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The Journal of Immunology, 2004, 173: 5064-5076.
Copyright © 2004 by The American Association of Immunologists

Identification of Seventeen New Simian Immunodeficiency Virus-Derived CD8+ T Cell Epitopes Restricted by the High Frequency Molecule, Mamu-A*02, and Potential Escape from CTL Recognition1

John T. Loffredo*, John Sidney{dagger}, Christina Wojewoda*, Elizabeth Dodds{ddagger}, Matthew R. Reynolds*,{ddagger}, Gnankang Napoé*, Bianca R. Mothé§, David H. O’Connor*, Nancy A. Wilson*, David I. Watkins*,{ddagger} and Alessandro Sette2,{dagger}

* National Primate Research Center, University of Wisconsin (WPRC), Madison, WI 53715; {dagger} La Jolla Institute for Allergy and Immunology, San Diego, CA 92109; {ddagger} Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, WI 53706; and § Department of Biological Sciences, California State University, San Marcos, CA 92096


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
MHC class I-restricted CD8+ T cells play an important role in controlling HIV and SIV replication. In SIV-infected Indian rhesus macaques (Macaca mulatta), comprehensive CD8+ T cell epitope identification has only been undertaken for two alleles, Mamu-A*01 and Mamu-B*17. As a result, these two molecules account for virtually all known MHC class I-restricted SIV-derived CD8+ T cell epitopes. SIV pathogenesis research and vaccine testing have intensified the demand for epitopes restricted by additional MHC class I alleles due to the shortage of Mamu-A*01+ animals. Mamu-A*02 is a high frequency allele present in over 20% of macaques. In this study, we characterized the peptide binding of Mamu-A*02 using a panel of single amino acid substitution analogues and a library of 497 unrelated peptides. Of 230 SIVmac239 peptides that fit the Mamu-A*02 peptide-binding motif, 75 peptides bound Mamu-A*02 with IC50 values of ≤500 nM. We assessed the antigenicity of these 75 peptides using an IFN-{gamma} ELISPOT assay with freshly isolated PBMC from eight Mamu-A*02+ SIV-infected macaques and identified 17 new epitopes for Mamu-A*02. The synthesis of five Mamu-A*02 tetramers demonstrated the discrepancy between tetramer binding and IFN-{gamma} secretion by SIV-specific CD8+ T cells during chronic SIV infection. Bulk sequencing determined that 2 of the 17 epitopes accumulated amino acid replacements in SIV-infected macaques by the chronic phase of infection, suggestive of CD8+ T cell escape in vivo. This work enhances the use of the SIV-infected macaque model for HIV and increases our understanding of the breadth of CD8+ T cell responses in SIV infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Increasing evidence suggests that CD8+ T cells play a major role in controlling HIV and SIV infection (1). Transient depletion of CD8+ cells in SIV-infected macaques results in increased viral loads (2, 3, 4, 5), while resolution of acute phase viremia correlates temporally with the appearance of virus-specific CD8+ T cells (6, 7, 8, 9, 10). It is now becoming clear that CD8+ T cells exert selective pressure on viral sequences in vivo, selecting for immune escape in both the acute (11, 12, 13, 14) and chronic states of HIV and SIV infection (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27). Additionally, an association exists between MHC class I and class II alleles for both slow and rapid HIV disease progression (28, 29, 30, 31, 32, 33, 34, 35). Therefore, it is likely that a successful HIV vaccine will need to elicit a CD8+ T cell response as well as a humoral component. Hence, the understanding of virus-specific CD8+ T cell responses will be central for vaccine development.

The SIV-infected Indian rhesus macaque is the best animal model for HIV infection, providing critical insights into immunodeficiency virus pathogenesis, viral evolution, and cellular and humoral immune responses (36, 37). The use of cloned viruses, controlled immunizations and infections, and the ability to precisely investigate disease progression from the acute phase through time of death in a reasonable period of time are several advantages that this model affords. This model is currently limited by the paucity of defined epitopes for MHC class I molecules that restrict SIV-specific CD8+ T cell responses. Recent investigations identified two MHC class I alleles associated with slow disease progression in SIV-infected macaques, Mamu-A*01 and Mamu-B*17 (38, 39, 40, 41, 42). Coincidentally, these are the only two Indian rhesus macaque MHC class I molecules with extensively characterized peptide-binding motifs. Because of already improved disease outcomes, animals expressing these two alleles may be less than ideal in evaluating vaccine efficacy, illustrating the need to expand our understanding of macaque MHC class I alleles. This is complicated by the large degree of MHC polymorphism, and the observation that between 4 and 12 different MHC class I alleles can be expressed in a given animal (43, 44).

The identification of peptide-binding motifs for Mamu-A*01 and Mamu-B*17 permitted comprehensive epitope screening to identify CD8+ T cell epitopes now used to evaluate the immunogenicity of potential SIV vaccines in macaques (45, 46, 47). To date, most research into SIV-specific CD8+ T cell responses has used Mamu-A*01+ macaques, mainly because 14 CD8+ T cell epitopes restricted by this allele are known (46). The resultant shortage of Mamu-A*01+ Indian rhesus macaques underscores the need to identify CD8+ T cell epitopes bound by other common MHC class I alleles. Besides the use of these epitopes for immunological techniques which detect cytokine production by Ag-specific CD8+ T cells, such as intracellular cytokine staining (ICS)3 and ELISPOT assay, the identification of minimal optimal epitopes allows design and synthesis of fluorescence dye-coupled tetrameric peptide-MHC complexes that directly detect virus-specific CD8+ T cells (10, 48, 49). Uncovering novel CD8+ T cell epitopes also facilitates viral evolution and escape studies. Finally, identification of these epitopes is important in the development of vaccine strategies designed to induce CD8+ T cell responses against SIV in rhesus macaques. Such knowledge allows for better use of the limited animal resources for SIV research while also providing a more complete description of the immune response.

