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GnRH tandem peptides for inducing an immunogenic response to GnRH-I without cross-reactivity to other GnRH isoforms
Vaccine 23 (2005) 4915–4920
GnRH tandem peptides for inducing an immunogenic response to GnRH-I without cross-reactivity to other GnRH isoforms J.A. Turkstra a,∗ , W.M.M. Schaaper a , H.B. Oonk b , R.H. Meloen a,c b
a Pepscan Systems, Edelhertweg 15, 8219 PH, Lelystad, The Netherlands Animal Sciences Group, Wageningen University and Research Centre, Edelhertweg 15, 8219 PH, Lelystad, The Netherlands c Academic Biomedical Centre, University Utrecht, Yalelaan 1, 3584 CL, Utrecht, The Netherlands
Received 29 December 2004; received in revised form 14 April 2005; accepted 20 May 2005 Available online 13 June 2005
Abstract Gonadotropin releasing hormone (GnRH) occurs in various isoforms in mammals, i.e. GnRH-I (mammalian GnRH), GnRH-II (chicken GnRH-II), GnRH-III (salmon GnRH) and two forms of lamprey GnRH. The function of the latter four molecules have only been partially investigated. Also not much is known about the physiological effects of GnRH-I immunization on the function of these GnRH isoforms. In order to avoid possible harmful side-effects due to undesired neutralization of GnRH isoforms, GnRH-I specificity of antibodies raised against a panel of alternative GnRH antigens was determined. The results show that GnRH antigens can be designed which generate antibodies that specifically bind GnRH-I, without cross-reacting with other GnRH isoforms. © 2005 Published by Elsevier Ltd. Keyword: GnRH-isoforms specificity vaccine
1. Introduction In mammals, the decapeptide gonadotropin releasing hormone (GnRH-I) triggers the synthesis and release of luteinising hormone (LH) and follicle stimulation hormone (FSH) from the pituitary. LH and FSH are responsible for steroidogenesis and gametogenesis. Recently, additional isoforms of GnRH were identified in mammals. GnRH-II, a molecule with an amino acid sequence similar to chicken GnRH-II, was cloned from a human genomic library [1]. A third GnRH isoform (GnRH-III), with a similar amino acid sequence as salmon GnRH, was extracted from the bovine and human brain [2]. Immunohistologically, the presence of GnRH isoforms (lamprey GnRH-I, lamprey GnRH-III) that were first detected in lamprey was demonstrated in the mammalian brain [3,4]. The function of the various isoforms in mammals, however, is ill defined. Most likely they play a role in fertility regulation. GnRH-II and GnRH-III are able to ∗
Corresponding author. Tel.: +31 320 237212; fax: +31 320 238120. E-mail address: [email protected] (J.A. Turkstra).
0264-410X/$ – see front matter © 2005 Published by Elsevier Ltd. doi:10.1016/j.vaccine.2005.05.020
induce the secretion of gonadotropines [2,5–8]. The presence of GnRH-II in the human endometrium and the fact that GnRH-II is released from the early human placenta indicates that it may be involved in embryo implantation and regulation of pregnancy [9,10]. It has also been suggested that the GnRH isoforms are involved in sexual behaviour [2,11,12]. In contrast to GnRH-II and GnRH-III, lamprey GnRH-III does not induce LH secretion. This isoform seemed to selectively activate FSH release, indicating that it could be the FSH-releasing factor [13–15]. However, this could not be confirmed by others [16]. GnRH-II is not only expressed in the brain, but also in tissues outside the brain. High concentrations were determined in the kidneys, bone marrow and prostate [1]. This indicates that GnRH-II may also have functions unrelated to fertility regulation. Indeed, neutralization of GnRH-II by active immunization, led to a slight increase in kidney weight suggesting a role for GnRH-II in kidney function [17]. Immunization against GnRH-I is shown to be an efficient method to block fertility and reproduction in male and female mammals [18–21]. In addition, immunization suppresses hor-
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mone dependent malfunctions, such as breast cancer and prostate cancer [22–26]. However, with the new insight, that several GnRH isoforms are present in mammals, it is not known which of the GnRH isoforms are targetted by GnRHI immunization. Although all GnRH isoforms seem to be involved in fertility, some may have additional functions unrelated to fertility. Possible undesired effects of GnRH-I neutralization on these unrelated functions must be circumvented. Therefore, the specificity of antibodies raised against a panel of alternative GnRH antigens for the GnRH isoforms was studied. Here we report that some GnRH antigens elicite antibodies which cross-react with most of the GnRH isoforms, while others are only directed against GnRH-I, indicating that small spectrum isoform specific antibodies or wide spectrum isoform non-specific antibodies can be generated as desired.
Table 2 Amino acid sequences of GnRH isoformsa Peptide
Amino acid sequence
Mammalian GnRH (GnRH-I) Chicken GnRH-II (GnRH-II) Salmon GnRH (GnRH-III) Lamprey GnRH-I (lamprey GnRH-I) Lamprey GnRH-III (lamprey GnRH-III)
pEHWSYGLRPGb pEHWSHGWYPGb pEHWSYGWLPGb pEHYSLEWKPGb pEHWSHDWKPGb
Underlined amino acid differ from GnRH-I amino acid sequence. a pE, pyroglutamic acid. b Amide.
2. Materials and methods
teine, subsequent conjugation to ovalbumine was achieved using N-ethyl-N -(3 -dimethyl-aminopropyl) carbodiimide. Antigens were administered with Specol adjuvant to young male pigs at 10 and 17 weeks of age. In the present study, only sera raised against five fully effective G6k-GnRHtandem dimer peptides are included [27]. Each group consisted of six (G6k) or seven (pE1A, S4A, R8A, G10A) pigs.
