Polysaccharide microparticles as an oral vaccine platform: proof-of-concept in male rats using a GnRH-targeting fusion antigen

Background Immunocastration through vaccination against gonadotropin-releasing hormone (GnRH) represents a humane alternative to surgical castration in animal production. However, oral delivery of protein antigens remains challenging due to gastrointestinal degradation and poor mucosal immunogenicity. Polysaccharide microparticles (PSMs), particularly alginate-chitosan systems, offer protection against gastric degradation and enhanced mucosal uptake. Objective To evaluate whether alginate-chitosan microparticles entrapping a recombinant GnRH-fusion protein (GF45) can elicit an immunocastrating response in male rats after oral administration. Methods GF45, a 45 kDa fusion peptide containing four tandem GnRH repeats fused to Rickettsia heat-shock protein fragments, was expressed in E. coli BL21(DE3) and purified under denaturing conditions followed by refolding. Microparticles were prepared by ionic gelation achieving 64% encapsulation efficiency. Male Sprague-Dawley rats (4 weeks) received oral prime-boost immunizations (weeks 4 and 7) with 200 µg GF45 in microparticles (n = 10). Controls included empty microparticles (NC, n = 10) and subcutaneous GF45 + Freund’s adjuvant (PC, n = 10). Serum testosterone and anti-GF45 IgG were monitored until week 16 via ELISA. Testes were weighed, volumetrically measured by Archimedes’ principle, and histologically examined using Johnsen’s scoring system. Results Oral GF45 reduced circulating testosterone by 95% (0.7 ± 0.3 vs 14.2 ± 1.8 ng dL -1 in NC; p < 0.0001) and induced anti-GF45 antibodies reaching 88.8% of the injectable response (titres log 10 4.1 ± 0.3 vs 4.4 ± 0.2; p = 0.08). Seroconversion began at week 6 post-prime, reaching 100% by week 16. Testis weight decreased by 52% and volume by 75% (both p < 0.01), accompanied by marked seminiferous-tubule atrophy (diameter reduced from 286 ± 9 µm to 124 ± 8 µm) and reduced Johnsen scores (3.2 ± 0.4 vs 9.1 ± 0.2; p < 0.001). Empty microparticles produced no effects. Conclusion Alginate-chitosan microparticles provide a practical, needle-free platform for oral immunocastration via induction of anti-GnRH autoimmunity. This polysaccharide-based system represents a versatile oral antigen carrier with potential applications in veterinary medicine and livestock production.