Pathogenesis and Immunity

Long-Term Control of HIV-1 in Hemophiliacs Carrying Slow-Progressing Allele HLA-B*5101

Yuka Kawashima, Nozomi Kuse, Hiroyuki Gatanaga, Takuya Naruto, Mamoru Fujiwara, Sachi Dohki, Tomohiro Akahoshi, Katsumi Maenaka, Philip Goulder, Shinichi Oka, Masafumi Takiguchi
DOI: 10.1128/JVI.00171-10

ABSTRACT

HLA-B*51 alleles are reported to be associated with slow disease progression to AIDS, but the mechanism underlying this association is still unclear. In the present study, we analyzed the effect of HLA-B*5101 on clinical outcome for Japanese hemophiliacs who had been infected with HIV-1 before 1985 and had been recruited in 1998 for this study. HLA-B*5101+ hemophiliacs exhibited significantly slow progression. The analysis of HLA-B*5101-restricted HIV-1-specific cytotoxic T-lymphocyte (CTL) responses to 4 HLA-B*-restricted epitopes in 10 antiretroviral-therapy (ART)-free HLA-B*5101+ hemophiliacs showed that the frequency of Pol283-8-specific CD8+ T cells was inversely correlated with the viral load, whereas the frequencies of CD8+ T cells specific for 3 other epitopes were positively correlated with the viral load. The HLA-B*5101+ hemophiliacs whose HIV-1 replication had been controlled for approximately 25 years had HIV-1 possessing the wild-type Pol283-8 sequence or the Pol283-8V mutant, which does not critically affect T-cell recognition, whereas other HLA-B*5101+ hemophiliacs had HIV-1 with escape mutations in this epitope. The results suggest that the control of HIV-1 over approximately 25 years in HLA-B*5101-positive hemophiliacs is associated with a Pol283-8-specific CD8+ T-cell response and that lack of control of HIV-1 is associated with the appearance of Pol283-8-specific escape mutants.

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells play a critical role in the control of HIV-1 infections (26, 5), but HIV-1 escape occurs during acute and chronic phases of an HIV-1 infection (6, 14). There are several mechanisms affording HIV-1 escape from the host immune system. They include the appearance of mutants that escape from HIV-1-specific cytotoxic T lymphocytes (CTLs) (6, 14) and neutralizing antibodies (27, 47, 48), impaired recognition of HIV-1-infected cells by HIV-1-specific CTLs due to Nef-mediated downregulation of HLA class I molecules (8, 42), and impaired function of HIV-1-specific T cells (3).

It is well known that long-term nonprogressors (LTNPs), who remain disease free and have very low or undetectable viral loads (VLs) in the absence of antiretroviral therapy (ART), exist as a very small population of HIV-1-infected individuals (7, 21, 38). A small minority of these LTNPs were infected by HIV-1 containing deletions in viral accessory molecules (10, 17, 24). HLA alleles such as HLA-B*57/5801, HLA-B*27, and HLA-B*51 are associated with slow progression to AIDS (19, 22, 37). Indeed, it is reported that many LTNPs carry these HLA alleles (31, 36). These findings imply that HIV-1-specific CTLs restricted by these alleles may play an important role in the control of HIV-1 replication in LTNPs. The mechanism of control of HIV-1 replication has been analyzed in LTNPs and slow progressors carrying HLA-B*57/5801, HLA-B*27, or HLA-B*13, and has been related to the Gag-specific CD8+ T-cell epitopes presented by these alleles (9, 11, 14, 16, 34). On the other hand, the mechanism underlying the association between HLA-B*5101 and slow progression remains unclear. To date, no study of the mechanism of control of HIV-1 in HLA-B*5101+ LTNPs has been reported.

Since the data indicate that HIV-1 replication can be controlled for more than 20 years in LTNP hemophiliacs, analysis of HIV-1-specific immune responses and HIV-1 in these patients is useful for investigating the immunological control of HIV-1. In Japan, HLA-B*57/58 and HLA-B*27 are very rare alleles (18). Therefore, it was speculated that only HLA-B*51 would play an important role in the control of HIV-1 replication in HIV-1-infected Japanese donors.

We showed previously that 2 Pol peptides and 1 Gag peptide were HLA-B*5101-restricted immunodominant CTL epitopes (45). Two Pol-specific CTLs are known to have strong abilities to suppress HIV-1 replication in vitro (43). Our recent study using 9 cohorts showed that of these T cells, Pol283-specific CTLs select mutations at position 8 (position 135 of reverse transcriptase [RT]) in the epitope (20). A Thr mutation at position 8 (8T) was found predominantly in HIV-1-infected HLA-B*5101+ donors, whereas the 8R, 8L, and 8V mutations were also found in these donors. The 8T, 8L, and 8R mutants had fitness similar to that of the wild-type virus, whereas the 8V mutation had a higher fitness cost than the others.

