Indian Journal of Expc:rimcntal I3iology Vol. 4 1, July 2003, pp. 682-693

Zona pellucida glycoproteins based immunocontraceptive vaccines: Strategies for development and their applications

Salish K Gupla *, Sangeela Choudhury, Nee lu Srivastava & Chitra Ravi

Gamete Ant igen Lahoratory, National Institute of Immunology, Aru na Asaf A li Marg, New D . lhi 11 0067, Imlla

The mammalian oocy te is su rrounded by an extra-ce ll ular matrix . the zona pellucida (ZP), composed of th rec major glycoproteins (ZP I , ZP2 and ZP3) . The ZP glycopro teins. by virtuc of their t issuc specifi city and criti ca l ro le during mammalian fcrtili zat ion, havc emcrgcd as potential candidatc antigens for the devclopment of [en imJl1unocontracepti ve vaccine. Molecular characterizat ion of Z P glycoproteins from scvera l species, reveal s a vGriable degree of homology among the dc:duced primary am ino acid sequc:nccs . which provided an opportunity to undertake act i ve Immunization studies in hetero logous animal Illodels. Active imlllunization of var ious animal spec ies with either nati ve ZP glycoproteins or those obtained by recombinant DNA technology k d to the inhibition of fertilit y . Thus ZP glyeoproteins based illlmunocontraccpti ve vaccines offer an allrGcti ve propositi on for controlling w ild l ife population. To mGke it a prac tical propositi on . additi onal research inputs arc requ ired to opt imi 7.e and devise novel strategies for va cine del ivery. Observed ovari an dysfunction. orten assoc iated with immuni zation by Z P glycoproteins is one of the major stumbling bl oeks ror thei r usc in humans. Ongoing studies to del im.:ate appropri ate B cell epitopes or ZP glycopro teins that arc devoid of oophoritogen ic T ·ccll epitopes. which will inhibit fertilit y w ithout concomitant oophoriti s, will be criti cal to delL-rmine their feasibility for human use .

Keywords: Immunocontracept ion. Ovary, Rc:coillbi nant proteins, Sy nthetic peptides, Zona pel luc ida glycoproteins

Il is projected that the globa l human population will be around ten billion by the year 2050. To control thi s increase in population , various contracepti ves such as oral pills, intrauterine devices, co ndoms etc are available in addition lo the terminal methods such as vasec tomy for malcs and tubectomy for females. With an ai 111 to provide a wider choice to end users, various groups ac ross the world , are working on the deve lopmen t of il11munocontraceptive vacc ines . Thi s entail s the generation of humoral and/or cell-med ialed immune responses against antigens that have crucial role(s) in the process of reproducli on. The proof of principle that il is feasible to design such a contraceptive vacc ine has been demonstrated by eX lended phase-II clini cal tri als, of a vacc ine based on the B-subunit of human chorionic gonadot rophin (~_hCG ) I , in women. This vaccine wa compri sed of a heterospecies dimer of ~-hCG annealed wilh the a-subunit of ovine luteini zing hormone (a-OLH) coupled to eilher tetanus toxoid (TT) or diphtheri a toxoid (DT) as allernate carri er prote in s. The vaccine was delivered with alum as an adju vant. In addition,

* For corrc»Jondencc : Telephone: 9 1 II 26 1(i228 1J26 183004 Ext 35 1. Fax: 9 1 11 26 162 125 E-mail: skgupta @nii .res .in

lhe first inj ecti on also contained a sodium phlhal yl deri vati ve of Ii popol ysaccharides (S PLPS) as an additiona l adju va nt.

Immuni zation of women with thi s vacc ine led to the generati on of anti-hCG antibodi es that neutrali zed the bi oactivity of hCG. Immuni zed women having circulating neutrali zing ant ibody titres above 50 ng/ml were protec t.ed against concept ion. Onl y one pregnancy was observed out of 1224 cyc les I. However, an antibody titre above SO ng/ml was observed onl y in 80% of the immuni zed women (n = 148). Currently , efforts are ongoing to impro ve lhe immunogen icily of the i3-hCG based vacc ine by incorporating "promiscuous" T non-B cell epilopes in stead of DTrrT as carrier prolein s. Further, employ ment of adjuvants, permi ss ible for human use, whi ch are more potent than alum will also help tn enhancing the immunogenicity of the vaccine.

The B-hCG based vacci ne is aimed to inlerfere duri ng implantati on - a post-fertili zation event. Another interest ing target for the developmcnl of an immunocontraceptive vaccine may be [() interfere during fertilization per se. To achieve lhi s, vari ous groups have been engaged in the charac teri zati on of sperm antigens and their eva luation as potenti al candidate I mmunogens fo r the deve lopment of

GUPTA el {fl.: ZONA PELLUCIDA GL YCOPROTEINS BASED IMMUNOCONTRACEPTIVE VACCINES 683

immunocontracepti ve vaccines. Alternately, it may be prudent to explore the potenti al of oocyte anti gens for development of immunocontracepti ve vaccines. The mammalian oocy te is covered by the zona pellucida (ZP), a thi ck, translucent. acellul ar glycoprotein matri x that plays an important rol e during fertili zation. Jt serves as the "docking" si te for the species-specific recogniti on and binding of the spermatozoa to the oocy te, induces acrosome- reac tion in the zona-bound spermatozoa, affec ts avo idance of polyspermy and protects the pre-implantati on blastocyst. ZP consists of three biochemica ll y di stinct glycoproteins, which have been class ified as ZP I, ZP2 and ZP] on the basis of their mobility on sodium dodecy l sul phate polyacrylamide gel electrophores is (SDS-PAGE).

