DNA vaccine targeting GnRH-receptor reduces testosterone and induces testicular atrophy in mice

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Silvia Reyes-Maya
Amanda Gayosso-Vázquez
Vianey Ramírez-Andoney
Pablo Pintor Ríos
María Luisa Escobar
Israel Muñoz-Velasco
Olga Margarita Echeverría
Carlos G. Gutiérrez
Rogelio Alejandro Alonso-Morales

Abstract

Reproductive control of invasive, feral, and domesticated animals is an urgent public health issue. Immunological vaccines are employed as an emerging strategy to target different components of the reproductive system, to achieve immunocastration. Successful use of immunogens against parts of the oocyte, spermatozoon, gonadotropin-releasing hormone (GnRH), and luteinizing hormone receptor (LHr) has been reported. In this work, a recombinant DNA vaccine was prepared (P2GnRHrP30) by the fusion of the first extracellular domain of the canine GnRH receptor (GNRHr) gene to the P2 and P30 tetanus toxoid epitopes. This construction was cloned in the pCI-NEO mammalian expression vector and evaluated as an immunocontraception DNA vaccine in male mice. The developed vaccine was demonstrated to be safe, and capable of reducing serum testosterone levels and causing testicular atrophy. Our work indicates that the first extracellular domain of the GnRHr gene may act as an immune target for reproductive control and potentially be used as a non-surgical sterilization procedure in mammals.

Keywords:
DNA vaccine, Canine GnRH-receptor, P2 P30 epitopes, Dog immunocontraception, Overpopulation control, Immunocontraception, Non-surgical sterilization, Reproductive immunology, Gene-based vaccine

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References

Alliance for Contraception in Cats and Dogs. Contraception and Fertility Control in Dogs and Cats [PDF]. A report of the Alliance for Contraception in Cats and Dogs. ACC&D. February 2013. https://static1.squarespace.com/static/60106baf6eaab30c510d352a/t/6049b81d48638940f786f028/1616300091169/ACCD-e-book.pdf

Clarke IJ, Pompolo S. Synthesis and secretion of GnRH. Animal Reproduction Science. 2005;88(1–2):29–55. doi: 10.1016/j.anireprosci.2005.05.003. DOI: https://doi.org/10.1016/j.anireprosci.2005.05.003

Millar RP. GnRHs and GnRH receptors. Animal Reproduction Science. 2005;88(1–2):5–28. doi: 10.1016/j.anireprosci.2005.05.032. DOI: https://doi.org/10.1016/j.anireprosci.2005.05.032

Siel D, Ubilla MJ, Vidal S, Loaiza A, Quiroga J, Cifuentes F, et al. Reproductive and behavioral evaluation of a new immunocastration dog vaccine. Animals. 2020;10(2):226. doi: 10.3390/ani10020226. DOI: https://doi.org/10.3390/ani10020226

Rydhmer L, Lundström K, Andersson K. Immunocastration reduces aggressive and sexual behaviour in male pigs. Animal. 2010;4(6):965–972. doi: 10.1017/S175173111000011X. DOI: https://doi.org/10.1017/S175173111000011X

Vargas-Pino F, Gutiérrez-Cedillo V, Canales-Vargas EJ, Gress-Ortega LR, Miller LA, Rupprecht CE, et al. Concomitant administration of GonaCon™ and rabies vaccine in female dogs (Canis familiaris) in Mexico. Vaccine. 2013;31(40):4442–4447. doi: 10.1016/j.vaccine.2013.06.061. DOI: https://doi.org/10.1016/j.vaccine.2013.06.061

Sandam N, Prakash D, Thimmareddy P. Immunocontraceptive potential of GnRH receptor-based fusion recombinant protein. Journal of Genetic Engineering and Biotechnology. 2021;19(1):63. doi: 10.1186/s43141-021-00164-9. DOI: https://doi.org/10.1186/s43141-021-00164-9

