Creating HIV-1 reverse transcriptase cytotoxic T lymphocyte target structures by HLA-A2 heavy chain modifications
Charles S. Dela Cruz,
Rusung Tan,
Sarah L. Rowland-Jones and
Brian H. Barber
Department of Immunology and Institute of Medical Science, University of Toronto, Medical Sciences Building, 1 King's College Circle, Ontario M5S 1A8, Canada
1 Department of Pathology & Laboratory Medicine, University of British Columbia, Vancouver, BC, V6T 1Z1, Canada
2 Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK
Correspondence to:
B. H. Barber
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Abstract
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Vigorous HIV-1-specific CD8+ cytotoxic T lymphocyte (CTL) responses play an important role in the control of HIV-1 replication and the induction of a strong, broadly cross-reactive CTL response remains an important goal of HIV vaccine development. It is known that the display of high levels of class I MHCviral peptide complexes at the cell surface of target cells is necessary to elicit a strong CTL response. We now report two strategies to enhance the presentation of defined HIV-1 epitope-specific CTL target structures, by incorporating subdominant HIV-1 reverse transcriptase (RT) CTL epitope sequences into the human class I MHC molecule HLA-A2. We show that either incorporation of HIV-1 CTL epitopes into the signal sequence of HLA or tethering of epitopes to the HLA-A2 heavy chain provide simple ways to create effective CTL target structures that can be recognized and lysed by human HLA-A2-restricted RT-specific CD8+ CTL. Moreover, cells expressing these epitope-containing HLA-A2 constructs stimulated the generation of primary epitope-specific CTL in vitro. These strategies offer new options in the design of plasmid DNA-based vaccines or immunotherapeutics for the induction of CTL responses against subdominant HIV-1 epitopes.
Keywords: cytotoxic T lymphocyte, HIV-1 HLA-A2, reverse transcriptase, subdominant epitopes
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Introduction
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Over the past decade, emerging data have strongly indicated that CD8+ cytotoxic T lymphocytes (CTL) play an important role in exerting significant immune pressure in HIV-1 infections (reviewed in 14). Virus-specific CTL have been shown to inhibit viral replication in autologous CD4+ T cells in vitro, either by the release of specific suppressive factors like ß-chemokines (5,6) or by their direct lysis of HIV-1-infected cells (7,8). The control of HIV-1 replication in vivo that often occurs during the period of primary infection coincides in time with the generation of HIV epitope-specific CTL (9,10). The strength of the CD8+ CTL response is inversely correlated with virus load and clinical outcome in chronically infected HIV patients (11,12). Moreover, vigorous HIV-1-specific CTL responses have been found in individuals who have either delayed disease progression by their long-term non-progressing status (1315) or who remain persistently HIV-1-seronegative despite frequent viral exposures (1619). In contrast, most infected individuals lose their HIV-1-specific CTL activity during progression to AIDS and this parallels the observed increase in their viral loads (2022). Gradual loss of epitope-specific CTL after antiretroviral therapy as measured by HLApeptide tetramers has recently been shown, which has led to an interest in immunotherapeutic strategies designed to boost the CTL response in treated patients (23). In addition, viral escape from cytotoxic T cell recognition has been shown to occur in untreated patients (24,25), which demonstrates the importance of inducing CTL responses to epitopes which are conserved because of their structural or functional importance to the virus. Viral escape has also been documented in mothers who are likely to transmit HIV-1 to their infants (26). Therefore, it is now clear that the in vivo induction of anti-viral CD8-mediated cytotoxic T cell responses to conserved epitopes could contribute significantly to the control of HIV-1 replication and should be an important component of any anti-HIV vaccine or immunotherapy strategies.
Naive precursor T cells are triggered to become activated effector CTL through their recognition of antigenic peptides bound to class I molecules of the MHC on the surface of antigen-presenting cells (APC), along with the necessary engagement of accessory signals through various co-stimulatory molecules (27,28). Eliciting a strong CTL response requires the display of high levels of class I MHCviral peptide complexes at the cell surface (2931). Therefore, the generation of higher numbers of a particular class I MHC and peptide as immunogens could be advantageous in eliciting strong CTL responses in vivo and could be used to divert the CTL response to conserved but subdominant epitopes. This is especially important when one considers what happens in HIV-1-infected patients. It is clear that CTL from infected HLA-A2-positive individuals respond more frequently to a dominant HIV-1 Gag p17 (SLYNTVATL; SL9) epitope, and in lesser degrees to other A2-restricted epitopes such as the reverse transcriptase (RT) epitopes ILKEPVHGV (IV9) and VIYQYMDDL (VL9) (32,33). This hierarchy of CTL responses, a phenomenon that is observed in other viral models, appears to arise from differences in the surface expression of corresponding class I MHCpeptide complexes (34). HIV-1-infected cells, for example, present the HLA-A2-restricted Gag SL9 epitope more abundantly compared to the RT IV9 epitope and are more readily lysed by Gag-specific, than RT-specific, CTL (31). The RT epitopes appear to be subdominant, as CTL responses to these peptides are less frequently observed in HIV-1 patients, but seen particularly if the immunodominant SL9 epitope of the virus mutates (32,33). The VL9 RT epitope is rarely recognized in vivo, possibly due to its relatively less efficient binding to HLA-A2 than the IV9 RT epitope (35). Moreover, others have observed that VL9HLA-A2 tetramers tend to be unstable (G. S. Ogg, pers. commun.). However, as this peptide contains the highly conserved YMDD sequence in the active site of the RT enzyme, anti-VL9 CTL responses could potentially be very valuable to the host. In support of this, the rare responders to this epitope tend to fall into the category of long-term non-progressors (36).
