Human leukocyte antigen B27 selects for rare escape mutations that significantly impair hepatitis C virus replication and require compensatory mutations

Authors


  • Potential conflict of interest: Nothing to report.

  • This project was funded, in part, with Federal funds from the National Institute of Allergy and Infectious Disease (NIAID) under grant R01-AI067926 (to T.M.A.) U19 AI082630 (to T.M.A. and G.M.L.), with funds from the Deutsche Forschungsgemeinschaft (Emmy Noether-Programm, NE 1567/1-1 to C.N.H.; KU 2250/1-1 to T.K.; and FOR1202 to V.L.), with funds from the Bundesministerium für Bildung und Forschung (BMBF 01EO0803 to C.N.H.), with funds from the French National Agency for Research on AIDS and Viral Hepatitis (ANRS; grant AO 2010/CSS4 to S.B.), and with funds from Association pour la recherche sur le cancer (ARC; postdoctoral fellowship to C.C.S.).

Abstract

Human leukocyte antigen B27 is associated with spontaneous viral clearance in hepatitis C virus (HCV) infection. Viral escape within the immunodominant, HLA-B27-restricted, HCV-specific, cluster of differentiation (CD)8+ T-cell epitope, nonstructural protein (NS)5B2841-2849 (ARMILMTHF), has been shown to be limited by viral fitness costs as well as broad T-cell cross-recognition, suggesting a potential mechanism of protection by HLA-B27. Here, we studied the subdominant HLA-B27-restricted epitope, NS5B2936-2944 (GRAAICGKY), to further define the mechanisms of protection by HLA-B27. We identified a unique pattern of escape mutations within this epitope in a large cohort of HCV genotype 1a–infected patients. The predominant escape mutations represented conservative substitutions at the main HLA-B27 anchor residue or a T-cell receptor contact site, neither of which impaired viral replication capacity, as assessed in a subgenomic HCV replicon system. In contrast, however, in a subset of HLA-B27+ subjects, rare escape mutations arose at the HLA-B27 anchor residue, R2937, which nearly abolished viral replication. Notably, these rare mutations only occurred in conjunction with the selection of two equally rare, and structurally proximal, upstream mutations. Coexpression of these upstream mutations with the rare escape mutations dramatically restored viral replication capacity from <5% to ≥70% of wild-type levels. Conclusion: The selection of rare CTL escape mutations in this HLA-B27-restricted epitope dramatically impairs viral replicative fitness, unless properly compensated. These data support a role for the targeting of highly constrained regions by HLA-B27 in its ability to assert immune control of HCV and other highly variable pathogens. (HEPATOLOGY 2011;)

The human leukocyte antigen (HLA) class I allele B27 has a protective role in both human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections,1, 2 two of the most highly variable pathogens, for which effective vaccines are still lacking. In HIV infection, HLA-B27 is associated with low viral loads, slow cluster of differentiation (CD)4+ decline, and delayed onset of acquired immunodeficiency syndrome (AIDS).1 Protection by HLA-B27 has been linked to the immunodominant targeting of a highly conserved CD8+ epitope in Gag, which requires a complicated pathway of viral escape to evade this response. Escape from this B27-KK10 response requires a multitude of mutations. First, an L268M mutation inside the epitope impairs T-cell recognition and also impairs dendritic cell (DC) function through increased interaction with an inhibitory receptor.3 A subsequent R264K mutation at the main HLA-B27-binding anchor of the epitope, which achieves more effective cytotoxic T lymphocyte (CTL) escape by abolishing epitope presentation, typically occurs much later in infection and is associated with disease progression.3 The late appearance of the main R264K escape mutation can be attributed to its substantial impact on viral replicative fitness when expressed alone, and thus it is commonly associated in vivo with a S173A compensatory mutation located 90 amino acids upstream of the epitope.3-5 Thus, the ability of HLA-B27 to impart substantial, durable control of HIV is the result, in part, of the requirement for a compensatory mutation to enable effective CTL escape from this dominant immune response.

