Cellular Response to Infection

Accumulation of Pol Mutations Selected by HLA-B*52:01-C*12:02 Protective Haplotype-Restricted Cytotoxic T Lymphocytes Causes Low Plasma Viral Load Due to Low Viral Fitness of Mutant Viruses

Hayato Murakoshi, Madoka Koyanagi, Takayuki Chikata, Mohammad Arif Rahman, Nozomi Kuse, Keiko Sakai, Hiroyuki Gatanaga, Shinichi Oka, Masafumi Takiguchi
Guido Silvestri, Editor
DOI: 10.1128/JVI.02082-16

ABSTRACT

HLA-B*52:01-C*12:02, which is the most abundant haplotype in Japan, has a protective effect on disease progression in HIV-1-infected Japanese individuals, whereas HLA-B*57 and -B*27 protective alleles are very rare in Japan. A previous study on HLA-associated polymorphisms demonstrated that the number of HLA-B*52:01-associated mutations at four Pol positions was inversely correlated with plasma viral load (pVL) in HLA-B*52:01-negative individuals, suggesting that the transmission of HIV-1 with these mutations could modulate the pVL in the population. However, it remains unknown whether these mutations were selected by HLA-B*52:01-restricted CTLs and also reduced viral fitness. In this study, we identified two HLA-B*52:01-restricted and one HLA-C*12:02-restricted novel cytotoxic T-lymphocyte (CTL) epitopes in Pol. Analysis using CTLs specific for these three epitopes demonstrated that these CTLs failed to recognize mutant epitopes or more weakly recognized cells infected with mutant viruses than wild-type virus, supporting the idea that these mutations were selected by the HLA-B*52:01- or HLA-C*12:02-restricted T cells. We further showed that these mutations reduced viral fitness, although the effect of each mutation was weak. The present study demonstrated that the accumulation of these Pol mutations selected by HLA-B*52:01- or HLA-C*12:02-restricted CTLs impaired viral replication capacity and thus reduced the pVL. The fitness cost imposed by the mutations partially accounted for the effect of the HLA-B*52:01-C*12:02 haplotype on clinical outcome, together with the effect of HLA-B*52:01-restricted CTLs on viral replication, which had been previously demonstrated.

IMPORTANCE Numerous population-based studies identified HLA-associated HIV-1 mutations to predict HIV-1 escape mutations from cytotoxic T lymphocytes (CTLs). However, the majority of these HLA-associated mutations have not been identified as CTL escape mutations. Our previous population-based study showed that five HLA-B*52:01-associated mutations at four Pol positions were inversely correlated with the plasma viral load in HLA-B*52:01-negative Japanese individuals. In the present study, we demonstrated that these mutations were indeed selected by CTLs specific for novel B*52:01- and C*12:02-restricted epitopes and that the accumulation of these mutations reduced the viral fitness in vitro. This study elucidated the mechanism by which the accumulation of these CTL escape mutations contributed to the protective effect of the HLA-B*52:01-HLA-C*12:02 haplotype on disease progression in HIV-1-infected Japanese individuals.

INTRODUCTION

Previous studies on HLA allele associations with progression to AIDS revealed that the presence of a particular HLA class I allele or haplotype is associated with the progression (1–6). HLA-B had the strongest effect on clinical outcome in 3 HLA class I loci (7). HLA-B*57 and -B*27 are well-known protective alleles that are associated with the control of HIV-1 or slow progression to the disease in Caucasians and Africans (8–12). The HLA-B*57-restricted responses toward the Gag epitopes TSTLQEQIAW (TW10; Gag 240 to 249) and KAFSPEVIPMF (KF11; Gag 162 to 172) have been linked to a substantially lower plasma viral load (pVL) in HLA-B*57+ subjects who respond to these epitopes than in other patients (13, 14). Similarly, the HLA-B*27-restricted response to the Gag epitope KRWIILGLNK (KK10; Gag 263 to 272) is associated with lower pVL in HLA-B*27-positive subjects than in other patients (15–17). These findings suggest that the CTLs restricted by these protective alleles strongly suppress HIV-1 in vivo.

In contrast, studies on HLA class I alleles or haplotypes in Asian populations are limited. A previous study on Japanese individuals, in whom HLA-B*57 and HLA-B*27 are absent and very rare, respectively, demonstrated that the HLA-B*52:01-C*12:02 haplotype and HLA-B*67:01 allele are significantly associated with a low pVL and high CD4 count (18). These results suggest that immune responses restricted by these HLAs have the ability to suppress HIV-1 replication. A recent study indeed demonstrated that HLA-B*52:01- and HLA-B*67:01-restricted T cells specific for five and three epitopes, respectively, are significantly associated with a low pVL and high CD4 count (19). Thus, these alleles have important roles in viral suppression via CTLs.

