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Journal of Virology, July 2009, p. 6566-6577, Vol. 83, No. 13
0022-538X/09/$08.00+0     doi:10.1128/JVI.00302-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Infection of HLA-DR1 Transgenic Mice with a Human Isolate of Influenza A Virus (H1N1) Primes a Diverse CD4 T-Cell Repertoire That Includes CD4 T Cells with Heterosubtypic Cross-Reactivity to Avian (H5N1) Influenza Virus{triangledown} ,{dagger}

Katherine A. Richards, Francisco A. Chaves, and Andrea J. Sant*

David H. Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical Sciences, Department of Microbiology and Immunology, University of Rochester, Rochester, New York 14642

Received 11 February 2009/ Accepted 14 April 2009


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ABSTRACT
 
The specificity of the CD4 T-cell immune response to influenza virus is influenced by the genetic complexity of the virus and periodic encounters with variant subtypes and strains. In order to understand what controls CD4 T-cell reactivity to influenza virus proteins and how the influenza virus-specific memory compartment is shaped over time, it is first necessary to understand the diversity of the primary CD4 T-cell response. In the study reported here, we have used an unbiased approach to evaluate the peptide specificity of CD4 T cells elicited after live influenza virus infection. We have focused on four viral proteins that have distinct intracellular distributions in infected cells, hemagglutinin (HA), neuraminidase (NA), nucleoprotein, and the NS1 protein, which is expressed in infected cells but excluded from virion particles. Our studies revealed an extensive diversity of influenza virus-specific CD4 T cells that includes T cells for each viral protein and for the unexpected immunogenicity of the NS1 protein. Due to the recent concern about pandemic avian influenza virus and because CD4 T cells specific for HA and NA may be particularly useful for promoting the production of neutralizing antibody to influenza virus, we have also evaluated the ability of HA- and NA-specific CD4 T cells elicited by a circulating H1N1 strain to cross-react with related sequences found in an avian H5N1 virus and find substantial cross-reactivity, suggesting that seasonal vaccines may help promote protection against avian influenza virus.


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INTRODUCTION
 
In recent decades, investigators studying both murine and human T-cell responses to influenza virus have succeeded in identifying peptide epitopes from immunized or vaccinated individuals that are the targets of CD4 T cells. These studies suggest a considerable diversity in CD4 responses. Epitopes derived from hemagglutinin (HA), neuraminidase (NA), nuclear protein (NP), polymerase (PB1 and PB2), matrix (M1), and nonstructural protein (NS1) have all been identified (9, 19, 25-28, 32, 61, 64, 85, 86). Our own laboratory previously analyzed the peptide specificity of CD4 T cells in the primary response of HLA-DR1 transgenic mice toward a human isolate of influenza virus and found that the CD4 T-cell repertoire specific for HA alone was diverse and encompassed at least 30 different peptide epitopes (63). In general, studies with humans have been much less systematic than those with the mouse because of the difficulty in obtaining lymphocyte samples from recently infected individuals and because of the complexity of major histocompatibility complex (MHC) molecules expressed in humans. However, recent studies with MHC class II tetramer reagents (19, 61, 64, 72, 86) have permitted the visualization of CD4 T cells specific for influenza virus directly ex vivo or after a brief (10- to 14-day) in vitro expansion. Those studies have led to the conclusion that the repertoire of CD4 T cells is more diverse than that of CD8 T cells and that CD4 T cells that are specific for most influenza virus proteins can be detected.

We have focused on the identification of the peptide specificity of CD4 T cells during the primary response to influenza virus infection using HLA-DR1 transgenic mice with several goals in mind. First, we seek to understand the intracellular events within influenza virus-infected antigen-presenting cells (APC) that shape the repertoire of the peptide:class II complexes expressed, because these events will play a pivotal role in determining the specificity of the anti-influenza virus CD4 T-cell response. Second, we expect these studies to provide significant new insight into the CD4 T-cell antigen repertoire that becomes established upon natural infection of humans with influenza virus. Finally, because HLA-DR1 is widely expressed in human populations, the results of our experiments and the corresponding peptide epitopes identified can immediately be utilized for analyses of human immune responses to influenza viruses and vaccines.

