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Short Communication |
Department of Virology and Postgraduate School of Molecular Medicine, Erasmus Medical Center, Rotterdam, The Netherlands
Correspondence
G. F. Rimmelzwaan
g.rimmelzwaan{at}erasmusmc.nl
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ABSTRACT |
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). Targets for CTL responses, such as the virus nucleoprotein (NP) and matrix protein, are considered as candidate vaccines because of their conserved nature. The use of conserved proteins could provide protective immunity against drift variants or viruses with novel subtypes (Townsend & Skehel, 1984; Yewdell et al., 1985). Indeed, the majority of CTL epitopes that have been identified are conserved (Parker & Gould, 1996). However, the use of prototypic strains in these studies may have resulted in a bias towards the preferential identification of conserved CTL epitopes (DiBrino et al., 1993, 1994, 1995; Gianfrani et al., 2000; Jameson et al., 1999; Macken et al., 2001; Man et al., 1995; Rohrlich et al., 2003; Townsend et al., 1986; Wedemeyer et al., 2001). In addition, functional constraints of these proteins may limit variation of epitopes and thus limit escape from virus-specific CTLs (Berkhoff et al., 2005; Rimmelzwaan et al., 2004a, 2005). We have recently shown that a number of influenza A virus epitopes are variable and that amino acid substitutions in these epitopes are associated with escape from virus-specific CTLs. An amino acid substitution at position 384 of the NP (R384G), which is at the anchor residues of the human leukocyte antigen (HLA)-B*2705-restricted NP383–391 and HLA-B*0801-restricted NP380–388 epitopes, abrogated the recognition of the epitopes by specific CTLs (Berkhoff et al., 2004; Gog et al., 2003; Rimmelzwaan et al., 2004b; Voeten et al., 2000). Other variable epitopes include the HLA-B*3501-restricted epitope NP418–426, which displayed extensive amino acid variation at T-cell receptor-contact residues (Boon et al., 2002b), and the NS1122–130 epitope (Macken et al., 2001; E. G. M. Berkhoff, unpublished observation). Because examples of mutations in influenza A virus CTL epitopes are still anecdotal, we wished to determine the extent of variation in CTL epitopes during influenza A virus evolution.
In a previous study, we showed that more non-synonymous mutations occurred in known influenza A virus CTL epitopes located in the NP than in the rest of this protein, which is suggestive of selective pressure on CTL epitopes (Berkhoff et al., 2005; Korber, 2001; Nei & Gojobori, 1986; Ota & Nei, 1994). In order to obtain an impression of the number of epitopes subject to amino acid variation that would affect their binding to their corresponding HLA molecules, predicted epitopes in a historic strain of influenza A virus (H3N2) were compared with their sequence in a more recent strain. To this end, the PB2, PB1, PA and NP sequence of influenza virus A/Hong Kong/1/68 (Macken et al., 2001) was used in the epitope-prediction algorithms BIMAS and SYFPEITHI (at http://bimas.dcrt.nih.gov/molbio/hla_bind/ and http://www.syfpeithi.de/ respectively). Epitopes were predicted for HLA alleles for which no epitopes had been described previously (non-HLA-A*0101, -A*0201, -B*0701, -B*0801, -B*2705, -B*3501). Three hundred and thirty-nine epitopes were predicted with a SYFPEITHI score of 
20 and 840 epitopes were predicted with a BIMAS ranking of 
10. These predicted epitopes were compared with their amino acid sequences of more recent influenza (H3N2) strains A/Shiga/25/97 (PB2, PB1, PA) and A/Christchurch/39/2000 (NP). Seventy-seven of the predicted epitopes displayed amino acid variation at anchor residues, resulting in a considerable decrease in score and/or ranking of these epitopes. Ten predicted wild-type epitopes were selected and synthesized as peptides, based on the availability of HLA-typed peripheral blood mononuclear cells (PBMCs) and the emergence of the variant epitope after 1980. However, none of these ten wild-type peptides was recognized by in vitro-expanded polyclonal T-cell populations specific for influenza virus A/Hong Kong/1/68, as measured in gamma interferon ELISPOT assays (data not shown). Unfortunately, there is a poor correspondence between predicted and experimental binding of peptides to major histocompatibility complex (MHC) class I molecules and it was reported that these programs could produce a considerable number of false positives (Andersen et al., 2000). In addition, only a minority of predicted epitopes proved to be immunogenic (Toebes et al., 2006). This approach would only allow the identification of amino acid substitutions at anchor residues. As it is known that virus also can escape from CTLs by mutations at T-cell receptor-contact residues (Boon et al., 2002b), we decided to follow an empirical approach as well.
In order to determine the frequency of virus-specific CTLs that lost recognition of their epitope during influenza A virus evolution, PBMCs of six HLA-typed study subjects of between 35 and 55 years of age were stimulated in vitro with an influenza virus that was isolated in 1981 [A/Netherlands/4791/81 (H3N2); 1981-virus]. This way, 1981-virus-specific CD8+ CTLs were expanded for 8 days in the presence of recombinant interleukin-2 as described previously (Boon et al., 2002a). We selected study subjects with HLA haplotypes (Table 1) for which no CTL epitopes had been described previously in order to prevent the identification of known conserved or variable epitopes. From the in vitro-expanded PBMCs, 304 virus-specific CTL clones were obtained by limiting dilution and subsequent non-specific stimulation with PHA (Table 1). Cells were cultured at a density of 0.3, 1 and 3 cells per well in the presence of irradiated allogenic PBMCs and selected Epstein–Barr virus-transformed B-lymphoblastoid cell lines (BLCLs) as described previously (Voeten et al., 2000). Next, all 1981-virus-specific CD8+ CTL clones were tested for recognition of autologous BLCLs infected with a virus that was isolated in 2003 [A/Netherlands/9/2003 (H3N2); 2003-virus]. Both the 1981-virus and the 2003-virus were propagated in cell culture only, excluding amino acid variation based on difference in passage history of these viruses. In four of the six study subjects, CTL clones were identified that were unable to recognize the 2003-virus-infected cells (Table 1). Eight of the 304 (2.6 %) 1981-virus-specific CTLs failed to recognize the recent strain of influenza A virus. Repeated infections in these individuals might have decreased the frequency of CTLs specific for the original version of variable epitopes relative to those specific for conserved epitopes that were boosted during repeated infections, which may result in an underestimation of the proportion of CTLs that originally lost recognition. Thus, the number of examples of variable CTL epitopes in the influenza A virus NP is increasing, indicating that there is more variation in these CTL epitopes than was thought previously (Parker & Gould, 1996).
