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Trichinella spiralis: Immunization of mice using monoclonal antibody affinity-isolated antigens
EXPERIMENTAL
PARASITOLOGY
Trichinella
59, 398-404 (1985)
spiralis:
Antibody
Immunization of Mice Using Monoclonal Affinity-Isolated Antigens H. R. GAMBLE
Animal Parasitology
Institute,
Agricultural Research Service, U.S. Department Beltsville, Maryland 20705, U.S.A.
(Accepted
for publication
14 January
of Agriculture,
1985)
GAMBLE, H. R. 1985. Trichinella spiralis: Immunization of mice using monoclonal antibody affinity-isolated antigens. Experimental Parasitology 59, 3988404. An antigen epitope was identified from the excretory-secretory products of Trichinella spiralis first-stage larvae using monoclonal antibodies, and the glycoprotein antigens bearing this epitope (Ts.49 and Ts.53) were isolated from the crude excretory-secretory preparation by affinity chromatography. In immunization experiments carried out in mice, antigen priming with Ts.49 and Ts.53 resulted in a reduction of muscle larvae resulting from a challenge infection at a level comparable to priming with crude excretory-secretory antigens. In addition, antigens Ts.49 and Ts.53 induced an accelerated expulsion of adult worms from the intestines of immunized mice and reduced the fecundity of remaining female worms. Q I985 Academic
Press, Inc.
DESCRIPTORS AND ABBREVIATIONS: Trichinellu spiralis; Ncmatodcs, parasitic; Mouse; Immunity, antihelminth, antiadult, antifecundity; Monoclonal antibodies; Antigens, epitopes, parasite, excretory-secretory (ES), affinity-isolated; Larva, first-stage muscle (L,). newborn (NBL); Hank’s balanced salt solution (HBSS); Dulbecco’s modified Eagle’s medium (DMEM); Phosphate-buffered saline (PBS); Enzyme-linked immunosorbent assay (ELTSA) INDEX
Immunity to reinfection with Trichinella can be readily developed in rodents by priming with a complete live infection (Culbertson 1942; Larsh and Race 1954) or abbreviated enteral or parenteral infections (James and Denham 1974, 1975; James et al. 1977; Bell and McGregor 1979a, b). This immunity is expressed by (1) an accelerated expulsion on adults from the intestine, (2) a reduction in numbers of newborn larvae produced by adult females (fecundity), and (3) reduced numbers of larvae accumulating in muscle. Likewise, antigens derived from different stages of the parasite produce immunity expressed against different stages. Immunization with muscle larval extracts or secretory preparations result in some acceleration of adult expulsion, a reduction in fecundity, and a reduction in muscle larvae
spiralis
burdens (Despommicr et al. 1977; Despommier 1981; Despommier and Lacetti 1981a, b). In contrast, immunization with an extract of adult T. spiralis results in a much higher percentage of adults expelled early, but without significant influence on the fecundity of female worms (Gamble 1985). The synergistic effects of combining larval and adult antigens results in high levels of immunity to challenge infection. Efforts to further refine T. spiralis antigens generally have used conventional biochemical separatory techniques (Despommier 1981; Despommier and Lacetti 1981a, b). However, Silberstein and Despommier (1984) have shown that antigens purified from a stichocyte fraction (S,) using monoclonal antibodies induced significant immunity in mice as assessed by reduced muscle larval burdens. Herein is reported immunity in mice gen398
0014-4894/85 $3.00 Copyright All rights
0 19S5 by Academic Prcrr, Inc. of reproduction in any form reserved.
Trichinella
spiralis:
EXCRETORY-SECRETORY
erated using an antigen isolated from T. spiralis L, ES products by monoclonal antibody affinity chromatography, including the effects of this antigen with respect to accelerated adult expulsion and reduced fecundity of female worms. The immunodiagnostic value of this antigen has been reported previously (Gamble and Graham 1984). MATERIALS
AND METHODS
Trichinella spiralis (Beltsville strain) was maintained by serial passage in female Sprague-Dawley rats. First-stage larvae were recovered from infected muscle by pepsin-HCl (1% each) digestion of eviscerated, ground rat carcasses and washed by settling through several changes of water. Adult worms were recovered from rat intestines 72 hr after inoculation. Briefly, intestines from rats inoculated with 6000-8000 L, were recovered, slit lengthwise, rinsed, and placed in a modified Baermann apparatus in HBSS at 37 C. After 2 hr, adult worms were harvested and washed several times in warm HBSS. For recovery of newborn larvae, adult worms were recovered 6 days after inoculation, as above, and incubated in DMEM supplemented with penicillin (100 units/ml) and streptomycin (100 kg/ml) for 24 hr at 37 C in 10% COz and air. Newborn larvae were then recovered by passage through a 60-km mesh screen and washed several times in warm HBSS. Crude homogenates were prepared from 7’. spiralis L, and adult worms by grinding in a Potter-Elvehjem Teflon tissue grinder on ice. Newborn larvae were ground in a glass tissue grinder with ground glass included. All homogenates were centrifuged at 20,000 g for 20 min at 4 C and the supernatants were recovered. Protein concentrations were determined by absorbance at 280 and 260 nm. Antigens were stored at - 20 C until used. ES products were collected from T. spiralis L, by maintenance (5000 worms/ml) in DMEM supplemented with 2 mM glutamine, 10 mM Hepes, and antibiotics (penicillin, 50 units/ml; streptomycin, 50 pg/ ml) (cDMEM) for a period of 18 to 20 hr at 37 C in 10% CO, and air. Culture fluid was harvested by filtration, dialyzed into PBS, and concentrated on a YM5 filter (Amicon) under pressure. Protein concentration was determined as above and antigen stored at - 20 C until used. The production and characterization of the hybridoma cell line designated 7C,C, has been described (Gamble and Graham 1984). The IgM antibody molecule secreted by 7CzC, recognizes an antigen epitope on T. spiralis L, ES proteins with apparent molecular
IMMUNOGENS
399