Our work (45, 57, 60, 68, 69) and the works of others (1, 18, 51, 58, 65, 71, 73, 75) regarding CD4 T-cell immunodominance in response to exogenous antigens indicate that CD4 T cells tend to focus on a limited number of peptides. Typical protein antigens that are taken up as a "pulse" by peripheral APC lead to CD4 T-cell priming that is very narrow in specificity, limited to usually only a few (less than five) epitopes. Our mechanistic studies (44, 68, 69) further indicate that immunodominant peptides characteristically display high-stability interactions with the MHC class II molecule. This selectivity in CD4 T-cell responses is at least in part due to DM editing within APC, where DM apparently removes the peptides that have low-stability interactions with class II molecules (44). Therefore, only a limited subset of antigenic peptides arrives at the cell surface at a sufficient density to recruit CD4 T cells.

The characteristics of influenza virus infection suggest that the immunodominance hierarchy might not follow the "rules" established for exogenous protein antigens. Because influenza virus is typically not a systemic infection, virus replication is normally restricted to the lung (3, 29, 33, 59). Therefore, the primary source of viral antigens available for CD4 T-cell priming may not be free virus particles but, rather, may be dendritic cells that become infected with influenza virus while in the lung and then migrate to the draining lymph node (4, 5, 33, 35, 48, 52). If so, then one might predict that the specificity of CD4 T cells could more closely resemble the repertoire that is elicited by "endogenous" antigens synthesized within the APC (21). Endogenous antigens that have ready access to the endosomally localized MHC class II molecules, because they are either membrane associated or secreted, are most efficiently presented by class II molecules (46, 53, 67, 84). For the influenza virus-infected dendritic cell, these preferences in antigen access would favor the presentation of peptides derived from HA and NA, leading to the selective priming of CD4 T cells that are reactive to these viral proteins.

Several critical questions remain with regard to the specificity of CD4 T cells that are elicited in response to influenza virus infection. The first question is how diverse the repertoire is, with regard to both peptide and protein specificities. The second issue is how the CD4 T-cell repertoire changes over time with repeated encounters with different strains of influenza virus, a common occurrence in humans. A final, very important question is whether CD4 T cells elicited during the primary response have equivalent potentials to promote protection against subsequent infection or if this potential is dependent on their antigen specificities. It is thought that the primary contribution of CD4 T cells to protective immunity is their role in facilitating the production of high-affinity neutralizing antibodies to HA and NA (38, 79). Recent studies by Sette and coworkers (74) suggest that for complex viral pathogens, the delivery of CD4 T-cell help for the production of high-affinity antibodies by B cells may require that the CD4 T cells share viral antigen specificity with the B cells. For influenza virus, the most useful CD4 T cells may therefore be those that are specific for the membrane glycoproteins HA and NA.

In the study reported here, we use an unbiased and comprehensive approach to evaluate the peptide specificity of CD4 T cells elicited after live influenza virus infection. We have focused on four viral proteins that have distinct intracellular distributions in infected cells: HA and NA, expressed at the plasma membrane of infected cells and on the exterior of the virion membrane; NP, expressed in the cytoplasm and nucleus of infected cells; and, finally, the NS1 protein, with a distribution similar to that of NP in infected cells but which is excluded from the virion particles. Our studies lead to the conclusion that influenza virus-specific CD4 T cells elicited during the primary response are distributed across all proteins studied and that the NS1 protein is particularly immunogenic. Because of the recent concern about pandemic avian influenza virus and because CD4 T cells specific for HA and NA may be particularly useful for promoting the production of neutralizing antibody, we have also evaluated the ability of HA- and NA-specific CD4 T cells elicited against a circulating H1N1 strain of influenza virus to cross-react with related sequences found in an H5N1 avian virus. We find that priming with an H1N1 virus elicits CD4 T cells that display a significant degree of cross-reactivity with influenza virus epitopes derived from avian viruses.


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MATERIALS AND METHODS
 
Cell lines and reagents. For the mapping of MHC class II restriction, DAP-3 fibroblast cells transfected with the genes encoding HLA-DR1, generously provided by E. Long, NIAID, NIH, were used. The cells were maintained as described previously (63). Cell lines similarly transfected with vector alone were used as a negative control for the determination of MHC restriction.

Mice. DR1 transgenic mice (B10.M/J-TgN-DR1), obtained from D. Zaller (Merck) through Taconic Laboratories, were maintained in the specific-pathogen-free facility at the University of Rochester according to institutional guidelines. Mice were used at 2 to 4 months of age.

