Heterosubtypic immunity in man has been suggested from epidemiologic studies of human outbreaks of influenza A [4, 5, 16]. Identification of the immune components necessary for a heterosubtypic immune response will be critical in the development of more broadly protective vaccines effective against influenza A virus. Both antibodies and cytotoxic T cells have been implicated in cross-protective immune responses in murine models of influenza infection, where the most often used end-point is mortality.
In the cotton rat model, we previously demonstrated that respiratory rate can be used as a measure of disease severity . Protection from tachypnea is observed in cotton rats immunized with one subtype of influenza A virus and subsequently challenged with another subtype, demonstrating a heterosubtypic immune response. This protection persists despite inhibition of the recruited memory response . The studies presented in this report show that protection is mediated by humoral immunity since passive transfer of immune serum from H1N1-immune animals is able to transfer components necessary for protection from H3N2-induced tachypnea. Protection correlates with HAI titer. While the HAI titer is a measure of a subtype-specific antibodies, it also reflects the total amount of antibody successfully administered during the passive transfer and is therefore likely to correlate with the amount of cross-reactive antibodies present in the serum. These antibodies are most likely specific for conserved epitopes of influenza A, and may include antibodies with specificity for NP, M2e or conserved HA peptides. Non-neutralizing HA-specific antibodies that may contribute to B cell-dependent, heterosubtypic protection against lethal infection by avian H5N1 influenza have been measured in the convalescent sera of mice . While there is good evidence that M2-specific antibodies are induced following infection , we were unable to measure anti-M2 titers in our cotton rat serum samples in an ELISA using M2e peptide to coat the plates. The poor sensitivity of this type of assay has been reported and it is known that functional M2e-specific antibodies are best detected using a cell-based expression system . While we do not know the fine specificities of antibodies present in convalescent cotton rat sera, our results show that M2e-specific but not NP-specific monoclonal antibodies can contribute to protection from influenza virus-induced tachypnea.
Further studies are needed to evaluate how antibodies contribute to cross-protection. They may reduce the amount of virus that can attach to cells by directing FcR-positive macrophages to the pathogen for uptake and degradation. A role for macrophages in heterosubtypic immunity is supported by the studies of Sambhara et al. . Alternatively, cross-protective antibodies may work in conjunction with NK cells as demonstrated for protection of mice by M2-specific antibodies . Our finding of antibody-mediated cross-protection against tachypnea in the cotton rat model is an important step toward recognition that this type of response is not limited to mice, and is therefore likely to be present in other animal species, including man.
Our results show that heterosubtypic immunity can be induced by vaccination with either live or inactivated virus that is administered intramuscularly. These results differ from those reported by Tumpey et al.  and Takada et al.  that show heterosubtypic protection in mice following vaccination with intranasal but not intramuscular-delivery of an inactivated virus vaccine. This latter failure to protect against challenge in mice is likely to reflect the relatively weak responses induced following parental immunization. In our studies three intra-muscular administrations of inactivated virus resulted in HAI titers similar to those obtained following infection; this vaccination regimen was sufficient for heterosubtypic protection supporting the idea that a mucosal IgA response is not necessary for this protection.
Increased respiratory rate is a single facet of influenza disease, and while an antibody-mediated mechanism protects against virus-induced tachypnea in cotton rats, it is likely that other immune mechanisms contribute to protection against other signs of disease. This may include cytokines that have antiviral activity or activate macrophages, and cytotoxic T lymphocytes that play a role in eradicating infected cells. Influenza vaccines that induce a broad range of mechanisms are likely to offer the most effective protection against all influenza A viruses, an important consideration in the development of vaccines designed to induce immunity against highly virulent H5N1 strains with potential for pandemic spread. Our results support the idea that antibodies specific for conserved epitopes play a role in protection from influenza induced disease and are therefore likely to contribute to vaccine efficacy, particularly when HA and NA components are poorly matched with circulating influenza A viruses.