Biochemical and Immunological Characterization ofthe Variant ...

INFECTION AND IMMUNITY, JUlY 1986, p. 166-172 0019-9567/86/070166-07$02.00/0 Copyright © 1986, American Society for Microbiology

Vol. 53, No. 1

Biochemical and Immunological Characterization of the Variant Surface Coat Glycoprotein Shed by African Trypanosomes PETER DIFFLEY1* AND DAVID C. STRAUS2

Department

of Biological

Sciences, University of Notre Dame, Notre Dame, Indiana 46556,1 and Department of Medical Microbiology, Texas Tech Health Sciences Center, Lubbock, Texas 794302 Received 16 December 1985/Accepted 28 March 1986

As the variant surface coat glycoprotein (VSG) was shed from Trypanosoma brucei rhodesiense into the blood of infected rats, it was biochemically characterized and compared with VSG that had been purified from trypanosomal homogenates. To determine if VSG was in association with lipid, membranes and lipoproteins in plasma of infected rats (IRP), VSG isolated from plasma (PVSG), and VSG isolated from trypanosomal homogenates (HVSG) were all concentrated by ultracentrifugation and assayed for the presence of VSG by radial immunodiffusion (minimum level of detection, 25 ,ug/ml) and by immunoelectroblots (minimum level of detection, 1 ,Ig/ml). Crimson red was used to detect lipid (minimum level of detection, 10 ,ug per sample) in electrophoresed samples. The VSG was neither concentrated with membrane or lipoprotein fractions nor stained by lipid crimson. Lipids from normal rat plasma, IRP, trypanosomal homogenates, HVSG, and PVSG were also extracted and separated by thin-layer chromatography (minimum level of detection, 20 ,ug of trypanosomal phospholipid per sample). The trypanosomal homogenates had five bands as detected by iodine vapors, of which three were phospholipids as detected by molybdenum blue. Both normal rat plasma and IRP had identical patterns of bands with a single phospholipid. The PVSG had one neutral lipid contaminant that apparently was not physically associated with the shed surface coat. The HVSG contained no lipids at all. Therefore, no evidence was obtained to implicate an association between membranes and VSG, once the latter had been shed into the blood of infected hosts. From immunoelectroblots of denatured material, it was determined that both HVSG and PVSG had the same reduced molecular weight. From molecular sieve column chromatography, however, it was determined that VSG released during the homogenization of trypanosomes is a noncovalently linked dimer, whereas that shed in the blood is apparently a trimer. This difference in native structure made no difference in immunological effect. Administered in a regimen that mimicked what the host encounters during a first peak of parasitemia, both HVSG and PVSG induced nonspecific proliferation of splenic lymphocytes and production of unelicited antibodies without the generation of nonspecific immunosuppression. This polyclonal activation of lymphocytes was not the result of contamination by exogenous pyrogen, because the activity was lost if VSG was immunologically absorbed from plasma. Furthermore, no carbon could be detected in hydrolyzed VSG that had been separated by thin-layer chromatography (minimum level of detection, 40 ng of lipopolysaccharide per sample). Finally, the results of the Limulus amebocyte lysate assay indicated that the VSG preparations contained less than 0.5 ng of endotoxin per jig of VSG. Administered in immunogenic dosages, VSG from either homogenate or plasma protected mice to the same degree from challenge with homologous trypanosomes.

The biochemical characteristics and the immunological effects of variant surface coat glycoprotein (VSG) as it exists after the mechanical disruption of Trypanosoma brucei rhodesiense have been studied extensively. It is a singlechained glycoprotein, with a molecular weight between 55,000 and 65,000 (10, 37), which exists primarily as a noncovalently linked dimer on the surface of the bloodstream trypanosome or in solution after disruption of the parasite (3, 38). VSG is an extrinsic protein covalently bound to membrane phospholipids (21). The glycoprotein apparently has a 3-h turnover rate on the bloodstream trypomastigote (5) and is applied (20) and lost (6) over the entire surface in a generalized fashion (i.e., unless the surface coat is cross-linked, no specialized organelles like the flagellar pocket are used, and no functions like capping are observed). The surface coat that is shed into the blood of infected hosts (18, 26, 40) has a half-life of less than 1 h in the blood before it is eliminated by the organs of the reticuloendothelial system (15). Isolated by ion-exchange column chromatography from trypanosomal homogenates (10, 16) *

and injected into mice at dosages encountered during acute infections, VSG causes a nonspecific proliferation of splenic lymphocytes and production of unelicited antibodies (11). At much higher dosages, soluble VSG elicits variant-specific immunity (10, 12, 37). VSGs isolated from trypanosomes that vary in virulence or variant antigen types have the same polyclonal and protective effects (12, 13). Mice that vary in resistance to African trypanosomiasis react differently to VSG treatments (13). The aforementioned immunological analyses were based on the assumption that the VSG isolated from trypanosomal homogenates and the VSG shed into the plasma of infected hosts have the same effects. One purpose of this study was to test that assumption. The second purpose was to biochemically characterize the VSG shed into the blood of infected rodents. All that is known about shed VSG is that it has the same isoelectric point (1) and variant antigenicity (18, 26, 40) that VSG from trypanosomal homogenates has. MATERIALS AND METHODS Source of VSG. T. brucei rhodesiense DTR11.1 is a virulent, pleomorphic African trypanosome. Its history of isola-

Corresponding author.