2.1. Antisera
2.2. GnRH isoform peptides
Antisera were raised in young male pigs and directed against different GnRH antigens. Table 1 gives an overview of the peptides used for immunization, including amino acid sequence in single letter code. Peptide synthesis, vaccine preparation and immunization procedure for mono and tandem has been described [13]. Briefly, the GnRH molecules synthesized with a C-terminal cysteine were conjugated to Keyhole Limpet Hemocyanin via maleinimidobenzoyl-hydoxysuccinimide ester. Conjugates were emulsified in complete freund adjuvant and incomplete freund adjuvant for the first and second immunization, respectively. The vaccines were administered to young male pigs at 10 and 18 weeks of age. Only sera from successfully treated pigs, as determined by undetectable serum testosterone levels and reduced testis weights were used in this study (3 and 5 pigs for the mono and the tandem group, respectively). Synthesis of the G6k-GnRH-tandem peptides has been described previously [27]. The peptides were brought in the tandem dimer form by dimerization via the C-terminal cys-
The peptides used for the competitive radioimmunoassay were synthesized and analyzed as described previously [27]. Peptides were at least 90% pure. Amino acid sequences of the synthesized peptides are given in Table 2 in a single letter code. 2.3. Competitive radioimmunoassay Serum samples were diluted in PBS with 0.4% BSA (dilution buffer). Fifty microliters serum dilution was put in microwell plates and pre-incubated with 25 l GnRH isoform peptide solution. An unrelated peptide was included as negative control. This mixture was allowed to incubate for 24 h at 4 ◦ C. The next day 25 l iodinated GnRH-I (approximately 13,000 cpm, Amersham Pharmacia Biotech, Buckinghamshire, England) was added to compete with the pre-incubated GnRH isoform peptides for binding to the antibodies. After incubation overnight (4 ◦ C) unbound peptide was separated from bound peptide using dextran-coated charcoal. After centrifugation (2000 × g, 15 min) supernatant
Table 1 Amino acid sequences and abbreviations of peptides used for immunizationa GnRH antigen
Amino acid sequence
Abbreviation
GnRH-monomer GnRH-tandem G6k-GnRH-tandem pE1A-G6k-GnRH-tandem S4A-G6k-GnRH-tandem R8A-G6k-GnRH-tandem G10A-G6k-GnRH-tandem
pEHWSYGLRPGCb pEHWSYGLRPGQHWSYGLRPGCb pEHWSYkLRPGQHWSYkLRPGCb c AHWSYkLRPGAHWSYkLRPGCb pEHWAYkLRPGQHWAYkLRPGCb pEHWSYkLAPGQHWSYkLAPGCb pEHWSYkLRPAQHWSYkLRPACb
Mono Tandem G6k pE1A S4A R8A G10A
k, d-lysine; G6k, Gly on position 6 in the native GnRH sequence substituted by d-lysine; Underlined amino acid differ from G6k-GnRH-tandem. a pE, pyroglutamic acid. b Amide. c Acetyl.
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was separated, counted and the percentage iodinated GnRH bound to the antibodies was calculated.
3. Results In a competitive radioimmunoassay displacement of iodinated GnRH-I by the GnRH isoforms (Table 2) was determined. Competition between the iodinated GnRH-I and 250 nM GnRH-I, resulted in the expected reduction of binding of iodinated GnRH-I by the antisera for all sera. Iodinated GnRH-I was displaced for 70–100% by GnRH-I (mean value per group >89%; Fig. 1a). Similar results were obtained with 25 nM GnRH-I (mean value per group >88%), whereas 2.5 nM GnRH-I showed a lower reduction of displacement (mean value per group >67%). In contrast, displacement of iodinated GnRH-I by 250 nM GnRH-II revealed clear differences between groups (Fig. 1b). Sera raised against pE1A and S4A, allowed hardly any displacement of iodinated GnRH-I by GnRH-II. Only one serum out of seven of both, the pE1A and the S4A vaccinated groups, showed a substantial displacement percentage (38 and 46%, respectively), resulting in a mean displacement percentage of 8 and 16% for pE1A and S4A, respectively. Sera raised against mono, tandem, G6k, R8A and G10A allowed greater displacement of iodinated GnRH-I by GnRHII (mean displacement per group 94, 79, 49, 74 and 68%, respectively). Competition with 25 nM and 2.5 nM GnRH-II
Fig. 1. Percentage of iodinated GnRH-I displaced by GnRH-I (a) and GnRHII (b) for sera raised against the different GnRH antigens as indicated on the x-axis and explained in Table 1, respectively GnRH mono, GnRH tandem, G6k (GnRH-tandem-dimer peptide with a d-lysine on position 6 of the native decapeptide), pE1A, S4A, R8A and G10A (alanine replacement variants of G6k). Each bar represents the mean value of 3–7 sera (see Section 2) raised against one antigen. The sera were obtained after the second immunization and diluted 1/10,000. The concentrations of GnRH-I and GnRH-II for displacement were: 250 nM (solid bars), 25 nM (dotted bars) and 2.5 nM (open bars).