In HCV genotype 1 infection, the majority of HLA-B27+ individuals are able to clear the virus spontaneously.2, 6 We have previously described several HLA-B27 restricted HCV-specific CD8+ T-cell epitopes.7 One of these epitopes, nonstructural protein (NS)5B2841-2849 (ARMILMTHF), is particularly immunodominant, because it is recognized in nearly all HLA-B27+ individuals with resolved HCV genotype 1 infection. In addition, nearly all HLA-B27+ patients who progress to chronic HCV genotype 1 infection display viral escape mutations in this epitope, suggesting that this response exerts significant selection pressure.7 Notably, this immunodominant HLA-B27-restricted CD8+ epitope is only present in HCV genotype 1, but absent from the other HCV genotypes, possibly explaining why HLA-B27+ individuals are not protected from chronic nongenotype 1 HCV infection.8 Importantly, viral escape within this epitope does not occur easily. Mutations experimentally introduced in replicon constructs at the HLA-B27-binding anchor residues of the epitope exhibit high fitness costs in vitro and have, therefore, not been found in viral sequences derived from patient specimens. In contrast, whereas mutations at T-cell receptor (TCR) contact residues within the epitope are tolerated by the virus, broad cross-recognition of these escape variants requires multiple “clusters” of these mutations to achieve efficient escape from this CD8+ T-cell response.9 The requirement for clustered mutations to escape from dominant HLA-B27-restricted CD8+ T-cell responses in both HIV and HCV infections suggests that HLA-B27 may be achieving immune control of these highly variable pathogens through the targeting of critical regions of these viruses in which viral escape is impaired.

To further explore this hypothesis, here, we have analyzed the patterns of viral escape in a second HLA-B27-restricted CD8 epitope, NS5B2936-2944 (GRAAICGKY). This CD8+ T-cell response also selects for viral escape mutations in the majority of patients with chronic HCV genotype 1a infection. We identified a subset of escape mutations within the epitope that nearly abolished viral replication and thus required additional, compensatory mutations located upstream of the epitope. This was in striking similarity to the escape pathways described for the immunodominant HLA-B27-restricted KK10 epitope in HIV Gag,3, 4 providing unique insight into the mechanisms by which some HLA alleles may be mediating their protective effect over HCV and other highly variable pathogens.

Abbreviations

AIDS, acquired immunodeficiency syndrome; CTL, cytotoxic T lymphocyte; DC, dendritic cell; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HLA, human leukocyte antigen; IC50, half-maximal inhibitory concentration; NS, nonstructural protein; PBMC, peripheral blood mononuclear cell; TCR, T-cell receptor.

Patients and Methods

Patients.

Viral sequences from 404, mostly (>90%) treatment-naïve, patients with chronic HCV genotype 1a infection were obtained previously10 (Kuntzen et al., manuscript in preparation). HLA class I typing was performed using standard molecular typing. A total of 19 of the 404 patients were positive for HLA-B27 (Supporting Table 1). In addition, peripheral blood mononuclear cells (PBMCs) were obtained from 14 HLA-B27+ patients with chronic HCV genotype 1a infection (10 patients from the above-mentioned cohort and 4 patients enrolled at the Freiburg University Medical Center, Freiburg, Germany), as well as from 2 HLA-B27+ patients with acute-resolving genotype 1a infection enrolled at the Freiburg University Medical Center. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the institutional review boards at Massachusetts General Hospital (Boston, MA) and the University of Freiburg, respectively. Written informed consent was obtained from each study participant.

Plasmid Construction, Electroporation, Transient-Replication Assays, NS5B Structure Analyses, Cellular T-Cell Assays, and HLA-B2705 Peptide-Binding Assay.

These methods are described in the Supporting Methods.

Statistical Analysis.

Frequencies of substitutions in HLA-B27+ and HLA-B27- patients (Table 1) were compared using the two-tailed Fisher's exact test (GraphPad Software, Inc). Replicative capacities of mutants and wild-type were compared using the paired t-test (GraphPad Prism 5.0; GraphPad Software, Inc., La Jolla, CA). P values <0.05 were considered significant.

Table 1. Amnio Acid Substitutions Compared to Consensus at Positions 2875, 2881, 2937, and 2940 in HLA-B27-positive and -negative Patients With Chronic HCV Genotype 1a Infection
ResidueSubstitutionB27+ (%) (n = 19)B27 (%) (n = 385)P Value
  1. Two-tailed Fisher's exact test comparing frequencies in B27+ versus HLA-B27 patients.