Biochemical and stmctural aspects of ZP glycoproteins

In spite 01" having very similar po lypeptide cores, diffe rences in the post-translati onal mod i ficati ons, including glycosy lati on leads to va ri ab ility in the mobility of ZP glycoproteins isolated from va ri oLl s spec ies on SDS-PAG E. In mice, under non-reducing condition s, ZP resolved as ZP I ( 180-200 kDa), ZP2 ( 120- 140 kDa) and ZP3 (83 kDa)2. Likewise, human ZP, under non-reducing conditions resolves into ZP I (90- 110 kDa), ZP2 (64-78 kDa) and ZP3 (57-73 kDa)3 The 2-D electrophores is revealed that ZP glycoprotein s are ac idi c in nature, and reso lve as several isoelectri c spec ies, a result of differenti QI glycosy lati on-l.

Analys is of genomic clones of ZP glycoproteins from vari ous species have revealed similar genomic organi zation of a given gene famil y. The ZP I famil y consists of ! 2 cxons, ZP2 famjl y 18 exons (human and cynomolgus monkey have an extra exon at the C­termi nal end) and ZP3 family 8 exons. The 12 exons in mouse ZP 1 (mZP 1) range from 82 -364 bp and encodes for a 623 amino ac id (aa) polypeptide. The organization of exons in human ZP I (hZP I) is similar to that in mZPI , spans II kb and encodes for a polypeptide of 540 aa5

. The exon size in mouse ZP2 (mZP2) ranges from 45- 1 <)0 bp, with the transcript of 220 I nucleotide (nt) encoding fo r a 7 13 aa polypeptide while the human ZP2 (hZP2) has 19 exons transcribed into 2235 bp mRNA coding for a 745 aa polypeptideo. Mouse ZP3 (mZP3) consists of 8 exons spanning 92-333 bp and encodes for a polypeptide of 424 a}. Human ZP3 (hZP3) also consists of 8 exons and encodes for a 424 aa polypeptide.

In additi on to the genomic clones, the cD A clones for the ZP glycoproteins from different species have been identifiedR

• The cDNA sequences have been characteri zed for mZPl, mZP2 , mZP3, hamster ZP3 , rabbit-55 kDa protein (homologue of mZP I), rabbit 75 kDa protein (homologue of mZP2), rabbit 45 kDa protein (homologue of mZP3), porcine ZP3a (homologue of mZPl), porcine ZP I (homologue of mZP2 ) and porcine ZP3~ (homologue of mZP3). In add ition, the marmoset ZP3, hZP1 , hZP2 , hZP3, dog and cat ZP I, ZP2, ZP3, bonnet monkey ZP I (bmZP I), ZP2 (bmZP2), and ZP3 (bmZP3) cD 'A clones have also been characterized. The deduced pri mary aa sequences of ZP I, ZP2 ~lIld ZP3 fro m va ri ous species share a certain degree of sequence identity with the respecti ve human counterparts as shown in Tab le I. The compariso n of the nt and deduced aa sequences of ZP glycoproteins fro m various species also revealed thar they possess certain common features:

i) Short 5' and 3' untrans lated regions. ii ) N-terminal signal peptide that directs the

proteins into the secretory pathway and gets cleaved-off from the mature protein .

iii ) Potential N- and O-linked glycosy lation sites. iv) C-terminal hyd rophobic transmembrane-like

domain that may playa ro le in the intracel lul ar trafficking of the proteins.

v) A potential tetra bas ic furin cleavage site, upstream of the transmembrane- like domain .

vi) A ZP-signature domain .

Even though the ZP glycoproteins have been class ified into three famili es, ZP I and ZP2 from different spec ies are more similar to each other as compared to ZP3. Comparison of the deduced aa sequence of bmZP 1 Vs bmZP2 revea led a region from aa residues 369 to 4 18 of bmZP1 , wh ich has 52% identity with bmZP2. Within 348 aa of mZP I (aa residues 268-623) that align with mZP2 (aa res idues 363-713 ), 32 % of the aa are identical'). This region is encoded by 8 exons in both mZP J and mZP2. This observa ti on suggests that these 8 exons may have ori gin ated from the same ancestral gene that has been duplicated and re-utili zed by exon shuffling. The conserved positi ons of 10 cysteine residues in mZPI and mZP2 suggest that the structural aspects with respect to thi s domain may be similar in these two proteins. Further, the ZP domain, comprising of 253-260 aa with 8 conserved cysteines res idues and additional conservation of hydrophobicity, polarity

684 INDIAN J EX P BIOL. JUL Y 20Q3

and turn-forming tendency have been recognized in all the members o f the ZP family except for cat ZP3 and mZP I . However, non-conformity of thi s domain in these two proteins is restricted to only one aa (aa res idue 459 in mZP I and aa residue 204 in cat ZP3). In addition LO members of the ZP fami ly, the ZP domai n is also present in extracell ular matrix proteins such as the 0.- and ~-tectorin s of the inner ear, renal uromodulin (Tamm-Horsfall protein) and cuticulin . Other members o f thi s family include membrane proteins, such as transforming growth fac tor-~­

receptor type 3 (TGFr3R-lIl ) and estrogen regulated gene type I (ERG I ). Recentl y, it has been demonstrated that the ZP domain is responsible for pol ymeri za tion of these protei ns into f i lamen ts and assembly of ZP glycoproteins into the ZP matri ces 10.