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VOL. 53, 1986

tion, cloning, cryopreservation, maintenance in rodents (16, 37), and the procedures for isolation of VSG from trypanosomal homogenates (HVSG) and plasma (PVSG) have been described in detail elsewhere (18, 16). Briefly, rats were treated with cyclophosphamide (17) and inoculated intraperitoneally with cryopreserved stabilates. When parasitemias were estimated to be in excess of 109 organisms per ml (23), blood was collected from the heart in sodium citrate (final dilution, 0.6%) and immediately centrifuged (1,500 x g for 10 min at 4°C). The plasma was removed and filtered (0.22-p.m pore size). Plasma from infected rats (IRP) was treated with tosylysylchloromethyl ketone (final concentration, 1 mM) and phenylmethylsulfonyl fluoride (final concentration, 0.4 mM) and either stored at -60°C until assayed or passed immediately through an antibody affinity column consisting of the immunoglobulin G (IgG) fraction of monospecific rabbit anti-VSG antibodies. PVSG was eluted with 5 M guanidine hydrochloride, dialyzed over 2 days against six changes of 10 mM sodium phosphate buffer, pH 8, and further purified by ion-exchange column chromatography. The PVSG was then dialyzed against changes of 0.085% saline for 2 to 3 days, concentrated 10-fold by lyophilization, and stored at -60°C until used. Neither the antibody affinity procedure used for PVSG nor the ionexchange chromatographic purification of HVSG affected the ability of VSG to elicit polyclonal (11) or protective (12) effects. To obtain HVSG, the parasites were separated from blood elements (14), treated with protease inhibitors, lysed, and centrifuged. The HVSG was separated from other trypanosomal proteins by ion-exchange column chromatography. It was then dialyzed and concentrated in the same manner as the PVSG had been. All chromatographic separations and dialysis were conducted under aseptic conditions at 4°C. In all cases, the concentration of protein was measured by the Folin-phenol method, using bovine serum albumin as a standard (28). The amount of VSG was determined by radial immunodiffusion with VSG standards and monospecific rabbit anti-VSG serum (18). The minimum level of detection was established at 25,ug of VSG per ml. The preparations were further tested for contamination with other trypanosomal and host antigens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoelectrophoresis, as previously described (14, 16). Over 98% of the protein in the HVSG preparations and about 40% of the protein in the PVSG preparations were VSG. The presence of endotoxin in VSG was tested with the Limulus amebocyte lysate assay. Following the instructions of the manufacturer (Sigma Chemical Co.), we determined that 1 pg of VSG had less than 0.5 ng of the activity of Shigella flexneri lipopolysaccharide (LPS). The presence of LPS was also tested by thin-layer chromatographic (TLC) separation of hydrolyzed lipids. To do this, we hydrolyzed 1 mg of Escherichia coli LPS (Difco Laboratories), 1.6 mg of HVSG, and 0.3 mg PVSG in 6 N HCI for 18 h at 100°C. Lipids were then extracted in chloroform-methanol and separated by TLC as described below. Hydrolyzed LPS generated two bands in iodine vapors (Rf, 0.74 and 0.91). From titrations of extracted LPS, we determined that the minimum level of detectability of the assay was 40 ng of LPS per sample. No bands appeared in either preparation of VSG, which indicated that endotoxin levels in HVSG if present at all were less than 0.0025% of the preparation. TLC. Lipids were extracted by the method outlined by Dixon and Williamson (19), described in more detail by

CHARACTERIZATION OF VSG SHED INTO PLASMA

167

Radin (34). Briefly, the lipids of 109 viable trypanosomes (separated from plasma proteins and cells), 1.6 mg of HVSG, 0.2 mg of PVSG, 1 ml of normal rat plasma, and 1 ml each of two IRP collections (parasitemias at collection were 8 x 108/ml of blood) were extracted in chloroform-methanol (2:1), filtered through 3-p.m-pore nitrocellulose membranes, and washed in Folch solution (chloroform-methanol-water, 3:48:47) with 0.74% KCI. The lower (organic) phase was allowed to evaporate, and lipids were suspended in chloroform. The medium was applied in 10- to 30-p.l samples to silica-coated glass plates (Sigma) and separated in a chloroform-methanol-water (65:25:4) solvent. Lipids were first detected in iodine vapors; phospholipids were detected by molybdenum blue (Sigma). From titrations of lipids extracted from trypanosomal homogenates (TH), the minimum level of detection was the lipid content of 108 trypanosomes, which contained about 20 ,ug of phospholipids (19). Ultracentrifugation. It has been reported that bloodstream trypanosomes form micelles or plasmanemes in vitro (41) and that they are covalently linked to membranes (21). Therefore, ultracentrifugation was used to determine if PVSG or HVSG were in association with membranes or smaller amounts of lipids. Each procedure was conducted at least twice and was performed in accordance either with instructions of the manufacturer (Beckman Instruments, Inc.) or with published standard procedures (27). To determine if VSG was concentrated with membranes, two 6-ml samples of IRP and HVSG (each 0.5 mg/ml of saline) were centrifuged at 60,000 rpm for 60 min at 18°C in a Beckman 70.1 fixed-angle rotor. The top 1 ml, the middle 4 ml (the cytosol fraction), and the bottom 1 ml with suspended pellet were collected separately and assayed for protein and VSG as described above. To determine if VSG was concentrated with the chylolipid fraction, IRP was centrifuged at 25,000 rpm for 80 min at 18°C in a Beckman 40.1 swinging-bucket rotor. The cloudy top 1 ml (the chylolipid layer), the middle 4 ml, and the bottom 1 ml with suspended pellet were collected separately and assayed for protein and VSG. To determine if VSG was concentrated with very lowdensity lipoproteins (VLDL) or with high-density lipoproteins (HDL), the 4-ml cystosol fraction was divided in half and mixed with 4 ml of either 195 mM NaCl or 195 mM NaCl plus 4.505 M NaBr. The continuous salt gradients were centrifuged at 40,000 rpm for 18 to 24 h at 18°C in a Beckman 70.1 fixed-angle rotor. The cloudy top 1 ml (the VLDL and HDL layers), the middle 4 ml and the bottom 1 ml with suspended pellet were collected separately, dialyzed against 0.085% saline for 24 h, lyophilized, and assayed for protein and VSG. Lipophilic stains. To further determine if lipid was bound to VSG, IRP, the HDL fraction of IRP, HVSG, and PVSG were stained with 1% lipid crimson as previously described (35) and electrophoresed in 3.1 to 7.5% linear-gradient, flatbed native polyacrylamide gels, as described in detail below. Raymond et al. (35) were able to detect less than 10 ,ug of serum lipoprotein per sample by this method. Immunoelectroblots. To compare the charge and size of VSG from plasma with that purified from plasma or from trypanosomal homogenates, IRP, PVSG, and HVSG were electrophoresed in vertical, flatbed, discontinuous native and SDS-PAGE and electrophoretically transferred to nitrocellulose paper (Western blot) according to standard procedures (16, 30, 37, 39). Normal rat plasma served as a negative control. For SDS-PAGE, the preparations were reduced, alkylated, and boiled before electrophoresis (30

