Construction of a GnRH mRNA Immunocastration Vaccine and Evaluation of Its Immunogenicity and Safety in Mice and Cats

Abstract Immunocastration has emerged as an alternative to surgical and chemical castration for managing reproductive function in animals, yet the development of safe and effective vaccines remains challenging. This study aimed to develop a gonadotropin-releasing hormone (GnRH)-based messenger RNA (mRNA) vaccine and systematically evaluate its immunogenicity, reproductive suppression efficacy, long-term durability, and biosafety in mice and cats. GnRH epitopes were fused to three carrier proteins, Fc, Foldon, and lumazine synthase nanoparticles (pLS) via a flexible linker. After identifying pLS as the optimal scaffold, three mRNA vaccine candidates (GnRH-3, GnRH-4, and GnRH-5) were generated with one, five, or ten tandem GnRH repeats, encapsulated in lipid nanoparticles (LNPs), and assessed in rodent and feline models. Immunogenicity was determined by enzyme-linked immunosorbent assay, gonadal histopathology, hormone measurements, transcriptomic analysis, and mating trials. Among the fusion partners, the pLS-based vaccine (GnRH-3) induced the strongest antibody responses and most pronounced reproductive suppression. Further optimization showed that GnRH-4, containing five tandem GnRH repeats, elicited the highest antibody titers, induced severe gonadal atrophy, and reduced litter size by 93.8% in mice. Transcriptomic analysis revealed that differentially expressed genes in males were enriched in spermatogenesis and motility pathways, whereas those in females were associated with RNA splicing and immune responses. In cats, the optimal regimen was a twoLdose schedule with 50Lμg per dose and a 21Lday interval, which induced robust antibody responses lasting at least 12 Lmonths and sustained reproductive suppression. HighLdose (500Lμg) administration showed no clinical toxicity or histopathological abnormalities, confirming favorable biosafety. This study successfully developed a pLSLbased GnRH mRNA vaccine (GnRH-4) with five tandem GnRH epitopes that demonstrates strong immunogenicity, longLlasting contraceptive effects, and excellent safety in both rodent and feline models, supporting its potential for clinical application in immunocastration.