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Journal of Virology, December 2008, p. 12241-12251, Vol. 82, No. 24
0022-538X/08/$08.00+0     doi:10.1128/JVI.01563-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Immunomic Analysis of the Repertoire of T-Cell Specificities for Influenza A Virus in Humans {triangledown} ,{dagger} ,{ddagger}

Erika Assarsson,1 Huynh-Hoa Bui,1 John Sidney,1 Qing Zhang,1 Jean Glenn,1 Carla Oseroff,1 Innocent N. Mbawuike,2 Jeff Alexander,3 Mark J. Newman,3 Howard Grey,1 and Alessandro Sette1*

Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California,1 Viral Respiratory Pathogens Research Unit, Departments of Molecular Virology and Microbiology, Baylor College of Medicine, Dallas, Texas,2 Pharmexa-Epimmune, San Diego, California3

Received 23 July 2008/ Accepted 22 September 2008


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ABSTRACT
 
Continuing antigenic drift allows influenza viruses to escape antibody-mediated recognition, and as a consequence, the vaccine currently in use needs to be altered annually. Highly conserved epitopes recognized by effector T cells may represent an alternative approach for the generation of a more universal influenza virus vaccine. Relatively few highly conserved epitopes are currently known in humans, and relatively few epitopes have been identified from proteins other than hemagglutinin and nucleoprotein. This prompted us to perform a study aimed at identifying a set of human T-cell epitopes that would provide broad coverage against different virus strains and subtypes. To provide coverage across different ethnicities, seven different HLA supertypes were considered. More than 4,000 peptides were selected from a panel of 23 influenza A virus strains based on predicted high-affinity binding to HLA class I or class II and high conservancy levels. Peripheral blood mononuclear cells from 44 healthy human blood donors were tested for reactivity against HLA-matched peptides by using gamma interferon enzyme-linked immunospot assays. Interestingly, we found that PB1 was the major target for both CD4+ and CD8+ T-cell responses. The 54 nonredundant epitopes (38 class I and 16 class II) identified herein provided high coverage among different ethnicities, were conserved in the majority of the strains analyzed, and were consistently recognized in multiple individuals. These results enable further functional studies of T-cell responses during influenza virus infection and provide a potential base for the development of a universal influenza vaccine.


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INTRODUCTION
 
Influenza virus infection causes an estimated 300,000 to 500,000 annual deaths worldwide (www.who.int). Certain groups are at higher risk for serious complications upon infection, including infants, the elderly, and people with chronic health conditions. Antibodies directed to the hemagglutinin (HA) protein can protect against infection and are the basis for the vaccine currently utilized for prevention against influenza A and B virus infection. The current vaccine is not, however, devoid of limitations, including the need for annual selection of new strains and generation and delivery of new vaccines.

T-cell responses have clearly been shown to reduce pathology and promote recovery in animal models of influenza virus infection (20, 31). Recent information supports a beneficial role in reducing human infections as well (17). Therefore, vaccination strategies aimed at generating T-cell-mediated immune responses directed toward conserved regions of the virus should be considered. This would allow for vaccinations with broad efficacy that could target most, if not all, strains of types A and B influenza virus. In this context, both CD8+ and CD4+ T-cell responses are of interest (8, 13). While CD8+ T cells are capable of directly eliminating infected cells and reducing viral replication (8), CD4+ T cells may exert antiviral effects by direct cytotoxicity and secretion of lymphokines, as well as by boosting and hastening the development of influenza virus-specific antibody responses and entering the generation of long-lasting memory (2). Besides their potential use in vaccines, influenza virus-derived epitopes are useful for evaluating the performance of different vaccine constructs and for basic studies of the role of cellular immunity and host-pathogen interactions during influenza virus infection.

In the present study, we have probed the repertoire of influenza virus-specific class I and class II HLA-restricted responses directed against influenza A virus epitopes in the human population. We were specifically interested in determining the breadth and diversity of the influenza virus-specific T-cell responses to influenza in humans. In recent years, various reports based on overlapping peptides, predicted epitopes, or expression libraries have revealed that immune responses in humans are very diverse and that immunodominance is much less strict than previously thought. Since most of these studies were performed utilizing relatively large and complex pathogens (18, 29), we wanted to determine whether this breadth of responses would also be observed in the context of a smaller pathogen, such as influenza virus.

