Immunology 1992 76 599-603
Bypass of carrier-induced epitope-specific suppression using a T-helper epitope S. SAD, K. RAO,* R. ARORA,* G. P. TALWAR & R. RAGHUPATHY* National Institue of Immunology and *International Centre for Genetic Engineering and Biotechnology, New Delhi, India Acceptedfor publication 6 April 1992
SUMMARY A gonadotropin-releasing hormone (GnRH)-based vaccine is being developed as a method for nonsurgical immunotherapy as immunization with this vaccine results in atrophy of the prostate. This vaccine, a conjugate of GnRH and diphtheria toxoid (DT), provides a unique hapten-carrier system for investigating the influence of carrier presensitization on antibody responses to self haptens. In a recent communication' we showed that preimmunization with carriers diphtheria toxoid and tetanus toxoid results in a strain-dependent inhibition of anti-GnRH responses in mice and that T cells from carrier-presensitized mice are responsible for anti-haptenic suppression. In the present report we describe a strategy for bypassing DT-induced epitopic suppression using a T-helper epitope from DT.
INTRODUCTION Gonadotropin-releasing hormone (GnRH) is a highly conserved decapeptide which plays a critical role in the process of reproduction. Immunization of rodents with GnRH-carrier conjugates leads to the production of anti-GnRH antibodies2'3 and the subsequent impairment of fertility.4 Equally interesting is the demonstration that immunization of male rats5'6 and monkeys (D. K. Giri and G. P. Talwar, unpublished observations) with this vaccine results in decreased testosterone levels, decreased testicular size and drastic atrophy of the prostate gland.5'6 This effect on the prostate has prompted us to investigate the immunotherapeutic value of this vaccine in hormone-dependent prostatic hypertrophy. GnRH being a self molecule, and a hapten, has to be chemically linked to foreign carriers such as diphtheria toxoid (DT) or tetanus toxoid (TT) in order to generate anti-GnRH antibodies. In studies designed to investigate the regulation of anti-hapten (GnRH) responses by the carrier (DT or TT), we have shown recently that pre-immunization with DT and/or TT can induce hyporesponsiveness to GnRH.' This presensitization effect is induced by immunizing animals sequentially with the carrier first and subsequently with the hapten-carrier conjugate. This phenomenon of carrier-induced epitope-specific regulation has been studied in a variety of hapten-carrier systems by several investigators7-'3 who have demonstrated that preimmunization with a carrier often results in an inhibitory effect on the production of antibodies to new epitopes linked to the same protein. Studies done so far show that epitope-specific suppression can be induced with diverse antigens administered under Correspondence: Dr R. Raghupathy, Immunogenetics Laboratory, National Institute of Immunology, New Delhi-i 10067, India.
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widely different immunization conditions in a variety of mouse strains. Equally interesting is the finding that while the antibody responses to the hapten are suppressed, anti-carrier responses proceed normally.7'-3 The universality of this phenomenon has been documented by studies in mice,7-9"2 rabbits'0 and humans.""3 Our studies on the GnRH-DT/TT system show that this effect is a strain-dependent one; presensitization with DT or TT results in a suppression of anti-GnRH titres in some, but not all, strains of mice tested.' Epitope-specific regulatory effects have been attributed by some workers to suppressor T cells''4"5 and by others to antigenic competition or clonal dominance of carrier-specific B cells. 16,17 If the former explanation is correct, suppression can be attributed to the possible existence of suppressor epitopes on DT or TT. If the latter is correct, competition can be attributed to the existence of too many competing determinants on DT or TT. Whatever the case, we speculated that epitope-specific suppression could be bypassed or circumvented by the use of a synthetic T-helper epitope from DT, as a carrier. In this report, we describe the delineation of one such T-helper epitope from DT and its usefulness in bypassing DT-induced GnRH-directed suppression.
MATERIALS AND METHODS Synthesis and conjugation of GnRH A modified GnRH decapeptide containing D-lysine (instead of glycine) at position six, attached to a linker E-amino caproic acid, was synthesized by the solid phase method using benzhydrylamine resin as the solid support.'8 This peptide was conjugated to DT by the glutaraldehyde method.2
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Selection ofpeptidesfrom DT Nine DT peptides spanning residues 23-40,43-58, 93-109, 118135, 142-159, 201-222, 284-300, 361-378 and 508-524 were predicted to be T-helper epitopes on the basis of high amphipathic scores obtained using the algorithm of Margalit et al.'9 These were subsequently found to contain the Rothbard and Taylor20 motif.
Synthesis of DTpeptides Peptides selected from DT were synthesized using the simultaneous multiple peptide synthesis technique originally described by Houghten.2' tBoc derivatized amino acids (Fisher Biotec, Pittsburg, PA) were used for accomplishing the synthesis on 4methylbenzhydrylamine resin (Nova Biochem. Ag, Basel, Switzerland). All peptides were synthesized on a 0 07 mmol scale and cleaved from the resin with anhydrous hydrogen fluoride in the presence of anisole, after which the crude peptides were purified by high-performance liquid chromatography (HPLC) using a linear gradient of acetonitrile, with 0-01 % trifluoracetic acid on a Vydac C18 column. Conjugation of GnRH to DTpeptide Peptide 201-222 was chosen as a carrier on the basis of its T-cell proliferative capacity (as discussed in Results). GnRH-peptide (201-222) conjugate was prepared by thiolating GnRH using a hetero-bifunctional reagent, succinimidyl 1-3-(2-pyridyl dithio) propionate (SPDP).22 GnRH in phosphate buffer was allowed to react with SPDP in dimethyl formamide, at room temperature for I hr, after which dithiothreitol (DTT) was added and the product purified by Sephadex G-10 chromatography. Peptide 201-222 which contains a cysteine at its N-terminal end was reduced with DTT prior to purification on a Sephadex G-10 column. The thiolated GnRH was allowed to react with the reduced peptide 201-222 at room temperature for 3 h and the conjugate so formed purified by Sephadex G-25 chromatography.