  2. Abbreviations: HLA, human leukocyte antigen; HCV, hepatitis C virus.

E2875Q0 (0.0)4 (1.0)1.0000
 K3 (15.8)1 (0.3)0.0003
 Any3 (15.8)5 (1.3)0.0043
P2881L4 (21.1)24 (6.2)0.0347
 Q1 (5.3)5 (1.3)0.2524
 S0 (0.0)2 (0.5)1.0000
 Any5 (26.3)31 (8.1)0.0183
R2937K5 (26.3)21 (5.5)0.0047
 G1 (5.3)1 (0.3)0.0920
 S1 (5.3)0 (0.0)0.0470
 Any7 (36.8)22 (5.7)0.0001
I2940T5 (26.3)16 (4.2)0.0017
 M0 (0.0)9 (2.3)1.0000
 V1 (5.3)1 (0.3)0.0920
 Any6 (31.6)26 (6.8)0.0020

Results

CTL Escape in the B27-GRAAICGKY Epitope Typically Occurs Through Conservative Mutations.

In a large-cohort analysis of 404 full-length HCV genotype 1a sequences, we identified that viral escape in the HLA-B27-restricted CD8+ T-cell epitope, NS5B2936-2944 (GRAAICGKY), was comprised of a unique array of CTL escape mutations. In the 19 HLA-B27+ individuals, the predominant escape mutations within this epitope arose at either the main B27 anchor residue (R2937K) or at a TCR contact site (I2940T) (Fig. 1). In each case, these mutations represented conservative substitutions with the swapping of positively charged residues at R2937K and the substitution of uncharged residues at I2940T. Notably, however, in 2 B27+ subjects, more rare escape mutations were observed, with R2937 replaced by the uncharged amino acids, serine (R2937S) or glycine (R2937G) (Fig. 1). In addition, in 1 B27-positive subject, the I2940 residue was replaced by a rare valine (I2940V) substitution in conjunction with the R2937K mutation (Fig. 1). Because of the more frequent development of the R2937K and I2940T mutations, we hypothesized that these more conservative substitutions might only minimally effect viral replication (viral fitness), whereas the more rare mutations might be associated with higher fitness costs.

Figure 1.

HLA-B27-driven sequence variation in the HLA-B27-restricted epitope, NS5B2936-2944. The HCV genotype 1a consensus sequence, the autologous viral sequence in 19 HLA-B27+ patients with chronic HCV genotype 1a infection, and the autologous viral sequence in 385 HLA-B27-negative patients with HCV genotype 1a infection are shown. Positions with HLA-B27-associated sequence polymorphisms (“footprints”; compare Table 1) are shown with a gray background.

Common Escape Mutations Within the B27-GRAAICGKY Epitope Have No Effect on Viral Replication.

To examine the effect of these CTL escape mutations on the virus, we constructed subgenomic HCV replicons bearing the different viral escape mutations. Replicative capacities of the mutants were then measured in a transient in vitro replication system in hepatoma cells using the luciferase reporter gene as a marker for HCV replication. As predicted, no significant differences in the replication capacity of either of the constructs containing the frequent mutations, R2937K or I2940T, were observed, as compared to the parental “wild-type” construct (Fig. 2). In fact, in this in vitro assay, the two variants even exhibited a slight, though not statistically significant, increase in replication. This unimpaired replication is in agreement with the observation that both mutations are more frequently observed in B27-negative subjects, with R2937K being the dominant viral species in 21 of 385 B27-negative patients (5.5%) and I2940T representing the dominant viral species in 16 of 385 B27-negative patients (4.2%) (Table 1).

Figure 2.

Rare escape mutations at position 2 of the HLA-B27-restricted epitope NS5B2936-2944 nearly abolish viral replication. A wild-type subgenomic replicon, a replication deficient variant (GND), and several variants containing escape mutations within the HLA-B27-restricted epitope NS5B2936-2944 were tested for replication capacity. Replication of the wild type was set at 100%. Means and standard errors of three individual experiments are shown. P values comparing replication of mutants and wild type were calculated by the paired t-test and are indicated as follows: *P < 0.05; **P < 0.005; ***P < 0.0005.

Rare Escape Mutations Within the B27-GRAAICGKY Epitope Substantially Impair Viral Replication.

In contrast to the common R2937K HLA-B27 anchor-binding mutation, both of the rare R2937S and R2937G mutations nearly completely abolished viral replication (Fig. 2), including the R2937G mutation in conjunction with I2940T, as observed in 1 patient (Fig. 1). The greater impact of these two rare escape mutations on viral replication is also in agreement with the absence of these mutations in HLA-B27-negative individuals; indeed, the R2937S mutation was not found in any of the 385 B27-negative patients in the cohort, whereas the R2937G mutation was only observed in a single B27-negative patient (0.3%; Table 1). In addition, the I2940V mutation, alone or in combination with mutation R2937K, was also found to impair viral replication, resulting in reductions in the replication of 15% and 24%, respectively, although these results did not reach statistical significance. Considering that the I2940V substitution is only rarely observed in B27-negative patients (1 of 385 patients, 0.3%; Table 1) suggests that even this moderate reduction in replication capacity may have a significant effect on the virus in vivo analogous to the effect of the M184V drug-resistance mutation in HIV.11

Mutations at Position E2875 and P2881 Coevolve With Rare Escape Mutations at Positions R2937 and I2940.

