Indian Patents. 217322:"A METHOD FOR PRODUCING AN IMMUNO-CONTRACEPTIVE VACCINE"

Title of Invention	"A METHOD FOR PRODUCING AN IMMUNO-CONTRACEPTIVE VACCINE"
Abstract	The present invention relates to a method for producing an immuno-contraceptive vaccine comprising the steps of isolating and preparing amino acid sequence comprising a peptide consisting of zona pellucida-1 (ZPl) shown in figure 3 mixing with the sequence with a pharmacologically-acceptable carrier, and optionally an adjuvant such as herein described in a ratio of 4:1.

Full Text	FIELD OF THE INVENTION relates to a method of inducing infertility in a mammal, a recombinant ZP1 protein or its fragments used for inducing the inferrility and a process of producing the recombinant ZPl protein and its fragments. Abbreviations used : A, absorbance (1cm): aa, amino acid(s); bp. base pairs IPTG. isopropyl-ß-D-thiogalactopyranoside; kb. kilobase(s) or l000bp: Ni-NTA. Nickle nitrilo-tri-acetic acid; nt, nucleotide(s); PCR, polymerase chain reaction: r-. recombinant ZP, zona pellucida; ZPl, zona pellucida glycoprotein-1. BACKGROUND AND PRIOR ART REFRENCES OF THE INVENTION The mammalian egg is surrounded by an acellular translucent envelope, the zona pellucida (ZP), comprised of acidic glycoproteins, which are synthesized and secreted during oocyte growth and follicular development (Wassarman. vol 57, .Ann Rev Biochem 415-442, 1988). ZP glycoproteins mediate critical steps in fertilization process including initial attachment followed by tight binding of spermatozoa to the ZP, induction of the acrosome reaction, and affecting a block to polyspermy. This critical role in reproduction together with their tissue specific nature have made the ZP glycoproteins potential candidate antigens for immunocontiaceptive vaccine. To test the efficacy of ZP glycoproteins for immunocontraception. female subjects from several species were immunized with pig zona pellucida glycoproteins isolated from ovaries. Such an immunization leads to block of fertility (Skinner et al., vol 115, Endocrinol 2418-2432, 1984, Mahi-Brown et al., vol 32. Biol Reprod 761-772, 1985; Dunbar et al, vol 52. Fertil Steril 311-318, 1989. Paterson et al. vol 46, Biol Reprod 523-534, 1992). However, it is invariably associated with either a transient or irreversible alteration in the cyclicity, hormonal profile and follicular development in the ovary Impairment of ovarian function is considerably reduced, if not completely eliminated, by the use of purified ZP glycoproteins along with permissible adjuvants such as alum, sodium phthalyl derivative of lipopolysaccharide and muramyl dipeptide instead of complete Freund's adjuvant (Sacco et al.. vol 21, Am J Rep Immunol 1-8, 1989; Bagavant et al.. vol 102, J Reprod Fertil 17-25, 1994). Active immunization studies with glycosylated and deglycosylated porcine ZP antigens, in rabbit model system have atrributed ovarian pathology to glycosylation (Keenan et al., vol 44, Biol Reprod 150-156, 1991; Jones et al, vol 95, J Reprod Fertil 513-525, 1992). Molecular characterization of ZP glycoprotems from various species led to the identification of three major proteins termed as ZP1 (ZPB), ZP2 (ZPA) and ZP3 (ZPC) (Harris et al.. vol 4, DNA Sequence 361-393, 1994; Castle and Dean, vol 50. J Reprod Fertil Supp 1-8. 1996). On the basis of the size of mRNA's ZP2 is the longest and ZP3 smallest [Harris et al., vol 4, DNA Sequence 361-393. 1994]. ZP1 has been cloned and sequenced from mouse (Epifano et al., vol 270, J Biol Chem 27254-27258. 1995), human (Hams et al.. vol 4. DNA Sequence 361-393. 1994), rabbit (Schwoebal et al.. Vol 266, J Biol Chem. 7214-7219. 1991). pig (Yurewicz et al., Vol 1174, Biochimica et Biophysica Acta 211-214, 1993) and cat (Harris et al., vol 4, DNA Sequence 361-393, 1994). In mouse, three zona transcripts have been detected uniquely in the ovary, where dieir expression is restricted to oocytes [Ringuette et al., vol 83, Proc Natl Acad Sci USA 4341-4345. 1986; Liang et al.. vol 10, Mol Cell Biol 1507-1515, 1990; Epifano et al.. vol 121. Development 1947-1956, 1995]. However, in cynomolgus monkey ZP2 and ZP3 transcript is present in oocytes at all stages of folliculogenesis including granulosa cells [Martinez et al.. vol 50, J Reprod Fertil Supp 35-41. 1996]. However. ZPI transcript is present in oocytes in secondary follicles and to lesser extend in tertiary follicles but absent in primordial, primary or antral follicles and granulosa cells [Martinez et al., vol 50, J Reprod Fertil Supp 35-41, 1996]. To avoid or minimize ovarian dysfunction for a ZP glycoprotein based immunocontraceptive vaccine for human application, it will be desirable that the candidate antigen is not present on the primordial/primary follicles and granulosa cells. To design a vaccine for human use, it will be desirable to either use human zona proteins or those having a high sequence homology with human zona proteins so as to elicit an immune response capable of inhibiting more efficiently the human sperm-egg interaction. So far full length ZPI gene has not been cloned from any . non-human primate. The present invention for the first time describes the full length ZPI cDNA clone isolation from bonnet monkey (Macaca radiaia) and its complete nucleotide and deduced ammo acid sequence. For immunocontraceptive puipose, large amount of ZP1 in a pure form without contamination of other ovarian associated antigens will be required. Toward this goal in this invention, we have establish a procedure for the expression of bonnet monkey ZP1 in E coli as a 6XHis fusion protein for its convenient purification. An ELISA system employing recombinant ZP1 coated microtitration plate for the quantitation of antibodies agains ZP1 has also been established. OBJECTS OF THE INVENTION To meet the above requirements, the objects of the present invention provide a method of inducing reversible or permanent infertility in a mammal employing recombinant ZP1 protein or its fragments. Therefore, one object of the invention relates to a method of inducing reversible or permanent infertility in a mammal. Another object of the invention provides use of a novel recombinant ZP 1 protein or its fragments to induce reversible or permanent infertility in a mammal. Further object of the invention relates to describing a novel full length ZP 1 cDNA clone isolated from bonnet monkey (Macaca radiata), its complete nucleotide and deduced amino acid sequence. Yet another object of the invention relates to immunizing a mammal with a dosage comprising ZP 1 protein or its fragments to induce reversible or permanent infertility. Another object of the invention relates to cloning a full length ZP1 gene from a non-human primate. Still another object of the invention relates to immunizing a mammal with a dosage comprising ZP1 protein or its fragments along with suitable adjuvants. diluents and carriers sufficient to induce reversible or permanent infertility. One more object of the invention provides a process for producing recombinant ZP1 or its fragments. DETAILED DESCRIPTION OF THE INVENTION The above objects are acheived by providing a method of inducing reversible or permanent infertility in a mammal which comprises immunization of the said mammal with a recombinant ZP 1 or its fragments derived from Macaca radium. The objects also achieved by providing a method of producing recombinant ZP1 protein or its fragments, said method comprising excising a ZP 1 transcript from pCRscript SK(+) vector and cloning into suitable expression vector such as pQE- 30. transferring the recombinant ZP1 pQE 30 vector obtained above in a host cell such as SG13009[pREP4], BL21(DE3) and BL21(plysS), expressing ZP1 protein . bv srrowina transfected host cells in suitable nutrient conditions and obtaining the ZP 1 or its fragments by any known method. The recombinant ZP1 or its fragments are derived by methods such as generic engineering or chemical synthesis. The invention providers a method for producing an immino contraceptive vaccine. as claimed in claim1 and a method for detecting antibedies as claimed in claim 6 Zona pellucida glycoproteins have been proposed as candidate antigens for immunocontraception Recent observations in cynomolgus monkey about the ptesence of ZP1 transcript only in the oocytes of secondary follicles and its absence in the primordial/pnmary follicles and granulosa cells [Martinez et al. vol 50. J Reprod Fertil Supp, 35-41, 1996J prompted us to clone and sequence the bonnet monkey ZP1 gene These studies can be facilitated by the availability of recombinant proteins A cDNA gtll library was constructed using poly-A~ mRNA isolated from bonnet monkey (Macaca radiata) ovaries and screened for bonnet ZP1 using 404bp (818-1221 nt) human ZP1 fragment as probe. Bonnet ZP1 cDNA comprises of 1617 nt and encode a polypeptide of 539 aa residues which shares 91.09% overall identity with human ZP1. The major difference between bonnet ZP1 versus human ZP1 is the deletion of 28 aa domain (100-127 aa residues corresponding to human ZP1) An intenal fragment (1317 bp) of bonnet ZP1. excluding the N-termmus signal sequence and the C-terrmnus transmembrane-like domain was amplified by PCR. The amplified Sac 1 and Kpn 1 restricted fragment was cloned m frame downstream of the T5 promoter under the lac operator control for expression m the pQE-30 vector Recombinant ZP1 (r-ZP1) was expressed as a poly-histidine fusion protein m E coh strains SG130009[pR£P4] and ompT and Ion proteases deficient BL21(DE3) and BL21(plysS). Partial ZPl sequence has also been reported from the Cynomolgous monkey [Harris et al., International Patent Publication Number WO 94/11019, 1994]. These investigators failed to isolate full length ZP1 cDNA clones inspite of exhaustive screening of the two cynomolgous monkey ovarian cDNA libraries with porcine ZP1 probe. An insert of 866 bp encoding a polypeptide of 270 aa corresponding to the C-terminus and approximately 50% of the expected full length sequence has been reported. Comparison of 270 aa corresponding to cynomolgous monkey ZP1 with bonnet ZP1 revealed changes in only 8 aa of which three are after the predicted furine cleavage site. Bonnet ZP1 has tri-basic (R-R-R) cleavage site beginning 464 aa which is similar to human ZP1. However, cynomolgous monkey ZP1 has only 2 instead of 3 arginine residues. On the basis of von Heijne's [vol 14, Nuc Acid Res 4683-4690, 1986] scoring method, bonnet ZP1 has a potential peptidase site after the 21st amino acid resulting in a N-terminal lysine. It does not have a 3' untranslated region, since the translation stop codon TAA coincides with the RNA polyadenylation signal, a feature common to the mRNA from human, mouse, rabbit, pig and cat. Comparison of the deduced aa sequence of precursor bonnet ZP 1 protein revealed highest sequence identity with human (91.09%) and lowest with mouse (36.55%; not shown in Fig. 2.]. The low sequence homology of bonnet ZP1 with mouse ZP1 is attributed to elongated exons 3 and 12 of mouse ZP1 [Castle and Dean, vol 50, J Reprod Fertil Suppl 1-8. 