In the present study, we analyzed the effect of HLA-B*5101 on clinical outcome in Japanese hemophiliacs infected with HIV-1. In addition, we investigated the role of HLA-B*5101-restricted HIV-1-specific CTLs in vivo in HLA-B*5101+ LTNP and slow-progressing Japanese hemophiliacs who had not been treated with antiretroviral therapy for approximately 25 years. Our results revealed a role for Pol283-8-specific HLA-B*5101-restricted HIV-1-specific CTLs in the long-lasting (approximately 25 years) control of HIV-1 replication.

MATERIALS AND METHODS

Patients.One hundred eight Japanese hemophiliacs who had been infected with HIV-1 before 1985, mostly around 1983, were recruited for the present study, which was approved by the ethics committees of Kumamoto University and the National Center for Global Health and Medicine. Written informed consent was obtained from all subjects according to the Declaration of Helsinki. Patient HLA type was determined by standard sequence-based genotyping. For sequence analysis, blood specimens were collected in EDTA. Plasma and peripheral blood mononuclear cells (PBMCs) were separated from heparinized whole blood.

Cells.C1R and 721.221 cells expressing HLA-B*5101 (C1R-B*5101 and 721.221-B5101, respectively) were generated previously (15, 33, 44). All cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and 0.15 mg/ml hygromycin B.

HIV-1 clones.An infectious proviral clone of HIV-1, pNL-432, and its mutant, pNL-M20A (containing a substitution of Ala for Met at residue 20 of Nef), were reported previously (1). Pol283-8 and Pol743-9 mutant (Pol283-8L, -8T, -8V, and -8R; Pol743-1I, -5I, and -4I5I) viruses were generated based on pNL-432 by using the GeneTailor site-directed mutagenesis system (Invitrogen).

HLA class I tetramers.HLA class I-peptide tetrameric complexes (tetramers) were synthesized as described previously (2). Four HIV-1 specific epitopes (Pol283-8, Pol743-9, Gag327-8, and Rev71-11) (45) were used for the refolding of HLA-B*5101 molecules. Phycoerythrin (PE)-labeled streptavidin (Molecular Probes) was used for the generation of the tetramers.

Flow cytometric analysis using tetramers.PBMCs were incubated with the tetramers at 37°C for 30 min. The cells were subsequently washed twice with RPMI-10% newborn calf serum (NCS) and were then stained with an anti-CD8 monoclonal antibody (MAb). Next, they were incubated at 4°C for 30 min and were then washed twice with RPMI-10% NCS. The cells were finally resuspended in phosphate-buffered saline (PBS) containing 2% paraformaldehyde, and then the percentage of tetramer-positive cells among the CD8+ population was determined by using a FACSCalibur flow cytometer (BD Bioscience, San Jose, CA).

Generation of CTL clones.Pol283-8-specific CTL clones and Pol743-9-specific CTL clones were generated from HIV-1-specific bulk-cultured T cells by limiting dilution in U-bottom 96-well microtiter plates (Nunc, Roskilde, Denmark) containing 200 μl of cloning mixture (about 1 × 106 irradiated allogeneic PBMCs from healthy donors and 1 × 105 irradiated C1R-B*5101 cells prepulsed with the corresponding peptide at 1 μM in RPMI 1640 supplemented with 10% human plasma and 200 U/ml human recombinant interleukin-2 [rIL-2]) (43).

CTL assay for target cells infected with HIV-1.The cytotoxicity of CTL clones for 721.221-B5101 cells infected with HIV-1 (>30% p24 antigen [Ag]-positive cells) was determined by the standard 51Cr release assay as described previously (42). The infected cells were incubated with 150 μCi Na251CrO4 in saline for 60 min, and then the infected cells were washed three times with RPMI 1640 medium containing 10% NCS. Labeled target cells (2 × 103/well) were added to each well of a U-bottom 96-well microtiter plate (Nunc, Roskilde, Denmark) with effector cells at an effector-to-target cell (E:T) ratio of 2:1. The cells were then incubated for 6 h at 37°C. The supernatants were collected and analyzed with a gamma counter.

Assay for suppression of HIV-1 replication by HIV-1-specific CTLs.The ability of HIV-1-specific CTLs to suppress HIV-1 replication was examined as previously described (42). CD4+ T cells isolated from PBMCs were derived from an HIV-1-seronegative individual with HLA-B*5101. After the CD4+ T cells had been incubated with the desired HIV-1 clones for 4 h at 37°C, they were washed three times with R10 medium. The HIV-1-infected CD4+ T cells were then cocultured with HIV-1-specific CTL clones. From day 3 to day 7 postinfection, culture supernatants were collected, and the concentration of p24 Ag in the supernatants was measured by an enzyme-linked immunosorbent assay (ELISA) (HIV-1 p24 Ag ELISA kit; ZeptoMetrix).

