No patient recognized a KF9 variant without recognition of the KF9 wild-type peptide. Roughly 90% (28/31) of the patients with KF9-specific immune responses also showed recognition of peptides comprising the M46I mutation (KF9-M2I, major drug resistance mutation for IDV, examples shown in Figs. 2A-C, patients 2, 4, and 5). In 12 of these 28 patients, recognition of KF9-M2I was attenuated in comparison with the recognition of the wild-type peptide (example shown in Fig. 2A patient 4). In 3 patients, the M46I mutation abrogated recognition completely (example shown in Fig. 2D, patient 6).
In contrast, peptides containing the M46L mutation (KF9-M2L) showed a good recognition in all patients tested so far (n = 13) (examples shown in Figs. 2A, C, D). In 61% of the patients recognizing KF9, recognition of the I47A (KF9-I3A) mutation was abrogated (examples shown in Figs. 2C, D: patients 2 and 6) or strongly attenuated in comparison with the wild-type KF9 peptide (examples shown in Figs. 2A, B, patients 4 and 5). In contrast, peptides containing the I47V mutation (KF9-I3V) were recognized in all tested samples (n = 26). In most of these patients (n = 22), recognition of peptides comprising the I47V mutation was comparable to the recognition of the wild-type peptide (examples shown in Figs. 2A, B). Only in 4 patients, recognition of the I47V mutation was reduced in comparison with the wild-type peptide (examples shown in Figs. 2C, D). The I50V mutation (KF9-I6V) was recognized in all but 2 samples (19/21), but in most tested samples, the functional avidity in peptide titration assays was reduced in comparison with the wild-type peptide (examples shown in Figs. 2A-D). Figure 2 shows the cross-recognition of variant peptides by KF9-stimulated CTL lines. Additional peptide titration assays were conducted using CTL lines generated by stimulation with peptides comprising the M46I, M46L, I47A, I47V, or I50V mutations. The obtained CTL lines showed similar recognition patterns in peptide titration assays as the KF9 stimulated CTL lines (data not shown).
The recognition pattern of the KF9 epitope was not influenced by the therapeutic regimen as each group included patients on PI and patients on PI-sparing therapies (data not shown). Recognition of KF9 and its variants by freshly isolated PBMC was analyzed by ELISPOT in 2 untreated PI-naive patients and in 13 patients on ART. Both untreated patients showed good recognition of KF9 (patient 2: 460 SFU per 1 × 106 cells; patient 5: 135 SFU per 1 × 106 cells) (Fig. 3). The analysis of the recognition of viral variants mirrored exactly the different recognition patterns, which were found in stimulated CTL lines (example given for patient 2 in Figs. 1B, 3B). Only in 2 of 13 patients on ART recognition of KF9 by freshly isolated PBMCs could be detected (70 SFU/1 × 106 cells and 150 SFU/1 × 106 cells, respectively).
Determination of HLA Restriction of Mutant KF9 Peptides
To rule out that mutations in the KF9 epitope did lead to the formation of a neoepitope binding to other HLA alleles, ELISPOT analyses were carried out using peptide-pulsed HLA-matched allogeneic B-LCL as target cells. These analyses verified that CTL lines stimulated with KF9-M2I, KF9-M2L, KF9-I3V, and KF9-I6V were also HLA-B*1501 restricted (data not shown). Restriction analysis for KF9-I3A could not be carried out due to the weak activity of cells stimulated with KF9-I3A.
Sequence Analysis of the PR
HIV-1 PR sequence analyses were carried out in 47 HLA-B*1501-positive patients and in 255 HLA-B*1501-negative patients. Our analysis showed that the frequency of patients with at least 1 of the potential CTL escape mutations (M46I, I47A, and G48V) in the KF9 epitope was very similar in HLA-B*1501-negative patients (19.6%) and HLA-B*1501-positive patients (19.1%); this was also true if each mutation was analyzed individually (see Table 2). As the HLA-A2 restricted epitope KI10 overlaps with the KF9 epitope, we performed an additional analysis, taking HLA-A2 into account. However, there was no evidence for selection of the CTL escape mutations by HLA-B*1501 and/or HLA-A2 (Table 2). Subanalysis of different patients groups, therapy-naive patients (n = 109; 9 patients HLA-B*1501 positive and 100 patients HLA-B*1501 negative), patients on therapies excluding PIs (n = 16), and patients on PI-containing therapies (n = 177; 34 patients HLA-B*1501 positive and 143 patients HLA-B*1501 negative) clearly showed that the potential CTL escape mutations are only associated with the usage of the respective PIs. Mutations in the KF9 epitope were only found in patients with current or previous PI-containing regimens. None of the therapy-naive patients showed amino acid substitutions in the KF9 epitope.
Considering these results, further analyses were restricted to the patients who had received PI treatment in the past. Protease sequence analysis in these patients revealed a slightly higher percentage of patients with at least 1 of the potential CTL escape mutations (M46I, I47A, and G48V) in HLA-B*1501-negative patients (35%; 50/143) than in the HLA-B*1501-positive patients (26.5%; 9/34). These differences were not statistically significant (Fisher exact test, P = 0.4). Numbers and percentages for the individual mutations are given in Table 2.