Two different strategies were investigated to enhance the presentation of subdominant peptides such as these two RT epitopes on the cell surface by increasing the likelihood of association between the viral peptide and its presenting MHC molecule. One approach was to incorporate the epitope into the leader sequence of the HLA-A2 mature heavy chain coding region. In the second strategy, we covalently coupled the same epitope to the N-terminus of the presenting HLA-A2 MHC molecule via a polypeptide linker. We show here that the expression of the MHC molecule on the cell surface was not adversely affected by either of these modifications. More importantly, stable transfectant lines expressing these HLA-A2-modified constructs were able to form effective CTL target structures, recognized by CTL of the appropriate specificity derived from HIV-1-infected individuals. The enhanced immunogenicity of these cell lines was demonstrated by their ability to stimulate the generation of epitope-specific CTL in vitro from the naive precursors of healthy HLA-A*0201+ human donors. The studies show the feasibility of the epitopeMHC coupling strategies for antigen presentation which could potentially be useful in the generation of epitope-specific vaccines to boost CTL responses to conserved but non-dominant epitopes.
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Methods
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Cell lines and antibodies
C1R (37), an MHC class I-deficient human EpsteinBarr virus (EBV)-transformed B cell line, and specific class I HLA transfectants were kindly provided by Dr P. Cresswell (Yale University). Cells were maintained in complete medium [RPMI 1640 supplemented with 10% FBS (v/v), L-glutamine, antibiotics, non-essential amino acids and sodium pyruvate]. C1R transfectants, C1R-A2 (38), were grown in 0.25 mg/ml hygromycin B (Sigma, St Louis, MO), whereas all other C1R transfectants generated were selected with 1 mg/ml G418 (Life Technologies, Grand Island, NY). B-LCL are autologous EBV-transformed B-lymphoblastoid cells (ATCC; CRL-1651). Anti-HLA-A2 antibodies, BB7.2 (HB82) (39) and PA2.1 (HB117) (40), both obtained from ATCC (Rockville, MD), as well as FMC5 (41), have been previously described. Isotype-matched control antibodies were specific to HLA-A3.
Vector constructions
Full-length HLA-A2 cDNA was generated by standard RT-PCR using mRNA isolated from HLA-A2 transgenic mouse splenocytes and cloned into the pcDNA3 eukaryotic expression vector (Invitrogen, San Diego, CA) as previously described (42). The pcDNA3 vector utilizes the CMV promoter for gene expression. To construct HLA-A2 with HIV RT CTL epitopes [RT476484 or ILKEPVHGV (IV9); RT346354 or VIYQYMDDL (VL9)] in the signal sequence, the first 24 nucleotides of the wild-type HLA-A2 leader sequence was replaced by RT-PCR using a PCR primer that encoded the sequences for either Met-IV9 or the Met-VL9. HindIII and XbaI restriction sites were incorporated into the 5' and 3' oligonucleotide primers respectively. These signal sequence constructs will be designated as ssIV9-A2 and ssVL9-A2. The constructs were ligated into the mammalian expression vector pcDNA3. To facilitate the construction of the epitopeHLA-A2 fusion proteins (IV9-L10-A2 or VL9-L10-A2), the pcDNA3-HLA-A2 plasmid vector was first modified by creating unique XhoI and NheI restriction sites between sequences that encode for the signal sequence and the coding region of HLA-A2 protein by gene sequence overlap extensions using PCR. Synthetic oligonucleotide containing the XhoI site, the sequence encoding for either the HIV RT IV9 or the VL9 CTL epitope and a 10 amino acid (GGSGGGASGG) linker, and the NheI site was made to be inserted into the modified HLA-A2 plasmid vector. All the vector constructs were characterized by restriction enzyme analysis, and confirmed by automated sequencing with T7 polymerase (Visible Genetics, Toronto, Ontario, Canada). The completed plasmid DNA were amplified in the JM109 Escherichia coli and purified using the EndoFree Megaprep kit (Qiagen, Chatsworth, CA).