Evidence that ZP glycoprotein genes in mammals have di versi fied from other species such as fi sh during evo lution has been accumul ating. The primary structure of medaka (O l),zins lalipes) egg vitelline envelope (VE) glycoprotein termed L-SF4 1 was found to be significantly similar to the ZP3 polypeptide ll

. Moreover, 10 of the invariant cys teine res idues ha ve been conserved in thi s species suggesting an overall conservat ion of the three­Jill1ensional st ructure of the ZP family through evolution. In Ilounders (P.I'e lldoplcllroll ecles all/erical/lls) the gene encod ing another VE protein ca lled wflcl11a ," shares considerab le sequence homology

with the ZP2 protein h nily'2. A nalys is of the sequence has revealed thaI exons 2-7 of thi s gene share homology with the rabbi t ZP I and mZP I . Furthermore, in the anuran (Xenop lls lac vis) the three cDNA cloned corresponding to the VE glycoproteins des ignated as gp3 7, gp41 and gp69 are homologues of the ZP glycoprote ins ZP I , ZP3 and ZP2 respectively". Compari son of the deduced aa sequence of gp37 with the ZP I sequence from human, porcine and mouse ZP I revealed a sequence identi ty of 4 1.6%, 4 1.7% and 36.8% respectively . The gp69 revealed an aa sequence identity of 28 .5%,27.6% and 26.9% with mouse, pig and human ZP2. The gp41 showed an aa sequence identity of 40.9%,40.0% and 40.8% with human, pig anci mouse ZP3 respect ively. Further analysis showed that they are not just related wi th respect to aa sequence homology observed wi th ZP glycoprote ins fro m mammals but also had conserved positions of cys teine res idues. Twenty out of 20 cys teine residues are conserved between gp37 and ZP I from pig and human, nineteen in case of mZPI. All 12 cyste ine residues were found to be conserved between gp4 1 and ZP3 from mice, pi gs and humans.

Functional aspects of ZJ> glycoproteins Stud ies from vari ous experimental an imal models

have led to the elucidation of various functions assoc iated w ith the ZP glycoproteins. In mice, hamster and humans, ZP3 acts as a prim;}ry receptor

Table 1- Characteristics or ZP glycoproteins

ZP Glycoprotei n

ZP 1 fami ly

M ouse ZP I (mZP I ) Pig ZPI Bonnet monkey ZP I (bmZP I ) Human Z PI (hZP I )

Z1'2 family

Mousc ZP2 (IllZP2) Pig ZP2 BOllnct monkey ZP2 (bmZP2) Human ZP2 (hZP2)

ZP3 family

M ousc Z P3 (mZP3) Pig ZP.1 Bonnet monkey ZP3 (bIllZP3) HUJll .1Il ZP3 (hZP3)

Genolllc size/ o. or

cxons

6.5 kb/12 0

NO IIkbJl2

12.1 kb! 18 25 kb/ I H

NO 13 kbJllJ

8.6 kb/8 0

NO 18.3 kb/8

Apparent molecular wt

(kOa)

t 80-200 -55 NO

90- 1 I ()

120-140 80-90 NO

64-78

83 -55 NO

57-73

Pcrce nt icientity or ci..:ciuccci aa sequcllcc with respecti vc human

homologuc

39 68 92

100

60 64 94.2

100

67 74 93.9

100

GUPTA el (/1.: ZONA PELLUCIDA GL YCOPROTEI S BASED IMM UNOCONTRACEPTIVE VACC INES 685

for sperm binding to the oocyte and induces the acrosome reacti on in the spermatozoa bound to Zp 14

-

15. Following the acrosome reaction, in mice, ZP2 acts as a secondary sperm receptor and helps in the binding of the acrosome- reacted spermatozoa to the oocyte l6

, Subsequent to fertili zati on, ZP2 undergoes limited proteolytic cleavage as a result of cortical reaction , resulting in the formation o f small molecular weight fragments that remain non-covalently bound l 7

In a mouse model , these changes accompanied by a loss of sperm receptor activity of ZP3 have been suggested to play an important role in preventing polyspermy , I n the mouse model. ZP J has been implicated to ha ve a role in maintaining the structural integrit y of the zona matrix by cross-linking ZP2-ZP3 heterodi mer 1 x.

[n the porcine model , high molecular weight hetero-complexes of ZP3 and ZP I binds w ith a very high affinity to boar sperm-assoc iated zona receptors whereas indi vidual glycoproteins failed to do so thereby suggesting that both the glycoproteins are invol ved in sperm binding l 9

, In the rabbit model , rec55 (a homologue of mZP I , expressed in baculovirus) has been shown to bind to spermatozoa ina dose-dependent manner~o , Both rec45 (a homologue of mZP3) and rec55 components o f rabbi t ZP have been shown to bind to recombinant Sp 17 (sperm autoantigen), suggesting the in vo lvement of a molecular mechanism similar to that found in the porcine system"I , Further, recombi nant bmZP I (1'­bmZP I ) expressed in E. coli binds to the principal segment of the acrosomal cap of the capacitated spermatozoa and the equatorial segment, post­acrosomal domain and mid piece of the acrosome­reacted spermatozoa, indicating a role for ZPl in

b' d' ~1 sperm In IIlg--,

Zona pellucida of mammalian oocyte as the target site for immunocontraception

The ZP glycopro teins, due to their crit ica l role in the fertili za ti on process, ti ssue specificity and accessibility to systemic antibod ies have emerged as potential candidates for regulation of fertility through immunologica l intervention.

