Optimization and immune recognition of multiple novel conserved HLA-A2, human immunodeficiency virus type 1-specific CTL epitopes

Sylvie Corbet1, Henrik Vedel Nielsen1, Lasse Vinner1, Sanne Lauemoller2, Dominic Therrien1, Sheila Tang1, Gitte Kronborg3, Lars Mathiesen4, Paul Chaplin5, Søren Brunak6, Søren Buus2 and Anders Fomsgaard1

1 Department of Virology, Statens Serum Institut, 5 Artillerivej, DK-2300 Copenhagen S, Denmark
2 Institute for Medical Microbiology and Immunology, University of Copenhagen, Denmark
3 Department of Infectious Diseases, University Hospital of Copenhagen, Denmark
4 Department of Infectious Diseases, University Hospital of Hvidovre, Denmark
5 Bavarian Nordic Research Institute, Martinsried, Germany
6 Center for Biological Sequence Analysis, BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark

Correspondence
Anders Fomsgaard
afo{at}ssi.dk


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
MHC-I-restricted cytotoxic responses are considered a critical component of protective immunity against viruses, including human immunodeficiency virus type 1 (HIV-1). CTLs directed against accessory and early regulatory HIV-1 proteins might be particularly effective; however, CTL epitopes in these proteins are rarely found. Novel artificial neural networks (ANNs) were used to quantitatively predict HLA-A2-binding CTL epitope peptides from publicly available full-length HIV-1 protein sequences. Epitopes were selected based on their novelty, predicted HLA-A2-binding affinity and conservation among HIV-1 strains. HLA-A2 binding was validated experimentally and binders were tested for their ability to induce CTL and IFN-{gamma} responses. About 69 % were immunogenic in HLA-A2 transgenic mice and 61 % were recognized by CD8+ T-cells from 17 HLA-A2 HIV-1-positive patients. Thus, 31 novel conserved CTL epitopes were identified in eight HIV-1 proteins, including the first HLA-A2 minimal epitopes ever reported in the accessory and regulatory proteins Vif, Vpu and Rev. Interestingly, intermediate-binding peptides of low or no immunogenicity (i.e. subdominant epitopes) were found to be antigenic and more conserved. Such epitope peptides were anchor-optimized to improve immunogenicity and further increase the number of potential vaccine epitopes. About 67 % of anchor-optimized vaccine epitopes induced immune responses against the corresponding non-immunogenic naturally occurring epitopes. This study demonstrates the potency of ANNs for identifying putative virus CTL epitopes, and the new HIV-1 CTL epitopes identified should have significant implications for HIV-1 vaccine development. As a novel vaccine approach, it is proposed to increase the coverage of HIV variants by including multiple anchor-optimized variants of the more conserved subdominant epitopes.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Specific MHC-I-restricted cytotoxic responses are considered a critical component of protective immunity against viruses, including human immunodeficiency virus type 1 (HIV-1) (McMichael & Rowland-Jones, 2001; Seder & Hill, 2000). Evidence includes the association of CTLs with virus clearance during acute HIV infection (Borrow et al., 1994; Koup et al., 1994), the inverse correlation between virus load and CTL frequency during the progression of infection (Carmichael et al., 1993; Klein et al., 1995), and the association of CTLs with resistance to infection in HIV-exposed but seronegative individuals (Rowland-Jones et al., 1993, 1995). It is generally recognized that the breadth of early CTL response is a critical factor in determining the rate of progression to disease (Chouquet et al., 2002; Pantaleo et al., 1997). Consequently, optimal design of broad CTL-based HIV-1 vaccines (Hanke & McMichael, 2000) depends on the ability to identify potent CTL epitopes conserved among the great diversity of HIV isolates circulating worldwide (Walker & Korber, 2001). CTL responses should be directed against accessory and early regulatory HIV-1 proteins. However, CTL epitopes in these proteins are rarely found. To facilitate the identification of new epitopes, computer-based predictions may be used. Sequence data obtained from naturally processed peptides bulk-eluted off MHC-I molecules have allowed the identification of MHC-binding motifs (Falk et al., 1991; Rammensee et al., 1995; Sette et al., 1989). Generally, algorithms based on the search for simple structural features or MHC-binding motifs are only moderately sensitive (Andersen et al., 2000; Gulukota et al., 1997; Kast et al., 1994). Although improved matrix-driven prediction methods have been developed that evaluate independently the influence of each position in a peptide (Schafer et al., 1998; Stryhn et al., 1996), they are being replaced by a new generation of more powerful methods based on artificial neural networks (ANNs) (Milik et al., 1998). Because ANNs analyse simultaneously the influence of all amino acids in a peptide, they can detect more subtle binding preferences and are able to predict more accurately in the entire spectrum from low to high affinity binding (Buus et al., 2003). We have, therefore, used such new ANNs able to quantitatively predict binding values of minimal epitope peptides to the HLA-A-*0204 molecules available to us in a large set of HIV-1 sequences (Human Retroviruses & AIDS, 1998). Epitopes were ranked according to their binding affinity, which may reflect their immunogenicity (Sette et al., 1994), and their sequence conservation among HIV-1 strains. Also, we predicted primary anchor-optimized peptides of intermediate binders to evaluate immunogenicity enhancement that could improve vaccines and provide better coverage of HIV-1 isolates (Berzofsky, 1993; Berzofsky et al., 1999). The new epitopes and anchor-optimized peptides were immunologically characterized further, including measurement of binding affinity to HLA-A*0204, immunogenicity and antigenicity in HLA-A2 transgenic mice (Shirai et al., 1995; Wentworth et al., 1996), and their CD8+ T-cell recognition in HIV-1-positive patients.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Quantitative predictions of HIV-1 peptide binding to HLA-A*0204.
An ensemble of seven ANNs was used to generate a prediction algorithm for quantitative peptide binding values to HLA-A2, as described (Buus et al., 2003). Briefly, seven different ANNs predicting the same peptide binding to HLA-A*0204 were developed and trained on separate sets of data. The ANNs were trained exclusively on non-HIV sequences. Each of these seven ANNs can be used to predict the outcome of a given peptide–HLA-A*0204 interaction, but we used the average of the seven ANNs as a more reliable prediction than any of the seven individual predictions. Candidate HLA-A*0204-binding CTL epitope peptides of eight and nine amino acids in length were then predicted from all full-length HIV protein sequences available in the 1998 Los Alamos database, 98Ladb (Human Retroviruses & AIDS, 1998). All Gag (n=96), Pol (n=86), Vif (n=265), Vpr (n=173), Vpu (n=156), Tat (n=101), Rev (n=105), Env (n=213) and Nef (n=251) sequences were scanned individually for HLA-A2-binding peptides.