Sequencing of proviral DNA or plasma RNA.Genomic DNA was extracted from PBMCs by using a QIAamp DNA blood minikit (Qiagen). Viral RNA was extracted from the plasma of HIV-1-infected individuals by using a QIAamp Mini Elute virus spin kit (Qiagen). cDNA was synthesized from the RNA with SuperScript II and random primers (Invitrogen). We amplified HIV RT and integrase sequences by nested PCR using RT-specific primers 5′-CCAAAAGTTAAGCAATGGCC-3′ and 5′-CCCATCCAAAGGAATGGAGG-3′ or 5′-CCTTGCCCCTGCTTCTGTAT-3′ for the first round of PCR and 5′-AGTTAGGAATACCACACCCC-3′ and 5′-GTAAATCCCCACCTCAACAG-3′ or 5′-AATCCCCACCTCAACAGAAG-3′ for the second round and integrase-specific primers 5′-ATCTAGCTTTGCAGGATTCGGG-3′ and 5′-CCTTAACCGTAGTACTGGTG-3′ or 5′-CCTGATCTCTTACCTGTCC-3′ for the first round of PCR and 5′-AAAGGTCTACCTGGCATGGG-3′ or 5′-TTGGAGAGCAATGGCTAGTG-3′ and 5′-AGTCTACTTGTCCATGCATGGC-3′ for the second round. PCR products were either sequenced directly or cloned by using a TOPO TA cloning kit (Invitrogen) and then sequenced. Sequencing was done with a BigDye Terminator cycle sequencing kit (version 1.1; Applied Biosystems), and sequences were analyzed by use of an ABI PRISM 310 genetic analyzer.

Cell surface staining and intracellular cytokine staining (ICC assay).PBMCs from HIV-1-infected individuals were stimulated with the desired peptide (1 μM) and cultured for 12 to 14 days. These cultured PBMCs were assessed for gamma interferon (IFN-γ)-producing activity as previously described (42). After C1R-B*5101 cells had been incubated for 60 min with epitope peptides (1 μM), they were washed twice with RPMI 1640 containing 10% FCS. These C1R cells and the cultured PBMCs were incubated at 37°C for 6 h at an effector-to-stimulator ratio of 2:1 or 4:1 after the addition of brefeldin A (10 μg/ml). Next, the cells were stained with an anti-CD8 MAb (Dako Corporation, Glostrup, Denmark), fixed with 4% paraformaldehyde at 4°C for 20 min, and then permeabilized at 4°C for 10 min with PBS supplemented with 0.1% saponin containing 20% NCS (permeabilizing buffer). The cells were resuspended in the permeabilizing buffer and were then stained with an anti-IFN-γ MAb (BD Bioscience Pharmingen, San Diego, CA). Finally, they were resuspended in PBS containing 2% paraformaldehyde, and then the percentage of CD8+ cells positive for intracellular IFN-γ was determined by using a FACSCalibur flow cytometer.

RESULTS

Association of HLA-B*5101 with long-term control of HIV-1 in HIV-1-infected Japanese hemophiliacs.We recruited 108 Japanese hemophiliacs who had been infected with HIV-1 before 1985. Eighteen of the patients had not been treated with any antiretroviral therapy (ART) and had CD4 counts of >350 (very-slow-progressor [VSP] group) by 1998, whereas the other 90 patients had been treated with ART and/or had a CD4 count of <350 (slow-progressor [SP] group). The frequency of HLA-B*5101 in the VSP group (9 of 18 donors [50.0%]) was higher than that in the SP group (15 of 90 donors [16.7%]), and the difference between these 2 groups was significant (P, 0.01). We analyzed the association of HLA class I alleles with disease progression during the years 1998 to 2007 in the VSP group. The 9 HLA-B*5101+ VSP hemophiliacs exhibited significantly slower progression of the disease over this period than the 9 HLA-B*5101− subjects (Fig. 1), and no other HLA-B alleles or HLA-A/DR alleles showed any significant influence on the progression of the disease in this group (not shown). One HLA-B*3501+ VSP hemophiliac was found in the HLA-B*5101+ group, but none were found in the HLA-B*5101− group, indicating that HLA-B*3501, which is associated with rapid progression to AIDS, did not affect the results for the 2 VSP groups. Other HLA-A/B/DR alleles were not associated with the HLA-B*5101+ or the HLA-B*5101− group (see Table S1 in the supplemental material). These results, taken together, show that the HLA-B*5101 allele was still associated with slow progression of the disease more than 20 years postinfection.

FIG. 1.
FIG. 1.