HIV-1-specific CTLs select for HIV-1 escape mutations (20–22). Since the specific CTLs fail to suppress the replication of escape mutant viruses, the patients have a higher pVL after the emergence of the mutant viruses (16, 17, 23). Some escape mutants have a low replication capacity (RC) (24–27), resulting in a low pVL in donors transmitting the mutant viruses. The most common escape mutation, Arg to Lys (R 264 K) at P2 in the HLA-B*27 KK10 epitope, causes not only the failure of KK10-specific T-cell recognition but also a reduction in RC (24). Consequently, the virus cannot accommodate this escape mutation without the compensatory S173A mutation (24). In the case of the HLA-B*57 p24 Gag epitopes, accumulating mutations in three of the epitopes contribute to the progressive loss of RC (25).

Numerous population-based studies on HLA-associated polymorphisms (HLA-APs) have been conducted in European, North American, Australian, African, and Asian cohorts and demonstrated HLA-associated mutations (28–34). Most of these mutations have not been confirmed as CTL escape mutations. Some HLA-associated mutations are significantly correlated with a low pVL in individuals who do not have the HLA allele identified in association with the mutations (32). These mutations may reduce the RC so that the individuals infected with the mutants have a lower pVL than do those infected with HIV-1 without the mutations, even in individuals without the particular HLA alleles.

A recent study on HLA-APs in the Japanese population showed that the breadth of HLA-B*52:01-associated Pol mutations was inversely correlated with the pVL in HLA-B*52:01− individuals (32), suggesting that the transmission of HIV-1 having these B*52:01-associated Pol mutations could modulate the pVL in this population. However, it is yet to be determined if they are selected by HLA-B*52:01-restricted CTLs and reduce RC. Therefore, in this present study we sought to investigate if these B*52:01-associated mutations could allow escape from specific CTLs and affect the RC. We first identified B*52:01-restricted CTL epitopes containing the positions where these B*52:01-associated mutations were found. We next evaluated the effects of these mutations on the recognition by specific CD8+ T cells to examine whether they were indeed escape mutations. Finally, we studied the effects of these mutations on the RC.

RESULTS

Identification of CTL epitopes including Pol positions at which HLA-B*52:01-associated mutations were found.We previously demonstrated HLA-B*52:01-associated mutations at four Pol positions, Pol 68, Pol 466, Pol 933, and Pol 935 (Table 1), and showed that the total numbers of the mutations are inversely correlated with pVL (32). However, there is no report of HLA-B*52:01-restricted CTL epitopes including these positions. We therefore sought to identify such CTL epitopes. We first attempted to identify a CTL epitope containing Pol 68 by using 11-mer Pol overlapping peptides generated in a previous study (19). Since HLA-B*52:01-binding peptides have the Q2 anchor residue (35), we selected two 11-mer overlapping peptides, Pol 11 to 30 (Pol11-30; ITLWQRPLVTI) and Pol11-31 (LWQRPLVTIKI) as candidate peptides. We analyzed T-cell responses by peripheral blood mononuclear cells (PBMCs) from a chronically HIV-1-infected individual, KI-647 (A*11:01/24:02, B*52:01/67:01, C*07:02/12:02), who showed a positive response in the enzyme-linked immunosorbent spot (ELISPOT) assay to the Pol 3 and 4 peptide cocktails including Pol11-30 and Pol11-31 (data not shown). The responses to Pol11-30 and Pol11-31 peptides were detected, though the response to Pol11-30 was much stronger than that to Pol11-31 (Fig. 1A), suggesting that these peptides included T cell epitopes. To identify the HLA restriction of these responses, we stimulated PBMCs from KI-647 with Pol11-30 or Pol11-31 peptide and cultured them for 2 weeks. T-cell responses to Pol11-30 or Pol11-31 peptide were tested by performing the intracellular cytokine staining (ICS) assay using 721.221-B5201 and .221-C1202 cells as stimulators. The result showed that both responses were restricted by HLA-B*52:01 (Fig. 1B). Gln is the P2 anchor residue for HLA-B*52:01 binding peptides, and LWQRPLVTI is overlapped between Pol11-30 and Pol11-31 peptides, suggesting that WI8 (WQRPLVTI) is a strong candidate for the HLA-B*52:01-restricted epitope. Indeed, the HLA-B*52:01-restricted T cells recognized WI8 more strongly than Pol11-30 (Fig. 1C). We further investigated the frequency of responders to WI8 in 80 HLA-B*52:01+ HLA-C*12:02+ individuals by using the ELISPOT assay. Five responders were found among these individuals (Fig. 1D), confirming that WI8 was a CTL epitope. All these responders shared only HLA-B*52:01 and HLA-C*12:02, supporting that WI8-specifc T cells are restricted by HLA-B*52:01.

