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Journal of Virology, September 2009, p. 9206-9214, Vol. 83, No. 18
0022-538X/09/$08.00+0     doi:10.1128/JVI.00932-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

T-Cell Tolerance for Variability in an HLA Class I-Presented Influenza A Virus Epitope{triangledown}

Angela Wahl,1 William McCoy,2 Fredda Schafer,1 Wilfried Bardet,1 Rico Buchli,3 Daved H. Fremont,2,4 and William H. Hildebrand1*

Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, 975 Northeast 10th Street, Oklahoma City, Oklahoma, 73104,1 Department of Pathology and Immunology,2 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110,4 Pure Protein L.L.C., 800 Research Parkway, Suite 340, Oklahoma City, Oklahoma 731043

Received 11 May 2009/ Accepted 21 June 2009


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ABSTRACT
 
To escape immune recognition, viruses acquire amino acid substitutions in class I human leukocyte antigen (HLA)-presented cytotoxic T-lymphocyte (CTL) epitopes. Such viral escape mutations may (i) prevent peptide processing, (ii) diminish class I HLA binding, or (iii) alter T-cell recognition. Because residues 418 to 426 of the hypervariable influenza A virus nucleoprotein (NP418-426) epitope are consistently bound by class I HLA and presented to CTL, we assessed the impact that intraepitope sequence variability has upon T-cell recognition. CTL elicited by intranasal influenza virus infection were tested for their cross-recognition of 20 natural NP418-426 epitope variants. Six of the variant epitopes, of both H1N1 and H3N2 origin, were cross-recognized by CTL while the remaining NP418-426 epitope variants escaped targeting. A pattern emerged whereby variability at position 5 (P5) within the epitope reduced T-cell recognition, changes at P4 or P6 enabled CTL escape, and a mutation at P8 enhanced T-cell recognition. These data demonstrate that substitutions at P4 and/or P6 facilitate influenza virus escape from T-cell recognition and provide a model for the number, nature, and location of viral mutations that influence T-cell cross-recognition.


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INTRODUCTION
 
Cytotoxic T-lymphocytes (CTL) kill virus-infected cells and release antiviral cytokines upon recognition of short viral peptides displayed on the cell surface by the class I HLA molecule (36). Virus-derived peptides are processed in the cytoplasm by proteasome degradation of viral proteins (25), shuttled into the lumen of the endoplasmic reticulum (ER) by the transporter-associated protein, and loaded into the basket-like groove of the class I molecule. Class I HLA molecules await peptide loading in the ER and demonstrate specificity for viral peptides with particular anchor residues representing a good fit for the class I HLA binding groove. Once stable class I HLA-peptide complexes are formed, the class I molecule and its peptide cargo are transported via the Golgi apparatus to the cell surface, where the complex is anchored to the plasma membrane (21, 36-38). CTL then survey class I HLA-presented peptides on the cell surface. Viral peptides must therefore be processed, specifically bound by class I HLA, and presented at the plasma membrane for CTL to distinguish infected cells from uninfected tissue.

A high mutation rate is one of many mechanisms utilized by viruses to escape detection by the immune system. Mutations within the genome allow viruses to accumulate and select for amino acid substitutions that (i) inhibit proteasome processing and viral peptide generation (2, 23), (ii) alter anchor residues within viral peptides to diminish class I HLA binding specificity (3, 14, 24, 32), or (iii) reduce immune recognition of the class I HLA-peptide complex by varying amino acids that come in contact with the T-cell receptor (6, 10, 27, 30, 35). While viral mutations might be advantageous for escaping immune detection, such flexibility can cost the virus in terms of replicative fitness. In order to maintain reproductive fitness and structural integrity, viruses must temper their use of genetic flexibility as a means of immune escape.

Influenza viruses have the well-documented ability to escape detection by various immune epitopes (3, 10, 27). A priori, investigators often assume that variable regions of the virus represent poor immune targets because such regions will not be consistently processed, presented, or recognized (15, 20). However, we along with others continue to find that a hypervariable stretch of the influenza virus nucleoprotein consisting of residues 418 to 426 (NP418-426) is presented to CTL by different HLA-B alleles (B*0702 and B*3501) in spite of extensive viral variability within this epitope (8, 10, 27, 34). Moreover, NP418-426 is a dominant immune epitope (8, 10, 27, 34). The consistent processing and presentation of NP418-426 by class I HLA can be explained by the finding that different influenza virus isolates cannot mutate the proline located at position 2 (P2) within the epitope because elimination of this proline reduces viral fitness (4, 5). Little to no variability is found at the methionine P9 anchor as well. These facts lead to the unique observation that strain-to-strain variability does not abrogate class I HLA presentation of the influenza virus NP418-426 epitope and that CTL respond to this consistently presented viral epitope in an immunodominant fashion.

