Biological Characterization And Efficacy Evaluation Of Monoclonal Antibodies Against The Hemagglutinin Of H7N9 Subtype Avian Influenza Virus

In 2013,H7N9 subtype avian influenza virus(AIV)emerged in the Yangtze River Delta region in the East of China,which has caused five waves of epedemic in humans so far.Among them,the fifth wave caused the largest number of infections,and highly pathogenic variant viruses appeared in this period,which caused huge losses to poultry industry.According to the statistics of Food and Agriculture Organization(FAO),H7N9 subtype AIV has resulted in 1568 laboratory-confirmed cases of human infection,and 616 cases died,with mortality rate as high as 40%,which poses a major threat to public health.H7N9 subtype AIV has the potential to cause the next influenza virus pandemic,therefore the development of highly effective antiviral agents is of great significance for the prevention and control of H7N9 pandemic.In addition,due to the immune pressure of vaccine and the frequent genetic reassortment of influenza virus,H7N9 subtype AIV has evolved significantly in genetic and antigenic levels.In addition,the virus has well-adapted in ducks,the "natural reservoir" of influenza virus,which may facilitate further variation of the virus.Therefore,understanding epitope characteristics of H7N9 virus is of great significance for the development and optimization of vaccines and antiviral agents.In recent years,monoclonal antibody(mAb)becomes an attractive measurement for the prevention and treatment of major viral infectious diseases of humans,and is also a powerful tool to characterize the antigenicity of viruses.In this study,biological activity of the mAbs against the hemagglutinin(HA)of H7N9 subtype AIV and their protective efficacy against H7N9 virus infection in a mouse model were evaluated.In addition,the epitopes recognized by the mAbs were identified and antibody engineering platform was established based on a cross-reactive mAb.The results showed that a mAb with strong virus neutralizing activity against H7N9 virus was screened,which had a high protective efficacy against a lethal infection with H7N9 virus in the mouse model.Three new epitopes in the HA protein were identified,and the mAb recognizing the conserved epitopes in the HA stem had a good cross-reactivity with different subtypes within group 1 and group 2.The sequences encoding the viraible regions of the heavey and light chains of this broadly-reactive antibody were determined and the antibody engineering platform was established.The isotype of this mAb was switched from IgG1 to IgG2a,underpinning further enhancement of efficacy of this antibody against H7N9 virus infection.Thus,our results provided a good mAb candidate for therapeutics of H7N9 virus infection and also presented new information for further understanding the epitope characteristics of H7N9 subtype AIV1.Biological characterization of the mAbs against the HA of H7N9 subtype AIVFour mAbs(1A1 1D7,1B10 1D1,4B7 4D5,5D3 1B5)were previously prepared against the HA protein of H7N9 virus(A/chicken/Guangdong/GD 15/2016,GD15).Their hemagglutination inhibition(HI)activity,virus neutralization(VN)activity and binding reactivity with the HA protein were determined.The results showed that the four antibodies had strong reactivity with the HA protein,as evidenced by high reactivity with the recombinant HA protein expressed in baculovirus and H7N9 virus-infected cells.4B7 4D5 showed potent HI and VN activities against H7N9 virus,with HI titer of 8 log2 and VN titer of 1:5120.4B7 4D5 only reacted with the H7 subtype viruses,suggesting its subtype specificity.The other three mAbs had neither HI nor VN activity,but exhibited cross-reactivity with different subtypes of influenza virus:1A1 1D7 and 1B10 1D1 reacted with the H7 and H15 subtypes,while 5D3 1B5 had a broad-spectrum reactivity with different subtypes of group 1 and group 2 influenza virus.Therefore,biological activities and cross-reactivity of H7N9 HA-specific mAbs were systematically analyzed,which laid a foundation for the subsequent epitope screening and in vivo efficacy evaluation of these antibodies.2.Evaluation of protective efficacy of the mAbs against the HA of H7N9 subtype AIVHerein,the protective efficacy of the four mAbs against H7N9 virus infection was evaluated through passive immunization in a BALB/c mouse model.A mouse-adapted H7N9 virus(JTC4 M11)obtained after 11 consecutive passage of the wild-type virus(JTC4)in BALB/c mice was used as the challenge strain.First,the efficacy of the antibodies was tested in a prophylactic setting,the results showed that the untreated mice showed rapid weight loss and severe clinical signs in a short time after infection,and all died within 14 days after challenge.1A1 1D7 and 5D3 1B5 could not provide protection,while 1B10 1D1(10,20,30 mg/kg)and 4B7 4D5(5,10,20,30 mg/kg)provided 100%protection against H7N9 virus infection,and virus isolation ratio was significantly lower than that of the