Before the potential utility of influenza virus-derived T-cell epitopes can be realized, significant challenges need to be met. A recent study demonstrated several knowledge gaps that hinder progress (3). For example, the known CD8+ and CD4+ T-cell epitopes are derived mostly from the HA and nucleoprotein (NP) antigens. Furthermore, the number of identified epitopes corresponding to sequences from avian strains is limited. Thus, more extensive epitope identification efforts seem desirable to detect responses against additional influenza virus antigens.

There are a number of additional challenges for the development of universal epitope-based vaccines and diagnostic tools, in particular the large strain-to-strain variability of the influenza virus. The selection of T-cell epitopes that are totally conserved across a wide range of virus strains (or panels of less conserved epitopes combined) is a possible avenue to meeting this challenge. In this context, the definition of a set of CD4+ and CD8+ T-cell epitopes that are broadly conserved would be of interest. Furthermore, it has been suggested that the concept of HLA supertypes (23, 26), whereby HLA molecules are clustered into sets with largely overlapping peptide binding repertoires, represents one solution to the challenge of HLA polymorphism, as it might provide high coverage across different ethnicities.

To identify a set of T-cell epitopes that could provide coverage against different influenza virus strains and among different ethnicities, we have devised the following experimental strategy. First, we included common human and selected avian influenza A virus subtypes to globally address variability. Second, to avoid an uneven representation of epitopes in terms of virus protein coverage, we broadly investigated 10 different influenza virus-derived antigens. Third, to tackle the challenge of HLA polymorphism, we considered six HLA class I supertypes and one class II (DR) supertype encompassing many common HLA antigens for peptide restriction. By following this strategy, we have identified a set of 54 T-cell epitopes derived from a wide range of virus antigens and that cover a wide range of influenza A virus strains and subtypes.


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MATERIALS AND METHODS
 
Influenza A virus strain selection and prediction of peptide/HLA binding. We assembled a set of target strains representative of the H1N1 and H3N2 subtypes that were utilized for vaccine development in the periods 1968 to 1975, 1976 to 1986, 1987 to 1989, 1989 to 1998, and 1999 to 2004 (Table 1). Various potentially pandemic viruses such as the H2N2, H5N1, H7N7, H9N2, and H6N1 subtypes were also included. To obtain complete sequences from all strains, 14 (of the 23) selected virus strains with incomplete sequences were sequenced at Baylor College of Medicine. The sequencing results were submitted to GenBank. The protein sequences were scanned for the presence of main anchor motifs associated with the A1, A2, A3, A24, B7, B44 (23, 24, 26) and DR supertypes (28), and quantitative prediction matrix analyses were run as previously described (5). Using our in-house major histocompatibility complex (MHC) binding database, supertype matrices were generated based on the family of HLA alleles associated with the seven prevalent supertypes. A cutoff value of 100 nM was used for selection. Peptide conservancy was then calculated. By combining motif scanning and quantitative analyses with a preference for higher conservancy levels, a total of 4,080 peptides (8-, 9-, 10- and 11-mers for class I and 15-mers for class II) were predicted and synthesized (see Table S1 in the supplemental material).


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  		TABLE 1. Set of influenza A virus strains included in the study

Peptide synthesis. Peptides utilized in the initial screening studies were purchased as crude material from Mimotopes (Minneapolis, MN). Peptides synthesized for use as radiolabeled ligands or utilized in the second round of screening were synthesized by A and A Labs (San Diego, CA) and purified to >95% purity by reverse-phase high-pressure liquid chromatography. Purity was determined using analytical reverse-phase high-pressure liquid chromatography and amino acid analysis, sequencing, and/or mass spectrometry. Peptides were radiolabeled by the chloramine T method (27).

Peptide/HLA binding assays. Quantitative assays to measure the binding affinities of peptides to HLA molecules are based on the inhibition of binding of a radiolabeled standard peptide and were performed as described in detail previously (9, 27).

Characteristics of the study population. Healthy males and females between 24 and 66 (average 35) years of age were used in this study. The ethnicities included were Caucasian, African-American, American Indian, Asian, and Hispanic. Exclusion criteria were a body weight of <45.4 kg and established pregnancy. Institutional review board approval and appropriate subject consent were obtained for this study.

PBMC isolation and HLA typing. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by gradient centrifugation with Histopaque-1077 (Sigma), and cells were resuspended in fetal bovine serum complemented with 10% dimethyl sulfoxide and cryopreserved in liquid nitrogen. PBMCs were typed for HLA-A, -B, and -DR by high-resolution PCR (Atria Genetics, San Francisco, CA).

