Two drugs, nifurtimox and benznidazole, have been shown to be effective at preventing fatal outcomes in acute- phase patients and at producing parasite ...
Vol. 62, No. 9
INFECTION AND IMMUNITY, Sept. 1994, p. 4072-4074
0019-9567/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Humoral Immune Response to the Trypanosoma cruzi Complement Regulatory Protein as an Indicator of Parasitologic Clearance in Human Chagas' Disease KAREN A. NORRIS,'* LUCIA M. C. GALVAO,1t JANE E. SCHRIMPF,1 JOAQUIM R. CAN(ADO,2 AND ANTONIANA U. KRETFLI3 Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261,1 and Faculdade de Medicina, Universidade Federale de Minas Gerais,2 and Centro de Pesquisas Rene Rachou, Fundapdo Oswaldo Cruz,3 Belo Horizonte, Brazil Received 21 December 1993/Returned for modification 11 February 1994/Accepted 14 June 1994
Immunoprecipitation of the purified 160-kDa complement regulatory protein of Trypanosoma cruzi by Chagas' disease patient sera was examined as a possible correlate of the complement-mediated lysis test and as an indicator of parasite clearance. The results presented demonstrate that assessment of the humoral response to this antigen is a useful indicator of parasite clearance and may be particularly helpful in the assessment of some patients for whom other serological tests produce ambiguous results.
Chagas' disease is endemic to Central and South America, and it is characterized by an acute parasitemic phase followed by a life-long chronic phase in which the paucity of bloodstream parasites makes their direct detection difficult. Parasitemia subsides 1 to 2 months after the initial infection, though conventional serologic tests using crude parasite extracts may remain positive throughout the lifetime of the patients. Two drugs, nifurtimox and benznidazole, have been shown to be effective at preventing fatal outcomes in acutephase patients and at producing parasite clearance in some of these patients (1). Long-term follow-up studies of some treated patients have demonstrated that conventional serologic tests may remain positive for several years in spite of repeated negative direct parasite detection tests, such as hemoculture or xenodiagnosis (2, 3, 7-9). An alternative to conventional serologic testing as an assessment of parasite clearance has been proposed by Krettli et al. (9), who have demonstrated that antibody-dependent, complement-mediated lysis (CML) of parasites by patient sera is a more reliable indicator of ongoing infection. In extensive long-term patient studies, it was observed that sera from patients with positive hemocultures supported lysis of 60% or more of the trypomastigotes in CML tests, whereas sera from patients with repeated negative hemocultures had diminished or no lytic capacity (3, 8, 9). It has therefore been proposed that the presence of lytic antibodies as determined by the CML test may be the best indicator of an ongoing infection and an indication of treatment failure in Chagasic patients (9). This test however requires the use of live, infectious parasites and is therefore not practical in the routine assessment of the chemotherapy and clinical management of Chagasic patients. One subset of anti-Trypanosoma cruzi antibodies which specifically mediates complement lysis is directed to a 160-kDa surface glycoprotein (10, 12). We have previously shown that * Corresponding author. Mailing address: Department of Molecular Genetics and Biochemistry, E1240 Biomedical Science Tower, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. Phone: (412) 648-8848. Fax: (412) 624-1401. Electronic mail address: KAN1@
vms.cis.pitt.edu. t Present address: Departamento de Parasitologia, Instituto de Ciencias Biol6gicas, Universidade Federal de Minas Gerais, Brazil.
