Construction And Immune Activity Evaluation Of Norovirus P Particle-based Influenza A Virus Multi-target Epitope Immunogen

Influenza causes significant public health and economic burdens globally.Currently,influenza vaccine is the most effective way to prevent and control its epidemic.The protective effect of seasonal influenza vaccine is limited,and the production process is complicated,time-consuming and has safety problems.Therefore,there is an urgent need for a universal influenza vaccine（UIV）that induce long-term protection against influenza A and B viruses and can be produced on a large scale.Currently,epitope vaccines that are easy to produce and precisely target key sites may be a promising strategy for developing safe and effective UIVs.The selection of epitopes is an important step in the epitope vaccine development,and the combination of multiple epitopes can enhance the broad spectrum of vaccine protection.Due to the small molecular weight of the epitope and low immunogenicity,it needs to be loaded into a suitable carrier to enhance the immunogenicity.Norovirus P particle has high stability,low production cost,and the spatial structure of three loop regions on its surface.No V P particle is beneficial to the presentation of exogenous antigens and the design of multivalent chimeric vaccines.Therefore,No V P particle has the potential as an ideal antigen or epitope delivery vehicle.The main purpose of this study is to design a novel multi-epitope immunogen by inserting the dominant linear epitopes of commonly used targets of UIV into the loop regions of NoV P particle.We look forward to developing candidate immunogens with strong immunogenicity and protection to lay a theoretical foundation for the development of a new generation of epitope-based UIV.In this study,we chose 3 potential candidate epitopes:1)H16:an H3N2 HA2（90-105）linear epitope that can induce a broad-spectrum neutralizing antibody response.2)M2e:Vaccines based on M2e（2-24）of H3N2 mainly induce Fc-mediated antibody-dependent cellular cytoxicity（ADCC）.Except for two amino acid residues,the conservation of amino acids in other positions in H3N2 is over 95%.3)NP9:NP（418-426）epitope can elicit cytotoxic T cell responses.First,we inserted H16,M2e,NP9 into the loop region of No V P particle individually or in combination of multiple epitopes,and prepared H16-PP,M2e-PP,NP9-PP and HMN-PP nanoparticles through E.coli expression system,respectively.SDS-PAGE,DLS and TEM confirmed the molecular weight,purity,size and structure of the protein.Secondly,in oreder to evaluate the immunogenicity of these nanoparticles,the immunogens were supplemented with aluminum adjuvant,and mixed in the volume ratio of 3:1.The mice were immunized three times subcutaneously with a dose of 30μg/200μL/mice,with an interval of 2 weeks.The results showed that H16-PP,M2e-PP,NP9-PP and HMN-PP produced epitope-specific Ig G antibody responses（Ig G titers were about 2.98×10~4～1.96×10~5）,and the antibody levels were stable in mice three months.H16-PP(ID₅₀:76.7～693.3)and HMN-PP(ID₅₀:43.3～346.7)could induce broad neutralizing responses against H3N2 and B viruses.The antibody serum induced by M2e-PP and HMN-PP showed significant ADCC responses against H3N2 virus.NP9-PP and HMN-PP induced high levels of CTL responses.HMN-PP also protected some mice from lethal infection by the A/Hong Kong/2671/2019（H3N2）virus.In conclusion,we successfully constructed an immunogen based on the dominant epitopes of influenza A virus HA2,M2e and NP using No V P particle as a vector,and evaluated the immunogenicity and protection.This study verifies the feasibility of the epitope-binding No V P particle vector technology,and also provides a reference for the subsequent development of UIV.