In this study we took advantage of the anchor residue conservation that prompts the NP418-426 epitope to be consistently presented to CTL by investigating the functional impact that influenza virus intraepitope variability has on CTL recognition. The amino acid alignment of human influenza A (H1N1 and H3N2) virus nucleoprotein molecules identifies 20 unique NP418-426 peptide sequences which demonstrate amino acid diversity between the anchors. We infected HLA-transgenic mice intranasally with influenza virus and tested CTL from these animals for their ability to recognize each of the 20 NP418-426 variants. These 20 NP418-426 sequences represent a natural "recombinant library" of viral epitopes that the immune system has and will face. The resulting data demonstrate a gradient of viral substitutions whereby CTL recognition diminishes depending upon the number of viral substitutions and their location within the epitope. Understanding how intraepitope variability impacts CTL recognition is discussed in terms of eliciting immune responses to variants of influenza.


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MATERIALS AND METHODS
 
Amino acid alignments. Full-length sequences of human influenza A (H1N1 and H3N2) virus NP were initially accessed from the NCBI Influenza Virus Resource database on 26 November 26 2007 (1). Following removal of identical protein sequences, the NP sequences were aligned, identifying 20 unique amino acid sequences at positions 418 to 426 of the NP molecule. Synthetic peptides were generated for the 20 NP418-426 amino acid sequences and single amino acid NP418-426 variants at ≥95% purity by the Molecular Biology Research Facility at the University of Oklahoma Health Sciences Center and Mimotopes and utilized in enzyme-linked immunospot (ELISPOT) and peptide binding assays.

A second amino acid alignment was performed on human influenza A (H1N1 and H3N2) virus NP full-length protein sequences accessed from the NCBI Influenza Virus Resource database on 17 April 2008 revealing an additional human influenza A (H3N2) virus NP418-426 sequence (LPFEKPTVM) (1).

Influenza PR8 virus infection of mice. All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Oklahoma Health Sciences Center. Six- to eight-week-old male and female HLA-B*0702 transgenic H-2KbDb double-knockout C57BL/6 mice (obtained from the laboratory of Francois Lemonnier at the Institut Pasteur) were inoculated intranasally with a 1,000 50% egg infectious dose (EID50) of mouse-adapted influenza A/Puerto Rico/8/34 (PR8) virus (kindly provided by the laboratory of David Woodland at the Trudeau Institute) or sterile endotoxin-free saline (mock infection) at a volume of 10 µl per nostril. Mice were monitored daily for weight loss. Splenocytes were harvested at 12 days postinfection and passed through a cell strainer, and lymphocytes were isolated by centrifuging cells over a lympholyte-M density gradient (Cedar Lane). Lymphocytes were stored at –180°C in 90% fetal bovine serum and 10% dimethyl sulfoxide.

Lymphocytes were measured for peptide specific gamma interferon (IFN-{gamma}) secretion via ELISPOT assay. Briefly, 105 lymphocytes were incubated with 10 µg/ml synthetic influenza virus peptide, medium alone (negative control), 10 µg/ml synthetic human immunodeficiency virus Nef peptide RPMTYKAAL (negative control), and 4 µg/ml concanavalin A (positive control) in RPMI 1640 culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin for 24 h at 37°C in triplicate wells of a 96-well membrane (polyvinylidene difluoride)-bottomed plate coated with anti-IFN-{gamma} antibody (Cell Sciences). The assay was performed according to the manufacturer's instructions. The number of IFN-{gamma} spots produced per well was enumerated with a Zeiss KS ELISPOT reader. The data generated are illustrated as the number of spot-forming units (SFU) per 105 lymphocytes.

Peptide binding assay. An HLA-B*0702 PolyScreen kit (Pure Protein) was used to determine peptide concentrations necessary to inhibit 50% polarization (IC50). Briefly, fluorescein isothiocyanate-labeled reference peptide and soluble HLA (sHLA) were incubated with each peptide until equilibrium of peptide replacement was reached. The fluorescent polarization of the control peptide as read on an Analyst AD plate reader (Molecular Devices) and a dose-response curve were used to calculate peptide IC50 values (11-13).

Molecular modeling. HLA-B structures from the Protein Data Bank (PDB) were first restricted to those bound to nonamer peptides. When multiple structures were available for a single HLA-B allele, the highest-resolution structure was selected. Five structures (1XR8-HLA-B*1501 [26], 1K5N-HLA-B*2709 [16], 2CIK-HLA-B*3501 [18], 1A1O-HLA-B*5301 [29], and 2BVP-HLA-B*5703 [31]; PDB codes precede the HLA designations) were loaded into InsightII (Accelrys), and each of the selected structures was used to build a model of HLA-B*0702 bound to the PR8 peptide LPFDRTTVM via the homology and consensus module. This module threads the unknown structure's amino acid sequence through the known structure without altering the position of the peptide backbone. Side chain clashes were minimized by varying rotamers. These models then underwent visual inspection of the PR8 peptide to eliminate side chain clashes, which were then minimized via manual or auto-rotamer adjustments of peptide or HLA groove residues. Models were compared to identify general trends in peptide conformation and side chain placement. The model that best accommodated the HLA-B*0702 PR8 sequence (1K5N-HLA-B*2709) (16) with a minimum of unfavorable peptide-HLA interactions was then exported as a PDB file. The final figure was generated using this PDB file imported into MacPyMOL (DeLano Scientific LLC).