Peptides. Peptides (17-mer) overlapping by 11 amino acids, encompassing the entire sequence of the HA and NA proteins from H1N1 influenza virus strain A/New Caledonia/20/99, the NS1 sequence from A/New York/444/2001, and NP from H1N1 influenza virus strain A/New York/348/2003, were used. The following reagents were obtained through the NIH Biodefense and Emerging Infections Research Repository, NIAID, NIH: peptide arrays for the influenza virus A/New Caledonia/20/1999 (H1N1) HA protein, NR-2602, and NA protein, NR-2606; peptide array for influenza virus A/New York/444/2001 (H1N1) NS1, NR-2612; and peptide array for influenza virus A/New York/348/2003 (H1N1) NP, NR-2611. The NP and NS1 sequences are highly conserved (98%) between virus strains A/New Caledonia/20/99, A/New York/444/2001, and A/New York/348/2003. For the H5N1-versus-H1N1 epitope comparison, peptides were made in our own peptide facility. All peptides were reconstituted at 10 mM in phosphate-buffered saline (PBS), with or without added dimethyl sulfoxide, to increase the solubility of hydrophobic peptides and at 1 mM dithiothreitol, for cysteine-containing peptides. Working stocks (100 µM or 1 mM) were prepared in complete Dulbecco's modified Eagle's medium, filter sterilized, and stored at –20°C, as were concentrated stocks.

Influenza virus infection of mice. Influenza A/New Caledonia/20/99 virus was produced as previously described (63). HLA-DR1 transgenic mice were infected intranasally at 50,000 50% egg infective doses, unless otherwise noted, in 30 µl of PBS after being anesthetized by intraperitoneal injection with tribromoethanol (Avertin; 250 to 300 µl per mouse). Eight to twelve days postinfection, mice were sacrificed, and spleen and mediastinal lymph nodes were excised and used as sources of CD4 T cells for enzyme-linked immunospot (ELISPOT) analyses. Lymphocytes from four to five mice were pooled, unless otherwise stated, and depleted of B cells, CD8 cells, and macrophages either by antibody-mediated complement lysis (63) or by negative selection using MACS depletion (Miltenyi Biotech, Gladbach, Germany), according to the manufacturer's instructions.

ELISPOT assays. ELISPOT assays were performed as previously described (45, 63). Briefly, 96-well filter plates (Millipore, Billerica, MA) were coated with 2 µg/ml purified rat anti-mouse interleukin-2 (IL-2) (clone JES6-1A12; BD Biosciences, San Jose, CA) in PBS, washed, and incubated with medium to block nonspecific binding. CD4 T cells (350,000 cells) were cocultured with DAP-3 fibroblasts expressing the HLA-DR1 MHC class II protein (35,000 fibroblasts) and with the indicated peptide or peptide pool at a final concentration of 10 µM each peptide in a total volume of 200 µl for 18 to 20 h at 37°C and 5% CO2. In most experiments, a previously defined immunodominant peptide (HA-75) was included in ELISPOT assays to control for the degree of CD4 T-cell priming in each experiment. Plates were processed to visualize IL-2-producing cells as described previously (63).


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RESULTS
 
Identification of the major epitopes recognized during the primary CD4 T-cell response to live influenza virus infection. We used two different strategies to identify HLA-DR1-restricted epitopes during the primary response to intranasal influenza virus infection (depicted schematically in Fig. 1) using overlapping peptides. For proteins that were large (HA, NP, and NA), peptide-pooling matrices were used, based on a recently described pooling strategy (80, 81) where peptide pools are constructed and arrayed in intersecting rows and columns, with no overlapping peptides in any given row or column. This strategy has several advantages. If there are very few immunodominant epitopes, the stimulatory peptides will be identified as those at the intersections of positive rows and columns. For proteins with more-broad immunodominance patterns, many rows and columns will be positive, making the intersecting points on the arrays less informative, but entire groups of peptides can be eliminated from further consideration if they are in a row or column that is nonstimulatory. If, in the remaining positive pools, the candidate peptides were few, single-peptide assays were employed for the final identification of epitopes. If the remaining number of potential peptide epitopes was high (>35 to 40 epitopes), the strongest candidates (at the intersecting columns and rows) were removed from the matrix for individual analyses, while the remaining peptides were arranged into a new secondary matrix. Peptides in negative pools were again eliminated, and the remaining peptides were tested as single peptides. In our experiments, every stage of the strategy was repeated at least twice. The second strategy employed to identify the HLA-DR1-restricted CD4 epitopes recognized in the primary response was used for small influenza virus proteins, usually of less than 400 amino acids (M1, M2, and NS1). Here, all peptides were tested as single peptides. In most experiments, known immunodominant peptides, typically including a dominant HA peptide ("63/75"), which was previously identified (63) were included in each ELISPOT assay to compare the magnitudes of responses to new CD4 epitopes relative to that of known immunodominant epitopes.


