| Influenza is an acute respiratory infection caused by influenza virus.It spreads quickly,is contagious,and has a certain seasonality.Influenza viruses cause a pandemic from time to time,leading to serious respiratory diseases and high mortality.Recombination of the complete gene fragment of influenza virus forms a new subtype,which causes a pandemic due to the lack of immunity of the population to the new variant strain.The influenza pandemics recorded in history are caused by recombinant strains.The occurrence of virus gene mutations and the emergence of new subtypes of viruses have raised concerns about health problems.For the treatment of influenza,influenza virus drugs,such as oseltamivir and zanamivir,have certain control effects on influenza virus,but due to the emergence of drug resistance,the influenza has not been effectively controlled,so vaccination is the main strategy for preventing and controlling influenza.Due to the constantly changing genes in the viral proteins that are easily recognized by the immune system,the virus is able to escape the antibody response produced by the vaccination.These genetic changes trigger new highly pathogenic strains that could lead to a pandemic.It is therefore necessary to update the vaccine frequently to ensure its effectiveness.Studies have shown that the rapid evolution of the HA head antigen may make its unique immune system ineffective for drifting strains that occur during the upcoming influenza season.In contrast,an immune response elicited by a highly conserved HA stem region epitope between subtypes can result in a broader cross-protective immunity.Therefore,a vaccine that concentrates the immune response on HA2 epitope will probably be the key to providing cross-reactive immunity to drifting influenza strains.And the highly conserved stem region of HA2 has recently been proposed as a useful strategy for designing universal influenza vaccines.Based on the above research background,the purpose of this study is to design a novel epitope polypeptide immunogen,which is highly conserved,contains a short linear dominant epitope,and has strong immunogenicity and better neutralizing activity.Conservative analysis,B cell epitope prediction,T cell epitope prediction and HA structure simulation were performed for the HA sequence from 1934 to 2016 in the H1N1 subtype of influenza A virus.A chimeric eight polypeptide sequence that is highly conserved and contains B,T cell dominant epitopes.Chemically coupled to the KLH vector and through a series of immunogenicity assays that coat different antigens,we identified three high immunogenic polypeptide immunogens.Based on the preliminary screening of the polypeptide sequence,the ferritin vector was further coupled by immunogen optimization design to further increase its immunogenicity.The ELISA results showed that the recombinant protein immune serum had a high binding activity.In vitro microneutralization experiments showed that the recombinant protein immune antibody serum had neutralizing activity against H1N1 subtype and H3N2 subtype virus.This cross-microneutralization proves the broad spectrum of our designed influenza epitopes indirectly.To sum up,this study has successfully screenned,designed,preparated and evaluated the conservative epitope peptide immunogen based on H1N1 HA,and the immunogen has high immunogenicity and the good neutralizing activity.In influenza vaccine application,the designed immunogen of this study can not only reduce the development cost,but can shorten the development cycle,effectively response to influenza pandemic.The designed epitope peptide based on high conservative HA2 stem region and the neutralizing activity detection can be laid a theoretical basis for the future development of broad spectrum influenza vaccine,and provide reference for clinical immunotherapy for influenza virus. |
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