In the present study, we chose the C57BL/6 (B6) mouse model to assess the extent of heterosubtypic immunity based on the following considerations: (1) Both CD8 and CD4 T cell epitope repertoire of influenza A virus have been extensively characterized in B6 mice and a Db
-restricted, immunodominant NP366
epitope is the major target to mediate a protective, memory CTL response after secondary infections with influenza A viruses 
. (2) Analysis of the deduced amino acid sequences of the NP gene from a large number of influenza A viruses (955 sequences), indicated that, certain amino acid residues within the NP366
CTL epitope undergo constant evolutionary change (), despite the overall conserved nature of this internal protein between different influenza A virus subtypes (~89% identity among sequences analyzed). A total of ten naturally occurring NP366
variants were identified, each representing a different subtype of influenza A virus. Of interest, all of the amino acid substitutions observed were located at the C-terminal bulge of the NP366
peptide backbone, the featured structural region of the NP366
complex exposed for recognition by the TCRs of CD8 T cell subset induced by influenza A virus infection 
. (3) Except for the NP366
epitope, each panel of heterosubtypic influenza A viruses used for priming and secondary challenge in the present study share identical immunodominant MHC class II T cell epitopes and all other known class I T cell epitopes (Table S1
and Table S2
). Such a combination of priming and challenge viruses offers an unique opportunity to dissect the impact of NP366
epitope sequence variation on memory CD8 T cell-mediated protective heterotypic immunity in B6 mice.
Naturally occurring NP366/Db CTL epitope variants of influenza A viruses1
Heterosubtypic protection is significantly decreased when challenge influenza A viruses do not share identical NP366/Db CTL epitopes as the priming virus
We first evaluated the extent of heterosubtypic protection against lethal challenge with viruses that do or do not share the identical NP366
CTL epitope as the priming viruses. As expected from previous studies 
, mice that were primed by intranasal infection with X31 virus (H3N2) did not show any body weight loss following intranasal challenge with a low lethal dose (3 LD50
) of heterosubtypic PR8 virus (H1N1) that shares complete sequence identity in the NP366
CTL epitope with X31 virus (, upper panel). To further evaluate the robustness of the heterosubtypic protection induced under this circumstance, a second group of X31-primed mice received a 10-fold higher lethal challenge dose (30 LD50
) of PR8 virus. Even in this case, X31-primed mice exhibited only modest and transient weight loss on day five after the lethal viral challenge (maximum mean weight loss: ~5%) and rapidly regained the weight to normal levels by day seven post-challenge. All X31-primed animals survived challenge with either lethal dose of PR8 virus. In contrast, mice that were primed by NT60 virus (H3N2) and then challenged with 3 LD50
of PR8 virus, lost approximately ~10% of body weight between day 7 and 9 post-challenge. When a second group of NT60-primed mice were challenged with high lethal dose (30 LD50
) of PR8 virus, mice experienced severe weight loss. Only 50% of the mice survived in this group of animals. Control mice challenged with either doses of PR8 virus experienced substantial and rapid weight loss and succumbed to between day five and seven post-challenge. Thus, compared with the control mice, priming with NT60 virus can confer substantial level of cross-protection when the challenge dose of the heterosubtypic PR8 virus was low. However, severe body weight loss was observed in the group of NT60 virus-primed animals when high lethal dose of the PR8 virus was administered. One explanation for the results described above is that the robust cross-subtype protection conferred by priming with X31 virus is simply due to the ability of the virus to replicate more extensively in the lung of B6 mice compared to NT60 virus (the mean peak lung virus titers are approximately 107
/lung, respectively), which may in turn stimulate a stronger memory immune response after priming. To rule out this possibility, we tested the ability of the X31 virus to induce cross-subtype protection against challenge with Taiwan virus (H1N1), where the NP366
epitope sequences between the two viruses differ by only one amino acid residue at the C-terminal bulge of the peptide-Db
ligand (). A different H3N2 priming virus, Memphis, was also used because it shares complete identity in NP366
epitope sequence with Taiwan virus and replicates to a similar extent in B6 mouse lungs as the NT60 virus (mean peak lung virus titer: 105
/lung). As shown in , lower panel, priming with the Memphis virus conferred robust protection against either a low (3LD50
) or high (9 LD50
) lethal dose challenge of the heterosubtypic Taiwan virus. Note that the latter is the maximum lethal challenge dose achievable for this virus. In contrast, cross-protection against the Taiwan virus was significantly reduced in X31-primed mice.