The observation that mutations R2937S and R2937G nearly completely impair viral replication was surprising, given their presence in subjects demonstrating significant viral loads (e.g., the B27-positive patient with mutations R2937G/I2940T had a viral load of 33.9 × 106 IU/mL, and the B27-negative patient with mutation R2937G had a viral load of 22.5 × 106 IU/mL). We, therefore, hypothesized that these mutations might occur in conjunction with one or several additional mutations that function as compensatory mutations to alleviate their high costs to viral fitness. In screening our cohort of 404 HCV genotype 1a–infected patients for additional substitutions coevolving with the rare HLA-B27-associated mutations, R2937G or R2937S, throughout the HCV polyprotein, we found coevolution (defined as a mutation present in at least 2 of the 3 patients with the R2937G or R2937S mutation, but not frequently occurring in other patients) at only two amino acid residues: E2875 and P2881 (Fig. 3). These two residues neither overlap with previously reported B27-restricted epitopes nor with predicted B27-restricted epitopes (T-cell epitope prediction algorithm: Immune Epitope Database and Analysis Research [IEDB]).

Figure 3.

Rare mutations R2937S, R2937G (with or without I2940T), and R2937K/I2940V within the HLAB27-restricted epitope, NS5B2936-2944, only occur in concert with an equally rare cluster of two mutations 71 and 65 amino acids upstream of the epitope, which restore a network of interactions in the NS5B thumb domain. (A) Autologous viral sequences are shown for all patients that harbor the E2875K mutation (4/404 patients in the cohort; compare table 1). All 4 patients also display one of the rare mutations at position 2937 (R2937S, R2937G, or R2940K/I2940V). In addition, all 4 patients display a mutation at position 2881 (P2881L or P2881Q). (B) Close-up of the epitope region in the three-dimensional structure of genotype 1a NS5B. NS5B is displayed as ribbons and colored by domains (red: fingers; yellow: palm; purple: thumb). The epitope is shown in green with the sites of rare escape mutations R2937 and I2940 displayed as sticks, colored by atom type (blue: nitrogens; green: carbons) and labeled. The sites of compensatory mutations P2881 and E2875 are similarly displayed (red: oxygens; cyan: carbons), as well as two other acidic amino acids (purple: carbons) also in direct interaction (dotted yellow lines) with R2937. The thumb's “beta-flap” is labeled in italics, as well as the linker (in light brown) C-terminal to the thumb. (C) Same view as in (B), but with mutations R2937S, E2875K, and P2881Q modeled into the structure (orange: carbons). E2875K compensates for the negative charge cluster at the base of the beta-flap that would be left by R2937S alone, restoring, in part, an orientation of the beta-flap that allows proper interaction with the linker. P2881Q completes the remodeling of the network of interactions between thumb and linker.