weights of 45K, 49K, and 53K (Ts.45, Ts.49, Ts.53) as assessed by immunoblots of L, ES proteins separated in sodium dodecyl sulfate-reducing gels and by precipitation reactions in similar gels. Monoclonal antibodies were partially purified from ascites fluid by precipitation in 50% ammonium sulfate, diluted to 2 mgiml in 0.1 M NaHCO, with 0.5 M NaCl (pH 8.3), and coupled to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer’s instructions. The column was preeluted with 0.2 M glycine (pH 2.0) and then equilibrated in 0.1 M Tris (pH 8.0). Antigen consisting of an ES preparation (4 mg in 4 ml) in 0.1 M Tris (pH 8.0) was passed over the column and eluted until the A,,, had returned to the original baseline. Specifically bound antigens were then eluted using a linear gradient from 0.1 M Tris (pH 8.0) to 0.2 M glycine (pH 2.0). Fractions containing the specifically bound peak were pooled, dialyzed into Dulbecco’s PBS (pH 7.2), and concentrated on a YM5 membrane under pressure. Protein concentration was determined by absorbance at 280 and 260 nm. Antigen was stored at - 20 C until used. An ELISA was performed as described previously (Gamble and Graham 1984) with minor modification. Briefly, 96-well microtiter plates (Cooke) were coated with antigen (10 pgiml) in 0.1 M carbonate buffer (pH 9.6) for 1 hr at 37 C. After coating and all subsequent reagent steps, wells were washed three times with PBS containing 0.05% Tween 20 (PBS-Tween). The following reagents were added sequentially (100 kliwell) and incubated for ‘/2 hr at room temperature: hybridoma cell supernatant, goat anti-mouse IgG and IgM (Kirkegaard and Perry) diluted 1:lCOOin PBSTween, and rabbit anti-goat IgG peroxidase conjugate (Sigma) diluted 1:lOOOin PBS-Tween. After the final wash, 100 ~1 of 5’-aminosalicylic acid (0.8 mg/ml) and 0.005% H,O, (pH 5.6) was added to each well. Plates were read on a Titertek Multiscan (Flow) at A,,, after 30 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed essentially by the methods of Laemmli (1970) in 5-20% acrylamide gradients. Samples were reduced in sample buffer containing 5% dithiothreitol by heating to 100 C for 90 sec. Gels were stained for proteins using Coomassie blue R-250 or for glycoproteins using the periodic acid-Schiff method. Groups of 20 female Swiss Webster mice (3-4 months of age) (Taconic Farms; Germantown, NY, USA) were immunized by intraperitoneal injection of antigens emulsified with complete Freund’s adjuvant at a 1:l ratio. Antigen was given in three doses at 2week intervals. The optimal antigen dose for L, ES antigen was previously determined at lo-100 l.tg (Gamble 1985). Antigens Ts.49 and Ts.53 were given in three doses of 5 pg each, which, based on their abundance as the percentage total ES protein recovered from affinity columns (lo%), fell within this optimal dose range. A control group received 0.85%
400
H. R. GAMBLE
NaCl in complete Freund’s adjuvant. One week after the last immunizing dose, all groups were challenged with 150 T. spirulis Lt. Adult worms were recovered 6 days after oral challenge of mice with Lt. Intestines were removed, slit lengthwise, rinsed briefly in HBSS, and cut into 3- to 4-m. lengths. Each intestine then was wrapped in a double thickness of cheesecloth, suspended in a 250ml beaker in HBSS, and incubated at 37 C for 2 hr with periodic agitation, After incubation, the intestines were removed and all but 50 ml of the fluid was aspirated; worms were counted directly in a gridded Petri dish. To determine female worm fecundity, three female worms were selected randomly from each intestinal recovery. Individual female worms were placed into 200 ul of cDMEM in 96-well flat-bottomed microtiter plates (Costar) and incubated for 24 hr at 37 C in 10% CO, and air. Newborn larvae were counted directly on an inverted microscope by superimposing the 96well plate over a gridded template. Muscle larvae were recovered from mice 3.5 days after challenge by digestion of individual, skinned, and eviscerated carcasses in pepsin-HCI (1% each) for 3 hr at 37 C. Aliquots of recovered larvae were counted. Differences among treatment groups were determined by analysis of variance and Duncan’s multiple range test at a probability level ~0.05. Reduction of worm burdens in immunized groups was determined as a percentage of worms recovered from control groups.
TABLE I Reactivity of Monoclonal Antibody 7CzC, with Antigens Derived from Different Life Cycle Stages of Trichinella spiralis Antibody Antigen Crude Larval Crude Crude
larval extract ES products adult extract newborn larval extract
P3X”
7C,C,
0.074b 0.065 0.069 0.068
0.780 0.731 0.089 0.072
0 Supernatant from an unfused myeloma cell line, P3X-Ag8.653, an inherent IgG,-secreting cell line. b Absorbance at 450 nm.
over a 7C,C, monoclonal antibody affinity column, only antigens Ts.49 and Ts.53 were recovered in the specifically bound peak (Fig. 2). These two bands were both doublets with molecular weights of 48SK and 49.5K (Ts.49) and 52.5K and 53.5K (Ts.53). Under nonreducing conditions, additional bands were present in sodium dodecyl sulfate-polyacrylamide gels of the affinity isolated antigens with approximate molecular weights of 92K, 98K, and 104K (Figs. 2 and 3). The appearance of these higher-molecular-weight bands was accomRESULTS panied by a decrease in the intensity of As previously reported (Gamble and Ts.49 and Ts.53. In addition, the molecular Graham 1984), the monoclonal antibody se- weights of the doublets of Ts.53 were recreted by hybridoma 7&C, binds to Tvi- duced by approximately 1000 to 5 1.5K and chinella spiralis L, ES proteins with molec- 52.5K; the molecular weight of Ts.49 was ular weights of 45K, 49K, and 53K, as de- also reduced to approximately 47.5K and termined under reducing conditions. This only a single band was apparent. Antigens Ts.49 and Ts.53 were compared monoclonal antibody does not bind in an ELISA to soluble extracts of adult para- to total L, ES products for ability to induce sites (3 days old) or NBL (Table I), indi- protection against challenge infection in cating the absence of a similar epitope in mice. Immunized animals were challenged and necropsied to determine accelerated these preparations. Total L, ES proteins were separated by expulsion of adult worms, female worm fesodium dodecyl sulfate-polyacrylamide gel cundity, and muscle larval burdens. Six electrophoresis and stained with Coo- days after the challenge inoculation, half massie blue R-250 or by the periodic acid- the animals from each group were killed Schiff method (Fig. 1). Antigens Ts.45, and adult worms were recovered from the Ts.49, and Ts.53 all stained intensely in the intestines (Table II). The crude L, ES anperiodic acid- Schiff reaction, indicating all tigens and antigens Ts.49 and Ts.53 gave comparable levels of protection as assessed three are glycoproteins. When total L, ES products were passed by the numbers of adult worms in the in-
Trichinella spiralis: EXCRETORY-SECRETORY
------_-’
IMMUNOGENS
401
rl L A600 ;\,\---I 1 J‘,,I, L’ \------A 525
FIG. 1. Densitometric scans of sodium dodecyl sulfate-polyacrylamide gels (5-20%) of T~ichinella spiralis L, ES products, separated under reducing conditions, and stained for protein with Coomassie blue R-250 and scanned at 600 nm (solid line). or for glycoprotein by the periodic-Schiff reaction and scanned at 525 nm (dashed line). Antigens Ts.45, Ts.49, and Ts.53 are indicated by arrows.
