Clin Exp Immunol 1994; 95:294-299

The significance of blood levels of IgM, IgA, IgG and IgG subclassesin Sudanese visceral leishmaniasis patients

A. M. S. ELASSAD*, S. A. YOUNISI, M. SIDDIG*, J. GRAYSON*, E. PETERSEN* & H. W. GHALIB*t*Medical Parasitology Project US NIH/Sudan MRC, tDepartment of Microbiology, College of Medicine, University of Juba,

and IDepartment of Zoology, Faculty of Science, University of Khartoum, Khartoum, Sudan

(Acceptedfor publication 21 September 1993)

SUMMARY

We developed an ELISA test using leishmania antigenic extracts to detect antigen-specific antibodyresponses, including subclass and isotype analysis, in visceral leishmaniasis (VL) patients from theSudan. A total of 92 parasitologically proven patients were compared with cutaneous leishmaniasis,schistosomiasis, malaria, onchocerciasis and tuberculosis patients, as well as with healthy endemicand non-endemic controls. Some VL patients were examined before and after chemotherapy. VLpatients showed significantly higher IgG responses compared with all other groups (93-4%sensitivity, 93 7% specificity), and higher (but not significantly) IgM responses. All groups showedlow IgA levels. All IgG subclasses, IgGl, 2, 3, and 4, showed higher levels in patients than all othergroups, with IgGl and IgG3 levels being significantly reduced following treatment. The rank orderfor specificity and sensitivity for IgG subclasses was IgG3 > IgGl > IgG2 > IgG4.

Keywords leishmaniasis Leishmania donovani ELISA IgG and IgG subclasses

INTRODUCTION

Visceral leishmaniasis (VL), one of the major tropical parasiticdiseases, is caused by an obligate intracellular parasitic protozoaof the Leishmania donovani complex. The disease is associatedwith severe morbidity and is fatal in untreated cases. It isendemic in the eastern and southern parts of Sudan, andfrequent epidemics were reported [1,2].

Definitive diagnosis of VL relies on the detection of theparasite amastigote form in lymph node, bone marrow and/orsplenic aspirates. These procedures are invasive, requiringtechnical expertise that is usually unavailable in isolatedendemic areas. A simple serologic test is still needed, especiallyin epidemic situations where parasitological diagnosis is notpractical. The disease is invariably associated with high leishma-nia-specific antibody levels [3]. The protective role of theleishmania-specific antibodies in human VL is controversial.High IgG and IgM antibody classes associated with comple-ment activation were reported in VL [4]. In animal models theywere associated with protection [5], and in vitro antibody-mediated cytotoxicity to promastigotes and amastigotes [6,7].Also, direct association between high antibody production andprogression of the disease was shown in murine leishmaniasis[8, 9].

Resolution of VL and cure in humans is associated withpredominant Thl response, good cell-mediated immunity(CMI), production of interferon-gamma (IFN-y) and macro-

Correspondence: H. W. Ghalib, PO Box 1270, Khartoum, Sudan.

phage activation [10]. VL disease (Kala-azar) is associated witha Th2 response, poor CMI and hypergammaglobulinaemia. TheTh2 response associated with active disease predominantlyexpresses and produces IL-4 and IL-10 cytokines [11]. IL-4, apredominant Th2 cytokine, has been shown to play an activerole in the progression of B cell activation and switching of theisotype response with a predominant IgE and IgGI productionin the human and mouse models [12,13]. On the other hand,IFN-y (Thl cytokine) down-regulates this activity [12,13].

In this study, an ELISA was developed to detect antigen-specific IgM, IgA, IgG and IgG subclass levels in Sudanese VLpatients. VL patients were compared with cutaneous andmucosal leishmaniasis, schistosomiasis, malaria, onchocercia-sis, and tuberculosis as well as healthy endemic and non-endemic controls. Some VL patients were examined before andafter successful chemotherapy, to examine the pattern andregulation of IgG and IgG subclass antigen-specific responses.Mechanisms underlying regulation of the subclasses will bediscussed in relation to the disease, cure and immune regulationin Th cell subsets.