168

DIFFLEY AND STRAUS

mA per gel) in 3 to 12% linear-gradient SDS polyacrylamide gels. Bovine serum albumin (67,000 daltons), bovine IgG heavy (50,000 daltons) light chains (23,000 daltons), and ovalbumin (OVA, 43,000 daltons) served as standards for mass. For native gels, the VSG preparations were diluted (2:1) with sample buffer consisting of 15% glycerol and 0.3% bromophenol blue in distilled water and electrophoresed (30 mA per gel) through a 3.125% stacking gel and 7.5% separating gel. After electrophoresis, gels were sandwiched with nitrocellulose paper, and proteins were electroblotted (10 V per gel) overnight in a 20 mM Tris base (150 mM glycine, 20% methanol [pH 8.3], blotting buffer). Unbound reactive sites on the nitrocellulose paper were then blocked with 1% gelatin in Tris-buffered saline (TBS) (20 mM Tris, 500 mM NaCl [pH 7.5]) for 1 h at 37°C. Rabbit anti-VSG serum, diluted 1:50 in 1% gelatin TBS, covered the paper for 90 min at room temperature on a shaker. The paper was then washed with five changes of 0.05% Tween 20 in TBS over a 30-min period on a shaker before adding 0.5% horseradish peroxidase-conjugated goat anti-rabbit IgG (Downington) diluted in 1% gelatin TBS. After the second incubation, the paper was washed as described above and soaked in color reagant (60 mg of 4-chloronaphthol, 20 ml of absolute methanol, 60 [L of 30% H202, 100 ml of TBS) until bands appeared. The paper was washed in cold tapwater and air-dried. From titrations of VSG, the immunoelectroblot could detect 1 ,ug of VSG per ml. Molecular sieve column chromatography. An ascending Sephadex G-200 column was used to determine native molecular weights of VSG in plasma and homogenates. Both trypanosomal extracts and plasma had been centrifuged and frozen but were otherwise untreated before chromatography. IRP (2 ml) was separated at 4°C on a Sephadex G-200 column (2.5 by 85 cm) equilibrated with 10 mM Tris hydrochloride buffer, pH 8 (flow rate, 10 ml/h). The column was standardized with aldolase (158,000 daltons), OVA (43,000 daltons), chymotrypsin (25,500 daltons), and RNase (13,700 daltons). Protein was detected at 280 nm in a flow-through UV light monitor, and eluent was collected in 4.6-ml fractions. The major protein peaks were pooled (peak 1: fraction no. 14 to 25; peak 2: fraction no. 26 to 33; peak 3: fraction no. 34 to 41; peak 4: fraction no. 42 to 54; peak 5: fraction no. 55 to 77; peak 6: fraction no. 78 to 90; and peak 7: fraction no. 91 to 101). VSG was detected in the fractions by gel diffusion (27) with rabbit anti-VSG serum. Pooled fractions positive for VSG (peaks 3 and 4) were either quantified by radial immunodiffusion (10) or rechromatographed under the same conditions, testing each fraction for the presence of VSG by gel diffusion reactions. Trypanosome homogenates (TH) were centrifuged (10,000 x g, 4°C, 30 min), and 20 mg of protein from trypanosomal extract (TE) was separated on the same column under the same conditions. Protein peaks were pooled (peak 1: fraction no. 19 to 30; peak 2: fraction no. 31 to 41; peak 3: fraction no. 42 to 65; peak 4: fraction no. 66 to 72; peak 5: fraction no. 73 to 88; peak 6: fraction no. 89 to 99) and assayed for VSG by gel and radial immunodiffusion. VSG-positive peaks were rechromatographed, and fractions were assayed for VSG by gel diffusion. Immunizations. Female, outbred, 12- to 16-week-old mice (Southern Animal Farms) were immunized with VSG as previously described (12). Briefly, six mice per group were intravenously injected with various concentrations of HVSG or PVSG (250 to 1,000 pLg of VSG per mouse) and challenged at 7 days with intraperitoneal injections of 5 x 103 homologous T. brucei rhodesiense DTR11.1. Infections were mon-

INFECT. IMMUN.