The Mamu-A*02 allele appears with high frequency (~20%), comparable to Mamu-A*01 expression (~22%), in Indian rhesus macaques (26, 50, 51). Previous research on the Mamu-A*02 molecule described two CD8+ T cell epitopes and proposed a tentative peptide-binding motif defining primary anchor residues that was derived from analysis of eluted natural ligands and in vitro binding assays (26, 50). However, several studies indicate that both primary and secondary anchors must be taken into account together with the use of in vitro peptide/MHC-binding assays to accurately identify potential T cell epitopes (52, 53, 54, 55). Once peptides binding with reasonable affinity are identified, they can be tested for CD8+ T cell recognition from infected animals. This method has proven successful for Mamu-A*01 and Mamu-B*17, resulting in the detection of 26 MHC class I-restricted SIV-derived epitopes (46, 47).

The aim of this study was to provide a detailed characterization of the peptide-binding motif of the high frequency MHC class I Mamu-A*02 allele, identify SIV-derived Mamu-A*02 CD8+ T cell epitopes, and ascertain the breadth of these responses in SIV-infected macaques. The binding motif allowed us to screen peptides derived from the SIVmac239 proteome, identifying 230 candidate Mamu-A*02 peptides, of which 75 peptides bound Mamu-A*02 with IC50 values of ≤500 nM. This study describes 17 new epitopes restricted by the Mamu-A*02 molecule. To determine which of these newly identified CD8+ T cell epitopes accrue variation consistent with CD8+ T cell escape, we sequenced 16 novel Mamu-A*02 epitope sites in six SIV-infected rhesus macaques (four Mamu-A*02+ and two Mamu-A*02–). Mamu-A*02-restricted viral mutations occurred within 2 of the 16 epitopes, indicating selective pressure in these CD8+ T cell epitopes. Peptide-binding studies revealed that these viral variants have decreased binding capacity relative to wild-type peptides, while ICS assays showed that the mutated epitopes induced a decreased IFN-{gamma} response in a majority of variants. Finally, we produced five Mamu-A*02 tetramers, using the newly discovered epitopes. The tetramers allowed us to uncover a discrepancy between CD8+ T cells which bind tetramer and those that produced IFN-{gamma}, indicating a functional impairment of several Mamu-A*02 CD8+ T cell responses in the chronic phase of SIV infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and viruses

The Indian rhesus macaques (Macaca mulatta) used in this study were identified as Mamu-A*01+ and/or Mamu-A*02+ by sequence-specific primer (SSP) DNA amplification (PCR-SSP), as previously described (51). Macaques were infected with a molecularly cloned virus, SIVmac239, or an engineered CD8+ T cell escape variant virus based on SIVmac239 (56). Animals 98015, 96115, and 98020 received the engineered CD8+ T cell escape variant virus. This virus contained point mutations in two known Mamu-A*01 epitopes (Tat28–35SL8 and Gag181–189CM9) along with one known Mamu-B*17 epitope (Nef165–173IW9). These mutations had minimal impact on the detection of positive responses in the IFN-{gamma} ELISPOT assay. Animal 2128 was vaccinated with an established prime-boost regimen (57) before SIVmac239 infection.

SIV-infected animals were maintained at the National Primate Research Center (University of Wisconsin, Madison, WI) and cared for in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals (58) and under the approval of the University of Wisconsin Research Animal Resources Center review committee.

Peptide synthesis

Peptides for screening were purchased from either Pepscan Systems (Lelystad, The Netherlands), Mimotopes (Clayton, Australia), synthesized at the Biotechnology Center at the University of Wisconsin, or at Epimmune (San Diego, CA) using standard tertiary butyloxycarbonyl or fluorenylmethoxycarbonyl solid phase methods (52). Peptides were resuspended at 4–20 mg/ml in 100% DMSO (Sigma-Aldrich, St. Louis, MO), then diluted to required concentrations in PBS or PBS + 0.05% Nonidet P-40. For ELISPOT assays, peptide stocks at 10 mg/ml in 100% DMSO were diluted to 1 mg/ml in HBSS (Invitrogen Life Technologies, Grand Island, NY). Peptides for use as radiolabeled probes were purified to >95% homogeneity by reverse phase HPLC, and composition ascertained by amino acid analysis, sequencing, and/or mass spectrometry analysis. Radiolabeling was done using the chloramine T method (59). SIV peptides were derived from the SIVmac239 sequence, GenBank accession M33262 (60).

MHC purification and peptide-binding assay

Stable transfectants expressing specific rhesus macaque MHC class I molecules for Mamu-A*02 were created in the HLA class I-deficient human B cell line 721.221, as described previously (44, 61). MHC class I molecules were purified from cell lysates using affinity chromatography as previously described with the anti-HLA class I (-A, -B, -C) Ab W6/32 (45, 59). Protein purity, concentration, and effectiveness of depletion steps were monitored by SDS-PAGE.

Quantitative assays for peptide binding to detergent solubilized Mamu class I 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 (59). Briefly, 1–10 nM radiolabeled peptide was coincubated at room temperature with 1 µM-1 nM purified class I molecules in the presence of 1 µM human {beta}2-microglubulin (Scripps Laboratories, San Diego, CA) and a mixture of protease inhibitors. The radiolabeled peptide used for Mamu-A*02 assays was the human J chain peptide 102–110 (sequence YTAVVPLVY).

After a 2-day incubation, binding of the radiolabeled peptide to the corresponding MHC class I molecule was determined by capturing MHC/peptide complexes on Greiner Lumitrac 600 microplates (Greiner Bio-one, Longwood, FL) coated with the W6/32 Ab, and measuring bound cpm using the TopCount microscintillation counter (Packard Instrument, Meriden, CT). In the case of competitive assays, the concentration of peptide yielding 50% inhibition of the binding of the radiolabeled probe peptide was calculated (IC50). Peptides were typically tested in three or more independent experiments. Under the conditions used, where [label] < [MHC] and IC50 ≥ [MHC], the measured IC50 values are reasonable approximations of the true Kd values (62). In each experiment, a titration of the unlabeled version of the radiolabeled probe was tested as a positive control for inhibition. The average IC50 of the human J chain 102–110 peptide was 4.0 nM.