Residue E2875 was found to be highly conserved, with 396 of 404 patients (98.0%) displaying the consensus residue at this position (Table 1). Only 8 patients carried sequence variations at this position: 4 B27-negative patients (0.8%) exhibited an E2875Q substitution, whereas this substitution did not occur in HLA-B27+ patients (not significant). Conversely, however, a substitution of the negatively charged amino acid, glutamate, by the positively charged amino acid, lysine (E2875K), occurred in 3 of the 19 HLA-B27+ patients (15.8%), notably more frequent than in the 0.3% (1/385) of B27-negative subjects (P = 0.0003, Table 1). Strikingly, as shown in Fig. 3A, all four E2875K substitutions occurred together with one of the rare mutations at position R2937 (R2937S or R2937G) or I2940 (I2940V). The coexpression of these very rare mutations indicated that E2875K may, indeed, be required for the development of these rare escape mutations, functioning to compensate for their impacts on viral replication. This hypothesis is confirmed by the structural analysis of genotype 1a NS5B. As shown in Fig. 3B, R2937's positively charged side chain inserts between three negatively charged side chains, of which the central one is that of E2875. Mutating R2937 to either S (Fig. 3C) or G (not shown) leaves a cluster of three negative charges, an unfavorable configuration that is compensated by the single E2875K mutation (Fig. 3C). Still, these changes would affect the conformation of the so-called “beta-flap,” a flexible beta-structure in NS5B's “thumb” domain, at the base of which are the three negatively charged residues (Fig. 3B). The beta-flap is one of two main anchors of the “linker,” a structural element whose movement has been proposed to be critical for NS5B's enzymatic activity.12 The other anchor is made by the outer faces of two helices in the thumb, harboring residue 2881 and the NS5B2936-3944 epitope, respectively. P2881, in particular, is sandwiched between two hydrophobic residues of the linker, an interaction that is changed by mutation to the bulkier L (not shown) or to the more polar Q (Fig. 3C). Substitutions at position 2881 are more commonly observed than at position 2875, with 36 of 404 patients (8.9%) showing a substitution of the consensus residue, proline (P) (Table 1); however, this substitution is also associated with HLA-B27 (5/19 HLA-B27+ patients [26.3%], compared to 31/385 HLA-B27 patients [8.1%]; P = 0.0183). Notably, three of the five substitutions in B27-positive patients occurred in those 3 patients with rare mutations at positions R2937 or I2940 (Fig. 3A).

Mutations at Positions E2875 and P2881 Restore the Fitness Defects of Rare Escape Mutations in the B27-GRAAICGKY Epitope.

To directly test whether the observed mutations at positions E2875 and P2881 could compensate for the effect on viral replication of the rare mutations at positions R2937 or I2940, we coexpressed the different mutations in the subgenomic replicon system. As shown in Fig. 4, coexpression of mutations R2937G, R2937G/I2940T, or R2937S with mutation E2875K led to a marked, though not complete, restoration of viral replication to levels of 39%, 28%, and 80%, compared to wild type, respectively. The same was true for the coexpression of the three respective R2937 mutations with mutations at P2881 (P2881L or P2881Q, respectively), resulting in viral replication levels of 17%-31%, compared to wild type. Only the combined coexpression of the position R2937 mutations, together with both candidate compensatory mutations (E2875K and P2881L/Q), led to a nearly complete restoration of viral replication, with replication levels of approximately 70% in the case of mutations R2937G and R2937G/I2940T and ≥100% in the case of mutation R2937S (Fig. 4). These results show clearly that the HLA-B27-associated substitutions at positions E2875 and P2881 are, indeed, compensatory mutations for the strong replicative defects caused by the substitution of arginine at R2937 by glycine or serine. When tested alone, the E2875K and P2881Q mutations were associated with a slightly reduced replication level (∼77% and 92% of wild type, respectively), as compared to wild type, explaining why these substitutions occur rarely in B27-negative patients (1/385 patients [0.3%] and 5/385 patients [1.3%], respectively; Table 1). The subgenomic replicon carrying the P2881L mutation alone, however, replicated at levels that even exceeded the wild-type replication level. This finding may, at least partially, be the result of the in vitro system used here. However, it is in agreement with the observation that the P2881L mutation is rather frequently observed in B27-negative patients in vivo (24/385 patients [6.2%]; Table 1).

Figure 4.

Viral replication capacity of the rare mutants R2937S, R2937G, and R2937G/I2940T can be restored by mutations E2875K and P2881L/Q. Replication levels of the wild-type subgenomic replicon, a replication deficient mutant (GND), mutants containing the rare escape mutations alone (R2937G, upper panel; R2937G/I2940T, middle panel; R2937S, lower panel), or in combination with mutations at position 2875 and/ or 2881 were compared. Replication of the wild-type was set 100%. Means and standard errors of three individual experiments are shown. P values comparing replication of mutants and wild-type were calculated by paired t test and are indicated as follows: *P < 0.05; **P < 0.005; ***P < 0.0005.

Escape Mutations Associated With a High Viral Fitness Cost May Be Required for Successful Escape From the B27-GRAAICGKY Response in Some Patients.

