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

Abstract

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.

A Strategy to Elicit M2e-Specific Antibodies Using a Recombinant H7N9 Live Attenuated Influenza Vaccine Expressing Multiple M2e Tandem Repeats

“…Although we did not study T-cell immunity here, our previous findings observed for similar chimeric H1N1+4M2e and H3N2+4M2e vaccines can be extrapolated to the H7N9+4M2e vaccine as well, suggesting weak induction of M2e specific T cell immunity [ 14 ]. Similarly, weak M2e-specific CD4+ T cell responses were detected in mice immunized with VLP-based M2e vaccine, even though it induced much higher levels of M2e-specific antibody [ 21 ].…”

“…It is well known that LAIVs induce cross-reactive immunity with a potential to protect against drifted influenza viruses in a variety of animal models as well as in human trials [ 15 , 42 , 43 , 44 ], and numerous studies have proven that LAIV is a promising viral vector for designing vaccines against other infectious pathogens [ 45 , 46 , 47 , 48 ]. Our recent study demonstrated that seasonal H1N1 and H3N2 LAIVs expressing four M2e tandem repeats within the chimeric HAs can elicit M2e-specific antibody and, to a lesser extent, T-cell responses which enhanced cross-protective potential of the vaccines [ 14 ]. With a view to the possible future clinical trials of LAIV+4M2e vaccines, it is important to minimize the interference of pre-existing immunity to seasonal influenza viruses with replication of the attenuated vaccine viruses in the upper respiratory tract of the volunteers; therefore, we developed a new M2e-based universal vaccine candidate based on pre-pandemic LAIV strain of H7N9 subtype.…”

“…Serum passive transfer experiment confirmed that the enhanced protection induced by the chimeric vaccine is mediated by antibody immune responses, most probably by anti-M2e antibodies. According to our previous findings, the M2e-targeted antibody can be actively secreted by B-cells in mediastinal lymph nodes (MLN) post-challenge, suggesting the major mechanisms of immune protection afforded by LAIV+4M2e vaccines [ 14 ].…”

“…Currently approved live attenuated influenza vaccines (LAIVs) are thought to stimulate mucosal and systemic IgA and IgG, as well as T-cell mediated immunity, nonetheless, similarly to natural virus infection, they are weak inducers of M2e-targeted antibody. We have recently developed a vaccine strategy that will result in enhanced M2e antibody responses after vaccinations by incorporation of four M2e tandem repeats into N-terminus of HA1 hemagglutinin subunit of seasonal LAIV strain [ 14 ]. A two-dose intranasal immunization of naïve BALB/c mice with the chimeric H1N1+4M2e or H3N2+4M2e elicited significant levels of M2e antibody which protected animals against heterologous influenza A virus challenge, as confirmed by serum passive transfer experiments.…”

Universal Live-Attenuated Influenza Vaccine Candidates Expressing Multiple M2e Epitopes Protect Ferrets against a High-Dose Heterologous Virus Challenge

“…Viruses 2021, 13, x FOR PEER REVIEW 9 of 25 cross-protection of immunized mice against heterosubtypic influenza viruses [6,7]. However, the inserted consensus M2e epitopes of human, swine and avian influenza viruses were designed based on previous publications and consensus sequences reported prior to the 2009 pandemic [17].…”