Active immunization with native ZP g[ycoproteins

(a) Total solubilized ZP

An early study demonstrated that immunization of mice wi th heat solubili zed hamster zonae generated antibodies against ZP and induced i nfertili ty23

Subsequently, porcine ZP became the antigen of choice due to its immunological cross-reacti vity wi th human ZP and easy access ibility from abattoir. Immuni zation of rabbits with the heat so lubili zed porcine ZP generated immunologica l cross- reactive antibodies leading to in ferti lit/ 4

, The in fertility was irreversible and histology of the ovari es revea led destruction of oocy tes in all the growing follicles and severe deplet ion of the pool of resting follicles, suggesting that the infertility was due to ovarian dystroph/5

, Immunizati on of female dogs wi th porcine ZP resulted in prolonged pro-estrus or estrus cyc les26

, Histolog ica l examination of ovaries from animals that generated high titres or antibod ies against the immunogen revea led depletion of the oocytes, Immunization of non-human primates with crude solubili zed porcine ZP al so resulted in in fertility that was irreversible and was accompanied by folli cular atres ia and abnormal hormonal profi les27

, The observed ovarian dystrophy follow ing immunizat ion with porcine ZP was attributed to the impuriti es presen t in the immunogens used.

(b) Pur!fied ZP givcoproleills To avoid the deleterious effects manifes ted due to

the contaminating ovarian antigens, further studies were done using purified porcine ZP glycoproteins as the immunogens. Immuni za tion of sq uirrel monkeys (Sc illliri scillreus) with purified porcine ZP3 ra

mixture of porcine ZP3a (-ZPI ) and ZP3~ (-ZP3) ! induced disturbances in the secreti on of ovarian steroid hormones initiall y and a histologica l examination of ovaries revealed interference in fo lli culogene is"x, The immunized animals remai ned infertil e during the study. In sp ite of high anti-porcine ZP3 antibody titres, the animals showed recovery of ovarian function after 10-15 months post­immunization, [n another study , female bonnet monkeys (Macaca radiara) immunized with purified porcine ZP3, employing adjuvants permi ss ible ro J' human use al so led to the inhibition of fertilit /'i, Immunized animals continued to have ovulatory cyc les, Laproscopic examination revealed norma l ovaries with developing follicles, Following the decline in antibody titres, 50% of the animals became pregnant. Ovari an histology of the animals that failed to regain fertility did not reveal any signs of inflammation or lymphocytic proliferation. There was also no observed increase in the number of atretic or degenerating follicles, These studi es suggest that some or the adverse effects on ovarian funct ions can

686 INDI AN J EXP B IOl. JUl Y 2003

be minimized subsequent to immuni za tion with the purified ZP glycoproteins.

In addition to the contaminating proteins in ZP glycoproteins, the nature of the adjuvants employed in ac ti ve immunization studi es w ill also influence the efficacy as well as the safety of the procedure. Use of complete Freund 's adjuvant (CFA) leads to ve ry high anti body titres but is also accompanied by granulomatous les ions at the site of injecti on and ovari an atrophy'°. Use of adju vants such as alumin ium hydrox ide gel (a lum), sod ium phthalyl derivative of lipopolysaccharides (SPLPS) and syntheti c mummyl dipeptide derivative (M DP) also generates an immune response to ZP an ti gens, capab le of blocking ferti li ty with less adverse side effects29

.l2

. Hence, there is a need to develop more potent and safer adjuvants.

Active immunization with recombinant ZP p.-oteins/ glycoproteins

Contaminati on of the ZP based antigens by other ovary associated proteins , one of the causes for ovarian pathology, has been circumvented by the use of recombi nant ZP protei ns/g lycopro tei ns. Immunization of marmosets (Callirhrix jncch lls) with the recombinant hZP3 (r-hZP3) expressed in CHO mammalian cel ls generated antibodies that led to long- term infertili ty'n . However, thi infertility was associated wi th ovari an pathology characte ri zed by deplet ion of primordial follicles . Immunization of fema le rabbits w ith recombi nant rabbit Z P I , expressed in eukaryotic ce ll s, caused infertility in 70% of the immunized animals34

. In the same study , female rabbits were also immunized with recombinant

myxoma virus harboring the ZP I gene followed by boosting with recombinan t ZPI, which resu lted in 80% infertility. The infertility was associated with follicu lar degeneration and depletion of primordi al 1'011 icles. I n another study, a comparati ve ac ti ve immunization of female cynomolgus monkey (Macaca fascicII/aris) and baboons (Papiu cY/l ocephalus) wi th purified r-hZP I , r-hZP2 and 1'­

hZP3 expressed in CHO cells revealed that those animals whi ch were imm Ul ni zed w ith r·hZP2 or 1'­

hZ P3 became pregnant b fore any of the animals illlmunized with r-hZP 1,5. The animals immunized with r-hZP I remai ned infertile for 9-35 months. During the ti me of high antibody titres, some animals experienced di sruption of the menstrual cyc le, which eventuall y returned to normal. These studies demonstrate that ZP I is a better candidate for fertili ty regulation as compared to ZP2 or ZP3. Our group has also shown that immuni za ti on of r male baboons (Papio anubis) with recombinant bmZ P I (r-bmZP I ) conj ugated to DT (r-bmZP I-DT) generated high antibody titres aga inst r_bmZPI 36

