| The flu epidemic causes about 3 to 5 million serious diseases worldwide and 0.25 to 0.5 million deaths each annually.Seasonal influenza viruses include influenza A and influenza B viruses.Influenza H1 and H3 viruses are the two major viral influenza A subtypes that currently circulate in humans around the world.In human history,influenza pandemics such as the Spanish H1N1 flu in 1918,caused more than 50 million deaths.Recently,the 2009 H1N1 swine flu spread more rapidly,sweeping over 200 countries and regions.In addition,the highly pathogenic avian influenza(HAPI)H5 subtype with high mortality rates up to 60%.Due to viral mutations and gene rearrangements,influenza vaccines need to be updated nearly every year,and drug resistance have been reported for new influenza virus treatments with ion channel inhibitors and neuraminidase(NA)inhibitors.The influenza virus surface glycoprotein hemagglutinin(HA)is the target of many broadly neutralizing antibodies(bnAbs)and is considered to be a key target for the development of novel drugs.The isolations and studies of bnAbs against influenza virus have brought new dawn to the development of influenza monoclonal antibody therapy and universal vaccine design.However,whether a broadly neutralizing antibody targeting the HA head region can be used to treat influenza is still unclear,how the epitope vaccine design based on their recognition epitopes for universal influenza vaccines has not clarified.This study used a cross-neutralizing antibodies against H1 and H5 subtypes,named 12H5,to further investigate the neutralization mechanism and therapeutic potential,and then determined the crystal structure and Cryo-Electron structure of its immune complex with influenza virus HA,and further use the epitope information to carry out preliminary design and feasibility study of epitope vaccine design.Firstly,this study carried out the research on the therapeutic potential and neutralization mechanism of 12H5 antibody.The human and mouse chimeric antibody C12H5 was successfully expressed and purified,and showed good reactivity and high affinity to HAs of seasonal H1 and pandemic H1,and recognized H5 strain.The binding reactivity was comparable to that of murine antibody.In vitro,C12H5 exhibits broader neutralization response than other H1 representative head region antibodies,and showed protective efficacy against seasonal and pandemic H1N1,and is superior to reported H1 subtype neutralizing antibodies.In addition,C12H5 can cross-neutralizes H5 stain,has a good prophylactic and therapeutic efficacy against H5 stain in mice.Furthermore,neutralization mechanism analyses revealed that C12H5 antibody exerts the antiviral reactivity via inhibits virus entry into cells against pandemic H1N1 and inhibits virus egress against seasonal H1N1 and H5N1 viruses.Secondly,this study further elucidated structural basis of the broadly neutralization of 12H5.This study was first to report the immune complex crystal structure and Cryo-EM structure of a broadly neutralizing antibody against H1N1 and cross-neutralizing H5N1 which targeted HA HA domain.We obtained a near-atom crystal structure of 12H5 with C A HA head region(pandemic H1N1)with a resolution of 3.15 (?) and the Cryo-EM structure of 12H5 with BJ HA trimer(seasonal H1N1)with a resolution of 6.95 (?).The binding mode of the Fab in the 12H5:CA crystal structure were basically consistent with the 12H5:BJ Cryo-EM in the trimer.The high-resolution immune complex structure revealed that 12H5 targets the HA receptor binding site(RBS),and mainly interacts with epitope of 130 loop,140 loop,150 loop and 190 helix.Sequence analysis showed the average conservation of this epitope on H1N1 was 89.2%.Again,this study continued to elucidate the molecular mechanism of H1 broadspectrum neutralization and H5 cross-neutralization.We carried out the alanine scanning and site-directed mutagenesis,and dementated that the eight sites of Y98,A137,H141,A142,G143,A144,W153,and D190 were critical for 12H5 recognition,with an average conservation of 90%.It was also verified by mutation that Q142,R144 and S145 are the specific key sites to H5 recognition.The average conservation of these three sites are 67%,so 12H5 can recognize part of H5N1 virus,achieving cross-subtype neutralization and protection.Further,the LCDR1 of antibody 12H5 can structurally accommodate 190 D and E polymorphism,which is a key site for viral receptor recognition transition,the ability of 12H5 antibody to bear the key mutation associated with virus receptor specificity and achieving cross-type neutralization was explained.Finally,this study was tried to explore epitope design based on the obtained broadspectrum and cross-neutralization epitope.The immune serum of peptide vaccine coupled to keyhole limpet hemocyanin(KLH)(named QH-KLH)had a better binding reactivity to 12H5 epitope,and had the cross-protection against H1 and H5 virus with the survival rates of 20%and 40%,respectively.The results above provided a reference for the design of epitope-based vaccines.In summary,with a combination of various methods of virology,structural biology techniques,biochemistry,molecular and cellular biology methods,we firstly and comprehensively characterized the most broadly neutralizing and protective antibodies against influenza H1N1 targeting HA receptor binding sites,which also has crossneutralization and protective efficacy against H5 stain.Those results demonstrated it has potential clinical application in future.Meanwhile,this study firstly elucidated the structural basis of the molecular mechanism of antibody neutralizing viral infection,and further,through the epitope design,we confirmed that this epitope can be used as a starting target for cross-vaccine development,and provides significant information for the molecular design of universal vaccine and development of therapeutic monoclonal antibodies or small molecule drugs. |
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