Schmitt, Matthias; Harrer, Ellen; Goldwich, Andreas; Bäuerle, Michael; Graedner, Irina; Kalden, Joachim R.; Harrer, Thomas
Monotherapy of HIV-1-infected patients with lamivudine rapidly selects mutations from M to V or M to I at amino acid position 184 within the catalytic site of reverse transcriptase (RT) [1,2]. These mutations are associated with high-level resistance to lamivudine and reduction of the sensitivity to didanosine, zalcitabine and abacavir [3–6]. Although the M184V and M184I mutations are rapidly induced in vitro by treatment with lamivudine [3,7], they are not observed in wild-type HIV-1 strains as they are associated with a reduction of viral fitness through functional impairment of the mutant RT  and with an increase in fidelity of RT . The lamivudine escape mutations are located within an HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope previously defined by us in a long-term non-progressing HIV-1-infected individual . CTL from this subject recognized the wild-type epitope VIYQYMDDL (RT 179–187), but not the variant peptide produced by the M184V mutation. This result indicated potential interactions between antiviral therapy and escape from CTL. As the M184V mutation is located within the T cell receptor recognition site of the epitope, it could potentially generate a new CTL epitope. To test whether HIV-1-infected patients can specifically target lamivudine-resistant viruses, we studied the CTL response against the RT sequence VIYQYVDDL containing the M184V mutation in a cohort of HLA-A2-positive HIV-1-infected patients.
Subjects and methods
Twenty-eight HLA-A2-positive HIV-1-infected patients were included in this study. The subjects gave written informed consent and the study was approved by the medical faculty's Human Studies Committee. Viral load was determined by b-DNA assay (Chiron, Ratingen, Germany) with a lower limit of detection of 500 copies/ml plasma. The median CD4 cell count of patients was 405.5 × 106 cells/l (range 18–846) and the median viral load was < 500 copies/ml (range < 500–287 000). At the time of the analysis, 26 patients (93%) received a combination therapy containing at least three antiretroviral drugs, including either one protease inhibitor or abacavir. One patient received a combination of lamivudine and stavudine and one patient still was untreated. All patients on therapy were treated with lamivudine for a median time of 11 months (range 4–48 months).
Subject EN is a 43-year-old male homosexual infected for at least 12 years. In 1990, when his CD4 cell count dropped to 336 × 106 cells/l, antiretroviral therapy was started first with zidovudine; in 1993 zalcitabine was added. In 1995, therapy was changed to a combination of zidovudine, lamivudine and saquinavir. In 1997, zidovudine was substituted by didanosine and stavudine. In January 1998, his CD4 cell count was 339 × 106 cells/l and his viral load was 2700 copies/ml.
HLA class I typing was performed using standard serological techniques according to the manufacturer's guidelines (Biotest AG, Dreieich, Germany). HLA-A2 subtyping in subject EN was performed using the DYNAL HLA-A2 SSP kit (Dynal AS, Oslo, Norway) according to the manufacturer's recommendations. The HLA-I type of subject EN was A2.1, A1, B62, B70, Cw9(3), Cw7.
Cell lines and culture media
Epstein–Barr virus-transformed B lymphoblastoid cell lines (B-LCL) were generated and maintained in R20 medium, consisting of RPMI 1640 medium containing 20% (v/v) heat-inactivated fetal calf serum (FCS) supplemented with l-glutamine (4 mmol/l), penicillin (50 U/ml), streptomycin (50 μg/ml) and Hepes (10 mmol/l), as described previously . CTL clones were cultured in RPMI 1640 containing 10% FCS (R10) supplemented with 10–100 U/ml recombinant interleukin-2 (Proleukin, Chiron). ELISPOT assays were conducted using R5AB media consisting of RPMI 1640 medium with supplements and 5% human AB serum (Sigma–Aldrich, Steinheim, Germany). For ELISPOT analysis, peripheral blood mononuclear cells (PBMC) were frozen with a freezing solution of RPMI 1640 medium , FCS (45% v/v) and dimethylsulfoxide (10% v/v) (Sigma-Aldrich).
The following synthetic HIV-1 peptides were synthesized by QCB (Hopkington, MA, USA) as crude peptides and as C-terminal acids: VL9: VIYQYMDDL; VL10: IVIYQYMDDL; VL10-V: IVIYQYVDDL; VL9-V: VIYQYVDDL; and VL9-I: VIYQYIDDL. Degree of purity was more than 75%. Lyophilized peptides were reconstituted at 2 mg/ml in sterile distilled water with 10% dimethylsulfoxide and 1 mmol/l dithiothreitol (Sigma-Aldrich). Stock solutions were stored at −80°C.
Isolation of peripheral blood mononuclear cells
PBMC were obtained by Ficoll–Hypaque density gradient centrifugation (Pharmacia, Uppsala, Sweden). For ELISPOT analysis, frozen PBMC were stored in liquid nitrogen.