PROCEDURE

RESULTS VSG Protein Ratio

CY-(AT

ug/ml mg/ml (iO13)

CITRATED BLOOD , I 1500xg/10'/4C ID

265

71

3.7

228

61

3.7

156 156

49 61 85

3.2

318 0

2.6 3.7

209 316

6 49 102

0

1

0 356

4 233 1.5

4.3 3.1

40k rpm/ 18-24 h/18C/70.1 in 195mM NaCI vI'

,in 195 mM NaCI &4.5 NaBr hdl

0 0 _ 309

4 2

22

198

1.6

FIG. 1. Ultracentrifugation of PVSG and HVSG. In two experiments, citrated blood from immunosuppressed rats was centrifuged. IRP was collected, filtered, and centrifuged to collect either chylolipids or cytosol. The cytosol was then subjected to a continuous salt gradient ultracentrifugation to collect either HDL or VLDL lipoproteins. The amount of VSG in each fraction was estimated by radial immunodiffusion; the amount of protein was estimated by the Folin-phenol method. All values are an average of the two experiments.

itored by microscopic examination of tail blood for 14 days, and parasitemias were measured at 24-h intervals (23). Polyclonal activation. Female, C57BL/6, 8- to 12-week-old mice (Cumberland View Farms) were treated with VSG in a regimen that mimicked levels encountered by the host during acute infections, as described in detail elsewhere (11). Briefly, two intravenous injections of increasing dosage (6 to 200 ,ug) of HVSG or PVSG were administered twice a day for 3 days. Negative control mice received equivalent amounts of OVA or were left untreated. Because the PVSG preparation contained rat plasma components, a second experiment was conducted to determine if they contributed to the polyclonal effects. This was tested by immunologic absorption of VSG from the preparations, which was found to significantly reduce the polyclonal effects of HVSG (11). Briefly, 10 mg of PVSG protein was absorbed with 2.5 mg of rabbit anti-VSG IgG and 200 ,ul of stock-fixed, protein A-containing Staphylococcus aureus (VSG [V] + antibody [A] + S. aureus [S]) (Calbiochem-Behring). The positive


VOL. 53, 1986

67

Om 50 ".

b*iw--4j*

HVSG PVSG

IRP

NRP

FIG. 2. SDS-PAGE of denatured HVSG and PVSG. NRP or IRP (20 ,ul), PVSG (5 ,ug) isolated from plasma by antibody affinity and ion-exchange column chromatography, and purified HVSG (20 ,ug) were reduced, alkylated, boiled, and electrophoresed in horizontal SDS-PAGE. Proteins were electroblotted onto nitrocellulose paper and immunologically identified with rabbit anti-VSG serum and horseradish peroxidase-labeled goat anti-rabbit IgG serum. The location of molecular weight markers were determined in Coomassie blue-stained duplicate gels.

control preparations contained PVSG mock-absorbed with S. aureus without antibody (V+S), whereas the negative control preparation was antibody with S. aureus and no VSG (A+S). The S. aureus with attached immune complexes was eliminated from the preparations in a Microfuge (Beckman) (6,000 x g for 2 min), and the supernatant fluid was filter sterilized (0.22-,um pore size) before injection. The polyclonal effects of VSG were assayed 5 days after the first injection as described (11). Briefly, to determine if VSG treatment caused splenomegaly, spleens were weighed and disassociated, and nucleated cells were counted in a hemocytometer. Macrophages were counted after ingestion of neutral red; T- and B-cell lymphocytes were identified with fluorescent antibodies. Production of unelicited polyclonal antibodies was measured in a modified Jerne plaque assay with sheep erythrocytes (SE) and trinitrophenylatedSE. Mitogen assays were used to detect nonspecific immunosuppression. Responses of cultured cells to concanavalin A, LPS, and phytohemagglutinin P were assayed by incorporation of tritiated thymidine into DNA.

RESULTS No evidence was obtained in three different approaches to indicate that VSG from TH or shed into plasma was associated with lipids. First, the ratio of VSG to protein did not change after the ultracentrifugation of IRP (Fig. 1), which indicated that VSG was not concentrated with the mem-