CD8+ cell depletion. To ensure class II restriction when measuring CD4+ T-cell responses, CD8+ cells were depleted from the PBMCs by negative selection using a Magnetic Cell Separation (MACS; Miltenyi Biotech, Bergisch Gladbach, Germany) purification system. When applicable, the CD8-depleted population was tested for the lack of recognition of a class I-restricted epitope previously identified in the same individual.

Ex vivo gamma interferon (IFN-{gamma}) enzyme-linked immunospot (ELISPOT) assay. A total of 4,080 peptides were synthesized and pooled (9 to 21 peptides per pool, with a majority of pools consisting of 20 peptides) by supertype, resulting in 208 pools (12 A1, 73 A2, 29 A3, 10 A24, 5 B7, 33 B44, and 46 DR supertypes). In initial experiments, PBMCs were incubated at 2 x 105 cells per well in the presence of peptide pools corresponding to the donor's haplotype. Peptide pools yielding positive responses (≥20 net spot-forming cells [SFC]/106, a stimulation index [SI] of ≥2, and a P value ≤0.05) were deconvoluted by subsequent testing of the PBMCs against each individual peptide at 10 µg/ml. The ELISPOT assays were performed as described previously (30). For negative control values, dimethyl sulfoxide alone was added at the same dilution as that present in the peptides/pools, and these values were subtracted from the experimental values. To assess statistical significance, a one-tailed Student t test was performed in which triplicate values of each condition were compared with those of the negative controls.


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RESULTS
 
Definition of a target set of influenza virus sequences and epitope prediction. As a first step in our study, we selected a panel of 23 representative influenza A virus strains to be used for epitope predictions. This approach avoided potential bias in the analysis, introduced by uneven representation of different subtypes in the sequence database available to us at the initiation of analysis (early 2005). To select virus strains that have been used for vaccination or are known to cause infection in the human population, representative strains for each of the H1N1 and H3N2 subtypes responsible for influenza virus infections from the periods 1968 to 1975, 1976 to 1986, 1987 to 1989, 1989 to 1998, and 1999 to 2004 were chosen (Table 1). In addition, we included representative strains from the 1918 (H1N1), 1957 (H2N2), and 1968 (H3N2) pandemics and from the avian subtypes H5N1, H7N7, H6N1, and H9N2.

To provide high coverage across different ethnicities, we considered six HLA class I supertypes (A1, A2, A3, A24, B7, and B44) (23, 24, 26) and a single HLA class II (DR) supertype encompassing many common alleles (DR1, DR2, DR4, DR7, DR8, DR9, DR11, DR13, DR51, and DR53) (10, 28, 32). Previous studies have shown that while the frequency of each allele in a supertype may vary dramatically among different populations, the frequency of each supertype is relatively constant (26). The combined coverage by the six HLA class I supertypes of the major ethnicities is >98% (24). Similarly, the DR supertype allows for 86.5 to 97.3% coverage (9, 28). For each of the HLA supertypes mentioned above, peptides were selected according to the presence of specific supertype motifs and average relative binding matrices generated by utilizing our in-house MHC binding database. A cutoff value of 100 nM for predicted affinity was used for selection. Approximately 17,000 peptides matching these characteristics were identified within the representative sequences. We next directed our selection procedure toward peptides with higher conservancy levels and chose 4,080 peptides for testing by immunogenicity assays (see Table S1 in the supplemental material). Reflecting our focus on conserved epitopes, almost all peptides with a conservancy level of ≥40% were synthesized and tested. A smaller sample of less-conserved peptides was also selected. The number of predicted peptides from the various supertypes varied according to the frequency with which the amino acids associated with each supermotif were found in proteins. For example, the A2 supertype peptides were most numerous, as the corresponding motif is composed of commonly found amino acids. Conversely, the B7 supertype peptides were the least numerous, as this supertype is characterized by a stringent requirement for the presence of a P residue in position 2 (26, 32). Likewise, the distribution of peptides predicted for each protein reflected the relative size of each protein, with the most peptides predicted for the PB1 and PB2 proteins (759 and 757 residues, respectively) and the least peptides predicted for the M2 and NS1 proteins (97 and 121 residues, respectively).