Fig. 2. Percentage of iodinated GnRH-I displaced by GnRH-III (a) lamprey GnRH-I (b) and lamprey GnRH-III (c) for sera raised against the different GnRH antigens, respectively GnRH mono, GnRH tandem, G6k (GnRH-tandem-dimer peptide with a d-lysine on position 6 of the native decapeptide), pE1A, S4A, R8A and G10A (alanine replacement variants of G6k). Each bar represents the mean value of 3–7 sera (see Section 2) raised against one antigen. The sera were obtained after the second immunization and diluted 1/10,000. The concentrations of GnRH-III, lamprey GnRH-I and lamprey GnRH-III for displacement were 250 nM (solid bars) and 25 nM (dotted bars).
showed a dose dependent reduction of the displacement of iodinated GnRH for sera raised against mono, tandem, G6k, R8A and G10A. Subsequently, displacement of iodinated GnRH-I by GnRH isoforms GnRH-III, lamprey GnRH-I and lamprey GnRH-III was investigated. An unrelated peptide served as a negative control (data not shown). Results are shown in Fig. 2. GnRH-III (250 nM) displaced iodinated GnRH-I in sera raised against mono, tandem, R8A and G10A, whereas 25 nM GnRH-III displaced similarly or slightly less as compared to the 250 nM dose. Lamprey GnRH-III displaced iodinated GnRH-I in a dose dependent manner, but displacement was not as high as for GnRH-III. GnRH-III and lamprey GnRH-III displaced iodinated GnRH-I in sera raised against G6k, but to a lesser extent than for the previous mentioned groups. No displacement of iodinated GnRH-I by GnRH-III and lamprey GnRH-III was seen in sera raised against pE1A and S4A, although low displacement percentages were measured for one serum raised against pE1A and two sera raised
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Fig. 3. Percentage of iodinated GnRH-I displaced by GnRH-I (a) and GnRHII (b) for sera raised against the different GnRH antigens, respectively GnRH mono, GnRH tandem, G6k (GnRH-tandem-dimer peptide with a d-lysine on position 6 of the native decapeptide), pE1A, S4A, R8A and G10A (alanine replacement variants of G6k). Each bar represents the mean value of 3–7 sera (see Section 2) raised against one antigen. The sera were obtained at time of second immunization and diluted 1/1000. The concentrations of GnRH-I and GnRH-II for displacement were: 250 nM (solid bars), 25 nM (dotted bars) and 2.5 nM (open bars).
against S4A. Lamprey GnRH-I and the control peptide were not able to displace iodinated GnRH-I. To study the development of the specificity of the sera during the immunization period, displacement of iodinated GnRH-I by GnRH-I and GnRH-II was also determined for pre-booster sera collected at time of second vaccination. As expected, iodinated GnRH-I was displaced by GnRH-I for pre-booster sera raised against all antigens (Fig. 3). However, in contrast to the observations for the post-booster sera, 250 nM GnRH-II was able to displace iodinated GnRH-I in pre-booster sera raised against all antigens, including pE1A and S4A, with lower mean displacement percentages per group, ranging from 41 to 65%. Lower concentrations of GnRH-II resulted in a decreased displacement of iodinated GnRH-I.
4. Discussion Recently, the presence of several GnRH isoforms in mammals has been demonstrated [1–4]. However, the functions of these GnRH isoforms in mammals have only partially been investigated. Most likely they all play a role in fertility regulation, but this role seems limited, as demonstrated in a mouse model. Mice which lack the GnRH-I gene do have GnRH-II producing cells as in normal mice but this is not sufficient to cause normal gonadal development in these mice [11]. The limited role of GnRH-II in fertility regulation is also shown in male rats [17]. Immunization against GnRH-II showed a
slight but significant decrease of the seminiferous tubules diameter. However, GnRH-II may have functions which are not related to reproduction, as is suggested by an increase in kidney weight of rats immunized against GnRH-II [17]. Indeed, high concentrations of GnRH-II were found in the kidneys and also in bone marrow and prostate [1]. Moreover, the widespread expression of the GnRH-II receptor mRNA may also suggest functions of GnRH-II which are not related to reproduction [28], making it even more desirable to direct the antigenic response of an immunocastration vaccine specifically towards GnRH-I. Immunization against GnRH-I in order to block GnRH-I induced gonadal steroid secretion, may induce antibodies that cross-react with other GnRH isoforms, which may result in undesired side-effects because of unknown functions of the GnRH isoforms. In this study, we evaluated the specificity of antibodies elicited by various GnRH antigens tested against different GnRH isoforms. The results show that GnRH-I specific antisera can be generated with dimerized G6k-GnRHtandem peptides. Specificity is achieved by replacing a single amino acid in the decapeptide sequence of the antigen. Substitution of pyroglutamine at position one or serine at position four by alanine resulted in an immunogenic antigen, which induces antibodies that neutralize GnRH-I and establishes immunocastration in male piglets, but did not cross-react with GnRH-II, GnRH-III or lamprey GnRH-III. We also conclude that the specificity of these antibodies is established after the second immunization, as antisera obtained at time of second immunization did not discriminate between the GnRH isoforms. The antisera raised against pE1A and S4A allow displacement only with GnRH-I, while antisera raised against other antigens, mono, tandem, G6k, R8A and G10A allow displacement by GnRH-I, GnRH-II, GnRH-III and lamprey GnRHIII. We suggest that this can be explained by the position of the amino acid substitutions in the antigen (Table 1). The results might indicate that two types of antibodies are generated, which define whether or not cross-reaction of the antisera with the GnRH isoforms occurs. One type of antibody binds the N-terminal part and the other type binds the C-terminal of the GnRH antigens. The antigens of which the N-terminal part does not contain any substitutions (i.e. mono, tandem, G6k, R8A and G10A, Table 3) generate antibodies against the N-terminus, which are able to bind GnRH isoforms without substitutions in the N-terminal part of the peptide (i.e. GnRH-I, GnRH-II, GnRH-III and lamprey GnRH-III). GnRH antigens with a substitution in the N-terminal part, i.e. pE1A and S4A, elicite antibodies against the N-terminus, which are not able to bind the N-terminal part of the GnRH isoforms. Antibodies raised against the C-terminus of pE1A and S4A only bind GnRH-I, resulting in GnRH-I specific antibodies. Lamprey GnRH-I did not bind either type of antibody as it has an amino acid substitution in both N- and C-terminal part of the peptide. Antisera raised against mono, tandem, G6k, R8A and G10A showed displacement of iodinated GnRH-I by GnRH-
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Table 3 Amino acid alterations in GnRH antigens and GnRH isoforms as compared to native GnRHa
a
Positions with amino acid replacements as compared with the native GnRH amino acid sequence are indicated with .