169

brane, chylolipid, HDL, or VLDL fractions. When HVSG was centrifuged to collect the cytosol fraction, a gradient was formed that ranged from 0.42 mg/ml in the top 1 ml to 1.14 mg/ml in the bottom ml. No VSG was detected in VLDL and HDL fractions of the cytosol by either radial immunodiffusion or immunoelectroblots. The immunoelectroblots can detect 1 ,ug of VSG per ml; 400 ,ug of PVSG and 1.8 mg of HVSG were ultracentrifuged in the salt gradient to obtain lipoproteins. Therefore, if lipid-associated VSG were present at all, it would represent less than 0.25% of the PVSG preparation and less than 0.05% of the HVSG preparation. Second, lipid crimson did not stain VSG in IRP or the HDL fraction of IRP, PVSG, or HVSG (gels not photographed). Lipid crimson can detect 10 ,ug of lipoprotein in an electrophoresed sample; 400 jig of PVSG and 1.8 mg of HVSG were ultracentrifuged in the salt gradient to generate the lipoprotein fractions. Therefore, if lipid-associated VSG were present, it would represent less than 2.5% of the PVSG and less than 0.5% of the HVSG preparations. Third, no lipids were detected in HVSG by TLC. The TH had 5 bands (Rf, two-run average, 0.32, 0.47, 0.56, 0.77, and 0.90), three of which were identified as phospholipids (Rf, 0.47, 0.56, and 0.77). From a titration of the lipids extracted from TH, we determined that the TLC could detect the lipids of 108 trypanosomes, or about 20 ,ug of phospholipid (19). Since 1.6 mg of HVSG was used for extraction, if lipidassociated VSG were present at all, it would represent less than 2.5% of the preparation. Five lipids were separated from plasma (Rf, average of three samples in one run, 0.41, 0.44, 0.50, 0.61, and 0.88), one of which was a phospholipid (Rf, 0.61). No differences in lipid content between normal and infected plasma were detected. Plasma-derived VSG had a single lipid that did not stain with molybdenum blue and that migrated to a spot (Rf, 0.86) similar to that of plasma lipids and dissimilar to that of TH lipids. Since lipids were extracted from 1 ml of IRP (containing 150 ,ug of VSG) and 250 ,ug of PVSG, if lipid-associated VSG were present at all, it would represent less than 8 to 13% of the preparation. PVSG and HVSG had the same reduced molecular weight, as indicated by immunoelectroblots, as did the denatured IRP, PVSG, and HVSG that had been electrophoresed in SDS-polyacrylamide gels (Fig. 2). In the three VSG preparations, a major band appeared at 55,000 daltons, while

BQG ---Mk

qw

PVSG VSG

0. .:

NRP IRP

FIG. 3. PAGE of native VSG from homogenates and plasma. Concentrations of antigens are the same as given in the legend for Fig. 2. Samples were not denatured before native PAGE. Immunoelectroblotting procedures are the same as was done for SDS-PAGE (see the text). The location of bovine gamma globulin (BGG) was determined by Coomassie blue-stained duplicate gels; bovine serum albumin migrated almost to the dye line and therefore did not appear in this figure.

170

DIFFLEY AND STRAUS

INFECT. IMMUN.

minor bands appeared at 45,000 daltons and at the dye line. Monoclonal antibodies generated against a variable epitope on VSG also detected minor bands, indicating that they were breakdown products of VSG (29, 32-33). These results not only indicated that the monomeric form of VSG was the same in plasma as that from homogenates, it also suggested that the procedures for the isolation of VSG from either plasma or homogenates did not alter the molecule. Electrophoretic mobility of nonreduced proteins through native polyacrylamide gels is a function of charge as well as size. In each of the VSG preparations, bands were immunologically identified as VSG (Fig. 3). Therefore, VSG shed into plasma had the same relative molecular weight and charge as VSG separated from homogenates. To establish the molecular weight of VSG in its native state, molecular sieve column chromatography was used. Protein profiles for the fractionation of TE and IRP are depicted in Fig. 4. Radial immunodiffusion of initially pooled protein peaks measured less than 25 pLg of VSG per ml in peak 4 of IRP and peak 2 of TE. Most of the VSG in IRP was detected in peak 3. In TE, most of the VSG was detected in peak 3. The VSG-positive fractions from IRP and TE were rechromatographed on the Sephadex G-200 column, and VSG-positive fractions (4.6 ml) are superimposed upon the protein profile of the initial fractionation (Fig. 4). Assuming a normal distribution, we calculated that HVSG was about 108,000 M,,1, twice that of the reduced form, and that PVSG was 167,000 M,,.. lThis difference in native structure had no effect upon biological activity. Mice immunized with either HVSG or PVSG were protected to similar degrees against challenge with homologous trypanosomes (Table 1). Because all animals died within 5 to 7 days postinfection, there was no evidence for partial immunity, nor was there time for a heterologous variant to arise. Surface coat isolated either from homogenates or from plasma also had similar polyclonal effects (Table 2). Mice injected with HVSG and PVSG had spleens that weighed 1.5 to 1.8 times more than OVA treated and untreated mice and had about 2 times more nucleated spleen cells (P < 0.025, DALTONS ( 10j

0.08

X

E. c

0

co

43 25 13.7

WHERE VSG []FRACTIONS WAS DETECTED. 16.

IRP 0.04-

158

0-

J *I08

0

10

First peak

50 FRACTION NO.

100

FIG. 4. Molecular sieve column chromatography of HVSG and PVSG. IRP (2 ml) and TE (20 mg) were run (10 ml/h) through a Sephadex G-200 column. Protein peaks were pooled and assayed by gel immunodiffusion for VSG. Positive fractions were rechromatographed, and each 4.6-ml fraction was assayed for presence of VSG. The positive fractions from the second run are superimposed upon the protein profiles of the first run.

