| Since its first appearance in the Yangtze River Delta region of China in 2013,the H7N9 subtype avian influenza virus(AIV)has caused serious human infections,resulting in a high mortality,which has posed a great threat to public health.At the end of 2016,the highly pathogenic(HP)H7N9 AIV appeared,causing outbreaks in chicken flocks,which brought huge economic losses to the poultry industry.The use of vaccines is one of the most effective measures to prevent and control AIVs.However,traditional inactivated vaccines used in clinic can only be immunized by intramuscular injection,which has some disadvantages,such as being time-consuming and laborious,only inducing humoral immunity,and causing stress reaction in poultry flocks.Newcastle disease virus(NDV)is an ideal vector for constructing a bivalent vaccine for poultry.It has good safety,high replication titer and can induce comprehensively immune responses,such as mucosal,humoral,and cellular immunity.Moreover,NDV can also be used for mass immunization through spraying,drinking water,etc.Therefore,the purpose of this study was to use the NDV as a vector to express the main protective antigenic protein of H7N9 AIV——hemagglutinin(HA),to construct a live vectored-vaccine and evaluate its efficacy.In addition,since the H7N9 AIV is a newly-emerged zoonoses,the research on epitopes of viral HA protein is not comprehensive and in-depth.Therefore,a series of H7N9 HA-specific monoclonal antibodies were used to map and analyze the B-cell antigenic epitopes of the HA protein and identify the immunodominant epitopes,which can provide a reference for vaccine design and modification.1.Construction and evaluation of a recombinant Newcastle disease virus-vectored vaccine expressing the HA gene of H7N9 subtype avian influenza virusIn this study,an attenuated genotype VII NDV(rAI4)was used as the backbone,and the H7N9 AIV strain(A/chicken/Guangdong/GD 15/2016,GD15)which has a good cross-reactivity with low pathogenic(LP)and high pathogenic(HP)strains of various years was selected as the donor of the HA gene.A recombinant NDV-vectored vaccine(rAI4HA)expressing the H7N9 HA gene was constructed by reverse genetics.In vitro,the basic biological characteristics of the recombinant vaccine rAI4HA are similar to the parental virus rAI4.Both of them have high yields in cells and ECEs,and show low pathogenicity to ECEs.In addition,the recombinant virus was able to stably express the HA protein after continuous passage in ECEs,and no genetic mutation was found,indicating that the recombinant virus has a good genetic stability.In vivo,106 50%embryo infectious dose(EID50)of rAI4HA was used to immunize 2-week-old SPF chickens via the nasal and ocular route.Two weeks after immunization,the recombinant virus could induce a high level of NDV-specific hemagglutination inhibition(HI)antibody in chickens.However,rAI4HA failed to induce satisfactory H7-specific HI antibody titers(mean titer:2 log2),only a 20%seroconversion rate(HI≥4 log2)was observed.Positive H7-specific HI and virus neutralization(VN)antibodies were not detected in most chickens.However,it is worth noting that a high level of H7-specific IgG antibodies was detected in the immune serum when using the ELISA method(mean titer:200).The challenge results showed that when chickens were challenged with a lethal dose of HP H7N9 subtype AIV,all unimmunized chickens were died in 3 days showing severe clincal symptoms,all rAI4-immunized chickens also died in 7 days,but all rAI4HA-immunized chickens survived without showing any clincal symptoms,and the level of virus shedding was significantly suppressed compared with rAI4-immunized chickens.The results show that vaccine candidate rAI4HA has good safety,high yield and genetical stability.It can provide 100%protection for HP H7N9 challenge and significantly inhibit the level of virus shedding after a single dose of immunization,indicating it is promising in controlling AIV.In addition,rAI4HA induced low titer of HI antibody but high titer of IgG antibody,suggesting at least in NDV-vectored H7N9 vaccines,traditional serological standards cannot accurately reflect the true immunity effect.2.Prediction and mapping of B cell epitopes of the H7N9 hemagglutininAlthough rAI4HA only induces low levels of H7-specific HI/VN antibodies,it can induce a high level of H7-specific IgG antibodies,the reason may be related to the special characteristics of H7N9 HA protein,which means the non-neutralizing B-cell epitopes in the HA protein becomes the immunodominant epitopes instead of traditional HI/VN epitopes.Therefore,in this study,multiple tools were used to predict the potential B-cell epitopes of the HA protein.After analyzing the prediction results comprehensively,19 peptides were designed and synthesized.Then 24 strains of H7N9 HA-specific monoclonal antibodies(mAbs)were used to react with the peptides to map linear B-cell epitopes of the HA protein,8 strains of mAbs were able to specifically bind to the peptides.Because some mAbs recognize the same epitope,3 new linear B-cell epitopes were screened.These three epitopes were located in the stem domain of HA protein,and none of the mAbs recognize them have the HI/VN activity.Then,the three peptides were truncated to identify the epitopes precisely.The results showed that the key motifs of Epitope-13 are 373-TAA-375 and the key amino acid of Epitope-15 is 459-E.When these key motifs were mutated,the reactivity of HA protein with corresponding antibodies was weakened significantly or lost completely.But truncated peptides of peptide-19 did not have the reactivity with the mAb,then phage display method was used to identify the epitope this mAb targets to.The results showed that the motif of the mimotope is HEMWGLHSFDMT.After analyzing the protein sequences,the mimotope is located in the stem domain of HA protein(428-450 aa).The Epitope-19 is a conformational B-cell epitope composed of discrete amino acids,and 429-E-431-W-445-H-448-D are the key motifs.3.Identification of immunodominant epitopes of the H7N9 hemagglutininThe NDV-vectored H7N9 vaccine mainly induces non-neutralizing antibodies,suggesting that epitopes recognized by these antibodies are immunodominant epitopes of HA protein.In order to identify these immunodominant epitopes,serum generated with different H7N9 vaccines were used to react with synthesized peptides by competitive ELISA method.The results showed that none of the 19 peptides reacted with the mice or chicken serum immunized with the NDV-vectored H7N9 vaccine and inactivated H7N9 vaccines.Only some of peptides that locate in the stem domain of HA had a reactivity with chicken serum generated by H7N9 infection,which indicating that serum generated with H7N9 vaccines may recognize conformational epitopes of HA protein.In addition,previous peptide-ELISA assay showed that most mAbs did not have a reactivity with linear B-cell epitopes,suggesting these mAbs may also recognize conformational epitopes.Therefore,the competitive ELISA method was performed to detect the reactivity of HA protein with these mAbs and the immune serum generated by NDV-vectored H7N9 vaccine.The results showed that 2B11 1F3 and 4B7 4D5 mAbs have significant competitive effects,which can reduce the binding ability of immune serum with HA protein by about 50%,indicating that these epitopes are immunodominant epitopes during antibody responses induced by the NDV-H7N9 vaccine.And when using the mixture of these two mAbs to compete with immune serum,we found that the inhibitory efficacy is not improved,indicating these two mAbs may bind the same epitope.Then we used the phage display method to identify the epitope recognized by 4B7 4D5,and the results showed that the mimotope was SNTALPSKFYGY and the key motif were 331-N-336-P-361-Y-362-G.These results showed that the antibody responses induced by H7N9 vaccines could not mimic the antibody responses during viral infection.The 331-N-336-P-361-Y-362-G motif in HA protein was identified as immunodominant epitope during antibody responses induced by the NDV-vectored H7N9 vaccine. |
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