Construction Of A Recombinant Chimeric Newcastle Disease Virus Vector And Its Application In The Development Of H7N9 And H9N2 Subtype Avian Influenza Vaccines

Avian influenza virus(AIV)of the H9N2 subtype is endemic in China.Recently,H9N2 viruses of the dominant S genotype frequently provide the internal gene cassette for the newly-emerged reassortant influenza viruses,including H5N1,H7N9 and H10N8 subtypes.These novel influenza viruses can infect birds and humans,and thus persistent spread of H9N2 AIV in poultry flocks is a dual threat for both poultry industry and public health.In addition,H7N9 AIV is hitting more and more areas in China and the emerged highly pathogenic variant of H7N9 virus has caused severe infection in poultry and human beings.Currently,inactivated vaccines are widely-used for prevention and control of AIVs,however,there are several disadvantages of inactivated vaccines,such as labor-intensive and time-consuming for vaccination,virulence residue and absence of cellular and mucosal immunity.Therefore,it is needed to develop new live influenza vaccines.In this study,two live vector vaccine candidates for H9N2 and H7N9 subtypes AIVs were generated,aiming at providing additional means for the prevention and control of AI of these two subtypes.Newcastle disease virus(NDV)belongs to Avian paramyxovirus serotype-1(APMV-1),which has been used as a vaccine vector for poultry disease to express foreign antigens from other pathogens and these vaccines are generally immunogenic and efficacious in chickens.However,a bottleneck for the application of NDV-vectored vaccines in commercial poultry flocks is due to the presence of high maternal Newcastle disease(ND)antibody.In order to circumvent this interference,a chimeric NDV vector was generated in which the ectodomains of the fusion(F)and hemagglutinin-neuraminidase(HN)proteins were replaced by those from antigenically-distant avian paramyxovirus serotype-2(APMV-2).In addition,the hemagglutinin(HA)gene of H9N2 or H7N9 AIV was inserted between the phosphoprotein(P)and matrix(M)genes of the chimeric virus vector and resulted in two recombinant vaccine candidates,which can serve as alternatives for prevention of H7N9 and H9N2 in commercial poultry flocks with high ND maternal antibody.1.Complete genome sequence analysis and characterization of an avian paramyxovirus serotype-2(APMV-2)isolateBased on the genomic sequences of APMV-2 strains available in GenBank,10 pairs of primers were designed to obtain the complete genome information of an APMV-2 isolate(APMV-2/T4).The whole-genome sequence homology between APMV-2/T4 and other APMV-2 strains was 68.1-77.6%.The amino acid sequence identities of the predicted F and HN proteins of T4 strain with other APMV-2 strains were 69.5-78.3%and 67.5-78.9%,respectively.The phylogenetic analysis of the whole genome,the F gene and HN gene indicated that APMV-2/T4 and three APMV-2 isolates from China belonged to genotype ? of APMV-2.The isolate APMV-2/T4 shared the similar gene structure and transcriptional regulatory elements with NDV.In addition,For the amino acid sequences spanning the F protein cleavage site(DKPASR ? F)of APMV-2/T4 contain non-consecutive basic residues,which is similar to those of lentogenic NDV strains.Biological characterization of the virus showed that the APMV-2/T4 strain effectively replicated in chicken embryos and is safe for chickens.Both commercial chickens and SPF chickens had comparable HI antibody levels after immunization with APMV-2/T4,and HI titers reached 7.7 log2 at 3 weeks after immunization,indicating that APMV-2/T4 can replicate effectively in chickens in the presence of ND antibodies and induce antibody response.2.Construction and immunization of a chimeric Newcastle disease virus vector in the presence of Newcastle disease maternal antibodyA chimeric NDV vector(AI4-T4FHN)based on an attenuated genotype ? virus backbone was generated in which the ectodomains of the fusion(F)and hemagglutinin-neuraminidase(HN)proteins were replaced by those from APMV-2/T4.After 10 passages,the rescued chimeric NDV vector virus AI4-T4FHN still can replicate efficiently in chicken embryos and is safe for chickens.The results of cross-HI test showed that antiserum of APMV-2/T4 had high HI titers against