| In practice,prevention of H7N9 subtype influenza viruses is usually achieved by vaccine immunization,where viral vector vaccines combine the advantages of recombinant DNA technology and live attenuated vaccines,allowing for rapid vaccine construction based on synthetic DNA and the induction of a full range of immune responses,including humoral,cellular and mucosal immunity,without the addition of adjuvants.However,it was found that a series of recombinant vector vaccines containing the HA protein of the H7N9 subtype of influenza virus,including but not limited to the Newcastle disease vector,induced the following immune response characteristics in mammals and birds:high levels of specific IgG antibodies and low levels of HI/VN antibodies;and high levels of protection against H7N9 attack.This immune profile cannot be adequately explained by current mechanisms for evaluating the immune effect of vaccines;therefore,this study will elucidate the immune mechanism of a vector vaccine containing H7N9 HA protein from two perspectives:the identification of the immunodominant antigenic epitope and the exploration of the main ways in which non-neutralizing antibodies play a role in the immune process.The ways and mechanisms by which non-neutralizing antibodies play a role in the immunization process of the H7N9 recombinant vector vaccine are explained through the two dimensions.1.Identification of the dominant antigenic epitope of the HA protein of H7N9 avian influenza virusIn this study,we analyzed and identified the immune sera of the H7N9 HA protein vector vaccine,the empty vector immune sera and the commercial inactivated vaccine,and the levels of HI antibodies against Newcastle disease in both the vector vaccine and the empty vector vaccine immune sera exceeded the detection limit of 32,while the levels of HI antibodies induced by the vector vaccine against H7N9 were much lower than the detection limit and the commercial inactivated vaccine sera,which reached 128.There was no significant difference between the total antibody levels induced by the vector vaccine and the commercial inactivated vaccine against H7N9.On this basis,a passive immunity model was developed to evaluate the protective effect provided by the vector vaccine immune serum.600 μl of vector vaccine immune serum,commercial vaccine immune serum and normal serum were injected into 7-day-old chicks,and strong H7N9 virus attack was carried out 1 h after injection,and the survival status of chicks was continuously observed.The results showed that both commercial vaccine sera with high levels of neutralizing antibodies and vector vaccine sera with low levels,of neutralizing antibodies and high levels of non-neutralizing antibodies provided 100%protection against the strong H7N9 virus.These results indicate that after excluding interference from cellular immunity,in a seropositive model of passive immunity,non-neutralizing antibodies not only protect but also play a very important role in the immunization of the vector vaccine against viral attack.On the basis of this study,a series of previously designed monoclonal antibodies targeting different sites of HA protein were used as competitive antibodies to bind competitively with H7N9 vector vaccine immune sera to the encapsulated antigen.A monoclonal antibody targeting the G151 site of HA protein was selected by comparing the competitive binding rates,with a competitive binding rate of 40%.After mutating this site,the mutant virus and the original virus were used as the envelope antigen,and different concentrations of monoclonal antibody were used to bind competitively to the vector vaccine immune serum.Further,ELISA experiments were performed with mutated viruses at adjacent sites S150 and S152 to compare the binding rates with the sera,and it was found that the viruses mutated at single sites G151,S150 and S152 respectively showed significantly lower binding rates with the vector vaccine sera compared to the original viruses.These results suggest that the G151,S150 and S152 loci are the dominant antigenic epitopes against which antibodies induced by the vector vaccine are directed.2.Development of a method for the detection of complement killing effect mediated by immunization with Newcastle disease virus vector vaccine containing H7N9 HA proteinThe study was conducted by constructing cell lines stably expressing HA protein to mimic target cells after viral infection,using vector vaccine immune serum as a provider of antibodies and complement,and measuring the extent of complement killing effect by detecting lactate dehydrogenase(LDH)release.A series of experiments were conducted to determine and optimize the series of indicators in the assay,including:the species of target cells,the concentration of target cells,and the concentration of serum in the experimental group,which was finally determined to be:293T,0.75x104/100μL,and a 1:10 dilution of 10μL.Meanwhile,the constructed lentiviral packaging plasmid Fv115-HA and the helper plasmid were used in a lentiviral packaging method 293T cells were co-transfected,and stable lentiviral-infected 293T cell lines were screened under puromycin stress.The results showed that the H7N9 HA protein was stably and efficiently expressed on the surface of 293T cells,and a 293T cell line with stable expression of HA protein was successfully constructed,providing a basis for subsequent experiments.3.Protection mechanism of Newcastle disease virus vector vaccine immune sera containing H7N9 HA proteinThe study focused on the complement killing effect of immune sera on target cells by measuring LDH release and on virus particles by indirect immunofluorescence and determination of viral TCID50.The experiments investigated the complement killing effect of heat-inactivated immune sera on target cells and virus particles,the complement killing effect of sera treated with the complement inhibitor sodium polyanethole sulfonate(SPS)on target cells and virus particles,and the complement killing effect of immune sera on viruses with mutated antigenic dominant epitopes and their infected cells,respectively.protection mechanism.The final results showed that the complement killing effect on target cells and virus particles was strongest when both complement and antibody were present in the serum;the killing effect of the immune serum on infected cells and particles was greatly reduced when only one complement or antibody was present;and the complement killing effect was largely lost after mutating the dominant antigenic epitope recognized by the immune serum on the H7N9 HA protein. |
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