Transfections
pcDNA3 plasmid vectors containing the different HLA-A2/HIV RT epitope constructs were transfected into COS-7 cells using DEAEdextran chloroquine and subsequent flow cytometry analysis was performed using anti-HLA-A2 antibodies to determine cell surface expression of HLA-A2 molecules on the transfectants. These plasmids were also transfected by electroporation into C1R B lymphoblastoid cells, that are negative for HLA-A2 expression. One milligram of PvuI-linearized plasmid was added to every 106 cells, washed twice in serum-free RPMI medium, in a 0.4 cm cuvette and pulsed at 700 V, 25 mF with a Gene Pulser apparatus (BioRad, Richmond, CA). Cells were incubated for 10 min at room temperature, after which 1 ml of complete medium was added and incubated for an additional 20 min. The transfectants were cultured overnight in normal medium, and transferred and plated at 110 cells/well and maintained in 1.5 mg/ml G418 selection medium for 34 weeks. Stable clones were selected for HLA-A2 cell surface expression analysis by flow cytometry.
Flow cytometry
Cells (106) (COS-7 or C1R transfectants) were incubated for 1 h with 23 mg of anti-HLA-A2 antibody in PBS containing 0.1% BSA and 0.02% sodium azide. Cells were washed twice and incubated for 1 h on ice with a secondary antibody, a goat anti-mouse IgGFITC conjugate (Sigma). Cells were then washed twice and resuspended in 0.5 ml PBS with 0.02% sodium azide. Samples were analyzed on a FACSCalibur flow cytometer, using CellQuest software (Becton Dickinson, San Jose, CA). All of the analyses shown were carried out on populations of cells gated by forward and side scatter to include only live cells.
Immunoprecipitation assays
The immunoprecipitations were performed as described (43). Briefly, 8x106 cells were washed and preincubated in methionine-free medium for 30 min. Subsequently, the cells were labeled for 5 min with 0.5 mCi [35S]methionine (Amersham, Arlington Heights, IL), and washed with cold PBS and later cold TBS (10 mM Tris, 150 mM NaCl, pH 7.4). Cells were then lysed on ice for 30 min using 1 ml of lysis buffer (0.5% NP-40, 10 mM Tris, 50 mM NaCl, 1 mM MgCl2, pH 7.4) which was supplemented with 1 mM PMSF and 5 mM iodoacetamide. The lysates were spun and the supernatants were precleared using normal mouse serum and fixed Staphylococcus aureus bacteria (Sigma) for 30 min on ice. Then 20 mg of mAb were added to the cleared lysates and samples were incubated overnight with rotation at 4°C. The antibodies were precipitated by adding 50 ml 25% (v/v) Protein Aagarose beads (Sigma) and incubated at 4°C for at least 2 h. Beads were washed 5 times with NTSE (0.5% NP-40, 10 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.4), and the protein was eluted by adding 60 ml SDSPAGE sample buffer and heating at 60°C for 5 min. All samples were boiled in the presence of 2-mercaptoethanol and run on SDSpolyacrylamide gels. Autoradiographic films were exposed for no more than 24 h before development.
CTL 51Cr-release assays
Peripheral blood mononuclear cells (PBMC) were separated from the blood of an HIV-1-infected hemophiliac (donor 008) with HLA-A*0201 and bulk cultures were established by re-stimulation with autologous phytohemagglutinin (PHA)-stimulated lymphoblasts as described previously (44). CTL were cultured in RPMI 1640 with 10% FCS, i.e. media and antibiotics for 1 week, with 10% Lymphocult T (Biotest, Solihull, UK) for the second week. Subsequently a CTL line specific for the A2-restricted RT peptide IV9 (residues 476484) was established by stimulating the bulk culture with the autologous irradiated B-LCL pulsed with the peptide at 50 µM and maintained in medium containing 10% Lymphocult T by weekly re-stimulations with autologous, irradiated peptide-pulsed B-LCL. Similarly, the CTL line specific for the HLA A*0201-restricted RT peptide VL9 (residues 346354) was derived from donor 868, an HLA-A*0201 HIV-1-infected patient, as previously described (45).
Standard 51Cr-release assays in microtiter plates were done using 100 mCi of Na2[51Cr]O4 (Amersham)-labeled 104 target cells per well, using the various C1R stable transfectants, together with HLA-A*0201-matched target B-LCL or C1R-A2 cells pulsed with the index HIV-1 RT epitope peptide at 50 µM. Epitope-specific, HLA-A*0201-restricted effector CTL cells were titrated, plated and incubated with the target cells at various E:T ratios at 37°C for 4 h. The assays were counted on a flat-bed scintillation counter (Wallac, Gaithersburg, MD). Percent lysis was calculated from the formula: 100x(E M/T M), where E is the experimental release, M is the release in the presence of R/10 medium and T is the release in the presence of 5% Triton X-100 detergent.