T-cell proliferation assays The T-cell proliferative activity of DT and its peptides was evaluated by immunizing mice in the foot pads with 10 pg of DT or 100 pg of peptide in 25 p1 saline emulsified with an equal volume of complete Freund's adjuvant (CFA). Eight days after immunization, the inguinal and popliteal lymph node cells (5 x 105 cells/well) were suspended in RPMI-1640 medium supplemented with 0-5% normal mouse serum and incubated in triplicate with various concentrations of DT or peptides in a 96well tissue culture plate. After incubation for 4 days in a humidified 5% C02 atmosphere, the cultures were pulsed for 18 hr with 1 pCi [3H]thymidine and harvested on glass fibre filters prior to liquid scintillation counting. Immunization In order to investigate whether the GnRH-peptide conjugate can bypass carrier-induced suppression, C3H/He mice (10/ group) were presensitized on Day 1 with DT (100 pg on calcium phosphate) or with saline, and then boosted on Days 30 and 60 with GnRH-DT or GnRH-peptide conjugate. Mice were bled on Day 75 and analysed for antibodies against GnRH. The effect of priming with T-helper epitope on the antibody response to GnRH was determined by priming C3H/He mice (10/group) on Day 1 with the peptide 201-222 (100 pg.
emulsified with CFA or adsorbed on calcium phosphate) and later boosted on Days 30 and 60 with GnRH-DT adsorbed on calcium phosphate. Mice were bled on Day 67 and analysed for anti-GnRH antibodies. Assay for anti-GnRH antibodies Anti-GnRH antibody titres were measured by radioimmunoassay and expressed as antigen-binding capacity. All individual sera were titrated simultaneously by the dilution method, using the same batch of the radioactive tracer. The assay protocol consisted of mixing 50 Mp each of normal horse serum [diluted 2 5 times in assay buffer, 50 mm phosphate-buffered saline (PBS) with 0 1 % bovine serum albumin], diluted antisera, ['25I]GnRH and assay buffer. After incubation for 18 hr at 40, the antibodybound fraction was precipitated by adding 1 ml of cold ethanol23 and separated by centrifuging at 1500 g for 15 min at 4°. ['25I]GnRH binding capacity (expressed in ng/ml serum) was calculated at a point at which proportionality between the dilution of the antiserum and ['251I]GnRH was obtained. The specific activity of the radiolabelled GnRH was used to calculate the antigen-binding capacity.
RESULTS T-cell proliferative response to DT peptides Of the strains susceptible to DT-induced suppression of antiGnRH titres, C3H/He mice showed maximal suppression.' In order to identify a T-helper epitope from DT, that would induce T-cell proliferation in C3H/He mice, the nine DT peptides synthesized were tested for their proliferative capacities in vitro. The capacity of DT peptides to induce T-cell proliferation was ascertained by measuring their capacity to induce the proliferation in vitro of lymph node cells from mice immunized in vivo with DT. C3H/He mice were immunized with 100 pg of DT in 25 p1 saline emulsified in CFA. Eight days later the inguinal and popliteal lymph node cells were incubated with various peptides of DT. Figure 1 depicts the T-cell proliferative response generated in C3H/He mice peptide 201-222 from DT. In order to determine whether in vivo immunization with peptide 201-222 can recruit T cells responsive to DT in vitro, C3H/He mice were injected with peptide 201-222 and their
40 PO
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07 15 31 62 125 25 50 Antigen concentration (pg/ml) T-cell 1. proliferative response of DT (0) and peptide 201-222 Figure (0) in C3H/He mice (five per group) immunized with DT. The background response in the control wells (without antigen) was in the
range of 1600-2400 c.p.m.
Bypass of epitope-specific suppression using T-helper epitope
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lymph node cells incubated with either DT or peptide 201-222. Figure 2 depicts the T-cell proliferative response generated by DT and peptide 201-222 in C3H/He mice.
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,_20
x
Bypass of DT-induced suppression 10
0 7 5 3 6 2 125 25 50 Antigen concentration (ug/ml)
Figure 2. T-cell proliferative response of DT (0) and peptide 201-222 (0) in C3H/He mice (five per group immunized with peptide 201-222). The background response in the control wells (without antigen) was in the range of 1100-2200 c.p.m.
