To develop safe and effective prophylactic strategies to combat human infections by both pandemic 2009 H1N1 virus and H5N1 virus, the highly conserved M2e of influenza A virus has proven to be a promising target antigen to produce cross-protective influenza vaccines. In this study, H5N1-M2e-MAP, whose cross-protection against different clades of H5N1 virus has already been established in our previous report [
18], was shown to confer heterosubtypic protection from lethal infection with the pandemic 2009 H1N1 virus. Remarkably, all mice vaccinated with H5N1-M2e-MAP plus Freund's adjuvant survived the lethal heterologous virus challenge. Although there is partial protection (80%) in the H5N1-M2e-MAP plus aluminum adjuvant group, H5N1-M2e-MAP vaccination in both Freund's and aluminum adjuvants demonstrated similar efficacy in limiting viral replication and attenuating virus-producing histopathological damage in lung tissues, thus showing the ability to control disease transmission.
Notably, it seems that higher titers of cross-reactive antibodies against H1N1-M2e result in better heterosubtypic protection from lethal virus infection (Figs.
2 and
4), implying that M2e-induced cross-reactive antibody is a crucial component in heterosubtypic protection in M2e-based vaccines. Other groups have reported that the antiviral effect of M2e-based vaccines was mediated by antibodies to M2e antigen and that its mechanism was antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-mediated cytotoxicity (CDC) [
20,
21]. Therefore, the cross-protection of the M2e vaccine was based on the premise that antibody responses with high levels of cross-reactivity were induced following vaccination. However, the difference of M2e amino acid sequences between human-type and avian-type viruses would affect their mutual recognition in varying degrees. Specifically, Fan et al. [
14] reported that antisera against human-type M2e sequence failed to react with avian-type M2e peptide. Also, Liu et al. [
22] indicated that mAb specific to a region (aa 6 - 13) of human-type M2e sequence can only weakly recognize, or not recognize at all, avian-type M2e sequence with variations in the same range. Nevertheless, in our studies, H5N1-M2e-MAP not only induced high titers of specific antibody against H5N1-M2e [
18] but also elicited potent and prolonged cross-reactive antibody recognizing H1N1-M2e (Fig.
2). Although it is not certain whether the only amino acid difference between the H1N1-M2e and H5N1-M2e (as indicated in Fig.
1) is located outside the region containing B-cell epitopes, the induction of cross-reactive antibody by H5N1-M2e-MAP against H1N1-M2e is obvious, and it is confirmed that H5N1-M2e-MAP can afford heterosubtypic protection against pandemic 2009 H1N1 virus (Figs.
3 and
4), regardless of the difference in M2e sequence between H1N1 and H5Nl by one amino acid.
Given that the immunization regimen consisting of H5N1-M2e-MAP and aluminum, the only adjuvant approved for use in humans, induced weaker immune responses (Fig.
2) and protective immunity (Fig.
4) than that is comprised of H5N1-M2e-MAP and Freund's adjuvant, it is urgently needed to develop more effective and safe adjuvant than aluminum for clinical use of human vaccines.