Identification Of A Linear Cross-reactive Antigenic Epitope In HA Fusion Peptide+helix A Of Type A Influenza Virus And Generation Of Its Recombinant Influenza Virus

Based on the antigenic properties of the viral surface glycoproteins,haemagglutinin(HA)and neuraminidase(NA),influenza A viruses(IAVs)can be subtyped into 18 HA subtypes and 11 NA subtypes.IAVs have a wide host ranges and can cause a respiratory synptom in human and animal.Human seasonal influenza,the occasional endemic of animal influenza or the crossing-host spread of animal influenza directly pose threats to human health and affect the sustainable development of animal husbandry every year.Although the vaccines and antiviral drugs have played important roles in controlling the influenza infections,the current vaccines mostly used for influenza virus are subtype or strain specificity,and need to be updated annually due to the antigenic drift and shift.Therefore,the study on universal or cross-protective influenza virus vaccines is one of hot spots.To identify the universal or cross-protective epitopes is extremely important for generating broad influenza vaccines.In this study,the HA fusion peptide+helix A fragments from human seasonal influenza virus H1N1 and human influenza virus H7N9 were cloned into pGEX-6P-1 respectively,and then expressed in BL21.BALB/c mice were immunized with the purified HA fusion peptide+helix A proteins and seven monoclonal antibodies(mAbs)specific to HA were generated.Epitope mapping for mAbs revealed that the C-ternimus of helix A carries a novel linear cross-reacted antigenic epitope.And a recombinant virus expressed this epitope in NA gene was successfully rescued and identified.1.Cloning and expression of HA fusion peptide+helix A in BL21In order to generate mAbs specific to HA fusion peptide+helix A,the HA fusion peptide+helix A fragments derived from human seasonal influenza virus H1N1 and human influenza virus H7N9 were cloned into pGEX-6p-1 and the recombinant plasmids were designed as pGEX-6P-1-GST-SH-HA2 and pGEX-6P-1-GST-CA-HA2 respectively.The GST-CA-HA2 and GST-SH-HA2 were expressed in the transformed BL21 by IPTG induction.SDS-PAGE showed GST-CA-HA2 and GST-SH-HA2 proteins were mainly expressed in the inclusion sediment.The GST-CA-HA2 and GST-SH-HA2 proteins were then denatured with 8 M carbamide and efficiently purified by GST agarose gel column.The generation of the purified GST-CA-HA2 and GST-SH-HA2 proteins provides the immunogen for the development of mAbs specific to HA fusion peptide+helix A.2.Identification of linear cross-reacted epitopes in HA fusion peptide+helix ATo identify the linear broad antigenic epitopes of HA fusion peptide+helix A,the BALB/c mice were immunized bv the purified GST-SH-HA2 and GST-CA-HA2 proteins,and seven mAbs specific to HA fusion peptide+helix A derived from H7N9 were generated through the fusion of Sp2/0 cells and splecocytes from the immunized mice,desgined as 1A9,1B11,1H8,2C7,2F9,3A11 and 4D11 respectively.Cross-reaction assays showed six mAbs could react with H3N2 and five mAbs could react with H9N2.Epitope mapping using peptide-based ELISA revealed that 5 of 7 mAbs could recognize the linear epitope in the C-terminal of helix A and the other 2 mAbs could recognize the linear epitope in the HA fusion peptide.Several peptides of C-terminal of helix A derived from different subtypes of influenza viruses were synthesized and their corresponding polyclonal antibodies were generated.ELISA test showed mAb 3A11 could react with C-terminal of helix A from different subtypes of influenza virus,and the serum against C-terminal of helix A derived from H3,H7 and H9 subtypes could cross-react with the C-terminal of helix A of HI,H3,H7 and H9 subtypes respectively.Notably,the serum against C-terminal of helix A of H5 could only react with the C-terminal of helix A derived from HI,H5 and H9 viruses,and that of H1 subtype had no reaction with the C-terminal of helix A from different subtypes of influenza viruses.All these demonstrate that C-terminal of helix A derived from some influenza viruses can be as universal or cross-reacted antigenic epitope.3.Generation of a recombinant virus expressing the C-terminal of helix ATo identify whether the C-terminal of helix A can provide universal or cross-protection against influenza viruses,the C-terminal of helix A of H7N9 was cloned into NA segment of PR8 through recombinase Exnase TM.And then a recombinant virus PR8-NA-helix A was rescued by the transfection of the seven plasmids of PR8 and the recombinated NA in the co-cultured 293T and MDCK cells.The rescued recombinant virus PR8-NA-helix A was identified by HA assay,RT-PCR and sequencing,and the expression of the C-terminal of helix A in PR8-NA-helix A was confirmed by Western blot.The generation of the recombinant PR8-NA-helix A which expressing the epitope of the C-terminal of helix A derived from H7N9 laid the foundation for further evaluate whether the epitope of C-terminal of helix A could provide broad or cross-protection against influenza viruses.