Proteins from Simian Immunodeficiency Virus of ...

This information is current as of June 13, 2013.

Characterization of the Peptide-Binding Specificity of Mamu-B*17 and Identification of Mamu-B*17-Restricted Epitopes Derived from Simian Immunodeficiency Virus Proteins Bianca R. Mothé, John Sidney, John L. Dzuris, Max E. Liebl, Sarah Fuenger, David I. Watkins and Alessandro Sette J Immunol 2002; 169:210-219; ; http://www.jimmunol.org/content/169/1/210

Subscriptions Permissions Email Alerts

This article cites 61 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/169/1/210.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscriptions Submit copyright permission requests at: http://www.aai.org/ji/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/cgi/alerts/etoc

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

Downloaded from http://www.jimmunol.org/ by guest on June 13, 2013

References

The Journal of Immunology

Characterization of the Peptide-Binding Specificity of Mamu-B*17 and Identification of Mamu-B*17-Restricted Epitopes Derived from Simian Immunodeficiency Virus Proteins1 Bianca R. Mothe´,*† John Sidney,‡ John L. Dzuris,‡ Max E. Liebl,* Sarah Fuenger,* David I. Watkins,*† and Alessandro Sette2‡

S

imian immunodeficiency virus-infected rhesus macaques represent an important disease model for the study of the pathogenicity of HIV and to test vaccine candidates (1). Several independent lines of evidence have implicated cellular immunity, in general, and CTL in particular, as playing a crucial role in the control of SIV and HIV viral replication (for reviews, see Refs. 2– 4). In this context, it is important to develop accurate methods to identify epitopes that bind and are presented to CTL by rhesus macaque MHC class I molecules. Several different methods have been used to predict and identify peptides bound by MHC class I molecules. Sequencing of pooled or individual naturally occurring ligands, together with definition of optimal T cell epitopes accurately identifies anchor positions and the most frequently used residues (canonical motifs) (5, 6) (see Ref. 7 for review). Peptide/MHC-binding assays with purified molecules usually reveal that a broader set of residues is permissible in the anchor positions (extended motifs) (8 –17). It has been commonly observed that motifs based solely on primary anchor positions are

*Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, WI 53715; †Department of Pathology, University of Wisconsin, Madison, WI 53706; and ‡Epimmune, San Diego, CA 92121 Received for publication February 4, 2002. Accepted for publication April 18, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by National Institutes of Health/National Institute of Allergy and Infectious Diseases Contract N01-AI-95362 (to A.S.) and National Institutes of Health Grant R24 RR15371 (to D.I.W.). 2 Address correspondence and reprint requests to Dr. Alessandro Sette, Epimmune, 5820 Nancy Ridge Drive, San Diego, CA 92121. E-mail address: asette@ epimmune.com

Copyright © 2002 by The American Association of Immunologists, Inc.

somewhat inaccurate in the sense that only a fraction of the motifbearing peptides actually binds the specified MHC molecule. Thus, to efficiently identify MHC-binding peptides, information from analysis of secondary anchor positions and the use of quantitative peptide/MHC-binding assays must be taken into account. To date, we have used this approach to refine motifs for over 20 different MHC class I molecules. To date, the most rigorously characterized rhesus macaque class I molecule is Mamu-A*01 (18 –30). Initial studies used elution of naturally processed peptides and live cell peptide-binding assays (30). Recently, the peptide-binding specificity of Mamu-A*01 was more rigorously characterized through the development of quantitative binding assays that used purified Mamu-A*01 molecules. These assays allowed analysis of the binding capacity of single substitution analogs of a model Mamu-A*01 ligand and of a large library of peptides corresponding to naturally occurring sequences (17). This detailed motif was used to identify 221 Mamu-A*01 motif-bearing peptides derived from SIV proteins. The binding capacity of these motif-positive peptides was determined using purified Mamu-A*01 molecules, and 37 were found to bind Mamu-A*01 with apparent Kd values of 500 nM or better. Interestingly, 21 of these peptides bound Mamu-A*01 with higher affinity than the previously identified immunodominant SIV epitope Gag181–189 (CM9). In a recent study, PBMCs from SIV-infected Mamu-A*01⫹ macaques recognized 14 Mamu-A*01-restricted CTL epitopes in ELISPOT, CTL, or tetramer analyses (18). Allen et al. (19) subsequently showed viral escape from one of these epitopes, Tat28 –35(SL8), early in the acute phase of viral infection. The Gag181–189(CM9) epitope has been the focus of several vaccine studies aiming at eliciting CTL responses (24, 31). Besides 0022-1767/02/$02.00


The SIV-infected rhesus macaque is an excellent model to examine candidate AIDS virus vaccines. These vaccines should elicit strong CD8ⴙ responses. Previous definition of the peptide-binding motif and optimal peptides for Mamu-A*01 has created a demand for Mamu-A*01-positive animals. We have now studied a second MHC class I molecule, Mamu-B*17, that is present in 12% of captive-bred Indian rhesus macaques. The peptide-binding specificity of the Mamu-B*17 molecule was characterized using single substitution analogs of two Mamu-B*17-binding peptides and libraries of naturally occurring sequences of viral or bacterial origin. Mamu-B*17 uses position 2 and the C terminus of its peptide ligands as dominant anchor residues. The C terminus was found to have a very narrow specificity for the bulky aromatic residue W, with other aromatic residues (F and Y) being only occasionally tolerated. Position 2 is associated with a broad chemical specificity, readily accommodating basic (H and R), bulky hydrophobic (F and M), and small aliphatic (A) residues. Using this motif, we identified 50 peptides derived from SIVmac239 that bound Mamu-B*17 with an affinity of 500 nM or better. ELISPOT and intracellular cytokine-staining assays showed that 16 of these peptides were antigenic. We have, therefore, doubled the number of MHC class I molecules for which SIV-derived binding peptides have been characterized. This allows for the quantitation of immune responses through tetramers and analysis of CD8ⴙ function by intracellular cytokine-staining assays and ELISPOT. Furthermore, it is an important step toward the design of a multiepitope vaccine for SIV and HIV. The Journal of Immunology, 2002, 168: 210 –219.


Materials and Methods Peptides Peptides for general screening were purchased as crude material from PepScan Systems (Lelystad, The Netherland) or synthesized at Epimmune (San Diego, CA) using standard tertiary butyloxycarbonyl or fluronylmethyloxycarbonyl solid phase methods (37). Peptides were resuspended at 4 –20 mg/ml in 100% DMSO, then diluted to required concentrations in PBS 0.05% Nonidet P-40. Peptides for use as radiolabeled probes were purified to ⬎95% homogeneity by reverse phase HPLC, and composition was ascertained by amino acid analysis, sequencing, and/or mass spectrometry analysis. Radiolabeling was done using the chloramine T method (38). SIVmac239 sequence(s) (accession no. M33262) was analyzed using the text string search software program Motifsearch 1.4 (D. Brown, San Diego, CA) to identify potential peptide sequences containing defined motifs.

