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*
Epimmune, San Diego, CA 92121;
New England Regional Primate Center, Southborough, MA 01772;
The Childrens Research Institute, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43205;
National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and
¶ Wisconsin Regional Primate Center, University of Wisconsin, Madison, WI 53719
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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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Previous studies using live cell binding assay systems were used to identify SIV epitopes presented by various rhesus macaque class I molecules (4). General motifs crucial for high affinity binding for five different Mamu class I molecules (A*01, A*11, B*03, B*04, and B*17) were also identified (5). The utility of this approach was emphasized by a recent study in which 27 new SIV-derived A*01-restricted epitopes were revealed, based on sequence motif and Mamu A*01 in vitro binding assays using purified MHC molecules.3
In this report, the peptide binding specificity of Mamu A*01 is more rigorously characterized through the 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. This analysis has allowed us to define the side chain specificity of both primary and secondary anchor residues.
It was previously shown that accuracy in epitope prediction can be greatly increased by developing detailed peptide binding motifs defining specificity at both primary and secondary anchor positions (6, 7, 8, 9, 10, 11, 12, 13). This knowledge allows for the rational design of optimized ligands. For example, natural sequences carrying suboptimal residues at primary and/or secondary positions can be identified. The suboptimal residues are then replaced with optimal anchors, generating epitopes with increased binding affinity (11, 14, 15). Following this modification, the wild-type peptides that were unable to elicit responses, or were poor immunogens, became highly immunogenic following analoging to increase their MHC binding affinity (14, 15, 16, 17, 18, 19). The CTL induced by such analogs were capable, in most instances, of recognizing target cells expressing wild-type Ag sequences. This phenomenon is likely to reflect less stringent epitope binding requirements for target cell recognition compared with that needed for stimulation of naive T cells to induce differentiation into effectors (20). In this respect, it has been noted that expression of as little as a single molecule of antigenic peptide complexed to class I molecules is sufficient for target cell recognition by effector CTL (21).
SIV encodes three structural retroviral genes (Gag, Pol, and Env), three early genes (Tat, Rev, and Nef), and four accessory genes (Tev, Vpu, Vpr, and Vif). The regulatory and accessory genes are transcribed early in viral replication as multiply spliced mRNAs and appear to be substantial virulence factors critical for the development of AIDS. They are important determinants of viral pathogenicity and are responsible for high titer virus replication in vivo (22, 23, 24, 25, 26, 27, 28, 29, 30). Tat regulates the high level transcription needed to maximize virus production during the short survival time of infected cells (26, 31), whereas Rev activates the export of unspliced RNA required for efficient expression of all viral genes (32). Nef has pleiotropic effects and appears to modulate the cellular membrane proteins that induce CD4 down-regulation (33, 34). Interestingly, infection with Nef-deleted strains of SIV leads to low viral loads, and simian AIDS does not develop in most monkeys. Variants of HIV isolated from long-term nonprogressors are often found with deletions in the Nef gene or defective Nef alleles (35).
Vpu also modulates cellular membrane proteins leading to down-regulation of CD4 (36). Vpu binds CD4 in the endoplasmic reticulum and targets it for proteolysis. The Vif protein must be present in the cells that produce virus, and its absence results in a block of infection soon after viral entry into target cells (37, 38). In addition, Vif may modulate virion assembly (39). HIV-1 and several strains of SIV contain the Vpr accessory gene, whereas HIV-2 and several other strains of SIV contain both Vpr and a highly homologous gene Vpx (40). Vpr appears to augment the importation of uncoated nucleoprotein complexes into the nucleus of the infected cell by interaction with cellular import factors (41). Vpr can also facilitate an increase in virus production by causing the infected cell to delay for extended periods of time in the G2 phase of the cell cycle, where the viral long terminal repeat (LTR) is more active (42). Thus, virus production is maximized in short lived T cells by reducing the time interval between the initial infection and the active production of new virions.