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RESULTS
 
The amino acid sequence of NP418-426 is highly variable in influenza A H1N1 and H3N2 virus strains. Data demonstrating that an epitope is presented by class I HLA in spite of viral divergence are scarce. Our direct epitope discovery data (Fig. 1), combined with CTL-driven studies in other laboratories, provide the rare demonstration that HLA-B molecules consistently present the NP418-426 epitope regardless of influenza virus strain-to-strain variability (9, 10, 27, 33, 34). Influenza virus variability does not impact proteolytic processing, translocation into the ER, or loading into class I HLA. Given steady epitope presentation, we took advantage of the considerable amino acid variability within NP418-426 of different influenza A virus subtypes to generate "nature's" recombinant NP418-426 epitope library for subsequent testing of CTL tolerance to various intraepitope substitutions. The data are positioned to provide fundamental insights for how efficiently CTL cope with viral epitope diversification.


Figure 1
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  		FIG. 1. Mass spectrometric identification of NP418-426 during infection with three influenza A virus strains. Class I HLA peptides eluted from naïve and influenza virus-infected HeLa cells were separated by reverse-phase high-pressure liquid chromatography, and fractions were sprayed via nanospray into a Q-Star Elite mass spectrometer. Naïve (data not shown) and infected mass spectrometric (MS) ion maps were aligned and visually compared to identify ions unique to the infected MS spectra. Despite tremendous amino acid variability within the influenza virus NP418-426 epitope, the NP418-426 peptide was eluted from sHLA-B*0702-transfected HeLa cells and identified during infection with three influenza virus A strains: PR8 (H1N1) (A), A/Oklahoma/7485/01 (7485; H1N1) (B), and A/Oklahoma/309/06 (309; H3N2) (C). In addition, the NP418-426 peptide was eluted from sHLA of 309-infected HeLa cells transfected with sHLA-B*3501, a HLA-B7 supertype allele (34) (D).

We began with an amino acid alignment of influenza A (H1N1 and H3N2) virus NP molecules from human isolates from 1918 to 2007 to assess the variety and nature of NP418-426 peptide sequences among influenza A virus subtypes (1). Alignments were compared to the PR8 virus. The alignment revealed 20 NP418-426 variant sequences (13 of subtype H1N1 and 11 of subtype H3N2) (Table 1). Four NP418-426 peptide sequences (LPFDRTTIM, LPFERATVM, LPFDKSTVM, and LPFDKSTIM) are apparent in both H1N1 and H3N2 influenza virus strains. The influenza A H1N1 virus isolates varied at P4 to P6 and at P8 within the NP418-426 epitope, and subtype H3N2 isolates had mutated amino acids at P4 to P9. P6 exhibited the greatest diversity in amino acids accommodated at a particular position within the NP418-426 ligand for both influenza A H1N1 and H3N2 virus isolates although H1N1 and H3N2 strains displayed a tendency for different amino acids at this position (in subtype H1N1, Thr; in subtype H3N2, Ser). In addition, influenza A H1N1 virus isolates demonstrated a propensity for Asp at P4 and Ile at P8 while reported influenza A H3N2 virus strains favored Glu at P4 and equally displayed Ile and Val at P8. Based on the frequency of particular amino acids at P4 to P8 within the NP418-426 epitope, influenza A H1N1 and H3N2 virus isolates exhibit the consensus sequence LPFDKTTIM (H1N1) and LPFEKST(I/V)M (H3N2) (substitutions are underlined). These data demonstrate the following: (i) the breadth of NP418-426 peptide sequences among human influenza A H1N1 and H3N2 virus isolates; (ii) the substantial variability at internal amino acid positions within the NP418-426 ligand; and (iii) despite 40 years of concurrent circulation within the human population, the segregation of the assortment of NP418-426 peptide sequences at P4, P6, and P8 between human influenza A H1N1 and H3N2 strains.