CTL induction using C1R transfectants
Human PBMC were prepared by the conventional Ficoll-Hypaque centrifugation method from heparinized peripheral blood of healthy donors who had been identified to be HLA-A*0201 by molecular HLA typing. Two different HLA-A*0201 donors were studied. Irradiated (4000 rad) C1R-ssIV9-A2 and C1R-IV9-L10-A2 cells were used as stimulator cells; irradiated autologous PBMC pulsed with 50 µg/mL of IV9 peptide were used as positive control, while unpulsed C1R-A2 cells were used as a negative control. Responder and stimulator cells were cultured in AIM-V medium supplemented with 10% pooled AB+ human serum (R & D Systems, Minneapolis, MN), L-glutamine, sodium pyruvate and non-essential amino acids (all from Life Technologies, Burlington, Ontario), and mixed together in a ratio of 2:1 (at 2x106 cells/ml), set-up in a 12-well tissue culture plate (Costar, Cambridge, MA). A final concentration of 10 ng/ml of recombinant human IL-7 (R & D Systems) was added at the initiation of the culture with 10 U/ml of recombinant human IL-2 (R & D Systems) added the next day and every 34 days thereafter. Responders were re-stimulated with the same stimulators at day 7 and recombinant IL-2 was added 1 day after each re-stimulation cycle. The cultures went through three rounds of in vitro stimulation over a period of 3 weeks. Cells were harvested to be tested for cytolytic activity 7 days after the last re-stimulation. Media from each well were replenished with fresh media when proliferating cells in the cultures became too dense. 51Cr-release assays similar to the one described above were performed using T2 cells (which are HLA-A2+) as target cells.
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Results
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Construction of HLA-A2 molecules with HIV peptide epitopes incorporated in the signal sequence
Mammalian expression vectors were constructed that encoded the human class I MHC HLA-A2 molecule modified to have the HLA-A2-restricted CTL epitope RT346354 (or VL9) or RT476484 (or IV9) from HIV-1 RT protein incorporated into the signal sequence of HLA-A2. These signal sequence-modified A2 constructs are designated as ssIV9-A2 and ssVL9-A2 (Fig. 1A
). It was determined previously that the N-terminal domain, or the first seven amino acids of the signal sequence (not including the methionine start site), of the heavy chain can be substituted with a stretch of nine amino acid CTL epitope from influenza nucleoprotein without affecting the signal peptide function (46). Using an RT-PCR strategy, we constructed recombinant HLA-A2 heavy chains containing different HIV reverse transcriptase CTL epitope sequences in place of the wild-type N-terminal domain in the signal sequence. A methionine residue was added for each construct in order to initiate protein synthesis. The wild-type A2 heavy chain, as well as the modified constructs, were cloned into the pcDNA3 eukaryotic expression vector.

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Fig. 1. HLA-A2 modified constructs. (A) Signal sequence constructs. The first seven amino acids of the wild-type signal sequence (ss) (excluding the initiation methionine residue) of HLA-A2 heavy chain were replaced with two different nine amino acid HLA-A2-restricted HIV-1 RT CTL epitopes; RT476484 or ILKEPVHGV (IV9) and RT346354 or VIYQYMDDL (VL9) (shown in bold). (B) Epitope-linked constructs. HIV-1 epitope (IV9 or VL9), along with a 10 amino acid glycine/serine linker, were inserted between the HLA-A2 signal sequence (ss) and its mature coding region. All the resulting modified HLA-A2 molecules were cloned into the CMV promoter-driven pcDNA3 vector.
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Construction of HLA-A2 molecules with covalently linked HIV peptide epitopes
We also constructed vectors that encoded the recombinant HLA-A2 molecules covalently attached to a single peptide. The pcDNA3 vector encoding the HLA-A2 protein was first modified to contain unique restriction sites to allow for the insertion by PCR of synthetic oligonucleotides encoding either the VL9 or IV9 HIV epitope, as well as the 10 amino acid Gly3Ser linker, between the signal sequence and the coding regions of the HLA-A2 heavy chain, thereby linking the HIV epitope to the N-terminus end of the HLA-A2 heavy chain. These epitopeHLA-A2 fusion constructs are designated as IV9-L10-A2 and VL9-L10-A2 (Fig. 1B
).