Recent data support the notion that immune responses directed against early SIV regulatory proteins might be extremely effective in controlling viral spread in the initial phases of infection (Ref. 43 ; see also Refs. 25, 44). Recognition of regulatory proteins might also be important to attack hidden latent reservoirs of SIV. Accordingly, these proteins are logical targets for the immune system and should be evaluated as vaccine immunogens. Based on the detailed Mamu A*01 motifs defined herein, we set out to identify natural sequences derived from SIV regulatory protein that could serve as CTL epitopes. Furthermore, we rationally designed a set of "fixed anchor" analog epitopes. Taken together, these studies should enable the design of vaccine constructs designed to elicit powerful CTL responses directed against SIV regulatory proteins.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SIVmac239 protein sequences (accession no. M33262) were analyzed using the text string search software program Motifsearch 1.4 (D. Brown, San Diego, CA) to identify potential peptide sequences containing defined motifs. Peptides were purchased as crude material from Chiron Mimotopes (San Diego, CA) or synthesized at Epimmune using standard tertiary butyloxycarbonyl or fluronylmethyloxycarbonyl solid phase methods as previously described (7). Peptides were resuspended at 4–20 mg/ml in 100% DMSO, then diluted to required concentrations in PBS.
Radiolabeled probe peptides were synthesized at Epimmune on a larger scale using standard tertiary butyloxycarbonyl or fluronylmethyloxycarbonyl solid phase methods. These peptides were subsequently purified to >95% homogeneity by reversed phase HPLC, and their composition ascertained by amino acid analysis, sequencing, and/or mass spectrometry analysis.
Mamu A*01 purification
721.221 cells transfected with Mamu A*01 cDNA were used as the source of Mamu A*01 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 (Hazelton Biologics, Lenexa, KS), and grown for large scale cultures in roller bottle apparatuses.
Mamu A*01 was purified from cell lysates using affinity chromatography (45). 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 A*01 was captured by
repeated passage over protein A-Sepharose beads conjugated with the
anti-HLA (A, B, C) Ab W6/32 (4). After 2–4 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 A*01 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 A*01 binding assay
Quantitative assays for the binding of SIV peptides to soluble
Mamu A*01 molecules was based on the inhibition of binding of a
radiolabeled standard probe peptide. These assays were performed using
the same protocol described for the measurement of peptide binding to
HLA class I molecules (45). Briefly, 1–10 nM of
radiolabeled probe peptide, a position 1 C
A analog of the SIV Gag
181-190 peptide (ATPYDINQML), was coincubated at room temperature with
1 µM to 1 nM of purified Mamu A*01 in the presence of 1 µM human
2-microglubulin (Scripps Laboratories, San
Diego, CA) and a cocktail of protease inhibitors. Following a 2-day
incubation period, the percentage of MHC-bound radioactivity was
determined by size exclusion gel filtration chromatography on a TSK
2000 column.
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. Because under the
conditions to be used, where [label] < [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,
and all different replicate observations were contained in a 4-fold
range. As a positive control, in each experiment the unlabeled version
of the radiolabeled probe was tested.
Sequence and binding analysis
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 assayed by dividing the
IC50 of the positive control for inhibition (SIV
Gag 181-190 C1A: 8.3 nM) by the IC50 for each
tested peptide. These values can subsequently be converted back into
IC50 nM values by dividing the average
IC50 nM of the positive control 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 (ARB)4 value, for all peptides of a
particular characteristic (7, 8, 9, 10, 11, 12, 46).