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  		TABLE 1. Relative binding affinity of influenza A virus NP418-426 peptides to sHLA-B*0702

NP418-426 variants exhibit various degrees of CTL cross-reactivity during PR8 virus infection of HLA-B*0702 transgenic mice. Upon identification of 20 NP418-426 peptide sequences among influenza A H1N1 and H3N2 strains, we tested splenocytes isolated from influenza PR8 (H1N1) virus-infected HLA-B*0702 transgenic H-2Kb–/–Db–/– mice for NP418-426-specific T-cell cross-reactivity measured by IFN-{gamma} production. Ten HLA-B*0702 transgenic mice were inoculated intranasally with 1,000 EID50 of murine-adapted PR8 and monitored daily for weight loss (data not shown), and splenocytes were isolated 12 days postinfection. Synthetic peptides were generated for 20 human influenza A virus NP418-426 sequences (nine H1N1 strain, seven H3N2 strain, and four H1N1/H3N2 strain sequences) (Fig. 2 and Table 1) and incubated at 10 µg/ml with 105 lymphocytes for 24 h in triplicate wells of an anti-IFN-{gamma} antibody-coated plate. Lymphocytes incubated with medium only and with 10 µg/ml HLA-B*0702 human immunodeficiency virus Nef peptide RPMTYKAAL (data not shown) served as negative controls, and lymphocytes cultured with 4 µg/ml concanavalin A served as a positive control.


Figure 2
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  		FIG. 2. IFN-{gamma} ELISPOT reactivity of 10 PR8-infected HLA-B*0702 transgenic mice to 20 NP418-426 variant peptides. IFN-{gamma} ELISPOT responses are illustrated for each mouse in SFU/105 lymphocytes. (A) The parental PR8 NP418-426 peptide (LPFDRTTVM) generated an average of 103.6 SFU/105 lymphocytes. The T-cell cross-reactive IFN-{gamma} response elicited to each H1N1 (B), H1N1/H3N2 (C), and H3N2 (D) variant NP418-426 peptide was compared to the parental PR8 NP418-426 peptide (shaded in gray). Three H1N1 restricted NP418-426 peptides (LPFDKTTIM, LPFDRPTIM, and LPFDKATIM) and three H1N1/H3N2 variants (LPFDRTTIM, and LPFDKSTIM, and LPFDKSTVM) elicited an average of at least 10 SFU/105 lymphocytes (shown in red).

The parental PR8 NP418-426 epitope (LPFDRTTVM) and six NP418-426 variant peptides (LPFDRTTIM, LPFDKTTIM, LPFDRPTIM, LPFDKSTIM, LPFDKSTVM, and LPFDKATIM) were clearly recognized, eliciting an average of at least 20 SFU/105 lymphocytes in 10 PR8 virus-infected mice (Fig. 2 and Table 1). Of these six NP418-426 variant peptides, three NP418-426 peptides are derived from H1N1 influenza A virus strains, and three NP418-426 ligands have been identified in both influenza A H1N1 and H3N2 strains. One NP418-426 variant, LPFDRTTIM, exhibited greater IFN-{gamma} T-cell reactivity than PR8 NP418-426 LPFDRTTVM, generating an average of 168.5 and 103.6 SFU/105 lymphocytes, respectively. Thirteen NP418-426 variant peptides (five H1N1 strain, seven H3N2 strain, and one H1N1/H3N2 strain peptides) generated between 4 and 10 SFU/105 lymphocytes during murine PR8 virus infection, including the NP418-426 variant found in the newly emerged H1N1 flu (swine flu) strain (LPFERATVM). All NP418-426 peptide sequences restricted to H3N2 influenza A virus strains elicited an average IFN-{gamma} T-cell response below 10 SFU/105 lymphocytes (Table 1).

In Table 1 H1N1, H1N1/H3N2, and H3N2 NP418-426 peptide sequences were aligned to the PR8 peptide (LPFDRTTVM) in ascending order by the average number of IFN-{gamma} spots (SFU/105 lymphocytes) for 10 PR8 virus-infected HLA-B*0702 transgenic mice, indicating amino acid positions involved in the PR8 NP418-426-specific T-cell response. Substantial NP418-426-specific T-cell cross-reactivity was observed only for NP418-426 variants derived from either influenza A H1N1 strains or H1N1/H3N2 strains. All NP418-426 peptides whose sequences were limited to influenza A H3N2 strains failed to generate a sizeable IFN-{gamma} T-cell response.