View this table:
TABLE 1

Four HLA-B*52:01-associated mutations

FIG 1
FIG 1

Identification of the HLA-B*52:01-restricted Pol WI8 epitope. (A) T-cell responses to two 11-mer overlapping peptides including the Pol68 position. The responses to the peptides at 1 μM were analyzed by using the ELISPOT assay. (B) HLA restriction of the T-cell responses to the 11-mer peptides. The responses of the bulk T cells stimulated with two 11-mer peptides to C1R-B5201, C1R-C1202, or C1R cells prepulsed with the peptides were analyzed by performing the ICS assay. (C) Identification of WI8 epitope-specific T-cell responses. The responses of bulk T cells stimulated with the 11-mer peptides to C1R-C1202 prepulsed with WI8 or Pol11-30 peptide were analyzed by using the ICS assay. (D) CTL responses to the WI8 epitope in HLA-B*52:01+ HLA-C*12:02+ individuals. The responses to the WI8 peptide at a concentration of 100 nM were analyzed by using the ELISPOT assay. A specific response (>200 spots/1 million CD8+ T cells) was detected in 5 of 80 HLA-B*52:01+ HLA-C*12:02+ individuals. The HLA haplotypes of the five responders are as follows: KI-647, A*11:01/A*24:02, B*52:01/B*67:01, C*07:02/C*12:02; KI-698, A*02:01/A*24:02, B*51:01/B*52:01, C*12:02/C*15:02; KI-740, A*24:02/−, B*07:02/B*52:01, C*07:02/C*12:02; KI-894, A*24:02/A*26:01, B*40:02/B*52:01, C*03:04/C*12:02; KI-906, A*02:06/A*11:01, B*15:01/B*52:01, C*04:01/C*12:02.

We next sought to identify CTL epitopes including Pol933 and Pol935. The P2 anchor residue for HLA-B*52:01 binding peptides, Gln, was found at 4 positions near Pol933 and Pol935. Therefore, we analyzed T-cell responses to seven 11-mer overlapping peptides (Pol11-462 to Pol11-468) carrying Gln at one or more than one position (Fig. 2A). We used PBMCs from KI-108 (HLA-A*24:02/−, HLA-B*52:01/−, and HLA-C*12:02/−), who responded to the Pol-47 peptide cocktail including those seven 11-mer peptides (data not shown). After PBMCs from this individual had been stimulated with this peptide cocktail and cultured for 2 weeks, bulk-cultured cells (“bulk cells”) were tested with those 11-mer overlapping peptides by performing the ICS assay, with cells of an autologous Epstein-Barr virus (EBV)-transformed B-cell line used as a stimulator. The results showed that the bulk T cells responded to three peptides, Pol11-463, -464, and -465 (Fig. 2A). These T-cell responses were restricted by HLA-B*52:01 but not by HLA-C*12:02 or HLA-A*24:02 (Fig. 2B). To identify the HLA-B*52:01-restricted epitope in these peptides, we generated eight truncated peptides (LI8, EI9, KI10, TK10, KR10, QF10, RF9, and KN8) covering the three overlapping peptides and investigated the recognition of these truncated peptides by bulk-cultured T cells induced by Pol11-463 or -465 peptides. The bulk T cells effectively recognized KI10, EI9, and LI8 but not TK10 (Fig. 2C, left). Further analysis using titrated peptides revealed that the T cells recognized LI8 more strongly than KI10 and EI9 (Fig. 2C, right), indicating that LI8 (LQKQITKI) was the epitope for the T cells. Pol QF10 was also recognized by HLA-B*52:01-restricted bulk-cultured cells stimulated with Pol11-465 peptide (Fig. 2C, left). Since Pol QF10 does not include the LI8 sequence, we speculated that another HLA-B*52:01-restricted epitope resided in these peptides. However, the QF10 peptide was not recognized at 100 nM concentration by the bulk-cultured cells (Fig. 2D), suggesting that the T cells cross-recognized Pol QF10 only at higher concentrations of the peptide. We next analyzed the frequency of responders to LI8 in 80 HLA-B*52:01+ HLA-C*12:02+ individuals by using the ELISPOT assay. The results showed 23 responders among these individuals (Fig. 2E), indicating that LI8 was presented as an immunodominant epitope by HLA-B*52:01.