Mamu-B*17 purification The 721.221 cells transfected with Mamu-B*17 cDNA were used as the source of Mamu-B*17 molecules. Cells were maintained in vitro by culture in RPMI 1640 medium (Flow Laboratories, McLean, VA); supplemented with 2 mM L-glutamine (Life Technologies, Grand Island, NY), 100 U (100 ␮g/ml) penicillin-streptomycin solution (Life Technologies), and 10% heat-inactivated FCS (Gemini Bio-Products, Calabasas, CA); and grown for large scale cultures in roller bottle apparatuses. Mamu-B*17 molecules were purified from cell lysates using affinity chromatography (38). Briefly, cells were lysed at a concentration of 108 cells/ml in 50 mM Tris-HCl, pH 8.5, containing 1% Nonidet P-40 (Fluka Biochemika, Buchs, Switzerland), 150 mM NaC1, 5 mM EDTA, and 2 mM PMSF. Lysates were then passaged through 0.45-␮M filters and cleared of nuclei and debris by centrifugation at 10,000 ⫻ g for 20 min, and MHC molecules were purified by affinity chromatography. For affinity purification, columns of inactivated Sepharose CL4B and protein A-Sepharose were used as precolumns. Mamu-B*17 was captured by repeated passage over protein A-Sepharose beads conjugated with the 3 Abbreviations used in this paper: ICS, intracellular cytokine-staining assay; ARB, average relative binding value; SFC, spot-forming cell.

anti-HLA (A, B, C) Ab W6/32. After two to four passages, the W6/32 column was washed with 10-column volumes of 10 mM Tris-HCl, pH 8.0, with 1% Nonidet P-40, 2-column volumes of PBS, and 2-column volumes of PBS containing 0.4% n-octylglucoside. Finally, Mamu-B*17 molecules were eluted with 50 mM diethylamine in 0.15 M NaC1 containing 0.4% n-octylglucoside, pH 11.5. A 1/26 volume of 2.0 M Tris, pH 6.8, was added to the eluate to reduce the pH to ⬃8.0. The eluate was then concentrated by centrifugation in Centriprep 30 concentrators at 2000 rpm (Amicon, Beverly, MA). Protein purity, concentration, and effectiveness of depletion steps were monitored by SDS-PAGE.

Mamu-B*17-binding assay Quantitative assays for the binding of peptides to soluble Mamu-B*17 molecules were based on the inhibition of binding of a radiolabeled standard probe peptide using the same protocol described for the measurement of peptide binding to HLA class I molecules (38). Briefly, 1–10 nM radiolabeled peptide was coincubated at room temperature with 1 ␮M to 1 nM of purified Mamu-B*17 in the presence of 1 ␮M human ␤2-microglobulin (Scripps Laboratories, San Diego, CA) and a mixture of protease inhibitors. The radiolabeled peptide used was a position 9W3Y analog of the SIV-derived epitope Nef165–173 (IW9; sequence IRFPKTFGY; see Refs. 36 and 39). After a 2-day incubation, the percentage of MHC-bound radioactivity was determined by size exclusion gel filtration chromatography on a TSK 2000 column. Alternatively, the percentage of MHC-bound radioactivity was determined by capturing MHC/peptide complexes on Optiplates (Packard Instrument, Meriden, CT) coated with the anti-class I mAb W6/32 and determining bound cpm using the TopCount microscintillation counter (Packard Instrument). In the case of competitive assays, the concentration of peptide yielding 50% inhibition of the binding of the radiolabeled probe peptide was calculated. Peptides were initially tested at one or two high doses. The IC50 of peptides yielding positive inhibition were then determined in subsequent experiments, in which two to six further dilutions were tested, since under the conditions to be used, in which radiolabeled peptide ⬍ MHC and IC50 ⱖ MHC, the measured IC50 values are reasonable approximations of the true Kd values. Each competitor peptide was tested in two to four completely independent experiments. As a positive control, in each experiment the unlabeled version of the radiolabeled probe was tested. For detailed analysis of the peptide-binding data, and to allow comparison of data obtained in different experiments, a relative binding value was calculated for each peptide by dividing the IC50 of the positive control for inhibition by the IC50 for each tested peptide. These values can subsequently be converted back into IC50 nM values by dividing the IC50 nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation has proved to be the most accurate and consistent for comparing peptides that have been tested on different days or with different lots of purified MHC. Standardized relative binding values also allow the calculation a geometric mean, or average relative binding value (ARB), for all peptides of a particular characteristic (14, 15, 37, 40 – 43). For library analyses, ARB values were standardized relative to the ARB of peptides carrying the residue at the same position associated with the best binding.

IFN-␥ ELISPOT assay Ninety-six-well flat-bottom plates (U-Cytech BV, Utrecht, The Netherlands) were coated with 5 ␮g anti-IFN-␥ mAb MD-1 (U-Cytech-BV) overnight at 4°C. The plates were then washed 10 times with PBST, PBS (Life Technologies) containing 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO), and then blocked with 2% PBSA, PBS containing 2% BSA (SigmaAldrich) for 1 h at 37°C. PBSA (2%) was discarded from the plates, and freshly isolated PBMC were added. Cells were resuspended in RPMI 1640 (Mediatech, Herndon, VA) supplemented with penicillin, streptomycin, and 5% FBS (Biocell, Rancho Dominguez, CA) (R05). The R05 also contained either 10 ␮g/ml Con A (Sigma-Aldrich), 10 ␮g/ml of each of the Mamu-B*17 peptides (see Table II), 10 ␮g/ml negative control influenza peptide (SNEGSYFF), or no peptide. Input cell numbers were 1.0 ⫻ 105 PBL in 100 ␮l/well, in triplicate wells. The plates were incubated with the cells overnight (16 h) at 37°C, 5% CO2. The cells were then removed by shaking them off the plates, and 200 ␮l/well ice-cold deionized water was added to lyse the remaining PBMC. The plates were incubated on ice for 15 min, after which they were washed 20 times with PBST. Next, 1 ␮g/well rabbit polyclonal biotinylated detector Ab solution (U-Cytech BV) was added and the plates were incubated for 1 h at 37°C. The plates were washed 10 times with PBST, after which 50 ␮l/well gold-labeled anti-biotin IgG solution (U-Cytech BV) was added. The plates were once again incubated for 1 h at 37°C and washed 10 times with PBST. A total of 30 ␮l/well activator mix (U-Cytech BV) was then


revealing an unprecedented complexity and diversity of anti-SIV CTL responses, the extensive characterization of SIV-derived peptides that bind Mamu-A*01 represented an important step toward the design of a multiepitope vaccine for SIV and HIV. Definition of a peptide-binding motif has facilitated the identification of minimal and optimal Mamu-A*01 epitopes, allowing for the quantitation of naturally occurring or vaccine-induced CD8⫹ responses using tetramers and the development of intracellular cytokine-staining assays (ICS)3 and ELISPOT assays (18 – 21, 24, 31, 32). However, this has created an intense demand for Mamu-A*01-positive animals, even though Mamu-A*01 is present in ⬃22% of the captive-bred Indian rhesus macaque (33). Only a very few SIV-derived epitopes that bind to other MHC class I molecules are known (34). In this respect, it was of interest to determine whether the remarkably broad repertoire associated with Mamu-A*01 is a general feature of SIV-specific CTL responses in macaques, or rather a peculiarity associated with the Mamu-A*01 allele. Previous studies using live cell-binding assay systems started to define the general peptide-binding motifs of four additional Mamu class I molecules (A*11, B*03, B*04, and B*17) (35). Mamu-B*17 is present in over 12% of the captive-bred Indian rhesus macaque population (36), making this class I molecule a good candidate for extensive characterization. In the present study, we have studied in detail the peptide-binding specificity of Mamu-B*17, identified SIV-derived Mamu-B*17 peptides, and probed the breadth of SIV-specific Mamu-B*17-restricted CTL responses in SIV-infected animals. We describe 16 epitopes that bind to Mamu-B*17. These epitopes will allow more accurate quantitation of SIV immune responses after natural infection or vaccination, with the synthesis of tetramers and in vivo functional assays such as ICS and ELISPOT.

211

212

MAMU-B*17 PEPTIDE-BINDING SPECIFICITY/SIV-DERIVED EPITOPES

added, and the plates were developed for ⬃30 min. The activator mix consists of a silver salt solution that precipitates at the sites of gold clusters (from the gold-labeled anti-biotin solution), visualizing the sites in which the IFN-␥ was secreted. Once these sites or black spots could be seen in the wells under an inverted microscope, the wells were washed with distilled water to stop development. The plates were then air dried. Wells were imaged with IP Lab Spectrum 3.23 software using a Hamamatsu C4880 series camera attached to a Nikon TE 300 inverted microscope. Spots were counted manually. A spot-forming cell (SFC) was defined as a large black spot with a fuzzy border (44). To determine significance levels, the average of the number of SFCs and SD for each peptide was calculated. Background (sample with no peptide) levels were subtracted from each peptide average. A response was considered positive if the number of SFCs exceeded twice the level of the sample with no peptide. Assay results are shown as SFC p/1 ⫻ 106 cell. Responses to Con A (positive control) were always greater than 1000 SFCs per 1 ⫻ 106 cells.