PATIENTS AND METHODS

Study populationThe study population consisted of 92 parasitologically provenVL patients. They were recruited from two main endemic areas:El Gedarif (Eastern Sudan) and Bentiu (Southern Sudan). Thefoci were previously described by Siddig et al. [14] and Zijlstra et

294

Blood levels ofIgM, IgA, IgG and IgG subclasses in Sudanese VL patients

al. [15], respectively. In 28 VL patients, sera were collectedbefore treatment and at the end of a 1 month treatment regimen.

A total of 171 individuals were included in the study ascontrols. They consisted of: 40 cutaneous leishmaniasis (CL)from Khartoum infected with L. major zymodeme (LON- 1),three mucosal leishmaniasis (ML) from eastern and westernSudan infected with L. donovani s.l., 20 schistosomiasis (SCH)from central Sudan infected with S. mansoni, 20 malaria (MAL)from eastern Sudan infected with Plasmodium falciparum, 19onchocerciasis (ONC) from eastern Sudan infected with 0.volvulus, 19 tuberculosis (TB) patients from Khartoum, 24healthy controls from El GedarifVL endemic area (EN) and 25healthy volunteers from Khartoum, which is non-endemic forVL (NE).

Clinical and parasitological examinationVL patients were selected according to criteria described bySiddig et al. [14]. Diagnosis was confirmed parasitologically bythe demonstration of leishmania amastigotes in impressionsmears obtained from lymph nodes, bone marrow and/orsplenic aspirates in the visceral form and in smears obtainedfrom the lesions in CL and ML. Other forms ofendemic diseaseswere confirmed parasitologically or bacteriologically, as in thecase of TB.

ChemotherapySpecific treatment was given to all patients included in the study.Adults and children with VL were treated daily with sodiumstibo-gluconate (Pentostam, Wellcome Ltd., London, UK) at adaily dose of 10mg/kg and 20 mg/kg body weight intravenously,respectively for 1 month.

Serum samplesBlood samples were collected from all the study groups. Serawere then separated and stored at -40'C until use.

Preparation of antigensLeishmania donovani s.t. zymodeme LON-48 and L. majorzymodeme LON-1 isolates, cultured from Sudanese patients,were used for the preparation of crude water-soluble antigens.The parasites were characterized by D. A. Evans (LondonSchool of Tropical Medicine and Hygiene, UK). Briefly,promastigotes were cultured for 4-5 days in RPMI medium(Sigma, St Louis, MO; no. 4SF-4607-1) supplemented withantibiotics and fetal calf serum (FCS). Cultures were thencentrifuged at 1700 g, washed two or three times in RPMI,sonicated and centrifuged again at 1600 g at 4°C. The proteincontent of the crude water-soluble antigen was assayed by theBiuret method, and the antigen was stored in small aliquots at-70°C until use.

ELISALeishmania-reactive IgM, IgA, IgG and IgG subclass antibodylevels were measured by ELISA using crude water-solubleantigen preparations. The ELISA test was performed accordingto Voller et al. [16].

The optimum working concentrations of antigens, sera andanti-human serum conjugates were determined by checkerboard titration method. Each well ofpolystyrene microtitre flat-bottomed plates (Nunc-immuno Plate 1; Nunc, Roskilde,Denmark) was coated with 1 5 pg ofleishmania antigen in 100 PI

of carbonate-bicarbonate buffer (pH 9-6). The plates wereincubated overnight at 4 C, then washed three times for 5 mineach with normal saline containing 0 05% Tween 20. Freebinding sites were blocked with 100 pl of 1% bovine serumalbumin (BSA) in coating buffer for 1 h at 37°C. The plates werethen washed three times. Sera diluted 1:100 in PBS buffer (pH7 4) with 0-05% Tween 20 were added in 100-pl aliquots usingduplicates for each sample.

