Construction And Immune Efficacy Of Recombinant Turkey Herpesvirus Expressing The Hemagglutinin Of H7N9 Subtype Avian Influenza Virus

The novel H7N9 subtype avian influenza viruses(AIV),which were firstly isolated from the Yangtze River Delta in 2013 in China,have already evolved into some highly pathogenic strains to poultry from the initially non-or low-pathogenic precursors during the past four years.Those variants contain an insertion of four consecutive basic amino acids(PKRKRTAR/GLF)at the HA cleavage site,consisting with the molecular characteristics of the highly pathogenic avian influenza(HPAI)viruses and posing a threat to poultry industry.The mutated HPAI will seriously undermine the poultry industry healthy growth.In addition,the H7N9 viruses can also infected human directly.Up to May 31,2017,the number of laboratory-confirmed human infection with H7N9 has accumulated to 1532 cases,of which 581 were dead,according to the latest statistics from World Health Organization.Although sufficient evidence of efficient transmission of H7N9 from person to person is still lacking,the risk that H7N9 could cause a potential pandemic is still high and its threat to public health should also not to be overlooked.Nowadays,vaccination is still one of the basic measures to prevent and control of HPAI in China.However,poultry-used H7N9 vaccines are still not available in clinical.Therefore,it is urgent to develop corresponding vaccines to reduce the damage to animal industry and public health caused by the infection of H7N9 viruses.Generally,traditional inactivated vaccines mainly stimulate humoral immunity but deficient of cellular immune response,while the immune effects are usually limited to factors such as the high level of maternal antibodies,poor antigen match between vaccine strains and circulating strains.In contrast to traditional inactivated vaccine,novel live vector vaccines have many advantages to overcome some of the shortcomings.For example,the turkey herpesvirus(HVT)vectored recombinant live vaccines,expressing main protective antigens,have the ability to induce both cellular and humoral immunity,possess a longer protection period and reduce virus shedding after infection.Therefore,it is a comparative ideal vaccine vector for poultry.However,as reported by colleagues,recombinant HVT rHVT-H7HA which was constructed by insertion of the primary protective antigen HA of AIV(H7N1 subtype)prevented only about 73%of the vaccinated SPF chickens from challenging with lethal dose of HPAI H7N1 virus.Moreover,a number of additional studies have shown that the choices of promoter and the immunogenicity of HA protein may be important factors affecting the immune efficacy of recombinant HVT.Therefore,we firstly applied the strategy of selecting different promoters and enhancing the antigenicity of HA in this study to construct a series of intermediate transfer plasmids.Subsequently,recombinant HVTs expressing the HA protein of H7N9 subtype AIV were further generated by homologous recombination and CRISPR/Cas9 mediated gene editing technique,and their immune efficacies were also evaluated in SPF chickens.In the present study,endogenous gB promoter(HgB)of HVT was firstly cloned,so was the exogenous CMV promoter serving as a contrast.Based on the HA gene from a strain of low pathogenicity H7N9 virus,codon optimization(OHA)to the bias of Gallus gallus was conducted to improve the efficiency amino acid translation.And two intermediate transfer plasmids of pHOH(HgB-OHA)and pVOH(CMV-OHA)containing HgB and CMV promoters were constructed,respectively.To further increase the presenting efficiency of antigenic peptide,signal peptide(MHCIss)and the transmembrane and cytoplasmic region(MITD)of MHC class I molecules were added and replaced to the N-termini and C-termini of OHA,respectively.Additionally,the WPRE(woodchuck hepatitis virus post-transcriptional regulatory element)sequence was fused to the C-termini of this modified OHA(OHAM)to enhance the protein translation efficiency.Accordingly,another two chimeric intermediate transfer plasmids of the pHMW(HgB-OHAM-WPRE)and pVMW(CMV-OHAM-WPRE)were constructed.By using commercial monoclonal antibodies against the HA of H7N9 virus,indirect immunofluorescence assay(IFA)and western blotting(WB)both demonstrated the correct expression of the HA protein at about 70 kDa.In order to further acquire the corresponding recombinant HVTs expressing HA,homologous recombination was executed on CEF cells via the calcium phosphate transfection method,by co-transfection of each of the four constructed intermediate transfer plasmids with the genome DNA of the previously generated recombinant HVT expressing GFP(rHVT-GFP with the insertion of GFP in the US2 non-essential region for replication of HVT),respectively.In consequence,two recombinant HVTs of rHOH and rHMW,both involving the HgB promoters,were obtained after screening,purification and identification.Meanwhile,we tried the CRISPR/cas9 technology to further improve the efficiency of homologous recombination.Another recombinant HVT of rVMW containing the CMV promoter was generated successfully.All of these three recombinant HVTs could properly express the exogenetic HA,as evidenced by IFA,WB and sequencing.Furthermore,the genetic stability and the localization of the expressed HA of rHOH and rHMW were measured on CEF cells.The results showed that both of the two recombinants could express the HA stably after 20 generations of passage,with no significant difference of the growth kinetics with the wild type HVT,respectively.As observed by laser confocal microscopy,the HA expressed from rHOH mainly located in the cytoplasm while the one from rHMW could also simultaneously located on the surface of the cell membrane in addition to cytoplasm.Finally,the immune efficacy of the generated rHOH and rHMW were evaluated by challenging the SPF chickens immunized at 1 day old with different doses,respectively.The results indicated that the average seroconversion percentages of different groups of rHMW and rHOH were 62.5%～72%and ≤62.5%on six weeks post immunization,respectively.As challenged with a HPAI H7N9 strain that were isolated in 2017 at the dosage of 105TCID50,the rHMW-vaccinated groups could provide an average clinical protection of about 77.8%.In summary,three recombinant HVTs expressing the HA protein of H7N9 subtype AIV were generated successfully and two of their immune efficacies on SPF chickens were evaluated in the current study.Although the clinical protective effect of rHOH and rHMW needs further improvement,the strategies for novel vaccine development involving selection of different promoters,codon optimization and addition of elements beneficial for enhanced protein translation,as well as the established CRISPR/Cas9 technology platform in the study,would lay a foundation for relevant research based on the vectors of HVT and other avian herpesviruses.