Viral Receptor Blocking Based Broad Antiviral Strategies Against CSFV And PRRSV

Viral infectious diseases seriously threat public health and safety.Conventional antiviral strategies mainly rely on inactivated and attenuated vaccines,which employ complete viruses,leading to biosafety risk in production and immunization.Besides,viral sequence variation and antigen drift hamper the cross protection with conventional vaccines against continuously evolving strains.As the viral life cycle divided into attachment,fusion,exudation,replication and package,interruption in virus-host interaction at the first step serves as one of the most effective antiviral strategies.From the 1970s,broad antiviral strategies,which target biological function of viral proteins and the interaction between virus and host receptor,has gradually become a hot topic in the field of antiviral research.Classical swine fever（CSF）and Porcine reproductive and respiratory syndrome（PRRS）are both highly contagious viral diseases,causing economically significant impacts on the swine industry.Enveloped protein E2 of CSFV is a key determinant for viral pathogenicity,target cell entry and neutralizing antibody elicition.Hence,E2 serves as an ideal model to study virus-targeting antiviral strategies.Meanwhile,due to the high variation in sequence,the delayed protective immune response and immunosuppression in hosts,most virus-targeting antiviral strategies against porcine reproductive and respiratory syndrome virus（PRRSV）are not effective.CD163 has been identified as an indispensable cellular receptor for PRRSV infection.Therefore,it provides a classic model for receptor-targeting antiviral strategies.These virus-host blocking strategies will pave ways for the complete prevention and elimination of CSFV and PRRSV.In this study,for virus-targeting active immunization strategy,CSFV E2ZJ subunit vaccine fused with a novel signal peptide was developed and evaluated.Further,as a virus-targeting passive immunization strategy,three broad neutralizing monoclonal antibodies（m Abs）against E2ZJ were screened and characterized to determine the efficacy in vitro and in vivo.Finally,to further improve the cross-protection of antiviral agents,CD163 receptor was studied for PRRSV receptor-targeting strategies.Two m Abs exhibited broad-spectrum antiviral activities against PRRSV were produced to evaluate antiviral activity,therapeutic and prophylactic treatment.The main studies are described below:1.Virus-targeting active immunization:development of a novel and effective CSFV E2ZJ subunit vaccineCSF live attenuated vaccines could not distinguish infected and vaccinated animals and glycoprotein E2 is the key determinant for immunity.Thus,we express E2 protein fused with a novel signal peptide,indicating that E2 protein secreted in culture supernatant was detected only with a novel signal peptide in insect cells.To identify the efficient signal peptide for E2 secretion,E2 protein was fused to a series of truncated candidates.Results showed that E2 secretion was effectively induced by the selected signal peptide,SPZJ（SP23）,with at least 50%increase as compared to any other signal peptides tested.Besides,SPZJ was considered to be a common signal peptide for variable E2s secretion.Recombinant E2s with the same dosage were further used to evaluate the immunogenicity in mice.As shown in IFA and ELISA,E2ZJ elicited CSFV antibody at the earlier stage than other E2 types tested.Meantime,higher level of neutralizing antibody with E2ZJ was detected as compared to other E2s and the neutralization titer of E2ZJ was up to 2000 at 28 dpi.Further,in piglets,the mean antibody blocking rates of piglets vaccinated with E2ZJ were more than the 75%at 28 dpi,which was significantly higher than that of non-ZJ E2 group.At the same time,E2ZJ successfully elicited high level of neutralizing antibodies against genotype 1 and genotype 2 strains.No typical temperature response,clinical symptoms,and histological changes were observed in all immunized groups after challenge,but not in PBS group.Importantly,a single dose of 5μg E2ZJ was sufficient to provide 100%protection against lethal virus challenge in piglets.These results indicated that,with advantages in expression and immunogenicity over counterparts,E2ZJ expressed in baculovirus expression system was a cost-effective and efficacious vaccine candidate against CSFV.2.Virus-targeting passive immunization:characterization of broad-spectrum neutralizing antibodies against CSFVBased on the immunogenicity of E2ZJ protein,a batch of monoclonal antibodies with neutralizing activity were screened by traditional method.In IFA,Western-blot and ELISA,m Abs 6D10,8D8 and 3C12 showed specific reactivity with different CSFV strains and strong binding reactivity with various CSFV E2 proteins.Moreover,m Abs 6D10,8D8 and 3C12 displayed broad-spectrum neutralizing activities against CSFV strains and powerful neutralizing activities in a dose-dependent manner,of which 6.25μg/m L,3.125μg/m L and 12.5μg/m L could completely neutralize CSFV of 100TCID₅₀.Further studies demonstrated that such neutralizing antibodies possessed multiple mechanisms,including blocking viral internalization and attachment steps in the viral life cycle.Reactivity with truncated E2 proteins and E2mutants conveyed that three linear neutralizing epitopes of m Abs were identified as¹⁴⁰TAVSPTTLR¹⁴⁸,⁸YRYAIS¹³ and ²⁵⁴HECLIG²⁵⁹,respectively.The latter two epitopes were reported for the first time.Sequence alignment revealed that the three epitopes were highly conserved in various CSFV strains,indicating that these m Abs have diagnostic potentials of CSFV.Spatial structure prediction and virus capture assay showed that ¹⁴⁰TAVSPTTLR¹⁴⁸ and ⁸YRYAIS¹³ epitopes were exposed on the surface of CSFV E2 protein.Recombinant polypeptides carrying epitopes successfully inhibit CSFV infection in a dose-dependent manner in PK-15 cells,indicating epitopes were located in receptor-binding domain（RBD）and involved in virus invasion.Further,both prophylactic and therapeutic functions of neutralizing antibody were evaluated in rabbits upon CSFV challenge,which further confirming the efficacy in vivo.These findings provide a new strategy for broad antiviral activity targeted CSFV and deepen the understanding in E2 function during CSFV invasion.3.Receptor-targeting antiviral strategy:broad antiviral strategy targeted CD163receptor against PRRSVCD163 receptor is the key receptor for PRRSV infection.Two m Abs（6E8 and9A10）against CD163 SRCR5-9 were selected based on the specific recognition of both native and recombinant CD163 in PAMs and Marc-145 cells.Both m Abs displayed an inhibitory effect on PRRSV infection in a dose-dependent manner and significantly broad anti-PRRSV activity in PAMs and Marc-145.In addition,as candidates for the prevention and treatment of PRRSV,both m Abs could successfully inhibit PRRSV infection and its related NF-κB pathway either pre-and post attachment.Besides,CD163 transcription in PAMs and Marc-145 was obviously down-regulated in m Abs-treated PAMs.It was potentially caused by the excessive accumulation of membrane associated CD163 and negative feedback inhibition of CD163 transcription due to the failure in CD163 cleavage with the antibody binding.Based on the reactivity of truncated SRCR5-9s and SRCR5-9 mutants,conformational epitopes targeted by 6E8 and 9A10 were identified to be spanning residues ⁵⁷⁰SXDVGXV⁵⁷⁶ in SRCR5 and Q⁷⁹⁷in SRCR7 respectively.The key residues were highly conserved in different species.Wild type CD163 and CD163with mutated epitopes overexpressed in 3D4 cells which did not support PRRSV infection were further verified the key role of these residues in PRRSV invasion.Besides,as a subunit vaccine targeted CD163 receptor,polypeptide of CD163 m Abs screened in the laboratory was preliminarily evaluated to inhibit PRRSV infection in vitro.These findings promote the understanding in the interaction between PRRSV and host receptor and provide novel broad antiviral strategies for PRRSV prevention and treatment.