Doseb (p.g VSG/mouse)

of

No. dead/total

Mean time of death" (days)

0

6/6

6

Trypanosomal homogenate (HVSG)

1,000 500 250

0/6 2/6 3/6

0 6 6

Plasma (PVSG)

1,000 500 250

0/6 1/6 2/6

0 7 6

Source of VSG

Unimmunized control

HVSG was purified from homogenates by ion-exchange chromatography; PVSG was purified by antibody affinity followed by ion-exchange chromatography. I VSG was injected intravenously. Mice were then challenged with 5 x 10i homologus T. brucei rhodesiense at 7 days postimmunization. " All mice that died during a first peak of parasitemia did so between 5 to 7 days postinfection.

Student t test). No significant differences were detected between the means of HVSG and PVSG treated mice or between OVA treated and untreated mice. The percentages of T cells (35%), B cells (45%), and macrophages (7%) did not vary significantly with treatment, indicating that proliferation occurred among all classes of nucleated splenocytes. The VSG treatments also elicited polyclonal antibodies to SE and TNP-SE without causing nonspecific immunosuppression. No significant differences were detected between the means of HVSG and PVSG treated mice or between OVA treated and untreated mice. If VSG was absorbed from the PVSG preparation (V+A+S), there was little proliferation of splenocytes and a significant reduction in production of polyclonal antibodies. No significant differences in mean spleen cell number or PFC response could be measured between the absorbed preparation and the negative control (A+ S). Mock absorbed PVSG (V+S) did elicit a polyclonal response that was significantly higher than the absorbed VSG (V + A + S) or negative control (A+ S).

DISCUSSION Trypanosomal membranes (presumably with attached surface coat) have been observed to be shed as plasmanemes in vitro (41). Membrane fractions (presumably with attached surface coat) and lipids purified from TH can cause proliferation among lymphocytes, nonspecific immunosuppression, or production of unelicited antibodies, or any combination of these (2, 9). Therefore, it was conceivable that VSG in homogenates and in plasma was associated with membranes and that the biological activity of VSG was in part due to lipids. No evidence, however, was gathered in this study to indicate that VSG was shed as part of a plasmaneme. No lipids were detected by TLC in HVSG. From measurements of the sensitivity of the TLC assay and from the amount of sample applied, if lipid-associated HVSG were present at all, it would represent less than 1% of the preparation. This does not mean that lipid-associated VSG does not exist under certain conditions. Membrane-bound VSG can be obtained by methods other than those used to isolate VSG in this study (21). One lipid was detected in PVSG. This lipid did not appear to be associated with VSG for the following reasons: (i) it was not a phospholipid; (ii) it appeared to be of

W~~~~~~~~~~~

d 0

0.01

TABLE 1. Protective effect of HVSG and PVSG'"


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171

TABLE 2. Polyclonal effects of HVSG and PVSG Treatment"

Avg (±SEM) spleen: No. of cells (106) Wt (mg)

HVSGf PVSGf OVA None

151 181 97 85

V+S' V+A+Sg A+S

189 ± 19 126 ± 17 108 ± 5

±6

± 21 ±6 ±4

130 144 76 72

±8

± 12 ± 10 ±8

127 ± 7 99 ± 5 85 ± 3

Acpm (103)6

Avg PFU/106 cells (±SEM) for: SE

6 7 1 1

± 1

± 1 ± 1 ± 1

14 ± 2 6± 1 3± 1

TNP-SE'

21 ± 18 ± 5± 1

3 2 1 1

ConAd

LPS

PHAe

62 61 54 56

45 65 22 32

31 44 15 17

21± 3 12± 3

8± 1

" Six mice were treated in two runs of three mice per group. VSG was absorbed from PVSG with anti-VSG IgG and fixed S. aureus (V+A+S). The positive control was mock-absorbed VSG with S. aureius (V+S), and the negative control was antibody and S. aureus (A+S). Mean Acpm of a double set of triplicate cultures are represented. TNP-SE, Trinitrophenylated sheep erythrocytes. d ConA, Concanavalin A. e PHA, Phytohemagglutinin P. f Mean (for all but ConA) is significantly higher (P c 0.025) than negative control means. g Mean is signficantly lower (P - 0.025) than mock-absorbed VSG (V+S).

host and not of trypanosomal origin; and (iii) VSG did not concentrate with membrane or lipoprotein fractions during ultracentrifugation. In view of the sensitivity of immunoelectroblots to VSG in lipoprotein fractions, if lipidassociated VSG were present at all, it would represent less than 3% of the preparation. The biological activity of VSG therefore appears to reside in the glycoprotein moiety. Where is the determinant on VSG that elicits polyclonal activation of lymphocytes? There is circumstantial evidence that it is located at the relatively constant carboxy-terminal end of the molecule. Four antigenically dissimilar VSG preparations purified from all three T. brucei subspecies have the same polyclonal effect (11, 13). Polyclonal activation can be induced without eliciting variant-specific immunity (11, 12). Furthermore, although the amino ends of VSG vary significantly in amino acid composition and sequence (8), the carboxy ends share amino acid sequences (24), sugars (25), configuration (36), and antigenic determinants (4, 22, 31). Direct evidence can be gathered if the epitope(s) that elicit variant specific immunity can be separated from the determinants that induce polyclonal activation of lymphocytes. Using cross-linking agents, Strickler and Patton (38) and Auffret and Turner (3) have determined that HVSG is primarily a noncovalently linked dimer. Data gathered from molecular sieve column chromatography and SDS-PAGE in this study support those findings. The VSG shed from the parasite during infection, however, was found to be primarily a trimer. This was not the result of differential handling of VSG. Both the IRP and TE were treated in the same manner before, during, and after molecular sieve chromatography. Perhaps the release of VSG from the membrane in blood is a process fundamentally different from that of mechanical disruption of the parasite. The continuous shedding of surface coat during infections may be similar to that observed when short-stumpy trypomastigotes transform into procyclic (insect midgut) forms (5) or when bloodstream forms change variant antigens (20). The continuous replacement of VSG on the bloodstream trypomastigote may be a multifaceted virulence factor. It is already known that VSG is the source of antigenic variation (10) and induces polyclonal activation of lymphocytes, in a manner that exhausts the immune system (11). It is also known that trypanosomes and VSG fix complement with or without variant-specific antibody (4). Perhaps trypanosomes evade complement-mediated