Cell surface expression of HLA-A2 molecules in transiently transfected COS-7
In order to determine that the modifications made to the HLA-A2 molecule did not affect its cell surface expression, we first transfected the signal sequence-modified HLA-A2 vectors, as well as the linker-modified HLA-A2 vectors, into mammalian COS-7 cells. Wild-type HLA-A2 vector was transfected into COS-7 cells to serve as positive control. Results show high surface expression of HLA-A2 in all HLA-A2 modified vector constructs as detected by flow cytometry using the anti-HLA-A2 mAb BB7.2 (Fig. 2B and C
). The levels of surface expression were comparable to those observed when wild-type HLA-A2 vector was used. Approximately 3050% of the COS-7 cells were transiently transfected with the vectors of interest (data not shown), when HLA-A2+ cells were compared with cells stained with the isotyped-matched control antibody (Fig. 2
).

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Fig. 2. Expression of HLA-A2 on the surface of transfected COS-7 cells. (A) Untransfected COS-7 cells and vector-transfected COS-7 cells. (B) COS-7 cells transfected with wild-type HLA-A2 and COS-7 cells transfected with either of the signal sequence-modified HLA-A2 (IV9-A2 and VL9-A2). (C) COS-7 cells transfected with wild-type HLA-A2 and COS-7 cells transfected with either of the epitopelinkerHLA-A2 fusion constructs (IV9-L10-A2 and VL9-L10-A2). Cells were stained for surface HLA-A2 expression using mAb BB7.2 and analyzed by flow cytometry. Dashed histograms represent control staining using an isotype-matched control mAb.
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Cell surface expression of HLA-A2 molecules in stable C1R transfectants
The various plasmid vectors encoding the different HLA-A2-modified molecules were stably transfected into the C1R MHC class I-deficient human B cell line by electroporation. The transfected clones were analyzed for cell surface expression of HLA-A2 by flow cytometry. The C1R-ssIV9-A2 and C1R-ssVL9-A2 lines stained positive for HLA-A2 using three different anti-HLA-A2 mAb; shown are staining with BB7.2 (Fig. 3B
). Likewise, the C1R-IV9-L10-A2 and C1R-VL9-L10-A2 cell lines showed similar positive staining patterns (Fig. 3C
). All the different transfectant clones used for subsequent experiments have comparable surface HLA-A2 expression. The results obtained from both COS-7 and C1R transfections clearly suggest that neither of the strategies to incorporate a CTL epitope into HLA-A2 significantly altered the capacity of the molecule to be exported to the cell surface, properly expressed and detected by flow cytometry using conformationally sensitive anti-HLA-A2 antibodies.

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Fig. 3. C1R B-lymphoblastoid transfectants with the modified HLA-A2 express high amounts of surface HLA-A2. (A) Untransfected C1R cells were stained with the HLA-A2-specific mAb BB7.2 and a FITC-labeled goat anti-mouse secondary conjugate (unfilled histograms). Cells were also stained with an isotype-matched control mAb (filled histograms). (B) C1R cells transfected with the two signal sequence-modified HLA-A2 constructs (C1R-ssIV9-A2 and C1R-ssVL9-A2). (C) C1R cells transfected with the two epitope-linker HLA-A2 fusion constructs (C1R-IV9-L10-A2 and C1R-VL9-L10-A2). Representative C1R transfectants are shown in (B) and (C), and were similarly stained for surface HLA-A2 expression using the mAb BB7.2 (unfilled histograms) or an isotype control mAb (filled histograms). Staining with other anti-HLA-A2 antibodies, PA2.1 and FMC5, showed similar results (data not shown).
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Determination of the integrity of the epitope-linked HLA-A2 molecule
Having established that the addition of a nine amino acid HIV peptide via a 10 amino acid linker to the N-terminus of the HLA-A2 heavy chain does not alter its cell surface expression, we set out to determine whether the peptide remains covalently attached to the HLA-A2 molecule in the epitopeHLA-A2 fusion transfectants. [35S]Methionine labeling and immunoprecipitation studies using anti-HLA-A2 mAb revealed that both the C1R-IV9-L10-A2 and the C1R-VL9-L10-A2 cell line lysates showed a single band that migrated at ~46 kDa, ~2 kDa larger in size than what was found when the cell lysates from the signal sequence transfectants, C1R-ssIV9-A2 and C1R-ssVL9-A2, were used. Shown are results for the IV9-containing constructs (Fig. 4
). It is expected that the epitope-linked transfected cell lines should have HLA-A2 molecules that are ~2 kDa larger than wild-type HLA-A2, a size difference that can be attributed to the additional mol. wt from the added epitope and linker. This larger band was not observed in cell lysates obtained from the signal sequence transfectants (Fig. 4
) or from C1R-A2 cells (data not shown), as a 44 kDa wild-type class I heavy chain was expected for both.