In analogy to the method described to determine secondary anchor effects influencing the capacity of peptide ligands to bind to HLA class I molecules (7, 8, 9, 10, 11, 12), maps of secondary interactions influencing peptide binding to Mamu A*01 were derived. All peptides of a given size (8, 9, 10, or 11 aa) and with at least one tolerated, and two preferred anchor residues, were selected for analysis. The binding capacity of peptides in each size group was further analyzed by determining the ARB values for peptides that contain specific amino acid residues in specific positions. For determination of the specificity at Mamu A*01 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. For example, an ARB value was determined for all 9-mer peptides that contain A in position 1, or F in position 7, etc. Because of the rare occurrence of certain amino acids, residues were grouped according to individual chemical similarities as previously described (7, 8, 9, 10, 11, 12, 46).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies using Edman degradation analyses of sets of pooled peptides eluted from Mamu A*01 indicated a dominant anchor specificity for proline in position 3 (4). Further analysis, using a live cell binding assay and panels of single substitution analogs, also implicated position 2 and the C terminus in determining Mamu A*01 binding capacity. In the present study, the Mamu A*01 binding capacity of the same panels of single substitution analogs was examined with an in vitro binding assay using purified Mamu A*01 molecules.
In the first panel, a nonconservative lysine (K) substitution was
introduced at every position of peptide 1279.06, a
C1, to A analog of the SIV Gag 181-190 epitope.
The results confirmed position 2 and the C terminus, in addition to
position 3, as critical for peptide binding (Table I
). In each of these cases, substitution
of the native peptide residue to K resulted in a 100-fold or greater
decrease in binding capacity. Substitutions at other positions were not
associated with significant decreases in binding capacity.
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It was found that at position 2, small and/or hydrophobic
residues A, P, and V were tolerated, with binding capacities 40- to
75-fold lower than the parent peptide (Table I
). Larger residues (Q, F,
and K) bound either not at all, or at least 100-fold less than the
parent peptide. On the basis of this data, and considering the chemical
similarity of different amino acid residues, the position 2 specificity
of Mamu A*01 was tentatively ascribed to a preference for the small
polar residues T and S. Small and/or hydrophobic residues, such as A,
P, G, L, I, V, and M, were defined as tolerated.
At position 3, all analogs tested were associated with at least
100-fold decreases in binding capacity (Table I). Interestingly, this
was true even for relatively conserved T and A substitutions, or the
semiconserved substitution to V. Thus, it was concluded that the
position 3 specificity of Mamu A*01 was solely for P.
At the C terminus, analogs carrying aliphatic and/or hydrophobic
residues such as I, M, or F (Table I) were associated with binding
capacities within 3-fold of the parent peptide, which carries L. The
small polar residue T was also tolerated, but was associated with a
100-fold decrease in binding capacity. Polar residues such as Q or K
were not tolerated. In summary, and in consideration of chemical
similarity, these data suggest a preference at the C-terminal anchor
position for aliphatic (L, I, V, and M) and aromatic residues (F, W,
and Y), with T also potentially being tolerated.
Detailed characterization of the Mamu A*01 primary anchor specificity
To define the Mamu A*01 motif in more detail, a library comprised of 714 peptides between 8 and 11 residues in length was tested for Mamu A*01 binding capacity. Each peptide represented a naturally occurring sequence of either viral or bacterial origin and carried residues conforming to the preliminary motif described above in at least two of the three primary anchor positions. The effect of specific amino acid residues 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 A*01 with an IC50 value of 500 nM or better was also calculated.
To determine in more detail the specificity at position 2, the binding
capacity of the library subset of peptides with P in position 3 and
aliphatic (L, I, V, M, and T) or aromatic (F, W, and Y) residues at the
C terminus was analyzed (Fig. 1).
Peptides with S in position 2 were, on average, the best binding
peptides. T was also preferred, with an ARB of 0.75, relative to
S2 peptides. In the case of both S and T, over
50% of the peptides bearing these residues bound Mamu A*01 with
affinities of 500 nM or better. Aromatic (F, W, Y), aliphatic (L, I, V,
M, N), or small (A, G, P) residues were tolerated, as defined above,
with ARB values in the 0.01–0.1 range. Large polar (Q) or charged
residues (D, E, R, K) or C were not tolerated, with ARB values of
<0.01.