control group.Although 1B10 1D1 protected the mice from death caused by H7N9 infection,high virus isolation ratio in the lung and turbinate as well as marked pathology in the lung were observed.In contrast,virus isolation ratio and severity of lung tissue damage of the mice treated by 4B7 4D5 were remarkably lower compared to the untreated and 1B101D1-treated mice,indicating a good efficacy of 4B7 4D5.Subsequently,the therapeutic effect of 4B7 4D5 against H7N9 virus was evaluated in a therapeutic setting,the result showed that administration of 4B7 4D5 at a high dose(30 mg/kg)12 h after infection provided a complete protection against H7N9 virus challenge,whereas the mAb at the same dose administered 24 h after infection only provided 40%protection.These results indicate that 4B7 4D5 can provide good protection against H7N9 virus infection in the prophylactic and the therapeutic setting when administered early(12 h).3.Identification of the epitopes recognized by the mAbs against the HA of H7N9 subtype AIVCharacterization of the epitopes in the HA protein is the key for understanding antigenic variation of H7N9 subtype AIV,and is also an important basis for developing vaccines and antiviral bioreagents.Herein,the epitopes targeted by three mAbs(1B10 1D1,4B7 4D5,5D3 1B5)were identified using peptide-based ELISA,phage display random peptide library screening or immune escape screening.Additionally,the variation as well as localization of the epitopes was analyzed by bioinformatics methods.Immune escape screening was used to map the epitope recognized by 4B7 4D5 because this mAb has strong VN activity.There was a significant decrease(8-fold)in HI titers of 4B7 4D5 against the screened immune escape viruses compared to that against the wild-type GD15 virus.G151E/R and I335V substitutions were identified in the HA gene of the escape mutants.The recombinant H7N9 viruses carrying artificial G151E and I335V substitutions were generated using reverse genetics and significant decreases in HI and VN reactivity with 4B7 4D5 were only seen for the virus harboring G151E substitution.These data suggested that 4B7 4D5 recognized HA G151 as the key amino acid of the epitope.In addition,the 431W-433Y-437L motif in the HA stem was identified as the epitope recognized by 5D3 1B5 using peptide-based ELISA and phage libray screening.The 220Q-225S-227G motif in the HA head was identified as the epitope recognized by 1B10 1D1 using the same strategy.Additionally,mutation of the identified epitopes significantly reduced the reactivity of H7N9 virus with the corresponding antibodies.Variation and localization analysis showed that the epitope(G151)recognized by 4B7 4D5 located in the antigenic site A in the HA head,which was highly conserved among the H7 subtype;the epitope targeted by 1 B10 1D1 located in the 220-loop near the receptor binding site in HA,which was highly variable among different subtypes.Notably,the epitope targeted by 5D3 1B5 located in the C-helix domain in the HA stem,which was highly conserved among influenza viruses of different subtypes.Therefore,three new epitopes in the HA protein of H7N9 virus,including two in the head and one in the stem,were identified,which provided new information for understanding the epitope characteristics of H7N9 subtype AIV4.Expression of subclass-switching chimeric antibody based on the mAb 5D3 1B55D3 1B5 has a broad cross-reactivity with different influenza subtypes of group 1 and group 2.The subclass of this mAb is IgGl and it doesn’t have VN activity and protective efficacy against H7N9 virus infection.Non-neutralizing antibodies are protective through activating Fc-dependent immune effector functions,and the affinity of the Fc with Fc receptors(FcR)on immune effector cells is a critical determinant for the efficacy.The murine IgG2a subclass has the highest affinity to FcR,while the IgGl subclass has the lowest affinity.Therefore,we aimed to enhance the efficacy of 5D3 1B5 by switching its subtype from IgG1 to IgG2a.The coding sequence of the variable region of the heavy(VH)and light chain(VL)was determined,the complementarity-determining regions(CDR)of the variable region were located,and molecular docking between the variable region and the HA protein was analyzed.The VH and VL genes were cloned into the expression vectors(pFUSEss-CHIg-mg2a and pFUSE2ss-CLIg-mK)that already contain murine IgG2a constant region.The plasmids were co-transfected into Chinese hamster ovary(CHO)cells and the expression of the chimeric antibody was measured by ELISA,IFA and Western blot.The results demonstrated that the chimeric antibody was successfully expressed after plasmid con-transfection,and the subclass was switched from IgG1 to IgG2a.The reactivity of the chimeric antibody witht the HA protein of H7N9 virus was comparable with that of the parental antibody 5D3 1B5.Therefore,subclass-switching chimeric antibody based on 5D3 1B5 was generated,which lays a foundation for the optimization of antibody efficacy.