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Fig. 4. C1R cells transfected with the epitopelinker constructs synthesize HLA-A2 protein of higher molecular mass. C1R cells transfected with the signal sequence constructs and C1R cells transfected with the linker constructs were radiolabeled for 10 min with [35S]methionine and chased for 1 h with cold methionine. HLA-A2 heavy chains were immunoprecipitated with either the anti-HLA-A2 mAb (BB7.2) (lanes 12) or with an isotype-matched control mAb (lanes 34). Reduced samples were run on a 12% SDSpolyacrylamide gel. Arrow depicts the location of the 44 kDa wild-type HLA-A2 mature heavy chain molecule.
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Coupling of peptide to HLA-A2 sensitizes C1R transfectants to cell lysis by peptide-specific HLA-A2-restricted CTL
We then determined whether the HLA-A2 molecules detected on the cell surface by flow cytometry were associated with their respective specific HIV-1 RT peptides. To test this, we assessed the ability of C1R cell lines transfected with the different HLA-A2epitope vectors to form CTL target structures in standard 51Cr-release assays. Human CTL lines derived from HIV-1-infected patients specific for each of these RT epitopes were used for these studies. Results obtained showed that the C1R-ssIV9-A2 (Fig. 5A
) and the C1R-ssVL9-A2 (Fig. 5B
) signal sequence transfectants were lysed at levels comparable or greater than cell lines expressing wild-type HLA-A2 pulsed exogenously with excess HIV-1 peptide. This CTL lysis was found to be HIV-1 epitope specific in that when a CTL line recognizing a different HIV epitope was used, only a background level of lysis was observed (Fig. 5C and D
). As a negative control, C1R-A2 cells alone without the added peptide exhibited only background lysis (Fig. 5A and B
). The epitope-tethered HLA-A2 transfectants were also able to be lysed by the corresponding CTL line. The C1R-IV9-L10-A2 transfectants (Fig. 5A
) created CTL target structures that could be recognized by the epitope-specific CTL at levels comparable to cells expressing wild-type HLA-A2 pulsed with exogenous free peptides. C1R-VL9-L10-A2 transfectants (Fig. 5B
), on the other hand, repeatedly exhibited very low to background levels of lysis, even though these transfectants expressed levels of surface HLA-A2 similar to C1R-IV9-L10-A2 transfectants (Fig. 3C
). Despite the failure of target formation with this particular construct, it can be clearly seen with the C1R-IV9-L10-A2 transfectants that proper target structures can be formed in a model that tethers an HIV epitope to the HLA-A2 heavy chain. As well, incorporating a relevant HLA-A2-binding HIV-1 epitope into the signal sequence also can be properly processed and presented with HLA-A2 to form an appropriately recognizable CTL target structure.

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Fig. 5. C1R cells transfected with signal sequence constructs or the epitopelinker constructs are lysed specifically by HIV-1 epitope-specific human CTL. Signal sequence and epitopelinker C1R transfectants were analyzed for presentation of A2-restricted CTL epitopes in 51Cr-release assays, using epitope-specific human A*0201-restricted CTL clones derived from HIV-1 patients. (A and C) Human CTL line specific for the A2-restricted RT peptide ILKEPVHGV (IV9) (derived from donor 008) was used as effectors, while a different human effector CTL line specific for the A2-restricted RT peptide VIYQYMDDL (VL9) (derived from donor 868) was used in (B) and (D). (A and B) C1R-A2 cells, expressing the wild-type unmodified HLA-A2 molecule, exogenously pulsed with RT peptides were used as positive control, while unpulsed C1R-A2 cells served as the negative control. Signal sequence C1R transfectants (C1R-ssIV9-A2 and C1R-ssVL9-A2) as well as the linker fusion C1R transfectants (C1R-IV9-L10-A2 and C1R-VL9-L10-A2) were tested as target cells. Results shown are mean of two different clones for each C1R transfectants, expressing similar levels of surface HLA-A2 (see Fig. 3 and text). (C and D) A*0201+ B-lymphoblastoid cell line pulsed with 50 µM of the RT peptides, served as positive control target cells. Shown are the specificities of the CTL lysis by the two human CTL lines.
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In vitro induction of epitope-specific CTL using IV9-containing C1R transfectants
With the knowledge that the various epitope-containing HLA-A2-modified C1R transfectants can properly present the HIV peptide in the context of HLA-A2 and be recognized for CTL lysis, we subsequently tested the immunogenicity of these transfectants by attempting to raise CTL by primary in vitro stimulation. Flow cytometric measurements of these C1R transfectants revealed that they expressed relatively high levels of both human CD80 (B7-1) and CD86 (B7-2) on their cell surface (data not shown), consistent with the cells' EBV-transformation status. Expression of these B7 co-stimulatory molecules on APC is known to be required for the potent stimulation of antigen-specific T cells (reviewed in 47). IV9-containing C1R transfectants, both the signal sequence variant as well as the epitopelinker fusion, were separately used to stimulate responder cells that were derived from the PBMC obtained by Ficoll-Hypaque density gradient centrifugation of blood from healthy HIV-1-negative donors HLA-typed by molecular analysis to be HLA-A*0201+. The cells were cultured in an IL-2 + IL-7-containing medium which is known to support CTL generation in vitro (48,49). Our findings show that primary, epitope-specific CTL effectors can be generated that are capable of lysing peptide-pulsed A2-expressing T2 cells when epitope-containing C1R transfectants were used as stimulators (Fig. 6A
). A similar result was observed when autologous cells pulsed with the specific IV9 peptide were used as stimulators (Fig. 6A
). In contrast, C1R-A2 cells when used alone as stimulators with no specific peptide added were not able to drive such effectors. As expected, a background level of specific lysis was observed when unpulsed T2 cells were used as target cells in the 51Cr-release assay (Fig. 6B
).