Computational peptide binding and bioinformatic analysis

For detailed analysis of the peptide binding data, binding values were standardized by calculation of a geometric mean, or average relative binding (ARB) value, for all peptides of a particular chemical specificity (47, 52, 54, 55, 63, 64, 65, 66). For peptides of identical size, binding capacity was further analyzed by determining the ARB values for all peptides that contain a particular amino acid residue in a specific position. For determination of the specificity at Mamu-A*02 main anchor positions, ARB values were standardized relative to the ARB values of peptides carrying the residue associated with the best binding. For secondary anchor determinations, ARB values were standardized relative to the ARB of the whole peptide set considered. ARB was derived for each residue considered individually when there were five or more occurrences of that residue in the specified position in the peptide library. When there were fewer than five occurrences (e.g., M, C, or W), ARB calculations include data obtained from a group of chemically similar amino acids as previously described (47, 52, 54, 55, 63, 64, 65, 66). Amino acids with ARB values ≥0.1 are considered preferred residues, 0.01–0.1 are defined as tolerated, and <0.01 indicate nontolerated.

IFN-{gamma} ELISPOT assay

PBMC were separated from whole heparin-treated blood by Ficoll-Paque PLUS (Amersham Biosciences, Uppsala, Sweden) density centrifugation. The PBMC were used directly in ELISPOT assays as previously described (46). Briefly, 96-well, flat-bottom, clear plate ELISPOT kits (U-Cytech, Utrecht, The Netherlands) were used for the detection of IFN-{gamma}. Cells were resuspended in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with L-glutamine (Mediatech, Herndon, VA), penicillin-streptomycin (Mediatech), and 5% FBS (FBS; HyClone, Logan, UT) (R05). The R05 also contained either 10 µg/ml Con A (Sigma-Aldrich), 10 µg/ml various Mamu-A*02-bound peptides, or no peptide. Input cell numbers were 1.0 x 105 PBMC in 100 µl/well in triplicate wells. Cells were then incubated 17–19 h (overnight) at 37°C in 5% CO2. When black spots appeared in the wells under an inverted microscope, the wells were washed with distilled water to stop development and then air-dried. The 75 peptides with IC50 values ≤500 nM were tested in all animals with the exception of animal 98020 where 28 of 75 binding peptides were selectively tested due to limited numbers of PBMC.

Wells were imaged with an AID ELISPOT reader (AID, Strassberg, Germany). Spots were counted by an automated system with set parameters for size, intensity, and gradient. Background (mean of wells without peptide) levels were subtracted from each well on the plate. A response was considered positive if the mean number of spot-forming cells (SFCs) of triplicate sample wells exceeded background plus two SDs. Assay results are shown as SFC per 1 x 106 cells. Responses <50 SFC per 1 x 106 cells are not considered positive because these counts are not significantly above background. Wells containing Con A (positive control) were always >1000 SFCs per 1 x 106 PBMC.

Amplification of viral RNA (vRNA) from plasma and nucleotide sequencing

SIVmac239 nucleotide sequencing was performed as previously described (14). Briefly, cell-free plasma was obtained by centrifugation of EDTA anti-coagulated whole blood on a Ficoll density gradient. vRNA was then extracted using the Qiagen QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions for large volumes. For each vRNA sample, 17 RT-PCRs were performed with the Qiagen One Step RT-PCR kit (Qiagen), generating overlapping amplicons spanning the open reading frames of SIVmac239. The amplified cDNA was purified using a QIAquick PCR purification kit (Qiagen). Both strands of each amplicon were directly sequenced as previously described (13). Nucleotide sequences were aligned pairwise to the GenBank SIVmac239 sequence (Accession no. M33262) (60) using alignment function in the MacVector 7.1.1 trial version (Accelerys, San Diego, CA). Amino acid replacements were derived from the nucleotide alignment using a program developed by Dr. D. O’Connor in Lasso Professional (Blue World, Bellevue, WA).

Mamu-A*02 tetramers

Stable transfectants expressing specific rhesus macaque MHC class I molecules for Mamu-A*02 were created in the HLA class I-deficient human B cell line 721.221, as described previously (44, 61). MHC class I molecules were purified from cell lysates using affinity chromatography as previously described with the anti-HLA class I (-A, -B, -C) Ab W6/32 (45, 59). Protein purity, concentration, and effectiveness of depletion steps were monitored by SDS-PAGE. Soluble Mamu-A*02 monomers, containing a C-terminal BirA tail (67), were collected from the supernatant of HLA class I-deficient human B cell line 721.221 stably transfected with Mamu-A*02 cDNA (N. Wilson and W. Rudersdorf, manuscript in preparation). The Mamu-A*02 sequence was determined in previously published work (43, 44). Fully folded, glycosylated monomers were affinity purified, biotinylated, then loaded with peptide. Tetramers were made with streptavidin PE (BioSource International, Camarillo, CA) and purified by size exclusion chromatography.