Figure 1
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  		FIG. 1. Schematic representation of the experimental approaches used to map influenza A virus protein epitopes. Depending on the size of the protein, one of two different strategies was used, as shown. After single-peptide epitopes were identified, MHC restriction was confirmed using DAP-3 fibroblast cells transfected with genes encoding HLA-DR1 alpha and beta chains or with cells transfected with HLA-DR alpha alone (DR1 surface negative). Only epitopes that were confirmed to be HLA-DR1 restricted were pursued for further studies.

Figure 2 and 3 show results of experiments that identified the immunodominant epitopes from NA. Figure 2 shows the composition of the peptide-pooling matrices and the ELISPOT results from primary and secondary matrices that allowed us to eliminate some peptides and test candidate positive epitopes. Single-peptide analyses are shown in Fig. 3 and summarized in Table S1 in the supplemental material. Approximately 12 of the total 76 individual NA peptides reproducibly elicited recall responses from CD4 T cells isolated from influenza virus-infected mice. There were three dominant epitopes, found in peptides 23, 44, and 53, and these epitopes stimulated approximately the same number of CD4 T cells as our prototypical immunodominant HA peptide 63/75 (63) (shown for comparison at the right of each data figure). Figure 8 shows that these epitopes are distributed across the entire NA protein and include both genetically conserved and nonconserved regions.


Figure 2
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  		FIG. 2. Screening of CD4 T cells for specificity for peptides in the neuraminidase protein. Mice were infected with A/New Caledonia/20/99 virus, and CD4+ T cells from the spleen of infected mice were isolated 8 days later. CD4 T cells were screened for reactivity (A) with pools of peptides (C). The averages of results from two independent experiments are shown, where the results are displayed as the numbers of IL-2 spots per million CD4 T cells, with standard errors of the means indicated. Shaded rows and columns were nonstimulatory, and those peptides were removed from further study. The boxed rows and columns (C) identify those peptide pools eliciting high levels of IL-2 responses. The intersections of those rows and columns correspond to the likely candidate epitopes (indicated in boldface type) and were removed from the matrix and tested as single peptides. The remaining 38 peptides were pooled into a new matrix (D), and each pool was tested in a secondary screening (B), further identifying some peptides (boldface type) and eliminating others (shaded). The immunodominant HA peptide (HA-75) was used as a positive control in each assay. Data are represented as the averages of two independent experiments, and the standard errors of the means are displayed.


Figure 3
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  		FIG. 3. Single-peptide epitopes from neuraminidase recognized in the primary CD4 T-cell response. CD4+ T cells isolated from spleen cells 10 days postinfection were screened for reactivity toward the indicated single 17-mer peptide at a final concentration of 10 µM. The results are shown as the averages of data from at least two independent experiments for each peptide, and all experiments were normalized to spot counts per million CD4+ cells. Each peptide was tested at least twice; the number of times a peptide was tested, as well as the sequence of the peptide, is shown in Table S1 in the supplemental material. The immunodominant HA peptide (HA-75), indicated by the gray bar, was used as a positive control in each experiment.


Figure 8
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  		FIG. 8. ClustalW sequence alignment of the H1N1 A/New Caledonia/20/99 and H5N1 A/Vietnam/1203/04 hemagglutinin and neuraminidase proteins. The identified epitopes are blue for minor, green for moderate, and red for dominant epitopes, based on the single-peptide analyses. Regions of homology are indicated by asterisks, and regions of variability are indicated by a colon or a period to indicate the extent of divergence.

This type of study was then initiated for NP. Peptides were arrayed into rows and columns, each containing seven to eight peptides, as shown in Fig. 4C. The ELISPOT assays and pools of the secondary matrix are shown in Fig. 4B and D, respectively. Collectively, these experiments revealed that of 80 potential CD4 T-cell peptide epitopes, approximately 18 peptides were stimulatory, 5 of which were overlapping (Fig. 5). Therefore, the number of epitopes in NP is thus likely to be approximately 14, 1 of which, contained in peptides 74 and 75, is consistently the most highly immunodominant epitope, typically stimulating approximately 500 CD4 T cells per million cells. The other epitopes in NP are intermediate or minor, but the minor epitopes are reproducibly positive (Fig. 5).


