T-cell-mediated immune responses play critical roles in host defenses against influenza A virus infection. The cross-reactive memory T cells, especially CTLs, are believed to improve virus clearance and reduce the severity of illness even in the absence of virus-specific antibodies, although they cannot prevent influenza infection (40
). The cross-reactivity of influenza A virus-specific T cells to heterosubtypic strains has been reported (5
). However, little is known about the cross-responses of seasonal influenza virus-specific CTLs against pdmH1N1. In this study, we further demonstrated that both bulk memory CTLs and epitope-specific CTLs established by seasonal human influenza A viruses or vaccine in healthy individuals had cross-responses against pdmH1N1 virus, as shown by the direct lysis of pdmH1N1-infected cells and expression of IFN-γ and TNF-α upon pdmH1N1 challenge. Thus, we clearly demonstrated the cross-cytotoxicity of the preexisting seasonal influenza A virus-specific CTLs against pdmH1N1.
It is known that CTLs may not provide sterilizing immunity but may promote viral clearance and alleviate illness severity, which have been extensively studied in animals (3
). There are more accumulating data showing that T-cell responses can protect against influenza. High levels of CD8 T-cell response are correlated with reduced viral shedding in persons without specific antibody (40
). Most importantly, the cross-protection of such CTLs has been demonstrated in humans in vivo
by the Cleveland family study, in which persons who had experienced symptomatic H1N1 influenza were found to be partially protected from the following pandemic H2N2 virus infection in 1957 (17
). Indeed, even for adults over 60 years old, the cross-reactive T-cell responses play some role despite the fact that they have some level of preexisting antibodies, as the cellular responses, but not antibodies, are correlated with protection against influenza illness among individuals older than 60 years (39
It is interesting that the disease severity seems not to be related to the preexisting cross-reactive memory T cells during infection with the highly pathogenic avian influenza virus, although cross-reactive memory T cells have been detected in healthy populations by determining the IFN-γ responses (29
). The fact that lethal H5N1 influenza viruses are resistant to the antiviral effects of IFN-α, IFN-γ, or TNF-α may partially explain this (49
pdmH1N1 has been shown to replicate more efficiently and cause more severe pathological lesions than the current seasonal influenza virus in animal models (28
). However, the illness caused by this novel pandemic pdmH1N1 in most patients is mild (9
). Recently, some degree of cross-reactive T-cell IFN-γ responses against pdmH1N1 virus was detected in a healthy population (21
). Here we provide further evidence of the preexistence of cross-reactively functional memory CTLs to pdmH1N1 in healthy individuals. In addition, these cross-reactive CTLs expressed CCR5 and CXCR3, which could help cross-reactive CTLs migrate to the sites of infection, as pdmH1N1-infected cells express the ligands of CCR5 (CCL3, CCL4, and CCL5) and CXCR3 (CXCL10) (57
). Our data suggest that the preexisting cross-reactive CTLs might contribute, at least in part, to the mild illness in this pandemic.
Since there is a very low frequency of influenza virus-specific T cells in human peripheral blood, most studies have used IFN-γ responses to investigate the cross-reactive T-cell responses or have used influenza virus peptide-pulsed cell lines or cell lines in which HA, NA, and M proteins are expressed as target cells to determine the cross-reactive CTLs of in vitro
-expanded CD8 T-cell lines (5
). However, these assays have some limitations. First, IFN-γ-secreting T cells are not necessarily capable of lysing the target cells (6
). Second, influenza virus peptides or HA, NA, and M proteins cannot fully represent the virus as a whole. To overcome these limitations, here we used the whole virus-infected and peptide-pulsed autologous primary cells as the target cells to examine the killing capacities of virus-specific CTLs. Although the assays we used here have obvious advantages, they still have limitations, as using in vitro
-amplified and purified virus-specific CD8 T cells to examine their cytotoxic activities may not fully represent the in vivo
situation. A prospective population-based case-control study may make it possible to further confirm our current findings.
Here, we have shown that 17 of 94 experimentally defined CD8 T-cell epitopes derived from the previous influenza A viruses are conserved in pdmH1N1, and more than half of these epitopes are derived from the M1 proteins. No conserved epitope was found in HA protein. In addition, we further demonstrated that about 65% (11/17) of the MHC class I-restricted CD8 T-cell epitopes in pdmH1N1 were completely conserved in seasonal influenza vaccine H1N1 strains during the last 20 years, and most of these invariant epitopes in vaccine strains are derived from M1 protein. Consistent with our findings, previous studies also demonstrated that the HLA-A2-restricted M158-66
epitope is the most dominant and frequently recognized CD8 T-cell epitope in humans, and memory responses to influenza virus are usually stronger in individuals with the HLA-A2 genotype than in those with other genotypes (2
). Importantly, we also found that seasonal influenza vaccination could expand the cross-reactively functional M158-66
epitope-specific memory CTLs in 20% (4 out of 20) HLA-A2-positive individuals. These findings suggest that vaccination with seasonal influenza vaccines might still be of benefit in this pandemic, at least in some cases, in terms of the induction of cross-reactive CTLs. Indeed, recent epidemiological studies also showed that seasonal trivalent inactivated influenza vaccine (TIV) could provide some protection (16
), although the seasonal influenza vaccine does not stimulate protective antibody responses to pdmH1N1.
The ability of commercially available influenza vaccines to induce human T-cell responses may vary depending on the internal protein contents of vaccines from different manufacturers (12
). Here we showed that TIV (GlaxoSmithKline) induced HLA-A2/M158-66
T cells in 20% of HLA-2-positive individuals, as this vaccine contains a relatively high level of M protein (11
). Consistent with our finding, recent studies also showed that TIV (Sanofi Pasteur) could induce HLA-A2/M1-tetramer+
T cells in 12 to 25% of HLA-2-positive individuals (45
). Live attenuated influenza vaccine (LAIV) is expected to induce T-cell responses more efficiently than TIV, as it contains all the viral internal proteins. He et al. reported that the frequency of influenza A virus-specific IFN-γ-producing CD4 and CD8 T cells significantly increased after LAIV but not TIV immunization in children of ages 5 to 9 years (26
). A larger proportion of elderly volunteers who received both LAIV and TIV than of those who received TIV alone experienced a postvaccination rise in anti-influenza A virus CTL activity (22
). These studies suggest that LAIV might have more benefits than TIV in providing cross-protection against variant influenza viruses, and this needs to be further confirmed by a well-designed case-control study in the future.
In conclusion, we clearly demonstrated that memory CTLs established by seasonal human influenza A viruses or vaccines could cross-react against pdmH1N1 virus. Our data suggest that individuals who were infected with seasonal human influenza A viruses previously or who received seasonal human influenza vaccines may derive benefit, at least in part, from the preexisting cross-reactive memory CTLs to reduce the severity of pdmH1N1 infection even without protective antibodies. These data may also provide some valuable insights for the future design of broadly protective vaccines to improve protection against influenza, especially pandemic influenza.