With a view to ascertain whether DT-induced suppression can be circumvented by the use of peptide 201-222 as a carrier, DTpresensitized mice were boosted with GnRH linked to peptide 201-222. This peptide was selected for use as a carrier as it elicited T-cell proliferation in C3H/He mice. Preimmunization with DT clearly inhibits the anti-GnRH antibody response in C3H/He mice (Fig. 3). There is a dramatic decrease in antiGnRH antibody titres from 23 ng/ml to 2 ng/ml GnRH binding capacity. Figure 3 shows that DT-induced suppression can be bypassed by using peptide 201-222 as a carrier. One injection of DT followed 30 days later by two GnRH-peptide boosters elevates the antibody response from 2 to 15 ng/ml. Injection of mice with GnRH-peptide conjugate (without preimmunization with DT) also elicits a moderate anti-GnRH antibody response. Immunization of mice with either GnRH or peptide 201-222 alone does not induce anti-GnRH antibody formation (data not shown). Effect of peptide priming on the antibody response to GnRH
DT DT G-DT G-DT G-P G-DT G-DT G-P
-
G-P G-P
Figure 3. Bypass of carrier induced epitope-specific suppression using a T-helper epitope from the carrier. C3H/He mice (10 per group) were primed on Day I with DT (100 pg on calcium phosphate) or saline, and later boosted on Days 30 and 60 with either GnRH-DT (G-DT) or GnRH-peptide 201-222 (G-P) emulsified with Freund's adjuvant. Values are expressed as mean + SE of the results obtained on Day 75.
In order to determine the influence of pre-existing immunity to the peptide on the antibody response to GnRH, mice were primed with peptide 201-222 from DT and later boosted with GnRH-DT. It is interesting to note that pre-existing immunity to this peptide does not result in inhibition of the anti-GnRH antibody response. Preimmunization with this peptide actually results in an enhancement of anti-GnRH antibody titres when subsequent immunization is done with GnRH linked to DT (Fig. 4). This enhancement of response from 10 ng/ml to 25 ng/ ml was observed when CFA was used as an adjuvant. However we failed to observe a similar effect when calcium phosphate was used as an adjuvant.
E
a
C
DISCUSSION
20
CM
- (FA)
G-DT G-DT
Peptide (FA) G-DT G-OT
(CP) PMde G-DT G-DT
Figure 4. Effect of peptide priming on the antibody response to GnRH. C3H/He mice (10 per group) were primed on Day I with the peptide 201-222 emulsified with Freund's adjuvant (FA) or adsorbed on calcium phosphate (CP) and later bossted on Days 30 and 60 with GnRH-DT adsorbed on calcium phosphate adjuvant. Values are expressed as mean + SE of the results obtained on Day 67.
It is now abundantly clear that the carrier can exert significant influences on responses to haptens linked to the carrier. The GnRH vaccine is a hapten-carrier conjugate which is intended for use in humans, where one aims for a universal response with minimal genetic restriction. It is important therefore, to investigate the influence of carriers on the antibody response to GnRH. From this point of view we felt it essential to study the effects of pre-existing immunity to the carrier on the antibody response to the hapten, GnRH. We have shown recently that preimmunization with DT and/or TT induces hyporeseponsiveness to GnRH.1 Experiments described here were designed to ascertain whether epitope-specific suppression could be bypassed using T-helper epitopes as carriers. Having shown that anti-GnRH responses can be suppressed by preimmunization with the carrier, we next addressed the question: is it possible to circumvent or bypass carrier-induced,
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epitope-specific suppression? We first set out to identify a Thelper epitope from DT that would be suitable for use as a carrier. The structural conformation of antigenic peptides bound to class II molecules is still not yet fully understood. However there is a lot of evidence suggesting that both a-helical and extended fl-sheet conformations would interact with major histocompatibility complex (MHC) as well as with T-cell receptor contact sites. Using the amphipathicity algorithm developed by Margalit et al.'9 we identified several putative Thelper epitopes from DT. Peptide 201-222 elicited T-cell proliferative response in C3H/He mice. In a reverse series of experiments, mice which were first immunized with the peptide 201-222, showed a T-cell proliferative response against DT in
REFERENCES 1. SAD S., GUPTA H.M., TALWAR G.P. & RAGHUPATHY R. (1991) Carrier induced suppression of the antibody response to a "self" hapten. Immunology, 74,223.
2. SAD S., TALWAR G.P. & RAGHUPATHY R. (1991) Influence of the genetic background and carrier protein on the antibody response to GnRH. J. reprod. Immunol. 19,197. 3. SHASTRI N., MANHAR S.K. & TALWAR G.P. (1981) Important role of the carrier in the induction of the antibody response without FCA against a self peptide LHRH. Am. J. reprod. Immunol. 1, 262. 4. SHAHA C., KAUL S., KINI M., CHAUDHURI M., ANAND R., DAS C. &
TALWAR G.P. (1986) Immunization against LHRH without the use of Freund's Complete Adjuvant. In: Immunological Approaches to Contraception and Promotion ofFertility (ed. G. P. Talwar), pp. 143. Plenum Press, New York.
vitro.