. The imlllunized animals ex hibited normal ovulatory cyc les. In the presence of high ant ibody titl-es, they remained infertile following mating with males of proven fert ili ty. However, subsequent to the decline in antibody titres the animals conceived. In addition, using a homologous animal model , female bonnet monkeys were also immuni zed with 1'- mZP I -DT and r-bIllZP2-DT conjugates37

. Immun ization led to th e generati on of antibodies against both the respective recombinant proteins as well as DT. The immuni zed females remained infertil e when mated with males of proven fertili ty in the presence of high antibody titres,

Table 2 - t:ITecl of immuni zalion with recoillbinani zona pellucida proleins on fcrtililY in fCI ale bonnel mon : cys

t:xp.:rimcn tal group

Control

Immunized with r-bmZPI -OT

Immunized wi lh r-bmZP2 -I)T

No. of ani mals uscd

4

5

4

"Adaptcd from Govind el 01. J7

StalUs of fcrt ility" Ovarian histology

Two females conceived ill firsl ovulatory cycle and remaining Normal folli cular deve lopment 2 in the second ovulatory cyc le.

CUlllulat ive infertility observed for 45 ovulalory cycles. Onc Follicu logcncsis dist urbed. animal concei ved during immunizalion when ant ibody litres Atrclie follicles showed werc low :lIld Olnolher conceivcd during reversa l slUdies. Threc dcgenerated oocyles w ith animals fa iled 10 conce ive evcn aner the decline of antibody disorganizcd ZP lilres to background

CUlllulat ive infertilil y observed for 32 ovulalory cyc les. One Same as wil li r-bmZPI -OT animal conceived during immunization when anlibody titres immunized grou, werc low and anolhcr conccivcd during rcvers;)1 slUdies. Two animals failed to conceive even aflcr the dcc l inc of ilillibody litrcs to background.

--(

GUPTA el al .. ZONA PELLUCIDA GL YCOPROTEI S BASED IMM U OCO TRACEPTIVE VACCINES 687

and fail ed to conceive even after the dec line of the anti-r-bmZP 1 and anti -r-bmZP2 antibody titres (Table 2). Ovarian histopathology of the immunized animals revealed the presence of atretic foil icles and degenerated oocytes, which may have been the principle cau se for the block in fertilit y.

Various rccombi nant fragments of mZP2 have al so been evaluated, in a homologous animal model, for their efficacy to block fertility. These studies revealed that immuni zation with the mZP2 fragment corresponding to aa res idues 35-637 led to a reducti on in fertility'x. The ovaries of the in fertile mice were histologicall y normal and produced a normal number of eggs suggesting th at the mouse ZP2 antibodies probabl y induce infertility by interfering with sperm­oocy te interac tions. Thi s study al so demonstrated the effi cacy of a chimeric recombinant protein comprising of mZP2 and sperm anti gen, Sp 17, to inhibit fertilit y.

Alternate modes of immunization A long wi th conventional illlmuni zati on, varIOUS

other modes and routes of del i very of Z P glycoproteins have been tri ed to generate more effecti ve immunocontracepti ve accines.

(aj Live recolllbinant vectors Oral immuni zati on of female mice with

recombinant Salmonella express ing mZP3 induced significant leve ls of IgG type of antibod ies against ZP in the seruny19. In additi on, significant levels of IgA antibodies aga inst ZP were al so observed in the vaginal secreti on. Infertility was observed in 3 out of 6 immun ized mice. In another study, ec tomelia virus - a natural pathogen for mice th at causes mouse pox­has been used as a li ve vec tor. A recombinant ectomelia virus expressing mZP3 was made-lu. Immunization of a group of mice w ith recombinant viruses led to a decrease in fertility as well as litter size compared to the group immuni zed w ith viru ses containing only the pl asmid. Immunizati on led to disruption of folli cul ar development but did not cause any ovarian oophoriti s.

(b) DNA vaccilles

not her alternati ve approach to conventional immuni zati on with protein is to use plasmid DN A encoding the said protein . The feas ibility of thi s concept was demonstrated for the first time by generating protec ti ve imlllunity aga inst influenza subsequent to immuniza ti on w ith a DNA vaccine-l i .

Since then. numerous studies have shown th at immuni zation w ith plasmid DNA encoding a variety of antigens corresponding to a wide spectrum of bacteria , viruses and protozoa leads to generati on of protecti ve humoral and/or cell-mediated immune

4' . I . responses -. A s compared to conventiona vaCCines, DNA vacc ines have several ad vantages. They are easier to produce and hence cheaper. Pl asmid D A is amenable to easy manipul ati on and hence D A vaccines can be modified with ease. They are quite stable at room temperature and may not require a cold chain. The injected DNA mostl y remains in the fo rm of an epi some, thu s avoiding the fear of integrati on into the host genome. Although the immune response to D A vacc ines is weak as compared to that induced by traditi onal vacc ines, it is excepti onall y long-I . 41 astlllg . .