Two microliters of Mamu-A*02 tetramers at 0.1 mg/ml stocks, were used in a total volume of 200 µl of R10 containing 0.5 x 106 to 1.0 x 106 Ficoll-purified PBMC. Freshly isolated PBMC were obtained from time points typically within 1 mo of the ELISPOT assays and no more than 6 mo later. The tetramer was incubated for 1 h at 37°C in a 5% CO2 incubator. Then the surface stain Abs were added, 3 µl of CD3 FITC (clone: SP34; BD Pharmingen, San Diego, CA), 6 µl of CD8 PerCP (clone: SK1; BD Biosciences, San Jose, CA), and 5 µl of CD4 allophycocyanin (clone: SK3; BD Biosciences). The stained cells were then left at room temperature in the dark. After 40 min, cells were washed twice with 1 ml of R10 ((RPMI 1640; BioWhittaker) supplemented with L-glutamine (Mediatech), penicillin-streptomycin (Mediatech), and 10% FBS (HyClone)) and then fixed in 150 µl of 2% paraformaldehdye (PFA; Sigma-Aldrich)/PBS solution. Data were acquired on a FACSCalibur (BD Biosciences). Sample analysis was completed using FlowJo software 4.4.4 (TreeStar, Ashland, OR). All samples were gated first on lymphocytes followed by the CD3+ cell population and then a dot plot of the CD8 vs Mamu-A*02 tetramer cell populations.

To identify the ratio of IFN-{gamma}-producing cells vs MHC class I tetramer-positive cells, we first calculated the percentage of CD8+ T cell SFCs in ELISPOT assays. This was achieved by multiplying the SFCs per 106 PBMC by an approximate percentage of CD3+CD8+ lymphocytes in the PBMC as identified by surface staining. This percentage was then divided by the percent of CD3+CD8+ lymphocytes with Mamu-A*02 tetramer staining.

ICS assay

PBMC were separated from whole EDTA-treated blood by Ficoll-Paque PLUS (Amersham Biosciences) density centrifugation. The PBMC were resuspended in RPMI 1640 (BioWhittaker) supplemented with L-glutamine (Mediatech), penicillin-streptomycin (Mediatech), and 10% FBS (HyClone) (R10) and used directly in IFN-{gamma} ICS assays as previously described (47). Briefly, each test contained 4 x 105 to 5 x 105 PBMC and was incubated with either staphylococcal enterotoxin B (10 µg/ml; Sigma-Aldrich) as a positive control, individual Mamu-A*02 peptides at 5 µg/ml or in serial 10-fold dilutions from 5 µg/ml to 5 pg/ml, or no peptide as a negative control. Between 1 x 105 and 2 x 105 lymphocyte-gated events were acquired on a FACSCalibur (BD Biosciences) and analyzed using FlowJo software 4.4.4 (TreeStar). All values are reported after subtraction of the background level staining (negative control of PBMC and media).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Determination of the primary anchor specificity for Mamu-A*02 using single substitution analogues of a peptide that binds with high affinity to Mamu-A*02

A preliminary Mamu-A*02-binding motif was previously described based on the sequence of seven endogenous peptides and in vitro binding assays. This motif suggested a preference for threonine (T) or serine (S) in position two, and a hydrophobic residue at the C terminus (position nine) (26). To determine a more detailed Mamu-A*02 peptide-binding motif, we tested a panel of single substitution analogues of the human J chain peptide (102–110) that binds Mamu-A*02 with an IC50 of 4.0 nM (Table I). First, we introduced a lysine (K) residue at each position of the peptide. Using this nonconservative substitution, we verified that positions two and nine (the C terminus) are the primary anchors. Lysine substitutions at either position completely abrogated binding with ARB values over 100-fold less than the best binding peptide. By contrast, at all other positions the lysine substitution was tolerated with ARB values in the 0.33–0.78 range when compared with the optimal index binding value of 1.0.


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  		Table I. Mamu-A*02 ARB of a panel of single substitution analogs of human J chain peptide 102–110

 
Additional single substitution analysis was performed with all 20 amino acids at positions two and nine of the index peptide. This analysis confirmed T and S as the preferred residues for Mamu-A*02 binding at the second position (P2). Amino acids valine (V) and glycine (G) also appear to be tolerated in this position, though with ARB values over 10-fold smaller relative to T (Table I). The specificity at the C terminus was found to be somewhat less restrictive. Methionine (M) and tyrosine (Y) were identified as the most preferred residues. Phenylalanine (F), leucine (L), tryptophan (W), and V were all tolerated with ARB values in the 0.01–0.1 range (Table I).

Primary and secondary anchor specificity based on analysis of binding data from a large library of peptide ligands

After defining the basic Mamu-A*02-binding motif using single substitution analysis, we scanned the SIVmac239 proteome to identify candidate ligands. For this search, a more permissive motif was used that included residues of similar chemical specificity to the residues identified as preferred or tolerated. This broader analysis reduced bias from a single reference peptide to identify 230 Mamu-A*02 motif-positive peptides (supplemental Table I).4 In addition, 267 peptides derived from influenza and other organisms (data not shown) were selected and included in the library. Together, binding analysis of these peptides was used to refine the Mamu-A*02 peptide-binding motif by calculating ARB values for all peptides carrying a particular residue at a specific position. This type of analysis is important because it identifies binding propensities in the context of a large number of different sequences and does not rely on a single prototype good binder.

As shown in Table II, Fine specificity at position two, this analysis identified V along with T and S as preferred at position two, while isoleucine (I), alanine (A), L, M, and G were tolerated. Analysis of the C terminus data suggested that I be included as a preferred residue with M, Y, F, W, L, and V. Peptides with A at the C terminus also bound, indicating that this residue is tolerated; although, it is associated with an ARB <10-fold of the optimal residue (Table II, Fine specificity at the C terminus).


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  		Table II. Primary features influencing Mamu-A*02 peptide-binding capacity

 
When analyzing binding capacity as a function of peptide size, we found that 9-mers and 10-mers are optimal ligand sizes with ARB values in the 0.4–1 range (Table II, Mamu A*02 binding as a function of peptide length). We examined the 9-mers and 10-mers in more detail to identify secondary influences on Mamu-A*02-binding capacity (Table III). The most notable secondary binding influences were associated with the charged amino acids aspartic acid (D) and glutamic acid (E). Both were highly deleterious to peptide binding at several different positions. In addition, position one appears to contribute prominently as a secondary anchor with preferences for basic residues, including arginine (R), and large hydrophobic residues, as tyrosine (Y), for binding in the A pocket. We observed this trend for both 9-mer and 10-mer peptides. The peptide library analysis for both primary and secondary anchor positions was summarized to provide a refined Mamu-A*02-binding motif (Fig. 1). These maps illustrate both the primary (position two and C terminus) and secondary binding influences for 9-mer and 10-mer peptides.