1996]. There are 7 aa residues which are unique in bonnet ZP1 (aa residues at position 2, 16, 120. 121, 455, 469 and 523 of precursor bonnet ZP1 protein) which are otherwise conserved in human, pig, rabbit and cat ZP1. There are 24 aa residues, identical and unique to both human and bonnet ZP1 (aa residues 12. 30. 77, 113, 116, 142, 146, 161, 187, 191, 217, 222, 266. 267, 268, 273, 282. 293. 430. 439, 451. 468, 479 and 506 numbered according to the bonnet 2P1 precursor protein) which otherwise are conserved in pig, rabbit and cat. At the moment, it is not clear whether repetition of -500 bp N-terminal fragment is an artifact introduced while packaging cDNA into A.gtl 1 or is due to a partial gene duplication. The 3' nt sequence of -500 bp fragment does not match with Xgtl 1 arm used for packaging and is likely not an artifact of packaging. In chicken, progesterone receptor A and B proteins are synthesized from the same mRNA by alternate initiation sites from two in frame AUG codons [Conneely et al., vol 264, J Biol Chem 14062-14064, 1989]. The chicken progesterone receptor B protein differs from A protein by an additional 128 aa located at the N-terminus. The evolutionary and functional significance of two forms of the steroid hormone receptor has been unclear. Whether such a possibility exist for expression of ZP1 in non-human primates is at best a speculation. To undertake feasibility studies of an immunocontraceptive vaccine based on bonnet ZP1, it will be desirable to have the antigen in a pure form without contamination of other ovarian associated antigens. Bonnet monkey ZP1 was expressed in E. coh as a 6XHis fusion protein for its convenient purification in a truncated form excluding the signal sequence (21 aa residues) and the C-terminal portion (77 aa residues) including the transmembrane-like domain, which will be processed by furine like proteases. On the SDS-PAGE the fusion protein showed an apparant molecular weight of 51 kDa Expression level was reasonably high (7.5 mg/L in a batch flask culture) though some amount of degradation of the expressed protein was evident in the Western blots. The degradation of r-ZPl was not either eliminated nor reduced with expression of the protein in a Ion and ompT protease deficient strain BL21(pIysS) indicating that some other and not these proteases were responsible for the degradation observed. Immunoblot with a murine monoclonal antibody, MA-410 (raised against porcine ZP3a - a homologue of bonnet ZP1, and crossreactive with bonnet zona pellucida) revealed a band of 51 kDa besides truncated fragments. Optimum expression of r-ZP1 was observed at 0.5 mM EPTG. Immunization of male rabbits with r-ZPl purified on Ni-NTA resin under denaturing conditions and female rabbits with r-ZP1 conjugated with diptheria toxoid (DT) generated antibodies reacnve with r-ZP1 m ELISA. The bonnet monkey r-ZPl was highly immunogenic in both male and female rabbits and anti-r-ZP 1 antibodies recognized the bonnet monkey zona pellucida. Protein expressed in a prokaryotic expression system are not glycosylated. Active immunization studies with glycosylated and deglycosylated porcine ZP antigens, in the rabbit model system have attributed ovarian pathology to glycosylation [Keenan et al., vol 44, Biol Reprod 150-156, 1991; Jones et aL vol 95, J Reprod Fertil 513-525, 1992]. Recently, cynomolgous monkeys had been immunized with the rabbit zona protein rc55 ( a homologue of bonnet monkey 2P1) expressed in a procaryotic expression system as cro-p-galactosidase fusion protein and coupled to KLH/Protein -A. Antibody response against the rc5$ inhibited homologous sperm-egg interaction without concomitant disturbance in cyclicity or any associated ovarian pathology [VandeVoort et al., vol 64. Fertil Steril 83£-847, 1995]. Moreover, polyclonal antibodies against bonnet r-ZPl when tested by indirect immunofluorescence on bonnet monkey ovarian sectioa showed positive fluorescence with zona pellucida. The information on sequence based on bonnet ZP1 and availability of recombinant protein will help in better understanding and evaluation of homologous immunization in a non-human primate model, the contraceptive potential of the immunocontraceptive vaccine of r-ZPl. Active immunization of primates such as female/bonnet monkeys and female baboons of proven fertility with r-ZP 1 conjugated to DT, tetanus toxoid (TT) or circumsporozoite protein are in progress. Therefore, the present invention provides a method of inducing reversible or permanent infertility in a mammal, which comprises immunization of the said mammal with a recombinant ZPl or its fragments derived from Macaca radiata. The mammal to be treated may include primates such as monkey, baboon and langoor, apes such as chimpanjee and humans. The invention also relates to a method of wherein ZP 1 protein or its fragments are obtained by any known method such as recombinant DNA technology or chemical synthesis. The invention, in addition, relates to administering a dosage of ZP1 or its fragments along with suitable adjuvants, diluents and. carriers to induce transient or permanent infertility in the mammal. The adjuvant is selected from sodium pthalyl derivative of lipopolysaccharide, Muramyl dipeptide, Squalene (C30H50) and Mannide monooleate and the earner is selected from DT. TT and circumsporozoite protein. Preferred diluents are saline, PBS with am suitable stabilizing agents. The preferred embodiments of the invention provides an isolated and purified DNA sequence encoding ZP1 of Macaca radiata as shown in Fig. 2 of the drawings and the DNA sequence is cloned into a sutiable cloning vector such as pCRscript SK(+). The polypeptide has the sequence as shown in Fig. 3 of the drawings or its truncated form being derived from the DNA sequence shown in the Fig. 2 of the drawings. The polypeptide or its fragments being expressed in suitable expression vectors such as pQE-30 and host cells such as SGI 3009[pREP4], BL21(DE3) and BL21(plys S). Another embodiment of the present invention relates to a method of producing recombinant ZP1 protein or its fragments, said method comprising: a. cloning a DNA sequence obtained from ovarian tissue of Macaca radiata encoding for ZP1 protein into pCRscript SK(-r) vector by any known method, b excising a ZPi transcript from pCRscript SK(-) vector and cloning into suitable expression vector such as pQE-30 in a manner known per se; c. transfecting the recombinant ZP1 pQE-30 vector obtained above in a host cell such as SG13009[pREP4], BL21(DE3) and BL21(plyS), d. expressing ZP1 protein by growing the transfected host cells in suitable nutrient conditions and obtaining the ZP1 or its fragments by any known method. Further embodiment of the invention relates to an ELISA to detect antibodies generated against ZP comprising of: i. ELISA plate coated with recombinant ZP 1 of Macaca radiata ii. sera from test species is reacted to the antigen coated ELISA plate, and iii. antibody is revealed using an antibody enzyme conjugate raised against that species. The preferred embodiments of the present invention are explained and illustrated in the following description with reference to the accompanying drawings. Hence, the scope of the present invention should not be restricted to the references to the drawings and illustrations. In the accompanying drawings : Fig. 1 Restriction map of clone 5 harbouring ZP1 insert in pCRScript SK(+) vector Purified plasinid from clone 5 was digested with Xhol and resolved by agarose gel electrophoresis. Lanes 1 and 3 represent A.Hind III and pGEM markers respecthely and lane 2 the restricted plasmid. The order of the various fragments in the clone 5 is shown in the upper part of me figure. Fig. 2 Nucleotide sequence of bonnet ZP1 cDNA clones The bonnet monkey ZP1 cDNA of clones 5 (BoC5) and 25 (BoC25) was sequenced using Sequenase Version 2 0 DNA sequencing kit (USB. Amersham Life Sciences Inc , Buckinghamshire. L'K) Line 2 under BoC5 represent 1-474 m repeat fragment which preceeds the rest of the sequence of BoC5 clone BoC25 sequence starts from 149 bp after ATG The putative 63 nt signal sequence is underlined and the arrow after 63 nt pomts to the predicted signal peptidase cleavage site. The predicted funn clea\age site is denoted by overline Asterisks represent conserved nucleotides with respect to clone 5 Fig. 3 Amino acid sequence of bonnet monkey ZP1 .Ammo acid sequence of the bonnet monkey was deduced from nucleotide sequence as shown m Fig.2.. The bonnet ZP1 comprised of 539, ammo acids Fig. 4 Comparison of the primary aa sequence of bonnet monkey ZP1 with ZP1 of 4 other species The aa are represented as single letter code Asterisks mdicate completely conserved aa with respect to bonnet ZP1 and potential N-lmked glycosylation sites (Asn-Xaa-Ser/Thr) are underlined. The signal peptidase cleavage site and tn-basic funne proteolytic processing signal (Arg-Arg-Arg) are mdicated by arrows and ovei lining respectively. The completeK conserved aa residues in the five species are underlined Fig. 5 Schematic representation of the plasmid pQE-ZPI The Sad and KpnJ digested 1317 bp ZP1 fragment amplified from the full length cD\A clone 5 in pCRScript SK(+) was cloned in frame downstream of a 6XHis tag in pQE-30 vector. T5, promotor of phage 15: Ampr, Ampicillin resistance marker; ORI. origin of replication. Fig. 6 Electrophoretic analysis and immunoblot of cell lysates prepared from SG 13009(pREP4] harbouring the pQE-ZPI plasmid. SGl3009[pR£P4] cells transformed with the pQE-ZPI r-plasmid were grown till A600 = 0-7 and induced with different concentrations of IPTG (lanes 2-5) for 2 hi". Cells were lysed by boiling for 5 min in SDS-PAGE buffer and electrophoresed as decribed in the Materials and Methods. Panel A represent Coomassie stained gel and Panel B an immunoblot of the same probed with MA-410. Lanes are represented as M, molecular weight marker (kDa); lane 1, uninduced cells and lanes 2, 3, 4 and 5 correspond to cells induced with IPTG at a concentrarion of 0.5. 