treatment with SAG.
Individually, three patients exhibited a decrease in the IgG titers,
whereas two showed an increase in the levels. After successful cure
with amphotericin B, however, the IgG antibodies in all of the patients
were significantly lowered, demonstrating a 96% reduction in the
levels in comparison to the unresponsive state (P < 0.05). Despite the decrease, strong IgG titers (1,350 ± 969)
persisted even after cure. In contrast, very low levels of LAg-specific
IgA isotypes were detected in untreated VL patients, with almost no
change after SAG and amphotericin B treatment and after cure (Fig. 1C;
Table 1). Low levels of IgM observed during disease on average remained
constant after unsuccessful SAG treatment, although some variation was
observed from patient to patient. Following treatment with amphotericin B, the IgM levels in all of the patients were reduced, and the average
reduction was statistically significant (Fig. 1B; Table 1). Levels of
IgE observed during disease remained almost constant after SAG
treatment, with only one patient showing a twofold increase in the
titer (Fig. 1D). After cure, however, a decrease in the level of IgE
was observed in all of the patient sera, and the percent reduction was
most significant for IgE (P < 0.001) (Table 1) in
comparison with other Ig isotypes.
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FIG. 1.
Levels of LAg-specific IgG (A), IgM (B), IgA (C), and
IgE (D) in the sera of Indian kala-azar patients before treatment and
following SAG unresponsiveness and cure. Symbols represent individual
patients, and the same symbols indicating the same patients are used in
Fig. 2. The heavy line represents the mean (± standard error) of the
Ig isotype level for five individuals.
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TABLE 1.
Changes in LAg-specific mean IgG, IgM, IgA, and IgG
subclass levels in kala-azar patients following drug resistance
and cure
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|
Analysis of the sera for their patterns of IgG subclass antibodies
reactive with LAg extracted from
L. donovani revealed
significantly
higher levels of IgG1 in comparison to IgG2, IgG3, and
IgG4 in
untreated VL patients (
P < 0.02) (Fig.
2). After unsuccessful
SAG treatment,
IgG1 increased significantly (
P < 0.01), by 2.2-fold
in comparison to the untreated state. The increase in IgG1 titer
was
observed in all five individual patients tested and reached
almost
equivalent levels (Fig.
2A; Table
1). In contrast, the
levels of IgG2
and IgG3 were reduced significantly (Fig.
2; Table
1). Of the five sera
tested, only one patient serum showed an
increase in the IgG2 and IgG3
levels after SAG treatment. The
anti-LAg IgG4 response, however,
remained almost constant with
antimony therapy, showing minimal
variation from patient to patient.
Following successful cure with
amphotericin B, the levels of all
of the IgG subclasses were reduced
significantly, with maximum
declines in IgG3 and IgG4 antibodies
(
P << 0.001). Nevertheless,
significant LAg-specific IgG1
titers (606 ± 499) persisted in
clinically cured individuals.
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FIG. 2.
Specific IgG subclass levels in kala-azar patient sera
before treatment and following SAG resistance and cure. The symbols and
the heavy line are as described for Fig. 1.
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|
Serological responses of VL patients successfully cured with
SAG.
Of the 15 patients investigated in this study, 10 responded
to the standard regimen of antimony with good clinical results and
parasitological cures. As observed earlier, before treatment was
started, all of the patients tested had strong anti-LAg IgG antibodies.
After cure, the level of IgG dropped significantly in all of the
patient sera (Fig. 3A; Table 1) but still
remained measurable (1,971 ± 834). A significant fall in IgE and
IgM levels was also observed in all patients except one, who showed an
increase in IgM titer (Fig. 3B; Table 1) at the end of treatment. In
contrast, the comparatively lower level of anti-LAg specific IgA
remained steady before and after therapy with SAG (Fig. 3C). Cure with antimony also corresponded with the most significant decline (97%; P << 0.001) in IgE antibodies.
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FIG. 3.
LAg-specific IgG (A), IgM (B), IgA (C), and IgE (D)
serum antibodies of kala-azar patients before and after SAG therapy.
Symbols represent individual patients, and the same symbols depict the
same patients in Fig. 4. The heavy line represents the mean (± standard error) of the Ig isotype level for 10 samples.
|
|
Analysis of the IgG subclass antibodies of the paired pre- and
posttreatment serum samples of 10 patients successfully cured
with SAG
revealed a significant decrease (
P < 0.01) in the
titers
of all of the IgG subclasses (Fig.
4; Table
1). This decline,
observed in
all of the patients, was most significant for IgG4
antibodies (
P
<< 0.001). Low but significant IgG1 titers (548
± 115)
remained after cure.
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FIG. 4.