testine. Female worms recovered from all immunized groups produced significantly fewer NBL than females recovered from control mice (Table II). However, this reduction was significantly greater using the L, ES antigen as compared to Ts.49 and Ts.53. Thirty-five days after challenge, the remaining mice from each group were killed and muscle larvae were recovered (Table II). The L, ES antigens and Ts.49 and Ts.53 produced significant and comparable levels of protection. DISCUSSION
In the present study, the protection-inducing properties of antigens isolated by monoclonal antibody affinity chromatography from the ES products of Trichinella spiralis L, have been examined. The antigen epitope recognized by monoclonal an-
FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel (j-20%) of Trichinella spiralis antigen fractions used to immunize mice. Gel was stained with Coomassie blue R-250. Lane 1, high-molecular-weight standards; lane 2, low-molecular-weight standards; lane 3, T. spirals L, ES products (reduced with 5% dithiothreitol); lane 4, antigens bound by affinity chromatography using monoclonal antibodies from hybridoma 7C,C, (reduced with 5% dithiothreitol); lane 5, antigens bound by affinity chromatography using monoclonal antibodies from hybridoma 7C,C, (unreduced); lane 6, T. spiralis L, ES products (unreduced).
tibodies produced by hybridoma 7&C, is present on three L, ES antigens with apparent molecular weights of 45K, 49K, and 53K, as determined under reducing conditions using several methods (Gamble and Graham 1984). These three antigens are glycoproteins based on their strong reaction when stained by the periodic acidSchiff method. Antigens Ts.45, Ts.49, and Ts.53, or precursor molecules, are present in the stichocyte cells of T. spiralis L, as determined by immunoperoxidase staining, and the antigens are elaborated orally by the parasite (Gamble and Graham 1984). The epitope recognized on these antigens is apparently restricted to the secretory products of the L, since 7C,C, monoclonal antibodies do
402
H. R. GAMBLE
from the intestine and reduced female worm fecundity. Muscle larval accumulation in mice immunized with Ts.49 and Ts.53 was reduced comparably to levels resulting from immunization with crude ES antigens. It is not clear whether any immune killing of NBL occurs after larvaposition; however, the levels of adult expulsion and reduced fecundity obtained here were sufficient to account for the reduced muscle larval burdens in animals immunized with either the crude ES antigens or antigens TS.49 and Ts.53. Other studies have identified antigens from T. spiralis L, using monoclonal antibodies. Ortegas-Pierres et al. (1984) produced two different monoclonal antibodies DISTANCE SCANNED which recognized four different-molecularFIG. 3. Densitometric scans of Trichinella spiralis afweight antigens from the surface of T. spirfinity-isolated antigens Ts.49 and Ts.53 separated in a sodium dodecyl sulfate-polyacryiamide gel (5-20%) alis L,. One of the antibodies (NIM-M2) under reducing (5% dithiothreitol) (A), and nonrebound to 47K and 90K surface glycoproducing (B) conditions (Lanes 4 and 5, Fig. 2). Gel was teins; although the antibody itself did not stained with Coomassie blue R-250 and scanned at 600 bind to the surface of intact L,, it did bind nm. to released ‘251-labeled antigens. Despite the molecular weight similarities between not bind to extracts of either adult worms antigens Ts.45, Ts.49, and Ts.53 and those of Ortega-Pierres et al. (1984) (i.e., glycoor newborn larvae in an ELISA. Using monoclonal antibody 7C2C, af- proteins, similar molecular weight, and refinity columns, only the 49K and 53K an- lease into culture media), there are obvious differences. First, under reducing conditigens were isolated. We have interpreted this to mean that, under nonreducing con- tions, there are no higher-molecular-weight ditions, the common epitope within the 45K bands present in affinity isolations using antigen is unavailable for antibody binding 7C,C, monoclonal antibodies; the bands of molecular weight 92K- 104K appear only and that the molecular weight modificaunder nonreducing conditions. Antigens tions, resulting in the higher-molecularweight species (49K and 53K), expose this 47K and 90K of Ortega-Pierres et al. (1984) do not alter molecular weight patterns epitope to the surface of the molecule. Under nonreducing conditions, the 49K under reducing and nonreducing condiand 53K antigens associate in part to form tions. Second, monoclonal antibody 7C,C, three higher-molecular-weight antigens distinctly stains stichocyte cells (Gamble and Graham 1984) and does not react with (92K, 98K, and 104K). Antigens Ts.49 and Ts.53 were capable the cuticle of T. spiralis L,. The antigens of inducing protection in mice against oral isolated by Ortega-Pierres et al. (1984) are challenge at levels comparable to those ob- apparently of cuticular origin based on iotained using crude ES antigens from the L,. dination procedures, although the antibody Immunization with Ts.49 and Ts.53 re- recognizing these antigens (NIM-M2) does sulted in significant immunity as assessed not bind to the cuticle. Silberstein and Despommier (1984) proby both early expulsion of adult parasites
Trichinella spiralis: EXCRETORY-SECRETORY IMMUNOGENS
403
TABLE II Parasite Recoveries from Mice Immunized with Crude ES Product Antigens from Trichinella spiralis L, or Affinity Isolated Antigens Ts.49 and Ts.53 Treatmenta
Adult wormsb
Newborn larvaeC
Muscle larvae“
Control ES (10 w) Ts.49, Ts.53 (5 pg)
47.3 f 2.7e 27.7 k 3.2f (41) 22.6 k 2.1f (52)
117.4 * 2.@ 74.6 i 3.9” (36) 98.5 t 2.7f (16)
20695 -c 2983e 7168 -c 801f (65) 6100 ‘-’ 1076’(71)
Note. Mice were challenged 1 week after the last immunization with 150 T. spiralis infective L,. a Antigen given three times at 2-week intervals at the doses indicated. b Adult worms recovered 6 days after challenge infection; values are means ? standard error for groups of 10 mice. Duncan ranking is indicated by superscript followed by the percentage reduction in worm numbers compared to the control group. c Newborn larvae produced by female worms recovered from intestines of mice and maintained in culture for 24 hr; values are means + standard error from 30 female worms per group. d Muscle larvae recovered 35 days after challenge infection; values indicated are means f standard error for 10 mice per group.