Amino acid substitutions at P4 to P6 decreased IFN-{gamma} T-cell reactivity during murine PR8 virus infection while exchanging Val at P8 for Ile increased the IFN-{gamma} ELISPOT response (Tables 1 and 2). IFN-{gamma} ELISPOT responses of PR8 virus-infected HLA-B*0702 transgenic mice to NP418-426 variant peptides indicate that amino acids at P4 to P6 and at P8 dictate antigen-specific T-cell cross-reactivity. The majority of amino acid substitutions at these positions decrease IFN-{gamma} T-cell reactivity; only a Val -> Ile substitution at P8 increased the NP418-426-specific IFN-{gamma} ELISPOT response. Tremendous amino acid variability is observed at P6 of the NP418-426 epitope. At P6 variant NP418-426 peptides exhibit both conservative (Thr -> Ser) and nonconservative (mutations of Thr to Ala, Pro, Gln, and Ile) amino acid substitutions. Considerably less amino acid variability is observed at P4, P5, and P8 in terms of the variety of different amino acids detected at these positions and the conservative nature of amino acid substitutions (Asp -> Glu at P4, Arg -> Lys at P5, and Val -> Ile at P8). The data generated indicate that both conservative and nonconservative amino acid substitutions within the NP418-426 epitope can impact T-cell recognition and that the influenza virus exhibits the most flexibility at P6.


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  		TABLE 2. Relative binding affinity of PR8 NP418-426 single mutant peptides to sHLA-B*0702

NP418-426-specific CTL cross-reactivity is not correlated with HLA-B*0702 binding affinity. We next determined whether the binding affinity of NP418-426 variant peptides to HLA-B*0702 correlated with the NP418-426-specific IFN-{gamma} T-cell responses observed above; it is possible that NP418-426 peptides exhibiting a higher affinity for HLA-B*0702 may be presented on the surfaces of target cells in greater abundance for CTL recognition. Therefore, we measured the relative HLA-B*0702 binding affinity of the 20 naturally occurring NP418-426 synthetic peptides in a competitive binding assay (11-13). Various concentrations of synthetic NP418-426 peptide were incubated with sHLA-B*0702 and an HLA-B*0702 fluorescein isothiocyanate-labeled reference peptide, and the IC50 of synthetic NP418-426 was recorded. Table 1 illustrates the relative HLA-B*0702 binding affinity of the synthetic NP418-426 variant peptides [high-affinity binders, log(IC50 nM) of <3.7; medium-affinity binders, log(IC50 nM) of 3.7 to 4.7, low-affinity binders, log(IC50 nM) of 4.7 to 5.5; very-low-affinity binders, log(IC50 nM) of ≥6.0]. The PR8 NP418-426 peptide LPFDRTTVM had the highest binding affinity for HLA-B*0702 [1.8 log(IC50 nM)]. The average HLA-B*0702 binding affinity for variants generating at least 10 SFU/105 lymphocytes during murine PR8 virus infection is equivalent to NP418-426 variants which elicited, on average, less than 10 SFU/105 lymphocytes, indicating that the binding affinity of the NP418-426 variant peptides did not correspond to a gain or loss in T-cell cross-reactivity. These data demonstrate that all variants tested have a high binding affinity and suggest that T-cell cross-reactivity to variant NP418-426 peptide sequences is heavily influenced by interaction with P4 to P6 and P8 of NP418-426.

Amino acid residues at P4 to P6 and P8 within the NP418-426 epitope dictate CTL recognition. Naturally occurring variants of NP418-426 often differ by multiple amino acid substitutions, making it difficult to ascertain how a given amino acid substitution or position within the epitope contributes to T-cell recognition. In order to unravel the individual impact that naturally occurring NP418-426 amino acid substitutions have upon T-cell cross-reactivity following PR8 virus infection, we synthesized epitopes differing by a single amino acid from the parental NP418-426. Lymphocytes isolated from the spleens of nine HLA-B*0702 transgenic mice inoculated intranasally with PR8 virus were assessed for IFN-{gamma} ELISPOT reactivity to NP418-426 peptides containing a single amino acid substitution at P4 to P6 with respect to the sequence of PR8 NP418-426 (Fig. 3A and B; Table 2). The introduction of these single amino acid substitutions into the PR8 NP418-426 epitope had a negligible impact on HLA-B*0702 binding (Table 2). Surprisingly, at P4 both conservative (Asp -> Glu) and nonconservative (Asp -> Gly) amino acid substitutions eliminated PR8 NP418-426 IFN-{gamma} ELISPOT cross-reactivity. On average, the LPFERTTVM and LPFGRTTVM (substitutions are underlined) synthetic peptides generated fewer than 10 SFU/105 lymphocytes. At P5, the conservative Arg -> Lys amino acid substitution dramatically reduced IFN-{gamma} ELISPOT reactivity but did not completely eliminate CTL recognition. Splenocytes incubated with the LPFDKTTVM synthetic peptide generated an average of 20.1 SFU/105 lymphocytes in comparison to PR8 NP418-426, which generated an average of 161.4 SFU/105 lymphocytes. As stated above, naturally occurring H1N1 and H3N2 NP418-426 variants exhibit the most variability at P6. The five P6 single amino acid NP418-426 variants varied in IFN-{gamma} ELISPOT reactivity. The conservative Thr -> Ser substitution did not substantially increase or decrease CTL recognition while the nonconservative substitutions of Ala, Pro, and Ile for Thr drastically decreased IFN-{gamma} ELISPOT reactivity, and the Thr -> Gln variant virtually eliminated IFN-{gamma} ELISPOT reactivity. Substitutions at P4 and P6 substantially altered the cross-recognition of natural NP418-426 variants.


