Recombinant Virus-Mediated Fusion Expression And Anti-PRRSV Activity Of Sn And CD163 Soluble Receptors

Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious swine disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),leading to huge economic losses to pig industry in China.The virus has complex infection and immune evasion mechanisms,and thus can be controlled mainly by vaccination.However,the inactivated vaccine is ineffective and the attenuated vaccine has the risk of virulence reversion.Therefore,development of the novel strategy for the disease control is urgently needed.Virus soluble receptors(VSR)can compete for virus binding with the cellular receptors,and thus can play the similar role as neutralizing antibodies.Sialoadhesin(Sn)and CD 163 are the two main receptors for PRRSV infection of macrophages.The first four Ig-like domains of Sn(Sn4D)and the 5-9 scavenger receptor cysteine-rich domains(SRCR59)of CD 163 contain PRRSV binding sites and thus can function as the two VSRs.In the previous study,two recombinant adenovirus(rAd)vectors separately expressing the two VSRs were shown to have the additive effect against PRRSV infection with a limited expression time in vivo.In the present study,we aimed at improving the VSR anti-PRRSV strategy by fusion expression of the two VSRs with different promoters and viral vectors,and by targeted mutation of the key amino acids in the Fc segment within the two VSRs.1.Study on recombinant AAV-mediated fusion expression of VSRs and their anti-PRRSV activitiesTo improve the practical application of the VRS anti-PRRV strategy,both Cap and Rep genes of AAV were cloned into baculovirus expression vector and the recombinant vector pBac-RC was constructed.The inverse terminal regions(ITR)of AAV were cloned into baculovirus vector and the resultant transfer vector was called pBac-ITR.Sn4D or Sn4D-Fc and SRCR59-Fc genes were linked by a GS linker or P2A self-cleavage peptide.After cloning into pBac-ITR vector,the two fusion expression vectors,namely pBac-Sn4D-SRCR59-Fc and pBac-SRCR59-Fc/Sn4D-Fc,were obtained.After co-transfection into insect cells with pBac-RC vector,two recombinant baculovirues,namely rAAV-Sn4D-SRCR59-Fc and rAAV-SRCR59-Fc/Sn4D-Fcand were generated.Under the optimized conditions for insect cell cultivation and rAAV transduction,the two rAAVs were amplified to high titers(8.4×109FFU/ml and 3.2×109FFU/ml)in 5-L bioreactor,with typical AAV morphology and structural proteins.After transduction into 3D4/21 cells,the two VSRs,namely Sn4D-SRCR59-Fc and SRCR59-Fc/Sn4D-Fc,were correctly expressed with the highest expression level of 168.9ng/mL or 158.1ng/mL,and the longest expression time of 120 h.The expressed SRCR59-Fc/Sn4D-Fc showed stronger anti-PRRSV activity than Sn4D-SRCR59-Fc,which was similar against four different PRRSV strains.After injection into mice,the expression times of the two rAAV vectors were prolonged by 21 days,as compared with rAd-SRCR59-Fc injection.Pigs were injected with rAAV-SRCR59-Fc/Sn4D-Fc and then housed with PRRSV-inoculated pigs.The results showed that rectal temperature,clinical symptoms,viremic titers,pathological lesions and organ virus loads of rAAV-injected pigs were significantly reduced.These data suggest that rAAVSRCR59-Fc/Sn4D-Fc vector can further developed as an anti-PRRSV agent.2.Study on VSR fusion expression strategies with different promoters and virus vectorsTo enhance the long-lasting and high-level VSR expression,porcine EF1α promoter was introduced into rAAV-SRCR59-Fc/Sn4D-Fc and the modified rAAVEF1α-SRCR59-Fc/Sn4D-Fc