Human T-cell responses to influenza virus infection are highly diverse. Human T-cell responses have been shown to be very diverse in the context of human immunodeficiency virus (HIV) (www.lanl.gov), cytomegalovirus (CMV) (29), and poxvirus (14, 18) infections. By contrast, based on a recent analysis of the published literature, a somewhat more limited breadth of responses has been characterized in the case of influenza virus (3). Therefore, we decided to investigate the influenza virus-induced responses in humans in more detail. Forty-four healthy volunteers between 24 and 66 years of age were recruited for leukopheresis or blood donations. HLA-typed, cryopreserved PBMCs were tested for recognition of supertype-matched pools comprising 10 to 20 peptides/pool in IFN-{gamma} ELISPOT assays. When CD4+ responses were measured, PBMCs were depleted of CD8+ cells to ensure class II restriction. Positive pools (SFC/106 ≥ 20, SI ≥ 2.0, and P ≤ 0.05) were deconvoluted to identify the individual peptides responsible for the responses. To ensure data quality and consistency, these peptides were resynthesized and purified and retested for all responding donors. To characterize these epitopes further, we measured their ability to bind to the most common HLA alleles representing the corresponding supertype (see Table S2 in the supplemental material). A peptide was considered a binder if it bound at least one allele with an affinity of ≤500 nM for class I and ≤1,000 nM for class II (25, 28). Altogether, these experiments identified 65 high-affinity binding epitopes that were found positive in at least two replicate assays using cells from the same individual. It is worth mentioning that additional epitopes were identified that generated reproducible T-cell responses but bound poorly to the HLA alleles included (data not shown). This might be explained by the fact that only the most common alleles within each supertype were tested for binding and that the binding of some peptides might have been initially mispredicted. However, as we do not know the HLA restriction of these peptides, we decided to exclude them from the remaining analyses.

To eliminate potential redundancies, we applied a cluster algorithm (http://tools.immuneepitope.org) to the set of 65 epitopes to identify nested or overlapping peptides, as well as homologous variants (≥80% identical), from different strains of influenza virus. Within a cluster, a representative optimal peptide was selected based on conservancy level, number of responding individuals, and magnitude of responses. It should be noted that if nested peptides were differentially recognized by the responding donors, each was maintained as a separate epitope. A total of 54 distinct epitopes (38 class I- and 16 class II-restricted epitopes) were identified accordingly (Table 2). Thus, this analysis reveals a heretofore unappreciated extreme breadth in the T-cell responses to influenza virus in humans. The epitopes varied in the frequency of their recognition by the panel of HLA-matched donors, from 3 to 47%, with an average of 2.5 class I- and 3.2 class II-restricted epitopes recognized by a given donor. In general, each individual donor recognized a different and unique set of epitopes. Thus, this analysis underlines the large donor-to-donor variation in the epitopes recognized in the influenza virus system.


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  		TABLE 2. Set of identified influenza A virus-derived HLA class I- and class II-restricted T-cell epitopes

We also wanted to compare this set of epitopes with previously published data. Of 56 unique HLA epitopes reported in the literature and restricted by the same alleles considered in our analysis, 16 were independently predicted and tested in our analysis (data not shown). The remaining 40 were mostly less conserved (29 peptides had a conservancy of <40%) and were therefore not included in our study. Of the 15 epitopes previously identified and tested in our study (6, 7, 11, 16), only 4 (VSDGGPNLY, GILGFVFTL, RMVLASTTAK, and TTYQRTRAL) were identified as epitopes in our donor population (Table 2).

PB1 and M1 are major targets of both CD4+ and CD8+ T-cell responses. A protein might be effectively targeted only at a certain stage of infection, when its expression is at a suitable level. It is therefore likely that immune responses targeting a broad range of viral proteins would be more efficient in combating a virus than those recognizing a single protein. However, the majority of the influenza A virus epitopes described in the literature are derived from the HA and NP proteins (3). The 4,080 peptides considered in this study were derived from 10 different influenza virus proteins, allowing examination of the extent to which additional antigens might also be targets of cellular immunity. Indeed, it was found that most proteins were recognized.

A somewhat uneven representation of peptides selected for testing was observed, with the PA, PB1, and PB2 antigens contributing the most and M2, NS1 and NS2 contributing the least. This uneven representation is due to differences in protein sequence variability (HA and neuraminidase are more variable than PA, PB1, and PB2) and size (M2, NS1, and NS2 are relatively small proteins). Epitopes were derived from all antigens except M2 and NS1, with PB1 contributing the highest number of epitopes for both CD4+ and CD8+ T-cell responses (Fig. 1A; Table 3). This is in contrast to the reported literature, where only 3% of the epitopes described were derived from PB1. The highly skewed distribution of previously reported class I- and class II-restricted epitopes in favor of HA and NP (3) likely reflects the recognition of more variable epitopes, as well as a bias in the antigens investigated.