Figure 3
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  		FIG. 3. IFN-{gamma} ELISPOT reactivity of PR8 virus-infected HLA-B*0702 transgenic mice to the PR8 NP418-426 variant peptide containing single amino acid substitutions at P4 to P9. IFN-{gamma} ELISPOT responses of splenocytes isolated from mice infected with 1,000 EID50s of PR8 virus generated against the P4 to P9 single mutant influenza A virus NP418-426 peptides are illustrated for each mouse in SFU/105 lymphocytes. (A) Nine PR8 virus-infected mice generated an average of 161.4 SFU/105 lymphocytes to the parental PR8 NP418-426 peptide (LPFDRTTVM). The T-cell cross-reactive IFN-{gamma} response elicited to synthetic PR8 NP418-426 peptides containing a single amino acid substitution at P4 to P6 (B) was compared to the parental PR8 NP418-426 peptide (shaded in gray). The IFN-{gamma} ELISPOT reactivity of lymphocytes isolated from the spleens of four PR8-infected mice to the PR8 NP418-426 (average, 137.9 SFU/105 lymphocytes) peptide (C) was compared to PR8 NP418-426 synthetic peptides containing a single amino acid substitution at P7 and P9 (D). NP418-426 single mutant peptides generating an average of greater than 10 SFU/105 lymphocytes are shown in red, and synthetic peptides that elicited less than 10 SFU/105 lymphocytes are shown in black.

Although the majority of the 20 naturally occurring H1N1 and H3N2 NP418-426 variant sequences exhibit variability at amino acids at P4 to P6 and P8, a conservative amino acid substitution is found at P7 and P9 in one variant each. The IFN-{gamma} ELISPOT response of lymphocytes isolated from the spleens of four PR8 virus-infected HLA-B*0702 transgenic mice indicates that the Thr -> Ile substitution at P7 does not substantially alter CTL recognition while the Met -> Ile amino acid substitution results in decreased IFN-{gamma} ELISPOT reactivity (Fig. 3C and D; Table 2). The paucity of NP418-426 variants containing a P7 Thr -> Ile substitution in nature is most likely due to the minimal impact of such a substitution on CTL cross-recognition. In contrast, although a Met -> Ile amino acid substitution at P9 decreases NP418-426 cross-reactivity (Fig. 3C and D; Table 2) and has not been shown to alter viral fitness (5), this substitution is not common in influenza H1N1 and H3N2 virus NP molecules (Fig. 4).


Figure 4
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  		FIG. 4. Molecular modeling of the HLA-B*0702 PR8 NP418-426 peptide complex. The orientation of the PR8 NP418-426 peptide (LPFDRTTVM) in the HLA-B*0702 binding groove was determined by molecular modeling. The position of LPFDRTTVM amino acid residues in the peptide binding groove are designated as follows: {uparrow}, up from the peptide binding groove; {downarrow}, down toward the peptide binding groove; ->, toward the alpha-1 helix; <-, toward the alpha-2 helix;, anchor residue. Molecular modeling indicates that amino acid residues at P4, P5, and P8 of the peptide point up from the peptide binding groove while residues at P6 point toward the alpha-1 helix. Single amino acid substitutions at P4 to P9 increase, decrease, or produce no change in IFN-{gamma} ELISPOT reactivity during PR8 murine infection.

These data indicate that amino acid residues at P4 to P6 and P8 of the NP418-426 epitope mediate CTL recognition. Amino acid substitutions located at P4 eliminated IFN-{gamma} ELISPOT cross-reactivity while variability at P5 drastically reduced CTL recognition. Conservative variability at P6 did not substantially impact CTL cross-reactivity while nonconservative amino acid substitutions substantially decreased or virtually eliminated IFN-{gamma} ELISPOT reactivity. Influenza virus strains containing the P8 Val -> Ile single amino acid substitution have been isolated in the human population and were shown to have increased IFN-{gamma} ELISPOT reactivity in the previous section (Fig. 2 and Table 1). CTL cross-recognition is largely mediated by the position and less by the nature of the amino acid substitution(s).