Body weight loss and survival after heterosubtypic influenza virus infections.
Together, these results support previous observations demonstrating that priming with one subtype of live influenza A viruses can results in significant protection against subsequent infections with a different subtype of influenza A viruses 
. Moreover, our results suggest that optimal protective heterosubtypic immunity conferred by CD8 T cells is only achieved when priming and challenge virus share the identical immunodominant CTL epitope(s). When the challenge viruses do not share the identical CTL epitopes with the priming viruses, the degree of heterosubtypic immunity against different subtypes of influenza A viruses appears to depend on the challenge dose. Complete cross-protection from death can be achieved, if the challenge dose is low, but protective heterosubtypic immunity is considerably reduced in the face of a high lethal challenge dose.
Decreased heterosubtypic immunity is not primarily correlated to mucosal and serum antibodies, but to CD8 T cell subset
It has become increasingly clear that both T cell and B cell arms of the immune system may contribute to heterosubtypic immunity after influenza A virus infection, depending on the experimental systems used. In the present study, we used immune competent B6 mice in conjunction with intranasal priming and challenge to assess the capacity of the heterosubtypic immunity. It is possible that the differential capacity of the heterosubtypic immunity observed under this circumstance may be attributed to either the B cell or T cell arm of the immune system, or both.
To distinguish these possibilities, we first examined the possible correlation between serum HA-specific antibodies and heterosubtypic immunity. As expected, priming of B6 mice with either X31 or Memphis virus intranasally resulted in a robust serum antibody response to the homologous strains of the H3N2 viruses (). Thirty-five days after the priming, serum HI geometric mean titers (GMT) to the homologous X31 and Memphis virus reached to 320 and 184, respectively. However, no subtype cross-reactive serum HI antibodies were detectable to the heterosubtypic Taiwan H1N1 virus following intranasal priming with either live H3N2 virus. Control mice did not show any detectable HI titers against any of the three influenza A viruses tested. These observations are consistent with the generally accepted understanding that serum HI and neutralizing antibodies to the HA glycoprotein of influenza A viruses are subtype-specific and their role in heterosubtypic immunity is minimal under normal circumstances.
Strain-specific and cross-reactive antibodies following lethal challenge of primed mice with heterosubtypic influenza A virus.
We next examined whether IgA antibodies on the surface of the upper and lower respiratory tract of the primed mice contributed to heterosubtypic immunity following a lethal challenge. shows that nasal washes obtained 35 days after priming with either X31 or Memphis virus contained IgA antibodies reactive to their homologous virus, although the GMT titers were low (32 and 8, respectively). However, cross-reactive nasal IgA antibodies to the heterosubtypic Taiwan H1N1 virus were not detected following intranasal priming with either H3N2 viruses. Similar results were obtained when lung IgG antibodies from the same animals were examined by a whole virus-ELISA, except that the amount of cross-subtype IgG antibodies to the Taiwan H1N1 virus were equally evident in the lung washes of both groups of the animals (). Given that HA and NA from X31, e.g. A/Aichi/2/68, and A/Memphis/102/72 virus share high sequence identity (HA1: 96.6%, NA: 94.4%, respectively), it is not surprise that approximately equivalent amount of cross-subtype IgG antibodies to the Taiwan H1N1 virus were detected in the lung washes of both groups of the animals (. GMT: 588 after priming with X31 versus 256 after priming with Memphis virus, respectively). Thus, neither nasal IgA nor lung IgG appeared to correlate with the differential capacity of the heterosubtypic immunity induced ().
Increasing evidence suggests that M2-specific antibodies induced by vaccination can provide cross-subtype protection against influenza A virus infections 
. We monitored the levels of serum anti-M2 antibodies after priming with either X31 or Memphis virus using an M2e peptide-based ELISA. As shown in , although M2-specific antibodies were detected, the GMT titers were low (35 after X31priming and only 5 after Memphis priming, respectively) and highly variable among individual mice. Therefore, we found no correlation between the levels of serum M2-specific antibodies induced by intranasal infection priming and the cross-subtype protection.
