Journal of Reproductive Immunology, 19 ( 1991 ) 197--207

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Elsevier Scientific Publishers Ireland Ltd.

JRI 00699

Influence of the genetic background and carrier protein on the antibody response to GnRH S u b a s h S a d , G . P . T a l w a r a n d Raj R a g h u p a t h y National Institute of hnmunolog.v, J.N.U. Complex, New Delhi-110067 (Indial (Accepted for publication 7 September 1990)

Summary A vaccine against the gonadotropin releasing hormone (GnRH) is being developed as an immunological method for the treatment of prostatic enlargement. The work described here was aimed at investigating the influence of the genetic background on immune responses to GnRH conjugated to diphtheria toxoid (DT). Mice of different strains were injected with the conjugate and the antibody levels against GnRH and DT quantitated in order to examine the effect of genetic background on immune responses to the hapten and the carrier. All immunized animals produced antibodies to DT. Anti-GnRH antibodies were generated by all strains of mice except 129. The low anti-GnRH response in the 129 strain mice did not appear to be MHC-linked, as C57BL/6 mice, which bear the same MHC haplotype as 129 mice, were able to generate a strong anti-GnRH response. The non-responsiveness to the hapten (GnRH) in 129 strain mice was overcome by the use of an 'alternate carrier' approach. Key words: anti-GnRH response; genetic control; hyporesponsiveness; alternate carrier.

Introduction The g o n a d o t r o p i n releasing h o r m o n e ( G n R H ) regulates a cascade o f events leading to the p r o d u c t i o n o f sex steroid h o r m o n e s and the product i on Correspondence to: Dr. Raj Raghupathy. 0165-0378/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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of gametes in both males and females. GnRH is critical for the normal functioning of the reproductive system and hence for fertility. Immunization with GnRH-carrier conjugates leads to the production of anti-GnRH antibodies and the subsequent impairment of fertility (Talwar et al., 1980). It has also been shown that the administration of either polyvalent (Fraser et al., 1973) or monoclonai (Talwar et al., 1985) anti-GnRH antibodies in vivo leads to infertility in male and female rats. Immunization of rats with GnRH conjugated to tetanus toxoid (TT) as a carrier results in decreased testicular size, cessation of spermatogenesis and a severe reduction in testosterone levels (Shastri et al., 1981; Shaha et al., 1986). A marked atrophy of the prostate gland was observed following immunization of rats with the GnRH conjugate (Jayashankar et al., 1989; Giri et al., 1990). The GnRH vaccine is currently in Phase I clinical trials for "immunological surgery" in patients with carcinoma of the prostate and benign prostatic hypertrophy. GnRH is by and large an evolutionarily conserved decapeptide in mammalian species. It is a hapten and a self antigen; thus, in order to obtain an antibody response to GnRH in the absence of Freund's complete adjuvant, it has to be linked to non-self carriers such as tetanus toxoid (Shastri et al., 1981) or diphtheria toxoid (DT) (Jayashankar et al., 1989). An important step in the development of a vaccine is the demonstration that the vaccine is sufficiently immunogenic in all individuals, irrespective of their genetic background. It is now well established that the major histocompatibility complex (MHC) plays crucial roles in the initiation and degree of immune responses (Benaceraff et al., 1978; Schwartz, 1978). Some non-MHC genes are also known to influence immune responses (Gammon et al., 1987). Hence an important prerequisite to large scale immunization with the GnRH vaccine is the investigation of potential genetically-influenced variations in anti-GnRH immune responses. One of the objectives of the work described here was to investigate the influence of the genetic background on immune responses to GnRH and to ascertain whether this response can be manipulated. This study also provides data on the influence of the carrier on anti-hapten (GnRH) responses. The basic strategy involves the immunization of different strains of mice with the GnRH conjugate and the measurement of the antibodies generated, both to GnRH and to the carrier protein. This report describes the influence of the genetic background and the carrier on the antibody response to GnRH and also an approach to elicit an anti-GnRH antibody response in a non-responder strain. Materials and methods

GnRH conjugates A modified GnRH containing D-lysine (instead of L-glycine) at position

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six attached to a linker, amino caproic acid, was synthesised by the solidphase method using benzylhydrylamine resin as the solid support. Both tBoc and Fmoc protective groups were employed for accomplishing the synthesis. The peptide was cleaved from the resin with anhydrous hydrogen fluoride in the presence of anisole, after which the crude peptide was purified by preparative H P L C (Waters Prep LC 3000 system) using a Vydac C18 column. The purified peptide was conjugated to diphtheria toxoid (DT) (Serum Institute, Pune, India) by the glutaraldehyde method (Avrameas, 1969). Briefly, 40 mg of GnRH in 5.7 ml of 0.1 M phosphate buffer (pH 7) was added to 28.2 mg of DT (60 ml, 0.1 M phosphate buffer (pH 7). Glutaraldehyde (234 /al) (Sigma Chemical Co.) in 45 ml of phosphate buffer was cooled and added gradually to the above mixture. The final concentration of glutaraidehyde in the reaction mixture was 0.1%. The reaction was carried out for 20 h at 4°C and then stopped by dialysis against phosphate buffered saline (PBS). The number of moles of GnRH conjugated per mole of DT was evaluated on a Waters PICO TAG amino acid analyzer. Briefly, a known amount of the conjugate was hydrolyzed with concentrated hydrochloric acid at 120°C for 17--18 h. After drying the resulting material, excess acid was neutralized with ethanol/water/triethylamine (2:1:1 by vol.). Derivatization was done with ethanol/water/triethylamine/phenyl isothiocyanate (7:1:1:1 by vol.) for 10 min at room temperature. The preparation was dried and dissolved in the PICO TAG sample diluent and loaded onto the column. The PTHderivatives of amino acids were eluted with acetonitrile gradient. The amount of amino caproic acid detected gives a direct estimation of the number of moles of GnRH conjugated per mole of DT. Native GnRH was conjugated to tetanus toxoid (TT) (Serum Institute, Pune, India) by the carbodiimide method (Shastri et al., 1981).