Molecular modeling of the HLA-B*0702 PR8 NP418-426 peptide complex. The data presented thus far indicate that amino acids at P4 to P6 and P8 within the NP418-426 ligand are key determinants of NP418-426-specific T-cell cross-reactivity. It is plausible that amino acids at these positions may influence T-cell cross-reactivity by directly contacting the T-cell receptor or by altering the conformation of the NP418-426 peptide in the class I HLA-B*0702 binding groove. To address the orientation of NP418-426 amino acid residues at P4 to P6 and P8, the PR8 NP418-426 ligand (LPFDRTTVM) was modeled in the HLA-B*0702 binding groove (Fig. 4). The PDB structures of five HLA-B molecules (HLA-B*2709, -B*1501, -B*3501, B*5301, and -B*5703) (16, 18, 26, 29, 31) were loaded into InsightII, and each structure was used to model the HLA-B*0702 LPFDRTTVM peptide complex via the homology and consensus module. Without altering the position of the peptide backbone, side chain clashes were minimized by manual or auto-rotamer adjustments of the peptide or HLA binding groove residues. The model that most favorably accommodated the HLA-B*0702 LPFDRTTVM sequence was based on the structure of HLA-B*2709 (PDB 1K5N) (16). Molecular modeling indicates that amino acids at P4, P5, and P8 are solvent exposed and point away from the peptide binding cleft while amino acids at P6 are directed toward the alpha-1 helix of the binding groove. Therefore, amino acid substitutions at P4, P5, and P8 are poised to dictate T-cell cross-reactivity by directly interacting with the T-cell receptor (TCR). This may explain why both nonconservative and conservative amino acid substitutions at these positions dramatically impact NP418-426-specific IFN-{gamma} ELISPOT cross-reactivity in the PR8 murine model. In contrast, a nonconservative substitution at P6 is more likely to shift the conformation of the NP418-426 peptide in the HLA-B*0702 binding groove by interacting with residues of the alpha-1 helix, indirectly altering T-cell recognition.


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DISCUSSION
 
CTL responses decrease morbidity and mortality and are key to viral clearance during human influenza A virus infection. As a foil to CTL detection, amino acid substitutions in peptide anchor positions and at CTL receptor contact residues hinder influenza virus class I HLA presentation and CTL recognition, respectively (3, 10, 27). Over time, influenza A viruses have emerged with anchor position substitutions in epitopes NP380-383/HLA-B*0801 and NP383-391/HLA-B*2705 that act to abolish class I HLA binding. Amino acid variability at P4 to P9 of the immunodominant HLA-B*0702 and -B*3501 influenza A virus NP418-426 epitope has been reported to modulate but not abolish recognition by CTL clones specific for older/newer influenza virus strains. Our laboratory has substantiated by direct peptide elution that hypervariability does not inhibit presentation of the NP418-426 epitope for three viral strains and two HLA-B alleles (34). Epitope NP418-426 represents a natural recombinant system that maintains processing, binding, and presentation so that the impact of intraepitope variability on CTL targeting can be examined. To our knowledge, no one has identified a consistently presented hypervariable epitope such that the give and take between CTL and natural viral mutations can be assessed.

The IFN-{gamma} ELISPOT reactivity of 20 natural NP418-426 influenza virus variants demonstrated that amino acid substitutions at P4 to P6 and P8 impact T-cell recognition. The contribution of individual amino acid substitutions at P4 to P9 of the NP418-426 peptide was determined by measuring the IFN-{gamma} ELISPOT reactivity of lymphocytes isolated from the spleens of PR8 virus-infected HLA-B*0702 transgenic mice incubated with synthetic PR8 NP418-426 peptides containing single amino acid substitutions. Data revealed that conservative and nonconservative amino acid variability at P4 eliminated CTL recognition. Likewise, conservative amino acid substitutions at P5 and P8 were able to dramatically modulate the NP418-426-specific IFN-{gamma} CTL response. The conservative Arg -> Lys P5 substitution substantially decreased CTL IFN-{gamma} cross-reactivity while the Val -> Ile P8 substitution increased CTL recognition following PR8 infection. In contrast, only nonconservative P6 substitutions impacted CTL cross-recognition. The conservative Thr-> Ser P6 substitution yielded no substantial in the NP418-426-specific IFN-{gamma} CTL response.

We were somewhat surprised that conservative biochemical substitutions such as Asp -> Glu at P4 or a P5 Arg -> Lys diminished recognition by the TCR. One can envision a scenario whereby a P4 Asp -> Glu substitution would not cost a pathogen much in terms of viral fitness or protein structure, yet this conservative substitution would provide the virus with a level of T-cell escape. The intraepitope location of a substitution is also key, and these data illustrate the dominant role that P4 plays in NP-specific immunity. Here, we see that subtle amino acid substitutions, when positioned correctly, can diminish immune recognition of a pathogen-derived epitope.

The results obtained here must be interpreted with caution as they may differ from studies performed with high-affinity T-cell clones. At 12 days postinfection, lymphocytes isolated from the spleens of infected mice represent a polyclonal population of T cells that contains few, if any, T cells with high-affinity TCRs. Also, our data using antigen-specific T cells obtained from the spleen may vary from that obtained from T cells harvested from draining mediastinal lymph nodes. Finally, these experiments must be repeated using T cells (preferably from the lungs) of HLA-B*0702-positive influenza virus-infected patients to confirm the patterns of NP418-426 T-cell cross-reactivity observed in mice.