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  		Table III. The relative influence of secondary anchor residues on Mamu-A*02-binding capacity

 


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  		FIGURE 1. Summary maps of the primary and secondary effects influencing the capacity of 9-mer (A) and 10-mer (B) peptides to bind Mamu-A*02 molecules. Secondary anchor positions illustrate the residues associated with 4-fold increases (preferred residues) or 4-fold decreases (deleterious residues) in Mamu-A*02-binding capacity compared with peptides of the same size carrying other residues, as described in Table III. Preferred and tolerated residues at the position two and C-terminal main anchor positions are as defined by the analyses summarized in Table II.

 
Identification of 75 SIVmac239-derived Mamu-A*02-binding peptides

As mentioned above, to identify Mamu-A*02-binding peptide ligands derived from the SIVmac239 proteome, the amino acid sequences of the SIVmac239 proteins were scanned using the more permissive Mamu-A*02 motif. Two-hundred-thirty SIVmac239 peptides were identified, synthesized, and tested for their capacity to bind purified Mamu-A*02 molecules. In total, 75 of the 230 peptides (32.6%) bound Mamu-A*02 with an IC50 of ≤500 nM (supplemental Table I). The 500 nM affinity threshold has previously been shown to be associated with T cell recognition in vivo in murine, human, and rhesus systems (46, 62, 68, 69, 70). Thirty-one peptides bound with an IC50 of ≤50 nM were classified as high-affinity binders. The 44 remaining peptides bound in the 51–500 nM range, classifying them as intermediate binders (52). The 75 binding peptides were derived from seven different SIVmac239 proteins. Interestingly, no Mamu-A*02-binding peptides were detected from Rev or Vpx proteins.

Analysis of eight Mamu-A*02+ SIV-infected rhesus macaques identifies 17 novel Mamu-A*02 peptide responses

To determine whether the 75 selected peptides were actually recognized in vivo, IFN-{gamma} ELISPOT assays were performed using freshly isolated PBMC from eight SIVmac239-infected, Mamu-A*02+ rhesus macaques. The 75 binding (IC50 ≤500 nM) peptides were tested in triplicate to evaluate immunogenicity. Positive responses exceeded the average background level plus two SDs. When possible, responses <100 SFC per 1 x 106 PBMC were repeated to confirm this response as positive.

IFN-{gamma} ELISPOT demonstrated functional reactivity to 21 of the 75 peptides characterized as binding Mamu-A*02 (10 of 31 high affinity binders and 11 of 44 intermediate binders) (Table IV). Responses to the previously characterized Mamu-A*02 epitopes Gag71–79GY9 and Nef159–167YY9 were detected in at least four of the eight Mamu-A*02+ animals, making them the two most frequent CD8+ T cell responses (supplemental Fig. 1). The remaining 19 peptides were previously undefined epitopes. However, two additional peptides were not considered new CD8+ T cell epitopes, Nef160–167TY8 and Nef169–178KV10. Nef160–167TY8 lies within the previously identified minimal optimal epitope Nef159–167YY9, while Nef169–178KV10 is one amino acid longer than Nef169–177KL9. Nef169–177KL9 appears to be the minimal optimal epitope with an IC50 over 10-fold greater than Nef169–178KV10 (6.3 nM compared with 81 nM, Table IV). Nef169–177KL9 also elicited responses in more Mamu-A*02+ animals (supplemental Fig. 1). Considerable variability existed among macaques regarding the peptides that were detected as well as the magnitude of responses. No single peptide was recognized by all of the animals tested. Animal 98020 produced the broadest repertoire of responses with nine, while animal 90098 had just one positive response. The number of SFCs per 1 x 106 PBMC, after subtracting background, detected against each positive responding peptide ranged from 60 (Env296–304RY9) to 1177 (Gag71–79GY9) per 1 x 106 PBMC (supplemental Fig. 1). Although considerable variability existed from animal to animal, replicate assays conducted on fresh PBMC from an individual macaque, in general, generated reproducible responses. Of the 17 newly identified CD8+ T cell responses, six were within the structural protein Env, while Pol contained two. The early protein Nef had five epitopes, while the late proteins, Vif and Vpr, contained three and one, respectively.


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  		Table IV. SIVmac239-derived peptides that bind to Mamu-A*02 with IC50 values ≤500 nM and are immunogenic in IFN-{gamma} ELISPOT assaysa

 
Overall, 11 of the 17 new Mamu-A*02 epitopes were recognized in two or more SIVmac239-infected macaques (Env788–795RY8, Nef169–177KL9, Nef221–229YY9, Env296–304RY9, Env760–768SY9, Env519–528GF10, Vif97–104WY8, Pol324–332FF9, Pol518–526LY9, Nef110–119TM10, and Env359–367QY9) (Table IV). As a negative control, we tested all 75 peptides in an ELISPOT assay in two SIVmac239-infected Mamu-A*02– Indian rhesus macaques (2161 and 2162). No reactivity was observed against any of these 75 peptides (data not shown). Two uninfected Mamu-A*02+ macaques were also tested in ELISPOT assays to test for peptide cross-reactivity with other non-SIV epitopes. None of the 75 peptides induced significant responses in either of these control animals (data not shown).