duced monoclonal antibodies against stichocyte antigens of T. spiralis L,. Two different hybridoma cell lines were identified which recognized stichocyte antigens with molecular weights of 48K (8A4.3.1.1) and 50-55K (7B2.3.1.1). The antigens, isolated by affinity chromatography using both antibodies, produced significant levels of immunity (81 and 59%, respectively) when used to actively immunize mice against oral challenge with T. spiralis Lt. The molecular weights of antigens isolated using monoclonal antibodies from hybridoma 7B2 (5055K) indicate similar binding specificities with monoclonal antibodies derived from hybridoma 7&C, (49K, 53K); both sets of antigens associate under nonreducing conditions to produce higher-molecular-weight proteins. However, antibodies produced by hybridoma 7C,C, recognize an additional antigen (45K) under reducing conditions; no additional antigens are bound by hybridoma 7B2 under reducing conditions. Because of the considerably different procedures used for the isolation of the starting material used in the present study (L, ES products) and that used by Silberstein and Despommier (1984) (S,, the large particle fraction of L, stichocytes), a direct comparison among the antibodies may not be possible until all the antibodies have been
evaluated against the different antigen sources. We are currently attempting to resolve the relationship of antigens Ts.45, Ts.49, and Ts.53 by analysis of the transcribed gene products of the Trichinella spivalis Lt. ACKNOWLEDGMENTS The author thanks Mss. J. S. Baker, E. Moore, and C. E. Graham for technical assistance, and Dr. K. D. Murrell: Dr. M. W. Fleming, and Dr. H. Alizadeh for comments on the manuscript.
REFERENCES BELL, R. G., AND MCGREGOR,D. D. 1979a. Trichinella spiralis: Expression of rapid expulsion in rats exposed to an abbreviated enteral infection. Experimental Parasitology 48, 42-50. BELL, R. G., AND MCGREGOR,D. D. 1979b. Trichineila spin&: Role of different life cycle phases in induction, maintenance, and expression of rapid expulsion in rats. Experimental Parasitology 48, 51-60. CAMPBELL.C. H. 1955. The antigenic role of the excretions and secretions of Trichinellu spirnlis in the production of immunity in mice. Journal of Purusitology 41, 483-491. CULBERTSON,J. T. 1942. Active immunity in mice against Trichinellu spiralis. Journal of Parasitology 28, 197-202. DESPOMMIER,D. D. 1981. Partial purification and characterization of protection-inducing antigens from the muscle larva of Trichinella spiralis by mo-
404
H. R. GAMBLE
lecular sizing chromatography and preparative isoelectric focusing. Purusite Immunology 3, 261-272. DESPOMMIER, D. D., CAMPBELL, W. C., AND BLAIR,
L. S. 1977. The in viva and in vitro analysis of immunity to Trichinella spiralis in mice and rats. Parasitology 74, 109-l 19. DESPOMMIER,D. D., AND LACETTI, A. 1981a. Trichinellu spiralis: Proteins and antigens isolated from a large particle fraction derived from the muscle larva. Experimental Parasitology 51, 279-295. DESPOMMIER:D. D., AND LACETTI, A. 1981b. Trichinella spiralis: Partial characterization of antigens isolated by immuno-affinity chromatography from the large particle fraction of the muscle larvae. Journal of Parasitology 67, 332-339. GAMBLE, H. R. 1985. Induction of immunity to Trichinellu spirdis in mice using antigens derived from different life cycle stages. Journal of Parasitology, Submitted for publication. GAMBLE, H. R., AND GRAHAM, C. E. 1984. Monoclonal antibody-purified antigen for the immunodiagnosis of trichinosis. American Journal of Veterinary Research 45, 67-74. JAMES, E. R., AND DENHAM, D. A. 1974. The stage specificity of the immune response to Trichinellu spiralis. In “Trichinellosis” (C. W. Kim, ed.), pp. 345-351. Intext Educational, New York. JAMES,E. R., AND DENHAM, D. A. 1975. Immunity to
Trichinella spiralis. VI. The specificity of the immune response stimulated by the intestinal stage. Journal of Helminthology 49, 43-47. JAMES, E. R., MOLONEY, A., AND DENHAM, D. A. 1977. Immunity to Trichinella spiralis. VII. Resistance stimulated by the parenteral stages of the infection. Journal of Parasitology 63, 720-723. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature (London) 227, 680-685. LARSH,J. E., AND RACE, G. J. 1954.A histopathologic study of the anterior small intestine of immunized and nonimmunized mice infected with Trichinellu spiralis. Journal of Infectious Diseases 94, 262272. ORTEGA-PIERRES, G., CHAYEN, A., CLARK, N. W. T., AND PARKHOUSE,R. M. E. 1984. The occurrence of antibodies to hidden and exposed determinants of surface antigens of Trichinellu spiralis. Parasitology 88, 359-369. SILBERSTEIN,D. S. 1983. Antigens. In “Trichinella and Trichinosis” (W. C. Campbell, ed.), pp. 309334. Plenum, New York. SILBERSTEIN,D. S., AND DESPOMMIER,D. D. 1984. Antigens from Trichinelln spiralis that induce a protective response in the mouse. Journul of Immunology 132, 898-904.