IgG subclass antibody titers of Indian kala-azar
patients before and after treatment with SAG. The symbols and the heavy
line are as described for Fig. 3.
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|
Immunoblot analysis of L. donovani antigens reacting
with Indian kala-azar sera.
Membrane antigens of L. donovani run on sodium dodecyl sulfate-polyacrylamide gels were
transferred to nitrocellulose for immunoblotting with pre- and
posttreatment kala-azar sera. Serum specimens from two patients, one
unresponsive to antimony and cured with amphotericin B and the other
successfully cured with SAG, were investigated for their reactivity
with LAg. Before therapy, both of the patients showed strong reactions
with more than 17 antigenic components ranging in molecular mass from
24 to 163 kDa (Fig. 5, lanes 1 and 4).
The major reactive components recognized by these untreated samples
were 130, 100, 72, 63, 52, 46, 39, 36, and 31 kDa. The serum specimen
from the patient unsuccessfully treated with SAG reacted with almost
all of the antigenic components recognized before therapy but with an
overall lower intensity (Fig. 5, lane 2). After successful cure, the
major polypeptides recognized by both the sera (130, 72, 63, 46, and 36 kDa) were similar (Fig. 5, lanes 3 and 5) and were fewer in number than the components of LAg reactive with pretreatment sera. However, the
extents of reactivity for both of the serum specimens were variable.
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FIG. 5.
Immunoblots of LAg with sera from untreated and cured
kala-azar patients. Lanes 1 to 3, reactivity with untreated,
SAG-unresponsive, and amphotericin B-cured sera of one kala-azar
patient at 0, 45, and 75 days posttreatment, respectively. Lanes 4 and
5, reactivity with untreated and SAG-cured sera of another kala-azar
patient at 0 and 50 days posttreatment, respectively. The reactivities
with the major bands are indicated on the right.
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| |
DISCUSSION |
Immunologic abnormalities and successful host defense in VL have
been shown to be associated with T-cell responses. The detection of
high titers of antileishmanial antibodies has been exploited mainly for
the development of serological tests for diagnosis, and very little
attention has been given to analysis of antibodies at the isotype and
subclass levels in relation to pathogenesis. Most of the serological
assays developed so far, however, fail to obviate the need for
microscopy as the "gold standard" for diagnosis. Based mainly on
the detection of IgG antibodies, these tests show cross-reactivity with
other pathogens, including those causing malaria, tuberculosis, or
leprosy, which are coendemic with kala-azar, and may remain positive
for cured VL sera for long periods of time (19, 22, 35, 38).
In a recent report we have demonstrated the Ig subclass distribution
and the specificity of IgG3 for the diagnosis of kala-azar
(5). In the present study we report the differential
response of Ig subclass antibodies after successful chemotherapy and
the distinct pattern for drug unresponsiveness in the sera of Indian
kala-azar patients.
Acute infection, as reported earlier (5), correlated with
elevated levels of IgG, IgM, IgE, and IgG subclasses in both SAG-responsive and unresponsive patients. Although some differences in
the levels of these isotypes were observed in the two groups, the
difference was statistically significant for only IgG3 and IgG4, their
titers being higher in the drug-resistant group. Successful drug
therapy demonstrated significant reductions in the levels of IgG, IgM,
IgE, and all IgG subclasses in both groups. This decrease in the
antibody levels was observed in all but one serum sample, for IgM. The
orders for the significance of reduction in the mean levels of antibody
responses, however, were IgE > IgG > IgM > IgA and
IgG4 > IgG1 > IgG3 > IgG2 for the SAG-cured patients
and IgE > IgM > IgG > IgA and IgG1 > IgG4 > IgG3 > IgG2 for the amphotericin B-treated individuals. This
difference arose because of the change in the pattern of the isotypes
following unsuccessful SAG treatment. Antimony unresponsiveness induced varying effects on the levels of IgG, IgA, IgG2, and IgG3 in the different patients, while the response was more consistent for IgG1,
IgG4, and IgE. Average titers revealed insignificant changes in the
levels of IgG, IgM, IgA, IgE, and IgG4 and a significant decrease in
the response of IgG2 and IgG3. The most noticeable effect, however, was
the increase in the titers of IgG1 antibodies observed in all of the
patients, rising to almost equal levels. Although it is not understood
how these changes in the isotype patterns occur from pretreatment to
drug unresponsiveness or cure, it appears that the enhancement of IgG1
after unsuccessful therapy may be a possible marker for the
identification of SAG resistance in VL patients. Since almost all of
the isotypes decrease markedly with resolution of disease, the steady
state of IgE, IgM, and IgG4 in SAG-unresponsive sera may serve as
additional markers for drug resistance. Conversely, successful therapy
could possibly be monitored by the decline especially in IgE and IgG4
and additionally in IgG1 in SAG-unresponsive amphotericin B-treated
individuals. These preliminary results are sufficiently promising to
warrant further validation to determine their potential as markers in kala-azar.