Intracellular IFN-␥ cytokine staining

Results Binding capacity of single substitution analogs of a MamuB*17-restricted CTL epitope Previously, the SIV Nef165–173 peptide (IW9; sequence IRFPKT FGW) was identified as a Mamu-B*17-restricted CTL epitope (36, 39). In the present study, panels of single substitution analogs of this epitope were examined for their capacity to bind purified Mamu-B*17 molecules. For these analyses, as in previous studies of HLA class I molecules (41), preferred anchor residues were defined as those whose binding capacity is 0.1 or better, relative to the binding capacity of the optimal residue. Residues whose binding capacity is between 0.01 and 0.1 were defined as tolerated. Finally, residues whose binding capacity is less than 0.01 were considered as nontolerated. The specificity at each position was investigated in detail using multiple analogs. For each position, at least one peptide representing a conservative, semiconservative, and nonconservative substitution was analyzed. As shown in Fig. 1A, at positions 1, 3, 4, and 8, none of the substitutions resulted in reductions in binding capacity of greater than 10-fold. At positions 5, 6, and 7, only one or two substitutions were associated with reductions in binding capacity in the 10- to 100-fold range; all other substitutions represented less than 10-fold reductions. These data suggest that the middle of the peptide may have some secondary influence on Mamu-B*17-binding capacity. At position 2, substitution of the positively charged and bulky residue R to the similar basic residue H or the bulky polar aliphatic residue Q was preferred, with relative binding values between 0.6 and 1.0. Interestingly, substitution to the small residues A, P, and S was also in the preferred range, with relative binding values

Detailed characterization of the preferred ligand size and primary anchor specificity of Mamu-B*17 A large library of peptide ligands carrying W at the C terminus was next analyzed to determine the correlation between peptide length (between 8 and 11 residues) and binding capacity. Each peptide represented a naturally occurring sequence of either viral or bacterial origin. It was found that 29 of 117 (24.8%) of the 9-mer peptides, 21 of 112 (18.8%) of the 10-mer peptides, and 4 of 23 (17.4%) of the 11-mer peptides bound Mamu-B*17 with IC50 of 500 nM or less. Peptides of 8 residues in length were only occasionally capable of binding. Specifically, 2 of 27 (7.4%) of the 8-mer peptides tested were Mamu-B*17 binders. In conclusion, this data have indicated that the optimal ligand size for MamuB*17 is between 9 and 11 residues in length. Subsets of the same large peptide library were next used to define the primary anchor specificity of Mamu-B*17. Library analyses offer the advantage that they avoid potential biases introduced by the use of analogs, in which the effect of a given residue is determined only in the context of a single amino acid sequence. The effect of a specific amino acid residue was determined by calculating the ARB value (see Materials and Methods) associated with each residue. The percentage of peptides bearing a specific residue at each anchor position that bound Mamu-B*17 with an IC50 of 500 nM or better was also calculated. As was done in the single substitution analyses, preferred residues were defined as those associated with an ARB between 0.1 and 1. Residues whose ARB was in the 0.02– 0.1 range were defined as tolerated. Finally, residues whose ARB was ⬍0.02 were considered nontolerated. The first library subset was used to probe the specificity at position 2 (Table I). Each peptide in the set analyzed was between 9 and 11 residues in length, and carried the aromatic residue W at the C terminus. W was identified in the single substitution analyses above as the optimal C-terminal anchor residue. It was found that the highest binding affinity was associated with the positively charged bulky residue H and the small aliphatic residue A, which had ARB of 1 and 0.6, respectively. The bulky residues F, R, and M were also preferred, with ARB between 0.1 and 0.2. It was


A total of 5 ⫻ 105 PBMC from three Mamu-B*17-positive animals, 2065, 2095, and 2129, was tested at 3 wk postinfection. Cells were incubated at 37°C for 1.5 h with anti-CD28 and anti-CD49d Abs (0.5 ␮g each Ab; BD PharMingen, San Diego, CA) and either staphylococcal enterotoxin B (10 ␮g/ml; Sigma-Aldrich), Mamu-B*17 peptides Nef165–173(IW9), Vif64 –73 (GW10), Vif66 –73 (HW8), or a negative control influenza peptide (SNEGSYFF) (1 ␮g each peptide/sample). A total of 10 ␮g/ml brefeldin A (10 ␮g/ml, Sigma) was added to inhibit protein trafficking, and cells were incubated a further 5 h at 37°C. Cells were then washed with FACS buffer (PBS ⫹ 2% FCS) and resuspended in 100 ␮l FACS buffer. Cells were surface stained with Abs specific for CD8␣-PerCP and CD4-APC (BD PharMingen) for 40 min at room temperature. Cells were washed twice with FACS buffer and fixed with 2% paraformaldehyde (PBS ⫹ 2% paraformaldehyde (Sigma-Aldrich)). Cells were placed at 4°C overnight. The following day, cells were washed once with FACS buffer and twice with permeabilization buffer (0.1% saponin (Sigma-Aldrich) in FACS buffer). Cells were then incubated in the dark for 50 min, stained with Abs specific for IFN-␥ FITC and for TNF-␣ PE (BD PharMingen) at room temperature. Cells were then washed two times with 0.1% saponin-buffer. Finally, a 100 ␮l cell suspension was fixed with 250 ␮l 2% paraformaldehyde. Acquisition was performed on a FACSCalibur flow cytometer collecting 100,000 – 200,000 lymphocyte-gated events per sample.

between 0.1 and 0.7. Substitution to other residues representing an assortment of chemical specificities, including G, K, E, L, T, and V, was tolerated, and associated with relative binding values between 0.01 and 0.07. All other substitutions tested were not tolerated, and associated with relative binding values lower than 0.01. In contrast to the rather permissive specificity of position 2, only the bulky aromatic residues W and F were allowed at the C terminus. W was the most preferred, while F was associated with a relative binding value of 0.15. None of the other residues tested were tolerated, being associated with relative binding values ⬍0.01. Single substitution analogs of a second Mamu-B*17 high affinity binder B35CON1 (sequence FPFKYAAAF) were also tested for their capacity to bind Mamu-B*17 (Fig. 1B). In agreement with patterns noted in the context of the IW9 analog panel, substitutions representing a wide range of chemical specificity were tolerated in position 2, and only a bulky aromatic residue was allowed at the C terminus. Significant decreases in Mamu-B*17 binding were similarly associated with substitutions in the middle of the peptide (positions 5, 6, and 7), underscoring the observation that the middle region of the peptide does have an important secondary influence on Mamu-B*17-binding capacity. Interestingly, and in contrast to the observations from the IW9 analysis, significant reductions in binding capacity were also noted at positions 1 and 3, suggesting that the residues in these positions are likely to be dominant secondary anchors.


