Displaying of GnRH Peptides on Bacteriophage T7 and Its Immunogenicity in Mice Model

Authors: Hai Xu, Yiwei Wang, Xi Bao, Bihua Deng, Pengcheng Li, Yu Lu

Abstract:

T7 phage could be used as a perfect vector for peptides expression and haptens presentation. T7-3GnRH recombinant phage was constructed by inserting three copies of Gonadotrophin Releasing Hormone (GnRH) gene into the multiple cloning site of T7 Select 415-1b phage genome. The positive T7-3GnRH phage was selected by using polymerase chain reaction amplification, and the p10B-3GnRH fusion protein was verified by SDS-PAGE and Western-blotting assay. T7-3GnRH vaccine was made and immunized with 10¹⁰ pfu in 0.2 ml per dose in mice. Blood samples were collected at an interval in weeks, and anti-GnRH antibody and testosterone concentrations were detected by ELISA and radioimmunoassay, respectively. The results show that T7-3GnRH phage particles confer a high immunogenicity to the GnRH-derived epitope. Moreover, the T7-3GnRH vaccine induced higher level of anti-GnRH antibody than ImproVac^®. However, the testosterone concentrations in both immunized groups were at a similar level, and the testis developments were significantly inhibited compared to controls. These findings demonstrated that the anti-GnRH antibody could neutralize the endogenous GnRH to down regulate testosterone level and limit testis development, highlighting the potential value of T7-3GnRH in the immunocastration vaccine research.

Keywords: Gonadotrophin releasing hormone, GnRH, immunocastration, T7 phage, phage vaccine.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1131103

References:

[1] Kirkpatrick JF, Lyda RO, Frank KM. Contraceptive vaccines for wildlife: a review. American journal of reproductive immunology. 2011; 66:40-50.
[2] Goodwin D, Varamini P, Simerska P, D'Occhio MJ, Toth I. Design, synthesis and evaluation of a gonadotropin releasing hormone-based subunit vaccine in rams (Ovis aries). Vaccine. 2015; 33:1453-8.
[3] Batorek N, Candek-Potokar M, Bonneau M, Van Milgen J. Meta-analysis of the effect of immunocastration on production performance, reproductive organs and boar taint compounds in pigs. Animal: an international journal of animal bioscience. 2012; 6:1330-8.
[4] Font IFM, Gispert M, Soler J, Diaz M, Garcia-Regueiro JA, Diaz I, et al. Effect of vaccination against gonadotrophin-releasing factor on growth performance, carcass, meat and fat quality of male Duroc pigs for dry-cured ham production. Meat science. 2012; 91:148-54.
[5] Han YG, Ye WJ, Liu GQ, Jiang XP, Ijaz N, Zhao JY, et al. Hepatitis B Surface Antigen S Gene is an Effective Carrier Molecule for Developing GnRH DNA Immunocastration Vaccine in Mice. Reproduction in domestic animals = Zuchthygiene. 2016; 51:445-50.
[6] Hashemi H, Pouyanfard S, Bandehpour M, Noroozbabaei Z, Kazemi B, Saelens X, et al. Immunization with M2e-Displaying T7 Bacteriophage Nanoparticles Protects against Influenza A Virus Challenge. PloS one. 2012;7.
[7] Tan GH, Yusoff K, Seow HF, Tan WS. Antigenicity and immunogenicity of the immunodominant region of hepatitis B surface antigen displayed on bacteriophage T7. Journal of medical virology. 2005; 77:475-80.
[8] Miller LA, Gionfriddo JP, Fagerstone KA, Rhyan JC, Killian GJ. The single-shot GnRH immunocontraceptive vaccine (GonaCon) in white-tailed deer: comparison of several GnRH preparations. American journal of reproductive immunology. 2008; 60:214-23.
[9] Quiniou N, Monziols M, Colin F, Goues T, Courboulay V. Effect of feed restriction on the performance and behaviour of pigs immunologically castrated with ImproVac®. Animal: an international journal of animal bioscience. 2012; 6:1420-6.
[10] Dunshea FR, Colantoni C, Howard K, McCauley I, Jackson P, Long KA, et al. Vaccination of boars with a GnRH vaccine (ImproVac®) eliminates boar taint and increases growth performance. Journal of animal science. 2001; 79:2524-35.
[11] Benka VA, Levy JK. Vaccines for feline contraception: GonaCon GnRH-hemocyanin conjugate immunocontraceptive. Journal of feline medicine and surgery. 2015; 17:758-65.
[12] Jinshu X, Jingjing L, Duan P, Zheng Z, Ding M, Jie W, et al. The immunogenicity of recombinant and dimeric GnRH vaccines incorporating a T-helper epitope and GnRH or repeated GnRH units. Journal of immunological methods. 2004; 289:111-22.
[13] Talwar GP, Raina K, Gupta JC, Ray R, Wadhwa S, Ali MM. A recombinant luteinising-hormone-releasing-hormone immunogen bioeffective in causing prostatic atrophy. Vaccine. 2004; 22:3713-21.
[14] Turkstra JA, Oonk HB, Schaaper WM, Meloen RH. The role of the individual amino acids of a GnRH-tandem-dimer peptide used as an antigen for immunocastration of male piglets determined with systematic alanine replacements. Vaccine. 2001; 20:406-12.