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* 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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To date, the most rigorously characterized rhesus macaque class I molecule is Mamu-A*01 (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 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 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, 19, 20, 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 µg/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-µM filters and cleared of nuclei and debris by centrifugation at 10,000 x 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
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 µM to 1 nM of purified
Mamu-B*17 in the presence of 1 µM human
2-microglobulin (Scripps Laboratories, San
Diego, CA) and a mixture of protease inhibitors. The radiolabeled
peptide used was a position
9W
Y
analog of the SIV-derived epitope Nef165–173
(IW9; sequence IRFPKTFGY; see Refs. 36, 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 
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, 41, 42, 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 µg 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 (Sigma-Aldrich) 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 µg/ml
Con A (Sigma-Aldrich), 10 µg/ml of each of the Mamu-B*17 peptides
(see Table II
), 10 µg/ml negative control influenza peptide
(SNEGSYFF), or no peptide. Input cell numbers were 1.0 x
105 PBL in 100 µl/well, in triplicate
wells.
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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 x 106 cell. Responses to Con A (positive control) were always greater than 1000 SFCs per 1 x 106 cells.
Intracellular IFN- cytokine staining
A total of 5 x 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 µg each Ab; BD PharMingen,
San Diego, CA) and either staphylococcal enterotoxin B (10 µg/ml;
Sigma-Aldrich), Mamu-B*17 peptides
Nef165–173(IW9), Vif64–73
(GW10), Vif66–73 (HW8), or a negative control
influenza peptide (SNEGSYFF) (1 µg each peptide/sample). A total of
10 µg/ml brefeldin A (10 µg/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 µl 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 µl
cell suspension was fixed with 250 µl 2% paraformaldehyde.
Acquisition was performed on a FACSCalibur flow cytometer collecting
100,000–200,000 lymphocyte-gated events per sample.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, the SIV Nef165–173 peptide
(IW9; sequence IRFPKTFGW) 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.
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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. 1
B). 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.
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 Mamu-B*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 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.
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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 preferred 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.
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 Mamu-B*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 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 x 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.
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. PBMC from Mamu-B*17-positive animals were tested at 3
wks postinfection using a subset of Mamu-B*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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 C-terminal preference for a specific hydrophobic residue will also 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 HLA-B*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 Mamu-B*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, 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 have been reported in humans in the case of influenza (50, 51), HIV (52, 53), HCV (54, 55, 56, 57, 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, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 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 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 Mamu-B*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.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alessandro Sette, Epimmune, 5820 Nancy Ridge Drive, San Diego, CA 92121. E-mail address: asette{at}epimmune.com
3 Abbreviations used in this paper: ICS, intracellular cytokine-staining assay; ARB, average relative binding value; SFC, spot-forming cell.
4 T. M. Allen, P. Jing, B. Calore, H. Horton, D. H. O’Connor, T. Hanke, M. S. Piekarczyk, R. A. Ruddersdorf, B. R. Mothé, 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.
Received for publication February 4, 2002. Accepted for publication April 18, 2002.
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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. 73:5466.1 helix of the HLA-B heavy chain can have an overriding influence on peptide-binding specificity. J. Immunol. 158:1660.[Abstract]
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