PARASITOLOGY
Trichinella
59, 398-404 (1985)
spiralis:
Antibody
Immunization of Mice Using Monoclonal Affinity-Isolated Antigens H. R. GAMBLE
Animal Parasitology
Institute,
Agricultural Research Service, U.S. Department Beltsville, Maryland 20705, U.S.A.
(Accepted
for publication
14 January
of Agriculture,
1985)
GAMBLE, H. R. 1985. Trichinella spiralis: Immunization of mice using monoclonal antibody affinity-isolated antigens. Experimental Parasitology 59, 3988404. An antigen epitope was identified from the excretory-secretory products of Trichinella spiralis first-stage larvae using monoclonal antibodies, and the glycoprotein antigens bearing this epitope (Ts.49 and Ts.53) were isolated from the crude excretory-secretory preparation by affinity chromatography. In immunization experiments carried out in mice, antigen priming with Ts.49 and Ts.53 resulted in a reduction of muscle larvae resulting from a challenge infection at a level comparable to priming with crude excretory-secretory antigens. In addition, antigens Ts.49 and Ts.53 induced an accelerated expulsion of adult worms from the intestines of immunized mice and reduced the fecundity of remaining female worms. Q I985 Academic
Press, Inc.
DESCRIPTORS AND ABBREVIATIONS: Trichinellu spiralis; Ncmatodcs, parasitic; Mouse; Immunity, antihelminth, antiadult, antifecundity; Monoclonal antibodies; Antigens, epitopes, parasite, excretory-secretory (ES), affinity-isolated; Larva, first-stage muscle (L,). newborn (NBL); Hank’s balanced salt solution (HBSS); Dulbecco’s modified Eagle’s medium (DMEM); Phosphate-buffered saline (PBS); Enzyme-linked immunosorbent assay (ELTSA) INDEX
Immunity to reinfection with Trichinella can be readily developed in rodents by priming with a complete live infection (Culbertson 1942; Larsh and Race 1954) or abbreviated enteral or parenteral infections (James and Denham 1974, 1975; James et al. 1977; Bell and McGregor 1979a, b). This immunity is expressed by (1) an accelerated expulsion on adults from the intestine, (2) a reduction in numbers of newborn larvae produced by adult females (fecundity), and (3) reduced numbers of larvae accumulating in muscle. Likewise, antigens derived from different stages of the parasite produce immunity expressed against different stages. Immunization with muscle larval extracts or secretory preparations result in some acceleration of adult expulsion, a reduction in fecundity, and a reduction in muscle larvae
spiralis
burdens (Despommicr et al. 1977; Despommier 1981; Despommier and Lacetti 1981a, b). In contrast, immunization with an extract of adult T. spiralis results in a much higher percentage of adults expelled early, but without significant influence on the fecundity of female worms (Gamble 1985). The synergistic effects of combining larval and adult antigens results in high levels of immunity to challenge infection. Efforts to further refine T. spiralis antigens generally have used conventional biochemical separatory techniques (Despommier 1981; Despommier and Lacetti 1981a, b). However, Silberstein and Despommier (1984) have shown that antigens purified from a stichocyte fraction (S,) using monoclonal antibodies induced significant immunity in mice as assessed by reduced muscle larval burdens. Herein is reported immunity in mice gen398
0014-4894/85 $3.00 Copyright All rights
0 19S5 by Academic Prcrr, Inc. of reproduction in any form reserved.
Trichinella
spiralis:
EXCRETORY-SECRETORY
erated using an antigen isolated from T. spiralis L, ES products by monoclonal antibody affinity chromatography, including the effects of this antigen with respect to accelerated adult expulsion and reduced fecundity of female worms. The immunodiagnostic value of this antigen has been reported previously (Gamble and Graham 1984). MATERIALS
AND METHODS
Trichinella spiralis (Beltsville strain) was maintained by serial passage in female Sprague-Dawley rats. First-stage larvae were recovered from infected muscle by pepsin-HCl (1% each) digestion of eviscerated, ground rat carcasses and washed by settling through several changes of water. Adult worms were recovered from rat intestines 72 hr after inoculation. Briefly, intestines from rats inoculated with 6000-8000 L, were recovered, slit lengthwise, rinsed, and placed in a modified Baermann apparatus in HBSS at 37 C. After 2 hr, adult worms were harvested and washed several times in warm HBSS. For recovery of newborn larvae, adult worms were recovered 6 days after inoculation, as above, and incubated in DMEM supplemented with penicillin (100 units/ml) and streptomycin (100 kg/ml) for 24 hr at 37 C in 10% COz and air. Newborn larvae were then recovered by passage through a 60-km mesh screen and washed several times in warm HBSS. Crude homogenates were prepared from 7’. spiralis L, and adult worms by grinding in a Potter-Elvehjem Teflon tissue grinder on ice. Newborn larvae were ground in a glass tissue grinder with ground glass included. All homogenates were centrifuged at 20,000 g for 20 min at 4 C and the supernatants were recovered. Protein concentrations were determined by absorbance at 280 and 260 nm. Antigens were stored at - 20 C until used. ES products were collected from T. spiralis L, by maintenance (5000 worms/ml) in DMEM supplemented with 2 mM glutamine, 10 mM Hepes, and antibiotics (penicillin, 50 units/ml; streptomycin, 50 pg/ ml) (cDMEM) for a period of 18 to 20 hr at 37 C in 10% CO, and air. Culture fluid was harvested by filtration, dialyzed into PBS, and concentrated on a YM5 filter (Amicon) under pressure. Protein concentration was determined as above and antigen stored at - 20 C until used. The production and characterization of the hybridoma cell line designated 7C,C, has been described (Gamble and Graham 1984). The IgM antibody molecule secreted by 7CzC, recognizes an antigen epitope on T. spiralis L, ES proteins with apparent molecular
IMMUNOGENS
399