Generation of specific cytotoxic T cell lines
PBMC (5 × 106) were stimulated with peptides at a final concentration of 2 μg/ml in 1 ml R10 medium supplemented with 10 U/ml interleukin-2. After 2 weeks, outgrowing cells were tested for specific recognition of peptide-pulsed B-LCL in a standard chromium-release assay. Peptide-specific CTL lines were restimulated every 2 weeks with peptide-pulsed irradiated B-LCL (irradiation with 60 Gy) and irradiated allogeneic feeder cells (irradiation with 40 Gy), as previously described .
B-LCL were sensitized with synthetic peptides as described, and tested in a 4-h chromium-release assay . For peptide titrations, chromium-labelled target cells were incubated with serial log dilutions of peptides in a 96-well plate for 1 h before adding effector cells.
Analysis of cells producing interferon-γ
ELISPOT analysis was performed with thawed PBMC that had a viability of more than 90%. Nitrocellulose filter-backed microtiter plates (96 well; MAHA-S-4510, Millipore, Molsheim, Germany ) were coated with 50 μl of interferon-γ antibody 1-D1K (Mabtech, Stockholm, Sweden) at a concentration of 10 μg/ml. After four washes with PBS and blocking with R5AB, 5 × 105 PBMC were added in 100 μl R5AB to the precoated wells in duplicate. Peptides were added directly to the wells at a final concentration of 40 μg/ml and the plates were incubated for 10 h at 37°C in 5% CO2. After six washes with PBS containing 0.05% Tween 20 (PBS/T 0.05%), 100 μl biotinylated interferon-γ monoclonal antibody 7-B6-1 (Mabtech) was incubated at a final concentration of 2 μg/ml for 2 h at 37°C in 5% CO2. After washing with 0.05% PBS/T, 100 μl avidin/peroxidase solution (Vectastain ABC-Kit, Vector Laboratories, CA, USA) was added to each well. After incubation for 1 h at room temperature and after three washes with 0.05% PBS/T and three washes with PBS, 100 μl AEC substrate (3-amino-9-ethylcarbazole, Sigma-Aldrich) containing 0.01% H2O2 was added as chromogen and spots developed within 4 min. The colorimetric reaction was stopped by washing the plates with distilled water and the plates were then air dried. Spots were counted using a stereomicroscope with a magnification of 20×.
Sequencing of autologous viruses
Autologous viruses were sequenced from phytohaemagglutinin (PHA)-stimulated PBMC and plasma. Viral RNA was isolated with Trizol LS Reagent (GIBCO BRL-Life Technologies, Eggenstein, Germany). The RNA pellet derived from 140 μl ethylenediamine tetraacetic acid-treated plasma was dissolved in 11 μl distilled water containing 0.1% diethylpyrocarbonate supplemented with 1 U/μl RNAsin (Promega, Mannheim, Germany) and then reverse transcribed using random hexamers and Superscript II reverse transcriptase (GIBCO BRL- Life Technologies).
QIAamp Blood Kit (Qiagen, Hilden, Germany) was used to isolate proviral DNA from cells generated by culture of PBMC for 6–7 days in R10 supplemented with 0.5 μg/ml PHA and 100 U/ml recombinant interleukin-2. The DNA was amplified by nested polymerase chain reactions (PCR). The primer pair 2509 (5′-GAAGAAATCTGTTGACTCAGMTTGG-3′) and 3769 (5′-AAGGGAGGGGTRYTRACAA-3′) were used in the first reaction. The PCR was completed after 40 cycles of 95°C for 20 s, 55°C for 30 s and 72°C for 90 s. The primer pair 2869 (5′-CAGT ACTGGATGTGGGCGATG-3′) and 3387 (5′-AG TGCTTTGGTTCCCCTAAGGAGTTTACA-3′) was used for the second reaction under the same conditions. For further sequencing, the PCR product was treated with Exo and CIAP (GIBCO BRL-Life Technol- ogies). In addition to the primers from the second amplification step, the forward primer 2929 (5′-ATACTGCATTTACCATACCTAGT-3′) and the reverse primer 3243 (5′-TTTRTCWGGRTGAGYCA TA-3′) were used together with the DNA Sequencing Kit (PE Applied Biosystems,Weiterstadt, Germany).
PBMC from 28 HIV-1-infected HLA-A2-positive patients were stimulated with the RT peptides VL9-V or VL10-V containing the M184V lamivudine-induced escape mutation. Only one subject, EN, demonstrated the vigorous outgrowth of a CTL line (TE-1) that recognized both the VL9-V and VL10-V peptides in chromium-release assays against peptide-sensitized autologous B-LCL. The CTL also recognized the VL9-I peptide containing the M184I mutation but not the wild-type peptides VL9 or VL10 (Fig. 1). The CTL line TE-1 was CD8 positive, as demonstrated by flow cytometry (data not shown). Recognition of VL9-V was restricted by HLA-A2, as shown by lysis of a panel of allogeneic B-LCL (Fig. 2). In peptide titration experiments, both the peptides VL9-V and VL9-I sensitized target cells for lysis with comparable efficiency at concentrations down to 100 ng/ml (Fig. 3).
