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Influenza viruses constantly evolve, reducing the overall protective effect of routine vaccination campaigns. Many different strategies are being explored to design universal influenza vaccines capable of protecting against evolutionary diverged viruses. The ectodomain of influenza A M2e protein (M2e) is among the most promising targets for universal vaccine design. Here, we generated two recombinant live attenuated influenza vaccines (LAIVs) expressing additional four M2e tandem repeats (4M2e) from the N-terminus of the viral hemagglutinin (HA) protein, in an attempt to enhance the M2e-mediated cross-protection. The recombinant H1N1+4M2e and H3N2+4M2e viruses retained growth characteristics attributable to traditional LAIV viruses and induced robust influenza-specific antibody responses in BALB/c mice, although M2e-specific antibodies were raised only after two-dose vaccination with LAIV+4M2e viruses. Mice immunized with either LAIV or LAIV+4M2e viruses were fully protected against a panel of heterologous influenza challenge viruses suggesting that antibody and cell-mediated immunity contributed to the protection. The protective role of the M2e-specific antibody was seen in passive serum transfer experiments, where enhancement in the survival rates between classical LAIV and chimeric H3N2+4M2e LAIV was demonstrated for H3N2 and H5N1 heterologous challenge viruses. Overall, the results of our study suggest that M2e-specific antibodies induced by recombinant LAIV+4M2e in addition to cellular immunity by LAIV play an important role in conferring protection against heterologous viruses.

Generation and Characterization of Universal Live-Attenuated Influenza Vaccine Candidates Containing Multiple M2e Epitopes