secretory antigen epitope

Inrerwtional

Pergamon

0020-7519(94)00073-5

JournaIfor Parasitology, Vol. 25. No. I, pp. 105-11 I, 1995 Copyright 0 1995 Australian Society for Parasitology Elsevier Science Ltd Printed in Great Britain. All rights reserved 002cL7519/95 $9.50 + 0.00

Idiotypic Replica of a Toxocara canis Excretory/Secretory Antigen Epitope R. BARDON,*

J. L. GUILLEN

and C. AGUILA

Departamento de Parasitologia, Facultad de Farmacia, Universidad Complutensede Madrid, 28040 Madrid, Spain (Received

18 August

1993; accepted

15 April

1994)

Abstract-Bardon R., Guillen J. L. and Aguila C. 1995. Idiotypic replica of a Toxocaru cunis excretory/secretory antigen epitope. International Journal of Parasitology 25: 105-111. This study describes the production, characterization and use of an anti-idiotype serum raised against the monoclonal antibody TC-1 which recognizes a T. canis excretory/secretory antigen (ES Ag) epitope. Anti-idiotypic (anti-Id or Ab2) antibodies were produced in rabbits using TC-1 F(ab’), fragments; these anti-Id inhibited ES Ag binding to biotinylated TC-1, and also inhibited a larval microprecipitation assay using TC-1. Assays show that the Ab2B or “internal image” of a T. canis ES Ag epitope was obtained. The antibodies have been wed as an idiotypic copy of ES Ag in a diagnostic ELISA for murine toxocariosis. Affinity-puritied anti-Id antibodies were used to raise a homologous anti-anti-Id (Ab3) response in rabbits. Antibody formation was followed in the sera of BALB/c mice inoculated with embryonated eggs of T. canis during a 12-month infestation. A 3week latency period was observed before speciiic anti-TC-1 epitope antibodies were detected. High levels were reached at 7 weeks post-inoculation with a maximum at the ninth month, and were then maintained until the end of the experiment. The results show the possible utility of anti-Id antibodies as an ES Ag molecular replica. Key words:

Toxocara

canis;

monoclonal antibody; anti-idiotype antibodies.

INTRODUCTION

Idiotype-anti-idiotype interactions allow the production and characterization of structures which mimic pre-selected antigenic determinants, particularly if the relevant idiotype is in or near the antigen-binding site. Several types of anti-idiotypic antibody have been defined, including o, B, and y antibodies, each with different characteristics (Jeme, Roland & Cazenave, 1982). An interesting feature of Ab2 antibodies is their ability to mimic the stereospecificity and function of the original antigen. However, not all anti-idiotypic antibodies behave as “internal *To whom all correspondence should be addressed at: Departamento de Parasitologia, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain.

images” of the epitope recognized by the originating antibody (Abl); this is a characteristic only of Ab2B antibodies. This method has potential use in parasitology; difficult-to-obtain or unstable antigens could be mimicked and applied to diagnosis of or protection against parasitic diseases (Sacks, Esser & Sher, 1982; Kresina & Olds, 1989). The larval migratory visceral syndrome is currently diagnosed in a larval ES Ag-based ELISA (Fenoy, Cuellar, Aguila & Guillen, 1992), the use of which has allowed the establishment of a specific, reliable technique. T. canis larvae survive for long periods in culture, excreting antigen into the medium. But difficulty in obtaining gravid females and the possible existence of parasite strains with antigen variants make the potential mimicry of 105

106

antigen epitopes useful, as it would reagent production.