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). Consistent with the single substitution
analysis, it was found that F, L, I, and M were preferred, with ARB
values in the 0.25–1.0 range (Fig. 2). A
preference for V, predicted on the basis of chemical similarity, was
also confirmed. The aromatic residues Y and W were tolerated, with ARB
values 0.070. The small residues T and A were also tolerated,
although somewhat less well, with ARB values 0.040.
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). The small residues T
and A were barely tolerated, with relative binding capacities in the
0.01–0.02 range. No other residue was associated with an ARB value of
>0.01.
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A library of peptide ligands carrying the Mamu A*01 motif defined
in Table II was next analyzed to determine the correlation between
peptide length (between 8 and 11 residues) and binding capacity. It was
found that 39.4% of the 9-mer peptides and 35.2% of the 8-mer
peptides bound with IC50 values of 500 nM or less
(Table III). Longer peptides were also
capable of binding, although somewhat less well. Specifically, 17.8%
of 10-mer and 21.8% of the 11-mer peptides had affinities of 500 nM or
better. In conclusion, this data has indicated that the optimal ligand
size for Mamu A*01 is 9 residues. Shorter peptides of 8 residues, or
longer peptides up to 11 residues in length, are also relatively well
tolerated.
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Each peptide sequence in the library was evaluated to determine
the number of preferred, tolerated, and nontolerated residues present
at position 2, position 3, and the C terminus. Peptides were then
grouped as shown in Table IV, and binding
data was evaluated as above. As expected, it was found that peptides
with preferred residues in each anchor position were overall the best
binders, binding more frequently (68.0%) and with higher ARB capacity
than other peptides in the library. Peptides with one tolerated anchor
(and two preferred anchors) bound with a frequency of (20.8%). The ARB
of these peptides was 25-fold less than peptides with all preferred
main anchor residues. Peptides with more than one tolerated anchor
residue, or with at least one nontolerated residue in an anchor
position, were found to be poor binders. These peptides bound with ARB
values in the 0.0025–0.0050 range, representing a 200- to 400-fold
reduction in binding capacity, compared with peptides with three
preferred anchors. Correspondingly, only between 3.6 and 5.8% of the
peptides were found to have binding affinities of 500 nM or better. In
conclusion, optimal binding is obtained, in general, when all three
anchor positions carry preferred residues. However, peptides with one
tolerated residue and two preferred residues may also be expected to
bind with reasonably high frequencies.
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In the next series of analyses, we sought to determine whether
secondary effects influencing peptide binding to Mamu A*01 could be
detected at positions other than the main anchors. All peptides of a
given size (8, 9, 10, or 11) with at least one tolerated and two
preferred anchor residues, were selected. The binding capacity of
peptides in each size group was further analyzed by determining the ARB
values for peptides that contain specific amino acid residues in
specific positions. The resulting relative binding values, by
corresponding residue/position pairs, for 8- to 11-mer sequences are
shown in Tables
V-VIII.
Summary maps are shown in Fig. 4, A–D.
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). Of note,
aromatic residues were often preferred in position 1, whereas negative
charges (D, E) and G were often deleterious at the same position.