Further immunological characterization of selected Mamu-A*02 CD8+ T cell epitopes

We initiated Mamu-A*02 tetramer production for further immunological characterization of selected Mamu-A*02 CD8+ T cell epitopes. Four of the tetramers were directed against new Mamu-A*02 epitopes (Env317–325KM9, Env788–795RY8, Nef221–229YY9, and Vif97–104WY8), and one previously identified epitope, Gag71–79GY9 (26). The Mamu-A*02 tetramers enable us to directly determine the frequency of Mamu-A*02-restricted Ag-specific CD8+ T cells without having to rely on indirect measurement by effector function, such as IFN-{gamma} secretion (Fig. 2A). When we correlated the levels of tetramer staining to the levels of IFN-{gamma}-producing cells detected for each of the five epitopes, we found a subset of the tetramer-positive cells did not produce IFN-{gamma} (Fig. 2B). We initially investigated this trend for CD8+ T cells directed against Gag71–79GY9. Gag71–79GY9 CD8+ T cell responses from these six responding animals displayed a previously observed discrepancy in the number of IFN-{gamma}-producing cells, as detected by ELISPOT, when compared with tetramer-positive cells (71). This ratio was between 0.17 and 0.91, and most animals had a ratio of <0.66, the ratio previously observed in chronic SIV infection and indicative of functional impairment of these cells (71). Similar findings have also been described in HIV-infected patients (72, 73, 74). Interestingly, animal 97086 had a ratio above 0.85 for Gag71–79GY9 and three other Mamu-A*02-restricted epitopes over a year postinfection (Fig. 2A). In addition, animal 97086 did surprisingly well, living >170 wk postinfection, even without expressing either of the two MHC class I alleles associated with slow disease progression, Mamu-A*01 and Mamu-B*17 (38, 39, 40, 41, 42).



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  		FIGURE 2. A, Representative dot plots of tetramer-positive lymphocytes in peripheral blood from five Mamu-A*02, SIV-specific CD8+ T cell responses indicate functional impairment of SIV-specific CD8+ T cells during the chronic phase of SIV infection. Percentages shown are for tetramer-specific CD3+CD8+ lymphocytes. Ratios below 1 indicate that some tetramer-positive cells are unable to produce IFN-{gamma} in response to specific peptide stimulation. B, Ratio of Gag71–79GY9-specific cells producing IFN-{gamma} to those binding the Mamu-A*02 Gag71–79GY9 tetramer.

 
Experiments suggest that high avidity CD8+ T cells can rapidly select for escape variants (14) and that avidity is an important determinant of CD8+ T cell suppression of virus replication in vivo (75). A vaccinated Mamu-A*02+ macaque (2128) challenged with SIVmac239 made several detectable Mamu-A*02 CD8+ T cell responses that could be evaluated for functional avidity. To evaluate functional avidity, we used ICS assays to measure IFN-{gamma} production in response to stimulation with different peptide concentrations. Ten-fold dilutions of peptides were tested over a range of 5 µg/ml to 5 pg/ml. We then identified the concentration of peptide that yielded 50% of the maximal IFN-{gamma} response. We compared our findings to the previously studied high avidity Nef159–167YY9 epitope (14). Besides Nef159–167YY9, Vif97–104WY8 was also shown to have a high functional avidity, <5 ng/ml necessary to induce 50% maximal IFN-{gamma} production (Table V). Interestingly, another Nef epitope, Nef221–229YY9, had a low functional avidity (>50 ng/ml) over 40 times less than Nef159–167YY9.


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  		Table V. Functional avidity of selected Mamu-A*02 CD8+ T cell responses

 
Analysis of viral variation in newly defined CD8+ T cell epitopes

To determine whether the CD8+ T cell responses against these novel epitopes select for viral escape, 16 Mamu-A*02 epitopes were sequenced in virus isolated from six SIVmac239-infected chronic stage Indian rhesus macaques, four Mamu-A*02+ animals, and two Mamu-A*02– animals. Two epitopes (Env788–795RY8 and Vif97–104WY8) showed amino acid variation within the Mamu-A*02 epitope regions of at least three of four Mamu-A*02+ macaques, possibly indicative of viral escape from CD8+ T cells (Fig. 3A). In the case of Env788–795RY8, position five mutates from serine (S) to leucine (L) in all four Mamu-A*02+ macaques, while position seven of Vif97–104WY8 substitutes asparagine (N) for aspartic acid (D) in three animals. In both instances, there are no detectable substitutions in the Mamu-A*02– animals. Less variation existed at additional Mamu-A*02 epitopes (supplemental Fig. 2). However, amino acid variation in 4 of the 17 novel Mamu-A*02 epitopes (Env296–304RY9, Env788–795RY8, Nef221–229YY9, and Vif97–104WY8) was detected at time of death in a separate study of genome-wide CD8+ T cell escape (76).



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  		FIGURE 3. Evidence for potential escape from CD8+ T cell recognition in the novel Mamu-A*02 epitopes, Env788–795RY8 and Vif97–104WY8, in SIVmac239 infection. The two Mamu-A*02-restricted CD8+ T cell epitopes, (A) Env788–795RY8 and (B) Vif97–104WY8, were directly sequenced from cell-free plasma from SIVmac239-infected Indian rhesus macaques at least 30 wk postinfection. Codons identical to the wild-type sequence are shown as dots. Complete amino acid replacements are shown as uppercase single letter amino acid codes; mixed populations are shown as lowercase single letter amino acid codes. "X" indicates that several amino acids could be encoded. Italicized animal numbers (2161 and 2162) are Mamu-A*02–. For the positional scanning, binding capacity is expressed as both the affinity value, IC50 (nM), and binding relative to the wild-type epitopes (sequence RTLLSRVY (C) and WTDVTPNY (D)). Dashes indicate a relative binding capacity relative to the wild-type peptide <0.01. Bold indicates primary anchor positions. Sequence EALLLRVY represents a potential variant in chronic SIV infection because multiple amino acids can be represented at position one (P1). A nonconservative amino acid substitution, E, was selected for analysis.