I, GnRH-II, GnRH-III and lamprey GnRH-III in a competitive radioimmunoassay. However, differences in displacement were seen. GnRH-I showed the highest displacement activity, followed by GnRH-III, whereas lower displacement was shown for GnRH-II and lamprey GnRH-III. This order of displacement activity of the GnRH isoforms correlates with the number of amino acid substitutions in the C-terminal part of these peptides: with 0, 2, 3 and 4 amino acid substitutions for GnRH-I, GnRH-III, GnRH-II and lamprey GnRH-III, respectively. All sera raised against the mono allowed displacement by GnRH-I, GnRH-II, GnRH-III and lamprey GnRH-III. These results are in contrast to those of Ferro et al. [17], who did not find any cross-reactivity of antibodies raised against a GnRH-I analogue with GnRH-II. This discrepancy can be explained by the fact that in their GnRH analogue the Nterminal pyroglutamine was substituted by a cysteine for conjugation purposes. This is in accordance with our results obtained with the pE1A and S4A antigens, which also generate antibodies that do not cross-react with GnRH-II. In this study, we show that we can direct the antibody response after immunization by appropriately engineered antigens in order to prevent the occurrence of possible harmful side-effects caused by cross-reaction of GnRH-I directed antibodies with other GnRH isoforms.
References [1] White RB, Eisen JA, Kasten TL, Fernald RD. Second gene for gonadotropin-releasing hormone in humans. Proc Natl Acad Sci USA 1998;95:305–11. [2] Yahalom D, Chen A, Ben-Aroya N, Rahimipour S, Kaganovsky E, Okon E, et al. The gonadotropin-releasing hormone family of neuropeptides in the brain of human, bovine and rat: identification of a third isoform. FEBS Lett 1999;463(3):289–94.
[3] Dees WL, Hiney JK, Sower SA, Yu WH, McCann SM. Localization of immunoreactive lamprey gonadotropin-releasing hormone in the rat brain. Peptides 1999;20(12):1503–11. [4] Urbanski HF, White RB, Fernald RD, Kohama SG, Garyfallou VT, Densmore VS. Regional expression of mRNA encoding a second form of gonadotropin-releasing hormone in the macaque brain. Endocrinology 1999;140(4):1945–58. [5] Lescheid DW, Terasawa E, Abler LA, Urbanski HF, Warby CM, Millar RP, et al. A second form of gonadotropin-releasing hormone (GnRH) with characteristics of chicken GnRH-II is present in the primate brain. Endocrinology 1997;138(12):5618–29. [6] Okada Y, Murota-Kawano A, Kakar SS, Winters SJ. Evidence that gonadotropin-releasing hormone (GnRH) II stimulates luteinizing hormone and follicle-stimulating hormone secretion from monkey pituitary cultures by activating the GnRH I receptor. Biol Reprod 2003;69(4):1356–61. [7] Densmore VS, Urbanski HF. Relative effect of gonadotropinreleasing hormone (GnRH)-I and GnRH-II on gonadotropin release. J Clin Endocrinol Metab 2003;88(5):2126–34. [8] Gault PM, Maudsley S, Lincoln GA. Evidence that gonadotropinreleasing hormone II is not a physiological regulator of gonadotropin secretion in mammals. J Neuroendocrinol 2003;15(9):831–9. [9] Cheon KW, Lee HS, Parhar IS, Kang IS. Expression of the second isoform of gonadotrophin-releasing hormone (GnRH-II) in human endometrium throughout the menstrual cycle. Mol Hum Reprod 2001;7(5):447–52. [10] Siler-Khodr TM, Grayson M. Action of chicken II GnRH on the human placenta. J Clin Endocrinol Metab 2001;86(2):804–10. [11] Chen A, Yahalom D, Ben-Aroya N, Kaganovsky E, Okon E, Koch Y. A second isoform of gonadotropin-releasing hormone is present in the brain of human and rodents. FEBS Lett 1998;435(2–3):199–203. [12] Temple JL, Millar RP, Rissman EF. Evolutionarily conserved form of gonadotropin-releasing hormone coordinates energy and reproductive behavior. Endocrinology 2003;144(1):13–9. [13] Yu WH, Karanth S, Walczewska A, Sower SA, McCann SM. A hypothalamic follicle-stimulating hormone-releasing decapeptide in the rat. Proc Natl Acad Sci USA 1997;94(17):9499–503. [14] Dees WL, Dearth RK, Hooper RN, Brinsko SP, Romano JE, Rahe H, et al. Lamprey gonadotropin-releasing hormone-III selectively releases follicle stimulating hormone in the bovine. Domest Anim Endocrinol 2001;20(4):279–88. [15] McCann SM, Karanth S, Mastronardi CA, Dees WL, Childs G, Miller B, et al. Control of gonadotropin secretion by follicle-
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[17]
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[19] [20] [21]