the T. cruzi 160-kDa protein functions to restrict alternative and classical complement activation and lysis of the parasites (11). The T. cruzi complement regulatory protein (CRP) binds to the complement proteins C3b and C4b, thus blocking the assembly and accelerating the decay of the C3 convertase, the central enzyme in the complement cascade (11, 13). Antibodies to the purified CRP have been shown to support CML of T cruzi trypomastigotes, presumably by blocking the trypomastigote's complement regulatory activity and allowing full complement activation and parasite killing (11, 12). This has been demonstrated in vitro by the inhibition of the binding of human C3b to the CRP in the presence of anti-CRP antibodies (11). In the present study, we sought to determine if immunoreactivity of patient sera with the CRP correlated with the results of CML tests and might therefore be useful as a specific indicator of drug efficacy and parasite clearance. The purification of the T. cruzi CRP was carried out by C3b-affinity chromatography as described previously (13), with the following modifications. Tissue culture-derived trypomastigotes (strain Y) were harvested from supernatants of infected NIH 3T3 cells grown in Dulbecco's modified Eagle medium (DMEM) (GIBCO) with 10% fetal calf serum (GIBCO), washed twice with phosphate-buffered saline (PBS)-1% glucose, and resuspended at 108 trypomastigotes ml in DMEM with 10 ,ug of ovalbumin per ml. Cells were labeled with [35S]methionine (50 ,uCi/ml) (Trans-label; ICN Biochemicals, Costa Mesa, Calif.) and incubated for 1 h at 37°C. The parasites were washed with PBS with 1% glucose and frozen at -80°C. Membrane preparations were made from frozen parasites at 4 x 108 parasites per ml as described previously (13), except that a solution containing 10 mM Tris-150 mM NaCl (Tris-buffered saline [TBS]; pH 7.5) and 2% Triton X-114 (Boehringer Mannheim) was used as the solubilization buffer. After membrane preparation and solubilization, lysates were centrifuged for 15 min at 13,000 x g, and the supernatant was collected. Detergent phase separation was carried out as described previously (15) by incubation of the supernatants for 30 min on ice followed by incubation for 3 min at 37°C and centrifugation for 3 min at 13,000 x g at 37°C. The aqueous phase was adjusted to 0.05% with Nonidet P-40 (NP-40) (Pierce Biochemical, Rockford, Ill.) and incubated for 1 h at 22°C with an equal volume of washed C3b-Affigel (10 mg of 4072
NOTES
VOL. 62, 1994 TABLE 1. Results of serology, CML test, immunoprecipitation, and hemoculture of Chagasic patients' sera Patient groupa
Patient
CRP CRP e
Hemoculture
-
-
-
-
-
-
-
-
-
% IIF %CMLtiter
tIF
4073
A Pa tient number
1 2 3 4
5
6 7 8
9 10 11 12 13 14
eactivityresult
CRP
Treated
Untreated, chronic phase
Treated; dissociated serum
3 4
0 0 0 0
5
0
-
-
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
0 1 89 77 90 73 85 70 65 2 0 6 1 0 6 0 0 5 2 0 0
-
-
-
1:320 1:320 1:640 1:160
-
-
+
+
+
+
+
+
+
+
1:320 1:160 1:640 1:320 1:320 1:320 1:160 1:640 1:160 1:160 1:640 1:160 1:160 1:40 1:160
+ + +
+ + +
-
-
-
-
-
-
-
-
1 2
a Serum was obtained from untreated, chronic-phase patients and patients treated with benznidazole and/or nifurtimox.
C3b per ml) prepared as described previously (13). The beads were washed three times with TBS-0.05% NP-40. Batch elution was carried out with an equal volume of 0.05 M NaHCO3, pH 10.5, at 22°C. Eluates were immediately neutralized with a 1/10 volume of 1 M phosphate buffer, pH 6.8, and NP-40 was added to a concentration of 0.05%. Immunoprecipitation of the purified CRP was carried out by incubation of 25 ,ul of serum (diluted 1:10 with TBS-0.05% NP-40) with 75 ,ul of CRP for 1 h at room temperature. Fifty microliters of a
1-mg/ml suspension of Staphylococcus aureus Cowan strain I (Sigma Chemical Co., St. Louis, Mo.) in TBS-0.02% NaN3 was added, and shaking was continued for 15 min. Mixtures were cleared by centrifugation for 3 min at 13,000 x g. Pellets were washed three times with TBS-0.05% NP-40 and resuspended in 20 R1 of TBS. Samples were prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography as previously described (11). In the present study, sera from 26 Chagasic patients were collected and processed over a period of 10 years at the Hospital das Clinicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. Serological tests and CML assays were carried out simultaneously on coded sera, and the results were later decoded. CML, indirect immunofluorescence (IIF), and hemoculture tests were carried out as described previously (3), and the results are summarized in Table 1. In all cases in which the CML test was positive, we detected the CRP by immunoprecipitation from membrane preparations (not shown) or from affinity-purified preparations (Fig. 1A, lanes 8 through 14). As previously described (9), treated patients whose sera did not support CML or IIF and failed to produce parasites by repeated hemoculture were considered treatment successes and were presumed cured. In all cases, sera from treated
CML
.+
+
+
+
+
+
+
B Patient number
15 16 17 18 19 20 21 22 23 24 25 26 11 12
CRP CML
-
++
FIG. 1. Immunoprecipitation of purified T. cruzi CRP with Chagasic patient sera. The result of a CML assay using a 1:2 dilution of the serum is indicated below each lane. Immunoprecipitation was carried out with a final serum dilution of 1:100. Patient numbers are those listed in Table 1. Six-day exposures of autoradiographs are shown.