213

noted that 38% or more of the peptides with these preferred residues in position 2 bound Mamu-B*17 with affinities of 500 nM or better. More specifically, 5 of 7 (71.4%) of the peptides with H, 6 of 10 (60.0%) with A, 4 of 7 (57.1%) with M, 4 of 10 (40%) with F, and 7 of 18 (38.9%) with R were Mamu-B*17 binders. Additional residues representing a broad range of chemical specificities were also found to be tolerated in position 2. These residues were associated with ARB in the 0.02– 0.08 range, and included the small residues P, S, C, T, and G; the aliphatic residues L and Q; the basic residue K; and the bulky aromatic residues W and Y. In general, 10 –25% of the peptides with these residues were Mamu-B*17 binders. Other residues, including the negatively charged residues D and E, the aliphatic/hydrophobic residues I and V, and the polar residue N, were associated with ARB ⬍0.02, and were not found in any peptide-binding Mamu-B*17 with affinities of 500 nM or better. With the exception of F, these data are largely in agreement with the patterns observed in the single amino acid substitution panels. It is also notable that, again, a broad chemical specificity could be associated with position 2. That is, the type of residues preferred or tolerated at position 2 represents a wide range of chemical types, including bulky hydrophobic (M, L), aromatic (F, W, Y), or basic residues (H, R, K), to small and/or aliphatic residues (A, P, S, Q, T, G). Next, the specificity at the C terminus was examined using a library comprised of 9- to 11-mer peptides that carried the pre-

ferred residues H, A, R, M, or F in position 2. When these peptides were tested for their Mamu-B*17-binding capacity, it was found that only W was associated with an ARB ⬎0.1; 26 of 52 (50%) of the peptides with W at the C terminus bound Mamu-B*17 with an IC50 of 500 nM or better (Table I). The aromatic residues F and Y were tolerated, with ARB in the 0.05– 0.08 range. However, it was noted that peptides with these residues were only infrequently Mamu-B*17 binders. Specifically, 1 of 9 (11.1%) of the peptides with F, and 1 of 36 (2.8%) of the peptides with Y had affinities better than 500 nM. Peptides with other residues were rarely found in Mamu-B*17 binders (3.7%), and were associated with ARB ⬍0.02. Thus, in agreement with the single amino acid substitution analyses, it is concluded that the bulky aromatic residue W is the preferred C-terminal main anchor specificity of Mamu-B*17, with Y and F residues being occasionally tolerated. Identification of SIVmac239-derived Mamu-B*17-binding peptides To identify peptide ligands derived from those proteins that could represent candidates for use in an SIV vaccine, sequences of SIVmac239 proteins were scanned for the presence of peptides with W at the C terminus. Peptides with the tolerated aromatic residues F and Y at the C terminus were also selected, despite the relative infrequency that these C-terminal anchor residues are associated with Mamu-B*17-binding peptides. A total of 460 sequences was identified.


FIGURE 1. Relative Mamu-B*17-binding capacity of single substitution analogs of A, SIV Nef165–173 (IW9; sequence IRFPKTFGW), and B, the nonnatural peptide B35CON2 (sequence FPFKYAAAF). Binding capacity is shown as a ratio relative to the analog with the highest binding affinity among the set of peptides with substitutions at the same position. Substitutions were described as preferred, tolerated, or nontolerated, as described in the text. The Mamu-B*17-binding capacity of SIV Nef165–173 IW9 and B35CON1 is 2.8 and 12 nM, respectively.

214


Table I. Analysis of the fine specificity of Mamu B*17 using peptide libraries

n

ARB

% Binding Peptides

Position 2a H A M R F

7 10 7 18 10

1.0 0.6 0.2 0.2 0.1

71.4 60.0 57.1 38.9 40.0

L P Q K S C W Y T G

16 32 18 22 9 3 9 12 14 12

0.08 0.05 0.04 0.04 0.04 0.03 0.03 0.03 0.02 0.02

18.8 28.1 22.2 13.6 22.2 0.0 22.2 16.7 14.3 8.3

N I E V D

8 13 19 8 5

— — — — —

0.0 0.0 0.0 0.0 0.0

Total C terminusb W

252

21.4

52

1.0

50.0

F Y

9 36

0.08 0.05

11.1 2.8

Other

27

—

3.7

Total

124

23.4

a The library subset analyzed was comprised of peptides 9 –11 residues in length, and that had W at their C terminus. A dash indicates ARB ⬍ 0.01. b The library subset analyzed was comprised of peptides 9 –11 residues in length, and that had H, A, M, R, or F in position 2. A dash indicates ARB ⬍ 0.01.

When the corresponding peptides were synthesized and tested for their capacity to bind Mamu-B*17, 50 were found to have affinities for Mamu-B*17 of 500 nM or less (Table II). Of the 50 binders, 13 bound with an IC50 of 50 nM or less, while the remaining 37 bound with intermediate affinity (IC50 in the 51–500 nM range). Not surprisingly, 46 of the 50 binders had W at the C terminus. It was also noted that 39 of the Mamu-B*17 binders are conserved between SIVmac239 and SIVmac251, while 11 others are unique to SIVmac239. Finally, the 50 peptides identified were derived from 7 different SIV proteins, including 20 peptides from Env, 10 each from Vif and Pol, 5 from Nef, 2 each from Vpx and Gag, and 1 from Vpr. Analysis of SIV-infected rhesus macaques identifies 15 novel Mamu-B*17-restricted responses Next, ELISPOT assays were performed to identify the peptides capable of eliciting Mamu-B*17-restricted responses. Specifically, it was assessed whether the selected peptides were actually recognized in vivo by fresh PBMC derived from SIV-infected MamuB*17-positive animals. As a control, responses to the peptides studied in one SIV-infected Mamu-B*17-negative animal (95084) were monitored. None of the 50 peptides induced responses in this animal (data not shown). Using IFN-␥ ELISPOT analysis of fresh PBMC derived from four SIV-infected Mamu-B*17-positive macaques, reactivity to 16

Discussion In this study, the molecular features of peptide ligands bound by the common rhesus macaque-derived MHC class I molecule Mamu B*17 are characterized in detail. Using this knowledge, we identified 15 new SIV-derived CTL epitopes that bind to MamuB*17. This is only the second MHC class I molecule for which SIV-derived binding peptides have been thoroughly investigated. Identification of these epitopes will allow for the accurate measurement and analysis of CTL responses after natural infection or immunization, through ICS, ELISPOT, and the synthesis of tetramers. As in the case of other macaque, mouse, and human MHC class I molecules, position 2 and the C terminus were found to be the main Mamu-B*17 anchor residues. In accordance with previous data (35), the C terminus was found to be associated with a very narrow specificity, with only the aromatic residue W being preferred. Other aromatic residues (Y and F) were only occasionally tolerated. In striking contrast, position 2 appears to have a broad chemical specificity. In this study, basic (H and R), bulky hydrophobic (F and M), and even small (A) residues were preferred. Positions 1 and 3 most likely function as dominant secondary anchors. Residues in the middle of the peptide (e.g., positions 5–7) may also have some secondary influence on Mamu-B*17-binding capacity. The very narrow specificity exhibited by Mamu-B*17 at the C terminus is relatively unique among the various Mamu, Patr, and HLA class I molecules studied to date. With the exception of HLA-A*0101, in most cases examined in detail a range of residues of similar chemical nature is typically tolerated at the C terminus. Thus, for example, most HLA-B molecules found to have a Cterminal preference for a specific hydrophobic residue will also


Residue

of these peptides was demonstrated (Fig. 2 and Table II). The number of SFCs detected against each peptide ranged from 40 to 497 per 1 ⫻ 106 cells. Not all peptides were recognized by all of the animals, and considerable variability existed from animal to animal with respect to the peptides recognized. Nine peptides were recognized in animal 95061, the animal with the largest repertoire of responses. This animal made a strong response to the previously described Nef165–173 (IW9) peptide (36, 39). The best response was detected in animal 95096, with 497 SFCs to Vif66 –73 (HW8). A response to this peptide was also seen in animal 95061. The other shared responses were to Nef195–203 (MW9) (animals 95061, 1937, and 96072), Nef165–173 (IW9) (animals 95061, 1937, and 95096), Nef165–175 (IW11) (animals 95061 and 95096), Nef194 –203 (LW10) (animals 95061, 1937, 95096, and 96072), Vif64 –73 (GW10) (animals 96072 and 1937), Vif44 –52 (HW9) (animals 95061 and 96072), and Env816 – 825 (LY10) (animals 95096 and 96072). Of the 16 CTL responses, 5 were from Nef, 5 from Vif, 3 from Env, and 3 from Pol. Fifteen of these epitopes are newly identified. To ascertain whether Mamu-B*17-restricted responses could be detected early in the acute phase of viral infection, and therefore possibly play a role in the initial control of viral replication, we performed ICS for IFN-␥. PBMC from Mamu-B*17-positive animals were tested at 3 wks postinfection using a subset of MamuB*17 peptides that included Nef165–173 (IW9), Vif64 –73 (GW10), and Vif66 –73 (HW8). We detected responses to all Mamu-B*17 peptides tested by ICS (Fig. 3). Thus, Mamu-B*17-restricted responses are present early in infection, thereby illustrating a potential role for Mamu-B*17-restricted responses in the initial containment of viral replication.