Our group has recentl y eva luated the potential of DNA vaccines with respect to immunocontracepti ve vacc ines . In thi s directi on. the cDNA corresponding to bmZP I , exc luding the N-termin al signal sequence and C- termin al transmembrane-like domain , was cloned in the mammal ian ex press ion vector V R 1020, downstream o f a ti ssue pl asminogen acti vator signal

d I . ~

sequence un er a cy tomega OV irus promoter . Immuniza tion o f male mice with the above pl asmid DNA vacc ine in sa line led to generati on of the antibodies aga inst £. coli expressed r-b lllZ P I . A dmini strati on of r-bmZPI in mice immunized with a DNA vacc ine encoding bmZP I , led to a further increase in the antibody titres. The antibodies th us generated recognized the nati ve ZP both from bonnet monkeys as well as humans. Interes tingly, the immune sera obtained from mice immuni zed w ith the D A vaccine showed si gni f icant in \'itro inhi biti on o f the binding of permatozoa to ZP in the hemi zona assay as compared to the immune sera obtained fro l11 mice immunized with the vec tor-l-l. Simil arl y, a D iA vaccine encoding dZP3 also eli cited an anti body response th at rccogni zcd the nati ve dog ZP345 A dominant IgG I iso type response was obse rved in animals immunized with the DNA vaccine using a gene gun as compared to a mi xed JgG 1-lgG2a iso type response when deli vered in sa line. Interes tin gly, female mice immuni zed with a DN A vacc ine using a gene gun al so generated antibodies that recognized 1'­

dZP3. as we ll as nati ve dog and bonnet monkey Z P. Keeping thi s in view, it may be or interest to

undertake ac ti ve i I11l11uni za ti on studies o f female dogs with the D A vacc ine encod ing dZ P3 to see its effec t on fe rtility.

6R8 INDI A N J EX P BIOL. JULY 2003

Relevance of ZP glycopl'otcins in controlling wild life population

Understanding the molecular bas is of fertili zation and relevance of ZP glycoproteins in thi s process will have direc t bea ring on dev ising strateg ies to conserve several spec ies th at arc on the verge of ex tincti on. A t the same time, an unchecked increase in the popul ation of some spec ies such as deer and elephants in fo res ts and stree t dogs has been observed . New legislations in many countri es, which fo rbid hunting and kil l ing of these ani mals, have further aggravated the si tu ati on. Hence, there is a need to develop new strateg ies to control an increase in wildlife popU lati on in a more humane way . A n immunocontracepti ve vacc ine for humans should be effecti ve in 100% of the rec ipients, should be potenti all y reversible, and free of side effects. For w ild li fe control, a vacc ine lead ing to an irreversible block in fertili ty may also be acceptable, and in some situati ons des irable, as it w ill be akin to an immunologica l castrati on. Further, such a vacc ine meant fo r female animals and hav ing an efficacy of about 50-70% w ill also be effecti ve in regulating wi ld li fe populati on.

In feral horse and donkey popUlati ons, a single annual booster o f solubi l ized porcine Z P was enough to prevent conception, w ithout affecting the complex social behavi or of the animals46

. Short-term treatment for up to 4 consecuti ve years did not result in any detec table debilitating side-ef fec ts and the contracepti ves effect was reversi ble whi Ie long-term treatment (5-7 years) was associated with few ovulation failures and depressed urinary es trogen l eve l s~ 6.

Out of the 74 spec ies of capti ve zoo animals immun ized with solubili zed porcine ZP, 27 species have shown distinct success in block of fertilitl 7

.

Immunizati on of white-tailed deer (Odocoileus vi rginiollus) with either nati ve porcine so lubili zed ZP or recombinant rabbit ZP glycoproteins led to a

signifi cant decrease in the fawning rates48 In a f ield trial carried out for control of elephant populati on. it was demonstrated th at out of 19 female elephants vacc inated w ith porcine ZP, 10 did not conceive. HO'vveve r, in the contro l group, 16 out of 18 animals became preg nant~ '). The effec ts of the vaccine were reversible, and had no deleteri ous ef fects on the ovary and i ts cyc licity. A single shot of porcine so lubili zed Z P using a l iposome deli v ry system decreased the birth o f pups in the gray se' ls (Ha/ihoert/ .I' g rv/Jil s) by 90 (7 . . . 1'0

/ 0 over a l i ve year pen oe ' . The potential of ZP glycoproteins as candidate

anti gens ha ve also been studied to inhi bit fertility in clogs. For thi s purpose, the cDN A encod ing dog ZP2 (cl ZP2) and dog ZP3 (dZ P3), excluding the N-

Suc I PSI I

1''1'7 , ~ lIisy dZP3 J-

71) hp

Id)a .\1

220 -97 -()() \.

-+5 -30 -2() --

Fig. I - Ex pression of recombinant dog Z P3 in E. coli . T he upper pancl is a schemati c represenLUtion of the construct of dZP3 in pQE-30 ex pression vector. The SG 130091 pR EP4] E. coli ce ll s we re used for the express ion of the recombinant prote in as polyhistidi ne fusion pro tein . The expressed r-dZP3 was puri fied on a nickel-nitril otri aceti c acid agarose affin ity column. The lower panel represents the SDS-PAGE profile of the purifi ed r-dZP3 stained wi th Coomassie bl ue. PT7, promoter of phage T7; Lane M , molecul ar weight markers; Larlc I , puri fied r-dZP3.