Wells containing pooled known positive and negative serawere included. Plates were incubated for I h at 37°C, thenwashed three times. Peroxidase-conjugated goat anti-humanIgG, IgM or IgA (Sigma no. A-8775, Sigma no. A-9290 and ICNno. 61-657-1 respectively) diluted 1: 1500, 1:2000 and 1:2000respectively, were added in I 00-,l aliquots. The plates were thenincubated for 1 h at 37°C. In case of IgG subclasses, mouseanti-human IgG subclass MoAbs (anti-lgGl, ICN no. 63-060;anti-lgG2, 69-061-1; anti-lgG3, 63-062; and anti-lgG4, 63-063)were used at an optimum concentration of 1: 2000. Plates werewashed three times. This was followed by 100 pl/well of anti-mouse IgG horseradish peroxidase-conjugate at 1: 4000 concen-tration. Finally, o-phenylenediamine (OPD) substrate diluted incitrate buffer (pH 5 0) and activated with 0-01% (v/v) H202 wasadded. The reaction was left to proceed in the dark for 20-30min, then stopped by addition of 100 Jul of 20% H2SO4. Thereaction was measured at 490 OD in an ELISA reader(Minireader 11 Dynatch product no. 011, 93-0800). The ODreadings were corrected by applying quality correction equation[16]:

Correcting factor=- fixed OD of reference positivemean OD of test reference positive

A reference positive serum was used in all plates, and onlyinterassay variation of less than 10% was accepted [17]. Thelower limit of positivity (cut off) was determined by the mean ofnegative control +2 s.d. [18].

Statistical analysisUnpaired Student's t-test, ANOVA and Scheffe test wereapplied using SPSS PC+ computer program.

RESULTS

Leishmania-specific antibody level in VL patients and controlgroupsVL patients showed significantly higher IgG responses com-pared with all other groups (93 4% sensitivity, 93-7% speci-ficity) and higher (but not significantly) IgM responses. Allgroups showed low IgA levels (Table 1). The ML patientsshowed higher IgG levels than VL patients, but were notincluded in the analysis due to the low number of subjects in thegroup.

Evaluation ofELISA as a diagnostic testfor VL patientsThe diagnostic power of the ELISA was measured by itssensitivity and specificity. VL patients showed a high significantIgG level (sensitivity of93-4% and specificity of 93-7%) when L.donovani was used as a capture antigen. Only six VL patientswere seronegative. When applying Scheffe test, the IgG responseof the VL patients was significantly different from all othergroups (P < 0-05). The mean level ofIgG in the CL patient groupwas below the cut-off value, and only 13/40 were positive

295

A. M. S. Elassad et al.

Table 1. Mean antibody levels (± s.d.) against Leishmania donovaniantigen of the different studied groups

Antibody levels (mean OD* + s.d.)No. of

IgG IgM IgA patients

VL 0 74+0 37 0 50+0-33 0 04+0 02 92CL 029+0 17 024+0 12 003+001 40ML 1 40+0 19 028+0 13 006+001 3MAL 0 06+0 09 0 43 +0 22 0 03 +0 03 20ONC 0-11+008 031+051 0-02+001 20SCH 0-18+007 0 19+0 19 002+001 20TB 008+007 021 +028 002+002 19EN 0 24+0-24 0 31 +0 15 0 01 +0 01 24NE 0 14+008 036+023 004+0-02 25

Total 290

VL, visceral leishmaniasis; CL, cutaneous leishmaniasis; ML,mucosal leishmaniasis; MAL, malaria; NE, healthy from non-endemicarea; ONC, onchocerciasis; SCH, schistosomiasis; TB, tuberculosis;EN, healthy from endemic area.

*Optical density (OD) read at 490 nm.

(Fig. 1). Eight of the other control individuals were falsepositive, including one MAL, two ONC, one NE and four EN(Fig. 1).

VL patients showed significantly higher IgG levels (96-6%sensitivity, 95-2% specificity) compared with CL patients whenL. major was used as a capture antigen in the ELISA test (Fig. 2).The mean IgG level of the VL group against L. donovani (meanOD = 0-74+ 0-37) was higher than their response to L. major(mean OD = 0-48 + 0-22) antigenic extract.