FIG 2
FIG 2

Identification of the HLA-B*52:01-restricted Pol LI8 epitope. (A) T-cell responses to 11-mer overlapping peptides including the Pol933 and/or 935 positions. The responses of bulk T cells stimulated with the 11-mer Pol-47 peptide cocktail to EBV-transformed cells established from KI-108 prepulsed with seven 11-mer overlapping peptides were analyzed by using the ICS assay. (B) HLA restriction of the responses to three 11-mer peptides. The responses of bulk T cells stimulated with an 11-mer peptide, Pol11-463, Pol-464, or Pol-465, to C1R-A2402, -B5201, or -C1202 cells prepulsed with the corresponding peptide were analyzed by performing the ICS assay. (C) Identification of the LI8 epitope. The responses of bulk T cells stimulated with Pol11-463 or Pol11-465 to C1R-B5201 prepulsed with each truncated peptide were analyzed by using the ICS assay. (D) Weak recognition of Pol QF10 by LI8-specific CD8+ T cells. The responses of T-cell clones specific for LI8 to C1R-B5201 prepulsed with QF10 or Pol11-465 peptide were analyzed by use of the ICS assay. (E) CTL responses to the LI8 epitope in HLA-B*52:01+ HLA-C*12:02+ individuals. The responses to the LI8 peptide at 100 nM were analyzed by performing the ELISPOT assay. A specific response (>200 spot/1 million CD8+ T cells) was detected in 23 of 80 HLA-B*52:01+ HLA-C*12:02+ individuals.

We finally sought to identify a CTL epitope containing Pol 466. We first tested the response of PBMCs from six chronically HIV-1-infected HLA-B*52:01+C*12:02+ individuals to five 11-mer overlapping peptides including Pol 466 (Pol11-229, -230, -231, -232, and -233) by using the ELISPOT assay. No individuals showed a clearly positive response (>200 spots), although KI-707 and KI-814 exhibited borderline responses (100 to 199 spots) to Pol11-233 (LD11, LKEPVHGVYYD) and Pol11-232 (EY11, EILKEPVHGVY), respectively (Fig. 3A). We therefore made 11-mer peptides between these two peptides overlapping by nine amino acids (Pol11-229/230, -230/231, -231/232, and -232/233) and then tested the possibility that one of these 11-mer peptides was a CTL epitope. Only one individual, KI-890, showed a positive response to Pol11-232/233 (IY11, ILKEPVHGVYY) in the ELISPOT assay (Fig. 3A). To confirm the responses to LD11, EY11, and IY11, we stimulated PBMCs from these individuals with the three peptides. After 2 weeks in culture, the responses of bulk cells to each peptide were tested. CD8+ T cells specific for these peptides were not detected in KI-707-derived bulk-cultured cells stimulated with LD11 or in KI-814-derived ones stimulated with EY11, whereas KI-890-derived bulk-cultured cells stimulated with IY11 recognized only IY11 (Fig. 3B). These results suggest that IY11 was the correct CTL epitope. To confirm the HLA restriction of the response to IY11, we tested the T-cell response to this peptide by using 721.221 cells expressing the same HLA carried by KI-890 (HLA-A*11:01/A*24:02, HLA-B*39:01/B*52:01, and HLA-C*07:02/C*12:02). IY11-specific T-cell response was detected only when 721.221-C1202 cells were used as stimulator cells (Fig. 3C), indicating that the T-cell response was restricted by HLA-C*12:02. We further analyzed the frequency of responders to IY11 in 80 HLA-B*52:01+ HLA-C*12:02+ individuals by using the ELISPOT assay. The results showed that 19 individuals responded to IY11 (Fig. 3D), indicating that IY11 was an immunodominant epitope restricted by HLA-C*12:02.

FIG 3
FIG 3

Identification of the HLA-C*12:02-restricted Pol IY11 epitope. (A) T-cell responses to 11-mer overlapping peptides including the Pol466 position. The T-cell responses in 6 HIV-1-infected HLA-B*52:01+ HLA-C*12:02+ individuals were analyzed by using the ELISPOT assay. The Pol466 position is highlighted in bold. The number indicates the number of spots/1 million CD8+ T cells. (B) Induction of IY11-specific CTLs. The responses were analyzed by using the ICS assay. The responses of bulk T cells stimulated with 1 of the 11-mer peptides, LD11, EY11, and IY11, to peptide-pulsed EBV-transformed cells established from individuals carrying HLA-B*52:01 and HLA-C*12:02 were analyzed by performing the ICS assay. (C) HLA restriction of the responses to the Pol IY11 epitope. The responses of bulk T cells stimulated with the IY11 epitope peptide to IY11-peptide-pulsed 721.221 cells expressing one of the HLA alleles carried by KI-890 (HLA-A*11:01/A*24:02, HLA-B*39:01/B*52:01, and HLA-C*07:02/C*12:02) were analyzed by using the ICS assay. (D) CTL responses to the IY11 epitope in HLA-B*52:01+ HLA-C*12:02+ individuals. The responses to the IY11 peptide at 100 nM were analyzed by performing the ELISPOT assay. The specific response (>200 spots/million CD8+ T cells) was detected in 19 of 80 HLA-B*52:01+ HLA-C*12:02+ individuals.