was generated.After transduction into PK-15 or 3D4/21 cells,the highest VSR expression level of rAAV-EF1α-SRCR59-Fc/Sn4D-Fc transduction group was 110.Ong/mL or 246.2ng/mL,which were significantly higher than that(72.2ng/mL and 168.9ng/mL)of rAAV-SRCR59-Fc/Sn4D-Fc transduction groups.After injection into mice,the VSR expression level of rAAV-EF1α-SRCR59-Fc/Sn4D-Fc was significantly higher than that of rAAV-SRCR59-Fc/Sn4D-Fc,which prolonged for 35 days.Then,the EF1αpromoter-driven expression cassette was cloned into adenovirus(Ad)vector,baculovirus(Bac)vector and porcine pseudorabies virus(PRV)vector,and the recombinant vectors were called rAd-EF1α-SRCR59-Fc/Sn4D-Fc,rBac-GED-EF1α-SRCR59-Fc/Sn4D-Fc or rPRV-EF1α-SRCR59-Fc/Sn4D-Fc,respectively.After transduction into PK-15 and 3D4/21 cells,rBac-GED-EF1α-SRCR59-Fc/Sn4D-Fc transduction groups showed the highest VSR expression levels of 202.1ng/mL and 402.8ng/mL,respectively,which were significantly higher than that of rAd-EF1α-SRCR59-Fc/Sn4D-Fctransduction groups(179.4ng/mL or 307.4ng/mL),rAAV-EF1α-SRCR59-Fc/Sn4D-Fc transduction groups(105.3ng/mL or 211.2ng/mL)and rPRV-EF1α-SRCR59-Fc/Sn4D-Fc transduction groups(44.5ng/mL or 103.6ng/mL).After transduction of PAM cells with the four viral vectors,PRRSV titer in rBac-GED-EF1α-SRCR59-Fc/Sn4D-Fc-transduced cells was significantly lower than that in other three transduction groups,with similar anti-PRRSV effects against four different strains.After injection into mice,rBac-GED-EF1α-SRCR59-Fc/Sn4D-Fc injection group showed the highest and longest VSR expression.These data suggest that EF1α promoter and rBac vector was the best combination for long-lasting expression of the two VSRs.3.Influence of key amino acid mutation in the Fc portion on half-life and anti-PRRSV effect of the two VSRsTo prolong the half-life of the VSR fusion in vivo,both M455L and N461S substitutions were introduced into the Fc portions within SRCR59-Fc/Sn4D-Fc by PCR cloning.The resultant rBac-GED-EF1α-SRCR59-lsFc/Sn4D-lsFc could correctly express the encoded VSR fusion in both insect and porcine cells,with higher expression level,longer expression time and stronger anti-PRRSV activity than rBac-GED-EF1α-SRCR59-Fc/Sn4DFc.After injection into mice,the VSR expression time in rBac-GED-EF1α-SRCR59-lsFc/Sn4D-lsFc-injected mice was significantly longer than that in rBac-GED-EF1α-SRCR59-Fc/Sn4D-Fc-injected mice.Compared to that(96 h and 7 days)of SRCR59-Fc/Sn4D-Fc,the half-lives of purified SRCR59-Fc/Sn4D-Fc in pig plasma and in pigs were prolonged by 20.8 h and 3.4 days,respectively.In addition,the in vivo bioavailability of SRCR59-Fc/Sn4D-Fc was increased by 3.6%,with stronger cytotoxicity in PRRSV-infected PAM cells.These data suggest that the key amino acid mutations of the Fc portions could improve both stability and anti-PRRSV activity of SRCR59-Fc/Sn4D-Fc.In conclusion,this study successfully expressed two fusion forms of the PRRSV soluble receptor with a single adeno-associated virus vector.The expression strategy showed that the SRCR59-Fc/Sn4D-Fc fusions had stronger anti-PRRSV activity and was suitable for the study of clinical trial.Further optimization of the expression strategy showed that the EF1αpromoter and rBac vector was the best combination to significantly prolong the fusion expression of the soluble receptor.In addition,the stability and anti-PRRSV activity of the soluble receptor can be improved by mutating key amino acids in the Fc portions,which provided a new strategy for the further study on PRRSV prevention and control.