Figure 4
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  		FIG. 4. CD4 T-cell epitope identification for influenza virus nucleoprotein. CD4+ T cells from the spleen of infected mice at 8 days postinfection were tested for reactivity with NP-derived peptides by 18-h coculture with DAP-3 DR1-positive transfectants cultured with the pools of peptides shown (C). Results of two ELISPOT assays are displayed as the numbers of IL-2 spots per million CD4 T cells (A). The strategy to identify candidate epitopes and to eliminate nonstimulatory peptides is described in the legend to Fig. 2. After the initial screening, 28 peptides were pooled into a new matrix (D), and each pool was tested in a secondary screening (B), further eliminating 13 peptides from study (shaded). The immunodominant HA peptide HA-75 was used as a positive control in each assay and is indicated by the gray bar. Data are represented as the averages of data from two independent experiments, and the standard errors of the means are displayed.


Figure 5
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  		FIG. 5. Single-peptide analyses of the CD4 response to influenza virus nucleoprotein. The number of IL-2-producing CD4+ T cells specific for each candidate peptide identified in the experiments shown in Fig. 4 was determined using CD4 T cells obtained from spleen 10 days postinfection using the single 17-mer peptide indicated below each bar. The results are shown as the averages of data from at least two independent experiments for each peptide, and all experiments were normalized to spot counts per million CD4+ cells. The number of times a peptide was tested, as well as the sequence of the peptide, is shown in Table S1 in the supplemental material. The immunodominant HA peptide (HA-75) was used as a positive control for each experiment and is indicated in gray.

NS1 is a relatively small protein, with only 230 amino acids, and, as a "nonstructural" protein, is expressed only in infected cells and not in the virion itself. Because of its small size, it was necessary to test only 37 candidate peptides. Figure 6 shows a composite of data from six independent experiments. These results show that NS1 is quite immunogenic for its size and contains at least eight different CD4 T-cell epitopes, three to four of which are major, eliciting more CD4 T cells (400 to 500 T cells/million cells) than the strongest HA-derived epitope (200 to 250 CD4 T cells/million cells).


Figure 6
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  		FIG. 6. Results from single-peptide screening of overlapping peptides from NS1 demonstrate its immunogenicity in the primary CD4 response to influenza virus infection. Mice were infected intranasally with A/New Caledonia/20/99 virus, and CD4 T cells were isolated from the spleen 10 days postinfection. The numbers of IL-2-producing CD4+ T cells that are reactive to the test single 17-mer peptide are shown. The results shown are the averages of data from six independent experiments, with the standard errors of the means shown. The response to the immunodominant HA peptide (HA-75) is shown in gray.

Our previous studies have characterized the primary response to HA (63) by using unpurified overlapping peptides of 18 amino acids offset by 11 amino acids. Figure S1 in the supplemental material shows the results of more-recent experiments using an alternative source of synthetic peptides, similar in size to and of the high purity used in this paper for the identification of epitopes from NA, NP, and NS1. HA contains many epitopes, consisting of four to six major, immunodominant peptides and as many as 40 intermediate and minor epitopes. Because epitopes were identified in overlapping peptides, they might consist of a single epitope shared between two overlapping peptides or could represent distinct registers in the two peptides.

Relative immunogenicity of each viral protein tested. The above-described data on epitope mapping were then analyzed in an additional way to gain insight into the relative immunogenicities of the different viral proteins. To make assessments of the potency of each protein in eliciting CD4 T cells, we averaged the spot count (n = 2 and, typically, n > 5 for each major peptide epitope) for each peptide epitope contained in each viral protein. These values, represented as average spot counts per 106 T cells, were summed, allowing us to estimate the total number of CD4 T cells dedicated to each viral protein during the primary response, which we refer to as the "relative immunogenicity" of that protein. The values for each protein are indicated in Table 1 and illustrated graphically in Fig. 7A. HA elicited an average of 2,400 CD4 T cells, NS1 elicited approximately 2,700 CD4 T cells, NP elicited approximately 2,000 CD4 T cells, and NA elicited approximately 1,100 CD4 T cells. We then adjusted these values for the molecular weight of each protein so that small and large proteins could be compared for immunogenicity. Accordingly, the total number of CD4 T cells specific for all of the peptides within a given protein was divided by the number of amino acids in that protein. These values, also shown in Table 1, were then represented graphically (Fig. 7B), which shows these data relative to the total number of CD4 T cells elicited (approximately 8,100 CD4 T cells per 106 CD4 T cells). Also shown in Fig. 7 is the theoretical graph that would have been obtained if the distribution of epitopes was based purely on size (Fig. 7C) or if the distribution of specificity was based on the known access of proteins to MHC class II (Fig. 7D), where we estimated a fivefold advantage in access to class II molecules for the membrane proteins HA and NA, which is almost certainly an underestimate (10, 12, 13, 67, 84). One can see from this analysis that although the primary CD4 response has T cells specific for peptides contained in all the viral proteins tested, NS1 appears to be the most "immunogenic" relative to its size.