Effect of HLA-B*52:01-associated mutations on the recognition by HLA-B*52:01- or HLA-C*12:02-restricted CTLs.To examine if these HLA-B*52:01-associated mutations were due to escape from two HLA-B*52:01-restricted CTLs or one HLA-C*12:02-restricted CTL, we investigated the recognition of the five mutant peptides by these CTLs. We first investigated the recognition of two mutant peptides, WI8-7I and WI8-7A, by WI8-specific HLA-B*52:01-restricted CTLs. The WI8-specific CTLs completely failed to recognize either peptide (Fig. 4A), indicating that 7I and 7A were escape mutations from the WI8-specific CTLs. These findings suggest that the 7I and 7A viruses were selected by WI8-specific CTLs.

FIG 4
FIG 4

Effects of four mutations on the recognition by specific CTLs. (A) Recognition of two mutant peptides by WI8-specific CD8+ T cells. The responses of bulk T cells stimulated with the WI8 epitope peptide to 721.221-B5201 cells prepulsed with the WI8, WI8-7A (7A), or WI8-7I (7I) peptide were analyzed by using the ICS assay. (B) Recognition of mutant peptides (left) and mutant virus-infected cells (right) by LI8-specific CD8+ T cells. The responses of LI8-specific CTL clones to 721.221-B5201 cells prepulsed with the LI8, LI8-6S (6S), or LI8-8L (8L) peptide or to those infected with NL4-3, NL4-3T933S (T933S), or NL4-3I935L (I935L) were analyzed by use of the ICS assay. The frequencies of 721.221-B5201 cells infected with NL4-3 (WT), T933S, and I935L were 37.7%, 32.4%, and 35.6%, respectively (infection rates: WT, 0.377; T933S, 0.324; I935L, 0.356). Absolute percentages of IFN-γ-producing cells among CD8+ T cells were as follows; WT, 28.2%; T933S, 18.2%; I935L, 0.44%. To normalize the response of CD8+ T cells to HIV-1-infected cells, relative percentages of IFN-γ-producing cells among CD8+ T cells were calculated as follows: absolute % of IFN-γ-producing cells among CD8+ T cells/(infection rate with WT or MT viruses/infection rate with WT virus), where MT is mutant. The differences in the T-cell responses between WT and the mutant were statistically analyzed by performing the unpaired t test. (C) Recognition of mutant peptides (left) and mutant virus-infected cells (right) by IY11-specific CD8+ T cells. The responses of bulk T cells stimulated with the IY11 peptide to 721.221-C1202 cells prepulsed with the IY11 or IY11-3R peptides and to those infected with NL4-3 (WT) or NL4-3K466R (K466R) were analyzed by using the ICS assay. The frequencies of 721.221-C1202 cells infected with WT and K466R are 10.2% and 15.0%, respectively (infection rates: WT, 0.102; K466R, 0.150). Absolute percentages of IFN-γ-producing cells among CD8+ T cells were as follows: WT, 65.7%; K466R, 51.5%. To normalize the response of CD8+ T cells to HIV-1-infected cells, the relative percentage of IFN-γ-producing cells among CD8+ T cells in the right figure was calculated as described for panel B. The differences in the T-cell responses between the WT and the mutant were statistically analyzed by performing the unpaired t test.

We next analyzed the recognition of LI8-6S and LI8-8L mutant peptides by LI8-specific HLA-B*52:01-restricted CTLs. The LI8-specific CTLs recognized these mutant peptides more weakly than the LI8 peptide (Fig. 4B, left), suggesting that these mutations may have affected the CTL recognition of target cells infected with the mutant viruses. We therefore generated the mutant viruses (NL-933S and NL-935L) and tested the CTL recognition of target cells infected with these mutant viruses. The CTLs failed to recognize target cells infected with the NL-935L virus, whereas they showed a significantly reduced recognition of target cells infected with NL-933S virus compared with those infected with NL4-3 wild-type virus (Fig. 4B, right). These results suggest that the 6S and 8L viruses were selected by LI8-specific CTLs.

We finally analyzed the recognition of the IY11-3R mutant peptide by IY11-specific HLA-C*12:02-restricted CTLs. The IY11-specific CTLs effectively recognized both IY11 and IY11-3R peptides, but significantly less so the IY11-3R peptide than the IY11 peptide at the concentrations of 0.01 and 0.03 nM (Fig. 4C, left), suggesting that this mutation may have affected the recognition by IY11-specific T cells of target cells infected with the mutant virus. To clarify the effect of the mutation on T-cell recognition of HIV-1-infected cells, we generated the K466R mutant virus and tested the recognition by CTLs of target cells infected with this mutant virus. The IY11-specific CTLs recognized target cells infected with the wild-type (WT) virus more than those infected with the K466R virus (Fig. 4C, right). These results suggest that the 3R mutation was selected by IY11-specific T cells.