Figure 3 shows that DT-induced suppression can be circumvented by supplying carrier information in the form of a peptide derived from the original protein. The administration of two boosters with GnRH-peptide elevates the antibody response from 2 to 15 ng/ml. Such carrier peptides may not necessarily be dominant, but they should be capable of using previously primed helper T cells to obtain the desired antibody response without interference from suppressive cells and be generally recongized by a large population. Animals immunized with GnRH-peptide 201-222 do not elicit antibodies to the peptide, and anti-DT sera do not recognize this peptide (data not shown) indicating that this peptide probably is devoid of B-cell determinants. This suggests, but does not prove, that this peptide bypasses suppression by avoiding antigenic competition or clonal dominance. However we have previously shown,' like others,'4 '5 that T cells appear to play an important role in mediating this phenomenon; the precise mechanism underlying this effect are yet to be unequivocally explained, at least in this particular system. It is interesting to note that pre-existing immunity to the peptide 201-222 does not result in inhibition of the anti-GnRH response. Preimmunization with the peptide actually results in an enhancement of anti-GnRH antibody responses when subsequent immunization is done with GnRH linked to DT. A similar strategy has been described by Etlinger et al.24 in a different hapten-carrier system. Since this vaccine is eventually intended for use in humans where one aims for a universal response, the use of a promiscuous T-cell epitope as a carrier would be ideal.2526 Preliminary results from our experiments with GnRH conjugated to known promiscuous T-cell epitopes from tetanus toxin and from the circumsporozoite protein of P. falciparum are encouraging. In conclusion our studies show that appropriate T-helper epitopes can be used to prime helper T cells and to eliminate the potential disadvantage of epitopic suppression resulting from carrier priming. The studies described here are aimed at optimizing and improving on hapten-carrier conjugate vaccines. These studies also pave the way for totally synthetic B-cell determinant-plus-T-cell epitope vaccines.
ACKNOWLEDGMENTS We would like to thank Ms P. V. Priya and Mr B. Kumar for technical help. This work was supported by research grants from the National Coordinated Project of the Department of Biotechnology, Govt. of India, the USAID-CD & RI Programme and the Rockefeller Foundation.
5. JAYASHANKAR R., CHAUDHURI M., OM SINGH, ALAM A. & TALWAR
G. P. (1989) Semisynthetic anti-LHRH vaccine causing atrophy of the prostate. Prostate, 14, 3. 6. GIRi D.K., CHAUDHURI M.K., JAYSASHANKAR R., NEELARAM G.S.,
7.
8.
9.
10. 11.
12. 13.
JAYARAMAN S. & TALWAR G.P. (1990) Histopathological changes in reproductive organs of male Wistar rats following active immunization with LHRH. Exp. Mol. Pathol. 52, 54. HERZENBERG L.A. & TOKUHISA T. (1982) Epitope-specific regulation. I. Carrier-specific induction of suppression for IgG antihapten antibody responses. J. exp. Med. 155, 1730. HERZENBERG L.A., TOKUHISA T., PARKS D.R. & HERZENBERG L.A. (1982) Epitope-specific regulation. II. A bistable, Igh-restricted regulatory mechanism central to immunologic memory. J. exp. Med. 155, 1741. HERZENBERG L.A., TOKUHISA T. & HAYAKAWA K. (1983) Epitopespecific regulation. Ann. Rev. Immunol. 1, 609. JACOB C.O., ARNON R. & SELA M. (1985) Effect of carrier on the immunogenic capacity of synthetic cholera vaccine. Molec. Immunol. 22, 1333. JOHN D.D., WASSERMAN S.S., TORRES J.R., CORTESIA M.J., MURILLO J., LOSONSKY G.A., HERRINGTON D.A., STURCHE D. & LEVINE M.M. (1989) Effect of priming with carrier on response to a conjugate vaccine. Lancet, 1, 1415. SCHUTZE M.P., LECLERC C., JOLIVET M., AUDIBERT F. & CHEDID L. (1985) Carrier induced epitopic suppression, a major issue for synthetic vaccines. J. Immunol. 135, 2319. ETLINGER H.M., FELIX A.M., GILLESSEN D., HEIMER E.P., JUST M., PINK J.R.L., SINIGAGLIA F., TAKACS B., TRZECIAK A. & MATILE H.
(1988) Assessment in humans of a synthetic peptide based against the sporozoite stage of the human malarial parasite Plasmodium falciparum. J Immunol. 140, 626. 14. TAGAWA M., TOKUHISA T., ONO K., TANIGUCHI M., HERZENBERG
L.A. & HERZENBERG L.A. (1984) Epitope-specific regulation. IV. In vitro studies with suppressor T cells induced by carrier/hapten-
carrier immunization. Cell. Immunol. 86, 327. 15. SCHUTZE M.P., LECLERC C., VOGEL F.R. & CHEDID L. (1987) Epitopic suppression in synthetic vaccine models: analysis of effector mechanisms. Cell. Immunol. 104, 79. 16. SCHUTZE M.P., DERIAUD G., PRZEWLOCK1 G. & LECLERC C. (1989) Carrier-induced epitopic suppression is initiated through clonal dominance. J. Immunol. 142, 2635. 17. LECLERC C., SCHUTZE M.P., DERIAUD E. & PRZEWLOCKI G. (1990) The in vivo elimination of CD4+ T cells prevents the induction but not the expression of carrier-induced epitopic suppression. J. Immunol. 145, 1343. 18. STEWARD J.M. & YOUNG J.D. (1984) Solid Phase Synthesis. Pierce Chemical Co., Rockford, IL. 19. MARGALIT H., SPOUGE J., CORNETTE J., CEASE K.B., DELIsi C. & BERZOFSKY J.A. (1987) Prediction of immunodominant helper T cell antigenic sites from the primary sequence. J. Immunol. 138, 2213.
