The C-terminal pentapeptide of LHRH is a dominant B cell epitope with antigenic and biological function
Introduction
Luteinizing hormone releasing hormone (LHRH) is secreted by the hypothalamus and initiates a cascade of endocrine events in all mammals that leads to reproductive competency. Immunisation against LHRH has been proven to be an effective method for blocking this hypothalamic pituitary axis (Fraser and Baker, 1978, Fraser et al., 1974, Talwar et al., 1992) and as consequence vaccines incorporating LHRH have been intensively studied for the control of fertility and hormone dependent cancers (Naz et al., 2005, Talwar, 1999). In most studies the immunogen consists of the LHRH decapeptide, EHWSYGLRPG. Within the native sequence of LHRH the N-terminal residue is pyroglutamic acid, a cyclized analogue of glutamic acid, and the C-terminus is in the amide (CONH2) form. Traditionally the peptide sequence is chemically conjugated to a protein carrier (Beekman et al., 1997, Beekman et al., 1999, Beekman et al., 2001, Ladd et al., 1990, Miller et al., 1997), incorporated into a recombinant immunogen (Jinshu et al., 2005, Talwar et al., 2004) or incorporated into a peptide vaccine containing CD4+ T cell epitopes for induction of T cell help (Ghosh and Jackson, 1999, Ghosh et al., 2001, Meloen et al., 1994, Oonk et al., 1998, Sad et al., 1992, Zeng et al., 2002). Unlike carrier proteins, the use of synthetic T helper (TH) cell epitopes has the potential advantage of by-passing carrier-induced epitope-specific suppression (Sad et al., 1992).
Our laboratory has been engaged in the preparation of totally synthetic contraceptive vaccines based on LHRH. We have shown that a totally synthetic peptide vaccine containing LHRH as the B cell epitope and a TH cell epitope elicits a potent antibody response against LHRH in mice and that these mice are rendered infertile (Ghosh and Jackson, 1999, Zeng et al., 2002, Zeng et al., 2005). We have used both full length LHRH and a homolog in which the N-terminal pyroglutamic acid residue is removed. In both cases the antibody responses induced were similar (Ghosh and Jackson, 1999, Zeng et al., 2001, Zeng et al., 2002, Zeng et al., 2005). An early report (Singh et al., 1985) indicated that antisera obtained following immunisation with an LHRH-tetanus toxoid conjugate were (i) able to inhibit follicular development and ovulation and (ii) reacted with full length LHRH but were devoid of reactivity with the peptide sequences SYG (LHRH4–6), LRPG (LHRH7–10) and with the free acid form of LHRH where the C-terminal glycine was in the carboxylate and not the amide form. On the other hand it has been reported that inoculation with LHRH-derived peptides in which His-2 and Trp-3 have been deleted are effective at inducing LHRH neutralising antibodies and causing immunocastration (Rigby et al., 1988).
These reports combined with our own results prompted us to investigate which part of the sequence of LHRH is the dominant B cell epitope and whether short sequences derived from LHRH could retain their immunogenic and antigenic properties when incorporated into peptide vaccines.
Section snippets
Reagents
Unless otherwise stated chemicals were of analytical grade or its equivalent. Dichloromethane (DCM), N,N′-dimethylformamide (DMF), piperidine, trifluoroacetic acid (TFA), O’benzotriazole-N,N,N’,N’-tetra methyl-uronium-hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt), diisopropylethylamine (DIPEA) and diisopropylcarbodiimide (DICI) were obtained from Auspep Pty Ltd (Melbourne, Australia) and Fluka (Buchs, Switzerland). Phenol and triisopropylsilane (TIPS) were from Aldrich (Milwaukee,
Inhibition of the binding of anti-LHRH antibodies to LHRH by peptide fragments
In this experiment we determined which part of the LHRH sequence was able to inhibit the binding of anti-LHRH antibodies raised against full length LHRH. Anti-LHRH antisera were obtained from mice inoculated with two doses of the peptide vaccine GALNNRFQIKGVELKS-LHRH1–10 in the presence of CFA on days 0 and 28. The sequence GALNNRFQIKGVELKS is a T helper cell epitope identified from the light chain (HA2) of influenza virus hemagglutinin (Jackson et al., 1995, Zeng et al., 2001). Anti-LHRH
Discussion
In this paper we have carried out a detailed study to determine whether or not there are any short dominant B cell epitopes within the LHRH hormone sequence. We found that an effective minimal B cell epitope is the C-terminal five-residue pentapeptide LHRH6–10. This pentapeptide not only efficiently inhibits the binding of LHRH1–10 to anti-LHRH1–10 antisera but also elicits a strong anti-LHRH antibody response in its own right when incorporated into a totally synthetic peptide vaccine.
Acknowledgements
This work was supported by the Cooperative Research Centre for Vaccine Technology and the National Health and Medical Research Council of Australia.
We would like to thank Dr. Lorena E. Brown for her useful suggestions and critical review of the manuscript.
References (33)
- et al.
Highly immunogenic and fully synthetic peptide-carrier constructs targetting GnRH
Vaccine
(1999) - et al.
Investigation into suitable carrier molecules for use in an anti-gonadotrophin releasing hormone vaccine
Vaccine
(1998) - et al.
A synthetic gonadotropin-releasing hormone (GnRH) vaccine for control of fertility and hormone dependent diseases without any adjuvant
Vaccine
(2005) - et al.
Efficient immunocastration of male piglets by immunoneutralization of GnRH using a new GnRH-like peptide
Vaccine
(1994) - et al.
Comparative efficacy of two immunocontraceptive vaccines
Vaccine
(1997) - et al.
New GnRH-like peptide construct to optimize efficient immunocastration of male pigs by immunoneutralization of GnRH
Vaccine
(1998) - et al.
Rational approaches to reduce adverse reactions in man to vaccines containing tetanus and diphtheria toxoids
Vaccine
(1998) - et al.
The antigenic and immunogenic properties of synthetic peptide immunocontraceptive vaccine candidates based on gamete antigens
Vaccine
(1999) - et al.
The dominant role of amide group at C-terminus for recognition by antibody in primates against gonadotropin releasing hormone
J. Steroid Biochem.
(1985) - et al.
A recombinant luteinising-hormone-releasing-hormone immunogen bioeffective in causing prostatic atrophy
Vaccine
(2004)