J.A. Turkstra et al. / Vaccine 23 (2005) 4915–4920 stimulating hormone-releasing factor, luteinizing hormone-releasing hormone, and leptin. Arch Med Res 2001;32(6):476–85. Amstalden M, Zieba DA, Garcia MR, Stanko RL, Welsh Jr TH, Hansel WH, et al. Evidence that lamprey GnRH-III does not release FSH selectively in cattle. Reproduction 2004;127(1):35–43. Ferro VA, Khan MA, Latimer VS, Brown D, Urbanski HF, Stimson WH. Immunoneutralisation of GnRH-I, without crossreactivity to GnRH-II, in the development of a highly specific antifertility vaccine for clinical and veterinary use. J Reprod Immunol 2001;51(2):109–29. Meloen RH, Turkstra JA, Lankhof H, Puijk WC, Schaaper WM, Dijkstra G, et al. Efficient immunocastration of male piglets by immunoneutralization of GnRH using a new GnRH-like peptide. Vaccine 1994;12(8):741–6. Erratum in: Vaccine 1994;12(12):1152. Millar LA, Johns BE, Elias DJ, Crane KA. Comparative efficacy of two immunocontraceptive vaccines. Vaccine 1997;15:1858–62. Thompson DL. Immunization against GnRH in male species (comparative aspects). Anim Reprod Sci 2000;60–61:459–69. Zeng XY, Turkstra JA, Tsigos A, Meloen RH, Liu XY, Chen FQ, et al. Effects of active immunization against GnRH on serum LH, inhibin A, sexual development and growth rate in Chinese female pigs. Theriogenology 2002;58(7):1315–26.
[22] Ladd A, Walfield A, Tsong YY, Thau R. Active immunization against LHRH alone or combined with LHRH-analogue treatment impedes growth of androgen-dependent prostatic carcinoma. Am J Reprod Immunol 1995;34(3):200–6. [23] Simms MS, Scholfield DP, Jacobs E, Michaeli D, Broome P, Humphreys JE, et al. Anti-GnRH antibodies can induce castrate levels of testosterone in patients with advanced prostate cancer. Br J Cancer 2000;83(4):443–6. [24] Talwar GP. Vaccines for control of fertility and hormone-dependent cancers. Immunol Cell Biol 1997;75(2):184–9. [25] Jacobs E, Watson SA, Michaeli D, Ellis IO, Robertson JF. Anti-gonadotrophin releasing hormone antibodies inhibit the growth of MCF7 human breast cancer xenografts. Br J Cancer 1999;80(3–4):352–9. [26] Ferro VA, Stimson WH. Immunoneutralisation of gonadotrophin releasing hormone: a potential treatment for oestrogen-dependent breast cancer. Eur J Cancer 1997;33(9):1468–78. [27] Turkstra JA, Oonk HB, Schaaper WM, Meloen RH. The role of the individual amino acids of a GnRH-tandem-dimer peptide used as an antigen for immunocastration of male piglets determined with systematic alanine replacements. Vaccine 2001;20(3–4):406–12. [28] Neill JD. GnRH and GnRH receptor genes in the human genome. Endocrinology 2002;143(3):737–43.
GnRH tandem peptides for inducing an immunogenic response to GnRH-I without cross-reactivity to other GnRH isoforms J.A. Turkstra a,∗ , W.M.M. Schaaper a , H.B. Oonk b , R.H. Meloen a,c b
a Pepscan Systems, Edelhertweg 15, 8219 PH, Lelystad, The Netherlands Animal Sciences Group, Wageningen University and Research Centre, Edelhertweg 15, 8219 PH, Lelystad, The Netherlands c Academic Biomedical Centre, University Utrecht, Yalelaan 1, 3584 CL, Utrecht, The Netherlands
Received 29 December 2004; received in revised form 14 April 2005; accepted 20 May 2005 Available online 13 June 2005
Abstract Gonadotropin releasing hormone (GnRH) occurs in various isoforms in mammals, i.e. GnRH-I (mammalian GnRH), GnRH-II (chicken GnRH-II), GnRH-III (salmon GnRH) and two forms of lamprey GnRH. The function of the latter four molecules have only been partially investigated. Also not much is known about the physiological effects of GnRH-I immunization on the function of these GnRH isoforms. In order to avoid possible harmful side-effects due to undesired neutralization of GnRH isoforms, GnRH-I specificity of antibodies raised against a panel of alternative GnRH antigens was determined. The results show that GnRH antigens can be designed which generate antibodies that specifically bind GnRH-I, without cross-reacting with other GnRH isoforms. © 2005 Published by Elsevier Ltd. Keyword: GnRH-isoforms specificity vaccine
1. Introduction In mammals, the decapeptide gonadotropin releasing hormone (GnRH-I) triggers the synthesis and release of luteinising hormone (LH) and follicle stimulation hormone (FSH) from the pituitary. LH and FSH are responsible for steroidogenesis and gametogenesis. Recently, additional isoforms of GnRH were identified in mammals. GnRH-II, a molecule with an amino acid sequence similar to chicken GnRH-II, was cloned from a human genomic library [1]. A third GnRH isoform (GnRH-III), with a similar amino acid sequence as salmon GnRH, was extracted from the bovine and human brain [2]. Immunohistologically, the presence of GnRH isoforms (lamprey GnRH-I, lamprey GnRH-III) that were first detected in lamprey was demonstrated in the mammalian brain [3,4]. The function of the various isoforms in mammals, however, is ill defined. Most likely they play a role in fertility regulation. GnRH-II and GnRH-III are able to ∗
Corresponding author. Tel.: +31 320 237212; fax: +31 320 238120. E-mail address: [email protected] (J.A. Turkstra).