IgG subclasses profile against L. donovani antigen in VL patientsAll IgG subclasses, IgGl, 2, 3 and 4, showed significantly higherlevels in VL patients than all other groups (P < 0-05) (Fig. 3,

1-8i

1 6

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0-4Cut-off (0.3) t -3---

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Fig. 1. IgG antibody response ofvisceral leishmaniasis (VL) patients andother control groups against Leishmania donovani antigen. Opticaldensity read at 490 nm. The horizontal lines mark the arithmetic mean ofeach group. CL, Cutaneous leishmaniasis; MAL, malaria; ONC,onchocerciasis; SCH, schistosomiasis; NE, non-endemid; EN, endemic.

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VL CL VL CLL. donovani L. major

Fig. 2. Comparison of sensitivity and specificity of the ELISA test invisceral leishmaniasis (VL) patients using the two antigens. *, Sensiti-vity; 0, specificity; VL, visceral leishmaniasis; CL, cutaneous leishma-niasis.

Table 2). IgG3 showed a mean OD level of 0 74+0 34 (100%sensitivity and 97-7% specificity); IgGI had a mean OD level of0-35 + 0-27 (96-4% sensitivity and 95-4% specificity); IgG2 had amean OD level of 0-29+0-29 (89% sensitivity and 94-3%specificity); and IgG4 had a mean OD level of 0 65 + 0 44 (67%sensitivity and 94 3% specificity). The rank order for specificityand sensitivity for IgG subclasses was IgG3 > IgGI > IgG2>IgG4.

Antibody response of VL patients before and after treatmentIgG and IgG subclass levels were determined longitudinally in28 VL patients, before treatment and following 1 monthtreatment with Pentostam. The mean IgG level did not changesignificantly following treatment (Table 3). Scheffe test wasapplied to analyse the significance of variation followingtreatment at the subclass level. IgGI and IgG3 subclasses weresignificantly reduced following treatment (P < 0 05) (Table 3).

Relationship between antibody level, sex, age and locality of thepatientsEleven males and eight females were included in this study. Theywere divided into five age groups: 5-10 years, 11-20, 21-30,31-40 and above 40. These were from two different endemicareas, eastern Sudan (El Gedarif) and southern Sudan (Bentiu).

The antibody response did not vary significantly accordingto sex or age of the patients. IgG, IgG I, IgG2 and IgG3 levelswere significantly higher in El Gedarif patients (eastern Sudan)than Bentiu (southern Sudan) patients. IgG4 levels were notsignificantly different between patient groups from the twoecologically distinct areas. Analysis of variance of the signifi-cance is shown in Table 4.

296

Blood levels ofIgM, IgA, IgG and IgG subclasses in Sudanese VL patients

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I~~~~~- I.

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Fig. 3. Scatter diagrams ofIgG subclasses (a, b, c, d) in visceral leishmaniasis (VL) patients, cutaneous leishmaniasis (CL) patients, otherdiseases, normal from non-endemic area (NE) and normal from an endemic area (EN). Optical density read at 490 nm. The horizontallines mark the arithmetic mean of each group.

Table 2. Specificity and sensitivity of IgG andIgG subclasses in visceral leishmaniasis (VL)

patients

Antibody Specificity (%) Sensitivity (%)

IgG 93 7 93 4IgG1 954 964IgG2 943 89-2IgG3 977 1000IgG4 94 3 67-0

Positive and negative individuals weredetermined according to the ELISA cut-offvalues of 030, 008,002,006 and 033 OD forIgGl, IgG2, IgG3 and IgG4 respectively.