Bypass of epitope-specific suppression using T-helper epitope 20. ROTHBARD J.B. & TAYLOR W.R. (1988) A sequence pattern common to T cell epitopes. EMBO J. 7, 93. 21. HOUGHTON R.A. (1985) General method for rapid solid phase synthesis of large numbers of peptides: specificity of antigenantibody interaction at the level of individual amino acids. Proc. nail. Acad. Sci. U.S.A. 82, 5131. 22. CARLSSON J., DREVIN H. & AXEN R. (1978) Protein thiolation and reversible protein-protein conjugation. Biochem. J. 173, 723. 23. JEFFCOATE S.L., FRASER H.M., HOLLAND D.T. & GUNN A. (1974) Radioimmunoassay of LHRH in serum from man, sheep and rat.
Acta. Endocrinol. (Copenh.), 75, 625. 24. ETLINGER H.M., GILLESSEN D., LAHM H.-W., MATILE H., SCHON-
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FELD H.-J. & TRZEC1AK A. (1990) Use of prior vaccinations for the development of new vaccines. Science, 249, 423. 25. PANINA-BORDIGNON P., TAN G., TERMIJTELEN A., DEMOTZ S., CORRADIN G. & LANZAVECCHIA A. (1989) Universally immunogeneic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur. J. Immunol. 19, 2237. 26. SINIGAGLIA F., GUTrINGER M., KILGUS J., DORAN D.M., MATILE H., ETLINGER H., TRZECIAK A., GILLESSEN D. & PINK R. (1988) A malarial T-cell epitope recognized in association with most mouse and human MHC class II molecules. Nature, 37, 778.
Bypass of carrier-induced epitope-specific suppression using a T-helper epitope S. SAD, K. RAO,* R. ARORA,* G. P. TALWAR & R. RAGHUPATHY* National Institue of Immunology and *International Centre for Genetic Engineering and Biotechnology, New Delhi, India Acceptedfor publication 6 April 1992
SUMMARY A gonadotropin-releasing hormone (GnRH)-based vaccine is being developed as a method for nonsurgical immunotherapy as immunization with this vaccine results in atrophy of the prostate. This vaccine, a conjugate of GnRH and diphtheria toxoid (DT), provides a unique hapten-carrier system for investigating the influence of carrier presensitization on antibody responses to self haptens. In a recent communication' we showed that preimmunization with carriers diphtheria toxoid and tetanus toxoid results in a strain-dependent inhibition of anti-GnRH responses in mice and that T cells from carrier-presensitized mice are responsible for anti-haptenic suppression. In the present report we describe a strategy for bypassing DT-induced epitopic suppression using a T-helper epitope from DT.
INTRODUCTION Gonadotropin-releasing hormone (GnRH) is a highly conserved decapeptide which plays a critical role in the process of reproduction. Immunization of rodents with GnRH-carrier conjugates leads to the production of anti-GnRH antibodies2'3 and the subsequent impairment of fertility.4 Equally interesting is the demonstration that immunization of male rats5'6 and monkeys (D. K. Giri and G. P. Talwar, unpublished observations) with this vaccine results in decreased testosterone levels, decreased testicular size and drastic atrophy of the prostate gland.5'6 This effect on the prostate has prompted us to investigate the immunotherapeutic value of this vaccine in hormone-dependent prostatic hypertrophy. GnRH being a self molecule, and a hapten, has to be chemically linked to foreign carriers such as diphtheria toxoid (DT) or tetanus toxoid (TT) in order to generate anti-GnRH antibodies. In studies designed to investigate the regulation of anti-hapten (GnRH) responses by the carrier (DT or TT), we have shown recently that pre-immunization with DT and/or TT can induce hyporesponsiveness to GnRH.' This presensitization effect is induced by immunizing animals sequentially with the carrier first and subsequently with the hapten-carrier conjugate. This phenomenon of carrier-induced epitope-specific regulation has been studied in a variety of hapten-carrier systems by several investigators7-'3 who have demonstrated that preimmunization with a carrier often results in an inhibitory effect on the production of antibodies to new epitopes linked to the same protein. Studies done so far show that epitope-specific suppression can be induced with diverse antigens administered under Correspondence: Dr R. Raghupathy, Immunogenetics Laboratory, National Institute of Immunology, New Delhi-i 10067, India.
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widely different immunization conditions in a variety of mouse strains. Equally interesting is the finding that while the antibody responses to the hapten are suppressed, anti-carrier responses proceed normally.7'-3 The universality of this phenomenon has been documented by studies in mice,7-9"2 rabbits'0 and humans.""3 Our studies on the GnRH-DT/TT system show that this effect is a strain-dependent one; presensitization with DT or TT results in a suppression of anti-GnRH titres in some, but not all, strains of mice tested.' Epitope-specific regulatory effects have been attributed by some workers to suppressor T cells''4"5 and by others to antigenic competition or clonal dominance of carrier-specific B cells. 16,17 If the former explanation is correct, suppression can be attributed to the possible existence of suppressor epitopes on DT or TT. If the latter is correct, competition can be attributed to the existence of too many competing determinants on DT or TT. Whatever the case, we speculated that epitope-specific suppression could be bypassed or circumvented by the use of a synthetic T-helper epitope from DT, as a carrier. In this report, we describe the delineation of one such T-helper epitope from DT and its usefulness in bypassing DT-induced GnRH-directed suppression.