R. Bardon

greatly simplify

MATERIAL AND METHODS Parasite and ES collection. Infective larvae were obtained from embryonated T. canis eggs and the ES antigen (Ag) by the De Savigny method (1975) as modified by Aguila, Cutllar, Fenoy, Guillen (1987) and Cdllar, Fenoy & Guillen (1990). Monoclonal antibody and immunogen preparation. The mAB TC-1 (IgG,-k) was obtained as described (Aguila, Cutllar & Guillen, 1988a); its antigen reactivity pattern was determined by immunoprecipitation and SDS-PAGE. TC1 reacts with a common determinant of the ES Ag in the 77-177 kDa range (Aguilla, Cutllar & Guillen, 1988b). Ascites fluid was produced in BALB/c mice. Immunoglobulin (Ig) was purified by double precipitation with caprylic acid and (NH&SO,, pepsin digested (Sigma 3,200 units/mg, enzyme/protein mass ratio 160) for 90 min at 37°C in 0.1 M citrate, pH 3.5; the F(ab’), fragments were separated from smaller peptides on an Ultragel ACA-44 column and characterized in SDS-PAGE (Laemmli, 1970). Production and purification of anti-idiotypic serum. Two New Zealand rabbits were immunized in multiple intraderma1 (i.d.) sites with 400 pg of purified TC-1 F(ab’), fragments in an equal volume of Freund’s complete adjuvant. Three intramuscular boosts (125 pg of antigen in incomplete adjuvant) were given 45, 60 and 95 days later. Animals were bled from an ear artery at the first boost and 22, 29, 57, 68 and 103 days later; serum samples were inactivated by heating. Most anti-isotype antibodies (Ab) present in the serum were absorbed on a Sepharose 4Bnormal mouse Ig affinity column; this material, called Ab2 reagent, was used for Ab2 antibody characterization. IgG was isolated from anti-Id serum on a Protein A-Sepharose 4B column, eluted with 0.05 M glycine-0.15 M NaCI, pH 2.3 and dialyzed overnight (o/n) against PBS. Assays ofanti-isotypic and anti-idiotypic activity in rabbit antisera. Anti-isotypic and anti-Id activity were measured in ELISA. In the first assay, plates were coated with normal mouse Ig and in the second, with mAb TC-I. Reactivity with TC2, another ES Ag-specific mAb, was also tested. Microtitre plates (96-well, Dynatech) were coated with 5 ug/ml of normal mouse Ig, TC-1 or TC-2, in 0.1 M carbonate buffer, pH 9.6, o/n at 4°C. Between assay-steps, plates were washed 3 times in PBS&OS% Tween 20 (PBS-T). Plates were blocked with 250 l&well of 0.1% bovine serum albumin (BSA) in PBS. Then 100 ~1 dilutions of each anti-Id Ab in PBS-T plus 0.1% BSA (PBS-T-BSA) were added and incubated 2 h at room temperature (RT). Appropriately-diluted peroxidase-conjugated goat antirabbit Ig (100 t.d/well, Tago) in PBS-T-BSA was added and incubated for 1 h at 37”C, followed by 100 pLywe of o-phenylene-diamine (OPD, Sigma) with 0.04% HZ02. The reaction was stopped with 3 N H,SO, and plates read at 492 nm. A normal rabbit serum control was used in the same conditions as the anti-Id serum. ELBA inhibition assays. The capacity of the anti-Id

et al. serum to inhibit ES Ag and TC-1 binding was tested. In the first assay, microtitre plates were coated with 100 pl of ES Ag and blocked as above. TC-1 dilutions in PBS-T-BSA with 1% negative mouse serum were preincubated alone or with a fixed concentration of Ab2 reagent at 37°C. After centrifugation at 5000 g for 3 min, 100 pl of supematant were transferred to Agcoated plates and incubated for 2 h at 37°C. The assay was completed as above. Results are shown as the percentage inhibition of TC-1 and ES Ag union. In the second assay, plates were coated and blocked as before. Biotin-labeled TC-1 in PBS-T-BSA was preincubated for 4 h at 37°C with doubling serial dilutions of ES Ag, negative rabbit serum and anti-Id IgG. After 3 min centrifugation at 5000 g, 100 pl of supematant were transferred to plates and incubated for 2 h at RT. Plates were developed with 100 @well streptavidin-peroxidase (StAPO, Sigma) in PBS-T-BSA for 45 min. Remaining steps and expression of results were as above. Larval microprecipitation inhibition. Tissue culture plates (96-well) were coated with 250 l&well of 1% BSA in PBS and incubated at 37°C for 2 h. The following reagents were co-incubated at 37°C with 1% negative mouse serum: doubling serial dilutions of TC-1 or TC-2 mAb, alone or with a fixed concentration of anti-Id serum, doubling serial dilutions of positive or negative mouse serum, alone or with fixed concentration of anti-Id serum, plate and larval viability controls (HBSS, anti-Id serum, positive and normal mouse sera). After centrifugation (2 min. 5000 g), supematants were collected and 100 ~1 added to the plates. From 20-25 larvae/well were added in 20 )LI of HBSS and incubated at 37°C. Wells were examined microscopically at 24 and 48 h, and the results interpreted as described (Guilltn, Cuellar & Aguila, 1987). Ab3 serum production. A New Zealand white rabbit was immunized i.d. with 390 pg anti-Id IgG, followed by three 120 pg i.m. boosts, using the same protocol as for Ab2 serum production. Ab3 serum was tested in direct ELISA against ES Ag. Induction of experimental murine toxocariosis and measurement of IgC and IgM anti-E/S antigen. BALBic mice were inoculated with 1000 embryonated T. canis eggs by oral administration with gastric tubing and were bled weekly from the retro-orbital plexus, under ether anaesthesia, for 7 weeks and then at month 9, 10 and 12 p.i. Specific IgG and IgM levels against ES Ag in experimental murine toxocariosis were assayed in a direct ELISA (CuCllar et al., 1990). Inhibition ELISA to determine TC-I-like antibody response. Plates were precoated o/n with 50 pl of Protein A-Sepharose 4B (5 &ml) in sodium carbonate buffer. Between steps, plates were washed as above. Wells were blocked with 50 pl of 0.1% BSA in PBS, and then sensitized o/n at 4°C with 50 ~1 of anti-Id serum in PBS-T-BSA. Next, 50 ~1 each of 1% negative rabbit and mouse serum in PBS-T-BSA were added consecutively, and each incubated for 1 h at RT. Then SO ~1 of antiToxocara-positive and negative control mouse sera (dil. l/l00 in PBS-T-BSA) were added and incubated for 90 min at 37°C. followed by 1 h at RT. Biotin-labeled TC-1 (50 &l/well) at an appropriate dilution in PBS-T-BSA with