Association of HLA-B*5101 with slow progression to AIDS. Kaplan-Meier survival analysis was used to estimate the time to the first CD4 cell count (24-week time-weighted average levels of CD4 cells) of <350/μl3 for 9 HLA-B*5101-positive (solid line) and 9 HLA-B*5101-negative (dashed line) hemophiliacs who had not been treated with antiretroviral therapy (ART) and who had a CD4 count of >350/μl in 1998.

Control of HIV-1 replication by HLA-B*5101-restricted CD8+ T cells.A previous study demonstrated that 2 types of HLA-B*5101-restricted CTLs, Pol283-8 (TAFTIPSI)-specific and Pol743-9 (LPPVVAKEI)-specific CTLs, suppressed HIV-1 replication in vitro much more strongly than did other HLA-B*5101-restricted CTLs (43), suggesting that these CTLs may play a key role in the control of HIV-1 in the HLA-B*5101+ SP group. To investigate the control of HIV-1 by these CTLs, we selected 10 HLA-B*5101-positive donors (8 VSPs and 2 SPs) who had not been treated with ART by 1998 and whose PBMC samples were available for analysis of HLA-B*5101-restricted CTLs (see Fig. S1 and Table S2 in the supplemental material). Three of the 8 VSP patients had VLs below 1,000 copies at all time points tested and were classified as LTNPs. We found that only 3 of the 108 HIV-1-infected hemophiliacs (KI-021, KI-051, and KI-124) were LTNPs for approximately 25 years and that all 3 of these LTNPs carried HLA-B*5101. We generated 4 HLA-B*5101 tetramers carrying Pol283-8, Pol743-9, Gag327-9, or Rev71-11, and we used them to determine the frequencies of HIV-1-specific CD8+ T cells among PBMCs from these 3 LTNPs (Table 1 and Fig. 2). KI-021 had both Pol283-8- and Pol743-9-specific CD8+ T cells but neither Gag327-9- nor Rev71-11-specific CD8+ T cells during the years 1997 to 2005 (Fig. 2A). KI-051 also had both Pol283-8- and Pol743-9-specific CD8+ T cells, whereas this patient had no Rev71-11-specific CD8+ T cells and a low number of Gag327-9-specific CD8+ T cells during the years 1999 to 2005 (Fig. 2B). KI-124 had Pol283-8-, Pol743-9-, and Gag327-9-specific CD8+ T cells (Table 1). These results suggest that the 2 Pol-specific CD8+ T cells may play an important role in the control of HIV-1 in these LTNPs carrying HLA-B*5101.

FIG. 2.
FIG. 2.

Longitudinal analysis of HLA-B*5101-restricted CD8+ T cells and Pol283 epitope sequences in 3 slow-progressing hemophiliacs. Four types of HIV-1-specific CD8+ T cells were detected by use of specific tetramers. PBMCs from KI-021 (A), KI-051 (B), and KI-127 (C) were analyzed by using Pol743-9-specific and Pol283-8-specific tetramers. The percentage of tetramer-positive cells among the CD8+ T-cell population is given in the upper right quadrant of each histogram. The sequence of the Pol283-8 epitope from each patient is shown. The detection limit of pVL was 400 copies/ml until 2000 and 50 copies/ml after 2000.

View this table:
TABLE 1.

Numbers of 4 types of HLA-B*5101-restricted CD8+ T cells among HLA-B*5101+ HIV-1-infected hemophiliacs

Selection of escape mutations of the Pol283-8 epitope in very slow progressors.Of the 8 HLA-B*5101+ VSP hemophiliacs, KI-127 had Pol283-8-specific CD8+ T cells at a low frequency in 1998, when the plasma viral load (pVL) was very low, whereas later this patient lost the response, and the pVL increased from an undetectable level to more than 103 copies (Fig. 2C). The other 4 VSPs, excluding 3 LTNBs, either had a low number of Pol283-8-specific CD8+ T cells or did not have any of these cells at any time points studied. These results suggest that Pol283-8-specific CD8+ T cells rather than Pol743-9-specific CD8+ T cells may control HIV-1 in vivo.

To clarify the role of these HLA-B*5101-restricted CD8+ T cells in the control of HIV-1 in vivo, we analyzed the correlation between the frequency of the HLA-B*5101-restricted CD8+ T cells and the pVL in 10 HLA-B*5101+ hemophiliacs. The frequency of Pol283-8-specific CD8+ T cells was negatively correlated with the pVL (P, 5.6 × 10−8), whereas the frequency of the other T cells was positively correlated with the pVL (Fig. 3). These results support the idea that Pol283-8-specific CD8+ T cells drive the suppression of HIV-1 replication in vivo.

FIG. 3.
FIG. 3.