phagocytosis by constantly replacing their surface coats. The coat is not replaced so quickly, however, as to evade variant-specific immunity (12). The addition of one monomeric unit to the native structure of VSG did not effect its biological activity. At pharmacological dosages, VSG from homogenates and plasma can elicit the same degree of variant-specific immunity. It is unlikely that an infected mouse would come in contact with 500 to 1,000 p.g of soluble VSG (7, 18). Therefore, the likely immunogenic form of VSG during infections is cell bound. Only 10 ,ug of VSG, fixed to SE, is required to elicit 100% protection against homologous challenge (12). Administered in a regimen that mimics what the host encounters during a first peak of parasitemia, VSG from homogenates or from plasma causes proliferation of splenic cells and production of unelicited antibodies. It is unlikely that this activity is caused by a pyrogenic contaminant such as LPS in the VSG preparations for the following reasons. (i) VSG is isolated and maintained aeseptically. (ii) Immunological absorption of VSG reduces biological activity (11; and this study). (iii) VSG treatments of an LPS nonresponder strain of mice (C3H/HeJ) still resulted in polyclonal activation (13). (iv) TLC analysis of hydrolyzed VSG indicated that if LPS were present at all, it would be in concentrations of less than 1 part to 1 x 104 to 4 x 104 parts VSG. (v) The Limulus amebocyte lysate assay indicated that VSG had less than 0.02% of the endotoxic activity of LPS. From this and previous studies (11-18, 26, 40) it can be concluded that VSG shed during infections is one virulence factor that induces polyclonal activation of lymphocytes. Now a system is available to establish the cellular and molecular basis for polyclonal activation as well as for its significance as an immunodysfunction. ACKNOWLEDGMENTS This study was supported by Public Health Service grant AI-22029 from the National Institute of Allergy and Infectious Diseases. We thank Kathleen Rasmussen for her technical assistance with immunoelectroblots and Khuzaima Mama for thin-layer chromatography. LITERATURE CITED 1. Allsopp, B. A., A. R Njogu, and K. C. Humphryes. 1971. Nature and location of Trypanosoma brucei subgroup exoantigen and

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its relationship to the 4S antigen. Exp. Parasitol. 29:271-284. 2. Assouku, R. K., I. R. Tizard, and K. H. Nielsen. 1977. Free fatty acids, complement activation, and polyclonal B-cell stimulation as factors in the immunopathogenesis of African trypanosomiasis. Lancet ii:956-959. 3. Auffret, C. A., and M. J. Turner. 1981. Variant specific antigens of Trypanosoma brucei exist in solution as glycoprotein dimers. Biochem. J. 193:647-650. 4. Barbet, A. F., and T. C. McGuire. 1977. Cross-reacting determinants in variant specific surface antigens of African trypanosomes. Proc. Natl. Acad. Sci. USA 75:1989-1993. 5. Barry, J. D. 1979. Capping of variable antigen on Trypanosoma brucei, and its immunological and biological significance. J. Cell Sci. 37:287-302. 6. Barry, J. D., and K. Vickerman. 1979. Trypanosoma brucei: loss of variable antigens during transformation from bloodstream to procyclic forms in vitro. Exp. Parasitol. 48:313-324. 7. Black, S. J., R. S. Hewett, and C. N. Sendashonga. 1982. Trypanosoma brucei variable surface antigen is released by degenerating parasites but not by actively dividing parasites. Parasite Immunol. 4:233-244. 8. Bridgen, P. J., G. A. M. Cross, and J. Bridgen. 1976. N-terminal amino acid sequence of variant specific surface antigens from Trypanosoma brucei. Nature (London) 263:613-614. 9. Clayton, C. E., D. L. Sacks, B. M. Ogilvie, and B. A. Askonas. 1979. Membrane fractions of trypanosomes mimic the immunosuppressive and mitogenic effects of living parasites on the host. Parasite Immunol. 1:241-249. 10. Cross, G. A. M. 1975. Identification, purification, and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei. Parasitology 71:393-417. 11. Diffley, P. 1984. Trypanosomal surface coat variant antigen causes polyclonal lymphocyte activation. J. Immunol. 131: 1983-1986. 12. Diffley, P. 1985. Trypanosoma brucei: immunogenicity of the variant surface coat glycoprotein of virulent and avirulent species. Exp. Parasitol. 59:98-107. 13. Diffley, P. 1985. Mice varying in resistance to African trypanosomiasis respond differently to treatments with variant surface glycoprotein. Eur. J. Immunol. 15:321-324. 14. Diffley, P., and B. M. Honigberg. 1977. Fluorescent antibody analysis of host plasma proteins on bloodstream forms of African pathogenic trypanosomes. I. Host specificity and time of accretion on Trypanosoma congolense. J. Parasitol. 63: 599-606. 15. Diffley, P., and A. N. Jayawardena. 1982. Surface coat variant antigen of Trypanosoma brucei brucei: its clearance from blood and concentration in organs of normal, infected, and immune mice. Infect. Immun. 35:173-178. 16. Diffley, P., and A. N. Jayawardena. 1982. Comparative analysis of procedures used to isolate variant antigen from Trypanosoma brucei rhodesiense. J. Parasitol. 68:532-537. 17. Diffley, P., and J. 0. Scott. 1984. Immunological control of