Considering that the rare mutations, R2937S, R2937G, and R2937G/I2940T, require a complicated pathway of compensatory mutations, it is surprising that these mutations nevertheless occur in a subset of HLA-B27-positive patients. This finding led us to the hypothesis that the ability of epitope-specific CD8+ T-cell responses to recognize the common escape mutations in the B27-GRAAICGKY epitope, in some subjects, may function to select for these rare, highly deleterious mutations. To better address this issue, we first determined the HLA-B27-binding capacity of the different epitope peptide variants. Indeed, a gradual effect of the different amino acid substitutions was observed (Table 2). Substitutions I2940T and I2940V did not show a substantial effect on HLA-B27 binding, consistent with the location of residue I2940 in the core region of the epitope (amino acid position 5), which likely is a TCR contact site, rather than an HLA-B27-binding anchor. The frequently observed R2937K substitution still showed some level of HLA-B27 binding, albeit at a strongly reduced level (half-maximal inhibitory concentration [IC50] of 1,893 nM, compared to the 31 nM observed for the wild-type peptide). The fact that this variant still showed some degree of HLA-B27 binding is in accord with the notion that lysine (K) has been found in a small portion of natural HLA-B27 ligands,13 and also with our previous finding that an arginine-to-lysine substitution in another HCV-specific, HLA-B27-restricted CD8+ T-cell epitope had no effect on HLA-B27 binding and only a minor effect on T-cell recognition.9 Peptides with the combined R2937K/I2940V mutations or the R2937S mutation showed only borderline HLA-B27-binding capacity (IC50, 4,668 and 4,815 nM, respectively), whereas peptides with the R2937G substitution showed no HLA-B27 binding at all (IC50, >75,000 nM). These results suggest that some of the epitope-specific escape mutations may still be able to be presented and cross-recognized by specific CD8+ T cells.

Table 2. Binding of Epitope Peptide Variants to HLA-B*2705
       PeptideIC50 (nM)
  1. Abbreviations: HLA, human leukocyte antigen; IC50, half-maximal inhibitory concentration.

        GRAAICGKY31
     -K---------1893
     ------T------122
     ------V-----32
     -K---V------4668
     -S----------4815
     -G---------->75000
     -G---T------>75000

To determine whether variant-specific CD8+ T-cell responses are capable of recognizing some of these escape mutations, we tested 2 B27-positive patients with an acute-resolving course of HCV genotype 1a infection and 12 B27-positive patients with a chronic HCV genotype 1a infection for responses against this epitope. We could establish peptide-specific CD8+ T-cell lines from both patients with acute-resolving infection as well as from 2 of the 12 patients with chronic infection. Of note, this low rate of detection in chronic HCV infection is similar to what has been observed for other dominant HCV-specific CD8+ T-cell responses.14 Next, the cross-recognition of different peptide variants by the wild-type–specific T-cell line was tested. As shown in Fig. 5, 1 of the 2 patients with acute-resolving infection showed a broad cross-recognition of the different peptide variants, including the common R2937K and I2940T mutations. Here, only the R2937G/I2940T double mutant led to a complete loss of cross-recognition. In the second acute-resolving patient, a rather narrow pattern of cross-recognition was observed, with only the R2937S, I2940V, and I2940T variant peptides showing a relevant level of cross-recognition. Finally, in both of the patients with chronic infection, a similarly narrow level of cross-recognition was observed (Fig. 5). In sum, these experiments suggest that in some patients, a broad degree of peptide-variant cross-recognition may drive the selection of rare, highly deleterious mutations, such as the R2937G/I2940T double variant, whereas in other patients, weak T-cell responses with a narrow degree of peptide-variant cross-recognition select for frequent, well-tolerated escape mutations or even allow replication of the wild-type sequence.

Figure 5.

NS5B2936-2944-specific CD8+ T-cell responses show varying degrees of variant cross-recognition in individual patients. Epitope-specific T-cell lines were derived from 2 HLA-B27+ patients with acute-resolving HCV genotype 1a infection (left panels) and from 2 HLA-B27+ patients with chronic HCV genotype 1a infection (right panels) and tested for interferon gamma production in response to wild-type peptide and variant peptides. Representative dot blots from the second patient with acute-resolving infection are shown below.

Discussion

HLA-B27 is associated with a high rate of spontaneous viral clearance in HCV genotype 1 infection.2, 8 We have previously suggested that the immunodominant, HLA-B27-restricted, HCV-specific CD8+ epitope, NS5B2841-2849 (ARMILMTHF), might have an important role in this protective effect,7, 9 because of the fact that viral escape from this epitope requires the evolution of several combined mutations within the epitope. In that epitope, because mutations at the main HLA-B27-binding anchors cannot occur as a result of viral fitness costs, mutations at T-cell contact residues are required for viral escape.9 However, because of a broad cross-recognition of these escape mutations, such TCR escape mutations need to occur in clusters to efficiently escape from this dominant CD8+ T-cell response.