Figure 1
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  		FIG. 1. PB1 and M1 are major targets for both CD4+ and CD8+ T-cell responses. Bars represent the fraction (%) of the total number of epitopes from the current study derived from each of the viral proteins (A); the recognition frequency (%) for the different viral proteins (number of epitopes recognized/number of peptides tested) (B); and the fraction (%) of the total number of epitopes from previously published studies derived from each of the viral proteins (C).


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  		TABLE 3. Antigen and HLA supertype distribution of influenza A virus-derived peptides

To normalize the data for the different numbers of peptides tested from the different proteins, a recognition frequency (hit rate) was determined as the number of peptides yielding positive responses divided by the number of peptides tested for each protein. The highest recognition frequency for both CD4+ and CD8+ T-cell responses was observed for M1. This also showed that even when compensating for the differences in the number of peptides tested, PB1 was second to M1 (Fig. 1B) in contributing the recognized epitopes. Interestingly, although PB2 is a subunit of the same RNA polymerase complex as PB1, the recognition rate for PB2 was eight times lower than that for PB1 (Table 3).

When comparing the antigen distribution of the epitopes identified herein with those reported in the literature, we found that while HA contributed to only a single class II-restricted epitope in our experiments (Table 3), about one-third of all published epitopes were derived from HA (Table 3; Fig. 1C). This discrepancy can be explained by the fact that HA is a protein commonly studied in influenza research, and therefore there is a higher probability that epitopes would be sought and identified from this protein. In addition, fewer HA-derived peptides were tested in this study as a consequence of our selection of peptides that were more than 30% conserved. However, despite testing 161 HA-derived predicted class I-restricted peptides, not a single epitope was identified for class I, suggesting that HA is not a common target for CD8+ T-cell responses (Table 3).

In conclusion, combining the current epitopes with those in the literature data set demonstrates that immunity to influenza virus infection in humans is very diverse and comprises responses to many different virus antigens.

Identified T-cell epitopes are highly conserved among various influenza virus strains and subtypes. Vaccination strategies based on conserved T-cell epitopes would be of great value to overcome seasonal variations in influenza virus antigens. This prompted us to focus our studies on the identification of highly conserved epitopes. Accordingly, the present study identified a number of well-conserved epitopes. Specifically, 18 of the 54 (33%) identified epitopes were conserved across the entire panel of 23 influenza virus strains considered. An additional eight (15%) epitopes were conserved in 80 to 96% of all strains (Table 2). A range of 36 to 44 different epitopes were totally conserved (100% identity) in a given influenza virus strain (data not shown). A similar result was observed when we focused on the avian subtypes H5N1, H6N1, H7N7, and H9N2, in which a range of 37 to 40 epitopes were found to be conserved in all strains within at least one of the avian subtypes, and 28 epitopes were conserved across all avian strains analyzed (Fig. 2A; data not shown). This implies that T-cell memory in influenza virus-infected and/or vaccinated individuals includes some reactivity against sequences also encoded in avian viruses, which further highlights the potential advantages of utilizing T-cell epitopes for vaccine development.


Figure 2
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  		FIG. 2. Identified T-cell epitopes are highly conserved among various influenza virus strains and subtypes. (A) The conservancy levels among the selected set of 23 influenza A virus strains for each epitope identified in previously published studies, or in the current study, and the conservancy level among influenza A virus strains of the H5N1, H6N1, H7N7, and H9N2 avian subtypes for each epitope identified in the current study are shown. (B) The conservancy levels among the more recent (2005 to 2007) influenza A virus strains of the H1N1, H3N2, or H5N1 subtypes are shown for each epitope identified in the current study.