Charged (K, R, H, or D, E) residues were also deleterious toward the
middle of the peptide (positions 5 and 6 for 8-mers; 5, 6, and 8 for
9-mers; 4, 5, 6, and 9 for 10-mers; 6 and 9 for 11-mers). Similarly, it
was noted that each particular size was associated with unique
preferences. For example, the position immediately preceding the C
terminus preferred aliphatic residues (L, V, I, or M) in the case of
8-mers, aromatic residues (Y, F, or W) in the case of 9-mers, G in the
case of 10-mers, and positive charges (K, H, or R) in the case of
11-mers. Strikingly, P is strongly preferred in position 4 only in the
case of 8-mer peptides, suggesting (because P is also an anchor in
position 3) that a double P might be important to achieve a favorable
conformation for binding for ligands of this size. In conclusion,
detailed maps of primary anchor preferences and secondary effects have
been defined for Mamu A*01 binding to peptides of 8, 9, 10, or 11
residues in length. Linear polynomial algorithms to predict Mamu A*01 binding
ARB values have been shown to represent effective coefficients for
use in designing polynomial algorithms for identifying peptide
sequences with a good probability of binding class I molecules with
high affinity (12). The basic premise of this predictive
methodology is the independent binding of peptide side chains, where
the stability contributed by a given residue at a given position is
independent of the nature of the residues at other positions. These
algorithms take into account both extended and refined motifs (7, 12) and are essentially based on the premise that the overall
affinity (
G) of peptide-MHC interactions can be approximated as a
linear polynomial function of the type G =
a1i x
a2i x
a3i ... ... x
ani where
aij is a coefficient that represents
the effect of the presence of a given amino acid (j) at a
given position (i) along the sequence of a peptide of
n amino acids. The crucial assumption of this method is that
the effects at each position are essentially independent of each other
(i.e., independent binding of individual side chains). When residue
j occurs at position i in the peptide, it is
assumed to contribute a constant amount ji
to the free energy of binding of the peptide, irrespective of the
sequence of the rest of the peptide. This assumption is justified by
the studies from our laboratories that demonstrated that peptides are
bound to MHC and recognized by T cells in essentially an extended
conformation.
A method for the derivation of specific algorithm coefficients
has been described by Gulukota et al. (Ref. 12 ; see also
Refs. 10, 11, 46). Briefly, for all i
positions, anchor and nonanchor alike, the geometric mean of the ARB of
all peptides carrying j is calculated relative to the
remainder of the group and used as the estimate of ji. To
calculate the algorithm score of a given peptide in a test set, the
geometric mean of all ARB values corresponding to the sequence of the
peptide is calculated. If the resulting score exceeds a chosen
threshold, the peptide is predicted to bind. Appropriate thresholds can
be chosen as a function of the degree of stringency of prediction
desired. For example, algorithm scores that allowed prediction of 90 or
75% of the binders in the data set analyzed are shown in Table IX.
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To identify peptide ligands derived from SIV proteins that could
represent candidates for use in an SIV vaccine, we next scanned
sequences of SIVmac239 early and late regulatory proteins for the
presence of peptides with the broadly defined Mamu A*01 binding motif
(Table II). Of the 51 wild-type sequences identified, 12 were found to
have the capacity to bind Mamu A*01 with an IC50
value of 500 nM or less (Table X). Four
of these peptides are conserved between SIVmac239 and SIVmac251, and
eight others are unique to SIVmac239. Overall, 23.5% of the
motif-positive peptides were Mamu A*01 binders. When the same SIV
proteins were analyzed using the algorithms described above, nine
sequences were identified that were predicted to be Mamu A*01 binders
on the basis of scores greater than or equal to the 75% cut-off
criteria. Seven (78%) of these peptides, representing 58% of all
binders, were found to bind Mamu A*01 with affinities of 500 nM or
better.
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, had previously
been identified as a Mamu A*01 binder and is also shown in Table IX.
However, it should be noted that Vif14 was the
only peptide among a panel of 39 SIVmac239 peptides with motifs not
described by Table II that bound Mamu A*01 with
IC50 values of 500 nM or better. In conclusion,
the motif described herein allowed the identification of 12 peptides
derived from SIVmac239 regulatory proteins that have the capacity to
bind Mamu A*01 with high affinity. An additional nonmotif-bearing
peptide with high affinity binding activity was also identified. Engineering fixed anchor analogs of SIV-derived peptides
In the same series of experiments, an additional 20 wild-type
peptides were identified that bound weakly, in the 500–30,000 nM
range. Nineteen of the weak binders were associated with suboptimal
motifs, carrying a tolerated residue at one of the three main anchor
positions (Table XI). For each
suboptimal sequence, an analog peptide was engineered by replacing
tolerated anchor residues with a preferred residue. For this study,
tolerated residues in position 2, position 3, and at the C terminus
were analogued to S, P, and L, respectively.