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), the regions in which the probable epitopes were located were identified. With partially overlapping and truncated synthetic peptides, the minimal epitopes were determined (Fig. 1a, d, g). For subject 0435, two HLA-B*4002-restricted CTL clones were obtained that were specific for the NP251–259 epitope AEIEDLIFS. Indeed, these clones (NP251–259/1981) failed to recognize the 2003-virus with the S259L mutation, as well as the mutant peptide AEIEDLIFL (data are shown for one of these clones; Fig. 1a–c). The HLA-B*1503-restricted CTL clone specific for the NP103–111 epitope KWMRELVLY (NP103–111/1981), obtained from subject 3909, failed to recognize the K103R mutant peptide RWMRELVLY (Fig. 1g, h). The other three CTL clones obtained from this study subject could not be tested further because of poor yields in cell number upon restimulation.
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, 2004). Therefore, we also stimulated PBMCs of subject 0435 with the 2003-virus and generated CD8+ CTL clones specific for the 2003-virus that were tested for their reactivity with the 1981-virus. Seven of 76 influenza virus A/Netherlands/9/2003-specific CTL clones failed to recognize the 1981-virus. Of special interest, one of these 2003-virus-specific CTL clones was specific for the HLA-B*4002-restricted NP251–259 variant epitope, which was not recognized by NP251–259-specific CTL clones directed to the 1981-virus (see above). This CTL clone cross-reacted with the 1981-variant of the peptide with 30-fold lower functional avidity than the homologous 2003-version of the peptide (Fig. 1d–f
). Most likely, the 1981-peptide concentration required for the activation of these 2003-virus CTLs is higher than that achieved during infection of target cells with the 1981-virus, which explains the observation that 1981-virus-infected cells are not recognized by this 2003-virus-specific CTL clone.
Thus, a mutant version of the epitope emerged that escaped from CTLs specific for the 1981-variant of the epitope and induced a CTL response that partially cross-reacted with the 1981-variant of the epitope. A similar finding was observed previously for the HLA-B*3501-restricted NP418–426 epitope, which displays extensive variation at T-cell receptor-contact residues (Boon et al., 2004). The other six 2003-virus-specific CTL clones were all directed to the HLA-A*6801-restricted NP145–152 epitope (DATYQRTR), which contained the T146A substitution. The full meaning of variation in this CTL epitope is unclear at present, as CTL clones specific for the 1981-variant of the epitope were not available.
As shown in Fig. 1(i), the amino acid substitutions in the NP251–259 epitope reached fixation rapidly during influenza A virus evolution and the 259L variant has been circulating since 1997. Variants of the NP103–111 epitope replaced each other more frequently; from 1968 until 1983 and from 1998 until 2001, the KWMRELVLY variant was most predominant, whereas the RWMRELVLY variant was found predominantly from 1983 until 1998 and from 2001 until 2004, and was subsequently replaced by the original variant of the epitope (Fig. 1j). The prevalence of the mutant viruses did not correlate with the phylogenetically distinct lineages of co-circulating influenza A viruses (Holmes et al., 2005). The rapid fixation of mutations in influenza A virus CTL epitopes was explained previously by small selective advantages and population dynamics in a theoretical model (Gog et al., 2003). The alternating fixation of wild-type and mutant variants of the NP251–259 and NP103–111 epitopes over time is of interest and may reflect a dynamic interplay between immune evasion and intrinsic virus fitness. It is possible that the relatively low prevalence of HLA-B*1503 and -B*4002 in the human population (0.87 and 2.94 % on average in the Caucasian population; Marsh et al., 2000) may have contributed to incomplete immune pressure on these viruses, allowing the wild-type variant to re-emerge repeatedly.
The antigenic changes in the NP responsible for escape from virus-specific CTLs may contribute to the successful persistence of influenza A viruses in the human population. Furthermore, these changes should be taken into account in the development of vaccines that aim at the induction of protective T-cell responses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 10 April 2006;
accepted 26 September 2006.
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) and the mutant version of the epitope derived from the 2003-virus (
) by ELISPOT assays (b, e, h) or chromium-release assays (c, f). Autologous BLCLs pulsed with serial dilutions of the respective peptides were used as stimulator cells in the ELISPOT assays (103 CTL clones stimulated with 2.5x104 stimulator cells) or as target cells in chromium-release assays (effector-to-target ratio of 10). Percentage specific lysis was calculated by the following formula: [(experimental release – spontaneous release)/(maximum release – spontaneous release)]x100 %. The prevalence of the NP251–259 and NP103–111 epitope variants during influenza A (H3N2) virus evolution is shown in (i) and (j). Indicated are the total numbers of NP251–259 (i) and NP103–111 (j) variant sequences per year available in the Influenza Sequence Database (