Here, we studied viral evolution in a second B27-restricted, HCV-specific CD8+ T-cell epitope in NS5B (NS5B2936-3944, GRAAICGKY). In this epitope region, viral sequence variations were strongly enriched in HLA-B27+ patients with chronic HCV genotype 1a infection, as compared to HLA-B27-negative patients. Although cross-sectional data from chronically infected patients cannot formally prove viral evolution, these data strongly indicate that these mutations are HLA-B27-driven viral escape mutations. Indeed, two different patterns of viral escape mutations were observed in patients chronically infected with HCV genotype 1a. In the majority of patients, viral escape was mediated by a conservative mutation, either at the main HLA-B27-binding anchor at position 2 (arginine to lysine) or at a TCR contact residue at position 5 (isoleucine to threonine). These mutations were also observed in a longitudinal sequence analysis of 2 HLA-B27+ patients with acute infection (Supporting Table 2), further supporting the notion that the sequence variations observed in chronically infected patients, indeed, reflect viral escape mutations. Importantly, these frequent mutations had no relevant effect on viral replication, as assessed in the subgenomic replicon model. In contrast, in a small subset of HLA-B27+ patients, nonconservative mutations occurred at the main HLA-B27-binding anchor at position 2, replacing the long, positively charged residue, arginine, by the short, uncharged residue, glycine or serine. These mutations led to a nearly abolished viral replication capability. The molecular basis for this effect is readily found by structural analysis of genotype 1a NS5B: These mutations of R2937 (position 2) disrupt a network of charge interactions at the base of NS5B's beta-flap, preventing correct interactions between the thumb domain and the linker element. These interactions have been proposed to be involved in the rate-limiting early steps of RNA synthesis by NS5B.12 Indeed, modulation of the interactions between thumb and linker through the mutation of R2937 was recently found to be involved in the replicative properties of strain JFH1 NS5B.15 Accordingly, detrimental escape mutations R2937S or R2937G could only coevolve in the genotype 1a context in concert with two additional, compensatory mutations located 61 and 55 amino acids upstream of the epitope. The former compensatory mutation, E2875K, is expected to restore charge complementarity at the base of the beta-flap, whereas the latter P2881L/Q would additively enhance interaction with the linker at its other binding site in the thumb. Although these observations provide an attractive explanation for the need of compensatory mutations in the presence of the R2937S and R2937G mutation, it is important to point out that conserved RNA structures in this region may also provide further functional restrictions on the evolution of viral escape mutations, as has been reported for the 3′ end of the RNA coding for NS5B.16 Evolution of certain amino acid mutations could further be complicated by the need to change two nucleotides from the consensus sequence. Importantly, mutation R2937S can be obtained by a single nucleotide change. The same is true for the R2937G mutation; however, the HLA-B27+ individual displaying this mutation had two nucleotide changes, whereas the HLA-B27-negative patient with the R2937G mutation had a single nucleotide change (Supporting Table 3).

The finding that the two compensatory mutations, E2875 and P2881, could restore the viral replication capacity from <5% to 70%-100% of wild-type level allows two important conclusions. First, it may help to explain why associations between certain protective HLA alleles, CD8+ T-cell responses or viral escape mutations and HCV viral loads, at least in the chronic phase, are hard to identify. Indeed, the high replication rate as well as the high mutation rate of HCV might allow the selection of compensatory mutations and restore replication levels similar to wild-type, even in the presence of mutations that are highly detrimental, if occurring alone. In agreement with this finding, the B27+ patient who displayed the rare R2937G/I2940T mutation in concert with the compensatory mutations, E2875K and P2881L mutation, even had an extraordinary high viral load (Supporting Table 1). Second, this finding points out that a modest viral fitness cost, such as a reduction of replication levels by 30%-50%, as observed in previous studies,9, 17 may still have a substantial effect in vivo. Indeed, this effect may be analogous to the benefit derived by maintaining HIV-positive patients on 3TC therapy, despite the presence of the M184V 3TC-resistance mutation because of the negative effect of M184V on viral replication.11 This is further supported by the notion that mutations that were associated with a modest viral fitness cost in the in vitro replicon assay (e.g., E2875K, P2881Q, and I2940V) were observed very rarely (0.3%, 1.3%, and 0.3%, respectively) in B27-negative patients (Table 1). In contrast, mutations that had no effect on viral replication levels (or replicated even at >100%, compared to wild type in the in vitro system) occurred more frequently in B27-negative individuals (e.g., P2881L, R2937K, and I2940T), although they were detected significantly more frequently in B27-positive patients. These findings are in line with the observation, in chimpanzees, that escape mutations with a low impact on viral replication are fixed over time, whereas escape mutations with a more dramatic effect on viral replication are not sustained and also revert in an in vitro model system.18 It is tempting to speculate that the well-replicating P2881L mutation may occur in a minor quasispecies in those patients that select for one of the rare, detrimental escape mutations, enabling sufficient replication until the additional E2875K mutations can evolve and to evade the CD8+ T-cell response.