HIV-1 epitope selection criteria.
Peptide-binding affinities to purified HLA-A*0204 molecules were measured using a biochemical binding assay, as described previously (Buus et al., 1995). The predicted epitope peptide binders (defined as IC50<500 nM) in the binding assay were then sorted according to percentage conservation among all HIV-1 protein sequences scanned. The global conservation cut-off was set at 8 % in order to include epitopes conserved in less represented subtypes. The intra-subtype conservation was set at 20 %. Anchor-optimization was designed by single or double amino acid substitution of P2 and P{Omega}. For the P2 residues, the preferred substitutions were L, M, I and Q; for the C-terminal P{Omega} residues, the preferred substitutions were V, L, I or A. The IC50 value was predicted for all possible modified peptides and only the peptide modification that lowered the IC50 (improved the affinity) the most was retained. Here, the cut-off was set at IC50<100 nM, expected to result in immunogenicity. A final criterion was novelty; that is, the epitopes were not found in the Los Alamos database (http://hiv-web.lanl.gov/immuno/index.html) or the MHCPEP database (http://wehih.wehi.edu.au/mhcpep) when the study started in 1998.

HLA-A2-binding assays.
Peptides were synthesized at Schafer-N. Purity was ascertained by reverse-phase HPLC and identity was confirmed by mass spectroscopy analysis. All peptides were dissolved in 100 % DMSO and diluted in RPMI-1640 to 4 mg ml-1. Each peptide solution was used fresh when immunizing mice or aliquotted and stored at -80 °C until further use in ELISPOT and CTL assays, respectively. Peptide-binding affinities to purified HLA-A*0204 molecules were measured using an in vitro biochemical binding assay (Buus et al., 1995). HLA-A*0204 molecules were chosen as a representative of HLA-A2 because this was the reagent available to our group. Briefly, the concentration of test peptide inducing 50 % binding inhibition (IC50) of a tracer peptide was measured. Thus, the lower the IC50 value, the higher the binding affinity.

HLA-A2 transgenic (C57-A2Kbtg) mice.
C57-A2Kbtg mice expressing a transgenic chimeric heavy chain of the MHC-I molecule (HLA-A2.1 {alpha}1 and {alpha}2, and H-2Kb {alpha}3 transmembrane and intracytoplasmic domains) were kindly provided by N. Holmes, University of Cambridge, Cambridge, UK (Epstein et al., 1989). HLA-A*0201 mice were chosen as representative of HLA-A2 because this was the strain available to our group. These mice were then backcrossed with C57Bl/6Bom mice (Bomholtgaard). HLA-A2.1 expression in offspring was assayed by indirect immunofluorescence using the PA2.1 (anti-HLA-A2) unlabelled monoclonal antibody (HB-117, ATCC) and FITC-conjugated rabbit anti-mouse immunoglobulin. Mice were maintained in groups of five per cage with food and water ad libitum and artificial light for 12 h per day. All cell lines were cultured in RPMI-1640 medium supplemented with 10 % FCS, 100 IU penicillin ml-1 and 100 µg streptomycin ml-1.

