Epitopes Mapping Of HA2 Protein In H5 Subtype Avian Influenza Virus By Using Peptide Microarray And Construction Of DIVA Vaccine Candidates

The H5 subtype avian influenza(AI)has been circulating in our country for a long time.It is one of the main poultry diseases causing high morbidity and mortality.Due to immune selection pressure,the evolution of H5 subtype avian influenza virus(AIV)becomes more and more complex,and antigens of different clades of H5 AIVs vary greatly.The updating cycle for vaccine is getting shorter accordingly.Differentiating infected from vaccinated animals(DIVA)vaccine can be used to distinguish between naturally infected animals and immunized animals,which brings convenience to monitoring and clean-up detection of H5 subtype AIV,However,common DIVA strategies are rarely applied to practice because of viral recombination and poor immunogenicity.Hemagglutinin(HA)is a glycoprotein which can induce neutralizing antibodies.Compared with HA1,HA2 is more conservative and has gradually become the main target protein in the study of universal epitopes.In view of high throughput,strong stability,and high sensitivity,peptide microarray has been well applied in clinical testing,pharmaceutical R&D or other fields.Therefore,epitopes mapping of H5 subtype AIV HA2 protein by using peptide microarray is expected to provide new directions for the design of DIVA vaccine.1.Epitopes mapping of HA2 protein in H5 subtype avian influenza virus by using peptide microarrayAccording to the HA2 protein of H5 subtype AIV A/Mallard/Huadong/S/2005,18 overlapping peptides(10 amino acids overlapped between two adjacent peptides)were synthesized and sampled onto iPDMS(Modified silica gel film)to prepare peptide microarray.First,the binding reaction conditions for serum and peptide microarray were determined.Then the binding activity of sera against different subtype AIVs to peptied microarray was deterimed.The result showed that the 14th peptide(KELGNGCFEFYHKCDNECME)could bind to immunized sera from 9 AIV subtypes(H1,H3,H4,H5,H6,H7,H8,H9 and H10).The 12th and 17th peptides(HDSNVKNLYDKVRLQLRDNA,QYSEEARLKREEISGVKLES)were identified as specific peptides which could only bind to immunized sera of H5 subtype AIV.2.Identification of a universal antigen epitope of avian influenza virus based on HA2 proteinTo confirm the broad-spectru.characteristics,the 14th peptide was further cut into four overlapping peptides(8 amino acids overlapped between two adjacent peptides)which were named 14-1(KELGNGCFEFY),14-2(GNGCFEFYHKC),14-3(CFEFYHKCDNE)and 14-4(FYHKCDNECME).The binding activity of sera against different subtype AIVs to peptides was determined.The result showed that the 14-4 peptide(485-FYHKCDNECME-495)could bind to immunized sera agaist Hl,H3,H4,H5,H6,H7,H8,H9,and H10 subtype AIVs.Meanwhile,14-1,14-2,and 14-3 peptide could only bind to few kinds of sera against AIVs.Based on the sequence of five amino acids(YHKCD)in the 14-4 peptide,16 mutation/deletion patterns were designed and recombinant AIVs were rescued by reverse genetic technique.The binding activities of rescued viruses to 14th peptide immune serum were analyzed by western-blot.The result showed that all AIVs with deletion patterns could not be rescued.Only AIVs substituted with a single amino acid(Y/H/K/D)to glycine(G)could be rescued.Rescued viruses were named S-Y-G,S-H-G,S-K-G,and S-D-G.Substitution of amino acids(K or D)in rescued viruses resulted in reduced binding activity of serum to HA2 protein.The Immune Epitope Database analysis revealed that the peptide also contained the T cell epitopes which were MHC-II restricted.Some research showed that they could affect the release of IL-2 and IFNg.All data suggested that 14-4 peptide might be a novel universal target for diagnostics.3.Construction of DIVA vaccine candidates based on specific epitope of HA2 protein in H5 subtype avian influenza virusSix H5 subtype AIV strains CK/JS/ZJ0104/16(2.3.2.1e5 ZJC),CK/JS/DT0303/16(2.3.2.1e,DTC),GS/JS/HJ0105/16(2.3.4.4c,HJG),GS/JS/YZ1111/16(2.3.4.4d,YZG),GS/GD/GD0531/17(2.3.4.4d,GDG)and CK/JS/YZ1111/16(2.3.4.4e,YZC),which were from different clades with stable hemagglutination(HA)titers,were selected to prepare immune sera.Vaccine candidate strain was screened by cross hemagglutination inhibition(HI)test of these immune sera.Since 12th peptide(HDSNVKNLYDKVRLQLRDNA)and 17th peptide(QYSEEARLKREEISGVKLES)were identified as specific epitopes for H5 subtype AIV.The recombinant viruses with substitution of these two epitopes by corresponding epitope of H1 or H3 subtype AIVs were designed and rescued.Results showed that the HI titers of DTC's immune serum against ZJ0104,DTC,HJG and GDG were higher than 7 log2,and the HI titers of that against YZG and YZC were 4 log2 and 21og2,respectively.Compared with other viruses,DTC had better broad-spectrum antigenicity and could be used as vaccine candidate strain of H5 subtype AIV.The recombinant vriuses DTC-Lo-121,DTC-Lo-171,DTC-Lo-173,DTC-Lo-121171 and DTC-Lo-121173 were successfully rescued by epitope substitution.Further microarray tests indicated that immune sera of DTC-Lo-121 with single-epitope substitution had positive responses both to the 12th peptide and 17th peptide.Immune sera of DTC-Lo-171 and DTC-Lo-173 didn't bind to 12th peptide and 17th peptide.Immune sera of DTC-Lo-121171 and DTC-Lo-121173 with double epitope substitution could bind to 12th or 17th peptide.Those results showed that DTC-Lo-171 and DTC-Lo-173 with single-epitope substitution had better DIVA characteristics,and could be used as DIVA vaccine candidates for protection of H5 subtype AI.This may provide possibility for further development of DIVA vaccine.