rgH3N2: 6:2 reassortant with WT HA and NA of A/Switzerland/2013. subtypes including H1N1, H3N2, H5N1, H7N9, and H9N2. The findings support a new approach to improve the efficacy of current vaccine platforms by recombinant influenza virus inducing immunity to HA and cross protective M2e antigens. Subject terms:Immunology, Microbiology, Diseases, Molecular medicine == Introduction == Influenza virus causes one of the most common respiratory diseases in humans, resulting LY 344864 S-enantiomer in significant public health concerns and deaths annually worldwide1,2. Influenza A virus is a negative-sense single-stranded RNA virus containing 8 segmented genomes, belonging to theOrthomyxoviridaefamily, and has an antigenic variety from 18 subtypes (H1H18) of hemagglutinin (HA) and 11 subtypes (N1N11) of neuraminidase (NA)3. Antigenic diversity is a challenging difficulty in preventing influenza. Vaccination has been the most effective preventive measure against influenza virus infection. The most common licensed platforms are inactivated influenza and live-attenuated influenza vaccines (LAIV). The overall vaccine effectiveness during 20052018 seasons is a wide range of low efficacy between 10 and 60% as estimated in the US Flu Vaccine Effectiveness Network4. Due to emergence of drifting mutations in circulating H3N2 strains, the vaccine effectiveness against H3N2 was estimated to be 6% during the 20142015 LY 344864 S-enantiomer season5,6. To overcome continued antigenic changes, universal vaccination strategies have been focused on inducing immunity to conserved epitopes and domains present in all influenza A viruses, including the M2 extracellular domain epitopes (M2e)7,8and the HA stalk domains9,10. Different platforms and vaccine adjuvants have been investigated to overcome low immunogenicity of M2e epitopes. M2e-based vaccine candidates include Hepatitis B virus core protein conjugates (M2e-HBc) with adjuvants11,12, virus-like particles (VLP) presenting M2e tandem repeats (5xM2e VLP)13, and flagellin conjugates (4.M2e-tFliC)14and fusion with oligomer stabilizing domains (M2e-tGCN4)15. M2e expressing viral vectored vaccines were reported using adenovirus16, modified vaccinia virus Ankara17, and a T7-bacteriophage18. These previous strategies inducing M2e immunity alone were insufficient LY 344864 S-enantiomer for conferring optimum protection and incompatible with current vaccine platforms. No universal vaccine against influenza is on the market. Vaccination of combined M2e VLP and inactivated influenza vaccines induced both cross protective M2e and strain specific HA immunity19,20. To enhance the cross protective efficacy by a strategy of utilizing current vaccine platforms, recombinant influenza H1N1 virus A/Puerto Rico/8/1934 (A/PR8) was engineered to express chimeric 4xM2e-HA where tandem M2e epitopes were inserted in the N-terminus HA21. A chimeric HA with a single M2e in the head site Ca was tested in inactivated recombinant A/PR8 virus inducing cross protection22. However, the protection was not tested against a wide range of different subtypes. In recent years, antigenic drifts have severely limited the effectiveness of the H3N2 component of LY 344864 S-enantiomer seasonal influenza vaccines. Here, using the reverse genetic (rg) technique, we generated reassortant seasonal influenza rgH3N2 4xM2e virus containing chimeric 4xM2e-HA in which the HA and NA genes were derived from A/Switzerland/9715293/2013 (H3N2) and the remaining 6 genes from the A/PR8 backbone. Reassortant rgH3N2 LW-1 antibody 4xM2e virus containing chimeric 4xM2e-HA was found to retain comparable growth properties but display highly attenuated phenotypes in mice. Intranasal single inoculation of mice with rgH3N2 4xM2e virus could effectively induce a broad range of enhanced cross protection against different influenza A virus subtypes including H1N1, H3N2, H5N1, H7N9, and H9N2. This study implicates a strategy of improving cross protection by utilizing currently licensed recombinant influenza vaccine platforms. == Results == == Generation of reassortant influenza H3N2 virus vaccine containing 4xM2e in an HA conjugate == Current strain specific HA-based influenza vaccine is less effective in conferring cross protection. M2e has been targeted to induce broad but weak cross protection. To induce immunity against both HA and highly conserved M2e epitopes, a seasonal A/Switzerland/2013 H3 HA gene conjugated with four tandem M2e (4xM2e) repeat was constructed (Fig.1a and Supplementary TableS1). Rescued viruses expressing wild type (WT) HA (rgH3N2) or chimeric HA (rgH3N2 4xM2e) were amplified in eggs and harvested. Antigenic characterization by ELISA showed that rgH3N2 4xM2e virus was highly reactive to M2e specific mAb 14C2 whereas rgH3N2 and A/PR8 virus controls did not show such M2e reactivity (Fig.1b). Both rgH3N2 and rgH3N2 4xM2e viruses displayed high antigenic reactivity to mouse antisera of rgH3N2 infection and goat antisera of A/Indiana/2011 (H3N2) immunization (Supplementary Fig.S1). These results support that 6:2 reassortant rgH3N2 4xM2e virus displays high reactivity to M2e specific 14C2 mAb and retains similar antigenicity to antisera of H3N2 viruses as compared to rgH3N2 virus. == Figure 1. == Reassortants rgH3N2 and rgH3N2 4xM2e virus expressing chimeric 4xM2e-HA are attenuated in vivo.(a) Diagram for chimeric 4xM2e-HA (H3) structures. The sequence H3 HA was derived from A/Switzerland/2013 (H3N2). SP: signal peptide, tandem repeat 4xM2e is composed of human, swine, and avian influenza A viruses. hM2e: SLLTEVETPIRNEWGSRSNDSSD, sM2e: SLLTEVETPTRSEWESRSSDSSD, aM2e: SLLTEVETPTRNEWESRSSDSSD. L and C represent linker (AAAGGAA) and connector (AAAPGAA) respectively. (b) 14C2 M2e.