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  		TABLE 1. Immunogenicities of the viral proteins testeda


Figure 7
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  		FIG. 7. Relative immunogenicities of HA, NA, NP, and NS1 during the primary CD4 T-cell response. For each positive peptide epitope identified, the number of CD4 T cells elicited per million cells was averaged among replicate experiments. The numbers of CD4 T cells elicited for all of the peptide epitopes within each protein were summed (Table 1) to yield an estimate of the total number of CD4 T cells that are reactive for each protein (A) (Table 1). This value was corrected for the total number of amino acids in the protein to compare the immunogenicity of each of the proteins (B). These values are compared to the theoretical value based on protein size (C) and the theoretical value based on accessibility to class II molecules (D).

Conservation of epitopes among H5N1 and H1N1 viruses. With the ongoing threat of new pandemic influenza virus strains, particularly avian influenza virus (14, 40, 49, 50, 55, 62, 83), one of the important questions to answer is whether previous confrontation with influenza virus infections or vaccines offers any significant protective immunity to distantly related strains of influenza virus. For CD4 T cells, we can now ask whether specificities elicited by these encounters with seasonal H1N1 viruses (or vaccines) offer any heterosubtypic immunity to avian influenza virus strains. The ability to control infection in murine models allows us to answer this question rigorously, with no uncertainty over infection or vaccination history, as one would have in human subjects. In Fig. 8, we have aligned the HA and NA protein sequences of the H1N1 strain of influenza virus used for this study (A/New Caledonia/20/99), which has been included in seasonal vaccines for the previous decade, and those from an H5N1 strain of influenza virus that has been isolated from humans in the last decade (A/Vietnam/1203/04). The DR1-restricted epitopes that we have defined in the current study are indicated by contrasting colors. For the intracellular proteins NP and NS1, there is a high degree of amino acid conservation, and CD4 T cells for these peptides should have almost-complete cross-reactivity. This type of conservation has long been noted in the literature (reviewed in reference 31) and is thought to be due to a lack of selective pressure for mutations. In contrast, as expected from their location at the virion membrane and, thus, their susceptibility to antibody recognition, the HA and NA proteins of these two strains display considerable sequence divergence. Interestingly, however, we did note that despite this amino acid diversity, there are some regions in each protein that are conserved between the two strains. A fraction of the HLA-DR1-restricted epitopes identified in our study are localized within these areas of relative conservation in HA and NA.

To test whether CD4 T cells elicited by seasonal vaccines or infections might be recalled by infection with avian influenza virus, a subset of peptides corresponding to the avian sequences were tested for cross-reactivity to A/New Caledonia/20/99 virus. HLA-DR1 mice were infected with A/New Caledonia/20/99, and CD4 T cells from the primed mice were compared for their abilities to recognize peptides representing the A/New Caledonia/20/99 sequence versus the sequence from A/Vietnam/1203/04. As shown in Fig. 9 and Table 2, a substantial fraction of the CD4 T cells initially elicited in response to the A/New Caledonia/20/99 virus can be recalled, with the peptides corresponding to the homologous sequences from the H5N1 virus. Despite some amino acid substitutions in these sequences, cross-reactive recognition occurred for both the HA and NA epitopes.


Figure 9
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  		FIG. 9. Cross-reactivity of CD4 T cells between H5N1 and H5N5 viruses. Mice were infected intranasally with A/New Caledonia/20/99 influenza virus, and the number of IL-2-producing CD4+ T cells was determined 10 days later by 18-h in vitro stimulation using either the H1N1 peptide epitope from A/New Caledonia/20/99 (closed bars) or the corresponding H5N1 peptide epitope from A/Thailand/4 SP-528 (open bars) at a final concentration of 10 µM. The results are shown as the averages of data from three independent experiments, with the standard errors of the means shown.