DISCUSSIONVisceral leishmaniasis patients have invariably high antibodytitres [3,19]. Irrespective of these antibodies the patients areunable to clear the infection. Protection from VL and ultimatecure from active disease is associated with good CMI, produc-tion ofIFN-y and macrophage activation [10]. It was shown, in amurine model infected with L. major, that resistance orsusceptibility are associated with two types ofT helper/inducercells differentiated by certain cytokine patterns. Resistant mice

Table 3. The significance of variation in the IgG and IgG subclass levelsof visceral leishmaniasis (VL) patients before and after treatment using

ANOVA and Scheffe test

Pre (n = 28) Post 4 weeks (n = 28) SignificanceImmunoglobulin mean OD + s.d. mean OD + s.d. (at 0 05)

IgG 0 77 +0 35 0 74+0 34 NSIgGI 0-35+0 27 0 20+019 SNIgG2 0 29+0 29 0 21 +0 15 NSIgG3 0 70+0 28 0 50+0 25 SNIgG4 065+044 051 +039 NS

NS, Not significant; SN. significant.

respond with Th 1 response pattern characterized by IFN-yproduction and healing, while susceptible mice respond withTh2 response characterized by predominant IL-4 and IL-10production and progressive disease [20]. Th2 pattern is asso-ciated with IL-4 and IL-10 cytokines, which play a role in B celldifferentiation, proliferation, and isotype regulation [13]. Thehigh, but not protective, leishmania-specific antibody levels seenin the active VL could be due to a mechanism similar to the Thpolarity seen in the mice model. The mechanism underlying theregulation ofthese patterns in human VL is not well understood.

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A. M. S. Elassad et al.

Table 4. ANOVA and Scheffe analysis for the association ofage, sex andlocality of visceral leishmaniasis (VL) patients with the IgG and IgG

subclasses

Age Sex Location

Immunoglobulin F P F P F P

IgG 0-612 0-234 0 291 0 600 5 658 0 035*IgGl 1-176 0 370 0-369 0 555 9-383 0-010*IgG2 0-228 0-917 0-116 0-740 8-168 0-014*IgG3 0-387 0-814 1-113 0-312 4-312 0-045*IgG4 0-572 0-688 0-648 0436 0 567 0-466

* Significant at 0 05 value.

The diagnosis of VL is still based on parasite detection inaspirates from lymph node, bone marrow and/or spleen. Thisparasitologic diagnosis is not sensitive and involves invasiveprocedures. Some VL patients are very anaemic, have coagula-tion disorders and are not fit for these invasive aspirationprocedures. Also, we previously reported failure to detectparasites in some clinically active patients [14,15]. All of thesefactors indicate difficulties associated with parasitological diag-nosis. The development of specific and sensitive serologic assaysor methods to detect parasite DNA using the polymerase chainreaction (PCR) technique will definitely increase the sensitivityofVL diagnosis. In this study, we utilized the characteristicallyhigh leishmania-specific antibody titres in VL to develop asensitive and specific ELISA assay. The use of the ELISA test todiagnose VL using L. donovani crude water-soluble antigenproved to be sensitive and specific. In our study, VL patients hadhigh leishmania-specific IgG levels compared with the othercontrol groups, which is consistent with previous reports inSudanese, Indian, Kenyan and American VL [4,21-23]. The fewreactive individuals in the control groups (malaria, onchocercia-sis and normal endemic control) could be due to an exposure toleishmania parasites. In fact, these individuals came from theeastern Sudan region which is endemic for VL, where they mayhave been exposed or were subclinically infected with leishma-nia at the time of sample collection. Such non-specific reactorsor early seroconverters were also observed in Brazil [24]. Thespecificity and sensitivity of diagnosis were further increasedwhen IgG subclasses were analysed. IgG3 and IgG1 werespecific, while IgG4 was not. Quantitative comparisons betweenisotype assays will be difficult to draw, since reagents may differin their sensitivity [25]. The high sensitivity and specificity of theELISA assay in the IgG3 subclass can be further utilized in thedevelopment of good diagnostic serologic assays for VL. Suchassays can be highly standardized using purified or clonedantigens. The protective role of the high and specific IgG3 andIgGl is not yet defined. These are associated with complexprotein antigen stimuli and hypergammaglobulinaemia [26,27],while IgG2 and IgG4 are associated with secondary antibodyresponse to polysaccharide antigens and protein antigens [28].The high but non-specific IgG4 levels seen in our patients were

also observed in onchocerciasis, bancroftian filariasis andschistosomiasis patients [29-31]. The role of IgG4 in theseparasitic infections is not clear, but was suggested to play a

blocking role in parasite killing and clearance. It has beenrecently reported that two different streptococcal protein anti-

gens elicit antibody response with markedly different subclassprofiles [25]. This indicates that antigens may regulate the typeof isotype or subclass.