Correlation of the number of HLA-B*5101-restricted CD8+ T cells with the viral load. The number of Pol283-8-specific (A), Pol743-9-specific (B), Gag327-specific (C), or Rev71-specific (D) CD8+ T cells among PBMCs from 10 HLA-B*5101+ hemophiliacs was measured at 1 time point or at 2 to 10 different time points (see Table 1) by using specific tetramers. The correlation of the median number of tetramer-positive cells with the median viral load was analyzed.

We speculated, therefore, that escape mutants within Pol283-8 epitopes were selected in slow progressors over a 25-year period, because these epitope-specific CTLs are thought to provide strong immune pressure on HIV-1. Two of the LTNPs had the Pol283-8V mutant, whereas the third had wild-type Pol283 in July 2002 but the 8V mutant in October 2006 (Table 2). As previously noted (34), Pol283-8-specific CTL clones showed the same killing activity toward target cells prepulsed with the Pol283-8V peptide as toward those prepulsed with the wild-type peptide. These T cells revealed similar killing activity toward 721.221-B*5101 cells infected with NL-432 carrying Pol283-8V (NL-Pol283-8V) as toward those infected with NL-432 (see Fig. S2A in the supplemental material) and only a marginally weaker ability to suppress the replication of NL-Pol283-8V (see Fig. S2B in the supplemental material). In contrast, the 5 VSPs and 2 SPs had Pol283-8T or Pol283-8R mutants (Table 2). Three Pol283-8-specific CTL clones failed to kill target cells infected with NL-432 carrying these mutants (NL-Pol283-8T and NL-Pol283-8R [see Fig. S2A in the supplemental material]) or to suppress the replication of these mutants (see Fig. S2B in the supplemental material), indicating that these were escape mutants.

View this table:
TABLE 2.

Sequences of Pol283-8 and Pol743-9 epitopes in HLA-B*5101+ HIV-1-infected hemophiliacs

Longitudinal analysis of KI-127 showed that the 8T mutant appeared in August 2000, when the VL had increased approximately 10-fold, whereas wild-type Pol283 was found in February 1998, when the VL was very low or undetectable (Fig. 2C). Previous population analysis using 9 cohorts showed strong association between HLA-B*51 and Pol283-8T (20). These observations together suggest that the 8T mutant is an escape mutant selected by Pol283-specific CTLs and implies that escape from this epitope reduces immune control of HIV-1.

In vitro selection of Pol283 escape mutants by Pol283-specific CTLs.The results shown in Fig. 4 suggested that Pol283-specific CTLs selected 8T, 8R, and 8L escape mutants. To further confirm the selection of these mutants by Pol283-specific CTLs, we investigated whether Pol283-specific CTLs selected these mutant viruses in vitro when the CTLs were cultured with HLA-B*5101-positive CD4+ T cells infected with NL-432 and the mutant virus together. Pol283-specific CTL clones selected these 3 mutant (8T, 8R, and 8L) viruses rapidly in this assay (Fig. 4A to C), supporting the notion that these mutants were selected as escape mutants by Pol283-specific CTLs.

FIG. 4.
FIG. 4.

In vitro selection of Pol283 escape mutants by a Pol283-8-specific CTL clone. T1 cells were infected with paired viruses (NL-432 [Pol283-8I] and a mutant virus [Pol283-8L, -8T, or -8R]) at a ratio of 9:1. The infected cells were incubated with Pol283-8-specific CTL clones at an E:T ratio of 1:0.05. The population change in the viral mixture was determined by the relative peak height on the sequencing electrogram. From day 4 to day 7 postinfection, culture supernatants were collected, and the concentration of p24 Ag in these supernatants was measured by an ELISA. The data obtained by using the mixture of Pol283-8T, -8L, or -8R with Pol283-8I are shown in panels A, B, and C, respectively.

Long-term maintenance of Pol283-8-specific memory CD8+ T cells and failure of induction of escape mutant-specific CD8+ T cells.If the Pol283-8T mutant was selected by Pol283-8-specific CTLs in donors first infected with HIV-1 carrying the Pol283-8 wild-type epitope, we can speculate that the donors had Pol283-8-specific memory CD8+ T cells but failed to elicit Pol283-8T-specific CD8+ T cells after the Pol283-8T mutation appeared. None of 4 HLA-B*5101+ hemophiliac donors carrying Pol283-8T (KI-032, KI-121, and KI-127 [Table 2] and 1 ART-treated hemophilic donor, KI-078 [data not shown]) had detectable Pol283-8-specific CD8+ T cells by analysis using the specific tetramers. But they may have had very small numbers of memory CD8+ T cells. To induce Pol283-8-specific CD8+ T cells from a possible Pol283-8-specific memory T-cell source, we stimulated PBMCs from these patients with the Pol283-8 peptide and then measured the number of Pol283-8-specific CD8+ T cells in 2-week cultures. The KI-127 and KI-078 cultures indeed showed the presence of Pol283-8-specific CD8+ T cells, but KI-127 lost the detectable memory response by April 2006 (Fig. 5), indicating that these 2 patients could maintain Pol283-8-specific memory CD8+ T cells for more than 20 years. In contrast, Pol283-8T-specific CD8+ T cells were not detected among PBMCs from any of these 4 donors after 2 weeks in culture (Fig. 5), indicating that the Pol283-8T escape mutant did not elicit specific CD8+ T cells in vivo. These results support the idea that the Pol283-8T mutant was selected by Pol283-8-specific CTLs in donors first infected with the wild-type virus. Similarly, Pol283-8R-specific CD8+ T cells were not detected in KI-007, although this patient had Pol283-8-specific memory CD8+ T cells (Fig. 5), supporting the notion that the 8R mutant was an escape mutant selected by Pol283-8-specific CTLs and failed to elicit these escape mutant-specific CTLs.