To investigate whether HLA-B*1501-restricted immune responses may influence viral replication in the context of KF9 resistance mutations, we analyzed VL at the time point of resistance testing in all 71 patients with mutations (M46I/L, I47A/V, G48V, I50V) in the KF9 epitope. All these 71 patients had a detectable VL ranging from 40 copies per milliliter to 1,120,000 copies per milliliter. Seventy of these patients were on a failing ART, and 1 patient (HLA-B*1501 positive; VL of 2562 copies/mL) was on a treatment interruption. VLs were significantly lower in the 11 HLA-*B1501-positive patients (median VL, 1015 copies/mL; range, 40-131,000 copies/mL) than in the 60 HLA-B*1501-negative patients (median VL, 9118 copies/mL; range, 378-1,120,000 copies/mL; Mann-Whitney U test, 2-tailed, P = 0.012). Lower VLs in HLA-B*1501-positive patients in comparison with the HLA-B*1501-negative patients could not be explained by the patterns of mutations within KF9 (data not shown).
Drug targets like the HIV-1 PR are exposed to both pharmacological selection pressure and immune selection exerted by CTL. In the past, several studies have provided evidence that selection pressure by CTL can interfere with the development of drug resistance mutations.12,14,18,19,20,31 To assess the interaction between pharmacological and immunological selection pressure, we performed a detailed study of CTL recognition of the HLA-B*1501-restricted KF9 epitope,18 which comprises several major PI resistance mutations (M46I/L, I47A/V, G48V, and I50V).5 The epitope was recognized by 80% of the HLA-B*1501-positive patients, and peptide stimulation induced a vigorous outgrowth of KF9-specific CTL lines in most of the patients, indicating that KF9 is a dominant CTL epitope in HLA-B*1501-positive patients. ELISPOT analyses using freshly isolated PBMCs demonstrated good recognition of KF9 and variant peptides in untreated patients, whereas patients on ART showed no or only weak recognition of the epitope. This is in accordance with earlier studies showing that the frequency of CTL decays after initiation of ART.27,28,29
Our analyses show that CTL of HLA-B*1501-positive patients can target important drug resistance mutations in the KF9 epitope, such as the M46I, M46L, I47A, I47V, and I50V, although there were strong differences between individual patients regarding the recognition of M46I and I47A. Interestingly, all patients failed to recognize the G48V mutation, which is a major mutation for SQV resistance. Despite the potential of the M46I, I47A, and G48V mutations for immune escape, we could not find evidence for CTL-mediated selection of these mutations in our cohort. We assume that the lack of immune selection of G48V in our cohort could be explained by the strong negative impact of this mutation on viral infectivity that has been described by Mammano et al.32
The second important mutation negatively affecting recognition by KF9-specific CTL was the LPV resistance associated I47A mutation. In contrast to the I47A mutation, I47V, also a major resistance mutation for LPV, was well recognized by KF9-specific CTL. Both mutations were only infrequently found in our cohort (I47V n = 9; I47A n = 4). In the HLA-B*1501-positive cohort, both mutations were only found in a single patient. This is arguing against a strong influence of HLA-B*1501 on the selection of the I47A mutation. The I47V mutation is induced by a single nucleotide A to G substitution, whereas the I47A mutation requires an additional U to C substitution. Thus, the I47V mutation is a necessary intermediate mutation in the virus for the development of the I47A mutation. As the I47V mutation is well recognized by KF9-specific CTL, the 2 nucleotide exchanges needed for the potential CTL escape mutation I47A could be a significant obstacle for a CTL-mediated immune selection of I47A.
The M46I mutation (major mutation for IDV; minor mutation for ATV, FPV, LPV, NFV), located at the P2 anchor position of the epitope, showed an unexpected recognition pattern. M46I abrogated (n = 3) or attenuated (n = 12) CTL recognition in about half of the HLA-B*1501-positive patients with recognition of the KF9 peptide (n = 31), but it was equally well recognized as KF9 in the other half of patients (n = 16). The decreased or abolished recognition of the M46I mutation in a subgroup of patients only does not seem to be the consequence of a lower peptide-binding affinity of the mutated peptide, given that in the majority of patients, peptide titration curves showed a similar recognition of the M46I-containing peptide and the wild-type KF9. Both a methionine and an isoleucine can serve as anchor for the HLA-B*1501 allele, and as all the tested patients carried the HLA-B*1501 allele, HLA-B15 polymorphism can be ruled out as a cause of weaker recognition of the M46I mutation. In addition, HLA restriction analyses of the mutated peptides demonstrated that the M46I-comprising peptide was actually presented by HLA-B*1501, thus excluding cross-presentation of the M46I mutation by other HLA alleles as the cause for the observed differences in the recognition of M46I. Therefore, we hypothesize that despite of its location in the P2-anchor groove of the HLA-B*1501 allele, the M46I mutation could induce changes in the conformation of the HLA-peptide complex, which may affect T-cell receptor (TCR) recognition depending on the patients' individual TCR. In contrast to the M46I mutation, M46L, which is a major mutation for IDV and a minor mutation for ATV, FPV, LPV, NFV, and TPV, was well recognized in all tested samples. Peptide titration curves showed a similar recognition of peptides including M46I or M46L. This could be a further hint that recognition of peptides with these mutations is dependent on the individual TCR.