Some of the effects observed in the subgenomic HCV replicon system might be the result of the specific sequence of the replicon used. Here, we used a genotype 1b (strain con1)-based replicon with NS5B from genotype 1a (strain H77). To exclude that the observed effects were caused by the genotype 1b proteins, we confirmed the observations made in this hybrid replicon in a replicon that only contained genotype 1a (strain H77) sequences. Importantly, similar effects of these mutations were observed in the genotype 1a replicon (Supporting Fig. 1). It is also noteworthy that previous studies have identified mutations that facilitate replication in the replicon system, though exhibiting a negative effect on viral replication, for example, in the chimpanzee model.19 Thus, it is important to interpret data obtained from the in vitro system in the context of data available in vivo. As described above, the viral fitness data observed for individual mutations in vitro corresponded well to their frequency observed in patients in vivo, indicating that they were not the result of cell-culture–specific phenomena.

With the majority of HLA-B27+ patients spontaneously clearing HCV genotype 1 infection,2, 8 these data may help to explain the protective effect of HLA-B27 in HCV infection. We have previously shown that a dominant response against the HLA-B27-restricted CD8+ T-cell epitope NS5B2841-2849 (ARMILMTHF) contributes to protection by HLA-B27, and could further demonstrate that a broad cross-recognition of viral escape mutations, as well as limitations of viral escape through fitness costs, are determinants of this protection.9 The requirement of clustered escape mutations in that epitope results in a complicated pathway of escape to evade immune response. During acute infection, this difficulty in rapidly achieving effective escape may provide the epitope-specific CD8+ T-cell response sufficient time to clear the virus before effective escape mutations can develop. In the present study, we reveal an equally complicated pathway of viral escape in a second neighboring HLA-B27-restricted CD8+ T-cell epitope that requires the evolution of a network of two compensatory mutations, in at least a subset of patients. This requirement for complex escape pathways for two epitopes restricted by HLA-B27 would hamper the development of effective escape mutations to both of these responses and thus prevent a major determinant of persistent infection.20-22

Of note, mutations that compensate for viral fitness costs associated with viral escape mutations have not been described previously for HCV, but have now been observed in the HLA-B27 restricted epitope described here, in an HLA-B57-restricted epitope (Oniangue-Ndza et al., personal communication), as well as in an HLA-A3-restricted epitope.23 Because all three HLA alleles have been described to be associated with spontaneous HCV clearance,2, 24 the requirement for compensatory mutations resulting from the high viral fitness costs of specific escape mutations may be a common characteristic of protective HLA alleles in HCV infection. As noted earlier, a similar requirement for compensatory mutations has been made for the immunodominant, HLA-B27-restricted KK10 epitope in HIV Gag,3, 4 as well as for the HLA-B57-restricted KF11 epitope in Gag,25 each of which are believed to contribute importantly to the immune control of HIV associated with each of these alleles.

In sum, our data suggest that CD8+ T-cell responses targeting the HLA-B27-restricted epitope, NS5B2936-2944 (GRAAICGKY), are uniquely capable of driving the selection of rare, highly deleterious mutations, in some HLA-B27-positive patients. These escape mutations require the evolution of a network of compensatory mutations located outside the epitope to restore viral replication. This complicated pathway of viral escape may significantly contribute to viral control in the acute phase of infection and thus contribute to protection by HLA-B27 observed in HCV infection.

Acknowledgements

The authors thank all participating patients, clinical collaborators, and collaborators at the Broad Institute of MIT and Harvard who conducted the full-genome virus sequencing.10 In addition, we thank Ralf Bartenschlager (University of Heidelberg) for providing plasmid I341PILuc_NS3_3ET as well as Huh7-Lunet cells and Charles Rice (Rockefeller University) for providing plasmid H77 (H/FL).

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