Replication capacity of HIV-1 carrying five Pol mutations.We investigated whether these mutations indeed reduced the replication capacity. First, we generated five single mutants of NL4-3 (NL4-3-T68A, NL4-3-T68I, NL4-3-K466R, NL4-3-T933S, and NL4-3-I935L) and then compared the RC of these mutant viruses with that of the wild-type virus by using a competitive replication assay. These mutants showed a weaker RC than the WT NL4-3 (Fig. 5A). A previous study showed an inverse correlation between the number of HLA-HLA-B*52:01-associated Pol mutations and pVL (32), suggesting that the accumulation of mutations at these four positions affected fitness in vivo. Therefore, we next generated two mutant viruses containing four mutations (NL4-3-T68A-K446R-T993S-I935L and NL4-3-T68I-K446R-T993S-I935L) and compared the RC of these mutant viruses with that of single mutant viruses. The results showed that the RC of these multiple mutant viruses was much lower than that of the single mutant viruses (Fig. 5B), indicating that the combination of these four mutations had a great effect on RC.

FIG 5
FIG 5

Competitive viral fitness analysis. Viral fitness levels of mutant viruses carrying each mutation (A) and four mutations (B) were compared with that of the wild-type virus. H9 cells were exposed for 6 h to the mixtures of paired virus stocks and then cultured. Every 7 days, the viruses in the culture supernatant were transmitted to new uninfected H9 cells. The supernatants collected on day 2 (passage 0) and at the end of each passage were subjected to direct DNA sequencing of HIV-1. Changes in the viral population were determined by the relative peak height in sequencing electrograms. The data are presented as means and SD (n = 3).

DISCUSSION

Although several population-based studies identified HLA-associated mutations in European, North American, Australian, African, and Asian cohorts (28–34), most of the mutations in these studies were not confirmed as escape mutations from CTLs. To identify CTL escape mutations among HLA-associated mutations experimentally, it was necessary first to identify CTL epitopes covering HLA-associated mutations and then to investigate whether the mutations reduced specific T-cell recognition. Although several hundreds of HLA-associated mutations have been identified (28–34), the majority are not located in known CTL epitopes. The identification of novel CTL epitopes is necessary to determine CTL escape mutations in most cases. That is, the identification of CTL escape mutations among HLA-associated mutations depends on the discovery of corresponding CTL epitopes. In the present study, we focused on five HLA-B*52:01-associated Pol mutations found at four positions. Since HLA-B*52:01-restricted epitopes including these positions have not been reported, we first sought to identify HLA-B*52:01-restricted epitopes containing these HLA-B*52:01-associated mutations. We employed 11-mer peptides overlapping by nine amino acids, which covered the four mutant positions, and successfully identified two HLA-B*52:01-restricted epitopes and one HLA-C*12:02-restricted one covering three and one HLA-associated mutations, respectively. IY11 was restricted by HLA-C*12:02 but not by HLA-B*52:01. Since HLA-C*12:02 and HLA-B*52:01 have very high linkage disequilibrium in Japan, it is likely that HLA-B*52:01-associated mutations can be escape mutations from HLA-C*12:02-restricted CTLs. We previously showed that IY10 (ILKEPVHGVY) is an HLA-C*12:02-restricted epitope (36). However, bulk T cells induced by IY11 recognized the IY11 peptide more than IY10 (Fig. 3B). In addition, a CTL clone previously established as an IY10-specific one recognized IY11 more than IY10 (data not shown). The analysis of the responses to IY10 in 80 HLA-B*52:01+ HLA-C*12:02+ individuals, conducted by using the ELISPOT assay, showed no responders to IY10 among these individuals (data not shown). These findings strongly suggest that IY11 is the CTL epitope but cannot exclude the possibility that IY10 is presented as a superimposed epitope within IY11 by HLA-C*12:02.