0264-410X/$ – see front matter © 2005 Published by Elsevier Ltd. doi:10.1016/j.vaccine.2005.05.020
induce the secretion of gonadotropines [2,5–8]. The presence of GnRH-II in the human endometrium and the fact that GnRH-II is released from the early human placenta indicates that it may be involved in embryo implantation and regulation of pregnancy [9,10]. It has also been suggested that the GnRH isoforms are involved in sexual behaviour [2,11,12]. In contrast to GnRH-II and GnRH-III, lamprey GnRH-III does not induce LH secretion. This isoform seemed to selectively activate FSH release, indicating that it could be the FSH-releasing factor [13–15]. However, this could not be confirmed by others [16]. GnRH-II is not only expressed in the brain, but also in tissues outside the brain. High concentrations were determined in the kidneys, bone marrow and prostate [1]. This indicates that GnRH-II may also have functions unrelated to fertility regulation. Indeed, neutralization of GnRH-II by active immunization, led to a slight increase in kidney weight suggesting a role for GnRH-II in kidney function [17]. Immunization against GnRH-I is shown to be an efficient method to block fertility and reproduction in male and female mammals [18–21]. In addition, immunization suppresses hor-
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mone dependent malfunctions, such as breast cancer and prostate cancer [22–26]. However, with the new insight, that several GnRH isoforms are present in mammals, it is not known which of the GnRH isoforms are targetted by GnRHI immunization. Although all GnRH isoforms seem to be involved in fertility, some may have additional functions unrelated to fertility. Possible undesired effects of GnRH-I neutralization on these unrelated functions must be circumvented. Therefore, the specificity of antibodies raised against a panel of alternative GnRH antigens for the GnRH isoforms was studied. Here we report that some GnRH antigens elicite antibodies which cross-react with most of the GnRH isoforms, while others are only directed against GnRH-I, indicating that small spectrum isoform specific antibodies or wide spectrum isoform non-specific antibodies can be generated as desired.
Table 2 Amino acid sequences of GnRH isoformsa Peptide
Amino acid sequence
Mammalian GnRH (GnRH-I) Chicken GnRH-II (GnRH-II) Salmon GnRH (GnRH-III) Lamprey GnRH-I (lamprey GnRH-I) Lamprey GnRH-III (lamprey GnRH-III)
pEHWSYGLRPGb pEHWSHGWYPGb pEHWSYGWLPGb pEHYSLEWKPGb pEHWSHDWKPGb
Underlined amino acid differ from GnRH-I amino acid sequence. a pE, pyroglutamic acid. b Amide.
2. Materials and methods
teine, subsequent conjugation to ovalbumine was achieved using N-ethyl-N -(3 -dimethyl-aminopropyl) carbodiimide. Antigens were administered with Specol adjuvant to young male pigs at 10 and 17 weeks of age. In the present study, only sera raised against five fully effective G6k-GnRHtandem dimer peptides are included [27]. Each group consisted of six (G6k) or seven (pE1A, S4A, R8A, G10A) pigs.
2.1. Antisera
2.2. GnRH isoform peptides
Antisera were raised in young male pigs and directed against different GnRH antigens. Table 1 gives an overview of the peptides used for immunization, including amino acid sequence in single letter code. Peptide synthesis, vaccine preparation and immunization procedure for mono and tandem has been described [13]. Briefly, the GnRH molecules synthesized with a C-terminal cysteine were conjugated to Keyhole Limpet Hemocyanin via maleinimidobenzoyl-hydoxysuccinimide ester. Conjugates were emulsified in complete freund adjuvant and incomplete freund adjuvant for the first and second immunization, respectively. The vaccines were administered to young male pigs at 10 and 18 weeks of age. Only sera from successfully treated pigs, as determined by undetectable serum testosterone levels and reduced testis weights were used in this study (3 and 5 pigs for the mono and the tandem group, respectively). Synthesis of the G6k-GnRH-tandem peptides has been described previously [27]. The peptides were brought in the tandem dimer form by dimerization via the C-terminal cys-
The peptides used for the competitive radioimmunoassay were synthesized and analyzed as described previously [27]. Peptides were at least 90% pure. Amino acid sequences of the synthesized peptides are given in Table 2 in a single letter code. 2.3. Competitive radioimmunoassay Serum samples were diluted in PBS with 0.4% BSA (dilution buffer). Fifty microliters serum dilution was put in microwell plates and pre-incubated with 25 l GnRH isoform peptide solution. An unrelated peptide was included as negative control. This mixture was allowed to incubate for 24 h at 4 ◦ C. The next day 25 l iodinated GnRH-I (approximately 13,000 cpm, Amersham Pharmacia Biotech, Buckinghamshire, England) was added to compete with the pre-incubated GnRH isoform peptides for binding to the antibodies. After incubation overnight (4 ◦ C) unbound peptide was separated from bound peptide using dextran-coated charcoal. After centrifugation (2000 × g, 15 min) supernatant
Table 1 Amino acid sequences and abbreviations of peptides used for immunizationa GnRH antigen
Amino acid sequence
Abbreviation
GnRH-monomer GnRH-tandem G6k-GnRH-tandem pE1A-G6k-GnRH-tandem S4A-G6k-GnRH-tandem R8A-G6k-GnRH-tandem G10A-G6k-GnRH-tandem
pEHWSYGLRPGCb pEHWSYGLRPGQHWSYGLRPGCb pEHWSYkLRPGQHWSYkLRPGCb c AHWSYkLRPGAHWSYkLRPGCb pEHWAYkLRPGQHWAYkLRPGCb pEHWSYkLAPGQHWSYkLAPGCb pEHWSYkLRPAQHWSYkLRPACb
Mono Tandem G6k pE1A S4A R8A G10A
k, d-lysine; G6k, Gly on position 6 in the native GnRH sequence substituted by d-lysine; Underlined amino acid differ from G6k-GnRH-tandem. a pE, pyroglutamic acid. b Amide. c Acetyl.