Leishmania-specific elevations of IgG subclasses in VL
patients have been reported for Somali, Sudanese, and Venezuelean
populations. Similar to our observations, Venezuelan and Somali patient
sera had dominant IgG1 antibodies (41, 47). However, in
Sudanese patients there was maximum generation of IgG3 and IgG4
production (18). Again, cure correlated with a decrease in
only IgG3 and IgG1 antibody titers in the Sudanese patient sera. The
discrepancies in these results may be due partly to ethnic variation
and differences in parasitic genotypes and partly to the specificities
of the antibodies for the antigens studied (21, 44). In
order to identify the immunodominant leishmanial proteins in our
antigen preparation and their involvement in the pathological
consequences, follow-up sera from two kala-azar patients were reacted
with immunoblots of LAg. Both of the acute-phase sera showed similar
reactivities to various leishmanial antigens. A number of the major
antigens recognized (130, 100, 72, 63, 52, 46, 39, 36, and 31 kDa) are expressed by different forms of leishmaniasis (9, 12, 14, 30) and recognize VL sera from different geographic regions (6, 23, 32, 33, 36, 38, 42). While SAG resistance did not
induce a major change in the antigenic reactivity, sera obtained after
successful therapy recognized fewer bands in LAg. Of interest is the
disappearance of the 39- and 31-kDa antigens, which were found to be
nonreactive in cured kala-azar patients and asymptomatic subjects by
other workers (32, 43).
Since the immunologic mechanisms underlying the profound stimulation of
the specific Ig isotypes in patients with kala-azar remain undefined,
the modulation of their response with chemotherapy also is not
understood. Cytokines produced by helper T cells have been implicated
in the regulation of isotype switching by activated B cells, and in
mice IFN-, preferentially secreted by the Th1 subset, has been shown
to stimulate the production of complement-fixing IgG2a and IgG3
antibodies (16, 45). The signature cytokines of Th2 cells,
IL-4 and IL-5, are recognized as helpers for B lymphocytes and
stimulate the production of high levels of IgE, IgM, and
non-complement-fixing IgG isotypes such as IgG1 in mice or its
homologue IgG4 in humans (1, 39). In murine models of
Leishmania infections, a Th2-IL-4-IgG1 response has been
associated with susceptibility and a Th1-IFN--IgG2a response has
been associated with protective immunity (2, 3, 11).
Similarly, significant stimulation of IgM, IgE, and IgG4 in kala-azar
patients may be correlated with the elevation of a Th2 response.
Simultaneous elicitation of IgG1, IgG3, and IgG2, however, is difficult
to explain. Since IgG1 and IgG3 have the greater ability to fix
complement in humans and to mediate inflammatory reactions
(24), which is also the principal function of Th1 cells,
elevation of these isotypes may reflect the activity of the Th1 subset.
The occurrence of both Th1 and Th2 subsets of T-helper cells during
active VL has been well documented through the detection of serum and
lesional cytokines (20, 26, 46, 48). Since a distinct
pattern of cytokines has not been identified with drug resistance and
even after cure, assessment of the immune status of patients through
cytokine analysis is still not feasible (7, 27, 28). In
contrast, a steady level of IgM, IgE, and IgG4 following unsuccessful
drug therapy may be correlated with disease persistence during clinical
relapse. The increase in IgG1 reflects its inability to down regulate
the disease-promoting effects of Th2 cells. Resolution of infection
corresponds to a decline most significantly of IgE and IgG4, which
further points to the significance of Th2 cells in the pathogenesis of
kala-azar and its regulation in the control of the disease.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge the patients of the School of Tropical
Medicine, Calcutta, India, who participated to this study. We thank J. Das and D. K. Ganguly, past and present directors, respectively,
of the Indian Institute of Chemical Biology, Calcutta, for supporting
this work.
This work was supported through grants from the CSIR and the DST,
Government of India, and the UNDP/World Bank/WHO Special Programme for
Research and Training in Tropical Diseases. K.A. is a research fellow
supported by ICMR.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Indian
Institute of Chemical Biology, 4, Raja S. C. Mullick Rd.,
Calcutta 700032, India. Phone: 91-33-473-3491/0492/6793. Fax:
91-33-473-0284/5197. E-mail: IICHBIO{at}GIASCL01.VSNL.NET.IN.
Editor:
S. H. E. Kaufmann
| |
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Infection and Immunity, December 1999, p. 6663-6669, Vol. 67, No. 12
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