 
To further understand the binding capacity of 8-mer peptides and investigate the effect the observed amino acid variation had on these two epitopes, we performed positional scanning of both 8-mer peptides. The positional scanning revealed that position two and the C terminus (position eight) are the main anchor residues, as is the case with the 9-mer and 10-mer peptides (Fig. 1). Changing these residues abolished binding by over 100-fold compared with the wild-type residues (Fig. 3). Position five also appears to function as an important secondary binding site; substitutions result in a 30- to 100-fold reduction in binding.

Next, we tested the binding of variant peptides observed in chronic SIVmac239 infection. When examining the Env788–795RY8 epitope, we observed dramatic reduction, >100-fold, when viral variation occurred at both positions two and five, as is observed in virus from animal 96020 (Fig. 3C). An amino acid substitution at position one decreased binding further. In the case of the Vif97–104WY8 epitope, we showed that while the position seven substitution (N to D), observed in animals 96020 and 97086, did not abrogate binding (Fig. 3D), dual substitutions at both position seven and eight, seen in 96031, caused a three-log reduction in binding capacity. For both epitopes, all variant peptides resulted in some level of reduced binding affinity.

We also performed IFN-{gamma} ICS assays to measure whether the mutant peptides could stimulate CD8+ T cells. Our findings summarized in Fig. 3 demonstrated that in most instances IFN-{gamma} cytokine production is diminished. Interestingly, despite reduced binding capacity, variant peptides, based on in vivo viral variation of the Vif97–104WY8 epitope, are still capable of eliciting IFN-{gamma} production in chronic SIV infection. This may be due to a multitude of factors including the immune responses changing over time to make responses against viral variants or some compensatory functions of the T cells, such as altered avidity or cross-reactivity to viral variants.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In both HIV and SIV infection, MHC class I-restricted CD8+ T cell responses are important for controlling virus replication (1). However, knowledge of the immunogenetics of the Indian rhesus macaque MHC is limited compared with humans. In this study, we refined the peptide-binding motif for a high frequency (>20%) MHC class I allele of Indian rhesus macaques, Mamu-A*02. A detailed characterization of the Mamu-A*02 peptide-binding motif enabled a SIVmac239 proteome scan to identify potential epitopes restricted by Mamu-A*02. Of the 230 peptides selected, 75 bound Mamu-A*02 with an IC50 of ≤500 nM, and 19 are antigenic minimal Mamu-A*02-restricted SIV CD8+ T cell epitopes. This investigation expands the feasibility of analyzing virus-specific CD8+ T cell responses in SIV-infected Indian rhesus macaque vaccine and challenge studies, and facilitates better use of this model for SIV/HIV research. In addition, this refined motif sets the stage for future proteome scans to identify novel epitopes from other category A-C pathogens where the Indian rhesus macaque is also an important biomedical model.

At the WPRC and other Indian rhesus macaque colonies, the Mamu-A*02 allele is expressed at a high frequency of over 20% (26, 50). Similar to the majority of human class I molecules, Mamu-A*02 binds peptides using the residues in position two and at the C terminus of the ligand as the main anchors. At position two, small amino acids are preferred, particularly the polar residues T and S, and the hydrophobic residue V (Fig. 1). The C terminus is much more flexible, preferring aromatic or hydrophobic residues as Y, F, M, L, W, V, or I. Peptides of 9–10 aa in length are optimal (Table II, Mamu A*02 binding as a function of peptide length); however, 8-mer peptides are also tolerated and immunogenic, representing 3 of 21 ELISPOT positive responses (Table IV). Interestingly, the peptide-binding motif of Mamu-A*02 is similar to that of alleles in the HLA A1 supertype (77). This observation suggests that if an overlap in peptide-binding repertoire can be established, Mamu-A*02 animals may allow the development of an animal model closely mimicking immune responses restricted by several of the most frequent class I alleles in humans.

Overall, 75 peptides derived from seven of the nine SIVmac239 proteins (Tat and Vpx were not represented) were identified that bind Mamu-A*02 with high affinity (supplemental Table I). Further analysis using the IFN-{gamma} ELISPOT assay revealed that 21 of these peptides can recall CD8+ T cell responses (Table IV). This provided 17 new Mamu-A*02-restricted SIV CD8+ T cell epitopes (Table IV) within five of the SIVmac239 proteins (Fig. 4). Our results also indicate that the previously identified Mamu-A*02 epitopes, Nef159–167YY9 and Gag71–79GY9 (26), elicited the most dominant responses (supplemental Fig. 1) and were two of the three highest binding peptides (Table IV). These 19 Mamu-A*02 epitopes continue to illustrate the broad CD8+ T cell repertoires against several viral proteins seen in both SIV-infected macaques and HIV-infected humans (46, 47, 78, 79), while further defining the SIV-infected rhesus macaque model for HIV.



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  		FIGURE 4. Location of Mamu-A*02-bound peptides recognized in SIVmac239-infected Indian rhesus macaques. Black boxes within each of the proteins correspond to the position of the Mamu-A*02-restricted CD8+ T cell epitopes which were identified by ELISPOT. Previously identified epitopes are gray boxes within the corresponding protein, and the sequence is in black text. Epitopes with functional Mamu-A*02 tetramers end with an asterisk (*), and epitopes with evidence of escape are italicized. In the case of Vif, three epitopes overlap by one amino acid each, and one large black marker represents these three continual epitopes.

 
Of the 17 epitopes, five were derived from Nef and three from Vif (Fig. 4). Nef is of particular interest because no immunogenic Mamu-A*01-restricted Nef epitopes have been identified to date (46). Because Nef is expressed early in each viral replication cycle of infection, it can be rapidly presented by MHC class I molecules and could be an attractive early target for vaccine development (80, 81, 82). Vif may also be an attractive target for future immunological analysis because of its role in modulating virion assembly (83, 84) and virus trafficking to the nucleus (85). In this study, positions 89–113 of Vif contained three contiguous Mamu-A*02 epitopes that were newly identified and only overlapped by a single amino acid, possibly a "hot spot" for CD8+ T cell responses.