Tab le 3 - Ef fec t of immunizati on with dog recombinant zona pellueida proteins on fertili ty in female dogs

Experimental group

Immuni led with DT

Imillun ized with r-c1ZP2-DT

I lllllluni zed w ith r-dZPJ-DT

No. of animats used

4

4

4

"Adapted from Srivasta va ('/ al. 5 1

Status of fertility" Ovarian histoloby

Out of 4 immunizcd animals, three became Normal folliculogenes is with pregnant on mating during estrus healthy oocytes

A ll the four immun ized female dogs conceived Normal foll iculogenes is on mating during estrus

Out of fou r immunized ani mats, three failed to A tl etic fo ll icles, degenerati ve conce ive when mated during estrus clu:nges in ZP

GU PTA el al. : ZONA PELLUCIDA GLYCOPROTEINS BASED IMM U 'OCONTRACEPT IVE VACC INES 689

terminal signal sequence and the C-tenninal transmembrane- like domain , were cloned in pQE-30 expression veetor under T7 promoter. The respecti ve protein was expressed as a polyhistidine fusion protein ill E. coli . The vector map and SDS-PAGE profil e of the purified r-dZP3 is shown in Figure I. The puri fi ed r-dZP3 and r-dZP2 were conj ugated to DT. The female dogs were immuni zed w ith r-dZP2-DT and r-dZP3-DT conjugates. Immuni 7.ati on led to the generati on of antibody response against the respecti ve ZP proteins as well as the carri er) ' . All the female dogs imllluni zed w ith r-dZ P2-DT conceived subsequent to mating (Table 3). Three out of four animals immunized w ith DT also conceived. However, 3 out of 4 animals immunized w ith r-dZP3-DT remained infertil e (Table 3). Ovarian histopathology of the animals immuni zed with 1'­

dZP3-DT revealed folli cul ar atres ia. These preliminary results indica te that the dog populati on can be controlled by immunizati on with r-dZP3, prov ided adequate antibody titres are achieved".

Prospecl" of developing a ZP glycoprotein based immuno-contraceptive vaccine for human lise

As ment ioned ea rl ier, it is i mperati ve th at ZP glycoproteins based contracepti ve vacc ines fo r humans should not have any side effects. The presence of oophoritogenic T cell epitope(s) in the ZP glycoproteins based immunogens has al so been impli cated as one of the fac tors responsible for the observed ch anges in sex-steroids hormonal pro file, disturbance in cyc li city and disrupted ovari an folli cle deve lopmelll, subsequent to immunization with ZP glycoproteins52 Hence, several groups are engaged in f inding the minimum effecti ve peptide o f the three ZP glycoproteins that could produce fertili zation­blocking antibodies w ithout undes irable side effects. The first ev idence to show its feas ibility was demonstrated by synthes izing a chimeri c peptide

compnslng of a " promiscuous" T-ce ll epitope of bovine RN ase (NCA YKTTQANK), co-linearl y synthesized with the minimal B-cell epirope of mZP3 corresponding to aa residues 335-342, with phenylalanine aa replaced by alanine (QAQIH GPR ) (T able 4). The peptide elicited antibodies in inbred mice of eight different haplotypes without acti vati on of oophoritogenic T cell s:u When mated, the l incr size of the immunized group was considerab ly lower as compared to that o f the contro l group. M oreover. the reducti on in the litter size correlated w ith the antibody titres. For the first time, these ex periments showed a meth od of overcoming the MHC dri ven non-responsiveness to a se l f-a nti gen and also avoiding sel f-pathogenic T cell responses. In analogy. cy nomolgus monkeys were immunized w ith a chimeric peptide compnslllg o f PlaslllodiulII f ({lcipanllll T ce ll helper epitope (aa res idues 326-337) and hZ P3 (aa res idues 334-342; RRQPH YMS). Antibodies thus eli cited, signifi cantl y inhibited the ill pilro binding of human sperm to antibody treated zona encased oocytes as compared to pre- immune sera54

. Results from our group also showed th at immuni zati on of female mice w ith a chimeri c pepti de corresponding to bmZP3 (aa res idues 334-343 ) synthes ized co- linearl y w ith a " promiscuous" T ce ll epitope o f circull1sporozoite protein (CSP, aa residues 378-398) of Plasmodiul1I Ia lciporulIl elicited antibodies that inhibited, 111 vilro, sperm-zona b· d' ss 111 Ing·· .

A s described above, immuni zat ion or female marmosets with r-hZ P3'.l and of female dogs wi rh 1'­

dZP3-DT51 led to in fertilit y concomitant w ith ovari an dysfuncti on. Immunization of female marmosets with recombinant marmoset Z P3 also leads to a block in fertility assoc iated with disturbances in ovarian folli cular development56 In contrast, no disrupti on or ovari an function was observed fo llow ing immunization of marmosets with sy ntheti c peptidcs

T ahle 4 - Zon:1 pel111cida glycopro lein -:I based sy nthelic pept ide imll1unogens

Syntheli c peplidc

mZP:l1.1~) .J~2 ""I Phe!."('1 rep laced by A la

M armoset ZP3IJII I .~211.1.11

An imal species

Micc

Marlllose t

130nnct nloli Key

OUIClI lliC or aClive immunizat ion

Block in rertilit y \Vi lhou l cO llcol1lil an t oophorit is

orma l ova ri an runct illns. A ntibodies aga illst pcpt ide sho\Vcd ill I'ilro

cllnt racepl ive c lTicacy.

BlOCK ill rc rti lit y. No disruplioll or cyc l icity. ol"l nal ro ll icul llgc lles is.

Referc llcc number

S3

56

57

690 INDIAN J EX P BIOL, JULY 2003

corresponding to human or marmoset ZP3 JJ·56

.

A ntibod ies against the marmoset ZP3 peptide. correspond ing to aa res idues 30 I-320, recognize both marmoset and human nati ve ZP and also reduced ill vitro human sperm-zona interacti on by 60% (Table 4). However. the ill vivo studies did not show consistent reduction in fertilit / 6

. Immuni za tion of f'cmaie bonnet monkeys with a syntheti c peptide corresponding to bmZ P3 (aa res idues 324-347) conjugated to DT led to high antibody titres against the peptide5

? Immunized animals continued to have ovu latory cycles (except summer amenorrhoea) and fa iled tn conceive when mated w ith males of pro ven fertilit y. No ovari an patho logy was observed in the immu ni zed animals (T ab le 4).