weights of 45K, 49K, and 53K (Ts.45, Ts.49, Ts.53) as assessed by immunoblots of L, ES proteins separated in sodium dodecyl sulfate-reducing gels and by precipitation reactions in similar gels. Monoclonal antibodies were partially purified from ascites fluid by precipitation in 50% ammonium sulfate, diluted to 2 mgiml in 0.1 M NaHCO, with 0.5 M NaCl (pH 8.3), and coupled to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer’s instructions. The column was preeluted with 0.2 M glycine (pH 2.0) and then equilibrated in 0.1 M Tris (pH 8.0). Antigen consisting of an ES preparation (4 mg in 4 ml) in 0.1 M Tris (pH 8.0) was passed over the column and eluted until the A,,, had returned to the original baseline. Specifically bound antigens were then eluted using a linear gradient from 0.1 M Tris (pH 8.0) to 0.2 M glycine (pH 2.0). Fractions containing the specifically bound peak were pooled, dialyzed into Dulbecco’s PBS (pH 7.2), and concentrated on a YM5 membrane under pressure. Protein concentration was determined by absorbance at 280 and 260 nm. Antigen was stored at - 20 C until used. An ELISA was performed as described previously (Gamble and Graham 1984) with minor modification. Briefly, 96-well microtiter plates (Cooke) were coated with antigen (10 pgiml) in 0.1 M carbonate buffer (pH 9.6) for 1 hr at 37 C. After coating and all subsequent reagent steps, wells were washed three times with PBS containing 0.05% Tween 20 (PBS-Tween). The following reagents were added sequentially (100 kliwell) and incubated for ‘/2 hr at room temperature: hybridoma cell supernatant, goat anti-mouse IgG and IgM (Kirkegaard and Perry) diluted 1:lCOOin PBSTween, and rabbit anti-goat IgG peroxidase conjugate (Sigma) diluted 1:lOOOin PBS-Tween. After the final wash, 100 ~1 of 5’-aminosalicylic acid (0.8 mg/ml) and 0.005% H,O, (pH 5.6) was added to each well. Plates were read on a Titertek Multiscan (Flow) at A,,, after 30 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed essentially by the methods of Laemmli (1970) in 5-20% acrylamide gradients. Samples were reduced in sample buffer containing 5% dithiothreitol by heating to 100 C for 90 sec. Gels were stained for proteins using Coomassie blue R-250 or for glycoproteins using the periodic acid-Schiff method. Groups of 20 female Swiss Webster mice (3-4 months of age) (Taconic Farms; Germantown, NY, USA) were immunized by intraperitoneal injection of antigens emulsified with complete Freund’s adjuvant at a 1:l ratio. Antigen was given in three doses at 2week intervals. The optimal antigen dose for L, ES antigen was previously determined at lo-100 l.tg (Gamble 1985). Antigens Ts.49 and Ts.53 were given in three doses of 5 pg each, which, based on their abundance as the percentage total ES protein recovered from affinity columns (lo%), fell within this optimal dose range. A control group received 0.85%
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H. R. GAMBLE
NaCl in complete Freund’s adjuvant. One week after the last immunizing dose, all groups were challenged with 150 T. spirulis Lt. Adult worms were recovered 6 days after oral challenge of mice with Lt. Intestines were removed, slit lengthwise, rinsed briefly in HBSS, and cut into 3- to 4-m. lengths. Each intestine then was wrapped in a double thickness of cheesecloth, suspended in a 250ml beaker in HBSS, and incubated at 37 C for 2 hr with periodic agitation, After incubation, the intestines were removed and all but 50 ml of the fluid was aspirated; worms were counted directly in a gridded Petri dish. To determine female worm fecundity, three female worms were selected randomly from each intestinal recovery. Individual female worms were placed into 200 ul of cDMEM in 96-well flat-bottomed microtiter plates (Costar) and incubated for 24 hr at 37 C in 10% CO, and air. Newborn larvae were counted directly on an inverted microscope by superimposing the 96well plate over a gridded template. Muscle larvae were recovered from mice 3.5 days after challenge by digestion of individual, skinned, and eviscerated carcasses in pepsin-HCI (1% each) for 3 hr at 37 C. Aliquots of recovered larvae were counted. Differences among treatment groups were determined by analysis of variance and Duncan’s multiple range test at a probability level ~0.05. Reduction of worm burdens in immunized groups was determined as a percentage of worms recovered from control groups.
TABLE I Reactivity of Monoclonal Antibody 7CzC, with Antigens Derived from Different Life Cycle Stages of Trichinella spiralis Antibody Antigen Crude Larval Crude Crude
larval extract ES products adult extract newborn larval extract
P3X”
7C,C,
0.074b 0.065 0.069 0.068
0.780 0.731 0.089 0.072
0 Supernatant from an unfused myeloma cell line, P3X-Ag8.653, an inherent IgG,-secreting cell line. b Absorbance at 450 nm.
over a 7C,C, monoclonal antibody affinity column, only antigens Ts.49 and Ts.53 were recovered in the specifically bound peak (Fig. 2). These two bands were both doublets with molecular weights of 48SK and 49.5K (Ts.49) and 52.5K and 53.5K (Ts.53). Under nonreducing conditions, additional bands were present in sodium dodecyl sulfate-polyacrylamide gels of the affinity isolated antigens with approximate molecular weights of 92K, 98K, and 104K (Figs. 2 and 3). The appearance of these higher-molecular-weight bands was accomRESULTS panied by a decrease in the intensity of As previously reported (Gamble and Ts.49 and Ts.53. In addition, the molecular Graham 1984), the monoclonal antibody se- weights of the doublets of Ts.53 were recreted by hybridoma 7&C, binds to Tvi- duced by approximately 1000 to 5 1.5K and chinella spiralis L, ES proteins with molec- 52.5K; the molecular weight of Ts.49 was ular weights of 45K, 49K, and 53K, as de- also reduced to approximately 47.5K and termined under reducing conditions. This only a single band was apparent. Antigens Ts.49 and Ts.53 were compared monoclonal antibody does not bind in an ELISA to soluble extracts of adult para- to total L, ES products for ability to induce sites (3 days old) or NBL (Table I), indi- protection against challenge infection in cating the absence of a similar epitope in mice. Immunized animals were challenged and necropsied to determine accelerated these preparations. Total L, ES proteins were separated by expulsion of adult worms, female worm fesodium dodecyl sulfate-polyacrylamide gel cundity, and muscle larval burdens. Six electrophoresis and stained with Coo- days after the challenge inoculation, half massie blue R-250 or by the periodic acid- the animals from each group were killed Schiff method (Fig. 1). Antigens Ts.45, and adult worms were recovered from the Ts.49, and Ts.53 all stained intensely in the intestines (Table II). The crude L, ES anperiodic acid- Schiff reaction, indicating all tigens and antigens Ts.49 and Ts.53 gave comparable levels of protection as assessed three are glycoproteins. When total L, ES products were passed by the numbers of adult worms in the in-
Trichinella spiralis: EXCRETORY-SECRETORY
------_-’
IMMUNOGENS
401
rl L A600 ;\,\---I 1 J‘,,I, L’ \------A 525
FIG. 1. Densitometric scans of sodium dodecyl sulfate-polyacrylamide gels (5-20%) of T~ichinella spiralis L, ES products, separated under reducing conditions, and stained for protein with Coomassie blue R-250 and scanned at 600 nm (solid line). or for glycoprotein by the periodic-Schiff reaction and scanned at 525 nm (dashed line). Antigens Ts.45, Ts.49, and Ts.53 are indicated by arrows.
