Identification of epitopes of monoclonal antibodies to porcine zona pellucida 3 beta glycoprotein, a homologue of the mouse/human sperm receptor.

PROBLEM: Immunization with zona pellucida (ZP) glycoproteins leads to a block in fertility with a variable degree of ovarian dysfunction. To avoid autoimmune oophoritis, synthetic peptides corresponding to B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. The main objective of the present study is to define the epitopes recognized by monoclonal antibodies (mAbs) generated against porcine ZP3 beta, a homologue of the designated primary sperm receptor in mice and humans. METHODS: A multipin synthetic peptides approach has been used to map the epitopes recognized by mAbs. Dodecapeptides with an overlap of 6 amino acids corresponding to a precursor pZP3 beta-deduced amino acid sequence (excluding the signal sequence) were synthesized on polypropylene pins and were tested for their reactivity with mAbs by enzyme-linked immunoadsorbent assay (ELISA). The ability of synthetic peptides corresponding to the identified epitopes to inhibit the binding of mAbs to pZP3 beta in a competitive inhibition ELISA was investigated to confirm the above findings. RESULTS: Reactivity of the mAbs with the pin-bound peptides in ELISA-identified epitopes for mAb-451 to EEKLVF (166-171) and mAb-462/470 to FKAPRP (250-255) amino acid residues. mAb-30 recognized QPVWQDEGQRLR (23-34) and VICRCC (316-321) amino acid residues. Competitive inhibition with synthetic peptides encompassing the motifs corresponding to 23-34 and 316-321 for binding of mAb-30 to pZP3 beta revealed the epitopic domain to be 23-34 amino acids. Synthesis of overlapping octapeptides further identified WQDE as the minimum motif for binding of mAb-30, and the replacement of one amino acid at a time with glycine revealed tryptophan as the critical residue. CONCLUSIONS: Collectively, these results describe peptide epitopes that will help in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZP3 beta or its homologues in other species.

Prospects of zona pellucida glycoproteins as immunogens for contraceptive vaccine.

The zona pellucida (ZP) surrounding a mammalian oocyte mediates the initial recognition and binding of spermatozoon to oocyte in a relatively species-specific manner and plays an important role in the subsequent activation events during the fertilization process. The ZP comprises three biochemically and immunologically distinct glycoproteins termed ZP1, ZP2 and ZP3. The critical role of ZP glycoproteins in reproduction together with their tissue-specific nature have led to their being considered as potential candidate antigens for immunocontraception. Immunization of females with ZP glycoproteins leads to a block of fertility in several animal models. However, it is invariably associated with either a transient or an irreversible alteration in the cyclicity, hormonal profile and follicular development in the ovary. To overcome these problems, attempts are being made to delineate relevant 'B' cell epitopes on ZP proteins so as to design immunocontraceptive vaccines based on synthetic peptides devoid of oophoritogenic 'T' cell epitopes. Monoclonal antibodies capable of inhibiting the gamete interaction are being employed to delineate such regions. Additionally, DNA-recombinant technology has made it feasible to obtain, in reasonably large quantities, the ZP glycoproteins from human and non-human primates. Availability of sequence information of these zona proteins and the availability of recombinant antigens (devoid of other ovarian-associated proteins) will further help in understanding more precisely their functions during fertilization and make it feasible to undertake immunization studies to determine their prospects as immunogens for fertility regulation.

Induction of native protein reactive antibodies by immunization with peptides containing linear B-cell epitopes defined by anti-porcine ZP3 beta monoclonal antibodies.