1, 2 and 3 mM respectively. The arrow in Panel A represent the recombinant 6XHis-ZPl fusion protein. Fig. 7 Expression of r-ZPl in BL21(plysS), an ompT and Ion protease mutant E. coli strain BL2l(plysS) cells and those transformed with the pQE-ZPI plasmid were grown till the A500 = 0.7 and induced with 0.5 mM IPTG for varying time. Cell lysate was resolved by SDS-PAGE and transferred to nitrocellulose membrane for Western blot analysis using MA-410 as a probe Lanes are represented as M, molecular weight markers (kDa), lane 2 wild type BL2l(plysS) cells induced with IPTG for 2 hr and lane 1 corresponding to uninduced control, lanes 4 and 6 BL21(plysS) harbouring the pQE-ZPl plasmid induced with IPTG for 1 and 2 hr respectively and lanes 3 and 5 are corresponding uninduced controls. Fig. 8 Reactivity of rabbit anti-r-ZPl serum with bonnet monkey zona peilucida by indirect immunofluorescence. Bonnet monkey ovarian cryosection (5 um) were incubated with 1:2000 dilution of rabbit anti-r-ZPl preunmune and immune sera followed by incubation with 1 1000 dilution of FITC-labeled anti- rabbit Ig as described in Materials and Methods. Representative results with the immune sera is shown. Top. monkey ovarian section as viewed under phase contrast (100X). Bottom, same section showmg specific fluorescence of ZP following treatment with immune serum (100X) PREFERRED MATERIALS AND METHODS INVOLVED IN THE INVENTION Bonnet monkey ovarian cDNA library, screening and sequencing of ZPl clones: Ovaries were collected from a 2-year old female bonnet monkey, snap frozen in liquid nitrogen and stored at -70°C until used. Total RNA was isolated from frozen ovaries and poly-A~ fraction purified by using the polyATtract mRNA isolation system (Promega, Madison, Wl. USA). cDNA was synthesized from the purified poly-A+ RNA by using Riboclone cDNA synthesis system (Promega. Madison. WI, USA) and cloned in gtll to construct a library (Packagene Lambda DNA Packaging System. Promega. Madison, WI. USA) The constructed bonnet monkey ovarian cDNA gtll library had 1.79x10^ independent plaques with 96% recombinant prior to amplification. The A.gt 11 ovarian cDNA library was screened using human ZP1 404bp (818-1221nt) fragment (provided by Dr. Jurien Dean, Laboratory of Cellular and Developmental Biology, NIH, Bethesda, USA). Six positive clones were obtained by plaque hybridization of amplified library with 404 bp fragment of human ZP1 cDN A. Insert from the positive clones was PCR amplified by means of A.gtl 1 forward (5' GGTGGCGACGACTCCTGGAGCCCG-3*) and reverse (5'- TTGACACCAGACCAACTGGTAATG-3') primers; this involved initial melt at 94°C for 10 min and 30 cycles of 94°C for 1 mm, 58°C for 2 min and 72°C for 3 min followed by a final extension at 72°C for 15 min. The PCR amplification was earned out in 50µ1 with Taq polymerase (Stratagene, La Jolla. CA. USA). The amplified cDNA fragment was resolved by electrophoresis and purified by use of the Genecleanll kit (Bio 101 Inc., La Jolla, CA. USA). The product was cloned in pCRScript SK(+) cloning vector (Stratagene, La Jolla, CA. USA) and sense strand sequenced using T3 (5'-AATTAACCCTCACTAAAGGG-3') and T7 (5- GTAATACGACTCACTATAGGGC-3') primers by the dideoxy chain termination method. The complete sequence of the insert was determined by following two different strategies i) Digesting the insert with EcoRl, cloning EcoRl digested fragments mto pCRScript SK(-) followed b> sequencing and ii) sequencing the full insert by designing and synthesizing the internal primers. Ammo acid sequence was derived using the DNASIS Program. Various characteristics of the ZP1 sequence was analyzed by PC/Gene (Intelligenetics. Inc.). The multiple alignment of the ZP1 aa sequence of different species with the bonnet monkey ZP1 sequence was carried out using die Clustal V Multiple Alignment Program [Higgins and Sharp, vol 5, CABIOS 151-153, 1989]. Amplification of the insert by PCR using A.gtl 1 forward and reverse primers and its analysis by electrophoresis revealed one clone (clone 5) to be 2 1 kb. two clones (clones 23 and 25) 1.6 kb and three clones (clones 22. 24 and 28) were smaller than 1.6 kb (Data not shown). The PCR amplified product from three clones (5, 23, 25) was cloned into pCRScript SK(+) vector. Sequences from both ends of these three fragments revealed that the clone 5 has a full length cDNA with both the translation initiation codon (ATG) and termination codon (TAA) along with the poly-A tail at die 3' end whereas clones 23 and 25 have the translation termination codon but not the translation initiation codon. Further mapping of the clone 5 wim EcoR I resulted in 4 inserts of ~ 750. -550. -500, and -250 bp. Nucleotide sequence revealed that both -500 (474 nt) and ~ 550 bp fragments have respective translation initiation codon and -500 bp fragment had identical sequence (except 42 nt at the 3'-end) as that of -550bp fragment. The -550 bp fragment had Xhol restriction site and one additional sue was also present in the pCRScript SK(-) vector. Digestion of clone 5 with Xhol. generated two fragments of 3.9 and 1.2 kb, indicating that -550 bp fragment is after -500 bp fragment (Fig. 1). Hence, the order of the various fragments in the clone 5 was N' - -500 bp- -550 bp- -750 bp- -250 bp- C. Clones 23 and 25 showed identical sequence and hence the sequence of only clone 25 is presented (Fig. 2). Clone 25 could only be read from 148bp after ATG and was almost similar to clone 5 except for 6 extra nucleotide at 521 nt position and deletion of three nucleotides after 1392 nt. It has a single open reading frame of 1617 nt, resulting in a polypeptide core of 539 aa in length with a calculated molecular mass of 59265 (Fig. 3). A potential signal peptidase site with aa at the -1 and -3 positions that are in accordance with the rule proposed by von Heijne [vol 14. Nuc Acid Res 4683-4690, 1986] is found after 21 aa in bonnet ZP1. Comparison of the deduced aa sequence revealed that bonnet monkey ZP1 protein had a 28 aa deletion from 100-127 aa as compared to human ZP1 (Fig. 4). The bonnet ZP1 aa sequence showed 91.09% identity with the human ZP1. The mature protein excluding the N-terminal signal sequence region and C-terminal transmembrane-like domain which would presumably be processed in a mature molecule had an identity of 94.4% with the corresponding human protein. Amino acid sequence identity with rabbit is 65.17%, pig 62.8%, and that with cat 64.75% (Fig. 4). Out of 539 aa, 249 aa are conserved in all the 5 species (shown in Fig. 4 by underline). The bonnet ZP1 polypeptide chain contains 5 potential N-linked glycosylation sites (Asn-X-Ser/Thr). Two potential cAMP and cGMP -dependent protein kinase phosphorylation sites at position 249 and 469 are found in bonnet ZP1 protein. It has 7 potential protein kinase C phosphorylation sites at positions 186, 212, 244, 245. 455, 463 and 480. The protein was also found to have 9 N-myxistoylation sites at positions 111. 166, 168. 182, 268, 273, 328. 511 and 520. The bonnet ZP1 polypeptide is rich in leucine (11.1%), serine (10.2%). valine (8.7%) and proline (7.7%). The conformation of bonnet ZP1 protein is 18.1% helical, 28.2% extended and 53.6% as coils. By Hopp and Woods [vol 78. Proc Natl Acad Sci USA 3824-3828, 1981] analysis, taking an average group of 6 aa residues three domain corresponding to 492-497 aa (.Ah = 2.0), 461-467 aa (Ah = 1.75) and 118-124 aa (Ah = 1.7) residues having highest hydrophilicity are observed. The analysis of bonnet ZP I sequence further revealed two predicted transmembrane segments corresponding to LLQCVLLCVSLSLVLSG (3-19 aa residues) and VLWVAGLSGTLILGGLV (506-528 aa residues) motifs [Klein et al.. vol 815, Biophys et Biochim Acta 468-476. 1985]. Cloning of bonnet monkey ZP1 cDNA in E. coli expression vector An internal fragment of the cDNA, excluding the signal sequence and the transmembrane-like domain, following the putative furine cleavage site, was amplified by PCR using the forward primer 5'- GGTGAGCTCAAGCCTGAGACCAGGT-3' mcorporating a Sad site and the reverse primer 5'-TCTGGTACCGAGATCAGGACAGGT-3' incorporating a Kpnl site. The PCR was done in a 50 µl volume using 50 pmole of each primer and Vent polymerase (NEB Inc., Beverly, MA, USA) for extension. The pBluescript- ZP1 (clone 5, 10 ng) was used as the template and was initially denatured at 95°C for 10 min. Amplification was carried out for 30 cycles of denaturation at 94°C for 1 min, primer annealing at 55°C for 2 min and extension at 72°C for 3 min followed by a final extension at 72°C for 15 min. The amplified 1.317 kb fragment was digested with Sad and Kpnl and cloned in frame downstream of a 6X histidine tag under the T-5 promoter-lac operator control in the pQE-30 vector (QIAexpress, Qiagen GmbH. Max-Volmer-Stra(3e4, Hilden, Germany). The design of the recombinant expression vector pQE-ZPl is given in Fig. 5. The pQE-ZPl plasmid was transformed in SG13009[pREP4] bacterial strains provided with the kit. In addition BL21(DE3) and BL21(plysS) cells, deficient in omp T and Ion proteases, were also transformed with the pQE-ZP 1 plasmid. It is also possible to employ any other suiatable host cells for obtaining the recombinant ZP1. For expression of recombinant ZP1 (r-ZPl) cloned in pQE-30 vector, a single transformed colony was inoculated and grown overnight at 37°C in 1 ml of Luria Broth (Difco Laboratories. Detroit. MI, USA) or any other suitable media containing 100 µg/ml of ampicillin and 25 µg/ml of kanamycin. Next rnomingf cells were subcultured 1:10 and grown until cell density reached an absorbance at 600 nm (A600) of approximately 0.6-0.7. The cells were further grown in the presence of IPTG to induce expression of the fusion protein under the T-5 promoter. Conditions with respect to the time and IPTG concentration used for induction of r-ZPl were standardized. The cells were collected by centrifugation at 13.000Xg for 60 sec and the resulting pellet was stored at -70°C until used. The cell pellet obtained from 1 ml culture was solubilized by boiling for 5 rnin in 100 p.1 of 2X sample buffer (0.0625 M Tns, pH 6.8, 2% SDS. 10% glycerol. 5% (3-mercaptoethanol and 0.001% bromophenol blue) and the proteins were resolved on a 0.1% SDS-10% polyacrylamide gel [Laemmeli, vol 227, Nature 680-685. 1970]. The proteins were electrophoretically blotted to 0.45 µm nitrocellulose membrane (Bio-Rad. Hercules, CA. USA) overnight at a constant voltage of 15 V in Tris glycine buffer with 20% methanol [Towbin et al, vol 76, Proc Natl Acad Sci USA 4350-4354, 1979]. For immunoblotting, nonspecific sites on the membrane were blocked by incubation with 5% BSA in 50 mM phosphate buffered saline (PBS), pH 7 4 for 1 h followed by 3 washes (15 mm each) with PBS containing 0 1% Tween-20 (PBST) For detection of bonnet monkey i-ZPl. a munne monoclonal antibody (MAb), MA-410 generated against porcine ZP3a {a homologue of bonnet monkey ZP1) and immunoIo with PBS The typical results obtained by 0 1%SDS-10%PAGE and irnmunoblot from one of the transformed clones is shown m Fig 6 The fusion protein had an apparent molecular weight of 51 kDa The optimum expression was obtained at 0 5 mM IPTG The expression of ZP 1 was tightly regulated as the same was not observed in the unmduced transformed bactenal cell (Fig 6) Higher IPTG levels seems to inhibit growth of transformed bactenal cells without any further mcrease in expression levels of r-ZPl. Cellular localization revealed that the fusion protein was present in the insoluble intracellular fraction and was not secreted in the periplasm (Data not shown) The r-ZPl was also expressed in BL21(plysS) cells deficient in the ompT and Ion pioteases The pattern obtained in the Western blot was comparable to that obtained with SG13009(pREP4) (Fig 7) Moreover. r-ZPl was observed in absence of IPTG suggesting that its expression is not highly regulated Purification and conjugation of r-ZPl with diphtheria toxoid and immunization. For purification, the r-ZPl fusion protein was expressed at the shake flask level (250 ml culture/flask; total volume 2 L). Cells were pelleted down at l,500Xg for 30 mm at 4°C and stored at -70°C till used. The cell pellet (1 g) was solubilized in 5 ml of buffer A (6 M Guanidine hydrochloride. 