Anti-M2e Abs are poorly induced in humans by infection or current vaccines 
, however several studies have shown the efficacy of anti-M2e Abs in protective anti-viral immunity. To improve anti-viral resistance, we evaluated a vaccine strategy with modified M2e-based multiple antigenic peptides. We investigated the nature and specificity of the M2e-specific B cell response using M2e-MAPs termed K2 and K3. Importantly, we assessed the ability of these M2e-MAPs to confer protection against viral challenge with homologous and M2-variant viruses and extended our studies to test the response in several inbred and outbred mouse strains.
Previously, immunization of BALB/c mice with a first-generation M2e-MAP, termed G40d, in combination with the adjuvants CpG and CT was shown to induce M2e-specific serum Abs and local immunity in the respiratory tract (RT) 
. We extended these studies using new M2e-MAPs that were generated using a simplified peptide synthesis that is more compatible with large-scale production and also incorporated promiscuous T helper epitopes that might be recognized by multiple haplotypes. Others have shown that the N-terminal 15 amino acid M2e peptide by itself is poorly immunogenic and failed to elicit a robust anti-M2e-Ab response in BALB/c mice in a prime-boost immunization 
. We show here that immunization of BALB/c mice with K2 and K3 in combination with the adjuvants, CpG and CT, induced high M2e-specific serum Ab titers (). Of note, these Ab titers exceeded the detected levels in previous studies with G40d 
, although the amount of anti-M2e Abs necessary for protection has not been determined. Anti-M2e Abs are not virus-neutralizing, but they can restrict viral replication and transmission, and passive transfer of anti-M2e monoclonal Abs in mice results in reduced viral titers, accelerated viral clearance and thus protection 
. Likewise, mice vaccinated with either K2 or K3 had significantly reduced viral titers when challenged with X31 virus and this protection extended to challenge with the more pathogenic PR8 virus ().
It has been hypothesized that the induction of local immunity at the site of virus entry, such as the mucosal surfaces of the nasal passages and the bronchial tree, is a critical determinant for protection to influenza virus infection (reviewed in 
). Work by several investigators has suggested that IgA in (upper) RT secretions in addition to IgG, the predominant isotype in the serum, plays a major role in antiviral immunity 
. We have compared the i.n. to s.c. route of K2 administration for their ability to induce M2e-specific IgA and IgG Abs and to promote protection. We show that the presence of M2e-specific IgA in BAL was dependent on i.n. administration of M2e-MAPs and correlated with IgA-producing cells in the RT (). On the other hand, s.c. immunization induced significantly higher Ab levels in the serum and was as efficient as i.n. immunization in inducing M2e-specific IgG in the BAL (). Our data are consistent with findings by other groups that i.n. vaccinations with live-attenuated influenza vaccine (LAIV), viral-like particles or inactivated viruses generate strong local Ab responses in the RT, in particular the induction of IgA, whereas parenteral/s.c. immunization elicits strong Ab responses in the serum but little IgA in the RT 
. However, in contrast to these studies, we demonstrate that the protection of i.n. and s.c. immunized mice to challenge with X31 virus was similar (), suggesting that the absence of M2e-specific IgA in BAL after s.c. immunization can be compensated for high titers of anti-M2e IgG in BAL and serum. Our findings support recent observations in IgA−/−
mice that suggest that IgG is sufficient for M2-induced anti-viral immunity 
Ideally, an influenza vaccine should induce broadly cross-reactive immunity and protection against variant viruses. We show that vaccination with K2 induced serum Abs that not only recognized the M2 sequence of PR8 used in the vaccine, but also bound efficiently to cells infected with influenza virus A/FM (), recognized M2e peptide from the pandemic H1N1 () and provided protection to challenge with the mutant viruses P10L and P10H (). These results suggest that immunization with the M2e-MAPs induces Abs that might also be cross-reactive against certain types of avian viruses that have similar exchanges at amino acid position 10, which have been observed among recent H5, H7 and H9 isolates 
. Our data corroborate the findings of other studies with M2e-based MAPs and contribute to the growing evidence that M2e-based vaccines are able to provide cross-reactive protection 
. Furthermore, we demonstrate that induction of cross-reactive Abs can depend on the route of immunization in that s.c. as compared to i.n. administration induced higher levels M2e-specific Abs () and thereby increasing the overall avidity of potentially cross-reactive anti-M2e Abs ().
Finally, we evaluated the response to K2 and K3 in other inbred and outbred mouse strains. In contrast to our findings in BALB/c mice, C57BL/6, C3H and the outbred mouse strains CD1/ICR and Swiss Webster exhibited poor anti-M2e Ab levels after vaccinations with K2 and K3 and were not protected from viral challenge (, data not shown and
). Likewise, others have reported a genetic restriction to M2e-based vaccines 
. Zhang et al. has demonstrated that the anti-M2e response in BALB/c mice was comprised of a set of highly restricted immunoglobulin genes usage 
. This finding could explain in part the low Ab titers after natural infection as well as the unresponsiveness we observed in other mouse strains. However, we demonstrate that multiple infections in outbred mice induced a small but detectable serum Ab response to M2e (). Moreover, M2e-specific Abs from both BALB/c and C57BL/6 allotypes were detected in vaccinated BALBxB6 F1 mice (), indicating that the lack of an Ab response to M2e-MAPs in C57Bl/6 mice was not due to an inherent failure of B cells from this background to recognize M2e.
A key feature of the modified M2e-MAPs used in this study is that they contain potentially universal T cell determinants. The T cell epitopes included in K2 and K3 vaccine constructs have previously been tested for promiscuity and were shown to bind many HLA-DR molecules 
. While efficacy in inducing T cell responses is variable and shows some genetic dependence, other studies demonstrated that the T cell epitope CS327–345
induces responses in C57BL/6 mice and non-human primates as a linear or branched peptide together with water-in-oil based adjuvants or CpG 
. Likewise, a MAP-combination of CS381–396
with the B cell epitope (NANP)3
induces responses in multiple inbred mouse strains 
. Additionally, polypeptide constructs with multiple T cell epitopes, including HA307–319
, and CS381–396
conjugated to Haemophilus influenzae
polysaccharide elicit Ab titers in CD1 outbred mice 
. However, we were unable to detect T cell responses in C57BL/6 and C3H mice after s.c. immunization with K2 and adjuvants (). Thus, the lack of a T cell response to the immunizing M2e-MAP peptide could explain the lack of measurable M2e-specific Abs.
A fully synthetic peptide vaccine based on a highly conserved viral antigen has the advantage that its preparation does not include undesirable components from the purification processes of live whole organisms and that it does not require yearly adjustments. However, the main question that still needs to be addressed for this vaccine approach is how to improve its immunogenicity. Although we have not formerly shown that the adjuvants used in this study, CpG and CT, have comparable effects in different mouse strains, studies by others suggest that their adjuvant activity is independent of genetic backgrounds 
. Embedding additional adjuvant sequences into MAPs as well as incorporating other conserved immunogenic viral target proteins, such as NP and HA, for a combination therapy have been shown to exceed protective efficacy compared to single antigens and may enhance their therapeutic potential 
. Rapid manufacturing of an influenza vaccine based on M2e-containing MAP constructs provided with optimal T cell help may be an attractive alternative for the production of current influenza vaccines.