To determine whether the five mutations were selected by CTLs specific to the three epitopes identified in the present study, we investigated the effects of these mutations at four positions on the recognition by the three kinds of CTLs. Two mutations (7I and 7A) in the WI8 epitope affected the recognition by WI8-specific HLA-B*52:01-restricted CTLs. This result strongly suggests that these mutant viruses were selected by WI8-specific CTLs. On the other hand, the two mutations (6S and 8L) at two positions reduced the recognition of peptide-pulsed cells by LI8-specific HLA-B*52:01-restricted CTLs. Although the 6S mutation weakly reduced CTL recognition of the peptide-pulsed cells, it significantly reduced CTL recognition of target cells infected with HIV-1. Furthermore, the 8L mutation also affected CTL recognition of HIV-1-infected cells. These findings suggest that these two mutations were selected by LI8-specific HLA-B*5201-restricted CTLs. The 3R mutation also reduced the recognition of peptide-pulsed and HIV-1-infected cells by IY11-specific HLA-C*12:02-restricted CTLs. Our previous study demonstrated that TI8-specific HLA-B*51:01-restricted CTL clones can suppress both wild-type and 8V mutant viruses, although the ability of the clones to suppress wild-type virus is slightly stronger than that to suppress the 8V virus, and that these clones select for the 8V virus in the competitive viral-suppression assay (37). This finding indicates that CTLs can select for mutant viruses even when the ability of CTLs to suppress wild-type virus is slightly stronger than that to suppress mutant viruses. These findings taken together suggest that the five mutations were selected by two kinds of HLA-B*52:01-restricted CTLs and one kind of HLA-C*12:02-restricted CTLs.

We analyzed T cell responses to the three epitopes in 80 HLA-B*52:01+ C*12:02+ individuals (Table 2). WI8-specific CTLs were found in only 5 of 80 individuals, whereas LI8-specific and IY11-specific CTLs were detected in 23 and 19 of 80 individuals, respectively. Fifty percent of these individuals showed T cell responses to at least one of these epitopes. Since WI8-specific CTLs completely failed to recognize the two mutant epitopes, cells infected with the mutant virus cannot stimulate the CTLs. The mutations in this epitope were detected in 47 of 96 HLA-B*52:01+ C*12:02+ individuals (Table 1). These findings may explain the low frequency of HLA-B*52:01+ C*12:02+ individuals having WI8-specific CTLs. In contrast, LI8-specific and IY11-specific CTLs may survive after the emergence of these mutant viruses since these CTLs cross-recognized the mutant epitopes.

View this table:
TABLE 2

Frequency of responders to epitopesa

Our previous study showed that an inverse correlation of these mutations at four Pol positions with pVL remained strongly detectable in HLA-B*52:01-negative individuals but not in HLA-B*52:01-positive individuals (32), suggesting that HLA-B*52:01-restricted Pol mutations possess fitness costs that manifest themselves in terms of a lower pVL in HLA-B*52:01-negative individuals. This suggestion was confirmed by the present study, which showed that each mutation had a weak effect on RC but that the four mutations together exhibited a stronger effect than each separately. In contrast, no such pVL effects were detectable in B*52:01-positive individuals (32), suggesting that the fitness costs of these mutations were outweighed by the advantages conferred by HLA-B*52:01- and/or HLA-C*12:02-restricted immune responses. Overall, the high fitness cost of HIV-1 having these mutations, together with the effect of HLA-B*52:01- and/or HLA-C*12:02-restricted immune responses, contributed to the protective effect of the HLA-B*52:01-C*12:02 haplotype on the disease progression.

In the present study, we demonstrated that five HLA-B*52:01-associated mutations at four Pol positions were selected by CTLs specific for two HLA-B*52:01-restricted epitopes and one HLA-C*12:02-restricted novel epitope. Furthermore, the in vitro competitive replication assay showed that these mutations reduced the RC. Thus, we clearly showed the mechanism accounting for the previous finding that the number of HLA-B*52:01-associated Pol mutations is inversely correlated with pVL in HLA-B*52:01− individuals. It is known that HLA-B*52:01-C*12:02 has a protective effect on the disease progression in HIV-1-infected Japanese individuals. Our recent study showed that HLA-B*52:01-restricted CTLs recognizing the five epitopes effectively suppress HIV-1 replication in the Japanese population (19). We showed here an additional mechanism for the protective effect of HLA-B*52:01-C*12:02 on the clinical outcome. Both mechanisms contribute to the protective effect of this haplotype on disease progression in the population.

MATERIALS AND METHODS

Subjects.HLA-B*52:01- and HLA-C*12:02-positive Japanese individuals chronically infected with HIV-1 clade B were recruited from the National Center for Global Health and Medicine. Informed consent was obtained from all individuals in accordance with the Declaration of Helsinki. This study was approved by the ethics committees of the National Center for Global Health and Medicine and Kumamoto University.

Peptides.Peptides were synthesized by utilizing an automated multiple peptide synthesizer and purified by high-performance liquid chromatography (HPLC). Their purity was examined by HPLC and mass spectrometry. Peptides with more than 90% purity were used in the present study.