215

Table II. SIVmac239-derived Mamu-B*17-binding peptides

Sequence

Protein

Position

Mamu B*17-Binding Capacity (IC50 nM)

Response to Peptide by ELISPOT

9 9 11 9 9 9 8 9 9 9 8 9 11 9 9 10 10 10 11 10 10 10 10 9 9 9 10 9 9 10 9 11 10 9 11 9 9 10 10 10 10 9 10 11 10 9 8 10 10 11

Nef Nef Nef Env Env Vif Vif Env Pol Pol Pol Pol Vpx Vif Gag Gag Env Env Env Vif Pol Env Nef Pol Env Vif Vif Env Vif Env Vif Env Env Pol Env Nef Env Env Vpx Env Pol Pol Vif Env Pol Vif Vpr Env Env Env

195 165 165 830 44 65 66 845 994 107 372 435 39 160 407 406 844 341 111 64 596 432 194 18 266 44 42 342 5 829 43 671 216 613 241 199 179 628 90 666 434 1002 13 228 604 135 12 816 265 264

2.8 7.6 9.9 10 13 16 16 22 28 34 40 43 44 51 55 76 100 101 103 116 122 133 139 143 162 180 181 183 200 206 250 251 260 280 292 305 321 321 323 331 345 351 360 378 401 427 435 439 443 447

⫹ ⫹ ⫹ ⫹

accept other conservative or semiconservative hydrophobic residues (41). Similarly, HLA-A molecules having a preference for the basic residue K will also accept the basic residue R (15). In the case of Mamu-B*17, the vast majority of peptides binding with high affinity had W at the C terminus, and those with other aromatic residues (F or Y) were only rarely found to be binders. The F pocket structure of Mamu-B*17 is similar to that of HLAB*1513, B*1516, B*1517, B*5701, and B*5801, all of which have a preference for bulky aromatic residues (45, 46). Interestingly, HLA-B*1513 appears to share a similarly narrow specificity focused on W. By contrasting the structure of the HLA-B*1513 and Mamu B*17 F pockets with those of other HLA or Mamu alleles associated with broader specificity, it might be possible to define a structural correlate of their narrow specificity. For diametrically opposed reasons, the specificity of MamuB*17 in position 2 is also somewhat peculiar. In this case, a large

⫹ ⫹

⫹ ⫹

⫹ ⫹ ⫹

⫹ ⫹

⫹ ⫹ ⫹

set of residues associated with a paradoxically broad range of chemical specificity is preferred or tolerated. Such an enigmatic specificity has not to our knowledge been demonstrated for any class I molecule in which position 2 is a primary anchor. In this respect, this finding represents a completely novel observation considering MHC class I molecules of humans, primates, or rodents. However, it should be noted that a similarly paradoxical specificity has been noted in the case of HLA-B*2705 (47, 48), in which both aromatic and basic residues have been noted at the C terminus of endogenously bound peptides. Analysis of the B pocket structure of Mamu-B*17 suggests a number of explanations for its unusual specificity. For example, both positions 63 and 70 are occupied by A. In the case of most HLA and Mamu-A and Mamu-B molecules, position 63 is very conserved, and is almost invariably occupied by E or N. Position 70 is somewhat more varied, but is typically occupied by a large polar residue (E, D, Q,


MHPAQTSQW IRYPKTFGW IRYPKTFGWLW FHEAVQAVW CATKNRDTW SHLEVQGYW HLEVQGYW LAGAWGDLW YREGRDQLW FAAPQFSLW MRHVLEPF FQWMGYELW HLPRELIFQVW RSQGENPTW RAPRRQGCW CRAPRRQGCW TLAGAWGDLW QAWCWFGGKW MRCNKSETDRW GSHLEVQGYW FHLPVEKDVW CHIRQIINTW LMHPAQTSQW KKTGMLEMW MMETQTSTW HFKVGWAWW VPHFKVGWAW AWCWFGGKW KRWIAVPTW YFHEAVQAVW PHFKVGWAW KLNSWDVFGNW IQESCDKHYW WQVTWIPEW LRCNDTNYSGF QTSQWDDPW KKKEYNETW LTPKWNNETW LGEGHGAGGW MYELQKLNSW PFQWMGYELW WKGPGELLW WRIPERLERW IRFRYCAPPGY VWEQWWTDYW CRFPRAHKY QREPWDEW LRTELTYLQY RMMETQTSTW TRMMETQTSTW

aa

216


N, or H). Thus, in the case of Mamu-B*17, the presence of the small residue A at positions 63 and 70 may explain the ability of its B pocket to accommodate bulky hydrophobic residues. The ability of Mamu-B*17 to accommodate basic residues may be explained by the presence of E in position 45. Position 45 is hypothesized to be located at the bottom of the B pocket of most class I molecules (49), and is suspected of influencing the overall pocket specificity. Interestingly, E occupies position 45 of HLA-B27 molecules that also bind peptides with basic residues in position 2. The conjunction of the narrow specificity at the C terminus with the broad specificity at position 2 suggests that selection has forced some type of compensatory mechanism. That is, if both pockets are very stringent or very loose, too few or too many peptides might bind. In this case, the extreme focus of one pocket is balanced by the promiscuous behavior of the other. In this respect, it is interesting to note that most HLA-B molecules with a narrow

specificity for P in position 2 have a considerably more broad specificity at their C terminus (41). Similarly, HLA-A*0101 appears to balance its narrow specificity for Y at the C terminus by using either or both position 2 and position 3 residues as anchors at the N terminus (14). We have been able to identify 50 peptides derived from 7 different SIV proteins (Env, Pol, Gag, Vpx, Vif, Nef, and Vpr) that bind Mamu-B*17 with high affinity. Further analysis using the ELISPOT assay and the ICS assay with PBMC from SIV-infected macaques reveals that 16 of these peptides have the capacity to elicit CTL responses. Responses were detected during both the chronic and the acute phases of viral infection, suggesting that Mamu-B*17 may play a role not only in the initial control of viral replication, but also throughout the course of infection. These data are in agreement with previous analyses of the breadth of repertoire of Mamu-A*01 responses (18). Similarly, broad repertoires


FIGURE 2. Detection of IFN-␥ production by PBMC using the ELISPOT assay. PBMC from various Mamu-B*17 SIV-infected animals were tested with the Mamu-B*17 peptides in 16-h ELISPOT assays. PBMC were plated in 96-well plates at 1 ⫻ 105 cells/well and stimulated with various peptides (10 ␮M concentration). Mean values from triplicate wells were averaged for each assay, and SFCs were enumerated, as described in Materials and Methods. Mean SFC values and SDs are shown for each peptide tested. Gray bars indicate positive responses (see Materials and Methods).