FIG. 5.
FIG. 5.

Induction of Pol283-8-specific CD8+ T cells from PBMCs of 2 very slow progressors and 3 slow progressors. PBMCs from 2 very slow progressors (KI-127 and KI-121) and from 3 slow progressors (KI-032, KI-007, and KI-078) were stimulated with the Pol283-8 epitope peptide or the Pol283-8T or -8R peptide and were then cultured for 12 to 14 days. The cultured cells were stimulated with C1R-B*5101 cells prepulsed with the peptide. IFN-γ-producing CD8+ T cells were measured by using flow cytometry. The percentages of IFN-γ-producing CD8+ T cells are given in the upper right quadrants.

DISCUSSION

It is well known that HLA-B*57 and -B*27 are associated with slow progression to AIDS (19, 37). HLA-B*57-mediated and HLA-B*27-mediated effects on disease progression are seen early and late, respectively, during an infection (6, 14). In the present study, we analyzed 108 HIV-1-infected Japanese hemophiliacs. In Japan, 1,439 patients had been infected with HIV-1 before 1985, mostly around 1983. At present, only 801 of these patients remain alive. Since they had not been treated with highly active antiretroviral therapy (HAART) before 1997, the survivors would seem to be slow progressors. This cohort does not include a large number of patients, because it is not easy to recruit a large number of HIV-1-infected hemophiliacs in Japan, where only 800 are still alive. We found that HLA-B*5101 had effects on the slow progression of the disease in the late phase (both in 1998 and during the years from 1998 to 2007), even when a small number of samples was analyzed. Our recent study also revealed that HLA-B*5101+ hemophiliacs had lower VLs and higher CD4 counts than HLA-B*5101− hemophiliacs but that only the CD4 count was significantly higher in HLA-B*5101+ than in HLA-B*5101− hemophiliacs (20). These findings support the idea that HLA-B*5101-restricted immune responses are associated with slow progression to AIDS.

Pol283-8, Pol743-9, and Gag327-9 are thought to be immunodominant HIV-1 epitopes, because CTLs specific for them were frequently detected in chronically HIV-1 infected HLA-B*5101+ individuals (45). A previous study demonstrated that Pol283-8-specific and Pol743-9-specific CTLs suppress HIV-1 replication strongly but that Gag327-9-specific CTLs suppress it only weakly in vitro (43), suggesting that HIV-1 replication can be suppressed in vivo by Pol283-8-specific and Pol743-9-specific CTLs. In the present study, we demonstrated that a higher number of Pol283-8-specific CD8+ T cells was detected predominantly in LTNPs, whereas Pol743-9-specific CD8+ T cells were found at higher levels in all 10 of the SP hemophiliac patients examined. ART-treated HLA-B*5101+ patients also carried Pol743-9-specific CD8+ T cells but not Pol283-8-specific CD8+ T cells (data not shown). The frequency of Pol283-specific CD8+ T cells was negatively correlated with the pVL, whereas the frequencies of the other 3 types of T cells were positively correlated with the pVL (Fig. 3). The longitudinal analysis of KI-127 showed that the VL increased after the 8T mutant appeared. This suggests that Pol283-specific CTLs may control HIV-1 in this patient, but the possibility that other CTLs also control HIV-1 cannot be excluded. These results support the notion that Pol283-8-specific CTLs play a key role in the control of HIV-1 in chronically HIV-1 infected HLA-B*5101+ hemophiliacs.