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  		TABLE 2. Cross-reactivity of CD4 T cells for epitopes derived from H1N1 and H5N1 virusesa


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DISCUSSION
 
Influenza virus is an immunologically complicated pathogen, particularly in humans, where a long life span allows multiple encounters with genetically variable influenza viruses and vaccines. The first encounter for most humans is through natural infection in early childhood, and therefore, the original memory compartment for influenza virus is based on priming through live infection. After this original contact, individuals encounter influenza virus and its proteins again periodically, perhaps every 2 to 3 years, through natural encounters with different subtypes or strains of influenza virus or through active vaccination (14, 38, 40, 54, 78). In order to understand how the influenza virus-specific CD4 T-cell memory compartment is shaped over time, it is necessary to first understand the diversity and specificity of the primary response. In this study, we have found that the primary CD4 T-cell response to live influenza virus infection is highly diverse and represented by T cells for each of the viral proteins tested: HA, NA, NP, and NS1. Surprisingly, we found that there was no selective enrichment for CD4 T cells specific for the membrane-associated antigens HA and NA and that of all the virus proteins examined, NS1 appeared to be the most immunogenic, recruiting more than 2,400 CD4 T cells per million CD4 splenic T cells. CD4 T cells specific for NP and HA were also abundant, with less representation of CD4 T cells specific for the membrane-associated NA protein.

Our results for CD4 T-cell responses suggest a much broader epitope distribution than was predicted from a previous study. The primary response of C57BL/6 (H-2b) mice to a mouse-adapted strain of influenza virus was found to be quite restricted, specific primarily for peptides from HA and NP, with two peptides from these proteins accounting for more than 30% of the total influenza virus-specific CD4 T cells from the lung (17). We performed a study similar to what we describe here using H-2b mice with strain A/New Caledonia/20/99 and found results similar to those described previously by Crowe et al. (17), with a highly restricted repertoire focused on NP (J. Nayak, K. Richards, and A. Sant, unpublished data). In contrast, during the primary response to A/New Caledonia in BALB/c mice, which express I-Ad and I-Ed, we find a highly diverse repertoire similar to what we have found with HLA-DR1 transgenic mice (A. Sant, unpublished data). We suspect that the pattern seen in the BALB/c and DR1 transgenic mice reflects the overall broad diversity of peptides that can be presented by these host class II molecules (I-Ad, I-Ed, and HLA-DR1) compared to that of I-Ab. We hypothesize that under conditions of relatively promiscuous capture of virus-derived peptides by class II molecules, the major force in driving CD4 T-cell specificity will be characteristics of the viral protein itself rather than stringent peptide selection by MHC class II molecules. Because humans express as many as 12 class II proteins, depending on the MHC haplotype expressed, heterozygosity at MHC loci, and options for cross-allelic mixed class II dimers, we expect that the results for the HLA-DR1 transgenic mice described here are more predictive of what will generally be found in the influenza virus-specific CD4 T-cell repertoire in humans. In fact, in a recent study using MHC-derived tetramers to detect influenza virus-specific T cells in human subjects (64), many of the HLA-DR1-restricted epitopes detected from peripheral blood of healthy human donors, including those from HA, NA, and NP, were identified in our studies. We have also confirmed a subset of these specificities in human CD4 T cells using ELISPOT assays (our unpublished results). The specificities detected for humans are indicated in Table S1 in the supplemental material. Although the diversity of CD4 T cells detected in the current study is surprising, several aspects of our experimental design as well as other ongoing work in our laboratory make us confident that the epitopes which we have defined all belong to the CD4 lineage, are HLA-DR1 restricted, and are not due to nonspecific activation by high concentrations of peptides used in the ELISPOT assays. With regard to the issue of CD4 T-cell specificity, the T-cell population used for all of our assays is rigorously (>99%) depleted of CD8 T cells, and all the peptides identified require HLA-DR1 expression to activate the CD4 T cells. In addition, we have produced CD4 T-cell hybridomas specific for a number of the epitopes identified here, and these T cells all display peptide specificity and HLA-DR1 restriction. Finally, we have no reason to suspect that the cytokine-producing cells are activated nonspecifically by peptides used at high concentrations. Lower concentrations of peptide (2 or 0.2 µM) are potent in activating many of the T cells identified here, and perhaps even more compellingly, in studying the response patterns of different MHC congenic strains of mice, using the same virus and same stock of purified peptides as those used here, we have discovered completely nonoverlapping patterns of epitope specificity of CD4 T cells.