MATERIALS AND METHODS Synthesis and conjugation of GnRH A modified GnRH decapeptide containing D-lysine (instead of glycine) at position six, attached to a linker E-amino caproic acid, was synthesized by the solid phase method using benzhydrylamine resin as the solid support.'8 This peptide was conjugated to DT by the glutaraldehyde method.2
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Selection ofpeptidesfrom DT Nine DT peptides spanning residues 23-40,43-58, 93-109, 118135, 142-159, 201-222, 284-300, 361-378 and 508-524 were predicted to be T-helper epitopes on the basis of high amphipathic scores obtained using the algorithm of Margalit et al.'9 These were subsequently found to contain the Rothbard and Taylor20 motif.
Synthesis of DTpeptides Peptides selected from DT were synthesized using the simultaneous multiple peptide synthesis technique originally described by Houghten.2' tBoc derivatized amino acids (Fisher Biotec, Pittsburg, PA) were used for accomplishing the synthesis on 4methylbenzhydrylamine resin (Nova Biochem. Ag, Basel, Switzerland). All peptides were synthesized on a 0 07 mmol scale and cleaved from the resin with anhydrous hydrogen fluoride in the presence of anisole, after which the crude peptides were purified by high-performance liquid chromatography (HPLC) using a linear gradient of acetonitrile, with 0-01 % trifluoracetic acid on a Vydac C18 column. Conjugation of GnRH to DTpeptide Peptide 201-222 was chosen as a carrier on the basis of its T-cell proliferative capacity (as discussed in Results). GnRH-peptide (201-222) conjugate was prepared by thiolating GnRH using a hetero-bifunctional reagent, succinimidyl 1-3-(2-pyridyl dithio) propionate (SPDP).22 GnRH in phosphate buffer was allowed to react with SPDP in dimethyl formamide, at room temperature for I hr, after which dithiothreitol (DTT) was added and the product purified by Sephadex G-10 chromatography. Peptide 201-222 which contains a cysteine at its N-terminal end was reduced with DTT prior to purification on a Sephadex G-10 column. The thiolated GnRH was allowed to react with the reduced peptide 201-222 at room temperature for 3 h and the conjugate so formed purified by Sephadex G-25 chromatography.
T-cell proliferation assays The T-cell proliferative activity of DT and its peptides was evaluated by immunizing mice in the foot pads with 10 pg of DT or 100 pg of peptide in 25 p1 saline emulsified with an equal volume of complete Freund's adjuvant (CFA). Eight days after immunization, the inguinal and popliteal lymph node cells (5 x 105 cells/well) were suspended in RPMI-1640 medium supplemented with 0-5% normal mouse serum and incubated in triplicate with various concentrations of DT or peptides in a 96well tissue culture plate. After incubation for 4 days in a humidified 5% C02 atmosphere, the cultures were pulsed for 18 hr with 1 pCi [3H]thymidine and harvested on glass fibre filters prior to liquid scintillation counting. Immunization In order to investigate whether the GnRH-peptide conjugate can bypass carrier-induced suppression, C3H/He mice (10/ group) were presensitized on Day 1 with DT (100 pg on calcium phosphate) or with saline, and then boosted on Days 30 and 60 with GnRH-DT or GnRH-peptide conjugate. Mice were bled on Day 75 and analysed for antibodies against GnRH. The effect of priming with T-helper epitope on the antibody response to GnRH was determined by priming C3H/He mice (10/group) on Day 1 with the peptide 201-222 (100 pg.
emulsified with CFA or adsorbed on calcium phosphate) and later boosted on Days 30 and 60 with GnRH-DT adsorbed on calcium phosphate. Mice were bled on Day 67 and analysed for anti-GnRH antibodies. Assay for anti-GnRH antibodies Anti-GnRH antibody titres were measured by radioimmunoassay and expressed as antigen-binding capacity. All individual sera were titrated simultaneously by the dilution method, using the same batch of the radioactive tracer. The assay protocol consisted of mixing 50 Mp each of normal horse serum [diluted 2 5 times in assay buffer, 50 mm phosphate-buffered saline (PBS) with 0 1 % bovine serum albumin], diluted antisera, ['25I]GnRH and assay buffer. After incubation for 18 hr at 40, the antibodybound fraction was precipitated by adding 1 ml of cold ethanol23 and separated by centrifuging at 1500 g for 15 min at 4°. ['25I]GnRH binding capacity (expressed in ng/ml serum) was calculated at a point at which proportionality between the dilution of the antiserum and ['251I]GnRH was obtained. The specific activity of the radiolabelled GnRH was used to calculate the antigen-binding capacity.
RESULTS T-cell proliferative response to DT peptides Of the strains susceptible to DT-induced suppression of antiGnRH titres, C3H/He mice showed maximal suppression.' In order to identify a T-helper epitope from DT, that would induce T-cell proliferation in C3H/He mice, the nine DT peptides synthesized were tested for their proliferative capacities in vitro. The capacity of DT peptides to induce T-cell proliferation was ascertained by measuring their capacity to induce the proliferation in vitro of lymph node cells from mice immunized in vivo with DT. C3H/He mice were immunized with 100 pg of DT in 25 p1 saline emulsified in CFA. Eight days later the inguinal and popliteal lymph node cells were incubated with various peptides of DT. Figure 1 depicts the T-cell proliferative response generated in C3H/He mice peptide 201-222 from DT. In order to determine whether in vivo immunization with peptide 201-222 can recruit T cells responsive to DT in vitro, C3H/He mice were injected with peptide 201-222 and their
40 PO
0
30-
CL 20-
E10
07 15 31 62 125 25 50 Antigen concentration (pg/ml) T-cell 1. proliferative response of DT (0) and peptide 201-222 Figure (0) in C3H/He mice (five per group) immunized with DT. The background response in the control wells (without antigen) was in the
range of 1600-2400 c.p.m.
