Construction And Immunological Study On A/B Chimeric Seasonal Influenza Aerosol Vaccine Candidates

Research backgroundThe 2009-H1N1 pandemic spread to 214 countries,infecting more than one million people.The continuous evolution of the antigens of the 2009 H1N1 influenza A virus brought it along with the 1968 H3N2 subtype of influenza.As seasonal influenza continues to circulate in human populations,resulting in a heavy global public health burden.One of the effective ways to prevent and control the spread of seasonal influenza virus is to vaccination.Currently,the commonly used seasonal influenza vaccines are divided into inactivated vaccine and live attenuated vaccine.Live attenuated influenza vaccine is widely used for its non-injection immunity and the characteristics of inducing high level mucosal and cellular immunity.However,there is a risk that live attenuated vaccine strains will reassort with circulating influenza strains.PurposesWe used influenza B virus,which with lower frequency of antigenic variation than influenza A virus,as the vector of vaccine candidate strains,constructed three A/B chimeric seasonal influenza attenuated strains expressing HA protein of H1N1/H3N2/B seasonal influenza virus as seasonal influenza vaccine candidate strains,and selected aerosolized immunization to vaccinate the candidates.On the basis of preserving the high level of mucosal immunity induced by influenza viruses,the recombination of vaccine candidates and epidemic strains was avoided by using the characteristics of influenza A and B viruses unable to recombine,and the safety,immunogenicity and challenge protection efficacy of seasonal influenza vaccine candidates were evaluated,which laid the foundation for the research and development of novel influenza vaccines.Methods and ResultsRescue of A/B chimeric attenuated Strain expressing HA protein of 2021-22 H1N1,H3N2 and B seasonal influenza viruses:A/B chimeric seasonal influenza attenuated strains expressing HA protein of H1N1/H3N2/B seasonal influenza were rescued by reverse genetics of influenza B virus.Morphological and sequence identification of three rescued strains were performed.The sequencing results showed that the sequences of the three virus strains were identified,and influenza virus-like particles were observed under electron microscopy.The hemagglutination titer of H1N1/B attenuated chimeric seasonal influenza strain r A/B-H1VEC-110 was 2⁶.It had low growth replication efficiency on MDCK cells,and the virus titer on chicken embryos was 10^5.5EID₅₀/m L.H3N2/B attenuated chimeric seasonal influenza strain r A/B-H3CAM-110had a hemagglutination titer of 2⁴and low growth replication efficiency on chicken embryos.The virus titer on MDCK cells was 10^4.50TCID₅₀/m L.The hemagglutination titer of r B-WA-110,attenuated chimeric seasonal influenza strain B,was 2⁷,and the virus titer was 10^4.25TCID₅₀/m L on MDCK cells and 10^7.25EID₅₀/m L on chicken embryos.Safety evaluation of three attenuated chimeric seasonal influenza strains A/B in mice:Three chimeric attenuated strains of r A/B-H1VIC-110(10⁵EID₅₀/50μL),r A/B-H3CAM-110(10⁴TCID₅₀/50μL)and r B-WA-110(10⁷EID₅₀/50μL)were selected as candidates for chimeric seasonal influenza aerosol vaccine.Mice were aerosol immunized,and the survival rate and body weight of mice were recorded for 14consecutive days.Virus titers and tissue tropism of mice were measured on the third day after immunization.The results showed that mice were immunized with the highest dose of aerosol with 3 chimeric attenuated strains,and the survival rate of mice was100%.The virus replication titers were 2.97 log₁₀EID₅₀/g and 2.88 log₁₀EID₅₀/g in the lungs of two mice in r A/B-H1VIC-110 group.A virus replication titer of 3.80log₁₀EID₅₀/g was detected in the lungs of one mouse in r B-WA-110 group.Immunogenicity evaluation test of A/B chimeric seasonal influenza aerosol vaccine candidate strains in mice:BALB/c mice were divided into single immunized group and combined immunized group.Mice in the single immunized group were immunized with three attenuated strains by aerosol.Mice in the combined immunized group were immunized with the aerosol of r A/B,a trivalent A/B chimeric seasonal influenza aerosol vaccine candidate.The results showed that three attenuated A/B chimeric seasonal influenza strains and trivalent A/B chimeric seasonal influenza aerosol vaccine candidate strains could induce the production of hemagglutination inhibition antibody,neutralizing antibody,specific Ig G antibody and s Ig A antibody in mice.The titer of hemagglutination inhibition antibody against H3 subtype in r A/B group was 1:1024.The titer of neutralizing antibody in r A/B group to H1 subtype could reach 1:1280.The influenza-specific Ig G antibody was 6400 times positive in the group B after combined immunization,but the Ig G antibody level in the r A/B group decreased somewhat.H3subtype group was still positive after 128 times dilution of alveolar lavage solution.The level of IFN-γcytokine secretion showed that the specific cellular immune response against seasonal influenza virus was more active in the mono-immunized mice.The results of IL-2 and TNF-αcytokine secretion showed that T cell efficent function was more active in the H3 immunized mice compared with the H1 and B groups.Challenge protection of A/B chimeric seasonal influenza aerosol vaccine candidate strains in mice:Mice immunized with the highest dose of nasal drops were constructed with double-protein chimeric mouse adapted influenza strains carrying HA and NA of H1N1/H3N2/B seasonal influenza as seasonal influenza homologous viruses,and mice were immunized with 1MLD₅₀nasal infection with mouse adapted strains H1N1-UI182and H3N2-S89 as homologous subtype heterogenic influenza viruses.To determine the challenge protection effect of A/B chimeric seasonal influenza aerosol vaccine candidate strains against homologous and heterologous influenza viruses.The results showed that combined immunization with A/B chimeric seasonal influenza vaccine candidate strains could help mice resist lethal doses of homologous and heterologous influenza viruses.Effect of amino acid substitution at site 212 in HA protein on saving live chimeric attenuated H1N1/B influenza vaccines:We used the reverse genetics to save two attenuated H1N1/B chimeric influenza strains expressing HA protein of 2009 H1N1influenza A as vaccine candidates using B/Yamagata/16/1988 as background,and found that chimeric HA protein had amino acid substitution at site 212 near RBS.This change in amino acid residues affects the rescue of H1N1/B chimeric viruses.Through computer analysis and simulation,it was found that the substitution of amino acid residues at site 212 changed the stability and receptor binding activity of chimeric HA protein.With the substitution of alanine residues at site 212 to threonine residues,the stability of chimeric HA protein decreased,and its binding activity with sialic acid receptor molecules decreased.These results indicated that substitution of A212T had a negative effect on the efficient rescue of H1N1/B chimeric influenza virus,which provided conditions for shortening the candidates rescue time and improving the rescue efficiency.ConclusionsIn summary,we saved three attenuated strains of A/B chimeric influenza virus,and as the candidates of A/B chimeric seasonal influenza aerosol vaccines.Saved strains have good safety in mice,and as candidate strains of A/B chimeric seasonal influenza aerosol vaccines,they can induce multiple immune responses in mice,which lays a foundation for subsequent research on novel influenza vaccines.