| Influenza virus(?)is an important respiratory pathogen that can cause seasonal pandemics.According to the nuclear protein(NP)of ?,there are 4 types of seasonal influenza viruses,types A,B,C and D.Influenza A and B viruses circulate and cause seasonal epidemics of disease.According to the differences of HA and NA,IAV can be divided into various subtypes.There have been three major influenza pandemics in the 20 th century-A/Spain/1918(H1N1),A/Asian/1957(H2N2)and A/Hong Kong/1968(H3N2).The ability to spread worldwide in a short period highlighted the public health threat and the global economy posed by novel influenza viruses.The drugs currently used to treat influenza viruses are mainly M2 ion channel inhibitors and neuraminidase(NA)inhibitors.The abuse of antiviral drugs could lead to drug resistance.The most effective way to prevent ? is vaccination because of the resistance and mutability.In order to prevent vaccine antigen mismatch leading to low efficacy,the WHO-GISRS continuously monitors the influenza viruses circulating in humans and updates the composition of influenza vaccines twice a year.A 4th component is recommended to support quadrivalent vaccine development and it is expected to provide wider protection against influenza B virus infections.Influenza vaccine could be ineffective when circulating viruses are completely mismatched with viruses contained in vaccines that may cause disastrous consequences and incalculable loss.Therefore,it is urgent to develop a broad-spectrum influenza vaccine to prevent the destruction of highly variable strains.As one of the important glycoproteins on the surface of ?,Hemagglutinin(HA)can induce a broad protective immune response,and the amino acid in the neck region of HA is highly conserved.Therefore,the development of broad-spectrum influenza vaccine based on HA protein is particularly concentrated.A short linear dominant epitope named 16 peptide for H3 subtype has been screened out in our early stage.Wehave coupled it to the MAP4 and norovirus vectors for prokaryotic expression and animal experiments.Due to the weak immunogenicity of MAP4 and the risk of norovirus prepreservation,the purpose of this study was to select a suitable vaccine carrier through the prokaryotic expression system in order to further enhance the immunogenicity of the 16 peptide and the safety of the vaccine carrier.Compared with traditional protein vaccines,DNA vaccine is more stable,safer,and more economical and convenient than eukaryotic protein expression.In order to further determine the immunogenicity of 16 peptides in eukaryotic system,this study intends to conduct the immunization of DNA vaccine at the same time.Ferritin proteins derived from Helicobacter pylori have thermal and chemical stability,24 ferritin single subunits can spontaneously form a 12 nm spherical structure that making it an efficient antigen-delivery nanometer platform.In this study,the 16 peptide was conjugated with the N-terminal domain of ferritin to construct the recombinant plasmid H16-ferritin.After the prokaryotic system was expressed,purified and reconstituted,supplemented with MF59 adjuvant,mice were immunized subcutaneous at the dose of 20?g/200?L for 4 times with an interval of 2 weeks.The results showed that immune serum had binding and neutralizing activities,but it was lower than other vaccine vectors,such as Nov particle,MAP4.MF59 as adjuvant of influenza vaccine had high potency and application prospect.In order to determine the immunogenicity of the 16-peptide in eukaryotic systems and the potential of ferritin as a vaccine vector,the recombinant plasmids H16-ferritin and HA-ferritin were constructed by conjugating the optimized 16 peptide and the full length of HA with Ferritin,respectively.The recombinant plasmids were immunized with 100?g by gene introduction instrument for 3 times with an interval of 3 weeks.The results showed that the immune serum of HA-ferritin group had high binding activity,neutralization activity and hemagglutination inhibition activity to H3N2,while the immune serum of 16 peptide group had no activity.In summary,this study aims to enhance the immunogenicity of the short epitope16 peptide and the safety of the vaccine carrier,fouces on the prokaryotic recombinant protein vaccine and eukaryotic DNA vaccine carried out by Ferritincarrier with epitope 16 peptide and full length HA.The study was successfully evaluated the immunogenicity of 16 peptides screened for H3 subtype ?,but the activity of 16 peptides was greatly influenced by antigen carrier and immune adjuvant.The results of DNA vaccine immunization showed that the immunogen with high molecular weight presented by ferritin as the carrier in eukaryotic system had a strong carrier advantage.If ferritin was used as the carrier to present the short peptide,the nature and combination of the short peptide should be fully considered in order to give full play to its immune advantages.DNA vaccines are more stable,cheaper and easier to produce than traditional protein vaccines.Despite the relatively low titer of DNA vaccines,the correct selection of vaccine sequence,carrier and adjuvant highlights its important role in future medical treatment.This study further optimized the preparation technology and quality evaluation method of broad-spectrum neutralizing influenza vaccine.The short HA2 neck epitope screened in this study has high broad-spectrum and low production cost,which lays a theoretical foundation for the research and development of broad-spectrum influenza vaccine.The preliminary exploration of DNA vaccine provides reference for the research and development of new vaccines. |
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