0 1 M NaHbPC^. 0 01 M Tns, pH 8.0). The suspension was centnfuged at 10,000Xg for 5 min at 4°C and the supernatant containing the r-fusion protem was mixed with gentle end to end shaking for 1 h at room temperature (RT) with the Ni-NTA resin (Qiagen GmbH, Max-Volmer-Strape 4, Hilden. Germany) The resin was loaded on a column and washed with 5 volumes of buffer A. The column was subsequently washed with 5 volumes each of buffers B and C which contamed 8 M Urea, 0.1 M NaH2PC4 and 0 01 M Tns and had successively reducing pH values of 8 and 6 3 The lecombinant fusion protein was eluted with buffers D and E in which the pH was further reduced to 5 9 and 4 5 respectively The eluted protein was concentrated ui an Amicon concentrator usmg a YM5 membrane (Amicon Corp., Lexington. MA. USA) and then dialyzed against 100 mM phosphate buffer pH 7.4 having 4 M urea. The purified protein was quantitated with bicinchoninic acid (Sigma Chemicals Co, St. Louis. MO, USA). From a 2 L batch culture, approximately 15 mg of purified r-ZPl was obtained and used to immunize male rabbits. Five milligram of r-ZPl was conjugated to 2.5 mg of diphtheria toxoid (DT; Serum Institute, Pune, India) using the "one step" glutaraldehyde coupling procedure [Avarameas, vol.6, Immunochem 43-53. 1969]. Conjugation was done in 100 mM phosphate buffer, pH 7.4 with 4 M urea using 0.1% glutaraldehyde. overnight at RT (room temperature) with gentle end to end mixing. Unreacted sites were blocked with 100 mM lysine for 3 h at RT. The conjugate was dialysed h against 10 mM PBS having 0.3 M urea. Au pat, Ahcti Two 6 montrrold male NZWJ rabbits (Small Experimental Animal Facility, National Institute of Immunology, New Delhi, India) were immunized with 200 g of r-ZPl emulsified in complete Freund's adjuvant intradermally at multiple sites. The animals were boosted after 4 weeks with an equivalent amount of r-ZP I in incomplete Freund's adjuvant intramuscularly. In addition, 2 six month old female NZW rabbits were also immunized with r-ZPl-DT conjugate (equivalent to 200 u.g r-ZPl/animal) following the procedure as described for the male rabbits, Seven days after the booster, animals were bled to characterize the antibody response. Reactivity of anti-r-ZPl antibodies was tested by a direct binding ELISA. Microtitration plates were coated with either 200 ng of r-ZPl or 500 ng of DT per well in 50 mM PBS, pH 7.4 for 1 h at 37°C followed by overnight at 4°C. All subsequent incubations were at 37°C for 1 h. After each incubation the plates were washed three times in 50 mM PBS having 0.05% Tween-20. Plates were blocked with 1% BSA. After blocking, 100 µ1 of doubling dilutions of the preimmune and immune serum samples were added in duplicate. HRPO conjugated goat anti-rabbit Ig (Pierce. Rockford. IL. USA) at 1:5000 dilution was used as revealing antibodies. Enzyme activity was revealed by adding 100 µl of 0.05% orthophenylene diamine and 0.06% H2O2 in 50 mM citrate phosphate buffer (pH 5.0) in each well and the reaction was stopped by adding 50 µl/well of 5 N H2SO4. A492 was read and annbody titres were calculated by regression analysis. Antibody titre is expressed as antibody unit i.e. dilution of serum giving an absorbance of 1.0. Immunization of male rabbits. R-136 and R-137, resulted in the generation of high antibody titres 926672 and 939283 antibody units against r-ZPl respectively (Table I). High antibody titres against r-ZPl and DT were also observed m the immunized female rabbits. However, in contrast to the male rabbits, 5 to 7 fold lower anti- r-ZP I antibody titres were observed in female animals. Table 1: Reactivity in ELISA of polyclonal antibodies generated against r-ZPl and r-ZPl-DT conjugate in rabbits with r-ZPl (Table Removed) Not Applicable Immunofluorescence on bonnet monkey ovarian sections. Normal cycling female monkey (10 years) was ovarectormsed, the ovaries snap frozen in liquid nitrogen and sections of 5 urn thickness were cut in a cryostat at —20°C and fixed for 20 min in chilled methanol. Sections passing through a follicle were selected, washed in PBS and blocked for 30 min in 5% normal goat serum. The sections were incubated at 37°C with 1:2000 dilution of rabbit preimmune and immune sera for 1 h, washed with PBS and incubated for 1 h with I 1000 dilution of goat anti-rabbit Ig-FITC conjugate (Pierce. Rockford, IL. L'SA) Slides were washed with PBS and mounted in Glycerol PBS (9 1) and examined under fluorescent microscope (Optiphot, Nikon, Chiyoda-Ku, Tokyo, lapan) For proving the authenticity of recombinant protein it is imperative to show that antiserum generated against the 4-ZP1 recognizes the native protein Rabbit antiserum against r-ZPl showed intense fluorescence with the zona pellucida on sections from the bonnet monkey ovary (Fig 8) The pre-immune serum failed to show any immunofluorescence (Data not shown) Moreover, the fluorescence was specific to the zona matrix and the antibodies did not react with other ovarian cell types Example: Preparation of Vaccine Zona pellucida (ZP), an extracellular matrix, surrounds the mammalian oocyte /P serves as a site for the species-specific recognition and binding of the spermatozoa to the oocyte, induces acrosome reaction in the ZP bound spermatozoa, atfects avoidance of polyspermy and protects the pre-implanted blastocyst The ZP is composed of three biochemically distinct glycoproteins, which have been classified as ZP1, ZP2 and ZP3 The cloning and characterization of bonnet monkey (Mac at a radiata) ZP1 (bmZPl) provided exciting and novel approaches for using bmZPl as an antigen to control fertility in humans and other mammals A vaccine tor fertility inhibition can be produced by combining either recombinant or synthetic bmZPl or a portion theieof with a suitable carrier protein for administration to a subject foi immunization The carrier protein help m providing 1 -cell help which in turn help in generating immune response against the ZP protein/peptide The carrier can bt selected from diphtheria toxoid or rabies glycoprotem-Cj Alternatively peptides corresponding to promiscuous T-cell non B-cell epitopes from malaria parasite tetanus toxoid etc can also be used as earner The bmZPl- carrier protein conjugate are used with or without adjuvant(s) to immunize female subjects (bonnet monkey oi other mammals) The antibody response thus generated is monitored by assay thai uses bmZPl as antigen Antibodies thus produced against bmZPl will bind to the oocyte and thus disrupt fertilization Alternatively, antibodies against bmZPl can lead to degenerative changes in ZP, inhibition in the growth of the tollicles and thus affecting ovulation These effects of immunization will lead to inhibition of fertility in the immunized animals It may be noted that the recombinant or synthetic bmZPl oi a portion thereof with a suitable carrier protein have a synergistic relationship in that the immunity to the animal is achieved only by a combination of these ingredients and not otherwise. Use of single ingredient gives no effect to the animal The availability of the bmZPl also allowed us to develop an homologous animal model system of using female bonnet monkey for evaluating efficacy, reversibility and safety of the immunocontraceptive vaccine based on bmZPl for fertility inhibition. Zona pellucida protein or peptide of the present invention can be combined with adjuvants, which leads to enhancement of immune response The mechanism of action of adjuvants is not fully understood The use of a combination of Squalene-Arlacel-A or Freund's adjuvants leads to a water-in-oil type of emulsion, which acts as a "depot" at the site of injection It helps in the slow release of the antigen and also protects the antigen from degradation. Along with the use of agents such as sodium phthalyl derivative of lipopolysacchande (SPLPS) stimulates the immune system non-specifically, probably by producing a battery of soluble peptide factors known as lymphokines. In turn, lymphokines stimulate the activity ot antigen processing cells directly There is a continuous rise in global human population In order to stabilize global human population by 2050, there is a need to develop new methods of contraception Another collateral issue is to find a more human way to control wild life population This has become imperative due to enactment of new legislations and awareness in the society that forbid killing of these animals In the present patent, we have proposed an immunocontraceptive vaccine aiming to interfere at the pre-fertihzation stage A translucent, non-cellular matrix termed as zona pellucida (ZP) surround the mammalian oocyte The ZP is composed of three major glycoproteins classified as ZP1, ZP2 and ZP3 Zona pellucida glycoproteins plays a critical role during fertilization In the present invention, we have optimized the cloning and expression of bonnet monkey (Macaca rathata) ZP1 and standardize various steps to make an immunocontraceptive vaccine We claim: 1. A method for producing an immune-contraceptive vaccine comprising: a. Isolating and preparing in a known manner an amino acid sequence comprising a peptide consisting of zona pellucida-1 (ZPl) shown in figure 3 and conservatively modified variants thereof; b. Mixing with the sequence of (a), a pharmacologically acceptable carrier, and optionally an adjuvant such as herein described in a ratio of 4:1, 2 . A method as claimed in claim 1 wherein the adjuvant is selected from Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane), Arlacel-A (mannide monooleate); sodium phthalyl derivative of lipopolysaccharide, complete Freund's adjuvant, incomplete Freund's adjuvant. and other related preparations. 3 . A method as claimed in claim 1 wherein the carrier is selected from diphtheria toxoid, rabies glycoprotein-G and peptides corresponding to promiscuous T non-B cell epitope. 4. A method as claimed in claim 1 wherein the amount of the ZPl protein is 50-500 )Ug/injection. 5. A method as claimed in claim 1 wherein the amount of the carrier is 50-500 jug/ inject ion. 6. A method for detecting antibodies that inhibit fertility comprising: a. providing a ZPl peptide (recombinant protein) as shown in figure 3; b. contacting an effective amount of the peptide with fluid obtained from a subject in an intmunoassay procedure for a sufficient period to form a complex between the pep tide and the antibody, and c. subjecting said complex to a detecting means, thereby detecting antibodies that inhibit fertilization in the subject. 7. A method as claimed in claim 6 wherein the immunoassay procedure is selected from a sandwich assay or a passive agglutination assay.