Compared to the epitopes already described in the literature, the epitopes identified in this study were significantly more conserved among the 23 strains included in the present study (Fig. 2A). However, in the context of vaccination strategies, it is also important that the epitopes remain conserved in newly emerging strains. To address this point, we determined epitope conservancy levels among a total of 57 H1N1, H3N2, and H5N1 strains that emerged between 2005 and 2007 and therefore were not initially included in this study. The conservancy level of the epitopes remained relatively constant; 25 out of the 54 epitopes were conserved in 80% of the strains (Fig. 2B). The oldest strain included in this study is the A/Brevig Mission/1/18 H1N1 strain, which was the causative agent of the Spanish influenza pandemic that occurred 90 years ago. Thus, it appears that some of the epitopes are derived from genomic regions that are completely conserved over long time periods and may therefore be of interest in the development of a universal vaccine.

We have reasoned that since humans are more likely to have been exposed to highly conserved peptides than to less well conserved peptides, especially if repeated infections throughout the years are taken into account, then this would mean that the hit rate for the recognition of a particular peptide would increase with its conservancy level. Accordingly, the fraction of epitopes identified (number of epitopes recognized/number of peptides tested) within each conservancy category was analyzed. The hit rate within the set of 100% conserved epitopes was higher (2.7%) than for peptides with a lower degree of conservation (1.0 to 1.1%). Similarly, the fraction of donors responding to a specific epitope also increased with the conservancy level of the epitope, with 8% responding to peptides <40% conserved and 14% responding to 100% conserved peptides. While epitope-specific T cells must have been primed by influenza virus infection of these subjects, we have not determined whether the epitope-specific T cells can also recognize target cells infected with the virus. As each individual generally recognized multiple epitopes, it is difficult to attribute the recognition of infected cells to any given epitope specificity. This could, however, be addressed by the derivation of epitope-specific T-cell lines by repeated in vitro stimulation. Such experiments are considered for future studies.

The set of identified epitopes provides broad population coverage. With regard to HLA supertype restriction, we have identified epitopes for all seven supertypes considered, including, to our knowledge, the first demonstration of A24-restricted influenza virus-derived epitopes. The distribution of the epitopes identified was uneven, with only one epitope restricted by B7 and 17 epitopes restricted by A2, reflecting the relatively uneven distribution of motifs. In fact, when the number of peptides yielding positive responses was divided by the number of peptides tested for each supertype, the hit rate was found to be relatively constant across the class I supertypes (0.9 to 1.6%) (Table 3).

Next, we addressed the extent to which the epitope sets identified would allow coverage of individuals in major ethnic groups. Based on the peptide binding data (see Table S2 in the supplemental material) for each of the different HLA supertype molecules and the reported frequencies of each HLA allele in different ethnic populations, we calculated theoretical population coverage for both the class I and class II epitope sets, using the population coverage calculation tool available through the Immune Epitope Database (4). For class I, the coverage was high throughout the major different populations, spanning from 90.5% of Australians to 99.9% of North Americans, with an average of 98.5% (Fig. 3A). On average, each individual was calculated to be capable of binding 6.5 epitopes. Similarly for class II, the average population coverage was 89.5%, spanning from 67.1% of South Americans to 96.0% of Europeans, and each individual had the capacity to bind 6.8 epitopes (Fig. 3B).


Figure 3
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  		FIG. 3. The identified epitopes provide broad population coverage. Based on the binding data for each class I-restricted (A) or class II-restricted (B) epitope, theoretical population coverage was calculated. The number of possible epitope-HLA allele combinations as a function of the fraction of each population (%) is shown. Horizontal dashed line indicates the number of possible epitope-HLA alleles presented to at least 80% of each population.

To further experimentally assess the issue of population coverage, we obtained PBMCs from an additional set of 20 untyped random human blood donors and tested them for their reactivity against peptide pools comprising the set of either class I or class II epitopes. A total of 15/20 (75%) individuals responded to the class I peptide pool (Fig. 4A) and 16/20 (80%) subjects responded to the class II-restricted epitope pool (Fig. 4B). Altogether, 17/20 (85%) subjects responded to either the class I and/or class II pools. The high population coverage observed is more impressive when considering the diverse infection histories of the individuals and would be expected to increase in a vaccination setting. In summary, these data suggest that the set of conserved epitopes identified herein provide a broad population coverage.


Figure 4
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  		FIG. 4. The majority of the individuals tested responded to both the HLA class I- and class II-restricted epitope sets. (A) PBMCs from 20 individuals were tested for the recognition of pools of the set of class I-restricted (A) and class II-restricted (B) epitopes in IFN-{gamma} ELISPOT assays. The average SI (specific response/nonspecific response) from three independent experiments is plotted for each donor. Gray bars, significantly positive responses; open bars, negative responses.