Previous studies showed that Gag-specific responses are negatively correlated with VL in chronically HIV-1 infected individuals (23, 25, 28, 49). Especially HLA-B*57/5801-, HLA-B*27-, HLA-B*13-, or HLA-B*63-restricted Gag-specific CD8+ T-cell responses are related to a low viral load (12, 16, 23, 34, 49). However, these studies had been performed with Caucasian and African cohorts. Since HLA-B*57/5801, HLA-B*27, and HLA-B*13 are very rare in Japan, Gag-specific CD8+ T-cell responses might not be related to a low pVL in Japanese patients. For the HLA-B*5101+ hemophiliacs studied here, it is striking that Pol283-specific CD8+ T-cell responses were much more effective in the control of HIV replication than Gag327-specific CD8+ T-cell responses. A previous study revealed that simian immunodeficiency virus (SIV)-infected cells are recognized earlier by Pol-specific T cells than by Nef-specific T cells (39). These results suggest that Pol-specific responses may be important in the control of HIV-1, and not only in the Japanese population. This is potentially an important result in relation to vaccine design and the specificity of the CD8+ T-cell responses that must be induced to achieve immune control of HIV.

Our recent study using 9 cohorts showed that there are 4 mutations (8T, 8R, 8L, and 8V) at position 8 of the Pol283 epitope, that the frequency of the 8T variant is significantly higher in HLA-B*5101+ donors than in HLA-B*5101− donors, and that some acutely infected HLA-B*5101+ subjects who had been infected with the wild-type virus had the 8T virus at only 6 or 12 months after the first test (20), indicating that the 8T mutant is selected by Pol283-specific CTLs. In the present study, we revealed that the Pol283-8T escape mutation was detected for the first time approximately 20 years post-HIV-1 infection in KI-127, indicating that this mutation had been slowly selected by Pol283-8-specific CTLs in this donor. Pol283-8R and Pol283-8L were also apparently escape mutants, because Pol283-8-specific CTLs failed to suppress the replication of HIV-1 carrying these mutants. However, the frequency of these mutations is not significantly higher in HLA-B*5101+ donors than in HLA-B*5101− donors (20), suggesting that other, non-HLA-B*5101-restricted CTLs may also select these particular mutants. Nonetheless, it is clear that the HLA-B*5101-restricted Pol283-specific CTLs select the 8R mutant, because KI-007, who had the 8R mutant virus, possessed Pol283-specific memory T cells (Fig. 5), and one HLA-B*5101+ subject with an acute HIV infection who had been infected with the wild-type virus had the 8R mutant 12 months after the first test (20).

The Pol283-8V mutant was found in only 6 of 60 HLA-B*5101+ donors, including 3 LTNP hemophiliacs (data not shown). Of the 3 nonhemophiliacs, 2 were progressors and 1 was a slow progressor. Since this mutation is rare and it is speculated that the mutations had not accumulated 25 years ago, it is unlikely that the 3 LTNP hemophiliacs had been infected with this mutant virus. On the other hand, the 3 nonhemophiliacs may have been infected with the 8V mutant. The 8V mutation did not influence the killing activity of Pol283-8-specific CTLs toward target cells infected with the HIV-1 mutant, whereas the ability of CTLs to suppress replication was significantly weaker for the Pol283-8V mutant than for the wild-type virus. Previous studies showed that HIV-1-specific CTL clones can partially suppress HIV-1 replication but fail to kill HIV-1-infected CD4+ T cells (42, 45), indicating that the replication suppression assay is more sensitive than the CTL assay. Since Pol283-8-specific CTLs cannot completely suppress the replication of the 8V mutant virus, and since the 8V virus has a higher fitness cost than the wild-type virus, the donors selecting this mutant virus can be LTNP hemophiliacs. However, it still remains unclear why the 8V virus appears in both LTNPs and progressors. We are now analyzing the HLA-B*5101+ nonhemophiliacs carrying the 8V mutants in order to compare them with the LTNPs carrying the 8V mutant.

Our previous study on the crystal structure of the HLA-B*5101-Pol283-8 peptide complex showed that the C-terminal anchor (PC) pocket is hydrophobic and relatively small compared with those of the serologically close alleles, HLA-B*3501 and -B*5301, whose C-terminal preferential amino acids include aromatic amino acids (30). Those findings explain why the PC residues for HLA-B*5101 are preferably aliphatic amino acids and not bulky aromatic amino acids. The PC residue is tethered with well-ordered polar and hydrophobic interactions, as observed in other major histocompatibility complex (MHC) class I molecules (Fig. 6A). Thus, the amino acid substitutions of the PC residue did not likely lead to large rearrangements of this network, and so the orientations of the side chains were presumably maintained. In the case of the 8R mutation, the PC pocket was not large enough to accommodate the Arg residue (Fig. 6B), conferring structural changes around the PC pocket that could possibly result in a lack of binding activity toward HLA-B*5101 (2). The 8L mutant exhibited slightly reduced binding activity toward HLA-B*5101 and CTL recognition for 8L peptide-pulsed target cells but no CTL response to 8L mutant-infected cells, suggesting that the mutation had a deleterious effect on antigen presentation in the system for export to the cell surface. The 8V mutation would delete only one methylene group from the Ile residue and thus would presumably have only a small influence on the binding to HLA-B*5101 as well as on its specific T-cell receptor (TCR) recognition. On the other hand, the Pol283-8T mutation likely introduces a hydrophilic OH group that probably is not appropriate for the hydrophobic pocket, resulting in diminished binding activity (43). Furthermore, the Pol283-8T mutation was detrimental to the CTL response and thus may also have induced a structural rearrangement that had a negative effect on TCR recognition.