testine. Female worms recovered from all immunized groups produced significantly fewer NBL than females recovered from control mice (Table II). However, this reduction was significantly greater using the L, ES antigen as compared to Ts.49 and Ts.53. Thirty-five days after challenge, the remaining mice from each group were killed and muscle larvae were recovered (Table II). The L, ES antigens and Ts.49 and Ts.53 produced significant and comparable levels of protection. DISCUSSION
In the present study, the protection-inducing properties of antigens isolated by monoclonal antibody affinity chromatography from the ES products of Trichinella spiralis L, have been examined. The antigen epitope recognized by monoclonal an-
FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel (j-20%) of Trichinella spiralis antigen fractions used to immunize mice. Gel was stained with Coomassie blue R-250. Lane 1, high-molecular-weight standards; lane 2, low-molecular-weight standards; lane 3, T. spirals L, ES products (reduced with 5% dithiothreitol); lane 4, antigens bound by affinity chromatography using monoclonal antibodies from hybridoma 7C,C, (reduced with 5% dithiothreitol); lane 5, antigens bound by affinity chromatography using monoclonal antibodies from hybridoma 7C,C, (unreduced); lane 6, T. spiralis L, ES products (unreduced).
tibodies produced by hybridoma 7&C, is present on three L, ES antigens with apparent molecular weights of 45K, 49K, and 53K, as determined under reducing conditions using several methods (Gamble and Graham 1984). These three antigens are glycoproteins based on their strong reaction when stained by the periodic acidSchiff method. Antigens Ts.45, Ts.49, and Ts.53, or precursor molecules, are present in the stichocyte cells of T. spiralis L, as determined by immunoperoxidase staining, and the antigens are elaborated orally by the parasite (Gamble and Graham 1984). The epitope recognized on these antigens is apparently restricted to the secretory products of the L, since 7C,C, monoclonal antibodies do
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H. R. GAMBLE
from the intestine and reduced female worm fecundity. Muscle larval accumulation in mice immunized with Ts.49 and Ts.53 was reduced comparably to levels resulting from immunization with crude ES antigens. It is not clear whether any immune killing of NBL occurs after larvaposition; however, the levels of adult expulsion and reduced fecundity obtained here were sufficient to account for the reduced muscle larval burdens in animals immunized with either the crude ES antigens or antigens TS.49 and Ts.53. Other studies have identified antigens from T. spiralis L, using monoclonal antibodies. Ortegas-Pierres et al. (1984) produced two different monoclonal antibodies DISTANCE SCANNED which recognized four different-molecularFIG. 3. Densitometric scans of Trichinella spiralis afweight antigens from the surface of T. spirfinity-isolated antigens Ts.49 and Ts.53 separated in a sodium dodecyl sulfate-polyacryiamide gel (5-20%) alis L,. One of the antibodies (NIM-M2) under reducing (5% dithiothreitol) (A), and nonrebound to 47K and 90K surface glycoproducing (B) conditions (Lanes 4 and 5, Fig. 2). Gel was teins; although the antibody itself did not stained with Coomassie blue R-250 and scanned at 600 bind to the surface of intact L,, it did bind nm. to released ‘251-labeled antigens. Despite the molecular weight similarities between not bind to extracts of either adult worms antigens Ts.45, Ts.49, and Ts.53 and those of Ortega-Pierres et al. (1984) (i.e., glycoor newborn larvae in an ELISA. Using monoclonal antibody 7C2C, af- proteins, similar molecular weight, and refinity columns, only the 49K and 53K an- lease into culture media), there are obvious differences. First, under reducing conditigens were isolated. We have interpreted this to mean that, under nonreducing con- tions, there are no higher-molecular-weight ditions, the common epitope within the 45K bands present in affinity isolations using antigen is unavailable for antibody binding 7C,C, monoclonal antibodies; the bands of molecular weight 92K- 104K appear only and that the molecular weight modificaunder nonreducing conditions. Antigens tions, resulting in the higher-molecularweight species (49K and 53K), expose this 47K and 90K of Ortega-Pierres et al. (1984) do not alter molecular weight patterns epitope to the surface of the molecule. Under nonreducing conditions, the 49K under reducing and nonreducing condiand 53K antigens associate in part to form tions. Second, monoclonal antibody 7C,C, three higher-molecular-weight antigens distinctly stains stichocyte cells (Gamble and Graham 1984) and does not react with (92K, 98K, and 104K). Antigens Ts.49 and Ts.53 were capable the cuticle of T. spiralis L,. The antigens of inducing protection in mice against oral isolated by Ortega-Pierres et al. (1984) are challenge at levels comparable to those ob- apparently of cuticular origin based on iotained using crude ES antigens from the L,. dination procedures, although the antibody Immunization with Ts.49 and Ts.53 re- recognizing these antigens (NIM-M2) does sulted in significant immunity as assessed not bind to the cuticle. Silberstein and Despommier (1984) proby both early expulsion of adult parasites
Trichinella spiralis: EXCRETORY-SECRETORY IMMUNOGENS
403
TABLE II Parasite Recoveries from Mice Immunized with Crude ES Product Antigens from Trichinella spiralis L, or Affinity Isolated Antigens Ts.49 and Ts.53 Treatmenta
Adult wormsb
Newborn larvaeC
Muscle larvae“
Control ES (10 w) Ts.49, Ts.53 (5 pg)
47.3 f 2.7e 27.7 k 3.2f (41) 22.6 k 2.1f (52)
117.4 * 2.@ 74.6 i 3.9” (36) 98.5 t 2.7f (16)
20695 -c 2983e 7168 -c 801f (65) 6100 ‘-’ 1076’(71)
Note. Mice were challenged 1 week after the last immunization with 150 T. spiralis infective L,. a Antigen given three times at 2-week intervals at the doses indicated. b Adult worms recovered 6 days after challenge infection; values are means ? standard error for groups of 10 mice. Duncan ranking is indicated by superscript followed by the percentage reduction in worm numbers compared to the control group. c Newborn larvae produced by female worms recovered from intestines of mice and maintained in culture for 24 hr; values are means + standard error from 30 female worms per group. d Muscle larvae recovered 35 days after challenge infection; values indicated are means f standard error for 10 mice per group.