Anti-idiotypic

antibodies

1% negative mouse serum was added and incubated o/n at 4°C. Finally, 50 *l/well StA-PO were added for 90 min at RT and the reaction developed with OPD. Results are shown as the per cent inhibition of the union between labeled TC-1 and Ab2 serum. RESULTS

Abl Due to its toxicity for T. canis larvae and its ability to produce a certain degree of passive in viva protection (Dominguez, Bardon, Aguila & Guillen, unpublished, IV National Congress of Parasitology, Salamanca, 1987) mAb TC-1 was selected to prepare anti-Id serum. To increase the relative concentration of idiotypic determinants in the immunizing material, TC-1 F(ab’), fragments were used. A62 To eliminate anti-isotypic antibodies from Ab2, hyperimmunized rabbit serum was absorbed on a normal mouse Ig-Sepharose 4B column. Absorbed serum was tested in ELISA for anti-isotypic activity (normal mouse Ig), anti-Id activity (TC-1 idiotype) and with another mAb (TC-2) specific for the same antigen (Fig. 1). The anti-Id activity was highly specific and was always stronger than that observed with normal mouse Ig. Ab2 serum crossreacted

in murine

toxocariosis

107

slightly with TC-2, indicating that TC-1 and TC-2 may recognize related epitopes. Both rabbit sera showed some anti-isotypic activity at low dilutions, which was higher in rabbit 2; rabbit 1 serum was thus used in the following experiments. Two ELISAs were used to test the inhibition of Abl binding to ES Ag. In the first, TC-1 concentrations from 0.01 to 3 &ml and anti-Id reagent at a l/2 dilution were tested initially; clear inhibition from the highest concentration was observed. Higher TC-1 concentrations were then tested. For an anti-Id reagent dilution of l/2 and TC-1 concentrations from 0.1 to 15 &ml, inhibition was detected from about 3 &ml. In further tests with doubling serial dilutions of anti-Id reagent from l/3 to l/48, inhibition was observed at lower TC-1 concentrations as serum dilutions increased (data not shown). In the second inhibition ELISA, ES Ag inhibitory activity was >98% at all concentrations tested. The Ab2 serum IgG fraction also had high inhibition percentages at concentrations >6 &ml; a control rabbit serum showed about 30% inhibition at the highest concentration (50 p&ml) (Fig. 2). This capacity of the Ab2 antibodies to block the binding site of the Abl idiotype to ES Ag may indicated the presence of Ab2B antibodies structurally similar to the ES Ag epitope recognized by TC-1.

A

B

O.D. 482 3-

-El-

8

TC-1 tAbI)

10000

10000 l/Dilution

TC-1 Mb11

Ab2

l/Dilution

Fig. 1. Anti-isotypic and anti-idiotypic activities as measured in ELISA. Panel A: rabbit serum No.2. Microtitre plates were coated with normal rabbit immunoglobulina (IgR), (Abl) (TC-I), or TC-2 monoclonal antibody (TC-2).