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).
This corresponds to an 89% success rate in engineering peptides with
significantly increased binding capacity. It was noted that
substitutions at any of the three anchor positions were equally
effective in generating higher affinity binding analogs. Lower incidence of Mamu A*01 binding in SIV early vs late regulatory proteins
When the protein of origin of the peptides described in the
preceding sections was examined, a bias toward late regulatory proteins
was observed (Table XII; see also
Tables IX and X). A total of 15 Vif-, 6 Vpx-, and 6 Vpr-derived
peptides were identified that bound with good
(IC50 
500 nM) or weak
(IC50 = 500–3000 nM) affinity. By comparison,
only one Rev-, one Nef-, and two (but overlapping) Tat-derived epitopes
were identified. This bias is not related to the different size of the
proteins considered. As shown in Table XII, Vif carried 7.0 binders/100
residues, Vpx 5.4, and Vpr 5.9 (average of 6.1 ± 0.8). By
comparison, the early regulatory proteins Tat, Rev, and Nef carried
1.5, 1.9, and 1.1 binders/100 residues (average of 1.5 ± 0.4),
respectively. This difference in frequency of binders is significant
beyond p = 0.001, as determined in a two-sample Z
test.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To more rigorously define the peptide binding specificity of Mamu A*01, the single substitution analysis was refined using a large peptide library. The use of large libraries allows the analysis of the effect of single residues on binding capacity in the context of multiple peptide sequences. This avoids potential biases and limitations presented by the use of single substitution analogs, where the effect of a specific residue is determined in the context of only a single amino acid sequence. Furthermore, the approach used herein offers an effective means of simultaneously probing for effects at both primary and secondary anchor positions. This method relies on the assumption that each position contributes independently to binding affinity. The validity of this assumption is supported by previous studies that compared neural network- and polynomial matrix-based methods for their success in predicting class I MHC binding capacity (12).
The preferred peptide size for Mamu A*01 binding was found to be 8 or 9 residues, although peptides of 10 or 11 residues could also bind with appreciable frequencies. This type of size specificity is somewhat different from what was previously noted in the case of most HLA class I molecules (47, 48, 49), which appear to prefer ligands of 9–10 residues in size. Future studies will be needed to determine whether this finding can be extended as a generalization for Mamu class I alleles. Interestingly, the one and only optimal epitope thus far identified for Mamu A*11 is indeed an 8-mer (5, 50). Additional analyses are necessary to determine whether shorter (7 residues), or longer (12 residues or above) peptides also bind Mamu A*01 with appreciable frequency.
This study represents the first in-depth analysis of secondary influences on peptide binding to rhesus macaque-derived class I molecules. Typically, for class I molecules, primary anchor residues are necessary for peptide binding (7, 51). However, other residues can act as secondary anchors providing supplemental binding energy. Alternatively, the presence of certain residues in specific positions may also have a negative effect on peptide binding capacity. Herein we demonstrate that the same type of influence is also apparent in the case of Mamu A*01 molecules. The fact that different secondary effects were associated with different ligand sizes explains why secondary anchor determination by sequencing of heterogeneous pools of acid-eluted natural ligands has thus far proved to be of limited success.
Primate lentiviruses may have evolved to exploit several viral and immunologic mechanisms that escape or weaken virus-specific immune responses. Indeed, despite vigorous virus-specific immune responses, HIV is able to establish chronic infection in most cases. Effective prophylactic vaccines against SIV or HIV may need to be targeted to proteins active very early in the course of infection (43). In the case of therapeutic vaccines, it might be crucial to target late regulatory proteins, involved in immune evasion and in maintaining reservoirs of latent infection.