Next we examined the effect of T cell subset depletion on the capacity of heterosubtypic immunity described above. As shown in , mock-primed animals experienced substantial body weight loss following lethal challenge with 9LD50 of Taiwan virus. All of the animals succumbed to infection by day 7 after lethal challenge, independent of CD8 or CD4 T cell depletion in vivo
. In contrast, consistent with the results shown in , priming with Memphis virus led to strong resistance to subsequent lethal challenge with 9 LD50
of Taiwan virus (). However, when the CD8 T cell subset was depleted in vivo
from the Memphis-primed animals, significant weight loss was observed on day 5 after lethal challenge (mean percentage of original body weight: 92.3% versus 82.9% when PBS control was compared with CD8-depleted group, p
0.0079). Depletion of CD4 T cell subset also resulted in slightly more severe weight loss compared to the PBS control group (90.2% versus 92.3%), but the difference was statistically not significant (P
0.0952). Note that when compared to PBS-treated naïve animals, mice that was primed with Memphis virus and depleted of memory CD8 T cells showed significantly higher degree of cross-protection on day 5 after challenge with heterosubtypic Taiwan H1N1 virus (Mean body weight loss: 77.4% versus 82.9%, respectively. P
0.0389). This is consistent with previous observation that primed CD4 T cell subset may also provide certain degree of cross-subtype protection under certain circumstances 
Effect of CD8- or CD4 T cell depletion on heterosubtypic immunity.
These results provide further evidence that multiple components of the immune system may be involved in the cross-subtype protection against heterosubtypic influenza A viruses, but CD8 T cell functions may be most closely correlated with heterosubtypic protection induced following intranasal inoculation with live influenza A virus.
Decreased capacity of the heterosubtypic immunity is correlated with significantly reduced magnitude of the cross-reactive memory CD8 T cell response to the NP366/Db variants
Early data obtained using 51
Cr-release assay revealed that influenza virus-specific CD8 T cells are in general highly cross-reactive 
. However, the magnitude of cross-reactive CD8 T cell responses to influenza virus CTL epitope variants has not been quantitatively studied in the context of heterosubtypic immunity in vivo
. We thus used a dual MHC class I tetramer technique to quantify the total numbers of the NP366
-specific CD8 memory effector cells in the lung and the spleen of the mice following the sequential influenza A virus infection. As shown in Fig. S1A
, flow cytometric analysis confirmed the specificity and cross-reactivity of these tetramers to homologous and heterologous NP366
variants. As shown in , priming with X31 virus followed by challenge with PR8 virus led to a massive expansion of the PR8-NP366
-specific CD8 T cells in the lungs of the animals on day 5 post-challenge (6.94×105
cell/lung). However, when NT60-primed mice were challenged with PR8 virus, the total number of tetramer-positive cells detected in the lungs was significantly lower (0.84×105
cell/lung) compared with those obtained following X31-PR8 virus sequential infection (p
0.0006). Similar results were obtained when X31-Taiwan virus sequential infection was performed. A detailed analysis of the compositions of the NP366
tetramer-positive memory effector cell populations showed that following X31-PR8 virus sequential infection, over 95% of the responding memory CD8 T cells were directed to the NP366
epitope shared by both virus strains ( and Fig S1B
). CD8 T cells cross-reactive to the NT60-NP366
variant were detectable, but at a very low frequency (<5%). In contrast, following either NT60-PR8 or X31-Taiwan sequential infection, the majority of the responding memory effector cells were cross-reactive to both the priming and challenge NP366
epitope. In either case, only a small proportion of the cells were specific for the respective priming and the challenge NP366
epitope. It is intriguing to note that the percentage of the CD8 T cells specific for the priming NT60-NP366
epitope was considerably higher than those specific for the challenge PR8-NP366
variant (24.5% versus 6.4%) following the NT60-PR8 sequential infection. However, such a biased memory CD8 T cell response was less pronounced following X31-Taiwan sequential infection (19.2% of X31-NP366
cells versus 14.2% of Taiwan-NP366
cells). These data clearly demonstrate that the quantity of the cross-reactive memory CD8 T cells is considerably diminished when priming and challenge viruses lack complete sequence identity of the NP366
CTL epitope. Preliminary data indicate that the functional quality of the different subsets of the NP366
memory effector cells may not differ considerably, as detection of intracellular IFNγ secretion following restimulation of the CD8 T cells with the homologous as well as the NP366
variant peptides did not reveal substantial differences for any population tested.(Fig. S2
Magnitude and composition of specific and cross-reactive CD8 T cells to wild-type and variant NP366/Db epitope after heterosubtypic influenza virus infections.