ELISPOT assay.PBMCs from HIV-1-infected individuals and peptides at a concentration of 1 μM were added to 96-well polyvinylidene plates (Millipore, Bedford, MA) that had been precoated with 5 μg/ml anti-gamma interferon (anti-IFN-γ) monoclonal antibody (MAb) 1-D1K (Mabtech, Stockholm, Sweden). The plates were then incubated for 16 h at 37°C and subsequently washed with phosphate-buffered saline (PBS) before the addition of biotinylated anti-IFN-γ MAb (Mabtech) at 1 μg/ml. After the plates had been incubated at room temperature for 90 min, they were washed with PBS and then incubated with streptavidin-conjugated alkaline phosphatase (Mabtech) for 60 min at room temperature. Following washes with PBS, individual cytokine-producing cells were visualized as dark spots after a 20-min reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium in the presence of an alkaline phosphatase-conjugated substrate (Bio-Rad, Richmond, CA, USA). The spots were counted with an Eliphoto-Counter (Minerva Teck, Tokyo, Japan). The number of spots was calculated per 106 CD8+ T cells by measuring the frequency of CD8+ T cells using flow cytometry. A mean + 3 standard deviations (SD) of the number of spots in samples from 10 HIV-1-infected antiretroviral therapy (ART)-naive HLA-B*52:01− C*12:02− individuals for these peptides was 142 spots/106 CD8+ T cells. Therefore, we defined >200 spots/106 CD8+ T cells as a positive response.

Cells.C1R cells expressing HLA-B*52:01 or HLA-C*12:02 and 721.221 cells expressing CD4 molecules as well as HLA-B*52:01 or HLA-C*12:02 were previously generated (36, 38, 39) and maintained in RPMI medium with 10% fetal calf serum (FCS) and 0.15 mg/ml hygromycin B.

Generation of epitope-specific CTL clones.Epitope-specific CTL clones were generated from epitope-specific bulk T cells by the limiting dilution method in 96-U plates, which contained 200 μl of cloning mixture (2 × 105 irradiated PBMCs from healthy donors, 3 × 104 irradiated C1R cells expressing each HLA molecule, and each epitope peptide at a concentration of 100 nM in RPMI 1640 containing 10% FCS, 200 U/ml recombinant interleukin-2 [rIL-2], and 2.5% phytohemagglutinin).

HIV-1 mutant clones.NL4-3 mutants (NL4-3PolT68A, T68I, K466R, T933S, I935L, T68A-K466R-T933S-I935L, or T68I-K466R-T933S-I935L) were generated by introducing the PolT68A, T68I, K466R, T933S, I935L, T68A-K466R-T933S-I935L, or T68I-K466R-T933S-I935L mutations, respectively, into NL4-3 by site-directed mutagenesis (Invitrogen).

Intracellular cytokine staining assay.The stimulator cells (C1R or 721.221 cell lines prepulsed with each peptide or 721.221 cells infected with each virus) and the effector cells (bulk-cultured cells or CTL clones) were added to a 96-well plate, and then the cells were incubated for 2 h at 37°C. Subsequently, brefeldin A (10 μg/ml) was added, and the cells were incubated further for 4 h. After having been stained with allophycocyanin (APC)-labeled anti-CD8 MAb (Dako, Glostrup, Denmark), the cells were fixed with 4% paraformaldehyde and incubated in permeabilization buffer (0.1% saponin and 5% FCS in PBS). Thereafter, the cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-IFN-γ MAb (BD Bioscience, CA). The percentage of IFN-γ-producing cells among the CD8+ T cell population was determined by flow cytometry.

Competitive HIV-1 replication assay.H9 cells (6 × 105) were exposed for 6 h to the mixtures of paired virus stocks (250 blue cell-forming units [BFU] in MAGIC-5 cells). Infected cells were washed twice with RPMI 1640 containing 10% FCS and then cultured as described previously (36, 40). Every 7 days, the viruses in the culture supernatant were transmitted to new uninfected H9 cells. The supernatants collected on day 2 (passage 0) and at the end of each passage were subjected to direct DNA sequencing of HIV-1. The change in the viral population was determined by the relative peak height in sequencing electrograms.

ACKNOWLEDGMENTS

We thank Sachiko Sakai for secretarial assistance.

This research was supported by a grant-in-aid (15fk0410019) for AIDS research from AMED and by a grant-in-aid (26293240, 15K19588) for scientific research from the Ministry of Education, Science, Sports, and Culture, Japan. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We declare that we have no financial conflicts of interest.

FOOTNOTES

    • Received 20 October 2016.
    • Accepted 21 November 2016.
    • Accepted manuscript posted online 30 November 2016.

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KEYWORDS

Genetic Fitness
HLA-B Antigens
Haplotypes
mutation
T-Lymphocytes, Cytotoxic
Viral Load
pol Gene Products, Human Immunodeficiency Virus
CTL
HIV-1
escape mutation

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