Construction And Evaluation Of Immunization Efficacy Of Recombinant Turkey Herpesvirus Expressing Mosaic HA Protein Of H5 Subtype Avian Influenza Virus

H5 subtype avian influenza is caused by member of influenza virus A genus of the Orthomyxoviridae family.The viruses are high pathogenic among poultry and mortality rate in infected flocks often approach 100%.The natural hosts of type A influenza viruses are aquatic birds,but the viruses expand their host range from bird to infect mammals,especially causing fatal infection in human,and spread to several continents as migratory birds migrating.Therefore,the effectiveness of prevention and control of H5 subtype avian influenza is vital to human public health.The H5 subtype avian influenza virus(AIV)surface glycoprotein hemagglutinin(HA)vary rapidly.The virus has continuously evolved into different genetic clades.In recent years,two branches of clade 2.3.4.4 and clade 2.3.2.1 of H5 AIV are prevalent in China,which were controlled with inactivated vaccines Re-8 and Re-6 respectively.At present,massive vaccination is the most effective way to prevent and control AIV outbreak.The outbreak of H5 AIVs is often due to failure of cross-protection of current vaccines.To achieve effective cross-protection,the antigenicity of a vaccine strain is required to match that of circulating strains.Unfortunately,the timely and accurate selection of vaccine strains is challenging because the influenza virus changes its antigenicity rapidly by either mutation or reassortment.As a result,AIV vaccines have to be updated every 2-5 years.Now,the development of broad-spectrum vaccines has become ne of the hotspots of H5 subtype AIV vaccine,including the design of vaccines against the conserved stem and the Mosaic vaccine.Meanwhile,cellular immunity plays an important role in the process of eliminating influenza viruses.Unlike live vaccines that stimulate both humoral and cellular immunity,the existing commercial vaccine is usually inactivated vaccine,which mainly stimulates humoral immunity.Recombinant live viral vector expressing AIV HA gene is one of the main research direction of the influenza virus vaccine.The common viral vector includes Turkey herpes virus,adenovirus,poxvirus,Newcastle disease virus and so on.Therefore,it is of practical significance to develop a live vaccine with better cross protection and simultaneously stimulating humoral and cellular immunity.1 Construction of recombinant transfer plasmid encoding Mosaic HA gene of H5 subtype avian influenza virusBased on the "Mosaic" method introduced in the field of human immunodeficiency virus(HIV)vaccinology,we try to design H5 broad-spectrum vaccine containing "Mosaic" HA proteins.First,we downloaded all H5 HA gene sequences available in the influenza virus database GISAID(http://platform.gisaid.org/epi3/frontend#48ef8b)and screened them to exclude the incomplete and repeated sequences.After sequence alignment,the phylogenetic tree of H5 gene was built.Only the sequences of clade 2 of H5 AIV HA genes were selected and translated into amino acid sequence.The protein sequences were uploaded into the Mosaic Vaccine Designer tool webpage(https://www.hiv.lanl.gov/content/sequence/MOSAIC/makeVaccine.html)to optimizae a vaccine cocktail with Mosaic tool using a genetic algortithm and to create a mosaic sequence cocktail,H5 mosaic protein sequence(H5M).The antigen epitope coverage rate of the Mosaic sequence was predicted by two online tools:Epitope Coverage Assessment Tool(Epicover)and Positional Epitope Assessment Tool(Posicover).The results showed that the antigen epitope coverage rate of H5M sequence was significantly higher than that of four H5 AIVs wild strains,SQ1402,YZ1111,Z6285,and 0520.For example,H5M protein perfectly(9/9)matched 72.99%of 9-mers potential epitopes in 4933 HA natural sequences;92.64%of potential epitopes matched 8 of 9 and 98.01%matched 7 of 9.In contrast,HA protein of four H5 AIV wild strains covered only 56.12%-56.3%(9 of 9),79.33%-83.98%(8 of 9)and 92.88%-95.16%(7 of 9).Thus,Mosaic sequences contain more antigenic epitopes than natural sequences.H5M gene sequence was obtained by optimizing H5M protein sequence using the Gallus gallus codon.The optimized H5M gene was synthesized commercially,and inserted into the plasmid under the control of strong Pec promoter or weak HgB promoter.In addition,HA genes of the representative strain of clade 2.3.2.1 SQ1402 and clade 2.3.4.4 YZ1111 were inserted into the plasmid using the same method as the control of the sequence of natural strains respectively.By identifying with sequencing and indirect immunofluorescence assay(IFA),six transfer plasmids expressing exogenous gene were obtained.2 Construction and evaluation of immunization efficacy of recombinant turkey herpesvirus expressing mosaic HA of H5 subtype avian influenza virusThe recombinant turkey herpesvirus(rHVT)was constructed as followed.Briefly,according to the principle of homologous recombination,genomic DNA of rHVT-GFP expressing green fluorescent protein was co-transfected with the transfer plasmid using calcium phosphate transfection method.After screening,purification and identification,four recombinant HVT(rHVT)obtained named as rHVT-H-H5M,rHVT-P-SQ1402,rHVT-H-SQ1402,rHVT-H-YZ1111 respectively.No rHVT was generated from two plasmids containing strong promoters.In addition,four rHVTs were continuously passaged on CEF cells to evaluate their heredity stability,in which expression of HA gene of every 5 generations of recombinant HVTs HA was detected by IFA.The results showed that four of recombinant HVTs could stably express the target gene after continuous passage for 15 generations.In order to further evaluate the immune protection of rHVT-H-H5M vaccine against H5 subtype AIV,1-day-old SPF chickens were immunized with 4 recombinant vaccines,rHVT-H-H5M,rHVT-P-SQ1402,rHVT-H-SQ1402 and rHVT-H-YZ1111,respectively.Serum of chicken in this experiment was collected for detection of HI titer 1,2,3,4,and 5 weeks post immunization.Five weeks post immunization,the H5 AIV strains 0520(clade 2.3.4.4)and Z6285(clade 2.3.2.1)were used to challenge the chickens in this experiment.The results showed that cross-protection of rHVT-H-H5M has an absolute advantage over that of rHVT-H-SQ1402 and rHVT-H-YZl 111 under the control of the same promoter.It can protect not only the challenge of clade 2.3.4.4,but also clade 2.3.2.1.The survial rate is 60%and 90%respectively.For the same exogenous gene,rHVT-P-SQ1402 vaccine under the control of strong promoter(Pec)induced higher HI antibody levels and provided better protection for the chicken than rHVT-H-SQ1402 vaccine under weak promoter(HgB).Although rHVT-P-SQ1402 vaccine provided 100%protection chicken from the challenge of clade 2.3.2.1 H5 AIV,it only provided 30%protection against clade 2.3.4.4.However,rHVT-H-H5M vaccine both provided higher clinical protection rate and significantly reduced the level of viral shedding.In summary,this study successfully constructed a recombinant turkey herpes virus rHVT-H-H5M expressing Mosaic H5 HA,which induced a certain cross protection against the two dominant circulating clades of H5 subtype avian influenza virus in China,clade 2.3.4.4 and clade 2.3.2.1.This research provides a new idea and method and lays a theoretical foundation for the development of broad-spectrum vaccine.