Together with the observations described above ( and ), these results clearly indicate that the capacity of the heterosubtypic immunity mediated by the NP366
CD8 T cells is closely associated with the sequence similarities between the priming and the subsequent challenge NP366
epitopes. If both epitopes bear the identical NP366
sequence, strong heterosubtypic immunity can be anticipated, primarily owning to a robust expansion of the memory CD8 T cells triggered by the homologous challenge NP366
epitope. When the challenge viruses do not share the identical sequence of the NP366
epitopes with the priming viruses, two important factors may contribute to the reduced hetersubtypic immunity. First, considerably reduced expansion of the cross-reactive NP366
cells triggered by the heterologous challenge NP366
variants (). Another factor may be a biased memory CD8 T cell response toward the priming NP366
, but not the challenge NP366
epitope, as was observed with the NT60-PR8 sequential infection (). The reason for this phenomenon is not clear at present. It is possible that CTL original antigenic sin may exist under this circumstance. So far, this phenomenon has been only documented after infection with lymphocytic choriomeningitis viruses 
or Dengue virus 
. More comprehensive studies are needed to ascertain whether the phenomenon observed after NT60-PR8 sequential infection represents a generic CTL original antigenic sin phenomenon in the influenza A virus system, as such a phenomenon was not observed following a sequential delivery of X31-NP366
, which differ in one amino acid residue at position 7 of the NP366
Limited plasticity of a dominant public TCR to the heterologous NP366/Db variants may contribute to the decreased expansion of the cross-reactive memory CD8 T cells
The data thus far suggest a limited TCR plasticity of the NP366
-specific memory CD8 T cells for heterologous NP366
variants. We and others have observed previously that public TCRs are strongly selected following a primary influenza A virus infection in B6 mice (up to 50%) 
. As a dominant public TCR that recognizes PR8-NP366
epitope has been functionally expressed in the form of a stable transfectant 
, this allowed us to dissect to what extent the public TCR can cross-react to the naturally occurring NP366
variants identified from the bank of NP sequences (). As shown in , with the exception of the NP366
ET-DN amino acid substitutions, all of the NP366
variants bound to the Db
molecule with similar high affinity. Surprisingly, when the ability of the public TCR to recognize these NP366
peptide variants was examined, only the cognate NP366
ligand derived from the NP of the PR8 virus was able to trigger the activation of the TCR transfectant (). Interaction between the nine NP366
peptide variants and the public TCR did not result in the production of IL-2 under the same experimental conditions. These results indicate that the high frequency of the public TCR in the memory CD8 T cell repertoire cannot tolerate any substitutions of the TCR contact residues within the original NP366
peptide sequence. Even a single conserved residue replacement at position 7 (D-E7) completely abolished the productive interaction between the TCR and the variant NP366
ligands, as observed in the case of the Taiwan-NP366
ligand (Table S1
). It is conceivable that such a stringent requirement for sequence identity between the public TCR and its original NP366
ligand may considerably reduce the cross-reactivity of memory CD8 T cells following re-infections with heterosubtypic influenza A viruses bearing NP366
Plasticity of a dominant public TCR to NP366/Db variants.
Taken together, our results reveal a significant limitation of an important immune effector responsible for heterosubtypic immunity to influenza A virus infection. We found that following primary infection, a high frequency of the NP366-specific memory T cells generated used the public TCRs that are strictly specific for the priming NP366/Db epitope sequence. Upon re-exposure to heterosubtypic viruses that share the identical NP366 epitope sequence with the priming strain, robust memory CD8 response to the initial NP366/Db ligand were obtained, leading to the generation of strong heterosubtypic immunity. However, if the challenge virus does not share the identical NP366 epitope sequence with the priming virus, expansion of the pre-existing, cross-reactive memory CD8 T cells may be limited, either due to their low frequency in the memory CD8 cell repertoire or possibly, a CTL original antigenic sin phenomenon under certain circumstances. Consequently, the heterosubtypic immunity generated is significantly reduced. This implies that efforts to promote CD8 T cell immunity-based vaccination strategies against heterosubtypic influenza A viruses may be most effective, only when the priming and the protective CTL epitopes targeted share the identical epitope sequences. Our findings re-emphasize that multiple immune components may be required for development of broad-protective influenza vaccines.