217

have been reported in humans in the case of influenza (50, 51), HIV (52, 53), HCV (54 –58), and hepatitis B virus (59) infections. Taken together, these results are in contrast with a view of immunodominance that postulates that only one or a few determinants are recognized. Indeed, it might be hypothesized that in several instances responses might appear narrowly focused only because a comprehensive analysis was not performed. Of the 16 epitopes identified, less than half are derived from the large structural proteins Env and Pol (3 epitopes each). The Gag protein did not contribute any epitopes. This is surprising given the large amount of known epitopes from SIV and HIV that are derived from these larger structural proteins (60). By contrast, a total of 10 epitopes was identified from the smaller regulatory and accessory proteins Nef and Vif. Specifically, 5 epitopes were identified from the early regulatory protein Nef, and 5 epitopes were identified from the accessory protein Vif. That the majority (10 of 16; 62.5%) of the epitopes identified were from Nef and Vif was unexpected, given the relatively small size of these proteins. Our results indicate that Nef and Vif contain at least twice as many Mamu-B*17 epitopes, on a per residue basis, than any of the other regulatory, accessory, or structural proteins investigated. The relatively large number of epitopes derived from Vif is perhaps explained by the fact that it is significantly ( p ⬍ 0.005) W enriched compared with other SIVmac239 proteins. About 5.6% of the residues in Vif are W. By comparison, only ⬃2.8% of the residues in other major regulatory, accessory, or structural proteins are W. Our results confirmed the immunodominance of the previously described Nef165–173 (IW9) epitope (36, 39) and identified four new CTL epitopes derived from this protein. Interestingly, Nef is produced early in the viral life cycle; thus, its peptides can be rapidly presented by MHC class I molecules (61). Nef may therefore be an appealing target for vaccine development. Curiously, Vif has also been noted to contain more Mamu-A*01 binders on a per residue basis than any of the other regulatory or

accessory proteins (17). Vif has been shown in other studies, in both SIV and HIV infection, as being preferentially targeted by CTL responses, despite its small size (32, 62). Because of its role in modulating virion assembly (63), Vif may be an important factor in viral pathogenicity. Indeed, Vif must be present in cells that produce virus, and its absence results in a block of infection soon after viral entry into target cells (64, 65). For similar reasons, Vif may also be crucial in maintaining viral reservoirs in latent infection. The significance of Vif as a potential target for SIV/HIV vaccines includes its primary function, which is to aid in the transport of virus trafficking to the nucleus (66). Vif interacts with intermediate filaments and mediates mobilization of the virion from the plasma membrane to the nuclear membrane (67). Thus, Vif may be more accessible to degradative pathways, as it is primarily present in the cytoplasm of infected cells. Accordingly, Vif may be an attractive target for the immune system, as it can be easily targeted by the class I Ag presentation pathway. Our results enable the use of Mamu-B*17-positive animals in SIV studies directed at understanding the role of CTL responses during SIV infection. To date, Mamu-A*01 has been the only MHC class I molecule intensely investigated4 (18 –30, 68), and has created an intense demand and shortage of Mamu-A*01-positive animals. The use of Mamu-B*17-positive animals should alleviate the demand for Mamu-A*01-positive animals, and broaden molecular analysis of immune responses to other Mamu types, affording more general insight on viral pathogenesis. The identification of Mamu-B*17-restricted epitopes is important in light of the very small number of known epitopes derived from SIV proteins (34). Given that vaccine constructs narrowly 4 T. M. Allen, P. Jing, B. Calore, H. Horton, D. H. O’Connor, T. Hanke, M. S. Piekarczyk, R. A. Ruddersdorf, B. R. Mothe´ , C. Emerson, N. Wilson, J. D. Lifson, J. A. Berzofsky, D. B. Allison, D. C. Montefiori, R. C. Desrosiers, S. Wolinsky, K.J. Kunstman, J. D. Altman, A. Sette, A. J. McMichael, and D. I. Watkins. Effects of CTL directed against a single SIV Gag CTL epitope on the course of SIVmac239 infection. Submitted for publication.


FIGURE 3. IFN-␥ production to Mamu-B*17 peptides during the acute phase of SIV. PBMC from Mamu-B*17-positive, SIV-infected animal 2095 were tested with Nef165–173 (IW9), Vif64 –73 (GW10), Vif66 –73 (HW8) peptides. Results depict the production of TNF-␣ and IFN-␥ to each peptide, the staphylococcal enterotoxin B-positive control, and the flu peptide-negative control for CD8⫹ lymphocytes.

218


focused toward one epitope have as yet been unable to elicit protective CTL responses that control viral replication (68),4 an alternative approach may be the inclusion of multiple CTL responses in candidate vaccines. The identification of SIV-derived MamuB*17-restricted CTL epitopes, in addition to the previously characterized Mamu-A*01-restricted responses, will further progress the development of a multiepitope CTL-based vaccine targeting multiple class I molecules. The current study thus allows the design and testing of specific vaccine constructs targeting CTL responses that are multispecific.

22.

23.

24.

References 25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.


1. Johnson, R. P. 1996. Macaque models for AIDS vaccine development. Curr. Opin. Immunol. 8:554. 2. Goulder, P., D. Price, M. Nowak, S. Rowland-Jones, R. Phillips, and A. McMichael. 1997. Co-evolution of human immunodeficiency virus and cytotoxic T-lymphocyte responses. Immunol. Rev. 159:17. 3. Letvin, N. L., J. E. Schmitz, H. L. Jordan, A. Seth, V. M. Hirsch, K. A. Reimann, and M. J. Kuroda. 1999. Cytotoxic T lymphocytes specific for the simian immunodeficiency virus. Immunol. Rev. 170:127. 4. Brander, C., and B. D. Walker. 1999. T lymphocyte responses in HIV-1 infection: implications for vaccine development. Curr. Opin. Immunol. 11:451. 5. Falk, K., O. Rotzschke, S. Stevanovic, G. Jung, and H. G. Rammensee. 1991. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature 351:290. 6. Van Bleek, G. M., and S. G. Nathenson. 1990. Isolation of an endogenously processed immunodominant viral peptide from the class I H-2Kb molecule. Nature 348:213. 7. Rammensee, H. G., T. Friede, and S. Stevanoviic. 1995. MHC ligands and peptide motifs: first listing. Immunogenetics 41:178. 8. Ruppert, J., J. Sidney, E. Celis, R. T. Kubo, H. M. Grey, and A. Sette. 1993. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 74:929. 9. Kast, W. M., R. M. Brandt, J. Sidney, J. W. Drijfhout, R. T. Kubo, H. M. Grey, C. J. Melief, and A. Sette. 1994. Role of HLA-A motifs in identification of potential CTL epitopes in human papillomavirus type 16 E6 and E7 proteins. J. Immunol. 152:3904. 10. Kubo, R. T., A. Sette, H. M. Grey, E. Appella, K. Sakaguchi, N. Z. Zhu, D. Arnott, N. Sherman, J. Shabanowitz, H. Michel, et al. 1994. Definition of specific peptide motifs for four major HLA-A alleles. J. Immunol. 152:3913. 11. Sidney, J., M. F. del Guercio, S. Southwood, V. H. Engelhard, E. Appella, H. G. Rammensee, K. Falk, O. Rotzschke, M. Takiguchi, R. T. Kubo, et al. 1995. Several HLA alleles share overlapping peptide specificities. J. Immunol. 154:247. 12. Sidney, J., S. Southwood, M. F. del Guercio, H. M. Grey, R. W. Chesnut, R. T. Kubo, and A. Sette. 1996. Specificity and degeneracy in peptide binding to HLA-B7-like class I molecules. J. Immunol. 157:3480. 13. Kondo, A., J. Sidney, S. Southwood, M. F. del Guercio, E. Appella, H. Sakamoto, E. Celis, H. M. Grey, R. W. Chesnut, R. T. Kubo, et al. 1995. Prominent roles of secondary anchor residues in peptide binding to HLA-A24 human class I molecules. J. Immunol. 155:4307. 14. Kondo, A., J. Sidney, S. Southwood, M. F. del Guercio, E. Appella, H. Sakamoto, H. M. Grey, E. Celis, R. W. Chesnut, R. T. Kubo, and A. Sette. 1997. Two distinct HLA-A*0101-specific submotifs illustrate alternative peptide binding modes. Immunogenetics 45:249. 15. Sidney, J., H. M. Grey, S. Southwood, E. Celis, P. A. Wentworth, M. F. del Guercio, R. T. Kubo, R. W. Chesnut, and A. Sette. 1996. Definition of an HLA-A3-like supermotif demonstrates the overlapping peptide-binding repertoires of common HLA molecules. Hum. Immunol. 45:79. 16. Sidney, J., S. Southwood, D. L. Mann, M. A. Fernandez-Vina, M. J. Newman, and A. Sette. 2001. Majority of peptides binding HLA-A*0201 with high affinity crossreact with other A2-supertype molecules. Hum. Immunol. 62:1200. 17. Sidney, J., J. L. Dzuris, M. J. Newman, R. P. Johnson, A. Kaur, K. Amitinder, C. M. Walker, E. Appella, B. Mothe, D. I. Watkins, and A. Sette. 2000. Definition of the Mamu A*01 peptide binding specificity: application to the identification of wild-type and optimized ligands from simian immunodeficiency virus regulatory proteins. J. Immunol. 165:6387. 18. Allen, T. M., B. R. Mothe, J. Sidney, P. Jing, J. L. Dzuris, M. E. Liebl, T. U. Vogel, D. H. O’Connor, X. Wang, M. C. Wussow, et al. 2001. CD8⫹ lymphocytes from simian immunodeficiency virus-infected rhesus macaques recognize 14 different epitopes bound by the major histocompatibility complex class I molecule Mamu-A*01: implications for vaccine design and testing. J. Virol. 75:738. 19. Allen, T. M., D. H. O’Connor, P. Jing, J. L. Dzuris, B. R. Mothe, T. U. Vogel, E. Dunphy, M. E. Liebl, C. Emerson, N. Wilson, et al. 2000. Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia. Nature 407:386. 20. Hel, Z., J. Nacsa, B. Kelsall, W. P. Tsai, N. Letvin, R. W. Parks, E. Tryniszewska, L. Picker, M. G. Lewis, Y. Edghill-Smith, et al. 2001. Impairment of Gag-specific CD8⫹ T-cell function in mucosal and systemic compartments of simian immunodeficiency virus mac251- and simian-human immunodeficiency virus KU2infected macaques. J. Virol. 75:11483. 21. Veazey, R. S., M. C. Gauduin, K. G. Mansfield, I. C. Tham, J. D. Altman, J. D. Lifson, A. A. Lackner, and R. P. Johnson. 2001. Emergence and kinetics of