Bypass of epitope-specific suppression using T-helper epitope
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lymph node cells incubated with either DT or peptide 201-222. Figure 2 depicts the T-cell proliferative response generated by DT and peptide 201-222 in C3H/He mice.
30 _
,_20
x
Bypass of DT-induced suppression 10
0 7 5 3 6 2 125 25 50 Antigen concentration (ug/ml)
Figure 2. T-cell proliferative response of DT (0) and peptide 201-222 (0) in C3H/He mice (five per group immunized with peptide 201-222). The background response in the control wells (without antigen) was in the range of 1100-2200 c.p.m.
With a view to ascertain whether DT-induced suppression can be circumvented by the use of peptide 201-222 as a carrier, DTpresensitized mice were boosted with GnRH linked to peptide 201-222. This peptide was selected for use as a carrier as it elicited T-cell proliferation in C3H/He mice. Preimmunization with DT clearly inhibits the anti-GnRH antibody response in C3H/He mice (Fig. 3). There is a dramatic decrease in antiGnRH antibody titres from 23 ng/ml to 2 ng/ml GnRH binding capacity. Figure 3 shows that DT-induced suppression can be bypassed by using peptide 201-222 as a carrier. One injection of DT followed 30 days later by two GnRH-peptide boosters elevates the antibody response from 2 to 15 ng/ml. Injection of mice with GnRH-peptide conjugate (without preimmunization with DT) also elicits a moderate anti-GnRH antibody response. Immunization of mice with either GnRH or peptide 201-222 alone does not induce anti-GnRH antibody formation (data not shown). Effect of peptide priming on the antibody response to GnRH
DT DT G-DT G-DT G-P G-DT G-DT G-P
-
G-P G-P
Figure 3. Bypass of carrier induced epitope-specific suppression using a T-helper epitope from the carrier. C3H/He mice (10 per group) were primed on Day I with DT (100 pg on calcium phosphate) or saline, and later boosted on Days 30 and 60 with either GnRH-DT (G-DT) or GnRH-peptide 201-222 (G-P) emulsified with Freund's adjuvant. Values are expressed as mean + SE of the results obtained on Day 75.
In order to determine the influence of pre-existing immunity to the peptide on the antibody response to GnRH, mice were primed with peptide 201-222 from DT and later boosted with GnRH-DT. It is interesting to note that pre-existing immunity to this peptide does not result in inhibition of the anti-GnRH antibody response. Preimmunization with this peptide actually results in an enhancement of anti-GnRH antibody titres when subsequent immunization is done with GnRH linked to DT (Fig. 4). This enhancement of response from 10 ng/ml to 25 ng/ ml was observed when CFA was used as an adjuvant. However we failed to observe a similar effect when calcium phosphate was used as an adjuvant.
E
a
C
DISCUSSION
20
CM
- (FA)
G-DT G-DT
Peptide (FA) G-DT G-OT
(CP) PMde G-DT G-DT
Figure 4. Effect of peptide priming on the antibody response to GnRH. C3H/He mice (10 per group) were primed on Day I with the peptide 201-222 emulsified with Freund's adjuvant (FA) or adsorbed on calcium phosphate (CP) and later bossted on Days 30 and 60 with GnRH-DT adsorbed on calcium phosphate adjuvant. Values are expressed as mean + SE of the results obtained on Day 67.
It is now abundantly clear that the carrier can exert significant influences on responses to haptens linked to the carrier. The GnRH vaccine is a hapten-carrier conjugate which is intended for use in humans, where one aims for a universal response with minimal genetic restriction. It is important therefore, to investigate the influence of carriers on the antibody response to GnRH. From this point of view we felt it essential to study the effects of pre-existing immunity to the carrier on the antibody response to the hapten, GnRH. We have shown recently that preimmunization with DT and/or TT induces hyporeseponsiveness to GnRH.1 Experiments described here were designed to ascertain whether epitope-specific suppression could be bypassed using T-helper epitopes as carriers. Having shown that anti-GnRH responses can be suppressed by preimmunization with the carrier, we next addressed the question: is it possible to circumvent or bypass carrier-induced,
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S. Sad et al.
epitope-specific suppression? We first set out to identify a Thelper epitope from DT that would be suitable for use as a carrier. The structural conformation of antigenic peptides bound to class II molecules is still not yet fully understood. However there is a lot of evidence suggesting that both a-helical and extended fl-sheet conformations would interact with major histocompatibility complex (MHC) as well as with T-cell receptor contact sites. Using the amphipathicity algorithm developed by Margalit et al.'9 we identified several putative Thelper epitopes from DT. Peptide 201-222 elicited T-cell proliferative response in C3H/He mice. In a reverse series of experiments, mice which were first immunized with the peptide 201-222, showed a T-cell proliferative response against DT in
REFERENCES 1. SAD S., GUPTA H.M., TALWAR G.P. & RAGHUPATHY R. (1991) Carrier induced suppression of the antibody response to a "self" hapten. Immunology, 74,223.