Full Text

FIELD OF THE INVENTION
relates to a method of inducing infertility in a mammal, a recombinant ZP1 protein or its fragments used for inducing the inferrility and a process of producing the recombinant ZPl protein and its fragments. Abbreviations used : A, absorbance (1cm): aa, amino acid(s); bp. base pairs IPTG. isopropyl-ß-D-thiogalactopyranoside; kb. kilobase(s) or l000bp: Ni-NTA. Nickle nitrilo-tri-acetic acid; nt, nucleotide(s); PCR, polymerase chain reaction: r-. recombinant ZP, zona pellucida; ZPl, zona pellucida glycoprotein-1. BACKGROUND AND PRIOR ART REFRENCES OF THE INVENTION The mammalian egg is surrounded by an acellular translucent envelope, the zona pellucida (ZP), comprised of acidic glycoproteins, which are synthesized and secreted during oocyte growth and follicular development (Wassarman. vol 57, .Ann Rev Biochem 415-442, 1988). ZP glycoproteins mediate critical steps in fertilization process including initial attachment followed by tight binding of spermatozoa to the ZP, induction of the acrosome reaction, and affecting a block to polyspermy. This critical role in reproduction together with their tissue specific nature have made the ZP glycoproteins potential candidate antigens for immunocontiaceptive vaccine.
To test the efficacy of ZP glycoproteins for immunocontraception. female subjects from several species were immunized with pig zona pellucida glycoproteins isolated from ovaries. Such an immunization leads to block of fertility (Skinner et
al., vol 115, Endocrinol 2418-2432, 1984, Mahi-Brown et al., vol 32. Biol Reprod 761-772, 1985; Dunbar et al, vol 52. Fertil Steril 311-318, 1989. Paterson et al. vol 46, Biol Reprod 523-534, 1992). However, it is invariably associated with either a transient or irreversible alteration in the cyclicity, hormonal profile and follicular development in the ovary Impairment of ovarian function is considerably reduced, if not completely eliminated, by the use of purified ZP glycoproteins along with permissible adjuvants such as alum, sodium phthalyl derivative of lipopolysaccharide and muramyl dipeptide instead of complete Freund's adjuvant (Sacco et al.. vol 21, Am J Rep Immunol 1-8, 1989; Bagavant et al.. vol 102, J Reprod Fertil 17-25, 1994). Active immunization studies with glycosylated and deglycosylated porcine ZP antigens, in rabbit model system have atrributed ovarian pathology to glycosylation (Keenan et al., vol 44, Biol Reprod 150-156, 1991; Jones et al, vol 95, J Reprod Fertil 513-525, 1992). Molecular characterization of ZP glycoprotems from various species led to the identification of three major proteins termed as ZP1 (ZPB), ZP2 (ZPA) and ZP3 (ZPC) (Harris et al.. vol 4, DNA Sequence 361-393, 1994; Castle and Dean, vol 50. J Reprod Fertil Supp 1-8. 1996). On the basis of the size of mRNA's ZP2 is the longest and ZP3 smallest [Harris et al., vol 4, DNA Sequence 361-393. 1994]. ZP1 has been cloned and sequenced from mouse (Epifano et al., vol 270, J Biol Chem 27254-27258. 1995), human (Hams et al.. vol 4. DNA Sequence 361-393. 1994), rabbit (Schwoebal et al.. Vol 266, J Biol Chem. 7214-7219. 1991). pig
(Yurewicz et al., Vol 1174, Biochimica et Biophysica Acta 211-214, 1993) and cat (Harris et al., vol 4, DNA Sequence 361-393, 1994). In mouse, three zona transcripts have been detected uniquely in the ovary, where dieir expression is restricted to oocytes [Ringuette et al., vol 83, Proc Natl Acad Sci USA 4341-4345. 1986; Liang et al.. vol 10, Mol Cell Biol 1507-1515, 1990; Epifano et al.. vol 121. Development 1947-1956, 1995]. However, in cynomolgus monkey ZP2 and ZP3 transcript is present in oocytes at all stages of folliculogenesis including granulosa cells [Martinez et al.. vol 50, J Reprod Fertil Supp 35-41. 1996]. However. ZPI transcript is present in oocytes in secondary follicles and to lesser extend in tertiary follicles but absent in primordial, primary or antral follicles and granulosa cells [Martinez et al., vol 50, J Reprod Fertil Supp 35-41, 1996]. To avoid or minimize ovarian dysfunction for a ZP glycoprotein based immunocontraceptive vaccine for human application, it will be desirable that the candidate antigen is not present on the primordial/primary follicles and granulosa cells. To design a vaccine for human use, it will be desirable to either use human zona proteins or those having a high sequence homology with human zona proteins so as to elicit an immune response capable of inhibiting more efficiently the human sperm-egg interaction. So far full length ZPI gene has not been cloned from any . non-human primate. The present invention for the first time describes the full length ZPI cDNA clone isolation from bonnet monkey (Macaca radiaia) and its complete nucleotide and deduced ammo acid sequence. For immunocontraceptive
puipose, large amount of ZP1 in a pure form without contamination of other
ovarian associated antigens will be required. Toward this goal in this invention, we
have establish a procedure for the expression of bonnet monkey ZP1 in E coli as a
6XHis fusion protein for its convenient purification. An ELISA system employing
recombinant ZP1 coated microtitration plate for the quantitation of antibodies
agains ZP1 has also been established.
OBJECTS OF THE INVENTION
To meet the above requirements, the objects of the present invention provide a
method of inducing reversible or permanent infertility in a mammal employing
recombinant ZP1 protein or its fragments.
Therefore, one object of the invention relates to a method of inducing reversible
or permanent infertility in a mammal.
Another object of the invention provides use of a novel recombinant ZP 1 protein
or its fragments to induce reversible or permanent infertility in a mammal.
Further object of the invention relates to describing a novel full length ZP 1 cDNA
clone isolated from bonnet monkey (Macaca radiata), its complete nucleotide and
deduced amino acid sequence.
Yet another object of the invention relates to immunizing a mammal with a
dosage comprising ZP 1 protein or its fragments to induce reversible or permanent
infertility.
Another object of the invention relates to cloning a full length ZP1 gene from a
non-human primate.
Still another object of the invention relates to immunizing a mammal with a
dosage comprising ZP1 protein or its fragments along with suitable adjuvants.
diluents and carriers sufficient to induce reversible or permanent infertility.
One more object of the invention provides a process for producing recombinant
ZP1 or its fragments.
DETAILED DESCRIPTION OF THE INVENTION
The above objects are acheived by providing a method of inducing reversible or
permanent infertility in a mammal which comprises immunization of the said
mammal with a recombinant ZP 1 or its fragments derived from Macaca radium.
The objects also achieved by providing a method of producing recombinant ZP1
protein or its fragments, said method comprising excising a ZP 1 transcript from
pCRscript SK(+) vector and cloning into suitable expression vector such as pQE-
30. transferring the recombinant ZP1 pQE 30 vector obtained above in a host cell
such as SG13009[pREP4], BL21(DE3) and BL21(plysS), expressing ZP1 protein .
bv srrowina transfected host cells in suitable nutrient conditions and obtaining the
ZP 1 or its fragments by any known method.
The recombinant ZP1 or its fragments are derived by methods such as generic
engineering or chemical synthesis.
The invention providers a method for producing an immino contraceptive vaccine.
as claimed in claim1 and a method for detecting antibedies as claimed in claim 6 Zona pellucida glycoproteins have been proposed as candidate antigens for immunocontraception Recent observations in cynomolgus monkey about the ptesence of ZP1 transcript only in the oocytes of secondary follicles and its absence in the primordial/pnmary follicles and granulosa cells [Martinez et al. vol 50. J Reprod Fertil Supp, 35-41, 1996J prompted us to clone and sequence the bonnet monkey ZP1 gene These studies can be facilitated by the availability of
recombinant proteins A cDNA gtll library was constructed using poly-A~ mRNA isolated from bonnet monkey (Macaca radiata) ovaries and screened for bonnet ZP1 using 404bp (818-1221 nt) human ZP1 fragment as probe. Bonnet ZP1 cDNA comprises of 1617 nt and encode a polypeptide of 539 aa residues which shares 91.09% overall identity with human ZP1. The major difference between bonnet ZP1 versus human ZP1 is the deletion of 28 aa domain (100-127 aa residues corresponding to human ZP1) An intenal fragment (1317 bp) of bonnet ZP1. excluding the N-termmus signal sequence and the C-terrmnus transmembrane-like domain was amplified by PCR. The amplified Sac 1 and Kpn 1 restricted fragment was cloned m frame downstream of the T5 promoter under the lac operator control for expression m the pQE-30 vector Recombinant ZP1 (r-ZP1) was expressed as a poly-histidine fusion protein m E coh strains SG130009[pR£P4] and ompT and Ion proteases deficient BL21(DE3) and BL21(plysS). Partial ZPl sequence has also been reported from the Cynomolgous monkey [Harris et al., International Patent Publication Number WO 94/11019,
1994]. These investigators failed to isolate full length ZP1 cDNA clones inspite of exhaustive screening of the two cynomolgous monkey ovarian cDNA libraries with porcine ZP1 probe. An insert of 866 bp encoding a polypeptide of 270 aa corresponding to the C-terminus and approximately 50% of the expected full length sequence has been reported. Comparison of 270 aa corresponding to cynomolgous monkey ZP1 with bonnet ZP1 revealed changes in only 8 aa of which three are after the predicted furine cleavage site. Bonnet ZP1 has tri-basic (R-R-R) cleavage site beginning 464 aa which is similar to human ZP1. However, cynomolgous monkey ZP1 has only 2 instead of 3 arginine residues. On the basis of von Heijne's [vol 14, Nuc Acid Res 4683-4690, 1986] scoring method, bonnet ZP1 has a potential peptidase site after the 21st amino acid resulting in a N-terminal lysine. It does not have a 3' untranslated region, since the translation stop codon TAA coincides with the RNA polyadenylation signal, a feature common to the mRNA from human, mouse, rabbit, pig and cat. Comparison of the deduced aa sequence of precursor bonnet ZP 1 protein revealed highest sequence identity with human (91.09%) and lowest with mouse (36.55%; not shown in Fig. 2.]. The low sequence homology of bonnet ZP1 with mouse ZP1 is attributed to elongated exons 3 and 12 of mouse ZP1 [Castle and Dean, vol 50, J Reprod Fertil Suppl 1-8. 1996]. There are 7 aa residues which are unique in bonnet ZP1 (aa residues at position 2, 16, 120. 121, 455, 469 and 523 of precursor bonnet ZP1 protein) which are otherwise conserved in human, pig, rabbit and cat ZP1. There are 24 aa
residues, identical and unique to both human and bonnet ZP1 (aa residues 12. 30. 77, 113, 116, 142, 146, 161, 187, 191, 217, 222, 266. 267, 268, 273, 282. 293. 430. 439, 451. 468, 479 and 506 numbered according to the bonnet 2P1 precursor protein) which otherwise are conserved in pig, rabbit and cat. At the moment, it is not clear whether repetition of -500 bp N-terminal fragment is an artifact introduced while packaging cDNA into A.gtl 1 or is due to a partial gene duplication. The 3' nt sequence of -500 bp fragment does not match with Xgtl 1 arm used for packaging and is likely not an artifact of packaging. In chicken, progesterone receptor A and B proteins are synthesized from the same mRNA by alternate initiation sites from two in frame AUG codons [Conneely et al., vol 264, J Biol Chem 14062-14064, 1989]. The chicken progesterone receptor B protein differs from A protein by an additional 128 aa located at the N-terminus. The evolutionary and functional significance of two forms of the steroid hormone receptor has been unclear. Whether such a possibility exist for expression of ZP1 in non-human primates is at best a speculation. To undertake feasibility studies of an immunocontraceptive vaccine based on bonnet ZP1, it will be desirable to have the antigen in a pure form without contamination of other ovarian associated antigens. Bonnet monkey ZP1 was expressed in E. coh as a 6XHis fusion protein for its convenient purification in a truncated form excluding the signal sequence (21 aa residues) and the C-terminal portion (77 aa residues) including the transmembrane-like domain, which will be processed by furine like proteases. On
the SDS-PAGE the fusion protein showed an apparant molecular weight of 51 kDa Expression level was reasonably high (7.5 mg/L in a batch flask culture) though some amount of degradation of the expressed protein was evident in the Western blots. The degradation of r-ZPl was not either eliminated nor reduced with expression of the protein in a Ion and ompT protease deficient strain BL21(pIysS) indicating that some other and not these proteases were responsible for the degradation observed.
Immunoblot with a murine monoclonal antibody, MA-410 (raised against porcine ZP3a - a homologue of bonnet ZP1, and crossreactive with bonnet zona pellucida) revealed a band of 51 kDa besides truncated fragments. Optimum expression of r-ZP1 was observed at 0.5 mM EPTG. Immunization of male rabbits with r-ZPl purified on Ni-NTA resin under denaturing conditions and female rabbits with r-ZP1 conjugated with diptheria toxoid (DT) generated antibodies reacnve with r-ZP1 m ELISA. The bonnet monkey r-ZPl was highly immunogenic in both male and female rabbits and anti-r-ZP 1 antibodies recognized the bonnet monkey zona pellucida. Protein expressed in a prokaryotic expression system are not glycosylated. Active immunization studies with glycosylated and deglycosylated porcine ZP antigens, in the rabbit model system have attributed ovarian pathology to glycosylation [Keenan et al., vol 44, Biol Reprod 150-156, 1991; Jones et aL vol 95, J Reprod Fertil 513-525, 1992]. Recently, cynomolgous monkeys had been immunized with the rabbit zona protein rc55 ( a homologue of bonnet monkey
2P1) expressed in a procaryotic expression system as cro-p-galactosidase fusion protein and coupled to KLH/Protein -A. Antibody response against the rc5$ inhibited homologous sperm-egg interaction without concomitant disturbance in cyclicity or any associated ovarian pathology [VandeVoort et al., vol 64. Fertil Steril 83£-847, 1995]. Moreover, polyclonal antibodies against bonnet r-ZPl when tested by indirect immunofluorescence on bonnet monkey ovarian sectioa showed positive fluorescence with zona pellucida. The information on sequence based on bonnet ZP1 and availability of recombinant protein will help in better understanding and evaluation of homologous immunization in a non-human primate model, the contraceptive potential of the immunocontraceptive vaccine of r-ZPl. Active immunization of primates such as female/bonnet monkeys and female baboons of proven fertility with r-ZP 1 conjugated to DT, tetanus toxoid (TT) or circumsporozoite protein are in progress.