chronic Trypanosoma brucei gainbiense in outbred rodents. Acta Trop. 41:335-342. 18. Diffley, P., J. E. Strickler, C. L. Patton, and B. H. Waksman. 1980. Detection and quantification of variant specific antigen in the plasma of rats and mice infected with Trypanosoma brucei brucei. J. Parasitol. 66:185-191. 19. Dixon, H., and J. Williamson. 1970. The lipid composition of blood and culture forms of Trypanosoma lewisi and Trypanosoma rhodesiense compared with that of their environment. Comp. Biochem. Physiol. 33:111-128. 20. Esser, K. M., and M. J. Schoenbechler. 1985. Expression of two variant surface glycoproteins on individual African trypanosomes during antigen switching. Science 229:190-193. 21. Ferguson, M. A. J., and G. A. M. Cross. 1984. Myristylation of the membrane form of a Trypanosoma brucei variant surface

INFECT. IMMUN.

glycoprotein. J. Biol. Chem. 259:3011-3015. 22. Hall, T., and K. Esser. 1984. Topographical mapping of protective and nonprotective epitopes on the variant surface glycoprotein of the WRATat 1 clone of Trypanosoma brucei rhodesiense. J. Immunol. 132:2059-2063. 23. Herbert, W. J., and W. H. R. Lumsden. 1976. Trypanosoma brucei: a rapid matching method for estimating the host's parasitemia. Exp. Parasitol. 40:427-431. 24. Holder, P. J., and G. A. M. Cross. 1981. Glycopeptides form variant surface glycoproteins of Trypanosoma brucei. Cterminal location of antigenically crossreacting carbohydrate moieties. Mol. Biochem. Parasitol. 2:135-150. 25. Johnson, J. G., and G. A. M. Cross. 1977. Carbohydrate composition of variant specific surface glycoproteins of Trypanosoma brucei. J. Protozool. 24:587-591. 26. Lanham, S. M., and A. E. R. Taylor. 1972. Some properties of immunogens (protective antigens) of a single variant of Trypanosoma brucei brucei. J. Gen. Microbiol. 72:101-116. 27. Lindgren, F. T., L. C. Jensen, and F. T. Hatch. 1972. The isolation and quantitative analysis of serum lipoproteins, p. 181-188. In G. J. Nelson (ed.), Blood lipids and lipoproteins: quantitation, composition, and metabolism. John Wiley & Sons, Inc., New York. 28. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265-272. 29. Lyon, J. A., J. M. Pratt, R. W. Travis, B. P. Doctor, and J. G. Olenick. 1981. Use of monoclonal antibody to immunochemically characterize variant-specific surface coat glycoprotein from Trypanosoma rhodesiense. J. Immunol. 126:134-137. 30. Maizel, J., Jr. 1971. Polyacrylamide gel electrophoresis of viral proteins. Methods Virol. 5:179-247. 31. Miller, E. N., L. M. Allen, and M. J. Turner. 1984. Mapping of antigenic determinants within peptides of a variant surface glycoprotein of Trypanosoma brucei. Mol. Biochem. Parasitol. 13:309-322. 32. Olenick, J. G., R. W. Travis, and S. Garson. 1981. Trypanosoma rhodesiense: chemical and immunological characterization of variant specific surface coat glycoproteins. Mol. Biochem. Parasitol. 3:227-238. 33. Pearson, T., and L. Anderson. 1980. Analytical techniques for cell fractions. XXVIII. Dissection of complex antigen mixtures using monoclonal antibody and two-dimensional electrophoresis. Anal. Biochem. 101:377-386. 34. Radin, N. S. 1969. Preparation of lipid extracts. Methods Enzymol. 14:245-255. 35. Raymond, S., J. L. Miles, and J. C. J. Lee. 1966. Lipoprotein patterns in acrylamide gel electrophoresis. Science 151:346-347. 36. Richards, F. F. 1984. The surface of the African trypanosomes. J. Protozool. 31:60-64. 37. Strickler, J. E., P. E. Mancini, and C. L. Patton. 1978. Trypanosoina brucei brucei: isolation of the major surface coat glycoprotein by lectin affinity chromatography. Exp. Parasitol. 46:262-276. 38. Strickler, J. E., and C. L. Patton. 1982. Trypanosomna brucei: Nearest neighbor analysis on the major variable surface coat glycoprotein-crosslinking patterns with intact cells. Exp. Parasitol. 53:117-132. 39. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 40. Weitz, B. 1960. The properties of some antigens of Trypanosoma brucei. J. Gen. Microbiol. 23:589-600. 41. Wright, K. A., W. H. R. Lumsden, and H. Hales. 1970. The formation of filipodium-like processes by Trypanosoma (Trypanozoon) brucei. J. Cell Sci. 6:285-297.