To identify pertinent target epitopes for contraceptive vaccine development, rabbit polyclonal antibodies were raised against four peptides synthesized from the deduced amino acid (aa) sequence of porcine zona pellucida macromolecule ZP3 beta and coupled to diphtheria toxoid (DT). Synthetic peptides consisted of: P1, 23-37 aa; P2, 164-179 aa with an additional C-terminal cysteine; P3, 246-263 aa with an extra C-terminal cysteine; and P4, 310-321 aa residues corresponding to pZP3 beta precursor protein. Selected sequences were based upon B cell epitopes identified previously by monoclonal antibodies. Immune sera reacted with their respective peptides and DT in an ELISA, and also recognized porcine SIZP and pZP3 beta both in ELISA and Western blot and zona pellucida of porcine oocytes in an indirect immunofluorescence assay. None of the four anti-peptide sera recognized pZP3 alpha in Western blot, emphasizing the specificity of these antibodies to pZP3 beta. The anti-peptide sera, individually, failed to inhibit in vitro attachment of boar sperm to antibody treated zona encased porcine oocytes. However, combinations of immune sera against peptides such as P1 + P4, P2 + P4 and P1 + P2 + P4, did significantly inhibit porcine sperm-oocyte interaction. These results identify combinations of peptides that could potentially be used in the design of an immunocontraceptive vaccine based upon synthetic peptides corresponding to pZP3 beta or its homologues in other species.

Expression of bonnet monkey (Macaca radiata) zona pellucida-3 (ZP3) in a prokaryotic system and its immunogenicity.

An internal fragment (978 bp) corresponding to the bonnet monkey (Macaca radiata) ZP3, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by PCR from a full-length cDNA clone. The amplified Bam HI and SacI restricted fragment was cloned in frame downstream of the T5 promoter under lac operator control for expression in the pQE-30 vector. Recombinant ZP3 (r-ZP3) was expressed as a poly-histidine fusion protein in E. coli strains SG13009[pREP4] and BL-21(DE3). Immunoblot with a murine monoclonal antibody, MA-451 (raised against porcine ZP3 beta-a homologue of bonnet ZP3, and cross-reactive with bonnet zona pellucida) revealed a predominant band of 50 kDa besides degraded fragments. Optimum expression of r-ZP3 was observed at 0.5 mM IPTG. Antisera generated in monkeys against synthetic peptides from the N-(23-45 aa residues) and C-(300-322 and 324-347 aa residues) termini of the deduced bonnet monkey precursor ZP3 sequence reacted with the r-ZP3 protein in ELISA. The r-ZP3 expressed in SG13009[pREP4] was purified on Ni-NTA resin under denaturing conditions and conjugated with diphtheria toxoid (DT). Immunization of a female rabbit and six female bonnet monkeys with the r-ZP3-DT conjugate generated antibodies reactive with r-ZP3 in ELISA. Rabbit r-ZP3 antiserum reacted with porcine ZP3 beta and bonnet r-ZP3 but failed to react with porcine ZP3 alpha in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida. The availability of r-ZP3 will further help in evaluating its efficacy for fertility regulation and understanding the autoimmune oophoritis associated with ZP3 immunization in nonhuman primates.

Immunoreactivity and in-vitro effect on human sperm-egg binding of antibodies against peptides corresponding to bonnet monkey zona pellucida-3 glycoprotein.

The following synthetic peptides were made as immunogens for development of a zona-based contraceptive vaccine: P1, KQPFWLLQGGASRAETSVQPVLVE [amino acids (aa) 23-45 with an additional K at the N-terminus]; P2, FSEEKLVFSLRLMEENC (aa 164-179 with an additional C at the C-terminus and T170 replaced by V); and P3, CSFSKSSNSWFPVEGPADICQCC (aa 300-322). The aa are numbered on the basis of bonnet monkey ZP3 precursor protein. Antibodies against an additional peptide P4, KGDCGTPSHSRRQPHVVSQWSRSA (aa 324-347), significantly inhibits human sperm-oocyte binding. In addition, antibodies against cocktail of peptide-diphtheria toxoid conjugates also significantly inhibit the binding of spermatozoa to zona pellucida in a hemizona assay. These results will further help in the design of an immunocontraceptive vaccine based upon synthetic peptides corresponding to ZP3.

Strategies for designing an immunocontraceptive vaccine based on zona pellucida synthetic peptides and recombinant antigen.