To enhance the immunocontracepti ve efficacy of anti -peptide antibodies, it Illay be prudent to des ign synthet ic ch imeri c i mmunogens encompass i ng multiple epitopes of a gi ven zona glycoprotein or mu ltiple zona glycoproteins. The above prem ise is deri ved from the observa ti ons th at a cock tail of anti­peptide antibod ies aga inst porcine ZP3~ (-ZP3) was effecti ve in inhibiting porcine sperm-oocy te attachment ill I'it ro whereas indi vidual anti-peptide an ti bodies fai led to do S051\ . On the other hand, female bonnet monkeys immuni zed with a physical cocktail of bmZP3 peptide, individually conjugated to DT, generared antibodies that signifi cantly inhibited human sperm-oocy te binding in l ' itro5~ . This was probab ly mediated by a cooperati ve effec t among the antibod ies pertaining to di ffe rent domains. Taking a cue from these observations, we have recentl y cloned and expressed a chimeri c recombinant protein encompass ing 8 ce ll epitopes of bmZP I (aa res idues 132- 147), bmZP2 (aa residues 86-11 3) and bmZP3 (aa res idues 324-347) in E. co/i60

. Female rabbits immunized with the chimeric recombinant protein conj ugated to DT generated antibodies that not only reacted w ith the respective recombinant proteins but also with the bonnet monkey and human nati ve ZP. The immune sera also inhibited the ill vit ro binding of human spermatozoa to the human zona in a hemizona assay. I n another study, female marmosets were immunized with a triple peptide vacc ine encompas­sing marmoset ZP3 epitopes (aa res idues 85-100, 24 1-260 and 30 1-320) including a "promiscuous" T -helper cell epitope

o, . Immuni zat ion of animals eli cited high antibody titres against regions corresponding to aa res idues 24 1-260 and 30 1-320. The third peptide from aa res idues 85-100 was poorl y immunogenic. Immune sera showed in vitro contraceptive efficacy. No loss of

ovari an functi on was observed in tht' immunized an imals.

Synthet ic peptides corresponding to ZP2 have also been targeted for the purposJ of immunocontraeeption. It was shown that rabbit polyc lonal antibodies aga inst hZP2 peptide corresponding to aa res idues 54 1-555 inhibited ill I' il ro human sperm binding to the human ZP by about 50%61. Immllnization of mice w ith a chimeric peptide comprising of mZP2 epitope (an residues 12 1- 140) and bOVIne RNase " promiscuous" T-cell epitope reacteu w ith nati ve mouse ZP and did not lead to oophoriti sr" . Rabbits immunized w ith an 18- lllcr synthetic peptide e rresponding to hZ P2 (aa rcs idues 50-(7) conjugated to DT not only recognized the nati ve human ZP but also effectively inhibited the ill vitro bind ing o f human sperm to human ZP6

.1. 6'\ .

To map the 8-cell ep itope on ZP I , we have employed two approache,·. I n the first approach. monoclonal an tibod ies were generated against r­bmZP I (,('. Those monoc lonal antibodie , which \vere capable of recognizing the nati ve human ZP and had a signifi cant inhi bitory effec t on the in vitro binding of human sperm to human zona, were selec ted. Epitope mapping studi es, using these monoc lonal antibodies, revea led a cOlllmon epitope, DAPDTDWCDSIP (aa residues 136- 147). I n the second approach, 4 peptides corresponding to bmZP I were synthesized on the basis of computational pred ictions of hydrophili city, surface probability and ar.ti geniciti 5

. Female mice were immuni zed with the synthetic peptides corresponding to bmZP I , conjugated indi vidually with DT. Characterization of the immune serum samples revea led that anti bodies against the bmZP I peptide correspond ing to aa res idues 251 -273 inhibited the binding of human spermatozoa to ZP in a hemi zona assai5

. These studies will help in lhe identification and characterization of syntheti c pepti de immunogens that may be devoid of oophoritogenic T­cell epitopes and ultimately lead to the design of immunogens encompass ing 8-cell ep itopes for better efficacy in inhibiting fertili ty.

Concluding ,comments The findings that immunization with either native

or recombinant ZP protf:ins/glycoproteins inhibits fertility, in several spec ies, may find applicati on in controlling wild li fe population. Novel strategies, such as DNA vacc ine or li ve vectors encoding ZP glycoproteins need further evaluati o . To make the use of such vaccines a prac tica l proposition, alternate routes of vaccine deli very , such as orally or by using

GUPTA 1'1 al. : ZONA PELLUC IDA GL YCO PROTEINS BASED IMM UNOCONTR ACEPT IVE VACC INES 69 1

darts have to be evolved. It may also be des irable to reduce the number of vacc ine inoculations. In order to develop a safer and effecti ve ZP glycoprotein based immunocontracepli ve vaccine for human use, it is imperative to have a comprehensive understanding of the mechanisms involved in gamete interac ti on. Thus , efforts to delineate new immunogens. compri sing o f B-cell epitopes of ZP glycoproteins and devo id of oophoritogenic T ce ll ep itopes must continue.

Acknowledgement This work has been supported by the grant s to SKG

from the Department o f Biotechnology ;.lI1d ICMR, New Delhi. The financial support under l ndo- S collaborati ve programmes on "Vacci ne Action Programme (V AP)" and "Contraception and Reproducti ve Health Research (CRHR)" IS also acknowledged.

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