2. SAD S., TALWAR G.P. & RAGHUPATHY R. (1991) Influence of the genetic background and carrier protein on the antibody response to GnRH. J. reprod. Immunol. 19,197. 3. SHASTRI N., MANHAR S.K. & TALWAR G.P. (1981) Important role of the carrier in the induction of the antibody response without FCA against a self peptide LHRH. Am. J. reprod. Immunol. 1, 262. 4. SHAHA C., KAUL S., KINI M., CHAUDHURI M., ANAND R., DAS C. &
TALWAR G.P. (1986) Immunization against LHRH without the use of Freund's Complete Adjuvant. In: Immunological Approaches to Contraception and Promotion ofFertility (ed. G. P. Talwar), pp. 143. Plenum Press, New York.
vitro.
Figure 3 shows that DT-induced suppression can be circumvented by supplying carrier information in the form of a peptide derived from the original protein. The administration of two boosters with GnRH-peptide elevates the antibody response from 2 to 15 ng/ml. Such carrier peptides may not necessarily be dominant, but they should be capable of using previously primed helper T cells to obtain the desired antibody response without interference from suppressive cells and be generally recongized by a large population. Animals immunized with GnRH-peptide 201-222 do not elicit antibodies to the peptide, and anti-DT sera do not recognize this peptide (data not shown) indicating that this peptide probably is devoid of B-cell determinants. This suggests, but does not prove, that this peptide bypasses suppression by avoiding antigenic competition or clonal dominance. However we have previously shown,' like others,'4 '5 that T cells appear to play an important role in mediating this phenomenon; the precise mechanism underlying this effect are yet to be unequivocally explained, at least in this particular system. It is interesting to note that pre-existing immunity to the peptide 201-222 does not result in inhibition of the anti-GnRH response. Preimmunization with the peptide actually results in an enhancement of anti-GnRH antibody responses when subsequent immunization is done with GnRH linked to DT. A similar strategy has been described by Etlinger et al.24 in a different hapten-carrier system. Since this vaccine is eventually intended for use in humans where one aims for a universal response, the use of a promiscuous T-cell epitope as a carrier would be ideal.2526 Preliminary results from our experiments with GnRH conjugated to known promiscuous T-cell epitopes from tetanus toxin and from the circumsporozoite protein of P. falciparum are encouraging. In conclusion our studies show that appropriate T-helper epitopes can be used to prime helper T cells and to eliminate the potential disadvantage of epitopic suppression resulting from carrier priming. The studies described here are aimed at optimizing and improving on hapten-carrier conjugate vaccines. These studies also pave the way for totally synthetic B-cell determinant-plus-T-cell epitope vaccines.
ACKNOWLEDGMENTS We would like to thank Ms P. V. Priya and Mr B. Kumar for technical help. This work was supported by research grants from the National Coordinated Project of the Department of Biotechnology, Govt. of India, the USAID-CD & RI Programme and the Rockefeller Foundation.
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carrier immunization. Cell. Immunol. 86, 327. 15. SCHUTZE M.P., LECLERC C., VOGEL F.R. & CHEDID L. (1987) Epitopic suppression in synthetic vaccine models: analysis of effector mechanisms. Cell. Immunol. 104, 79. 16. SCHUTZE M.P., DERIAUD G., PRZEWLOCK1 G. & LECLERC C. (1989) Carrier-induced epitopic suppression is initiated through clonal dominance. J. Immunol. 142, 2635. 17. LECLERC C., SCHUTZE M.P., DERIAUD E. & PRZEWLOCKI G. (1990) The in vivo elimination of CD4+ T cells prevents the induction but not the expression of carrier-induced epitopic suppression. J. Immunol. 145, 1343. 18. STEWARD J.M. & YOUNG J.D. (1984) Solid Phase Synthesis. Pierce Chemical Co., Rockford, IL. 19. MARGALIT H., SPOUGE J., CORNETTE J., CEASE K.B., DELIsi C. & BERZOFSKY J.A. (1987) Prediction of immunodominant helper T cell antigenic sites from the primary sequence. J. Immunol. 138, 2213.
Bypass of epitope-specific suppression using T-helper epitope 20. ROTHBARD J.B. & TAYLOR W.R. (1988) A sequence pattern common to T cell epitopes. EMBO J. 7, 93. 21. HOUGHTON R.A. (1985) General method for rapid solid phase synthesis of large numbers of peptides: specificity of antigenantibody interaction at the level of individual amino acids. Proc. nail. Acad. Sci. U.S.A. 82, 5131. 22. CARLSSON J., DREVIN H. & AXEN R. (1978) Protein thiolation and reversible protein-protein conjugation. Biochem. J. 173, 723. 23. JEFFCOATE S.L., FRASER H.M., HOLLAND D.T. & GUNN A. (1974) Radioimmunoassay of LHRH in serum from man, sheep and rat.
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FELD H.-J. & TRZEC1AK A. (1990) Use of prior vaccinations for the development of new vaccines. Science, 249, 423. 25. PANINA-BORDIGNON P., TAN G., TERMIJTELEN A., DEMOTZ S., CORRADIN G. & LANZAVECCHIA A. (1989) Universally immunogeneic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur. J. Immunol. 19, 2237. 26. SINIGAGLIA F., GUTrINGER M., KILGUS J., DORAN D.M., MATILE H., ETLINGER H., TRZECIAK A., GILLESSEN D. & PINK R. (1988) A malarial T-cell epitope recognized in association with most mouse and human MHC class II molecules. Nature, 37, 778.