Substantial changes were observed in the IgG subclassantibody levels of VL patients following treatment. IgGl andIgG3 followed the same pattern of decline, similarly IgG2and IgG4. A significant decline (P < 0 05) was observed in IgG 1and IgG3 antibody response titres after 4 weeks of treatment.The fall in antibody titres, in particular IgG3 and IgG l, could beused as a serological marker for the determination of cure in VLpatients, as suggested earlier in India and Kenya [32,33].However, long term follow up of patients following successfultreatment is essential to validate these observations.

We measured the total amount of lgG subclasses by radialimmunodiffusion in the serum of some VL patients before andafter treatment, and noticed a decline in their total protein levels(data not shown), which again reflects a down-regulation in thehypergammaglobulinaemia associated with the disease.

The biological significance of this decline is not clear. Theseantibodies could be involved at the site of parasite clearance inthe reticuloendothelial system since they are good complement-fixing antibodies [34] and their Fc regions facilitate immune celladherence; or they could be complexed with antigens released bydestroyed parasite.

The decline in the IgGl and IgG3 subclass level is probablydue to the immune regulation at the T helper cell level.Treatment might have lowered the parasite load and allowed theimmune response to switch from Th2 to Thl cytokine patternresponse. IL-4, the predominant Th2 cytokine, is associatedwith isotype switching of IgE and IgG1, while IFN-y (Thlcytokine) suppresses this activity and enhances IgG2a responsesin mice [12,13]. We observed in parallel studies that VL patientsusually had poor cellular proliferative response to leishmaniaantigen before treatment, which improved significantly at theend of 4 weeks of treatment [35]. These patterns of change inantibody level or CMI suggest regulation at the T helper leveland cytokine production. We have recently reported a pre-dominant in vivo and in vitro production of IL-10 in VLlymphoid tissue and in VL peripheral blood mononuclear cell(PBMC) proliferative response to L. donovani antigen. We alsofound predominant IL-4 in infected lymphoid tissues. Theseimportant Th2 cytokines correlated with pathology related toL. donovani in humans [11]. IL-10 and IL-4 disappeared follow-ing successful treatment, while IFN-y was consistently producedfollowing therapy [11]. This regulation pattern seen in Th2 andTh I cytokine patterns in Sudanese VL patients could beresponsible for the down-regulation of parasite-specific IgG 1

and IgG3 antibody levels.Our results indicated that age and sex had no effect on the

humoral antibody response in all patients studied. We observeddifferences in antibody levels related to geographical locationsin the Sudan. El Gedarif (eastern region) patients had higherantibody titres than Bentiu (southern region) patients. Thisdifference could be related to ethnic and genetic background or

strain variation. In fact, the strain used in the ELISA test was

L. donovani s.l. zymodeme LON 48, which we originally isolatedfrom the eastern region, while the strains prevailing in thesouthern region were found to be dimorphic and includedzymodemes of L. donovani s.l. MON 82 and 30 or MON 18[36,37]. Badaro et al. had indicated similar serological regionaldifferences in Brazilian patients [38].

298

Blood levels ofIgM, IgA, IgG and IgG subclasses in Sudanese VL patients 299

Further studies on the antibody response ofVL patients andimmune individuals at the subclass level will definitely improvediagnostic assays and help to evaluate B cell and T cell responsesassociated with infection or immunity in visceral leishmaniasis.

ACKNOWLEDGMENTS

We thank Professor A. M. El Hassan and Dr S. M. Sulaiman forreviewing the manuscript, and for their valuable suggestions, andAngela Fuhlman for typing. This work was supported by the US NIH/Sudan MRC Medical Parasitology Research Project, NIH grant no.AI-16312.

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