Peptide immunization and CTL/ELISPOT assay of C57-A2Kbtg mice.
C57-A2Kbtg mice, 6–12 weeks old, were injected subcutaneously with 100 µg peptide plus 120 µg of an I-Ab-binding synthetic T-helper peptide from hepatitis B virus (TPPAYRPPNAPIL), emulsified in 100 µl of incomplete Freund's adjuvants. At day 10 post-infection, spleen cells from primed mice were re-stimulated using peptide-loaded mitomycin C-treated C57-A2Kbtg B-cell blasts (ratio of splenocytes : blasts was 2·5 : 1). Briefly, 5x107 spleen cells were cultured with 2x107 peptide-pulsed LPS-induced C57-A2Kbtg lymphoblasts in complete RPMI. LPS blasts were obtained by culturing naive spleen cells in complete RPMI medium containing 25 µg LPS ml-1 (L-2387, Sigma) and 7 µg dextran sulphate ml-1 (Pharmacia). After 3 days in culture, the B-cell blasts were loaded with 10 µg peptide ml-1 and 50 µg mitomycin C ml-1 for 1 h at 37 °C. The cells were then washed twice and incubated with immunized spleen cells, as described above. The culture medium was supplemented with 5 IU recombinant human IL-2 ml-1 (Roche) at day 3 post re-stimulation. After 5 days of re-stimulation, effector cells were washed and re-suspended to 5x106 cells ml-1. A standard 5 h 51Cr-release CTL assay was performed. Effector cells were assayed against either peptide-pulsed or unpulsed HLA-A2*0201/h{beta}2m-transfected EL4 target cells (HHD-EL4S3-Rob) kindly provided by F. Lemonnier, Institut Pasteur, Paris, France (Pascolo et al., 1997). This cell line was chosen because it expresses no mouse H-2Kb class I molecules at the cell surface. Both the HHD-EL4S3-Rob and the EL4 (mouse H-2Kb) cell lines were used as target cells in cytotoxicity assays.

Target cells were labelled (30 µCi Na251CrO4 per 106 cells) and peptide-pulsed (20 µg ml-1) for 1 h at 37 °C, then washed three times and re-suspended to 5x104 cells ml-1. Target cells (100 µl) were then mixed with effector cells at effector : target cell ratios (E : T) of 50 : 1, 25 : 1, 12·5 : 1 and 6·25 : 1 in triplicate. After incubation for 5 h at 37 °C in 5 % CO2, 51Cr-release was measured in 30 µl supernatant using LumaplateTM-96 (Packard) and a microplate scintillation counter (Topcount-NTX, Packard). Spontaneous and total 51Cr-release were measured by adding growth medium or 1 % Triton X-100, respectively. The percentage of specific lysis was calculated as 100x(experimental release-spontaneous release)/(total release-spontaneous release).

For ELISPOT assays, spleen cells were plated on 96-well nitrocellulose microtitre plates (MAHA S45 10, Millipore) that had been coated overnight at 4 °C with 50 µl per well of anti-IFN-{gamma} monoclonal antibody (18181D, Pharmingen) at 0·5 µg ml-1 in 0·1 M sodium bicarbonate buffer, pH 9·6. Four serial twofold cell dilutions (500 000–62 500 cells per well) were performed in duplicate and peptides were added to a final concentration of 10 µg ml-1 in complete RPMI-1640 medium supplemented with 5 IU recombinant IL-2 ml-1. Plates were incubated at 37 °C and 5 % CO2 overnight and processed as described (Nielsen et al., 1999). The number of specific IFN-{gamma}-secreting T-lymphocytes was counted by direct visualization, calculated by subtracting the negative control value, and expressed as the number of spot-forming units (s.f.u.) per 106 input cells. The threshold for being considered positive was above or equal to 10 s.f.u. per 106 input cells after subtraction of the negative control value and at least two times greater than the mean background activity.

