title: Canine zona pellucida glycoprotein-3: up-scaled production, immunization strategy and its outcome on fertility.
abstract: Zona pellucida (ZP) glycoproteins based contraceptive vaccines have been proposed for the management of wildlife population. In the present study, a fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by a dilysine linker and an ectodomain of dog ZP3 (ZP3; aa residues 23-348) without any affinity tag (TT-KK-ZP3) has been expressed in Escherichia coli. The recombinant protein was successfully produced in fed-batch fermentor and purified. The average yield of purified refolded protein was 12.20 ± 0.61 mg/2g wet cell pellet. Female FvB/J mice immunized with the varying doses of recombinant TT-KK-ZP3 supplemented with alum/PetGel A as adjuvants following a three injection schedule, showed dose dependent increase in serum IgG titer. Antibodies against TT-KK-ZP3 recognized native mouse/dog ZP and significantly inhibited mouse in-vitro fertilization (p=0.012). Immunized mice showed significant reduction in fertility (p<0.05). Higher antibody titers were associated with a decrease in the number of pups born to the immunized female mice. To reduce the number of injections, two injection schedule using various dose combinations of TT-KK-ZP3 supplemented with alum revealed lower immunogenicity and contraceptive efficacy as compared to the three injection schedule. To overcome this, CpG motif was included in addition to alum and both intraperitoneal and intranasal route of immunization following the two injection schedule was investigated. Inclusion of CpG significantly enhanced the antibody titer and improved contraceptive efficacy. In the mice immunized following intraperitoneal route, serum/vaginal IgG and in the mice immunized through intranasal route, vaginal IgA seemed to be important for curtailment in fertility. To conclude, the recombinant protein described herein may be a good candidate for developing contraceptive vaccine for the wildlife population management, in particular street dogs.
authors.affiliation: Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
authors.forename: Abhinav
authors.initials: A
authors.lastname: Shrestha
authors.affiliation: Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
authors.forename: Sudeepa
authors.initials: S
authors.lastname: Srichandan
authors.affiliation: Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
authors.forename: Vidisha
authors.initials: V
authors.lastname: Minhas
authors.affiliation: Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
authors.forename: Amulya Kumar
authors.initials: AK
authors.lastname: Panda
authors.affiliation: Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India. Electronic address: skgupta@nii.ac.in.
authors.forename: Satish Kumar
authors.initials: SK
authors.lastname: Gupta
mesh_terms: D000276:Adjuvants, Immunologic / Q000008:administration & dosage; D000818:Animals; D000906:Antibodies / Q000097:blood; D003001:Cloning, Molecular; D004285:Dogs; D004527:Egg Proteins / Q000008:administration & dosage / Q000276:immunology* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005260:Female; D005298:Fertility; D015870:Gene Expression; D007114:Immunization / Q000379:methods*; D007074:Immunoglobulin G / Q000097:blood; D008098:Litter Size; D008562:Membrane Glycoproteins / Q000008:administration & dosage / Q000276:immunology* / Q000302:isolation & purification*; D051379:Mice; D011956:Receptors, Cell Surface / Q000008:administration & dosage / Q000276:immunology* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000276:immunology / Q000302:isolation & purification; D016896:Treatment Outcome; D022642:Vaccines, Contraceptive / Q000008:administration & dosage / Q000276:immunology* / Q000302:isolation & purification; D000072216:Zona Pellucida Glycoproteins
chemical_list: D000276:Adjuvants, Immunologic; D000906:Antibodies; D004527:Egg Proteins; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D022642:Vaccines, Contraceptive; D000072216:Zona Pellucida Glycoproteins; C000606521:Zp3 protein, mouse
keywords: Adjuvant; CpG motif; Immunocontraception; Recombinant protein produced in fermentor; Zona pellucida glycoprotein-3