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was separated, counted and the percentage iodinated GnRH bound to the antibodies was calculated.
3. Results In a competitive radioimmunoassay displacement of iodinated GnRH-I by the GnRH isoforms (Table 2) was determined. Competition between the iodinated GnRH-I and 250 nM GnRH-I, resulted in the expected reduction of binding of iodinated GnRH-I by the antisera for all sera. Iodinated GnRH-I was displaced for 70–100% by GnRH-I (mean value per group >89%; Fig. 1a). Similar results were obtained with 25 nM GnRH-I (mean value per group >88%), whereas 2.5 nM GnRH-I showed a lower reduction of displacement (mean value per group >67%). In contrast, displacement of iodinated GnRH-I by 250 nM GnRH-II revealed clear differences between groups (Fig. 1b). Sera raised against pE1A and S4A, allowed hardly any displacement of iodinated GnRH-I by GnRH-II. Only one serum out of seven of both, the pE1A and the S4A vaccinated groups, showed a substantial displacement percentage (38 and 46%, respectively), resulting in a mean displacement percentage of 8 and 16% for pE1A and S4A, respectively. Sera raised against mono, tandem, G6k, R8A and G10A allowed greater displacement of iodinated GnRH-I by GnRHII (mean displacement per group 94, 79, 49, 74 and 68%, respectively). Competition with 25 nM and 2.5 nM GnRH-II
Fig. 1. Percentage of iodinated GnRH-I displaced by GnRH-I (a) and GnRHII (b) for sera raised against the different GnRH antigens as indicated on the x-axis and explained in Table 1, respectively GnRH mono, GnRH tandem, G6k (GnRH-tandem-dimer peptide with a d-lysine on position 6 of the native decapeptide), pE1A, S4A, R8A and G10A (alanine replacement variants of G6k). Each bar represents the mean value of 3–7 sera (see Section 2) raised against one antigen. The sera were obtained after the second immunization and diluted 1/10,000. The concentrations of GnRH-I and GnRH-II for displacement were: 250 nM (solid bars), 25 nM (dotted bars) and 2.5 nM (open bars).
Fig. 2. Percentage of iodinated GnRH-I displaced by GnRH-III (a) lamprey GnRH-I (b) and lamprey GnRH-III (c) for sera raised against the different GnRH antigens, respectively GnRH mono, GnRH tandem, G6k (GnRH-tandem-dimer peptide with a d-lysine on position 6 of the native decapeptide), pE1A, S4A, R8A and G10A (alanine replacement variants of G6k). Each bar represents the mean value of 3–7 sera (see Section 2) raised against one antigen. The sera were obtained after the second immunization and diluted 1/10,000. The concentrations of GnRH-III, lamprey GnRH-I and lamprey GnRH-III for displacement were 250 nM (solid bars) and 25 nM (dotted bars).
showed a dose dependent reduction of the displacement of iodinated GnRH for sera raised against mono, tandem, G6k, R8A and G10A. Subsequently, displacement of iodinated GnRH-I by GnRH isoforms GnRH-III, lamprey GnRH-I and lamprey GnRH-III was investigated. An unrelated peptide served as a negative control (data not shown). Results are shown in Fig. 2. GnRH-III (250 nM) displaced iodinated GnRH-I in sera raised against mono, tandem, R8A and G10A, whereas 25 nM GnRH-III displaced similarly or slightly less as compared to the 250 nM dose. Lamprey GnRH-III displaced iodinated GnRH-I in a dose dependent manner, but displacement was not as high as for GnRH-III. GnRH-III and lamprey GnRH-III displaced iodinated GnRH-I in sera raised against G6k, but to a lesser extent than for the previous mentioned groups. No displacement of iodinated GnRH-I by GnRH-III and lamprey GnRH-III was seen in sera raised against pE1A and S4A, although low displacement percentages were measured for one serum raised against pE1A and two sera raised
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Fig. 3. Percentage of iodinated GnRH-I displaced by GnRH-I (a) and GnRHII (b) for sera raised against the different GnRH antigens, respectively GnRH mono, GnRH tandem, G6k (GnRH-tandem-dimer peptide with a d-lysine on position 6 of the native decapeptide), pE1A, S4A, R8A and G10A (alanine replacement variants of G6k). Each bar represents the mean value of 3–7 sera (see Section 2) raised against one antigen. The sera were obtained at time of second immunization and diluted 1/1000. The concentrations of GnRH-I and GnRH-II for displacement were: 250 nM (solid bars), 25 nM (dotted bars) and 2.5 nM (open bars).
against S4A. Lamprey GnRH-I and the control peptide were not able to displace iodinated GnRH-I. To study the development of the specificity of the sera during the immunization period, displacement of iodinated GnRH-I by GnRH-I and GnRH-II was also determined for pre-booster sera collected at time of second vaccination. As expected, iodinated GnRH-I was displaced by GnRH-I for pre-booster sera raised against all antigens (Fig. 3). However, in contrast to the observations for the post-booster sera, 250 nM GnRH-II was able to displace iodinated GnRH-I in pre-booster sera raised against all antigens, including pE1A and S4A, with lower mean displacement percentages per group, ranging from 41 to 65%. Lower concentrations of GnRH-II resulted in a decreased displacement of iodinated GnRH-I.