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Fig. 6. In vitro induction of primary HIV-1 IV9 epitope-specific CTL from A*0201+ donor using IV9-containing C1R transfectants. Responder cells were derived from Ficoll gradient-purified PBMC of an HIV-negative A*0201+ donor. C1R-A2, C1R-ssIV9-A2 and C1R-IV9-L10-A2 cells were used as stimulators. In addition, as positive control, responder cells were also stimulated with autologous PBMC pulsed with IV9 peptide. Responder and stimulator cells (in a 2:1 cell number ratio) were incubated in the presence of IL-7 on day 1, with IL-2 added on day 2, and were re-stimulated 3 times, with media replacement every 34 days. In vitro stimulated cells were harvested for detection of epitope-specific CTL in a 51Cr-release assay, using T2 (which is HLA-A2+) pulsed with the IV9 peptide as target cells (A). Unpulsed T2 cells were used as negative control targets (B). Shown are results from one of the donors tested. Similar results were obtained from repeat experiments.
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Discussion
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In an effort to enhance the presentation of defined CTL target structures, we report the creation of HIV-1 epitope-specific CTL targets by coupling the HIV-1 RT CTL epitope sequences with the human class I MHC (HLA-A2) molecule. We chose two antigenic peptides derived from HIV-1 RT protein as our model peptides in our epitopeHLA-A2 coupling system for several reasons. Epitopes derived from RT exhibit relatively minor variation among differing HIV isolates as the HIV-1 RT protein itself is functionally essential for the virus' own replication. The two HIV-1 RT CTL epitopes (RT346354 or VL9 and RT476484 or IV9) conform to the HLA-A2 consensus motif (50), are known to bind HLA-A*0201 and can trigger favorable anti-HIV-1 CTL activity in patients (31,36). In addition, RT is a target for virus-specific CTL at various stages of HIV-1 infection (51). Therefore, it is reasonable to expect that viral mutations in the RT-derived CTL epitopes would be deleterious to HIV-1 replication and, in populations not exposed to anti-retroviral therapy (as is the case in the developing world), would represent good targets for CTL-based HIV-1 vaccine design.
Interestingly, however, more than two-thirds of HLA-A*0201 HIV-1+ individuals recognize the SL9 Gag epitope, while most of the remainder respond to the IV9 RT epitope, with even fewer responding to the VL9 RT epitope (32,33), despite the apparent advantages of its critical position in an enzyme central to viral replication. In support of these observations, SL9-specific CTL clones have also been found to be more efficient at controlling HIV-1 replication in vitro than IV9-specific clones (52). The immunodominant nature of the SL9 epitope in vivo seems to correlate well with its relative abundance in infected cells over the RT epitopes (31). Such a hierarchy of epitope-specific CTL responses has been observed with other viruses and appears to be due to differences in class I MHCpeptide densities on the cell surface (34).
These observations suggest that subdominant, but potentially valuable epitopes, could be made more immunogenic if they are more abundantly associated with its MHC on the cell surface. Our experiments with the various HLA-A2-modified constructs yielded three conclusions. First, we showed that the incorporation of HIV-1 CTL epitopes into the N-terminus of the HLA-A2 signal sequence does not adversely affect signal peptide function and still allows for proper HLA-A2 expression at the cell surface. Physical addition of the epitope to the HLA-A2 molecule through a linker also did not adversely affect the expression of the class I molecule. Transient COS-7 and stable C1R human B cell transfectants of the various constructs all showed high levels of cell surface HLA-A2 expression using different HLA-A2-specific mAb by flow cytometry. Second, the presence of the specific epitope associated with the HLA-A2 was confirmed when these transfectants were tested for their ability to be recognized and lysed by human HLA-A2-restricted HIV-reactive CD8+ CTL (derived from HIV-1 patients) specific for the HIV-1 respective RT epitopes. C1R cells transfected with the different epitope-HLA-A2 constructs (signal sequence and linker versions) were as effective or better targets than unmodified C1R-A2 cells given exogenous peptides as determined by 51Cr-release assays. We find that the HIV-1 CTL epitope embedded within the signal sequence of the class I heavy chain, once cleaved, can be a source of peptide for efficient presentation to class I-restricted T cells. Likewise, when the CTL epitope was physically linked to the class I heavy chain via a flexible polypeptide linker, no exogenous peptide was required to sensitize the cells for lysis by specific CTL. Although we did not quantitate the number of peptideMHC complexes at the cell surface, it has been shown by our group and others that only small numbers of class I MHCpeptide complex are required to sensitize a cell for T cell-mediated lysis (numbers ranging from a few hundred to <10) (5355). The epitope coupling modifications we made to the HLA-A2 molecules provide ways to potentially improve the density of the chosen MHCpeptide complex, thereby improving its antigenicity and immunogenicity. However, the precise levels and stability of the MHCpeptide displayed on the surface of our transfectants remain to be determined. Finally, the signal sequence-modified HLA-A2, as well as the epitopeHLA-A2 fusion constructs, are immunogenic in vitro as they can stimulate the generation of epitope-specific CTL in culture settings.
