Our results indicate that M2 vaccination can induce cross-reactive antibody responses, virus-specific T-cell responses, and protection against challenge with lethal heterologous virus. As has been found in previous studies of M2-based vaccines, we found strong antibody responses to the conserved M2e region. Anti-M2 antibodies in the serum of mice immunized with M DNA suggest expression of M2 from this plasmid (not shown). However, reactivity could be due to the 9 amino acid portion shared with the M1 sequence, and we did not explore this possibility. Fan et al. used A/PR/8 and Aichi M2e-carrier conjugates for immunization, and the resulting antibodies did not cross-react with the avian M2e sequences tested (6
). We found cross-reactivity with M2e peptides that had considerable sequence divergence from the human influenza M2 consensus. Immunization with M2e-con/KLH induced antibodies that were reactive with M2e-H5(SP-83) peptide but less reactive with M2e-H5(HK). However, immunization with M2e-H5(HK)/KLH induced antibodies that were reactive with all the M2e peptides. This pattern parallels the results of Liu et al. (14
). However, neither Fan et al. nor Liu et al. investigated protection against challenge with H5N1 subtype.
In our lethal challenge studies, M2e peptide conjugates protected against not only a 1934 subtype H1N1 virus (A/PR/8) but also a 1947 subtype H1N1 virus (A/FM). The latter virus, which is virulent in mice, has an M2e sequence with 3 amino acid differences from the consensus and thus is as divergent from the consensus sequence as some M2-H5 sequences. Encouraged by this broad cross-reactivity and cross-protection, we expanded the study to DNA vaccination and DNA prime–Ad boost regimens. These approaches have the advantage of providing more epitopes than peptide immunization and relevant T-cell immunity.
Using M2 consensus DNA vaccination with or without Ad boost, we again saw cross-reactivity on avian peptides M2e-H5(SP-83) and M2e-H5(HK), although cross-reactivity was low on the HK peptide. T-cell responses to M2 peptides were detected by ELISPOT.
Several studies have shown that M2e-specific antibodies can mediate protection against influenza infection in vivo (9
). In agreement with those studies, we found that serum antibodies induced by peptide conjugates or by prime-boost vaccination could transfer protection to naive recipients. We found that T cells were also important because depletion of CD4+
T cells during the challenge period reduced protection against a higher challenge dose. This could reflect M2e-specific memory T cells, which we have demonstrated in spleen and peripheral blood by ELISPOT, or a concurrent T-cell response to challenge virus supplementing the protective effects of antibodies.
In lethal challenge studies, the M2 consensus DNA and rAd constructs could protect against not only A/PR/8 but also against A/FM, a virus quite divergent in the M2e sequence. Furthermore, they could protect mice against challenge with SP-83 (H5N1) isolated from a fatal human case, at a dose lethal to control mice. Virus replication in lungs and illness reflected by loss of body weight were also reduced by M2 immunization. Protection against challenge with other H5N1 subtypes remains to be explored, and serologic results on M2e-H5(HK) peptide suggest results of such studies might differ on the basis of sequence variations.
M2 expression constructs with various M2e sequences could be used as vaccines. Our observation of protection across substantial sequence divergence means that H5-derived vaccines might also protect against circulating H1N1 and H3N2 subtypes. An additional advantage of protection across substantial divergence is potential protection by an M2 vaccine against an unexpected subtype that could cause a pandemic.
One concern about M2 vaccines is the possibility of escape mutants. A study of forced escape mutants found limited diversity (13
), which indicates that structural constraints, perhaps due to requirements of the M1 structure encoded by the same segment, may limit drift.
The cross-reactivity and protective efficacy of M2-specific antibodies suggest that M2-specific MAbs could be useful for antiviral therapy. These features, combined with constraints on M2 structure, highlight the potential of M2-specific MAbs to inhibit replication of influenza viruses, including some H5N1 strains. Although traditional M2-directed drugs (e.g., amantadine) have led to drug resistance, the mutations that confer resistance are within the transmembrane region (22
), which may have fewer structural constraints than the ectodomain.
An M2 prime-boost regimen is intended to be combined with vaccination against additional antigens rather than acting as a standalone vaccine. For example, prime-boost vaccination against conserved NP is highly protective ([18
]; ). The use of multiple antigens has several advantages: reduced likelihood of escape mutants, better coverage of human leukocyte antigen haplotypes in the genetically diverse human population, and a broader spectrum of immune response mechanisms (with antibodies perhaps dominating for M2 and cytotoxic T lymphocytes for NP).
Vaccines based on conserved antigens are not intended to replace strain-matched vaccines that induce neutralizing antibodies and thus prevent infection. However, strain-matched vaccines may be difficult to produce in adequate quantities in short time periods, and continued antigenic drift may render them ineffective. Vaccinations as described here, based on M2, might reduce deaths and severity of disease while strain-matched vaccines were being prepared and could enhance protection afforded by inactivated vaccines. Immunogenicity and safety studies in people are needed to evaluate this approach.