Ab2

serum no.1; Panel B: rabbit TC-1 monoclonal antibody

108

R. Bardon et al. tested in a direct ELISA. Specific anti-ES Ag activity increased in Ab3 serum over the immunization period; it thus was an anti-anti-Id serum recognizing an antigen with which the animal had no contact (Fig. 4).

% INHIBITION

751 50

-

/ + 25

+

+

r

Conc.).ag/ml

Fig. 2. ELISA to test inhibition of Abl binding to ES Ag. Biotinylated mAb TC-I was preincubated with several concentration of ES antigen (ES Ag), normal rabbit serum (NRS) and the IgG fraction of the anti-idiotypic reagent (Ab2). The larval microprecipitation technique was used to confirm this hypothesis. Its advantage lies in the anti-Id Ab competes with ES Ag produced by T. canis larvae during the assay. Ab2 was tested for its ability to inhibit T. canis larval microprecipitation of TC-1. Anti-Id serum was incubated with its idiotype TC- 1 or with TC-2 at concentrations from 2.7 to 350 p&ml, and with mouse serum in dilutions from l/2 to l/256. Inhibition of microprecipitation was observed in the lowest TC-1 dilution tested; TC-2 did not show the same ability to inhibit microprecipitation. Mouse serum showed the same reactivity pattern against Toxocaru larvae in the presence of Ab2. Assay of TC-I concentrations from 0.03 to 20 pg/ml confirmed this data; Ab2 inhibited TC-1 microprecipitation from 2.5 kg/ml. Positive control mouse serum maintained its reactivity against ES Ag (data not shown). These data show the anti-idiotypic activity of the Ab2 reagent in a natural system (Fig. 3). Ab3 If Ab2P antibodies mimic an ES Ag epitope, they could be used to generate Ab3 which react as the original Abl. The Ab3 response was induced by immunization with anti-Id IgG. The presence in Ab3 serum of antibodies reactive with the ES Ag was

Immune response study Specific IgG and IgM against ES products in BLABlc mice were studied. IgM was detected in the first week p.i., and increased rapidly week 2; high levels were maintained throughout the study. IgG was detected in week 2 p.i., increasing progressively until peaking at month 10 (Fig. 5). In the inhibition assay used to detect Ab to the TC-1 epitope, a 3-week latency period was observed, with a rapid increase in week 4 p.i. High inhibition levels were reached at week 7, peaked in month 9 (45% inhibition) and were maintained to the end of the study (Fig. 6). The dynamics of this TC-l-like Ab differ from the IgG and IgM responses described earlier, with a 3-week latency period. The highest Ab levels were detected from month 9 p.i., and maintained in both cases to the end of the study. The specific humoral response against the epitope recognized by TC-1 is thus principally IgG, as shown by its late appearance. DISCUSSION

The Abl antibody used to generate an anti-Id response can be purified directly from ascites by passage over affinity columns (Sacks et al., 1982; Kresina & Olds, 1989); enzyme digestion to generate F(ab’), fragments, followed by affinity purification is not essential. However, the relative increase in specific Id determinants in this material with respect to the total number of antigenic determinants present in an Ig molecule may induce higher-titer antisera (Palomo, Albar, Garcia-Barren0 & Melero, 1990); the reduced anti-isotypic response also simplifies purification of anti-Id Ab. The anti-Id Ab obtained here were highly specific, and the remaining anti-isotypic activity of the Ab2 was low. The crossreaction between TC-2 and the Ab2 confirms previous immunoprecipitation studies in which both mAb recognized the same fraction in SDS-PAGE (Aguila et aZ., 1988b). Inhibition assays similar to those of Grzych, Capron, Lambert, Dissou, Torres & Capron (1985), Kresina & Olds (1989) and Palomo et al., (1990), confirm that Ab2 serum competes in liquid medium with ES Ag for TC-1 binding, proving that these at Ab2P. Olson’s larval microprecipitation technique ( 1960), despite its difficulty, is the reference method for specific Ab detection in toxocariosis. It was used