4. Discussion Recently, the presence of several GnRH isoforms in mammals has been demonstrated [1–4]. However, the functions of these GnRH isoforms in mammals have only partially been investigated. Most likely they all play a role in fertility regulation, but this role seems limited, as demonstrated in a mouse model. Mice which lack the GnRH-I gene do have GnRH-II producing cells as in normal mice but this is not sufficient to cause normal gonadal development in these mice [11]. The limited role of GnRH-II in fertility regulation is also shown in male rats [17]. Immunization against GnRH-II showed a
slight but significant decrease of the seminiferous tubules diameter. However, GnRH-II may have functions which are not related to reproduction, as is suggested by an increase in kidney weight of rats immunized against GnRH-II [17]. Indeed, high concentrations of GnRH-II were found in the kidneys and also in bone marrow and prostate [1]. Moreover, the widespread expression of the GnRH-II receptor mRNA may also suggest functions of GnRH-II which are not related to reproduction [28], making it even more desirable to direct the antigenic response of an immunocastration vaccine specifically towards GnRH-I. Immunization against GnRH-I in order to block GnRH-I induced gonadal steroid secretion, may induce antibodies that cross-react with other GnRH isoforms, which may result in undesired side-effects because of unknown functions of the GnRH isoforms. In this study, we evaluated the specificity of antibodies elicited by various GnRH antigens tested against different GnRH isoforms. The results show that GnRH-I specific antisera can be generated with dimerized G6k-GnRHtandem peptides. Specificity is achieved by replacing a single amino acid in the decapeptide sequence of the antigen. Substitution of pyroglutamine at position one or serine at position four by alanine resulted in an immunogenic antigen, which induces antibodies that neutralize GnRH-I and establishes immunocastration in male piglets, but did not cross-react with GnRH-II, GnRH-III or lamprey GnRH-III. We also conclude that the specificity of these antibodies is established after the second immunization, as antisera obtained at time of second immunization did not discriminate between the GnRH isoforms. The antisera raised against pE1A and S4A allow displacement only with GnRH-I, while antisera raised against other antigens, mono, tandem, G6k, R8A and G10A allow displacement by GnRH-I, GnRH-II, GnRH-III and lamprey GnRHIII. We suggest that this can be explained by the position of the amino acid substitutions in the antigen (Table 1). The results might indicate that two types of antibodies are generated, which define whether or not cross-reaction of the antisera with the GnRH isoforms occurs. One type of antibody binds the N-terminal part and the other type binds the C-terminal of the GnRH antigens. The antigens of which the N-terminal part does not contain any substitutions (i.e. mono, tandem, G6k, R8A and G10A, Table 3) generate antibodies against the N-terminus, which are able to bind GnRH isoforms without substitutions in the N-terminal part of the peptide (i.e. GnRH-I, GnRH-II, GnRH-III and lamprey GnRH-III). GnRH antigens with a substitution in the N-terminal part, i.e. pE1A and S4A, elicite antibodies against the N-terminus, which are not able to bind the N-terminal part of the GnRH isoforms. Antibodies raised against the C-terminus of pE1A and S4A only bind GnRH-I, resulting in GnRH-I specific antibodies. Lamprey GnRH-I did not bind either type of antibody as it has an amino acid substitution in both N- and C-terminal part of the peptide. Antisera raised against mono, tandem, G6k, R8A and G10A showed displacement of iodinated GnRH-I by GnRH-
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Table 3 Amino acid alterations in GnRH antigens and GnRH isoforms as compared to native GnRHa
a
Positions with amino acid replacements as compared with the native GnRH amino acid sequence are indicated with .
I, GnRH-II, GnRH-III and lamprey GnRH-III in a competitive radioimmunoassay. However, differences in displacement were seen. GnRH-I showed the highest displacement activity, followed by GnRH-III, whereas lower displacement was shown for GnRH-II and lamprey GnRH-III. This order of displacement activity of the GnRH isoforms correlates with the number of amino acid substitutions in the C-terminal part of these peptides: with 0, 2, 3 and 4 amino acid substitutions for GnRH-I, GnRH-III, GnRH-II and lamprey GnRH-III, respectively. All sera raised against the mono allowed displacement by GnRH-I, GnRH-II, GnRH-III and lamprey GnRH-III. These results are in contrast to those of Ferro et al. [17], who did not find any cross-reactivity of antibodies raised against a GnRH-I analogue with GnRH-II. This discrepancy can be explained by the fact that in their GnRH analogue the Nterminal pyroglutamine was substituted by a cysteine for conjugation purposes. This is in accordance with our results obtained with the pE1A and S4A antigens, which also generate antibodies that do not cross-react with GnRH-II. In this study, we show that we can direct the antibody response after immunization by appropriately engineered antigens in order to prevent the occurrence of possible harmful side-effects caused by cross-reaction of GnRH-I directed antibodies with other GnRH isoforms.
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