Immunization of female bonnet monkeys (Macaca radiata) with pig zona pellucida glycoprotein ZP3 using adjuvants permissible for human use leads to infertility. Fifty per cent of the animals conceived after antibody titres declined. Animals that failed to regain their fertility did not show any obvious ovarian changes. As part of the attempt to design an effective immunocontraceptive vaccine based on synthetic peptides, the epitopes recognized by monoclonal antibodies (mAb) against pig ZP3 alpha and ZP3 beta and possessing contraceptive efficacy in vitro were mapped. These studies identified an N-blocked decapeptide from the N-terminus corresponding to 23-32 amino acids of the precursor protein of pig ZP3 beta. As an alternative approach to using synthetic peptides, DNA encoding bonnet monkey ZP3 was cloned and sequenced. The deduced primary amino acid sequence revealed an overall similarity of 93.9% with human ZP3. Bonnet monkey ZP3 corresponding to an internal 975 nucleotide fragment excluding the N-terminus signal sequence and the C-terminus transmembrane domain has been expressed in Escherichia coli. These results will assist in the design of an immunocontraceptive vaccine based on either synthetic peptides or recombinant glycoproteins or proteins corresponding to ZP.

PIP: Previous studies have demonstrated the efficacy, in mice, of synthetic peptides derived from zona pellucida (ZP) glycoprotein in blocking fertility without ovarian dysfunction. This study used bonnet monkeys (closely related to humans in the primate evolutionary tree and less susceptible to summer amenorrhea than rhesus monkeys) to explore the design of an immunocontraceptive vaccine based on synthetic peptides, recombinant glycoproteins, or proteins corresponding to ZP. Immunization of female monkeys with pig ZP3 glycoprotein using adjuvants permissible for human use produced infertility. Although only half the animals conceived after antibody titres declined, monkeys that failed to conceive did not show any obvious ovarian changes. Mapping of the epitopes recognized by monoclonal antibodies against ZP3 alpha and beta and possessing contraceptive efficacy in vitro identified an N-blocked decapeptide from the N-terminus corresponding to 23-32 amino acids of the precursor protein of pig ZP3 beta. When DNA encoding bonnet monkey ZP3 was cloned and sequenced, the deduced primary amino acid sequence showed a 93.9% similarity with human ZP3. Bonnet monkey ZP3 corresponding to an internal 975 nucleotide fragment excluding the N-terminus signal sequence and the C-terminus transmembrane domain has been expressed in Escherichia coli.

Localization of epitopes for monoclonal antibodies at the N-terminus of the porcine zona pellucida glycoprotein pZPC.

Zona pellucida glycoproteins play an important role in fertilization. In this study, attempts have been made to identify and define epitopes of monoclonal antibodies (mAbs) possessing contraceptive efficacy in vitro. The porcine zona glycoprotein pZPC, a homologue of mouse/human ZP3, was reduced and alkylated and subsequently digested with trypsin. Reverse-phase HPLC of the tryptic digest yielded twenty two peaks (T1-T22). When tested against mAbs reactive against sequential determinants on pZPC, T11 was immunoreactive with two mAbs, mAb-455 and mAb-467, as shown by antigen inhibition ELISA. IC50 values of 3.1 nM and 8.6 nM were recorded versus mAb-455 and mAb-467 respectively, and approximated the IC50 values obtained with intact pZPC. Amino acid analysis, Edman degradation, and FAB-MS identified T11 as the N-blocked decapeptide pyro-Gln-Pro-Val-Trp-Gln-Asp-Glu-Gly-Gln-Arg derived from the N-terminus of pZPC. Synthesis of overlapping octapeptides further identified VWQDE and WQDE as the minimum motifs with antigenic activity for mAb-455 and mAb-467, respectively. Glycine replacement peptides confirmed residues W,Q,E as critical for binding mAb-455 and W,Q,D,E as critical for binding mAb-467. Both mAbs inhibited binding of boar sperm to zona-encased porcine oocytes. These results, the first to define peptide epitopes of porcine zona glycoprotein, will assist in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZPC or its homologues in other species.