patients who had presumably cleared the parasites failed to precipitate the CRP, in agreement with the CML test results (Fig. 1A, lanes 1 through 7). Studies to assess the efficacy of chemotherapeutic agents in Chagas' disease have led to the observation that approximately 30% of the treated patients maintain positive serological results while parasites are not detected and CML tests are repeatedly negative (3, 8, 9). Patient sera with this reactivity profile have been characterized as dissociated (3, 8, 9). We examined dissociated sera from various patients and found that the loss or diminution of the capacity to precipitate the CRP correlated with a negative CML test and a negative hemoculture, even though conventional serology results (as measured by a high IIF titer) remained positive (Fig. 1B, lanes 15 through 26). In these patients, the IIF titers ranged from 1:40 to 1:640, though the precipitation of the CRP was greatly decreased. Patient samples 11 and 12 in Fig. 1, panel B are the same as serum samples 11 and 12 in panel A and were used as exposure controls. These results show that the immunoreactivity of the patient sera with the CRP antigen correlates with the results of the CML tests. Whereas serologic testing is useful as a diagnostic tool for Chagas' disease, it is not satisfactory for the assessment of clearance and the efficacy of drug treatment (8). Recent work of Gazzinelli and coworkers regarding the mechanisms responsible for the lack of correlation in some cases between conventional serologic data and parasite clearance has begun to clarify this phenomenon (4). They have shown that the continued positive serologic titers in treated and presumably cured patients are the result of cross-reactivity between natural anti-galactosyl a 1-3 galactose antibodies and the epitope galactosyl a 1-3 galactose which is present in the parasite extracts used for conventional serologic tests. Microorganisms of the normal flora as well as many pathogens share this epitope, and it is proposed that bacteria releasing this antigen may continue to stimulate B cells, whose numbers had been initially expanded by the T. cruzi galactosyl 1-3 galactose epitopes, even after the parasites have been cleared. In support of this hypothesis, these authors demonstrated that the absorp-
4074
INFECTr. IMMUN.
NOTES
tion of dissociated patient sera with melibiose, a carbohydrate which contains the epitope recognized by anti-galactosyl 1-3 galactose antibodies, significantly reduced reactivity of the sera to T. cruzi antigens in enzyme-linked immunosorbent assays (ELISAs) (4). Alternatively, anti-idiotypic epitopes which mimic parasite epitopes have been observed and may be responsible for prolonging the immune response in some patients in the absence of continuing parasite antigenic stimulation (5). In either case, it is clear that biochemically and immunologically defined antigens are necessary to provide a definitive assessment of parasite clearance in Chagasic patients. We believe that the T cruzi CRP will be particularly useful in this regard, since this protein is a specific target of antibodies involved in CML of the parasites (12). Recently, it has been shown that the immunoreactivity of patient sera with proteins shed from trypomastigotes correlated with positive CML test results in ELISAs (4). We have previously purified the CRP from shed supernatant proteins (11), and we and others have reported that the shed proteins contain the complement decay activity (6, 11, 14). Taken together, these results indicate that the purified CRP may be useful in an ELISA system such that patient sera, especially those exhibiting dissociated characteristics, could be readily screened. Although the CML test has been shown to be an excellent measure of parasitologic clearance, the test is difficult and not practical for general use, since it requires the careful handling of infectious trypomastigotes. We have previously demonstrated that immunoreactivity to the 160-kDa CRP is the likely basis for the high degree of CML in these assays by using antibodies to the purified protein, and the present results with patient sera support this conclusion. This study was supported by Public Health Service grant A132719 from the National Institute of Allergy and Infectious Diseases and by UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases grant TDR90032.
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