FIG. 6.
FIG. 6.

Binding model of HLA-B*5101 mutant peptides. (A) Polar interactions around the PC residue in the HLA-B51-Pol283-8 complex. The Pol283-8 peptide and the HLA-B51 heavy chain are shown as yellow and cyan stick models, respectively (N and O atoms are shown as blue and red, respectively). The dotted lines indicate hydrogen bonds or salt bridges. (B) (Left) Surface representation (gray) of the HLA-B51 heavy chain with the stick model of the Pol283-8 peptide (with the same coloring as in panel A). 8I (PC) penetrates into the small pocket. (Right) The sliced image of the small PC pocket (right) explains why bulky and long amino acids are not preferential.

A higher accumulation of Pol283-8 escape mutations is found in the Japanese population than in other populations, because the frequency of HLA-B*51 is much higher in Japan than in other countries (20). The fitness of the 8T, 8R, and 8L viruses is similar to that of the wild-type virus, and these escape mutants do not revert to wild-type viruses in HLA-B*5101− donors (20). The donors with escape mutant viruses failed to elicit escape mutant-specific CTLs. These findings suggest a difficulty in controlling the replication of these mutant viruses in HLA-B*5101+ individuals initially infected with the mutant virus. We showed previously that recently infected HLA-B*5101+ donors have no advantage in the control of HIV-1 (20). Thus, the association between HLA-B*5101 and slow progression to AIDS may disappear in newly HIV-1 infected Japanese donors.

HLA-B*57-mediated immune pressure early selects an escape mutant of the TW10 epitope, which has a low viral fitness (29, 32). Escape mutations (K, G, Q, and T at position 242) of the KK10 epitope selected by HLA-B*27-mediated immune pressure impair viral replication, but the compensatory S173A mutation restores viral replication (40, 41). Pol283-8 escape mutations (T, L, and R) are different from those escape mutations, because these Pol283-8 mutations do not influence viral fitness (43). HLA-B*5701 is highly associated with LTNPs, but the mechanism of suppression of HIV-1 replication by epitope-specific CTLs still remains unknown (35, 36). On the other hand, several reports indicate that epitope-specific CTLs in HLA-B*57+ LTNPs have the ability to cross-recognize variant epitopes (4, 13, 46), suggesting the control of escape mutants by these CTLs. In the present study, we demonstrated the selection of escape mutations by HLA-B*5101-mediated immune pressure and showed that 2 kinds of mutations, escape mutations for slow progressors and a mutation reducing viral fitness and weakly affecting T-cell recognition for LTNPs, were selected in slow-progressing and LTNP hemophiliacs.

In the present study, we showed that HLA-B*5101+ hemophiliacs exhibited significantly slow progression during the years 1998 to 2007. Furthermore, we demonstrated that the control of HIV-1 over approximately 25 years in HLA-B*5101-positive hemophiliacs was associated with a Pol283-8-specific CD8+ T-cell response. This is the first study finding that a Pol-specific CTL response is more effective in the control of HIV-1 than a Gag-specific CTL response. Our findings provide a novel mechanism for understanding the long-term control of HIV-1 in LTNPs and slow progressors.

ACKNOWLEDGMENTS

We thank Manami Satoh for technical assistance and Sachiko Sakai for secretarial assistance.

This research was supported by the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases and by the Global COE program “Global Education and Research Center Aiming at the Control of AIDS,” launched as a project commissioned by the Ministry of Education, Science, Sports, and Culture of Japan; by a grant-in-aid for scientific research from the Ministry of Health of Japan; by a grant-in-aid (1839014120390134) for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan; and by a grant from the Japan Health Science Foundation.

FOOTNOTES

    • Received 25 January 2010.
    • Accepted 12 April 2010.
  • ↵*Corresponding author. Mailing address: Division of Viral Immunology, Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan. Phone: 81-96-373-6529. Fax: 81-96-373-6532. E-mail: masafumi{at}kumamoto-u.ac.jp

  • ↵‡ Y.K., N.K., and H.G. contributed equally to this work.

  • ↵▿ Published ahead of print on 21 April 2010.

  • ↵† Supplemental material for this article may be found at http://jvi.asm.org/.

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