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DISCUSSION
 
In the present study, we have probed the repertoire of influenza virus-specific class I and class II immune responses of humans directed against influenza A virus epitopes. We were specifically interested in determining how diverse the T-cell repertoire is in humans. Since previous analysis of published data suggested a preponderance of responses in humans directed against the NP and HA proteins, we were also interested in determining whether responses against other virus proteins could be identified. Because of their relevance to vaccine development, we were further interested in defining a set of CD4+ and CD8+ epitopes that are broadly conserved and would provide coverage for a large fraction of the population, irrespective of ethnicity.

A set of 54 influenza virus-derived T-cell epitopes that are recognized by healthy human individuals was identified. Several reasons can account for what appears to be a low yield of epitopes from the pool of candidate peptides. For example, it has been recently demonstrated in a mouse model of vaccinia virus infection that one out of seven high-affinity binding peptides is generated by natural processing and that one out of two peptides can elicit a T-cell response upon single peptide immunization; only 1/10 of these possible epitopes is in fact recognized during infection in vivo (1). This might be due to some immunoregulatory mechanisms which remain to be understood. In addition, some epitopes might have been overlooked in our rather stringent experimental setting. Nevertheless, most of the identified epitopes were recognized by more than one individual, but the responses varied significantly, both in magnitude and frequency. Moreover, the apparent diversity of the immune responses is likely to represent an underestimation. For example, highly variable sequences not investigated in the current study probably encode additional epitopes. Furthermore, additional epitopes are likely to exist that are not restricted by the alleles considered in the current analysis. Indeed, of the 54 epitopes identified herein, only 4 (VSDGGPNLY, GILGFVFTL, RMVLASTTAK, and TTYQRTRAL) had been previously described (www.immuneepitope.org) (6, 11, 16). These results underline the extreme breadth of responses in the influenza virus system in humans. The HLA-A*0201-restricted epitope GILGFVFTL has been described as the most immunodominant influenza virus-derived epitope in humans (15) and was also the most frequently recognized epitope in this study. This parallels the case of other immunodominant epitopes such as the hepatitis B virus core 18-to-27 peptide epitope, which is also restricted by HLA-A*0201 and recognized in about 60% of individuals (32). While the molecular bases of immunodominance are not completely understood, it is likely that a combination of high precursor frequencies of T cells specific for the epitope, high MHC binding affinity, and efficient generation of the epitope as a result of cellular processing all contribute to the effect (1, 15, 33). Despite the immunodominance of the GILGFVFTL epitope, the individual responses to that epitope varied significantly. One possible explanation for the lower recognition rate observed for our study than for some previous studies (11, 22) is that those studies performed in vitro restimulation of the T cells, which would increase the frequency of influenza virus-specific T cells and thereby enhance the sensitivity of their detection. For four of the donors included in our study, the GILGFVFTL epitope was the most highly recognized, whereas for the other six donors, the responses varied between the top 20% and the top 90% of the epitopes recognized. Based on these data, it would appear that in the case of influenza virus infection, immunodominance can be seen at the individual level in the sense that a given set of epitopes dominates the response but less so at the population level in the sense that the dominant epitope(s) tends to differ between individuals. This observation is in agreement with results for the vaccinia virus and cytomegalovirus systems, where a remarkably diverse pattern of immune recognition has been described in humans (18, 19, 29). Furthermore, several hundred CD4+ and CD8+ T-cell epitopes have been identified in HIV-infected individuals (www.lanl.gov), suggesting that a large breadth of responses in humans characterizes disparate types of viruses, such as DNA and RNA viruses with either small or large genomes.

In contrast to the published data that suggested that the HA and NP proteins were recognized predominantly, we detected responses broadly directed against most proteins, with the exception of the small M2 and NS1 proteins. Interestingly, despite testing a total of 199 predicted HLA-binding peptides from HA, we did not identify a single HA-derived class I-restricted epitope and only a single class II-restricted epitope. This might be attributable, at least in part, to the conservancy threshold used in our study, since HA is highly variable. Also, in the case of NP, relatively fewer epitopes were identified in this study than in previous studies. Therefore, the highly skewed distribution of previously reported epitopes in favor of HA and NP proteins (3) is likely to reflect the recognition of less-conserved epitopes and a bias in favor of investigations related to these antigens. Another striking observation was the high number of PB1-derived epitopes discovered. While only about 3% of previously reported class I-restricted epitopes are derived from PB1, as many as one out of three epitopes identified in this study were derived from this antigen. In fact, PB1 contributed the highest number of epitopes for both class I- and class II-restricted responses in this study. This suggests that PB1 must be considered for vaccine development.