duced monoclonal antibodies against stichocyte antigens of T. spiralis L,. Two different hybridoma cell lines were identified which recognized stichocyte antigens with molecular weights of 48K (8A4.3.1.1) and 50-55K (7B2.3.1.1). The antigens, isolated by affinity chromatography using both antibodies, produced significant levels of immunity (81 and 59%, respectively) when used to actively immunize mice against oral challenge with T. spiralis Lt. The molecular weights of antigens isolated using monoclonal antibodies from hybridoma 7B2 (5055K) indicate similar binding specificities with monoclonal antibodies derived from hybridoma 7&C, (49K, 53K); both sets of antigens associate under nonreducing conditions to produce higher-molecular-weight proteins. However, antibodies produced by hybridoma 7C,C, recognize an additional antigen (45K) under reducing conditions; no additional antigens are bound by hybridoma 7B2 under reducing conditions. Because of the considerably different procedures used for the isolation of the starting material used in the present study (L, ES products) and that used by Silberstein and Despommier (1984) (S,, the large particle fraction of L, stichocytes), a direct comparison among the antibodies may not be possible until all the antibodies have been
evaluated against the different antigen sources. We are currently attempting to resolve the relationship of antigens Ts.45, Ts.49, and Ts.53 by analysis of the transcribed gene products of the Trichinella spivalis Lt. ACKNOWLEDGMENTS The author thanks Mss. J. S. Baker, E. Moore, and C. E. Graham for technical assistance, and Dr. K. D. Murrell: Dr. M. W. Fleming, and Dr. H. Alizadeh for comments on the manuscript.
REFERENCES BELL, R. G., AND MCGREGOR,D. D. 1979a. Trichinella spiralis: Expression of rapid expulsion in rats exposed to an abbreviated enteral infection. Experimental Parasitology 48, 42-50. BELL, R. G., AND MCGREGOR,D. D. 1979b. Trichineila spin&: Role of different life cycle phases in induction, maintenance, and expression of rapid expulsion in rats. Experimental Parasitology 48, 51-60. CAMPBELL.C. H. 1955. The antigenic role of the excretions and secretions of Trichinellu spirnlis in the production of immunity in mice. Journal of Purusitology 41, 483-491. CULBERTSON,J. T. 1942. Active immunity in mice against Trichinellu spiralis. Journal of Parasitology 28, 197-202. DESPOMMIER,D. D. 1981. Partial purification and characterization of protection-inducing antigens from the muscle larva of Trichinella spiralis by mo-
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lecular sizing chromatography and preparative isoelectric focusing. Purusite Immunology 3, 261-272. DESPOMMIER, D. D., CAMPBELL, W. C., AND BLAIR,
L. S. 1977. The in viva and in vitro analysis of immunity to Trichinella spiralis in mice and rats. Parasitology 74, 109-l 19. DESPOMMIER,D. D., AND LACETTI, A. 1981a. Trichinellu spiralis: Proteins and antigens isolated from a large particle fraction derived from the muscle larva. Experimental Parasitology 51, 279-295. DESPOMMIER:D. D., AND LACETTI, A. 1981b. Trichinella spiralis: Partial characterization of antigens isolated by immuno-affinity chromatography from the large particle fraction of the muscle larvae. Journal of Parasitology 67, 332-339. GAMBLE, H. R. 1985. Induction of immunity to Trichinellu spirdis in mice using antigens derived from different life cycle stages. Journal of Parasitology, Submitted for publication. GAMBLE, H. R., AND GRAHAM, C. E. 1984. Monoclonal antibody-purified antigen for the immunodiagnosis of trichinosis. American Journal of Veterinary Research 45, 67-74. JAMES, E. R., AND DENHAM, D. A. 1974. The stage specificity of the immune response to Trichinellu spiralis. In “Trichinellosis” (C. W. Kim, ed.), pp. 345-351. Intext Educational, New York. JAMES,E. R., AND DENHAM, D. A. 1975. Immunity to
Trichinella spiralis. VI. The specificity of the immune response stimulated by the intestinal stage. Journal of Helminthology 49, 43-47. JAMES, E. R., MOLONEY, A., AND DENHAM, D. A. 1977. Immunity to Trichinella spiralis. VII. Resistance stimulated by the parenteral stages of the infection. Journal of Parasitology 63, 720-723. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature (London) 227, 680-685. LARSH,J. E., AND RACE, G. J. 1954.A histopathologic study of the anterior small intestine of immunized and nonimmunized mice infected with Trichinellu spiralis. Journal of Infectious Diseases 94, 262272. ORTEGA-PIERRES, G., CHAYEN, A., CLARK, N. W. T., AND PARKHOUSE,R. M. E. 1984. The occurrence of antibodies to hidden and exposed determinants of surface antigens of Trichinellu spiralis. Parasitology 88, 359-369. SILBERSTEIN,D. S. 1983. Antigens. In “Trichinella and Trichinosis” (W. C. Campbell, ed.), pp. 309334. Plenum, New York. SILBERSTEIN,D. S., AND DESPOMMIER,D. D. 1984. Antigens from Trichinelln spiralis that induce a protective response in the mouse. Journul of Immunology 132, 898-904.