Samoylov A, Napier ID, Morrison A, Cochran A, Schemera B, Wright J, et al. DNA vaccine targeting gonadotropin-releasing hormone receptor and its application in animal contraception. Molecular Biotechnology. 2019;61:73–83. doi: 10.1007/s12033-018-0137-9. DOI: https://doi.org/10.1007/s12033-018-0137-9

Samoylov A, Cox N, Cochran A, Wolfe K, Donovan C, Kutzler M, et al. Generation and characterization of phage-GnRH chemical conjugates for potential use in cat and dog immunocontraception. Reproduction in Domestic Animals. 2012;47(Suppl 6):406–411. doi: 10.1111/rda.12061. DOI: https://doi.org/10.1111/rda.12061

Dalum I, Jensen MR, Gregorius K, Thomasen CM, Elsner HI, Mouritsen S. Induction of cross-reactive antibodies against a self protein by immunization with a modified self protein containing a foreign T helper epitope. Molecular Immunology. 1997;34(16–17):1113–1120. doi: 10.1016/s0161-5890(97)00147-8. DOI: https://doi.org/10.1016/S0161-5890(97)00147-8

Ramírez V, Gayosso A, Pintor JP, Vázquez JE, Alonso RA. Chimeric myostatin-tetanic toxin epitopes and heterologous prime-boost immunization improve immune response stimulating muscle growth in mice. Biotechnology and Bioprocess Engineering. 2019;24:773–781. doi: 10.1007/s12257-019-0092-8. DOI: https://doi.org/10.1007/s12257-019-0092-8

Gerloni M, Xiong S, Mukerjee S, Schoenberger SP, Croft M, Zanetti M. Functional cooperation between T helper cell determinants. Proceedings of National Academy of Sciences. 2000; 97(24):13269–13274. doi: 10.1073/pnas.230429197. DOI: https://doi.org/10.1073/pnas.230429197

Valmori D, Pessi A, Bianchi E, Corradin G. Use of human universally antigenic tetanus toxin T cell epitopes as carriers for human vaccination. Journal of Immunology. 1992; 149(2):717–721. doi: 10.4049/jimmunol.149.2.717. DOI: https://doi.org/10.4049/jimmunol.149.2.717

Guevara GI. Desarrollo de una vacuna de ADN para la castración inmunológica en caninos [tesis de maestría]. México: Universidad Nacional Autónoma de México; 2015.

Chung HO, Yang Q, Catt KJ, Arora KK. Expression and function of the gonadotropin-releasing hormone receptor are dependent on a conserved apolar amino acid in the third Intracellular loop. Journal of Biological Chemistry. 1999;274(50):35756–35762. doi: 10.1074/jbc.274.50.35756. DOI: https://doi.org/10.1074/jbc.274.50.35756

Vázquez-Nin GH, Escobar ML, Echeverría OM, Ortiz R, Márquez HG, Juárez S. Combined method for the evaluation of the initial and late phases of the apoptosis process in prepubertal rat testes. Journal of Advances in Biology. 2014;5(1):594–602. https://rajpub.com/index.php/jab/article/view/5353/pdf_62

Fang F, Li H, Liu Y, Zhang Y, Tao Y, Li Y, et al. Active immunization with recombinant GnRH fusion protein in boars reduces both testicular development and mRNA expression levels of GnRH receptor in pituitary. Animal Reproduction Science. 2010;119(3–4): 275–281. doi: 10.1016/j.anireprosci.2010.01.003. DOI: https://doi.org/10.1016/j.anireprosci.2010.01.003

Rebourcet D, O'Shaughnessy PJ, Monteiro A, Milne L, Cruickshanks L, Jeffrey N, et al. Sertoli cells maintain Leydig cell number and peritubular myoid cell activity in the adult mouse testis. PLoS One. 2014;9(8):e105687. doi: 10.1371/journal.pone.0105687. DOI: https://doi.org/10.1371/journal.pone.0105687