| Seasonal influenza causes severe health impacts and economic burdens worldwide each year.In addition,global influenza pandemics occasionally occur when a new of influenza A viru strain spreads from an animal host to humans and acquires the ability to transmit from person to person.Pandemic influenza poses a more serious public health and socioeconomic threat than seasonal influenza.Currently,annual influenza vaccination is the best way to prevent and control seasonal influenza.These seasonal vaccines mainly induce neutralizing antibodies against the head domain(HA1)of the influenza virus hemagglutinin(HA),preventing the virus from attaching to cells and prematurely interrupting the viral life cycle.However,HA1 is highly plastic,and the presence of neutralizing antibodies can also cause selective pressure on influenza viruses,resulting in persistent mutations in HA1 that allow the virus to evade immune recognition by the body.Therefore,neutralizing antibodies targeting HA1 are usually strain-specific,and seasonal influenza vaccines must be updated annually.Notably,currently available vaccines are ineffective against potential pandemic influenza.To overcome the limitations of seasonal influenza vaccines and enhance pandemic preparedness,there is an urgent need to develop universal influenza vaccines(UIVs)that induce broad and durable immune protection against antigenically distinct influenza viruses.HA is the most abundant protein on the surface of influenza virus,consisting of a globular head domain HA1 and a stalk domain HA2.HA2 is highly conserved among different influenza virus subtypes,and antibodies elicited by HA2 have been shown to provide cross-reactive protection against multiple influenza virus strains.The matrix protein 2 extracellular domain(M2e)is an evolutionarily conserved region in influenza A viruses.M2 e consists of 24 amino acids and its amino-terminal epitope SLLTEVET(residues 2-9)is fully conserved in all influenza A viruses,with only minor mutations observed in the distal part.In addition,the antibodies targeting M2 e have been shown to be protective against different influenza A virus strains.To date,various vaccine platforms and technologies have been developed,including DNA/RNA vaccines,virus-like particle(VLP)vaccines,and self-assembling nanoparticle(SANP)vaccines.Ferritin is a SANP composed of 24self-assembled subunits with strong thermal and chemical stability.Ferritin has been used to display antigens associated with many infectious diseases,such as influenza virus,human immunodeficiency virus(HIV),hepatitis B virus,Epstein-Barr virus and severe acute respiratory syndrome coronavirus 2(SARS-Co V-2).The main purpose of this study is to combine the dominant epitopes of influenza virus with ferritin nanotechnology to develop UIVs with strong immunogenicity,ability to induce cross-immune responses and protection,and rapid mass production.This research includes the following three aspects:Part 1: Design and immune effect evaluation of nanovaccine based on H1N1 HA trimerTo develop an effective vaccine with rapid production,robust immunogenicity,and high protective efficiency,we designed a DNA vaccine,termed HA-F,by fusing influenza virus HA with self-assembled ferrittin nanoparticles.The vaccine candidate was prepared and purified in a 293-6E cell eukaryotic expression system,and characterized in terms of particle structure and size.After three immunizations of BALB/c mice with 100 μg of HA-F plasmid DNA,HA-F elicited significant HA-specific humoral and T-cell immune responses.The HA-F DNA vaccine also protected mice from lethal infection by the homologous A/17/California/2009/38(H1N1)virus.Part 2: Design and biological activity of nanovaccine targeting influenza virus HA2 epitopeVarious vaccine strategies have been developed to provide broad protection against a variety of influenza viruses.HA2 is the main potential target for these vaccines.Enhancing immunogenicity and eliciting cross-protective immune responses are critical for HA2-based vaccine design.In this part of the study,the A helix(Ah)and CD helix(CDh)of H3N2 HA2 were fused to ferritin individually or in tandem to produce Ah-f,CDh-f and(A+CD)h-f nanoparticles,respectively.These nanoparticles were prepared by the prokaryotic expression system.After three subcutaneous immunizations of BALB/c mice with these nanoparticles,CDh-f and(A+CD)h-f induced strong humoral and cellular immune responses.Furthermore,CDh-f and(A+CD)h-f nanovaccines provided complete protection against lethal infection by H3N2 virus,while no obvious immune response and protection were detected in the Ah-f group.Part 3: Design and biological activity of multi-epitope influenza nanovaccineCurrently,combining multiple epitopes into a vaccine is preferable to using a single antigen to develop UIV.However,epitopes generally induce weaker immune responses.Although the use of adjuvants can overcome this obstacle,it may raise new problems.Efficient antigen delivery vehicles that can act as antigen carriers and built-in adjuvants are highly desirable for vaccine development.In this part of the study,we report a biepitope nanovaccine(3MCD-f)that elicited complete protection against H3N2 virus and partial protection against H1N1 virus in mice.3MCD-f consists of two conserved epitopes(M2e and CDh)that are presented in a sequence tandem on the surface of ferritin.Subcutaneous immunization with 3MCD-f without adjuvant induced strong humoral and cellular immune responses.In conclusion,in the first part of the study,we report a DNA vaccine strategy of HA trimer-conjugated ferritin nanoparticles.This vaccine induced stronger humoral and cellular immune responses than other forms of HA trimer vaccines,including high levels of neutralizing antibodies,T cell proliferation,and cytokine secretion.Rapid evaluation of this vaccine strategy could accelerate the approval and clinical use of various promising vaccines.However,the HA-F DNA vaccine has certain limitations.Since the full length HA1 is retained,the vaccine can only provide protection against specific strains.Based on this,we focus the target of vaccine development on the more conserved HA2 region,hoping to develop nanovaccine that induces broad immune responses.Therefore,in the second part of the study,based on the high conservation of HA2 epitopes Ah and CDh,we successfully prepared three epitope-conjugated ferritin nanoparticle vaccines.Microneutralization(MN)assays showed that CDh-f and(A+CD)h-f elicited cross-reactive neutralizing antibodies against H3N2 and B viruses.Furthermore,sera from CDh-f and(A+CD)h-f immunized mice elicited ADCC responses to H3N2 virus.These functional antibodies may play a key role in preventing viral infections.Cellular immune responses were assessed by cytokine tests and lymphocyte proliferation assays,and the results showed that CDh-f and(A+CD)h-f induced strong T-cell responses.These results suggest that CDh-based nanovaccine represents a promising influenza vaccine platform,while epitope combined with ferritin nanotechnology may be a potential vaccine strategy against other infectious viruses such as SARS-COV-2.In the third part,to further enhance the protective breadth of the nanovaccine,we prepared a biepitope 3MCD-f nanovaccine by introducing the M2 e epitope on the basis of CDh-f,which induced cross-neutralizing antibodies against H3N2 and B viruses,and the antibody serum showed significant ADCC activity against H1N1 and H3N2 viruses.These results suggest that the 3MCD-f nanovaccine may be an ideal candidate for preventing future influenza pandemics. |