The I50V is a major resistance mutation for FPV and darunavir and a minor resistance mutation for ATV, TPV, and NFV. Peptides comprising this mutation were well recognized by the majority of patients, although the I50V-containing peptide exhibited a lower peptide-sensitizing capacity than the wild-type peptide.
The HLA-B*1501-restricted epitope KF9 is located within the known HLA-A2-restricted CTL epitope KMIGGIGGFI.30 In contrast to the strong response against KF9 in HLA-B*1501-positive patients, KI10 was recognized only by 40% of HLA-A2-positive patients, and the KI10 peptide usually possessed only a weak stimulatory capacity for the induction of KI10-specific CTL in PBMCs from HLA-A2-positive donors in comparison with the good stimulatory capacity of the KF9-peptide in PBMCs from HLA-B*1501-positive patients. This demonstrates that there are strong functional differences between different HLA alleles regarding the capacity to mount CTL responses against epitopes that are located in the same sequence area. The observed stronger stimulatory capacity of the HLA-B*1501-restricted KF9 peptide in comparison with the HLA-A2-restricted KI10 peptide is in accordance with an earlier study from Kiepiela et al8 reporting on a substantially greater induction of CTL by HLA-B alleles than by HLA-A alleles.
Despite strong CTL activity against the KF9 epitope, sequencing of HIV-1 PR genes failed to provide clear evidence for CTL-mediated immune selection within the KF9 epitope in HLA-B*1501-positive patients in our cohort. None of the mutations, which acted as CTL escape mutations in the ELISPOT analysis (M46I, I47A, G48V), occurred at higher frequency in HLA-B*1501-positive patients than in HLA-B*1501-negative patients. Although the M46I, I47A, and G48V mutations can strongly impair CTL recognition, the KF9 is a highly conserved CTL epitope and especially mutations at position 47 and 48 occur only infrequently. We assume that the rarity of mutations is due to strong functional constraints for mutations within the KF9 epitope as it is located in a region of the HIV-1 PR with important functional activity. The amino acids comprising KF9 are all located within the flap region of the PR. Several studies have shown that the flexible flaps participate in the binding of the substrate and therefore are strongly conserved. Amino acids 45, 46, and 47 are located in the flap tips of the PR, and amino acids 48 and 50 are substrate cleft residues.33-38 There is a high genetic barrier for most of the major drug resistance mutations within the KF9 epitope. In the past, several studies have indicated that major drug resistance mutations in functionally important regions of the PR require compensatory mutations in other regions of the PR or of Gag, which compensate for the functional impairments induced by the major resistance mutations.39-42
All patients in our cohort with mutations in the KF9 epitope were on antiretroviral regimens selecting for the respective mutations, indicating that PIs exert greater selection pressure than HLA-B*1501-restricted CTL responses. Nevertheless, the frequency of resistance mutations within KF9 was low even in viremic patients on PI treatment.
Together with several other rare HLA-B15 subtypes, HLA-B*1501 represents the serologically defined HLA-B62 group. In an earlier study by Hentges et al,43 HLA-B62 was associated with prolonged clinically asymptomatic HIV-1 infection. So far, it is unknown, whether and to what extent CTL recognition of the KF9 epitope has a beneficial effect on the course of HIV-1 infection in HLA-B*1501-positive patients. Although there are a number of HLA-B*1501-restricted CTL epitopes in various parts of the HIV-1 genome, we conclude from our study that the KF9 epitope is an important epitope for HLA-B*1501-positive patients that should be considered in the design of preventive and therapeutic HIV-1 vaccines. From our sequence analysis, we could find no evidence that HLA-B*1501 contributes to the development of drug resistance mutations in the KF9 epitope. Rather, the frequency of mutations in KF9 was slightly higher in HLA-B*1501-negative patients (35%) than in HLA-B*1501-positive patients (26.5%) on PI therapy (not statistically significant). Therefore, we hypothesize that the cross-recognition of drug resistance mutations by KF9-specific CTL could inhibit the emergence of drug resistance mutations in HLA-B*1501-positive patients on PI therapy. This would be in line with our observation that HLA-B*1501 was associated with lower VLs in patients failing PI therapy with mutations in the KF9 epitope.
Prospective studies in larger cohorts of patients on PI therapy are needed to assess the role of HLA-B*1501-restricted CTL responses for the success or failure of PI therapy.
The authors thank all patients participating in this study. They also thank the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, for providing the HIV-1 Consensus B Pol (15-mer) peptides, complete set.
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Keywords:© 2011 Lippincott Williams & Wilkins, Inc.
CTL; drug resistance; protease inhibitor