Recombinant B2L And Kisspeptin-54 DNA Vaccine: Effect On Reproductive Function,meat Quality And Immunogenicity Against Orf Virus

Orf is a highly contagious and zoonotic disease of sheep and goats caused by Parapoxvirus.Despite there are currently live attenuated and inactivated orf virus(ORFV)vaccines,these vaccines are unable to induce long-lasting immunity,and cannot protect hosts from re-infection.Kisspeptin,encoded by the KISS1 gene with its cognate receptor G Protein-Coupled Receptor(GPR54),is recognized as an upstream orchestrator in the hypothalamic-pituitary-gonadal(HPG)axis.Although kisspeptin role as a gatekeeper of gonadotropin-releasing hormone(GnRH)secretion is well recognized,the effects of kisspeptin immunization on fertility and meat quality in goats are not yet studied.This study aimed to construct a DNA vaccine that produces a fusion peptide composed of the ORFV envelope gene(B2L)and Kisspeptin-54 and evaluate its immunogenicity against orf virus and immunocastration effect in rats and goats,and its effect on growth,carcass characteristics and meat quality in goats.(1)To construct PVAX-B2L-asd(PB2L-asd)and recombinant PVAX-B2L-Kisspeptin-54-asd(PBK-asd)DNA vaccines,the sequences of ORFV envelope gene(B2L)and Kisspeptin-54 were retrieved and designed.Thereafter,the 3D structure and physicochemical properties of the fusion protein were predicted using immunoinformatic tools.The designed plasmids were then synthesized and cloned into the PVAX-asd vector.The transcription and translation of the fusion plasmid were detected by reverse transcriptase-polymerase chain reaction(RT-PCR)and western blot,respectively.The results showed that PB2L-asd and recombinant PBK-asd plasmids were successfully constructed as confirmed by restriction digestion and sequencing.The immunoinformatic analysis classified these vaccines as antigenic,non-allergen and stable.Furthermore,the results indicate that the plasmid can be successfully expressed in HeLa cells.(2)For this experiment,24 eight weeks male Sprague-Dawley rats were used and randomized into the treatment and control groups,which were,respectively immunized by PBK-asd vaccine(100 ?g/dose)and PVAX-asd empty plasmid via intramuscular route.The anti-B2L antibody response,cell proliferation rate,CD4+and CD8+content and antigen-specific cytokines were measured.The result showed that from the 4th weeks after the first immunization to the end of the experiment(12 weeks),the anti-B2L antibody in the PBK-asd immunized group was significantly higher than the control group(p<0.05).Splenocytes from PBK-asd treated rats proliferated more in response to B2L stimulation than the control group.Besides,CD4+T cells and Thl helper cytokine markers(IL-2,INF-?,TNF-?)in the PBK-asd-immunized group were significantly higher than the control group.(3)Fifteen 5-month-old Yiling male goats were randomly divided into three groups:PBK-asd immunization,PB2L-asd immunization and PVAX-asd(control).The vaccine was given intramuscularly(lmg/dose)and boosted twice at 21 days interval.Compared to the control group,PBK-asd and PB2L-asd immunization induced serum anti-B2L antibodies and Thl cytokine markers(IL-2,INF-?,TNF-?).Interestingly,the anti-B2L antibody response of PBK-asd immunized goats was significantly higher than the PB2L-asd immunized goats from 8th weeks of initial immunization to the end of the experiment(14 weeks)(p<0.05).(4)For this study,36 Sprague-Dawley male rats,aged 8 weeks were randomly divided into three groups:PBK-asd immunization,PVAX-asd treatment(control),and surgical castration,and immunized as described above.The results showed that PBK-asd immunization significantly stimulates serum anti-kisspeptin antibody response(p<0.05).The serum testosterone and luteinizing hormones(LH)concentrations significantly reduced in immunized rats.However,the serum LH levels were significantly higher in the surgical castration group than the control group.The current recombinant vaccine markedly reduced testicular growth and inhibited spermatogenesis in vaccinated rats.(5)Fifteen Yiling male goats,aged 5 months were randomly divided into three groups:PBK-asd immunization,PVAX-asd treatment(control)and surgical castration,and immunized as described above.Immunization of goats with PBK-asd vaccine significantly stimulate serum anti-kisspeptin antibody,and reduced serum follicle-stimulating hormone(FSH),luteinizing hormone(LH)and testosterone hormones(p<0.05).Notably,the PBK-asd vaccine reduced testicular growth and prevented spermatogenesis in vaccinated goats.Furthermore,goats immunized with the PBK-asd vaccine showed significantly smaller scrotal circumferences and lower libido behaviors.Compared with the control group,hypothalamic KISS1 mRNA expression was significantly increased in both immunized and surgical castrated groups(p<0.05).However,PBK-asd immunization inhibited mRNA expression of hypothalamic androgen receptor(AR),GPR54 and GnRH.Moreover,mRNA expression of pituitary GnRH receptor,follicle-stimulating hormone beta(FSH?),and luteinizing hormone beta(LH?)as well as,testicular LH receptor and FSH receptor,were down-regulated in PBK-asd vaccinated group.In contrast,surgical castration markedly up-regulated mRNA expression of all reproductive genes detected on the HP axis except AR(p<0.05)(6)The growth performances,carcass characteristics,meat quality and safety of Yiling goats immunized with PBK-asd vaccine,PVAX-asd empty plasmid and surgical castrated were investigated.The results showed that immunization with PBK-asd vaccine had no significant effect on growth hormone,body weight,average daily gain(ADG)and carcass weight of vaccinated goats.However,the ADG,body weight and carcass weight of surgical castration group were significantly lower than the PBK-asd immunization and control groups(p<0.05).Interestingly,PBK-asd plasmid did not integrate into the host genome,indicating that the meat from vaccinated goats is safe to consume.There were no significant differences among treatment groups in terms of carcass characteristics(dressing percentage,loin area,and fat thickness)and meat quality traits(pH,color,cooking loss,drip loss,and shearing force)(7)In conclusion,PBK-asd DNA vaccine can induce humoral and cellular immune responses against ORFV envelope protein(B2L),suggesting that it has immunogenicity against orf virus.Immunization of goats with PBK-asd vaccine produces a robust anti-kisspeptin antibody that neutralizes the intrinsic kisspeptin thereby,the normal hypothalamic-pituitary-testicular signaling pathway disrupted,eventually immunocastration achieved.Furthermore,the results indicate that the PBK-asd DNA vaccine is safe and does not affect carcass and meat quality traits of goats.Above all,these studies indicate that the recombinant B2L and Kisspeptin-54 DNA vaccine is a promising candidate vaccine for goat immunocastration without negatively affecting carcass and meat quality and has a potential to induce humoral and cellular immune responses against ORFV.To assess the protective efficacy of this vaccine against ORFV,further virus challenge studies in goats are needed.