simian immunodeficiency virus-specific CD8⫹ T cells in the intestines of macaques during primary infection. J. Virol. 75:10515. Chen, Z. W., A. Craiu, L. Shen, M. J. Kuroda, U. C. Iroku, D. I. Watkins, G. Voss, and N. L. Letvin. 2000. Simian immunodeficiency virus evades a dominant epitope-specific cytotoxic T lymphocyte response through a mutation resulting in the accelerated dissociation of viral peptide and MHC class I. J. Immunol. 164:6474. Schmitz, J. E., M. J. Kuroda, R. S. Veazey, A. Seth, W. M. Taylor, C. E. Nickerson, M. A. Lifton, P. J. Dailey, M. A. Forman, P. Racz, et al. 2000. Simian immunodeficiency virus (SIV)-specific CTL are present in large numbers in livers of SIV-infected rhesus monkeys. J. Immunol. 164:6015. Allen, T. M., T. U. Vogel, D. H. Fuller, B. R. Mothe, S. Steffen, J. E. Boyson, T. Shipley, J. Fuller, T. Hanke, A. Sette, et al. 2000. Induction of AIDS virusspecific CTL activity in fresh, unstimulated peripheral blood lymphocytes from rhesus macaques vaccinated with a DNA prime/modified vaccinia virus Ankara boost regimen. J. Immunol. 164:4968. Furchner, M., A. L. Erickson, T. Allen, D. I. Watkins, A. Sette, P. R. Johnson, and C. M. Walker. 1999. The simian immunodeficiency virus envelope glycoprotein contains two epitopes presented by the Mamu-A*01 class I molecule. J. Virol. 73:8035. Egan, M. A., M. J. Kuroda, G. Voss, J. E. Schmitz, W. A. Charini, C. I. Lord, M. A. Forman, and N. L. Letvin. 1999. Use of major histocompatibility complex class I/peptide/␤2M tetramers to quantitate CD8⫹ cytotoxic T lymphocytes specific for dominant and nondominant viral epitopes in simian-human immunodeficiency virus-infected rhesus monkeys. J. Virol. 73:5466. Kuroda, M. J., J. E. Schmitz, W. A. Charini, C. E. Nickerson, C. I. Lord, M. A. Forman, and N. L. Letvin. 1999. Comparative analysis of cytotoxic T lymphocytes in lymph nodes and peripheral blood of simian immunodeficiency virus-infected rhesus monkeys. J. Virol. 73:1573. Kuroda, M. J., J. E. Schmitz, D. H. Barouch, A. Craiu, T. M. Allen, A. Sette, D. I. Watkins, M. A. Forman, and N. L. Letvin. 1998. Analysis of Gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex. J. Exp. Med. 187:1373. Chen, Z. W., L. Shen, M. D. Miller, S. H. Ghim, A. L. Hughes, and N. L. Letvin. 1992. Cytotoxic T lymphocytes do not appear to select for mutations in an immunodominant epitope of simian immunodeficiency virus gag. J. Immunol. 149: 4060. Allen, T. M., J. Sidney, M. F. del Guercio, R. L. Glickman, G. L. Lensmeyer, D. A. Wiebe, R. DeMars, C. D. Pauza, R. P. Johnson, A. Sette, and D. I. Watkins. 1998. Characterization of the peptide binding motif of a rhesus MHC class I molecule (Mamu-A*01) that binds an immunodominant CTL epitope from simian immunodeficiency virus. J. Immunol. 160:6062. Hanke, T., R. V. Samuel, T. J. Blanchard, V. C. Neumann, T. M. Allen, J. E. Boyson, S. A. Sharpe, N. Cook, G. L. Smith, D. I. Watkins, et al. 1999. Effective induction of simian immunodeficiency virus-specific cytotoxic T lymphocytes in macaques by using a multiepitope gene and DNA prime-modified vaccinia virus Ankara boost vaccination regimen. J. Virol. 73:7524. Mothe, B. R., H. Horton, D. K. Carter, T. M. Allen, M. E. Liebl, P. Skinner, T. U. Vogel, S. Fuenger, K. Vielhuber, W. Rehrauer, et al. 2002. Dominance of CD8 responses specific for epitopes bound by a single major histocompatibility complex class I molecule during the acute phase of viral infection. J. Virol. 76:875. Knapp, L. A., E. Lehmann, M. S. Piekarczyk, J. A. Urvater, and D. I. Watkins. 1997. A high frequency of Mamu-A*01 in the rhesus macaque detected by polymerase chain reaction with sequence-specific primers and direct sequencing. Tissue Antigens 50:657. Allen, T. M., and D. I. Watkins. 1999. SIV and SHIV CTL epitopes identified in macaques. In HIV Molecular Immunology Database. B. T. Korber, C. Brander, B. F. Haynes, R. A. Koup, C. L. Kuiken, J. P. Moore, B. D. Walker, and D. I. Watkins, eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, p. IV. Dzuris, J. L., J. Sidney, E. Appella, R. W. Chesnut, D. I. Watkins, and A. Sette. 2000. Conserved MHC class I peptide binding motif between humans and rhesus macaques. J. Immunol. 164:283. Horton, H., W. Rehrauer, E. C. Meek, M. A. Shultz, M. S. Piekarczyk, P. Jing, D. K. Carter, S. R. Steffen, B. Calore, J. A. Urvater, et al. 2001. A common rhesus macaque MHC class I molecule which binds a cytotoxic T-lymphocyte epitope in Nef of simian immunodeficiency virus. Immunogenetics 53:423. Ruppert, J., J. Sidney, E. Celis, R. T. Kubo, H. M. Grey, and A. Sette. 1993. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 74:929. Sidney, J., S. Southwood, C. Oseroff, M. F. del Guercio, A. Sette, and H. M. Grey. 1998. Measurement of MHC/peptide interactions by gel filtration. Curr. Prot. Immunol. 18:3.1. Evans, D. T., D. H. O’Connor, P. Jing, J. L. Dzuris, J. Sidney, J. da Silva, T. M. Allen, H. Horton, J. E. Venham, R. A. Rudersdorf, et al. 1999. Virusspecific cytotoxic T-lymphocyte responses select for amino-acid variation in simian immunodeficiency virus Env and Nef. Nat. Med. 5:1270. Kondo, A., J. Sidney, S. Southwood, M. F. del Guercio, E. Appella, H. Sakamoto, E. Celis, H. M. Grey, R. W. Chesnut, R. T. Kubo, et al. 1995. Prominent roles of secondary anchor residues in peptide binding to HLA-A24 human class I molecules. J. Immunol. 155:4307. Sidney, J., S. Southwood, M. F. del Guercio, H. M. Grey, R. W. Chesnut, R. T. Kubo, and A. Sette. 1988. Specificity and degeneracy in peptide binding to HLA-B7-like class I molecules. J. Immunol. 157:3480.