In considering the CD4 T-cell specificities reported here, it is intriguing that NS1 and NP appear to be highly immunogenic during the primary response to influenza virus despite their localization in the cytosol and nucleus, sites that are thought to have relatively inefficient access to MHC class II molecules. This finding requires the consideration of alternative factors that may control epitope dominance in the response to influenza virus. The absolute abundance of the viral protein within APC may play a critical role in determining its access to MHC class II molecules, as might the kinetics of viral protein expression. In most cells studied, influenza virus infection essentially shuts down host cell gene expression within a few hours of infection (41, 42). Because the MHC class II-restricted presentation of antigen utilizes primarily newly synthesized class II molecules (reviewed in references 11, 15, 39, and 68), it is possible that the earliest-synthesized and most-abundant proteins will most efficiently access MHC class II molecules in endosomal compartments. Although the kinetics of influenza virus protein synthesis in cultured cell lines suggest that NS1 is among the earliest proteins synthesized (66, 76, 88), little is known about the kinetics of individual influenza virus proteins in dendritic cells, the relevant cell type in CD4 T-cell priming known to have distinctive characteristics of influenza virus infection compared to those of other cell types (5, 37). With regard to the restrictions imposed by subcellular localization, it is possible that the cytosolic and nuclear antigens NP and NS1 gain access to endosomal compartments of APC via the process of autophagy, an intracellular process that allows the engulfment and ultimate delivery of cytosolic and nuclear proteins to lysosomal compartments by the fusion of membrane vesicles derived from the endoplasmic reticulum (16, 20, 47, 70, 77). Autophagy rates increase upon virus infection (47, 70, 87), and it is possible that this alternative mechanism allows the needed access of viral NS1, NP, and other cytosolic/nuclear proteins to the MHC class II loading compartments.

Independently of the mechanisms that control antigen presentation of influenza virus peptides by MHC class II molecules and, thus, the repertoire of elicited CD4 T cells, it is important to consider the implications of our findings for host defense. The most important function of CD4 T cells for protection from influenza virus is thought to be the provision of "help" for the production of high-affinity neutralizing antibodies to HA and NA (reviewed in reference 38). Recent data suggest that for some viruses, there may be an obligate link between the specificity of CD4 T cells and the antigen-specific B cells (74). If this model is correct, then the most useful CD4 T cells may be those that are specific for HA and NA. CD4 T cells that are specific for intracellular NP, polymerase, and NS1 proteins may be of more-limited value in facilitating antibody responses. If true, then the implications for heterosubtypic immunity are profound. Because of the high degree of genetic drift within influenza viruses (6, 8, 22, 24, 31, 34, 36), intermittent encounters with influenza virus strains in humans may preferentially boost T cells that are specific for conserved peptides enriched in nucleoprotein and polymerase proteins (2, 7, 22, 23, 30, 43, 56, 82). However, if these CD4 T cells have limited contributions in facilitating antibody responses, they may not be particularly valuable for protection. Instead, heterosubtypic immunity will depend on the cross-reactivity of CD4 T cells specific for the HA and NA epitopes that are shared among seasonal viruses and newly emerging viral strains. Because of the importance of this issue, we evaluated a subset of these HA- and NA-derived epitopes, localized primarily to genetically conserved regions of these proteins, for cross-reactivity with homologous sequences from a human isolate of an H5N1 virus. Our study revealed a substantial degree of cross-reactivity between H1N1 and H5N1 sequences. It is likely that for those peptides that have amino acid differences within a peptide epitope, the amino acid substitutions may be located at MHC anchor sites rather than T-cell contact sites. Anchor substitutions typically have very little negative impact on T-cell reactivity (44, 45). Our results are encouraging and suggest that although substantial numbers of CD4 T cells that are specific for internal viral proteins are elicited in the primary response to live influenza virus infection, the CD4 T cells primed during seasonal encounters with influenza virus or through vaccination with seasonal vaccines may prime CD4 T cells that are reactive with HA and NA which have the potential to be reelicited and expanded upon challenge with a heterosubtypic strain of influenza virus. CD4 T cells of this specificity may be effective in promoting more-rapid and more-robust antibody responses to heterosubtypic challenge than would occur in a nonvaccinated individual.


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ACKNOWLEDGMENTS
 
This work was supported by awards HHSN266200700008C, R01 AI051542-0641, and 5R21AI69372 to Andrea J. Sant from the National Institutes of Health.


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FOOTNOTES
 
* Corresponding author. Mailing address: David H. Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical Sciences, Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14642. Phone: (585) 275-9798. Fax: (585) 273-2452. E-mail: Andrea_Sant{at}urmc.rochester.edu Back

{triangledown} Published ahead of print on 22 April 2009. Back

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/. Back


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Journal of Virology, July 2009, p. 6566-6577, Vol. 83, No. 13
0022-538X/09/$08.00+0     doi:10.1128/JVI.00302-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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