It should be noted that our approach might fail to detect CD8+ T cell responses that bind with low affinities. Additionally, responses detected during chronic SIV infection may not include those which escape during acute infection such as Nef159–167YY9 in Mamu-A*02 (14, 26) due to reduced CD8+ T cell frequencies. To address this issue, we comprehensively sequenced virus from four Mamu-A*02+ macaques during chronic infection but did not see consistent amino acid substitutions expected by escape from strong, acute phase CD8+ T cell responses (data not shown). This sequence analysis also enabled us to determine whether CD8+ T cell responses to a given epitope were able to select for escape mutations in SIVmac239. Direct sequencing of viral amplicons across the regions of two novel epitopes (Env788–795RY8 and Vif97–104WY8) showed amino acid replacement in these epitopes by the chronic phase of infection. Additionally, we showed that all variant peptides had reduced binding affinity and the majority of these peptides impair both binding and recognition, as measured by IFN-{gamma} production, when compared with the wild-type peptides (Fig. 3). Overall, our sequence analysis suggests that these 17 novel Mamu-A*02-specific CD8+ T cell responses exert differential selective pressure on the virus (13, 14, 26).

Because Mamu-A*01 and Mamu-A*02 have similar binding specificity at position two and the C terminus, there is the potential for cross-reactivity and/or restriction of Mamu-A*02-binding peptides to Mamu-A*01. Interestingly, none of the 19 identified Mamu-A*02 epitopes have proline (P) in the third position, the dominant Mamu-A*01 anchor (45). However, three peptides, Env359–367QY9, Nef20–28LY9, and Pol324–332FF9, induced responses in three Mamu-A01+ Mamu-A*02– animals (data not shown). Previous investigations illustrate that such cross-reactivity has occurred in humans including HLA-B57 and HLA-B58, HLA-A31 and HLA-A33, along with HLA-B*3501 and HLA-B*5101 (86, 87, 88, 89). Further investigation of these three epitopes may verify cross-reactivity or Mamu-A*01 restriction. Although possible, it is unlikely that another identified molecule besides Mamu-A*02 and/or Mamu-A*01 restricts these responses. Two Mamu-A*01– Mamu-A*02– animals (2161 and 2162) were tested for the panel of 75 Mamu-A*02 binders. Neither animal made responses detectable by IFN-{gamma} ELISPOT (data not shown).

The identification of minimal optimal CD8+ T cell epitopes will facilitate future cellular studies involving both vaccination and challenge in the SIV-infected rhesus macaque model. These Mamu-A*02 SIV-derived CD8+ T cell epitopes can now be used for a variety of applications including the testing of vaccine strategies, detection of viral escape mutations (13, 20, 26), or the creation/development of MHC class I tetramers (10, 48, 49). The synthesis of tetramers allows not only for the quantification of Ag-specific CD8+ T cell immune responses but for in situ detection in tissues (78) and Ag-specific cellular sorting for the development of new functional assays. These investigations are now feasible in Mamu-A*02+ rhesus macaques with the construction of five Mamu-A*02 tetramers (Fig. 2), while additional Mamu-A*02 tetramers are currently in development.

The SIV-infected Indian rhesus macaque is the best animal model to address questions regarding HIV pathogenesis, virology, and immunology (36, 37). Mamu-A*01 is one of only two MHC class I alleles extensively investigated in the Indian rhesus macaque, resulting in a shortage of Mamu-A*01+ macaques due to SIV vaccine and challenge studies. Use of Mamu-A*02+ animals greatly increases the pool of MHC-defined animals for which an extensive number of SIV CD8+ T cell epitopes are known because Mamu-A*02 is expressed at a high frequency. Elucidation of epitopes that bind other MHC class I molecules, such as Mamu-A*02, may alleviate the intense demand for Mamu-A*01+ animals while shedding additional light on the complexity and breadth of SIV-specific CD8+ T cell responses. This study represents another important step toward facilitating more comprehensive testing of vaccine approaches in the SIV-infected Indian rhesus macaque model for application to HIV infection in humans. As a result, more MHC class I-restricted CD8+ T cell responses can be observed when studying the interaction between SIV and the Indian rhesus macaque immune system, increasing the utility of this model for HIV investigations.


   Acknowledgments
 
We thank Tim Jacoby and William Rehrauer for the PCR-SSP typing, Richard Rudersdorf for construction of the Mamu-A*02 transfectant, and Shari Piaskowski, Sarah Martin, and Jacque Miller for immunological assay assistance. We also thank Adrian McDermott, Thomas Friedrich, and Sarah Martin for helpful discussion and the Immunology and Virology Core Laboratories at the National Primate Research Center (University of Wisconsin) for technical assistance.


   Footnotes
 
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 by National Institutes of Health Grants R24 RR015371 and 5P51 RR000167 (to D.I.W.) and National Institutes of Health/National Institute of Allergy and Infectious Diseases Contracts NO1-AI-95362 and HHSN266200400006C (to A.S.). D.I.W. is a recipient of an Elizabeth Glaser scientist award. Back

2 Address correspondence and reprint requests to Dr. Alessandro Sette, La Jolla Institute for Allergy and Immunology, 3030 Bunker Hill Street, Suite 326, San Diego, CA 92109. E-mail address: alex{at}liai.org Back

3 Abbreviations used in this paper: ICS, intracellular cytokine staining; SSP, sequence-specific primer; ARB, average relative binding; SFC, spot-forming cell; vRNA, viral RNA. Back

4 The on-line version of this article contains supplemental material. Back

Received for publication January 20, 2004. Accepted for publication August 3, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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