CD8+ T-lymphocyte reaction to new epitopes in HIV-positive patients.
Blood was collected from HIV-positive patients at two University Hospitals in Denmark. These patients, who were without highly active antiviral therapy, demonstrated CD4 cell counts of >300 µl-1 and virus loads of 1000–100 000 RNA copies ml-1. Intracellular IFN-{gamma} (IC-IFN-{gamma}) reaction in CD8+CD3+ T-lymphocytes to 43 individual peptides was measured by flow cytometry. Cells were stimulated with 2 µg peptide, as described (Kern et al., 1998), except that 400 µl of whole heparinized blood was used instead of purified PBMCs. Blood was treated with red blood cell-lysing solution and lymphocyte permeabilization solution (Becton Dickinson Immunocytometry System), according to the manufacturer's instructions. Cells were stained with appropriate antibodies for 30 min at room temperature and fixed in 2 % paraformaldehyde. Mouse anti-human CD28, anti-human CD49d, FITC-conjugated anti-human IFN-{gamma}, PE-conjugated anti-human CD3, PerCP-conjugated anti-human CD8 and mouse immunoglobulin G1 and G2a isotype controls were obtained from Becton Dickinson. Five-parameter flowcytometric analysis was performed on a FASCAN (Becton Dickinson) using 150 000–400 000 events per analysis. CD3+CD8+ cells were examined for IC-IFN-{gamma} and data analysed using CELLQUEST software. Control conditions were established using autologous PBMCs stimulated with a mouse OVA-Kb-restricted epitope peptide (SIINFEKL). The negative background activities thus obtained were used to set the cell populations boundaries (gating) for exclusion of 99·97 % of the control lymphocytes, as described earlier (Altfeld et al., 2001b). Thus, the background activity of the OVA-Kb peptide itself was adjusted to 0·03 % IFN-{gamma} cells in the CD3+CD8+ population in each case. Positive reactions to cytomegalovirus (CMV) and HIV-1 peptides were recorded if the percentage of positive cells gated in this way were equal to or above 0·09 % (greater than three times the value of the negative OVA-Kb control). HLA-A and HLA-B genotypes of patients were examined using a genotypic sequencing kit (Applied Biosystems), according to the manufacture's protocol.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Selection of new predicted HLA-A2-binding epitopes
Initially, a total of 4215 HLA-A2 HIV-1 epitopes were predicted quantitatively. However, many of these were found only in one HIV-1 strain. Thus, to select vaccine-relevant epitopes, we applied a criterion of global conservation (>8 %) to include epitopes conserved in less represented subtypes as well as an intra-subtype conservation (>20 %). Peptides were grouped according to their binding affinity as ‘good binders' (predicted IC50<100 nM) and ‘intermediate binders' (predicted 100 nM<=IC50<=500 nM). It is assumed that good binders would be both antigenic and immunogenic, intermediate binders would be antigenic but less immunogenic and low binders would be neither antigenic nor immunogenic (Sette et al., 1994). Since improvement of peptide–MHC binding by optimizing primary anchor residues may enhance immunogenicity (Berzofsky, 1993; Berzofsky et al., 1999), MHC-I binding after anchor-optimization was also predicted quantitatively. The natural intermediate binders that could be predicted as improvable to an IC50<100 nM by modifying one or two of the primary anchor residues were retained for further investigation. A total of 150 of the 4215 predicted epitopes fulfilled our four selection criteria: (1) novelty; (2) predicted IC50<500 nM; (3) possible anchor-optimization for intermediate binders to reach an IC50<100 nM; and (4) a global conservation (>8 %) and intra-subtype conservation (>20 %). We aimed first to select a maximum of 10 epitopes from each of the nine HIV-1 proteins (‘cover all’); however, due to the scant number of epitopes predicted in the non-structural proteins (Vif, Vpr, Vpu, Tat, Rev and Nef) that fulfilled the requirements, only 52 natural epitopes were obtained for subsequent synthesis and analyses. We predicted that 42 of the 52 natural epitopes could be anchor-improved and thus these additional 42 peptides were also synthesized and analysed further.

In vitro HLA-A2 binding of epitopes
Our ANNs predicted that 82 % of the HLA-A2-restricted HIV CTL epitopes described previously would have a significant binding affinity (IC50<500 nM). Indeed, 25 peptides derived from the 98Ladb and MHCPEP database (http://wehih.wehi.edu.au/mhcpep/) were synthesized for an initial validation and all tested positive at IC50<500 nM (data not shown). Thus, using 500 nM as a cut-off value, we would only have missed 18 % of the HLA-A2-restricted CTL epitopes reported. Some of these missed epitopes were synthesized and actually showed a low measured binding affinity to HLA-A*0204: for example, Pol334 (VIYQYMDDL), Env108 (IISLWDQSL) and Nef190 (AFHHVAREL) had a measured IC50 of 770 nM, 1000 nM and 65 000 nM, respectively. This indicates that our ANNs identified these epitopes correctly as low or non-binders.

The measured binding affinity of each selected new HIV-1 CTL epitope peptide to HLA-A*0204 was measured and compared with the predicted values (Table 1). Of the 52 natural epitopes tested, 36 (69 %) did in fact bind (IC50<500 nM) (Table 1). Of these, 22 epitopes were measured as good binders (IC50<100 nM) and 14 epitopes were measured as intermediate binders (100 nM<IC50<500 nM). The remaining 16 epitopes had a measured IC50>500 nM and were considered as low or non-binders. The correlation between logarithm of predicted versus logarithm of measured binding of our ANNs was analysed more accurately by linear regression using the full set of unselected data points (n=397). The regression line thus obtained is y=0·99x–0·02 (CPearson=0·87, P<0·001; Buus et al., 2003).