Therefore, the present invention provides a method of inducing reversible or permanent infertility in a mammal, which comprises immunization of the said mammal with a recombinant ZPl or its fragments derived from Macaca radiata. The mammal to be treated may include primates such as monkey, baboon and langoor, apes such as chimpanjee and humans.
The invention also relates to a method of wherein ZP 1 protein or its fragments are obtained by any known method such as recombinant DNA technology or chemical synthesis.
The invention, in addition, relates to administering a dosage of ZP1 or its fragments along with suitable adjuvants, diluents and. carriers to induce transient or permanent infertility in the mammal. The adjuvant is selected from sodium pthalyl derivative of lipopolysaccharide, Muramyl dipeptide, Squalene (C30H50) and Mannide monooleate and the earner is selected from DT. TT and circumsporozoite protein. Preferred diluents are saline, PBS with am suitable stabilizing agents.
The preferred embodiments of the invention provides an isolated and purified DNA sequence encoding ZP1 of Macaca radiata as shown in Fig. 2 of the drawings and the DNA sequence is cloned into a sutiable cloning vector such as pCRscript SK(+). The polypeptide has the sequence as shown in Fig. 3 of the drawings or its truncated form being derived from the DNA sequence shown in the Fig. 2 of the drawings. The polypeptide or its fragments being expressed in suitable expression vectors such as pQE-30 and host cells such as SGI 3009[pREP4], BL21(DE3) and BL21(plys S).
Another embodiment of the present invention relates to a method of producing recombinant ZP1 protein or its fragments, said method comprising: a. cloning a DNA sequence obtained from ovarian tissue of Macaca radiata encoding for ZP1 protein into pCRscript SK(-r) vector by any known method, b excising a ZPi transcript from pCRscript SK(-) vector and cloning into suitable expression vector such as pQE-30 in a manner known per se;
c. transfecting the recombinant ZP1 pQE-30 vector obtained above in a host
cell such as SG13009[pREP4], BL21(DE3) and BL21(plyS),
d. expressing ZP1 protein by growing the transfected host cells in suitable
nutrient conditions and obtaining the ZP1 or its fragments by any known method.
Further embodiment of the invention relates to an ELISA to detect antibodies
generated against ZP comprising of:
i. ELISA plate coated with recombinant ZP 1 of Macaca radiata
ii. sera from test species is reacted to the antigen coated ELISA plate, and
iii. antibody is revealed using an antibody enzyme conjugate raised against that
species.
The preferred embodiments of the present invention are explained and illustrated
in the following description with reference to the accompanying drawings.
Hence, the scope of the present invention should not be restricted to the references
to the drawings and illustrations. In the accompanying drawings :
Fig. 1 Restriction map of clone 5 harbouring ZP1 insert in pCRScript SK(+)
vector
Purified plasinid from clone 5 was digested with Xhol and resolved by agarose gel
electrophoresis. Lanes 1 and 3 represent A.Hind III and pGEM markers respecthely
and lane 2 the restricted plasmid. The order of the various fragments in the clone 5
is shown in the upper part of me figure.
Fig. 2 Nucleotide sequence of bonnet ZP1 cDNA clones
The bonnet monkey ZP1 cDNA of clones 5 (BoC5) and 25 (BoC25) was sequenced using Sequenase Version 2 0 DNA sequencing kit (USB. Amersham Life Sciences Inc , Buckinghamshire. L'K) Line 2 under BoC5 represent 1-474 m repeat fragment which preceeds the rest of the sequence of BoC5 clone BoC25 sequence starts from 149 bp after ATG The putative 63 nt signal sequence is underlined and the arrow after 63 nt pomts to the predicted signal peptidase cleavage site. The predicted funn clea\age site is denoted by overline Asterisks represent conserved nucleotides with respect to clone 5 Fig. 3 Amino acid sequence of bonnet monkey ZP1
.Ammo acid sequence of the bonnet monkey was deduced from nucleotide sequence as shown m Fig.2.. The bonnet ZP1 comprised of 539, ammo acids Fig. 4 Comparison of the primary aa sequence of bonnet monkey ZP1 with ZP1 of 4 other species
The aa are represented as single letter code Asterisks mdicate completely conserved aa with respect to bonnet ZP1 and potential N-lmked glycosylation sites (Asn-Xaa-Ser/Thr) are underlined. The signal peptidase cleavage site and tn-basic funne proteolytic processing signal (Arg-Arg-Arg) are mdicated by arrows and ovei lining respectively. The completeK conserved aa residues in the five species are underlined
Fig. 5 Schematic representation of the plasmid pQE-ZPI
The Sad and KpnJ digested 1317 bp ZP1 fragment amplified from the full length
cD\A clone 5 in pCRScript SK(+) was cloned in frame downstream of a 6XHis
tag in pQE-30 vector. T5, promotor of phage 15: Ampr, Ampicillin resistance
marker; ORI. origin of replication.
Fig. 6 Electrophoretic analysis and immunoblot of cell lysates prepared from
SG 13009(pREP4] harbouring the pQE-ZPI plasmid.
SGl3009[pR£P4] cells transformed with the pQE-ZPI r-plasmid were grown till
A600 = 0-7 and induced with different concentrations of IPTG (lanes 2-5) for 2 hi".
Cells were lysed by boiling for 5 min in SDS-PAGE buffer and electrophoresed as
decribed in the Materials and Methods. Panel A represent Coomassie stained gel
and Panel B an immunoblot of the same probed with MA-410. Lanes are
represented as M, molecular weight marker (kDa); lane 1, uninduced cells and
lanes 2, 3, 4 and 5 correspond to cells induced with IPTG at a concentrarion of 0.5.
1, 2 and 3 mM respectively. The arrow in Panel A represent the recombinant
6XHis-ZPl fusion protein.
Fig. 7 Expression of r-ZPl in BL21(plysS), an ompT and Ion protease mutant
E. coli strain
BL2l(plysS) cells and those transformed with the pQE-ZPI plasmid were grown
till the A500 = 0.7 and induced with 0.5 mM IPTG for varying time. Cell lysate
was resolved by SDS-PAGE and transferred to nitrocellulose membrane for
Western blot analysis using MA-410 as a probe Lanes are represented as M,
molecular weight markers (kDa), lane 2 wild type BL2l(plysS) cells induced with
IPTG for 2 hr and lane 1 corresponding to uninduced control, lanes 4 and 6
BL21(plysS) harbouring the pQE-ZPl plasmid induced with IPTG for 1 and 2 hr
respectively and lanes 3 and 5 are corresponding uninduced controls.
Fig. 8 Reactivity of rabbit anti-r-ZPl serum with bonnet monkey zona
peilucida by indirect immunofluorescence.
Bonnet monkey ovarian cryosection (5 um) were incubated with 1:2000 dilution
of rabbit anti-r-ZPl preunmune and immune sera followed by incubation with
1 1000 dilution of FITC-labeled anti- rabbit Ig as described in Materials and
Methods. Representative results with the immune sera is shown. Top. monkey
ovarian section as viewed under phase contrast (100X). Bottom, same section
showmg specific fluorescence of ZP following treatment with immune serum
(100X)
PREFERRED MATERIALS AND METHODS INVOLVED IN THE
INVENTION
Bonnet monkey ovarian cDNA library, screening and sequencing of ZPl
clones: Ovaries were collected from a 2-year old female bonnet monkey, snap
frozen in liquid nitrogen and stored at -70°C until used. Total RNA was isolated
from frozen ovaries and poly-A~ fraction purified by using the polyATtract mRNA isolation system (Promega, Madison, Wl. USA). cDNA was synthesized
from the purified poly-A+ RNA by using Riboclone cDNA synthesis system (Promega. Madison. WI, USA) and cloned in gtll to construct a library (Packagene Lambda DNA Packaging System. Promega. Madison, WI. USA) The constructed bonnet monkey ovarian cDNA gtll library had 1.79x10^ independent plaques with 96% recombinant prior to amplification. The A.gt 11 ovarian cDNA library was screened using human ZP1 404bp (818-1221nt) fragment (provided by Dr. Jurien Dean, Laboratory of Cellular and Developmental Biology, NIH, Bethesda, USA). Six positive clones were obtained by plaque hybridization of amplified library with 404 bp fragment of human ZP1 cDN A.
Insert from the positive clones was PCR amplified by means of A.gtl 1 forward (5'
GGTGGCGACGACTCCTGGAGCCCG-3*) and reverse (5'-
TTGACACCAGACCAACTGGTAATG-3') primers; this involved initial melt at
94°C for 10 min and 30 cycles of 94°C for 1 mm, 58°C for 2 min and 72°C for 3 min followed by a final extension at 72°C for 15 min. The PCR amplification was earned out in 50µ1 with Taq polymerase (Stratagene, La Jolla. CA. USA). The amplified cDNA fragment was resolved by electrophoresis and purified by use of the Genecleanll kit (Bio 101 Inc., La Jolla, CA. USA). The product was cloned in pCRScript SK(+) cloning vector (Stratagene, La Jolla, CA. USA) and sense strand sequenced using T3 (5'-AATTAACCCTCACTAAAGGG-3') and T7 (5-
GTAATACGACTCACTATAGGGC-3') primers by the dideoxy chain termination method. The complete sequence of the insert was determined by following two different strategies i) Digesting the insert with EcoRl, cloning EcoRl digested fragments mto pCRScript SK(-) followed b> sequencing and ii) sequencing the full insert by designing and synthesizing the internal primers. Ammo acid sequence was derived using the DNASIS Program. Various characteristics of the ZP1 sequence was analyzed by PC/Gene (Intelligenetics. Inc.). The multiple alignment of the ZP1 aa sequence of different species with the bonnet monkey ZP1 sequence was carried out using die Clustal V Multiple Alignment Program [Higgins and Sharp, vol 5, CABIOS 151-153, 1989].
Amplification of the insert by PCR using A.gtl 1 forward and reverse primers and its analysis by electrophoresis revealed one clone (clone 5) to be 2 1 kb. two clones (clones 23 and 25) 1.6 kb and three clones (clones 22. 24 and 28) were smaller than 1.6 kb (Data not shown). The PCR amplified product from three clones (5, 23, 25) was cloned into pCRScript SK(+) vector. Sequences from both ends of these three fragments revealed that the clone 5 has a full length cDNA with both the translation initiation codon (ATG) and termination codon (TAA) along with the poly-A tail at die 3' end whereas clones 23 and 25 have the translation termination codon but not the translation initiation codon. Further mapping of the clone 5 wim EcoR I resulted in 4 inserts of ~ 750. -550. -500, and -250 bp. Nucleotide sequence revealed that both -500 (474 nt) and ~
550 bp fragments have respective translation initiation codon and -500 bp fragment had identical sequence (except 42 nt at the 3'-end) as that of -550bp fragment. The -550 bp fragment had Xhol restriction site and one additional sue was also present in the pCRScript SK(-) vector. Digestion of clone 5 with Xhol. generated two fragments of 3.9 and 1.2 kb, indicating that -550 bp fragment is after -500 bp fragment (Fig. 1). Hence, the order of the various fragments in the clone 5 was N' - -500 bp- -550 bp- -750 bp- -250 bp- C.
Clones 23 and 25 showed identical sequence and hence the sequence of only clone 25 is presented (Fig. 2). Clone 25 could only be read from 148bp after ATG and was almost similar to clone 5 except for 6 extra nucleotide at 521 nt position and deletion of three nucleotides after 1392 nt. It has a single open reading frame of 1617 nt, resulting in a polypeptide core of 539 aa in length with a calculated molecular mass of 59265 (Fig. 3). A potential signal peptidase site with aa at the -1 and -3 positions that are in accordance with the rule proposed by von Heijne [vol 14. Nuc Acid Res 4683-4690, 1986] is found after 21 aa in bonnet ZP1. Comparison of the deduced aa sequence revealed that bonnet monkey ZP1 protein had a 28 aa deletion from 100-127 aa as compared to human ZP1 (Fig. 4). The bonnet ZP1 aa sequence showed 91.09% identity with the human ZP1. The mature protein excluding the N-terminal signal sequence region and C-terminal transmembrane-like domain which would presumably be processed in a mature molecule had an identity of 94.4% with the corresponding human protein. Amino
acid sequence identity with rabbit is 65.17%, pig 62.8%, and that with cat 64.75% (Fig. 4). Out of 539 aa, 249 aa are conserved in all the 5 species (shown in Fig. 4 by underline). The bonnet ZP1 polypeptide chain contains 5 potential N-linked glycosylation sites (Asn-X-Ser/Thr). Two potential cAMP and cGMP -dependent protein kinase phosphorylation sites at position 249 and 469 are found in bonnet ZP1 protein. It has 7 potential protein kinase C phosphorylation sites at positions 186, 212, 244, 245. 455, 463 and 480. The protein was also found to have 9 N-myxistoylation sites at positions 111. 166, 168. 182, 268, 273, 328. 511 and 520. The bonnet ZP1 polypeptide is rich in leucine (11.1%), serine (10.2%). valine (8.7%) and proline (7.7%). The conformation of bonnet ZP1 protein is 18.1% helical, 28.2% extended and 53.6% as coils. By Hopp and Woods [vol 78. Proc Natl Acad Sci USA 3824-3828, 1981] analysis, taking an average group of 6 aa residues three domain corresponding to 492-497 aa (.Ah = 2.0), 461-467 aa (Ah = 1.75) and 118-124 aa (Ah = 1.7) residues having highest hydrophilicity are observed. The analysis of bonnet ZP I sequence further revealed two predicted transmembrane segments corresponding to LLQCVLLCVSLSLVLSG (3-19 aa residues) and VLWVAGLSGTLILGGLV (506-528 aa residues) motifs [Klein et al.. vol 815, Biophys et Biochim Acta 468-476. 1985].
Cloning of bonnet monkey ZP1 cDNA in E. coli expression vector
An internal fragment of the cDNA, excluding the signal sequence and the
transmembrane-like domain, following the putative furine cleavage site, was
amplified by PCR using the forward primer 5'-
GGTGAGCTCAAGCCTGAGACCAGGT-3' mcorporating a Sad site and the reverse primer 5'-TCTGGTACCGAGATCAGGACAGGT-3' incorporating a Kpnl site. The PCR was done in a 50 µl volume using 50 pmole of each primer and Vent polymerase (NEB Inc., Beverly, MA, USA) for extension. The pBluescript-
ZP1 (clone 5, 10 ng) was used as the template and was initially denatured at 95°C
for 10 min. Amplification was carried out for 30 cycles of denaturation at 94°C for
1 min, primer annealing at 55°C for 2 min and extension at 72°C for 3 min
followed by a final extension at 72°C for 15 min. The amplified 1.317 kb fragment was digested with Sad and Kpnl and cloned in frame downstream of a 6X histidine tag under the T-5 promoter-lac operator control in the pQE-30 vector (QIAexpress, Qiagen GmbH. Max-Volmer-Stra(3e4, Hilden, Germany). The design of the recombinant expression vector pQE-ZPl is given in Fig. 5. The pQE-ZPl plasmid was transformed in SG13009[pREP4] bacterial strains provided with the kit. In addition BL21(DE3) and BL21(plysS) cells, deficient in omp T and Ion proteases, were also transformed with the pQE-ZP 1 plasmid. It is also possible to employ any other suiatable host cells for obtaining the recombinant ZP1.