Anti-idiotypic

antibodies in murine toxocariosis

A

109

B

Fig. 3. T. canis larvae. Panel A: microprecipitate formed around the larval oral orifice, caused by the reaction between ES Ag and mAb TC-1. Panel B: no microprecipitates were formed in the presence of anti-Id Ab. here to show the ability of anti-Id Ab to compete with the antigen produced by T. canis larvae during the assay. The results confirm, in a natural ES Ag production system, the possible presence of an Ab2P or “internal image” of the epitope recognized by TC-I. However, other anti-Id Ab (AbZy) reacting with idiotypic determinants near the Ag combining site of Abl would also inhibit binding of the original mAb to the antigen (Jerne et al., 1982). We thus tested the capacity of the Ab2 reagent to produce anti-anti-Id antibody (Ab3) which reproduces the antigen recognition characteristics of Abl . The Ab2 recognizes the ES Ag, with which the animal had no contact. Palomo et al. (1990) induced an Ab3 which recognized human respiratory syncytial virus in ELISA and was also active in a viral microneutralization test; Kresina & Olds (1989) detected Ab3 in animals immunized with anti-Id serum. The immune response results are comparable to those of Bowman, Mika-Grieve & Grieve (1987), who detected IgM from week 1 p.i. in BALB/c mice,

with a peak at 36 weeks; our maximum IgM level wa at 9-10 months pi. They detected IgG from the week 2 pi., peaking at 6-8 weeks, while we detected the highest levels in month 9, as for IgM. Robertson, Burkot, Gillespie, Kennedy, Wambai & Maizels (1988) determined the anti-epitope immune response in an inhibition assay with 2 antiT. canis ES Ag mAb, Ten-2 and Ten-3. In ES Agcoated ELISA plates, they measured specific Ab to the epitopes recognized by Ten-2 and Ten-3 in sera from rabbits inoculated with embryonated parasite eggs. Antibodies to the Ten-2 (IgM) epitope were clearly positive by day 14 p.i., maintained maximum activity until day 42, declined and were undetectable after day 70. Anti-Ten-3 epitope Ig was detected from week 4, increased rapidly to maximum activity at week 5 and maintained high levels to the end of the 14-week study. The 3-week latent period observed for detection of specific IgG against the TC-1 epitope coincides with that observed from Tcn3. Although the peak appeared later (month 9 p.i., rather than week 5), high levels were maintained to

110

R. Bardon

1

O-8

O.D.

r

et al

492

% INHIBITION 50

c

036

30

0,4

20

1

O-2

10

OL 10

1

l/serum

0 L

1000

100 dilution

/

0

1w

2w

3w

4w

5w

I

6W

J

I

I Pm

7w

10m

12m

TIME

Fig. 4. ES Ag recognition by Ab3 antibodies in ELISA. NRS: negative rabbit serum. Ab3 IS: serum from the first bleed, Ab3 2s: serum’ from the second bleed.

hD.p/O.D.c

t

Fig. 6. Dynamics of TC-l-like antibodies in BALB/c mice inoculated with embryonated T. canis eggs. Microtitre plates were coated with Ab2. w = weeks, m = months.

the end of the study in both cases. Individual dynamics thus vary in the specific Ab response to different ES Ag epitopes (recognized by Ten-2, Ten-3 and TC-I), but a steady polyclonal response is seen. The possible utility of anti-Id Ab as molecular replicas of the ES Ag is thus clear, although antigenie replicas of all immunodominant epitopes would be necessary. An anti-Id serum against a specific polyclonal serum could be obtained; however, a superior alternative would be to have all the monoclonal anti-Id Ab required to mimic the most representative ES Ag epitopes. As DNA technology cannot yet mimic glycosylated antigen epitopes, anti-Id methods may be of use in their production, particularly for the many highly-glycosylated parasite antigens, such as the T. canis ES Ag.

Index

2c

16

10

a 6 4 2 0

L

I--” 0

1w

2w

3w

4w

5w

6W

7w

1

I Pm

IOm 12m

TIME

Fig. 5. Dynamics of IgG and IgM immune responses in BALBlc mice inoculated with embryonated T. canis eggs. Microtitre plates were coated with ES Ag. The O.D.p/O.D.c index is the optical density of the test serum divided by the optical density of the control. w: weeks, m: months.

Acknowledgements-We thank Femandez, Head, Departamento Facultad de Farmacia, U.C.M., facilities, and Dr J. P. Albar, Dr for help and useful discussion in

Dr

A. de

R. Martinez Parasitologfa, Madrid for advice and F. Bolas and C. Mark the preparation of this

paper.

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Anti-idiotypic

antibodies in murine toxocariosis

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Sacks D. L., Esser K. M. & Sher A. 1982. Immunization of mice against trypanosomiasis using antiidiotypic antibodies. Journal of Experimental Medicine 155: 1108-1119. Savigny D. De 1975. “In vitro” maintenance of Toxocara canis larvae and a simple method for the production of Toxocara antigen for use in serodiagnosis test for LMV. Journal

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