Because of their small size, these proteins contain relatively few binding peptides. However, by virtue of refined motifs, we have been able to identify four peptides derived from early, and nine from late, SIV regulatory proteins that bind Mamu A*01 with high affinity. In addition, we have also been able to design 17 additional potential epitopes, by replacing suboptimal anchor residues with optimal ones. The identification of these epitopes is important given the very small number of known epitopes derived from the early and late regulatory proteins. Thus, this study allows the design of specific vaccine constructs targeting early and late SIV regulatory proteins. In this light, we have recently described the engineering and testing of experimental multiepitope MiniGene vaccines in HLA-transgenic animals (52) and rhesus macaques (43).3
Finally, our exhaustive analysis revealed a striking difference in the number of Mamu A*01 binding epitopes contained in early, compared with late, regulatory SIV proteins. A possible explanation of this observation is that these different frequencies are the result of selective pressure from immune responses, resulting in viral escape and reduced numbers of class I binding peptides. These results are also in agreement with a recent study by Allen et al. that underlined the crucial role of Tat epitopes in controlling acute infection, and demonstrated immune pressure and viral escape in the case of the Mamu A*01-restricted Tat 27 epitope but not in the case of other Vif- or Vpx-derived epitopes (43).
Alternative explanations should also be considered. It is possible, for example, that this observation might reflect selection for structural reasons against proline residues in these early regulatory proteins. This explanation appears unlikely in light of the fact that no significant difference exists in the overall frequency of proline in early (7.3 ± 4.2) or late (8.2 ± 2.8) regulatory proteins or in structural proteins (5.2 ± 1.9). It should also be noted that SIVmac239 was derived after serial passage in macaques of a virus originally derived from sooty mangabeys. Thus, these results might reflect selection in sooty mangabeys with alleles binding a motif similar to Mamu A*01. In this light, it is intriguing to point out that in previous studies (53) one of the original epitopes recognized by sooty mangabeys carried a proline in position 3 and isoleucine in position 9.
These results are also reminiscent of earlier studies by Berzofsky and associates (54) that had suggested a lower number of class I motif-positive peptides as one of the structural features of HIV-1. However, actual binding capacity of the motif-containing peptides was not measured. This might explain why, in that case, differences among early and late regulatory proteins were not reported. In future studies, analysis of regulatory proteins might be expanded to other class I and class II specificities of both human and rhesus macaque origin. A similar analysis of the frequency of binding peptides in the structural proteins Gag, Pol, and Env could also be performed. Additional studies addressing viral selection have been presented by other groups (55, 56).
In conclusion, our experiments provide an in-depth look into the
interactions between peptide ligands and Mamu A*01. Primary and
secondary anchor maps allowed for efficient identification of peptides
binding Mamu A*01 and allowed for the design of ligands with enhanced
binding capacity. Using the simple primary anchor motif, 25% of the
predicted peptides bound with high affinity, whereas with the improved
algorithm 75% of the predicted peptides bound well. The converse
study, to determine how many good binders from all possible 8- to
11-mer peptides encoded SIV regulatory genes are effectively predicted,
was not performed. Previous studies analyzing HLA class I binding and
human papillomavirus E6 and E7 proteins indicated that over 90% of the
binders identified carry the appropriate motif. We have applied this
knowledge to the identification and engineering of epitopes derived
from SIV regulatory proteins, thus enabling the design of vaccine
constructs aimed at focusing immune response directed against
these Ags.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alessandro Sette, Epimmune Inc., 5820 Nancy Ridge Drive, Suite 100, San Diego, CA 92121.
3 T. M. Allen, 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. CD8+ lymphocytes from SIV-infected rhesus macaques recognize 27 different epitopes bound by the MHC class I molecule Mamu A*01: implications for vaccine design and testing. Submitted for publication.
4 Abbreviation used in this paper: ARB, average relative binding.
Received for publication June 30, 2000. Accepted for publication September 6, 2000.
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