AI4-T4FHN.In contrast,antiserum of NDV exhibited low HI titers against AI4-T4FHN.There was an apparent antigenic variation between the chimeric virus and NDV.The influence of ND antibody on immunogenicity of the chimeric NDV vector was examined in 1-day-old commercial chickens and SPF chickens.No clinical symptoms and pathological damage were observed after inoculation.The virus was quickly cleared,confirming its safety as a vaccine vector.The results of seroconversion showed that the level of HI antibody induced by LaSota and the chimeric virus AI4-T4FHN was comparable in SPF chickens,but in commercial chickens,HI antibody titers in the AI4-T4FHN-immunized birds were significantly higher than those in LaSota-immunized group.Therefore,the immune efficacy of LaSota was strongly affected by ND maternal antibodies,while the AI4-T4FHN could effectively induce the corresponding antibody in commercial chickens with high level ND maternal antibodies.Taken together,the chimeric NDV generated in this study is a promising vector to develop live avian vaccines that can evade the interference of ND maternal antibody.3.Construction and evaluation of chimeric Newcastle disease virus vector vaccine expressing the HA protein of H7N9 subtype avian influenza virusThe HA gene of the H7N9 virus was inserted between the P and M genes of the chimeric NDV(AI4-T4FHN)backbone or the attenuated NDV(AI4)backbone.Meanwhile,the NDV gene-end(GE)-like sequence in the HA gene(1185AGAAAA AA1192)was modified by silent mutagenesis.The rescued recombinant viruses AI4-T4FHN-H7 and AI4-H7 could replicate efficiently in chicken embryos,and the HA titer reached 10 log2.The results of Western blot and indirect immunofluorescent assay(IFA)showed that the HA protein can be stably expressed in NDV vectors.To enhance immune response against H7N9,two different vaccination strategies were applied,1-day-old chickens were primed with the chimeric vector vaccine AI4-T4FHN-H7,and boosted with the homologous vector vaccine(AI4-T4FHN-H7)or the heterologous vector vaccine(AI4-H7).At 2 weeks post boost,heterologous prime-boost vaccination induced higher HI antibody titers(1.5-fold)compared to homologous prime-boost vaccination,whereas HI titers induced by these two vaccinations were low.However,high levels of total H7N9-specific IgY titers were elicited as measured by ELISA.Vaccination of commercial chickens with these two programs conferred a complete protection against challenge with highly pathogenic H7N9 virus.However,a significant decrease in the titer of the shedding virus from the challenged birds was observed in heterologous prime-boost group compared with homologous prime-boost group.The results showed that prime with the chimeric vaccine AI4-T4FHN-H7 and boost with AI4-H7 can protect commercial chickens against infection with highly pathogenic H7N9 virus.4.Construction and evaluation of chimeric Newcastle disease virus vector vaccine expressing the HA protein of H9N2 subtype avian influenza virusThe HA gene of the H9N2 virus was inserted between the P and M genes of the chimeric NDV(AI4-T4FHN)or the attenuated NDV(AI4)backbone.The rescued recombinant AI4-T4FHN-H9 and AI4-H9 showed high levels of replication in chicken embryos and are safe for chickens.The expression of the HA protein was detected by Western blot and IFA in the cells infected with the recombinant viruses.AI4-T4FHN-H9 induced High levels of H9N2-specific HI antibody in chickens with high levels of pre-existing ND antibody.Additionally,results of challenge-protection experiments showed that AI4-T4FHN-H9 immunization significantly reduced the amount of shedding virus and alleviate the pathological damages.In order to investigate the immunogenicity and efficacy of AI4-T4FHN-H9 in conmmercial chickens,10-day-old commercial chickens were immunized with AI4-T4FHN-H9 or AI4-H9,respectively.AI4-T4FHN-H9 can overcome the interference with ND MDA and induce high level of vector-specific antibody.The level of H9-specific HI antibody induced by AI4-T4FHN-H9 was significantly higher than that induced by AI4-H9 at 3 weeks after immunization.These findings suggested that the chimeric vectored H9N2 vaccine can alleviate the interference of ND MDA,induce antibody response and provide good protection against H9N2 challenge.