For expression of recombinant ZP1 (r-ZPl) cloned in pQE-30 vector, a single transformed colony was inoculated and grown overnight at 37°C in 1 ml of Luria Broth (Difco Laboratories. Detroit. MI, USA) or any other suitable media containing 100 µg/ml of ampicillin and 25 µg/ml of kanamycin. Next rnomingf cells were subcultured 1:10 and grown until cell density reached an absorbance at 600 nm (A600) of approximately 0.6-0.7. The cells were further grown in the
presence of IPTG to induce expression of the fusion protein under the T-5 promoter. Conditions with respect to the time and IPTG concentration used for induction of r-ZPl were standardized. The cells were collected by centrifugation
at 13.000Xg for 60 sec and the resulting pellet was stored at -70°C until used.
The cell pellet obtained from 1 ml culture was solubilized by boiling for 5 rnin in 100 p.1 of 2X sample buffer (0.0625 M Tns, pH 6.8, 2% SDS. 10% glycerol. 5% (3-mercaptoethanol and 0.001% bromophenol blue) and the proteins were resolved on a 0.1% SDS-10% polyacrylamide gel [Laemmeli, vol 227, Nature 680-685. 1970]. The proteins were electrophoretically blotted to 0.45 µm nitrocellulose membrane (Bio-Rad. Hercules, CA. USA) overnight at a constant voltage of 15 V in Tris glycine buffer with 20% methanol [Towbin et al, vol 76, Proc Natl Acad Sci USA 4350-4354, 1979]. For immunoblotting, nonspecific sites on the membrane were blocked by incubation with 5% BSA in 50 mM phosphate
buffered saline (PBS), pH 7 4 for 1 h followed by 3 washes (15 mm each) with PBS containing 0 1% Tween-20 (PBST) For detection of bonnet monkey i-ZPl. a munne monoclonal antibody (MAb), MA-410 generated against porcine ZP3a {a homologue of bonnet monkey ZP1) and immunoIo with PBS
The typical results obtained by 0 1%SDS-10%PAGE and irnmunoblot from one of the transformed clones is shown m Fig 6 The fusion protein had an apparent molecular weight of 51 kDa The optimum expression was obtained at 0 5 mM IPTG The expression of ZP 1 was tightly regulated as the same was not observed in the unmduced transformed bactenal cell (Fig 6) Higher IPTG levels seems to inhibit growth of transformed bactenal cells without any further mcrease in expression levels of r-ZPl. Cellular localization revealed that the fusion protein was present in the insoluble intracellular fraction and was not secreted in the periplasm (Data not shown)
The r-ZPl was also expressed in BL21(plysS) cells deficient in the ompT and Ion pioteases The pattern obtained in the Western blot was comparable to that obtained with SG13009(pREP4) (Fig 7) Moreover. r-ZPl was observed in absence of IPTG suggesting that its expression is not highly regulated Purification and conjugation of r-ZPl with diphtheria toxoid and immunization.
For purification, the r-ZPl fusion protein was expressed at the shake flask level (250 ml culture/flask; total volume 2 L). Cells were pelleted down at l,500Xg for
30 mm at 4°C and stored at -70°C till used. The cell pellet (1 g) was solubilized in 5 ml of buffer A (6 M Guanidine hydrochloride. 0 1 M NaHbPC^. 0 01 M
Tns, pH 8.0). The suspension was centnfuged at 10,000Xg for 5 min at 4°C and the supernatant containing the r-fusion protem was mixed with gentle end to end shaking for 1 h at room temperature (RT) with the Ni-NTA resin (Qiagen GmbH, Max-Volmer-Strape 4, Hilden. Germany) The resin was loaded on a column and washed with 5 volumes of buffer A. The column was subsequently washed with 5 volumes each of buffers B and C which contamed 8 M Urea, 0.1 M NaH2PC4 and
0 01 M Tns and had successively reducing pH values of 8 and 6 3 The lecombinant fusion protein was eluted with buffers D and E in which the pH was further reduced to 5 9 and 4 5 respectively The eluted protein was concentrated ui an Amicon concentrator usmg a YM5 membrane (Amicon Corp., Lexington. MA.
USA) and then dialyzed against 100 mM phosphate buffer pH 7.4 having 4 M urea. The purified protein was quantitated with bicinchoninic acid (Sigma Chemicals Co, St. Louis. MO, USA).
From a 2 L batch culture, approximately 15 mg of purified r-ZPl was obtained and used to immunize male rabbits.
Five milligram of r-ZPl was conjugated to 2.5 mg of diphtheria toxoid (DT; Serum Institute, Pune, India) using the "one step" glutaraldehyde coupling procedure [Avarameas, vol.6, Immunochem 43-53. 1969]. Conjugation was done in 100 mM phosphate buffer, pH 7.4 with 4 M urea using 0.1% glutaraldehyde. overnight at RT (room temperature) with gentle end to end mixing. Unreacted sites were blocked with 100 mM lysine for 3 h at RT. The conjugate was dialysed h against 10 mM PBS having 0.3 M urea. Au pat, Ahcti Two 6 montrrold male NZWJ rabbits (Small Experimental Animal Facility,
National Institute of Immunology, New Delhi, India) were immunized with 200 g of r-ZPl emulsified in complete Freund's adjuvant intradermally at multiple sites. The animals were boosted after 4 weeks with an equivalent amount of r-ZP I in incomplete Freund's adjuvant intramuscularly. In addition, 2 six month old female NZW rabbits were also immunized with r-ZPl-DT conjugate (equivalent to 200 u.g r-ZPl/animal) following the procedure as described for the male rabbits, Seven days after the booster, animals were bled to characterize the antibody response.
Reactivity of anti-r-ZPl antibodies was tested by a direct binding ELISA. Microtitration plates were coated with either 200 ng of r-ZPl or 500 ng of DT per well in 50 mM PBS, pH 7.4 for 1 h at 37°C followed by overnight at 4°C. All
subsequent incubations were at 37°C for 1 h. After each incubation the plates were washed three times in 50 mM PBS having 0.05% Tween-20. Plates were blocked with 1% BSA. After blocking, 100 µ1 of doubling dilutions of the preimmune and immune serum samples were added in duplicate. HRPO conjugated goat anti-rabbit Ig (Pierce. Rockford. IL. USA) at 1:5000 dilution was used as revealing antibodies. Enzyme activity was revealed by adding 100 µl of 0.05% orthophenylene diamine and 0.06% H2O2 in 50 mM citrate phosphate
buffer (pH 5.0) in each well and the reaction was stopped by adding 50 µl/well of 5 N H2SO4. A492 was read and annbody titres were calculated by regression
analysis. Antibody titre is expressed as antibody unit i.e. dilution of serum giving an absorbance of 1.0.
Immunization of male rabbits. R-136 and R-137, resulted in the generation of high antibody titres 926672 and 939283 antibody units against r-ZPl respectively (Table I). High antibody titres against r-ZPl and DT were also observed m the immunized female rabbits. However, in contrast to the male rabbits, 5 to 7 fold lower anti- r-ZP I antibody titres were observed in female animals.
Table 1: Reactivity in ELISA of polyclonal antibodies generated against r-ZPl and r-ZPl-DT conjugate in rabbits with r-ZPl
(Table Removed)
Not Applicable Immunofluorescence on bonnet monkey ovarian sections.
Normal cycling female monkey (10 years) was ovarectormsed, the ovaries snap frozen in liquid nitrogen and sections of 5 urn thickness were cut in a cryostat at
—20°C and fixed for 20 min in chilled methanol. Sections passing through a follicle were selected, washed in PBS and blocked for 30 min in 5% normal goat serum.
The sections were incubated at 37°C with 1:2000 dilution of rabbit preimmune and immune sera for 1 h, washed with PBS and incubated for 1 h with I 1000 dilution of goat anti-rabbit Ig-FITC conjugate (Pierce. Rockford, IL. L'SA) Slides
were washed with PBS and mounted in Glycerol PBS (9 1) and examined under fluorescent microscope (Optiphot, Nikon, Chiyoda-Ku, Tokyo, lapan)
For proving the authenticity of recombinant protein it is imperative to show that antiserum generated against the 4-ZP1 recognizes the native protein Rabbit antiserum against r-ZPl showed intense fluorescence with the zona pellucida on sections from the bonnet monkey ovary (Fig 8) The pre-immune serum failed to show any immunofluorescence (Data not shown) Moreover, the fluorescence was specific to the zona matrix and the antibodies did not react with other ovarian cell types
Example: Preparation of Vaccine
Zona pellucida (ZP), an extracellular matrix, surrounds the mammalian oocyte /P
serves as a site for the species-specific recognition and binding of the spermatozoa to the oocyte, induces acrosome reaction in the ZP bound spermatozoa, atfects avoidance of polyspermy and protects the pre-implanted blastocyst The ZP is composed of three biochemically distinct glycoproteins, which have been classified as ZP1, ZP2 and ZP3 The cloning and characterization of bonnet monkey (Mac at a radiata) ZP1 (bmZPl) provided exciting and novel approaches for using bmZPl as an antigen to control fertility in humans and other mammals A vaccine tor fertility inhibition can be produced by combining either recombinant or synthetic bmZPl or a portion theieof with a suitable carrier protein for administration to a subject foi immunization The carrier protein help m providing 1 -cell help which in turn help in generating immune response against the ZP protein/peptide The carrier can bt selected from diphtheria toxoid or rabies glycoprotem-Cj Alternatively peptides corresponding to promiscuous T-cell non B-cell epitopes from malaria parasite tetanus toxoid etc can also be used as earner The bmZPl- carrier protein conjugate are used with or without adjuvant(s) to immunize female subjects (bonnet monkey oi other mammals) The antibody response thus generated is monitored by assay thai uses bmZPl as antigen Antibodies thus produced against bmZPl will bind to the oocyte and thus disrupt fertilization Alternatively, antibodies against bmZPl can lead to degenerative changes in ZP, inhibition in the growth of the tollicles and thus affecting ovulation These effects of immunization will lead to inhibition of fertility in the immunized animals It may be noted that the recombinant or synthetic bmZPl oi a
portion thereof with a suitable carrier protein have a synergistic relationship in that the immunity to the animal is achieved only by a combination of these ingredients and not otherwise. Use of single ingredient gives no effect to the animal
The availability of the bmZPl also allowed us to develop an homologous animal model system of using female bonnet monkey for evaluating efficacy, reversibility and safety of the immunocontraceptive vaccine based on bmZPl for fertility inhibition. Zona pellucida protein or peptide of the present invention can be combined with adjuvants, which leads to enhancement of immune response The mechanism of action of adjuvants is not fully understood The use of a combination of Squalene-Arlacel-A or Freund's adjuvants leads to a water-in-oil type of emulsion, which acts as a "depot" at the site of injection It helps in the slow release of the antigen and also protects the antigen from degradation. Along with the use of agents such as sodium phthalyl derivative of lipopolysacchande (SPLPS) stimulates the immune system non-specifically, probably by producing a battery of soluble peptide factors known as lymphokines. In turn, lymphokines stimulate the activity ot antigen processing cells directly
There is a continuous rise in global human population In order to stabilize global human population by 2050, there is a need to develop new methods of contraception Another collateral issue is to find a more human way to control wild life population This has become imperative due to enactment of new legislations and awareness in the society that forbid killing of these animals In the present patent, we have proposed an immunocontraceptive vaccine aiming to interfere at the pre-fertihzation stage A translucent, non-cellular matrix termed as zona pellucida (ZP) surround the mammalian oocyte The ZP is composed of three major glycoproteins classified as ZP1, ZP2 and ZP3 Zona pellucida glycoproteins plays a critical role during fertilization In the present invention, we have optimized the cloning and expression of bonnet monkey (Macaca rathata) ZP1 and standardize various steps to make an immunocontraceptive vaccine