An important consideration in our investigations was to determine whether we could detect responses directed against broadly conserved epitopes, as these sequences might be of particular interest for influenza vaccination. Conserved sequences might be of interest also for monitoring influenza virus responses elicited by different strains and subtypes. Due to the selection criteria used in our analyses, a large fraction of the 54 epitopes were totally conserved in all 23 influenza virus strains considered, including the pandemic H1N1 strain from 1918. More importantly, similar conservancy levels were observed when assessing the conservancy in more recent strains (from the period 2005 to 2007). This points to the presence of completely conserved regions in the proteome that are being recognized by T cells. We also found that the frequency of recognition of a certain epitope among different individuals increases with its conservancy level.

It should be noted that the maximum number of SFC in the ELISPOT assay for several of the epitopes is rather low, in some instances less than 50/106 PBMCs (Table 2). In this respect, it might be advisable to utilize these epitopes as a pool in future studies analyzing influenza responses. The relatively low reactivity to several epitopes is also consistent with the notion that the reactivity naturally observed against conserved influenza virus-derived epitopes may be low and insufficient to provide universal coverage against different variants, unless it is boosted by deliberate vaccination with the conserved epitopes.

A large proportion of the set of identified epitopes was also conserved among the avian strains included in the analysis; each of the five avian strains contained between 37 and 40 of the epitopes at 100% identity. This suggests that due to previous infection and/or vaccination with heterologous influenza virus strains, T-cell immunity directed against sequences found in avian strains commonly resides in the general population. Strategies aimed at selectively amplifying these responses are of interest in the context of concerns for a potential avian flu pandemic.

Additional experiments are required to determine whether the conserved epitopes alone may be adequate for protection or whether less conserved epitopes would be required. It is also possible that vaccination with these conserved epitopes might provide a more vigorous response against conserved regions of the virus and, when used in conjunction with more conventional antibody-inducing vaccines, offer partial protection against new pandemic strains. Our screening strategy was designed to identify epitopes derived from various HLA supertypes and, accordingly, provide coverage of the human population. We show here that the set of epitopes identified have the potential of binding to a diverse set of HLA molecules and that the set of class I and class II epitopes were recognized by 75 to 80% of a set of 20 blood donors. These frequencies reflect memory T-cell responses in individuals with diverse infections and/or vaccination history. The coverage would be expected to increase in a vaccination setting, where individuals would be exposed to only the vaccine epitopes, eliminating the potential for competition with the many epitopes occurring in the course of natural infection. In addition, because of herd immunity, the total population protection is expected to be higher than the actual vaccine coverage (21 and reviewed in reference 12). Although the epitope sets were tested only in a donor population recruited from the San Diego, CA, area, the selection process utilized and the calculations based on HLA binding data and HLA frequencies in different ethnicities predict their potential utility irrespective of the ethnicity considered.

In summary, we report a large breadth of human responses directed against influenza A virus epitopes representing most influenza virus antigens. A set of 54 influenza virus-derived epitopes has been identified in this study. This epitope set is highly conserved in a diverse set of human and avian influenza virus strains and subtypes and affords high coverage among the most common ethnicities. This study provides a useful tool for functional studies of T-cell responses during influenza virus infection and a potential base for the development of universal influenza vaccines.


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ACKNOWLEDGMENTS
 
We thank Josephine Babin, Lori Giancola, and Jean Glenn for experimental assistance; Louis Huynh, Carrie Moore, Amiyah Steen, and Sandy Ngo for performing the MHC binding assays; and Robert Couch and Valerie Pasquetto for helpful discussions.

This study was supported by National Institutes of Health contract N01-AI30039. E.A. was supported by a fellowship from the Wenner-Gren Foundation.


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FOOTNOTES
 
* Corresponding author. Mailing address: La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037. Phone: (858) 752-6916. Fax: (858) 752-6985. E-mail: alex{at}liai.org Back

{triangledown} Published ahead of print on 8 October 2008. Back

{ddagger} Kirin publication number 1036. Back

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


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Journal of Virology, December 2008, p. 12241-12251, Vol. 82, No. 24
0022-538X/08/$08.00+0     doi:10.1128/JVI.01563-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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