It was interesting to note that for one of our epitopeHLA-A2 fusion constructs, VL9-L10-A2, even though the transfectants were able to express the HLA-A2 molecule on the surface, they could not present the epitope for efficient recognition and lysis by the specific effector cells. Both the C1R-VL9-L10-A2 and C1R-IV9-L10-A2 linker transfectants express comparable levels of HLA-A2 on the cell surface as determined by flow cytometric analysis and both contain a higher mol. wt A2 heavy chain product when immunoprecipitated. When the VL9 epitope was incorporated into the signal sequence of HLA-A2, we observed that the transfectants, C1R-ssVL9-A2, efficiently formed CTL target structures, in fact more so than C1R-A2 cells pulsed with the VL9 peptide. Thus, the difference between the two linker transfectants cannot be due to the fact that the VL9 CTL epitope bind less well to the HLA-A2 molecule than IV9. The difference may be due to a peptide specific level of conformational distortion in the peptideA2 complex conferred by the covalent linkage that results in an unrecognizable target.
It is known that peptides presented by MHC class I molecules come from proteins degraded in the cytosol of the cell and these peptides gain access to the endoplasmic reticulum via transporter-associated with antigen presentation (TAP) proteins where they then associate with newly synthesized class I molecules. However, signal peptide sequences can require TAP for efficient class I MHC presentation. Our group has previously demonstrated that the presentation of an optimal influenza nucleoprotein CTL epitope inserted into mouse Db heavy chain signal sequence requires TAP in order to be processed and presented for T cell recognition (46). Thus, the signal sequence-containing constructs used in this study would probably utilize such TAP-dependent pathway for peptide presentation.
Recent reports have shown that covalent coupling of a CTL peptide epitope to its MHC class I molecule, either through class I heavy chain (56,57) or through its ß2-microglobulin (58,59), can result in the generation of CTL targets, in accordance with the findings we report here. By coupling subdominant HIV CTL epitopes to its MHC, we were able to effectively form stable CTL target structures and show they can be immunogenic in vitro. It has become clear that HIV-1-specific Th cell responses are important at initiating and sustaining a good anti-HIV-1 CTL response (60,61). In HIV-1-infected individuals, vigorous HIV-1-specific CD4+ T cell responses are associated with stronger virus-specific CTL responses (62) and better control of the virus (62,63). CD4+ T cell epitopes can additionally be incorporated in the vaccine constructs to provide the necessary help to further enhance CTL induction. A similar peptideMHC coupling can theoretically be done for class II MHC to allow for better presentation of weaker binding helper epitopes. The HIV epitopeclass I HLA linkage strategies which we have described here should offer new options in the design of vaccines or immunotherapeutics against HIV-1. For example, these constructs could be of particular use to elicit epitope-specific CTL responses in vivo using plasmid DNA-based or cellular-based immunogens expressing these modified class I HLA molecules.
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Acknowledgments
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This work was supported by the Canadian Universities Research Program and the Medical Research Council of Canada (C. S. D. C. and B. H. B.), and by the UK Medical Research Council (R. T. and S. L. R.-J.). C. S. D. C. is the recipient of an MRC MD/PhD Scholarship award. The authors would like to thank R. Kim for his technical assistance and C. Smith for her assistance with the flow cytometry.
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Abbreviations
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APC antigen-presenting cell |
CTL cytotoxic T lymphocyte |
EBV EpsteinBarr virus |
PBMC peripheral blood mononuclear cell |
PHA phytohemagglutinin |
RT reverse transcriptase |
TAP transporter-associated with antigen presentation |
 |
Notes
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Transmitting editor: A. McMichael
Received 10 December 1999,
accepted 23 May 2000.
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