Immunization Four to six-week-old CBA (H-2k), AKR (H-2k), C3H (H-2k), BALB/c (H2 d) C57BL/6 (H-2b), 129 (H-2b), SJL (H-2 ~) and FVB (H-2 q) mice were used for immunization studies. Ten adult mice per group were immunized with 3 monthly injections of the GnRH conjugate. Animals were injected intramuscularly with 200/al of the conjugate containing 10/~g of GnRH, adsorbed on alum. Immunized animals were bled 7 days after the third injection and analysed for antibodies to the hapten (GnRH) and to the carrier. lodination of GnRH Synthetic G n R H was labelled with '25I by the IODO-GEN method following the procedure of Fraker and Speck (1978). The iodinated material was purified on a Sephadex G-25 column (1 x 40 cm). Fractions (0.5 ml) were collected and the radioactivity estimated in a gamma counter (LKB 1260 II). Two distinct peaks were obtained. The second peak contained the radiola-

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belled hormone which was used in the assay after dilution with buffer to approximately 10,000 cpm/50/al.

Assay for anti-GnRH antibodies Anti-GnRH antibody titers were assayed by radioimmunoassay and expressed as antigen binding capacity (Abc). 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/al each of normal horse serum (as carrier protein) diluted 2.5 times in 50 mM phosphate buffered saline with 0.1°70 BSA diluted antiserum, [~2~]GnRH and assay buffer. After incubation for 18--20 h at 4°C, the antibody bound fraction was separated by the method of Jeffcoate et al., (1974). Antigen binding capacity (expressed in ng/mi) was calculated at a point at which proportionality between the dilution of the antiserum and [~251]GnRH binding was obtained. Assays for anti-carrier antibodies Anti-carrier (DT and TT) antibodies were detected by ELISA. Briefly, 1 /ag protein in 100/al phosphate buffer (50 raM, pH 7.4) was coated onto each well of Nunc (Denmark) ELISA plates. This was followed by incubation with 100/al of diluted antisera and subsequently with 100/al of anti-mouse immunoglobulin antibodies conjugated to horse radish peroxidase. Each incubation lasted for 1 h at 37°C and was followed by three 5-min washes with the washing buffer (PBS, pH 7.4 with 0.2O7,o Tween 20). Colour was developed by adding 50/al of ortho-phenylenediamine (Sigma Chemical Co.) as the substrate. The reaction was stopped after 20 min with 50/al of 5 N sulphuric acid and the absorbance measured at 490 nm. Results

Characteristics o f the GnRH-D T conjugate Amino acid analysis of the GnRH peptide revealed that the amino acid composition of the purified peptide corresponded to its constituent amino acids. Figure 1 shows the H P L C profile of a purified preparation of the GnRH-DT conjugate. As is evident, the conjugate does not contain any free DT or GnRH and elutes earlier than DT or GnRH. The degree of conjugation of GnRH to DT was estimated by Pico Tag method exploiting the presence of amino caproic acid in GnRH. Immune response to the GnRH-DTconjugate Anti-GnRH response. Eight strains of mice were immunized with the GnRH-DT conjugate. Mice of all strains tested, except the 129 strain, generated antibodies to GnRH (Fig. 2). The response to the modified vaccine was

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substantially higher in magnitude as well as in the range of strains in which it is immunogenic as compared to the response elicited by the native GnRH-TT (unpublished observations). Mice of the FVB strain produced high antiGnRH antibody titres whereas 129 mice showed a poor anti-GnRH response. The other strains tested generated a moderate anti-GnRH response. A n ti-D T r e s p o n s e

Figure 3 depicts the anti-DT response in eight strains of mice after immunization with GnRH-DT. SJL appears to be a high responder strain whereas CBA and AKR are low responders. There appeared to be no correlation between responses to the hapten (modified GnRH) and to the carrier (DT). Strains which respond well to GnRH (AKR, CBA) were relatively low res-

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Fig. 2. Anti-GnRH antibod.~ response in different strains of mice (ten mice,'group) after three injections of G n R H - D T ( 10 ~g) at monthly interx.als. Values are expressed as mean __. S.E.

ponders to DT, while the 129 strain, which was a low responder to G n R H , presented a good response to DT.

Alternate carrier approach As is evident in Fig. 2, the G n R H - D T conjugate elicited a good response in every strain tested except the 129 strain. This conceivably could be due to a lack of appropriate T cell help by the carrier, DT, or due to a gap in the B cell repertoire to G n R H . To test the former hypothesis, we used an "alternate carrier a p p r o a c h " to determine whether DT is responsible for the low response to G n R H in the 129 strain. The same mice that had failed to exhibit high levels of anti-GnRH antibodies after three immunizations with G n R H linked to DT were immunized twice with G n R H linked to a different carrier, namely tetanus toxoid (TT). On altering the carrier, a strong anti-GnRH response was generated in the 129 mice (Fig. 4). In a separate group of mice two further immunizations with G n R H linked to DT did not raise the level of anti-GnRH antibodies to an appreciable level. However, two immunizations with G n R H - T T resulted in a substantial a n t i - G n R H response. Figure 5 depicts the anti-DT and anti-TT antibody titres in this group of mice.

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Influence of the genetic background and carrier protein on the antibody response to GnRH.

A vaccine against the gonadotropin releasing hormone (GnRH) is being developed as an immunological method for the treatment of prostatic enlargement. ...
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