Molecular modeling of the NP418-426 peptide in context of the HLA-B*0702 binding groove provides a model for how immune receptors interact with this epitope. Amino acids located at P4, P5, and P8 are solvent exposed and available for direct interaction with the TCR. In contrast, residues at P6 are directed toward the alpha-1 helix of the peptide binding groove and are less accessible to the TCR. Nonconservative amino acid substitutions at P6 may slightly alter the conformation of the peptide in the HLA-B*0702 peptide binding cleft, but it is unlikely that P6 changes directly interact with the TCR. These data suggest that viruses modulate CTL recognition by accumulating one or two conservative amino acid substitutions at peptide positions in direct contact with the TCR. However, it should be noted that the majority of naturally occurring human H1N1 and H3N2 NP418-426 peptide sequences that influence CTL recognition exhibit variability at two or more peptide positions. Factors that influence viral mutagenesis must extend well beyond the selective pressures exerted by T-cell recognition.

It is interesting that during 40 years of concurrent circulation in the human population, influenza A H1N1 and H3N2 virus strains have evolved a propensity for different amino acids at P4 of the NP418-426 epitope (for H1N1, Asp; for H3N2, Glu). Aspartic acid has not been reported at P4 of the NP418-426 sequence in influenza A H3N2 strains isolated in the United States since 1978, 10 years after the introduction of the H3N2 subtype into the human population in 1968. Glutamic acid has not been absent at P4 in H1N1 isolates from the United States, its appearance in human circulation has been sporadic at best (1). As demonstrated in this study, variability at P4 eliminates IFN-{gamma} CTL cross-reactivity. Based upon these observations, we hypothesize that viral strain specificity at P4 deters CTL cross-reactivity between the H1N1 and H3N2 influenza A virus subtypes and that amino acid variability at P5, P6, and P8 modulates CTL recognition within a subtype.

We have so far focused upon viral substitutions within, and CTL recognition of, an influenza virus NP epitope. What can we learn from HLA molecules of the B7 supertype that interact with this epitope? HLA-B*0702 is found in roughly one-fourth of the population (26% and 16% of the Caucasian and African American populations of the United States, respectively), and presentation by other members of the B7 supertype family would extend presentation of NP418-426 to one-third or more of the population (22, 28). Indeed, we find that epitope NP418-426 is presented by HLA-B*3501, a member of the B7 supertype and an allele found at a phenotype frequency of 12% and 10% in Caucasian and African American populations of the United States, respectively (22). Additional experiments will need to confirm presentation by all alleles within the B7 supertype, yet the extraordinary variability that influenza virus strains exhibit within this epitope (others have classified this region as hypervariable) (8) certainly suggests that the cellular immune system has focused considerable pressure on this segment of the virus. It is important for vaccine architects to recognize that variable sequences bracketed by conserved anchors can indicate robust class I HLA-presented immune targets.

In summary, observations including direct HLA-B*0702 epitope elution following infection by divergent viral strains justifies a detailed characterization of the consistently presented influenza A virus epitope NP418-426. This particular epitope cannot avoid class I HLA-B7 presentation, and CTL cross-recognition experiments demonstrate that influenza virus utilizes substitutions at P4 to P6 and P8 within this epitope to diminish and/or abrogate immune targeting. The natural variants tested indicate that the virus is constrained in its ability to substitute amino acids, yet by accumulating one or two conservative intraepitope substitutions, the influenza virus is able to thwart cross-targeting of NP418-426. These data additionally show that particular substitutions do not disrupt CTL recognition, which is useful information for the design of CTL-eliciting therapeutics. Finally, it is conceivable that other class I HLA-B alleles present immunodominant epitopes from regions of variability; the HLA-B locus plays a critical role in controlling several viruses (7, 9, 17, 19), and future testing is needed to determine the presentation of epitopes spanning more variable regions to CTL.


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ACKNOWLEDGMENTS
 
We thank Gillian Air and Sherry Crowe for their insight on the human influenza A virus and PR8 mouse model of influenza virus infection, respectively.

This work was supported by National Institutes of Health Contract HHSN266200400027C (W.H.H.) and National Institute of Allergy and Infectious Disease institutional training grant A1007633-006 (A.W.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, 975 NE 10th Street, Oklahoma City, OK 73104. Phone: (405) 271-1203. Fax: (405) 271-3117. E-mail: William-Hildebrand{at}ouhsc.edu Back

{triangledown} Published ahead of print on 24 June 2009. Back


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Journal of Virology, September 2009, p. 9206-9214, Vol. 83, No. 18
0022-538X/09/$08.00+0     doi:10.1128/JVI.00932-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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