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Table 1. HLA-A2 binding and immunogenicity of novel CTL epitopes in HLA-A2 transgenic mice and HLA-A2-positive HIV patients

 
Immunogenicity of natural HLA-A2 HIV-1 CTL epitopes in HLA-A2 transgenic mice
The ability of the 36 new natural binders (measured IC50<500 nM) to induce specific cell-mediated immune responses was evaluated in groups of C57-A2Kbtg mice. We also tested six of the 16 low binders (measured IC50>500 nM). Table 1 summarizes the specific immune responses induced by each of these 42 peptides tested as the number of positive mice and the percentage of specific lysis of peptide-pulsed HLA-A2*0201/h{beta}2m-positive EL4 target cells (HHD-EL4S3-Rob). Of the E : T ratios examined, only the 50 : 1 CTL results are shown, as well as the numbers of IFN-{gamma}-producing cells per 106 splenocytes (s.f.u.). Since the ELISPOT assay was performed with bulk spleen cells, we cannot exclude that, in some cases, IFN-{gamma}-producing cells also derived from mouse MHC-I or -II activation. We found that 25 of the 36 (69 %) natural binders (IC50<500 nM) were immunogenic in transgenic mice, inducing CTL and/or IFN-{gamma}. Also, 13 of the 22 (59 %) good binders (measured IC50<100 nM) induced HLA-A2-restricted cytolytic response in at least one mouse and 5 of the 14 (36 %) intermediate binders induced CTLs, whereas only 1 of the 6 (17 %) low or non-binders (IC50>500 nM) induced CTLs (Table 1).

CD8+ T-lymphocyte reactivity to novel epitopes in HIV-positive patients
We also examined specific CD8+ T-cell reactivity to the 36 new, natural HLA-A2 HIV-1 epitope peptides with confirmed MHC-I binding (IC50<500 nM) in immune-competent HIV-1-infected patients controlling their virus without anti-viral therapy. The patient characteristics are provided in Table 2 along with their HLA-A and HLA-B genotypes. Five of the patients (nos 24, 25, 37, 41 and 43) were infected for more than 13 years and may be classified as long-term non-progressors. Among the 17 HLA-A2-positive patients, specific CD8+ T-cell reactivity could be demonstrated against 23 of the 36 new natural epitopes. For comparison, specific CD8+ T-cell reactivity could be demonstrated against five of the seven natural epitopes known (Table 3). Of the 17 patients, 11 reacted to a CMV CTL epitope. These patients were also anti-CMV IgG antibody positive, indicating a previous or chronic CMV infection. Each HLA-A2-positive patient recognized at least one of the new HIV-1 epitopes. An average of four HIV-1 epitopes were recognized per patient. One patient (no. 21) who did not react to any of the seven epitopes known reacted to two of the new CTL epitopes. Seventeen of the 25 epitopes that were immunogenic in mice were also immunogenic in HIV-1-infected patients (Table 1). Thus, a total of 31 new HIV-1 CTL epitopes with measured IC50<500 nM were immunogenic in HLA-A2 mice or humans.


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Table 2. Characteristics of HLA-A2-positive HIV-1-positive patients

 

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Table 3. Immune recognition of new and known HIV-1 T-cell epitopes in HLA-A2 positive HIV-1 patients

 
Conservation and cross-reaction of natural and natural variant HIV-1 epitopes
Many of the new epitopes are highly conserved among described HIV-1 strains and subtypes (Table 4). Moreover, some epitopes with a relatively low global conservation could be highly conserved among certain subtypes [for example, Vif101(9M) or Env67(2I)]. A few natural epitopes differed from each other at one of the primary anchor residues [for example, Vif101(9L) versus Vif101(9M), Vif23(9V) versus Vif23(9I), Env67(2I) versus Env67(2V) and Gag362(9V) versus Gag362(9A) (Table 4)] and, for example, epitope Vif101(9L) is found mainly in clades B and D, whereas the related epitope Vif101(9M) is more conserved in clades C, D, H and J (Table 4).


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Table 4. Conservation of immunogenic new HIV-1 HLA-A2 epitopes

 
Since an optimal immunogen should generate an immune response against as many HIV-1 strains as possible, we investigated whether T-cell responses raised against one variant epitope could cross-react against another. To investigate this we selected the variant that showed the best HLA-A2 binding as the immunogen. An example is shown in Fig. 1(a). Mice immunized with Vif101(9L) (IC50=3 nM) mounted a strong CTL response against that peptide and raised a somewhat stronger CTL response against epitope Vif101(9M) (IC50=135 nM) (Fig. 1a, left panel) than mice immunized with Vif101(9M) (Fig. 1a, right panel). Also, a high frequency of peptide-specific IFN-{gamma}-producing precursors was observed in mice immunized with epitope Vif101(9L) (Fig. 1b and Table 5). In fact, T-cell cross-recognition of variants was observed in all cases (Table 5, var).



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Fig. 1. T-cell cross-recognition of natural variant and modified designer epitopes with the parent epitopes was measured in CTL and ELISPOT assays. (a, left panel) Peptide-specific CTL responses observed by a classical 51Cr-release assay in one representative C57-A2Kbtg mouse immunized with Vif101(9L), reacting against both Vif101(9L) ({blacksquare}) and the variant Vif101(9M) ({blacktriangleup}). (a, right panel) One representative mouse immunized with Vif101(9M) reacts with only 10 % lysis at an E : T ratio of 50 : 1 against Vif101(9M) ({blacklozenge}). (b) Frequency of IFN-{gamma}-producing cells per 106 splenocytes (s.f.u. per 106 spleen cells) after re-stimulation with medium alone or with the specified peptide in two representative C57-A2Kbtg mice immunized with either Vif101(9L) or Vif101(9M). (c) Example of immunogenicity observed by immunization with an anchor-optimized designer epitope. (Left panel) A representative C57-A2Kbtg mouse immunized with the anchor-optimized epitope Vpu66mod reacts in CTL 51Cr-release assay against HHD-EL4S3-Rob cells pulsed with Vpu66mod ({blacksquare}) and Vpu66 ({blacktriangleup}). (Right panel) A representative mouse immunized with Vpu66 shows <10 % lysis at an E : T ratio of 50 : 1 against Vpu66 ({blacklozenge}). Unpulsed HHD-EL4S3-Rob cells were used as negative control targets cells ({square} and {lozenge}).

 

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Table 5. Immunogenicity and cross-reaction of primary anchor-optimised epitope peptides

Immune responses below the threshold of positivity (<10 % specific lysis at an E : T ratio of 50 : 1 and <10 s.f.u. per 106 cells).

 
Immunogenicity of primary anchor-optimized designer epitopes
To evaluate further the influence of primary anchor modifications on T-cell recognition of HIV-1 CTL epitopes, we tested the ability of anchor-optimized designer epitopes to induce cross-reacting CTL responses to the parent epitopes. Improved binding by anchor-optimization was predicted for 42 of the 52 natural epitopes. However, only 21 of these 42 anchor-optimized peptides showed actual improvement in measured binding as compared to their natural epitopes (Table 5). These were tested subsequently in HLA-A2 transgenic mice. Fig. 1(c) shows an example of a CTL rescue by anchor-optimization of epitope Vpu66. Whereas Vpu66 induced no CTLs to Vpu66-pulsed target cells (Fig. 1c, right panel), Vpu66mod induced a strong CTL response to both Vpu66mod and Vpu66 (Fig. 1c, left panel). Fifteen of the 21 improved epitopes (71 %) were capable of generating a strong CTL response by either specific lysis of HHD-EL4S3-Rob target cells pulsed with the anchor-optimized peptide or specific IFN-{gamma} production. Furthermore, 10 of these 15 (67 %) peptides triggered either a clear cytotoxic response or IFN-{gamma} production against the parent natural epitope. Some examples of clear improvement in immunogenicity were seen. Whereas none of the natural-occurring epitopes, Vpu66, Nef68, Nef188 and Pol606, could induce detectable specific immune responses either in mice or in patients (Table 1), clear CTL responses were produced against them when using the anchor-optimized epitopes as immunogens (Table 5). On the other hand, examples were also found in which anchor substitution totally abrogated the T-cell recognition of the natural epitopes that were already immunogenic [for example, Pol59, Vif149 and Rev66 (Table 5)].


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Using new quantitative ANNs (Buus et al., 2003), we have predicted 4215 candidate HLA-A2 HIV-1 CTL epitopes from full-length HIV protein sequences. As expected, most of these epitopes (71 %) were located in the structural genes Env (50 %), Pol (12 %) and Gag (9 %). However, a large number of epitopes (29 %) was predicted also in the accessory and regulatory proteins Vif (8 %), Vpu (8 %), Nef (6 %), Rev (4 %), Vpr (2 %) and Tat (1 %). Unexpectedly, far fewer epitopes were predicted in Tat, which is of similar genetic diversity and length as Rev and Vpu. If we consider the number of sequences scanned from each protein and their protein lengths, then Vpu, Rev and Env generated the highest numbers of epitopes, which may be explained by the high genetic diversity of these proteins. Recently, special attention has been paid to the CTL responses against accessory and early regulatory proteins in HIV-1-infected patients (Addo et al., 2001; Altfeld et al., 2001a; Novitsky et al., 2001), which emphasizes the need to include such epitopes in candidate vaccines. However, despite the more than 150 HIV-1-specific CTL epitopes now defined (Korber et al., 2000), neither Tat, Vif, Vpu nor Rev epitopes restricted to HLA-A2 have been identified. Despite Tat being targeted frequently by CTL responses in HIV-1-infected patients, no responses to Tat were defined for HLA-A2 patients (Addo et al., 2001; Novitsky et al., 2001). In agreement, our data suggest that Tat proteins would not provide a broad panel of HLA-A2 targets as other HIV-1 accessory and regulatory proteins. Instead, our data points at Vpu as being the most immunogenic and antigenic target. Although Vpu seems to be targeted infrequently by host cellular responses (Addo et al., 2002), the low sequence conservation of most Vpu epitopes may have rendered their identification particularly difficult.

The cut-off value of predicted MHC-I binding was set at IC50=500 nM, expected to be the threshold for immunogenicity (Sette et al., 1994), although exceptions are noted (for example, Table 1, Pol334). In agreement, we found that 25 of the 36 (69 %) natural binders tested (IC50<500 nM) were immunogenic in groups of C57-A2Kbtg mice inducing CTLs and/or IFN-{gamma}. Moreover, a high binding affinity to MHC-I was associated with immunogenicity. For example, 36 of the 47 peptides binding <100 nM were immunogenic in HLA-A2 transgenic mice, whereas only 9 of the 18 peptides binding >100 nM were immunogenic (0·02>P>0·01, chi-squared test). Similar findings have been reported earlier (Sette et al., 1994) and may reflect immune dominance. Although immunogenicity in HLA-A2 transgenic mice generally overlaps with antigenicity for human HLA-A2-restricted CTLs, some differences have been reported (Wentworth et al., 1996). Therefore, we also examined specific CD8+ T-cell reactivity to the 36 HLA-A2 binding (IC50<500 nM) new natural epitopes in immune-competent HIV-1-infected patients controlling their virus without anti-retroviral therapy. Here, we demonstrated specific CD8+ T-cell reactivity for 23 of the 36 new and 5 of 7 known epitopes tested in 17 HLA-A2-identified patients (Table 3). At least one new HIV-1 epitope and an average of four HIV-1 epitopes were recognized per patient. A suggested association between numbers of different proteins or epitopes recognized and years of virus control (Chouquet et al., 2002) was not clear from our limited data. More epitopes and more patients are needed to clarify this. Two of the seven known HLA-A2 HIV epitopes tested in patients were not recognized. One of these, Nef190 (AFHHVAREL), did show a very low binding to HLA-A2 (IC50=65 000 nM). In this respect, our data support the debated non-HLA-A2-binding nature of the Nef190 peptide (Hunziker et al., 1998). However, the patient study demonstrates proper antigen processing, peptide antigenicity and immunogenicity induced during a natural HIV infection of these 23 new HIV CD8+ T-cell epitopes. Only 17 of the 25 epitopes that were immunogenic in mice were also immunogenic in the natural infections, as evidenced by the 17 patients examined. The lack of epitopes in the patients' HIV-1 strains, lack of proper epitope processing and/or the immune dominance of other epitopes could cause this. As more patients, especially from other parts of the world with higher frequencies of HIV-1 non-B subtypes, are investigated, the remaining eight peptides may also turn out to be immunogenic.

Interestingly, six epitopes that tested negative in mice reacted positively in patients (Table 1), which may indicate some limitation in the transgenic mice model used (Wentworth et al., 1996). Thus, a total of 31 new HIV CTL epitopes (IC50<500 nM) were immunogenic in HLA-A2-positive mice and/or humans. This significantly increases the numbers of HLA-A2 HIV-1 CTL epitopes identified and includes the first ever reported in the accessory and regulatory proteins Vif, Vpu and Rev. For practical reasons, we only tested about a third of the epitopes that matched our selection criteria and we can, therefore, project that the real number of novel epitopes may be as much as three times higher. Interestingly, some of the new immunogenic HIV-1 epitopes were, in some cases, also recognized by non-HLA-A2 patients (data not shown). Such cross-reactions of epitopes among different HLA types are seen commonly and may imply a broader HLA coverage of vaccines designed from such HIV-1 CTL epitopes.

Many of the new epitopes are surprisingly conserved among all HIV-1 strains and subtypes, whereas other epitopes with a relatively low global conservation could be highly conserved among certain subtypes (Table 4). A few natural epitopes differ from each other at one of the primary anchor residues and, for example, epitope Vif101(9L) is found mainly in clades B and D, whereas the related epitope Vif101(9M) is conserved in clades C, D, H and J (Table 4). Thus, another aspect of our approach is that it identifies epitopes, which could be of potential value in the design of vaccines tailored at the HIV-1 subtypes found frequently in the non-industrialized world (Table 4). Despite the serious health problems of HIV infection in these parts of the world, little research has, until now, focused on these subtypes. An optimal immunogen should generate an immune response against as many HIV-1 strains as possible and we found that T-cell cross-recognition of variants was in fact observed in all cases (Table 5, var). The apparently improved immunogenicity of the variants Vif101(9L) and Vif23(9V) may be regarded as natural ‘anchor-optimized’ epitopes (Table 1). This demonstrates that it may be important to include in a vaccine the variant CTL epitope with the highest binding affinity even though is may appear to have a lower conservation. The cross-reaction demonstrated among natural variants increases the repertoire of target HIV-1 strains and represents natural examples of the proposed strategy of anchor improvement. In our study, globally conserved epitopes (>8 %) represented less than 7 % of all the 4215 HIV-1 epitopes predicted. Interestingly, more than 70 % of these were predicted as intermediate binders and are, therefore, probably subdominant epitopes. The prerequisites for selecting a subdominant epitope as a vaccine candidate will be to demonstrate that this HIV-1 epitope, although it may not be immunogenic, it is at least antigenic, and then improve its immunogenicity in order to induce a strong consistent CTL response in vivo. One approach to enhance immunogenicity is to improve the peptide–MHC-binding capability by optimizing primary anchor residues in designer epitope peptides (Berzofsky, 1993; Berzofsky et al., 1999). Therefore, primary anchor-optimization was performed and 15 of the 21 optimized designer epitopes (71 %) were capable of raising a strong homologous CTL response. Importantly, 10 of these 15 (67 %) peptides triggered either a clear cytotoxic response or IFN-{gamma} production against the parent natural epitope. A more accurate quantification of any improvement of immunogenicity would have required peptide titrations; however, some examples of clear improvement in immunogenicity were seen. On the other hand, there were also examples that anchor substitution could totally abrogate the T-cell recognition of the natural epitopes that were already immunogenic [for example, Pol59, Vif149 and Rev66 (Table 5)]. In the latter cases, the substitution of the primary anchor residues most probably led to a conformational change in the peptide destroying the T-cell receptor recognition. Taken together, anchor improvements might enhance both immunogenicity and the degree of HIV-1 vaccine coverage. Also, optimization of a secondary anchor position (P1) may be considered (Firat et al., 2001).

CTL responses against a few immune-dominant epitopes have been reported recurrently in HIV-1 patients. This has lead to the inclusion of these epitopes as vaccine candidates (Hanke & McMichael, 2000). However, the protective role of such epitopes in HIV infection remains to be demonstrated. It is particularly noteworthy that CTL responses in exposed seronegative people and long-term non-progressors are not directed towards immune-dominant CTL epitopes in rapid progressors (Kaul et al., 2001; Rowland-Jones et al., 2001). In this context, it is very interesting that subdominant CTL epitopes have shown themselves to be as potent as immune-dominant epitopes in inducing protection against lymphocytic choriomeningitis virus infection (Gegin & Lehmann-Grube, 1992; Rodriguez et al., 2001; van der Most et al., 1997) and virus-induced tumours (Feltkamp et al., 1995). Also, in the macaque model, the inclusion of subdominant epitopes in vaccinations leads to a broader CTL response after challenge (Santra et al., 2002). Thus, we propose that novel HIV vaccines should contain multiple subdominant epitopes that are globally more conserved, within certain subtypes or within a defined geographical area, and that the inclusion of anchor-improved variants should be considered. Moreover, turning antigens (Vpu66, Nef68, Nef188 and Pol606) into immunogens (Vpu66mod, Nef68mod, Nef188mod and Pol606mod) may render these immunologically silent HIV-1 epitopes as a complete new vaccine target. The advantage of such a strategy would be that far more virus epitopes would be available and the likelihood of generating a more successful multi-epitope approach should, therefore, be improved considerably. The disadvantage is that the identification of subdominant HIV-1 epitopes requires a ‘reverse immunology’ approach; however, this is exactly what the quantitative ANN-driven predictions of antigen presentation used in this study may facilitate.

In conclusion, we identified 31 new HIV-1 CTL epitopes immunogenic in HLA-A2 mice and/or patients, four antigenic, but not immunogenic, epitopes, plus 10 anchor-optimized epitopes that are being evaluated for vaccine candidates. Of the naturally occurring immunogenic epitopes, 13 were found in accessory and early regulatory proteins, including the first HLA-A2 minimal epitopes described in Vif, Rev and Vpu. The new HIV CTL epitopes characterized should have implications for further patient studies and HIV vaccine design. Intermediate-binding epitopes of low or no immunogenicity and, therefore, subdominance were found to be antigenic and more conserved. Such epitope peptides could be anchor-optimized to improve immunogenicity and to increase further the number of potential vaccine epitopes. As a novel broad vaccine approach, it is proposed to include multiple subdominant epitopes that are more conserved as anchor-optimized variants.


   ACKNOWLEDGEMENTS
 
We acknowledge the technical assistance of Ms Kirsten Boedker, Ms Nina Frølund, Ms Irene Jensen, Ms Gerda Jensen, Ms Birgit Knudsen and Ms Anne Lyhning. The authors are grateful to Drs N. Holmes and F. Lemonnier for providing the HLA-A2 transgenic mouse and the HHD-EL4S3-Rob cell line, respectively. This work was supported by grants from the Danish Research Council's ‘Technology by Highly Oriented Research’ (THOR) programme, the Danish AIDS Foundation and the EU 5FP QLGT-1999-00713.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 10 February 2003; accepted 1 May 2003.



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