57.

58.

59. 60.

61.

62.

63.

64.

65.

66.

67. 68.

phocytes specific for hepatitis C virus: identification of multiple epitopes and characterization of patterns of cytokine release. J. Clin. Invest. 96:2311. Scognamiglio, P., D. Accapezzato, M. A. Casciaro, A. Cacciani, M. Artini, G. Bruno, M. L. Chircu, J. Sidney, S. Southwood, S. Abrignani, et al. 1999. Presence of effector CD8⫹ T cells in hepatitis C virus-exposed healthy seronegative donors. J. Immunol. 162:6681. Cooper, S., A. L. Erickson, E. J. Adams, J. Kansopon, A. J. Weiner, D. Y. Chien, M. Houghton, P. Parham, and C. M. Walker. 1999. Analysis of a successful immune response against hepatitis C virus. Immunity 10:439. Chisari, F. V., and C. Ferrari. 1995. Hepatitis B virus immunopathogenesis. Annu. Rev. Immunol. 13:29. Brander, C., and P. J. R. Goulder. 2000. The evolving field of HIV CTL epitope mapping: new approaches to the identification of novel epitopes. In HIV Molecular Immology Database. B. T. M. Korber, C. Brander, B. F. Haynes, R. Koup, C. Kuiken, J. P. Moore, B. D. Walker, and D. Watkins, eds. Theoretical Biology and Biophysics Group, Los Alamos, p. I. Ranki, A., A. Lagerstedt, V. Ovod, E. Aavik, and K. J. Krohn. 1994. Expression kinetics and subcellular localization of HIV-1 regulatory proteins Nef, Tat and Rev in acutely and chronically infected lymphoid cell lines. Arch. Virol. 139:365. Altfeld, M. A., B. Livingston, N. Reshamwala, P. T. Nguyen, M. M. Addo, A. Shea, M. Newman, J. Fikes, J. Sidney, P. Wentworth, et al. 2001. Identification of novel HLA-A2-restricted human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte epitopes predicted by the HLA-A2 supertype peptidebinding motif. J. Virol. 75:1301. Borman, A. M., C. Quillent, P. Charneau, C. Dauguet, and F. Clavel. 1995. Human immunodeficiency virus type 1 Vif-mutant particles from restrictive cells: role of Vif in correct particle assembly and infectivity. J. Virol. 69:2058. Von Schwedler, U., J. Song, C. Aiken, and D. Trono. 1993. Vif is crucial for human immunodeficiency virus type 1 proviral DNA synthesis in infected cells. J. Virol. 67:4945. Gabuzda, D. H., K. Lawrence, E. Langhoff, E. Terwilliger, T. Dorfman, W. A. Haseltine, and J. Sodroski. 1992. Role of vif in replication of human immunodeficiency virus type 1 in CD4⫹ T lymphocytes. J. Virol. 66:6489. Karczewski, M. K., and K. Strebel. 1996. Cytoskeleton association and virion incorporation of the human immunodeficiency virus type 1 Vif protein. J. Virol. 70:494. Miller, R. H., and N. Sarver. 1997. HIV accessory proteins as therapeutic targets. Nat. Med. 3:389. Allen, T. M., L. Mortara, B. R. Mothe, M. Liebl, P. Jing, B. Calore, M. Piekarczyk, R. Ruddersdorf, D. H. O’Connor, X. Wang, et al. 2002. Tatvaccinated macaques do not control simian immunodeficiency virus SIVmac239 replication. J. Virol. 76:4108.


42. Gulukota, K., J. Sidney, A. Sette, and C. DeLisi. 1997. Two complementary methods for predicting peptides binding major histocompatibility complex molecules. J. Mol. Biol. 267:1258. 43. Southwood, S., J. Sidney, A. Kondo, M. F. del Guercio, E. Appella, S. Hoffman, R. T. Kubo, R. W. Chesnut, H. M. Grey, and A. Sette. 1998. Several common HLA-DR types share largely overlapping peptide binding repertoires. J. Immunol. 160:3363. 44. Klinman, D. M. 1994. ELISPOT assay to detect cytokine-secreting murine and human cells. Curr. Prot. Immunol. 6:1. 45. Barber, L. D., L. Percival, K. L. Arnett, J. E. Gumperz, L. Chen, and P. Parham. 1997. Polymorphism in the ␣1 helix of the HLA-B heavy chain can have an overriding influence on peptide-binding specificity. J. Immunol. 158:1660. 46. Falk, K., O. Rotzschke, M. Takiguchi, V. Gnau, S. Stevanovic, G. Jung, and H. G. Rammensee. 1995. Peptide motifs of HLA-B58, B60, B61, and B62 molecules. Immunogenetics 41:165. 47. Rotzschke, O., K. Falk, S. Stevanovic, V. Gnau, G. Jung, and H. G. Rammensee. 1994. Dominant aromatic/aliphatic C-terminal anchor in HLA-B*2702 and B*2705 peptide motifs. Immunogenetics 39:74. 48. Jardetzky, T. S., W. S. Lane, R. A. Robinson, D. R. Madden, and D. C. Wiley. 1991. Identification of self peptides bound to purified HLA-B27. Nature 353:326. 49. Madden, D. R. 1995. The three-dimensional structure of peptide-MHC complexes. Annu. Rev. Immunol. 13:587. 50. Jameson, J., J. Cruz, and F. A. Ennis. 1998. Human cytotoxic T-lymphocyte repertoire to influenza A viruses. J. Virol. 72:8682. 51. Gianfrani, C., C. Oseroff, J. Sidney, R. W. Chesnut, and A. Sette. 2000. Human memory CTL response specific for influenza A virus is broad and multispecific. Hum. Immunol. 61:438. 52. McMichael, A. J., and R. E. Phillips. 1997. Escape of human immunodeficiency virus from immune control. Annu. Rev. Immunol. 15:271. 53. Rowland-Jones, S. L., T. Dong, K. R. Fowke, J. Kimani, P. Krausa, H. Newell, T. Blanchard, K. Ariyoshi, J. Oyugi, E. Ngugi, et al. 1998. Cytotoxic T cell responses to multiple conserved HIV epitopes in HIV-resistant prostitutes in Nairobi. J. Clin. Invest. 102:1758. 54. Chang, K. M., N. H. Gruener, S. Southwood, J. Sidney, G. R. Pape, F. V. Chisari, and A. Sette. 1999. Identification of HLA-A3 and -B7-restricted CTL response to hepatitis C virus in patients with acute and chronic hepatitis C. J. Immunol. 162:1156. 55. Rehermann, B., K. M. Chang, J. McHutchinson, R. Kokka, M. Houghton, C. M. Rice, and F. V. Chisari. 1996. Differential cytotoxic T-lymphocyte responsiveness to the hepatitis B and C viruses in chronically infected patients. J. Virol. 70:7092. 56. Koziel, M. J., D. Dudley, N. Afdhal, A. Grakoui, C. M. Rice, Q. L. Choo, M. Houghton, and B. D. Walker. 1995. HLA class I-restricted cytotoxic T lym-

219