ELISPOT analysis of antigen-specific interferon-γ secretion of single cells was performed with PBMC of this patient obtained from the same blood sample that had been used for the generation of the CTL line TE-1. We failed to detect any cells recognizing the VL10 wild-type peptide, but we observed CTL specific for the VL10-V peptide and even more frequently CTL with specificity for the VL9-V peptide (Fig. 4). The patient displayed a stable low viral load ranging from 7800 copies/ml in February 1997 to 2700 copies/ml in January 1998, the time of the generation of the TE-1 CTL line. To test whether the patient's viruses carried the M184V mutation, we evaluated the sequences from autologous virus derived from plasma samples obtained in August of 1997. Because of the low plasma viraemia, viral sequences of later time points could be obtained only from PHA-stimulated PBMC. Both in plasma and PBMC samples, molecular analysis revealed the presence of the M184V mutation together with a conservative V to I substitution at position RT179 (Table 1).
Emergence of drug resistance is a critical problem in antiviral drug therapy for HIV-1 infection. HIV-1 can acquire mutations in RT and protease that confer resistance to all the currently available RT- and protease inhibitors [13–15]. Risk factors for the development of drug resistance are advanced stages of HIV-1 infection, high viral load and poor compliance . So far, few data are available regarding potential influences of the immune system on the development of drug resistance and the efficacy of drug therapy.
Here, we report a patient receiving treatment with a lamivudine-containing antiretroviral combination regimen who could generate HIV-1 RT-specific CTL that specifically targeted lamivudine-resistant viruses. These CTL recognized both the lamivudine-escape variants M184V and M184I but failed to recognize the wild-type sequence VIYQYMDDL. ELISPOT analysis performed using PBMC from this patient confirmed the presence of VL9-V- and VL10-V-specific CTL; the PBMC of this patient failed to recognize the VL10 peptide containing the wild-type sequence IVIYQYMDDL. The ELISPOT assay detected a higher frequency of VL9-V-specific T cells than VL10-V-specific T cells, although both peptides were recognized with the same efficiency by the CTL line TE-1 in the chromium-release assay using as target cells B-LCL sensitized at a high peptide concentration. Because of shortage of TE-1 cells, the sensitizing capacity of the two peptides in peptide titration experiments could not be compared. As the VL9-V peptide, which is nine amino acid residues long, presumably is the optimal peptide for this HLA-A2-restricted epitope, we suggest that the lower frequency of VL10-V-specific CTL in the ELISPOT resulted from a weaker sensitization capacity of this peptide in comparison with that of VL9-V. This would lead to a weaker stimulation of specific T cells by the VL10-V-presenting cells within the PBMC. Alternatively, it could be speculated that the PBMC contained another CTL clone recognizing only the peptide VL9-V, but not the peptide VL10-V.
CTL against the VIYQYMDDL epitope were first reported by us in a long-term non-progressing HIV-1-infected individual . In contrast to the patient in this study, the CTL in that long-term non-progressor only recognized the wild-type epitope not the M184V-mutation. Although recognition of the VIYQYMDDL epitope seems to be infrequent, it is not restricted to long-term non-progressors and has been described also in patients with progressive disease [16–18].
Viruses with mutations at residue 184 of RT only arise under drug pressure; they are not seen in wild-type viruses as these mutations adversely affect the function of RT and the replicative capacity of HIV-1 [8,9]. The fact that the patient was able to mount a specific CTL response against lamivudine-resistant viruses demonstrates that, even in advanced stages of HIV-1 infection, at least some patients are able to recruit and expand CTL that express T cell receptors with specificity for the newly emerging epitope. It is not known whether these CTL in this 43-year-old patient were already present in the peripheral T cell repertoire or whether they were generated from lymphoid stem cells de novo. Recently, it has been shown that highly active antiretroviral drug therapy is associated with a rapid and sustained increase of generation of new naive T cells . This finding suggests that the HIV-specific T cell response can adapt to viral sequence variation, at least in patients on antiretroviral therapy.
Sequencing of autologous viruses from plasma and PHA-stimulated PBMC revealed the presence of the M184V mutation in the patient's viral quasispecies, suggesting that the mutated viruses had recruited and expanded the M184V-specific CTL in this donor. Although drug therapy was not able to suppress HIV-viraemia in this patient, plasma viral load remained stable at low levels and even declined over time gradually without change in antiretroviral therapy. This indicates that, in addition to drug therapy, the HIV-1-specific immune response contributed to the control of HIV-1 in this patient. So far, we only can speculate whether the variability in the clinical response to antiretroviral therapy could result from potential influences of the HIV-1-specific immune response targeting epitopes with drug-escape mutations. Such epitopes could represent attractive new targets for HIV-1-specific immunotherapeutic and even preventive vaccines. Further studies are necessary to examine whether the induction of CTL against drug-escape variants can help to delay or even prevent the emergence of drug-resistant HIV-1 strains.
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