We claim:
1. A method for producing an immune-contraceptive vaccine comprising:
a. Isolating and preparing in a known manner an amino
acid sequence comprising a peptide consisting of zona
pellucida-1 (ZPl) shown in figure 3 and
conservatively modified variants thereof;
b. Mixing with the sequence of (a), a pharmacologically
acceptable carrier, and optionally an adjuvant such
as herein described in a ratio of 4:1,
2 . A method as claimed in claim 1 wherein the adjuvant is
selected from Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane), Arlacel-A (mannide monooleate); sodium phthalyl derivative of lipopolysaccharide, complete Freund's adjuvant, incomplete Freund's adjuvant. and other related preparations.
3 . A method as claimed in claim 1 wherein the carrier is
selected from diphtheria toxoid, rabies glycoprotein-G and peptides corresponding to promiscuous T non-B cell epitope.
4. A method as claimed in claim 1 wherein the amount of
the ZPl protein is 50-500 )Ug/injection.
5. A method as claimed in claim 1 wherein the amount of
the carrier is 50-500 jug/ inject ion.
6. A method for detecting antibodies that inhibit
fertility comprising:
a. providing a ZPl peptide (recombinant protein) as
shown in figure 3;
b. contacting an effective amount of the peptide
with fluid obtained from a subject in an

intmunoassay procedure for a sufficient period to form a complex between the pep tide and the antibody, and
c. subjecting said complex to a detecting means, thereby detecting antibodies that inhibit fertilization in the subject.
7. A method as claimed in claim 6 wherein the immunoassay procedure is selected from a sandwich assay or a passive agglutination assay.

Documents:

346-del-2001-abstract.pdf

346-DEL-2001-Claims.pdf

346-del-2001-complete specification (as, files).pdf

346-del-2001-complete specification (granted).pdf

346-del-2001-correspondence-others.pdf

346-del-2001-correspondence-po.pdf

346-DEL-2001-Description (Complete).pdf

346-del-2001-drawings.pdf

346-del-2001-form-1.pdf

346-del-2001-form-13.pdf

346-del-2001-form-19.pdf

346-DEL-2001-Form-2.pdf

346-del-2001-form-26.pdf

346-del-2001-form-3.pdf

346-del-2001-petition-138.pdf

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Patent Number

217322

Indian Patent Application Number

346/DEL/2001

PG Journal Number

15/2008

Publication Date

11-Apr-2008

Grant Date

26-Mar-2008

Date of Filing

23-Mar-2001

Name of Patentee

NATIONAL INSTITUTE OF IMMUNOLOGY

Applicant Address

ARUNA ASAF ALI MARG, NEW DELHI 110 067,INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	RACHNA BISHT	GAMETE ANTIGEN LOBORATORY,NATIONAL INSTITUTE OF IMUNOLOGY ARUNA ASAF ALI MARG,NEW DELHI 110 067,INDIA
2	ARUNA BEHRA	GAMETE ANTIGEN LOBORATORY,NATIONAL INSTITUTE OF IMUNOLOGY ARUNA ASAF ALI MARG,NEW DELHI 110 067,INDIA
3	MANJU SHARMA	GAMETE ANTIGEN LOBORATORY,NATIONAL INSTITUTE OF IMUNOLOGY ARUNA ASAF ALI MARG,NEW DELHI 110 067,INDIA
4	SATISH KUMAR GUPTA	GAMETE ANTIGEN LOBORATORY,NATIONAL INSTITUTE OF IMUNOLOGY ARUNA ASAF ALI MARG,NEW DELHI 110 067,INDIA.
5	RENUKA KAUL	GAMETE ANTIGEN LOBORATORY,NATIONAL INSTITUTE OF IMUNOLOGY ARUNA ASAF ALI MARG,NEW DELHI 110 067,INDIA

PCT International Classification Number

C07K 14/47

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA