Two MHC class I molecules associated with elite control of immunodeficiency virus replication, Mamu-B*08 and HLA-B*2705, bind peptides with sequence similarity.
ABSTRACT HLA-B27- and -B57-positive HIV-infected humans have long been associated with control of HIV replication, implying that CD8(+) T cell responses contribute to control of viral replication. In a similar fashion, 50% of Mamu-B*08-positive Indian rhesus macaques control SIVmac239 replication and become elite controllers with chronic-phase viremia <1000 viral RNA copies/ml. Interestingly, Mamu-B*08-restricted SIV-derived epitopes appeared to match the peptide binding profile for HLA-B*2705 in humans. We therefore defined a detailed peptide-binding motif for Mamu-B*08 and investigated binding similarities between the macaque and human MHC class I molecules. Analysis of a panel of approximately 900 peptides revealed that despite substantial sequence differences between Mamu-B*08 and HLA-B*2705, the peptide-binding repertoires of these two MHC class I molecules share a remarkable degree of overlap. Detailed knowledge of the Mamu-B*08 peptide-binding motif enabled us to identify six additional novel Mamu-B*08-restricted SIV-specific CD8(+) T cell immune responses directed against epitopes in Gag, Vpr, and Env. All 13 Mamu-B*08-restricted epitopes contain an R at the position 2 primary anchor and 10 also possess either R or K at the N terminus. Such dibasic peptides are less prone to cellular degradation. This work highlights the relevance of the Mamu-B*08-positive SIV-infected Indian rhesus macaque as a model to examine elite control of immunodeficiency virus replication. The remarkable similarity of the peptide-binding motifs and repertoires for Mamu-B*08 and HLA-B*2705 suggests that the nature of the peptide bound by the MHC class I molecule may play an important role in control of immunodeficiency virus replication.
- SourceAvailable from: Henrik N. Kløverpris[Show abstract] [Hide abstract]
ABSTRACT: Recent studies in the SIV-macaque model of HIV infection suggest that Nef-specific CD8+ T-cell responses may mediate highly effective immune control of viraemia. In HIV infection Nef recognition dominates in acute infection, but in large cohort studies of chronically infected subjects, breadth of T cell responses to Nef has not been correlated with significant viraemic control. Improved disease outcomes have instead been associated with targeting Gag and, in some cases, Pol. However analyses of the breadth of Nef-specific T cell responses have been confounded by the extreme immunogenicity and multiple epitope overlap within the central regions of Nef, making discrimination of distinct responses impossible via IFN-gamma ELISPOT assays. Thus an alternative approach to assess Nef as an immune target is needed. Here, we show in a cohort of >700 individuals with chronic C-clade infection that >50% of HLA-B-selected polymorphisms within Nef are associated with a predicted fitness cost to the virus, and that HLA-B alleles that successfully drive selection within Nef are those linked with lower viral loads. Furthermore, the specific CD8+ T cell epitopes that are restricted by protective HLA Class I alleles correspond substantially to effective SIV-specific epitopes in Nef. Distinguishing such individual HIV-specific responses within Nef requires specific peptide-MHC I tetramers. Overall, these data suggest that CD8+ T cell targeting of certain specific Nef epitopes contributes to HIV suppression. These data suggest that a re-evaluation of the potential use of Nef in HIV T-cell vaccine candidates would be justified.PLoS ONE 01/2013; 8(9):e73117. · 3.53 Impact Factor
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ABSTRACT: Major histocompatibility complex (MHC) gene products control the repertoire of T cell responses that an individual may create against pathogens and foreign tissues. This text will review the current understanding of MHC genetics in nonhuman primates, with a focus on Mauritian-origin cynomolgus macaques (Macaca fascicularis) and Indian-origin rhesus macaques (Macaca mulatta). These closely related macaque species provide important experimental models for studies of infectious disease pathogenesis, vaccine development, and transplantation research. Recent advances resulting from the application of several cost effective, high-throughput approaches, with deep sequencing technologies have revolutionized our ability to perform MHC genotyping of large macaque cohorts. Pyrosequencing of cDNA amplicons with a Roche/454 GS Junior instrument, provides excellent resolution of MHC class I allelic variants with semi-quantitative estimates of relative levels of transcript abundance. Introduction of the Illumina MiSeq platform significantly increased the sample throughput, since the sample loading workflow is considerably less labor intensive, and each instrument run yields approximately 100-fold more sequence data. Extension of these sequencing methods from cDNA to genomic DNA amplicons further streamlines the experimental workflow and opened opportunities for retrospective MHC genotyping of banked DNA samples. To facilitate the reporting of MHC genotypes, and comparisons between groups of macaques, this text also introduces an intuitive series of abbreviated rhesus MHC haplotype designations based on a major Mamu-A or Mamu-B transcript characteristic for ancestral allele combinations. The authors believe that the use of MHC-defined macaques promises to improve the reproducibility, and predictability of results from pre-clinical studies for translation to humans.ILAR journal / National Research Council, Institute of Laboratory Animal Resources 01/2013; 54(2):196-210. · 1.05 Impact Factor
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ABSTRACT: The development of HIV vaccines has been hampered by the lack of an animal model that can accurately predict vaccine efficacy. Chimpanzees can be infected with HIV-1 but are not practical for research. However, several species of macaques are susceptible to the simian immunodeficiency viruses (SIVs) that cause disease in macaques, which also closely mimic HIV in humans. Thus, macaque-SIV models of HIV infection have become a critical foundation for AIDS vaccine development. Here we examine the multiple variables and considerations that must be taken into account in order to use this nonhuman primate (NHP) model effectively. These include the species and subspecies of macaques, virus strain, dose and route of administration, and macaque genetics, including the major histocompatibility complex molecules that affect immune responses, and other virus restriction factors. We illustrate how these NHP models can be used to carry out studies of immune responses in mucosal and other tissues that could not easily be performed on human volunteers. Furthermore, macaques are an ideal model system to optimize adjuvants, test vaccine platforms, and identify correlates of protection that can advance the HIV vaccine field. We also illustrate techniques used to identify different macaque lymphocyte populations and review some poxvirus vaccine candidates that are in various stages of clinical trials. Understanding how to effectively use this valuable model will greatly increase the likelihood of finding a successful vaccine for HIV. Curr. Protoc. Immunol. 102:12.14.1-12.14.30. © 2013 by John Wiley & Sons, Inc.Current protocols in immunology / edited by John E. Coligan ... [et al.] 01/2013; 102:12.14.1-12.14.30.
Two MHC Class I Molecules Associated with Elite Control of
Immunodeficiency Virus Replication, Mamu-B*08 and
HLA-B*2705, Bind Peptides with Sequence Similarity1,2
John T. Loffredo,* John Sidney,†Alex T. Bean,* Dominic R. Beal,* Wilfried Bardet,‡
Angela Wahl,‡Oriana E. Hawkins,‡Shari Piaskowski,* Nancy A. Wilson,*
William H. Hildebrand,‡David I. Watkins,*§and Alessandro Sette3†
HLA-B27- and -B57-positive HIV-infected humans have long been associated with control of HIV replication, implying that CD8?T
cell responses contribute to control of viral replication. In a similar fashion, 50% of Mamu-B*08-positive Indian rhesus macaques
control SIVmac239 replication and become elite controllers with chronic-phase viremia <1000 viral RNA copies/ml. Interestingly,
Mamu-B*08-restricted SIV-derived epitopes appeared to match the peptide binding profile for HLA-B*2705 in humans. We therefore
defined a detailed peptide-binding motif for Mamu-B*08 and investigated binding similarities between the macaque and human MHC
class I molecules. Analysis of a panel of ?900 peptides revealed that despite substantial sequence differences between Mamu-B*08 and
HLA-B*2705, the peptide-binding repertoires of these two MHC class I molecules share a remarkable degree of overlap. Detailed
knowledge of the Mamu-B*08 peptide-binding motif enabled us to identify six additional novel Mamu-B*08-restricted SIV-specific
CD8?T cell immune responses directed against epitopes in Gag, Vpr, and Env. All 13 Mamu-B*08-restricted epitopes contain an R at
the position 2 primary anchor and 10 also possess either R or K at the N terminus. Such dibasic peptides are less prone to cellular
degradation. This work highlights the relevance of the Mamu-B*08-positive SIV-infected Indian rhesus macaque as a model to examine
elite control of immunodeficiency virus replication. The remarkable similarity of the peptide-binding motifs and repertoires for Mamu-
of immunodeficiency virus replication. The Journal of Immunology, 2009, 182: 7763–7775.
munodeficiency virus replication. Human elite controllers (EC)4
are a rare population of individuals that control HIV replication to
extremely low levels without medical intervention (4). Similarly, a
n light of recent phases II and III HIV vaccine trial failures
(1–3), it may be useful to place additional emphasis on un-
derstanding the immune correlates involved in control of im-
small number of macaques control pathogenic SIV replication and
become EC (5, 6). These unique animals are a valuable resource to
complement human studies in understanding immunodeficiency
virus pathogenesis and immunity.
Recently, we identified an MHC class I allele in Indian rhesus
macaques, Mamu-B*08, that is enriched in EC cohorts and asso-
ciated with reduced chronic phase plasma virus concentrations of
the pathogenic strain SIVmac239 (6). Over 50% of Mamu-B*08-
positive, SIV-infected macaques become EC. We then identified
seven CD8?T cell responses in SIV-infected EC macaques that
were restricted by Mamu-B*08 (7). All seven SIV epitopes con-
tained R at position 2 and L at the C terminus. Interestingly, these
epitopes appeared to match the peptide binding profile of HLA-
B27 (8–11), an allele associated with slow disease progression in
humans (12–17). Furthermore, in both HLA-B27-positive humans
and Mamu-B*08-positive macaques, virus-specific CD8?T cells
have been implicated in controlling immunodeficiency virus rep-
lication (7, 13, 15, 18–21).
The apparent similarities between Mamu-B*08 and HLA-B27
warranted a more thorough comparison of these two alleles. We
also investigated Mamu-B*03 due to its sequence similarity with
Mamu-B*08 (22) as well as its association with slow SIV disease
progression (23). Although Mamu-B*08 and Mamu-B*03 exhibit
sequence similarity in their ?1 and ?2 domains, the sequences of
these macaque alleles and HLA-B*2705 are considerably different
(Fig. 1). Despite this fact, a previous study demonstrated that
Mamu-B*03 could bind several endogenous HLA-B27 ligands
In this study, we examined binding similarities between Mamu-
B*08, Mamu-B*03, and HLA-B*2705. Using ligand elution and
positional scanning combinatorial library (PSCL) approaches, we
*Department of Pathology and Laboratory Medicine, University of Wisconsin, Mad-
ison, WI 53706;†Division of Vaccine Discovery, La Jolla Institute for Allergy and
Immunology, La Jolla, CA 92037;‡Department of Microbiology and Immunology,
University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; and
§Wisconsin National Primate Research Center, University of Wisconsin, Madison,
Received for publication January 13, 2009. Accepted for publication April 14, 2009.
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.
1This research was supported by National Institutes of Health (NIH)/National Insti-
tute of Allergy and Infectious Disease Grants N01-AI-40023, N01-AI-40024, and
HHSN266200400006C (to A.S.), and HHSN266200400088C (to D.I.W.), as well as
NIH Grants R01 AI049120, R01 AI052056, R24 RR015371, and R24 RR016038 (to
D.I.W.). Additionally, this publication was made possible in part by Grant P51
RR000167 from the National Center for Research Resources, a component of the
NIH, awarded to the Wisconsin National Primate Research Center, University of
Wisconsin Madison. This work was conducted in part at a facility constructed with
support from Research Facilities Improvement Program Grant nos. RR15459 and
RR020141 (Wisconsin National Primate Research Center).
2This publication’s contents are solely the responsibility of the authors and do not
necessarily represent the official views of NCRR or National Institutes of Health.
3Address correspondence and reprint requests to Dr. Alessandro Sette, Division of
Vaccine Discovery, La Jolla Institute for Allergy and Immunology, 9420 Athena
Circle, La Jolla, CA 92037. E-mail address: email@example.com
4Abbreviations used in this paper: EC, elite controller; PSCL, positional scanning
combinatorial library; SF, specificity factor; SFC, spot-forming cell.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
first defined a detailed peptide-binding motif for Mamu-B*08,
along with Mamu-B*03, and compared it to that of HLA-B*2705.
We then tested the capacity of a panel of ?900 peptides to bind
these MHC class I molecules. This analysis revealed that despite
minimal sequence similarity in the peptide-binding groove among
the human molecule, HLA-B*2705, and the Indian rhesus ma-
caque molecules, Mamu-B*08 and Mamu-B*03, these molecules
bound the same peptides.
We also examined the chronic-phase breadth and magnitude of
SIV-specific responses restricted by Mamu-B*08. Using the
Mamu-B*08 peptide-binding motif, we found ?200 peptides that
bound to Mamu-B*08 with IC50s ?500 nM. The peptides were
subsequently tested in IFN-? ELISPOT assays. In addition to the
seven previously described Mamu-B*08-restricted epitopes, we
identified six novel SIV-specific CD8?T cell responses restricted
by Mamu-B*08. Four epitopes were located in Env, while two
were located in Gag and Vpr. Interestingly, the region contain-
ing the SIV Gag epitope is conserved between SIVmac239, and
HIV-2 and also contains the immundominant HLA-B27-re-
stricted, HIV-1 epitope Gag263–272KK10. However, unlike the
situation in HLA-B27-positive HIV-infected humans, Gag263–271YL9
is subdominant and infrequently targeted in Mamu-B*08-positive
SIV-infected macaques. In addition, although viral escape is pre-
dominantly detected in the HLA-B27-restricted Gag263–272KK10
response in HIV-infected humans (13, 18, 19), we found that
viral variation in Mamu-B*08-positive macaques typically oc-
curs in a number of other CD8?T cell epitopes. Interestingly,
most of the SIV variants are located in Vif and Nef. Therefore,
although HLA-B*2705 and Mamu-B*08 are similar in function
and human and macaques expressing these two MHC class I
alleles can control immunodeficiency virus replication, individ-
uals expressing these alleles typically target different areas of
the virus. This suggests that control of immunodeficiency virus
replication is due to shared characteristics among viral epitopes.
Materials and Methods
MHC class I typing of macaques
Indian rhesus macaques (Macaca mulatta) were genotyped for Mamu-
B*08 (6) along with the following MHC class I alleles, Mamu-A*01,
-A*02, -A*08, -A*11, -B*01, -B*03, -B*04, -B*17, and -B*29 using PCR
amplification with sequence-specific primers (PCR-SSP) as described pre-
Animals and virus infections
Macaques were infected with the pathogenic molecular clone SIVmac239
(26) (www.ncbi.nlm.nih.gov/GenBank/index.html; GenBank accession no.
M33262) with the exception of macaque r99006, which was infected with
an SIVmac239 recombinant virus bearing escape mutations in three CD8?
T cell epitopes (27). SIV viremia was monitored in the infected macaques
by quantitative PCR as described previously (6, 28).
Seven macaques at varying disease courses in the chronic phase of SIV
infection (?35 wk post-SIV infection) were utilized in this study. Four of
the macaques were considered ECs with viral loads ?1,000 vRNA cop-
ies/ml during this study (r00032, r02019, r98016, and r99006). The con-
troller animal, r01027, maintained viral loads ?20,000 vRNA copies/ml or
a log less than the viral set point of typical progressor macaques (6), while
macaque r91003 displayed a typical progressor disease course with viral
loads ?0.5 ? 106vRNA copies/ml. Animal r00078 was previously an EC
that had steadily increasing viral loads since 1 year after depletion of its
CD8?cells (28). At the time of the initial ELISPOT assays, the plasma
virus concentrations in r00078 was ?30,000 vRNA copies/ml.
All SIV-infected animals were maintained at the National Primate Re-
search Center (University of Wisconsin Madison) and cared for according
to the regulations and guidelines of the University of Wisconsin Institu-
tional Animal Care and Use Committee.
Creation of stable MHC class I transfectants
A soluble Mamu-B*08 transfectant was produced in the MHC class I-de-
ficient EBV-transformed B lymphoblastoid cell line 721.221. Briefly, a
pcDNA 3.1?vector (Invitrogen) encoding soluble Mamu-B*08 was cre-
ated by removing the cytoplasmic and membrane domains with PCR mu-
tagenesis. This construct was then transfected into the 721.221 cell line
(29). A previously created membrane-bound Mamu-B*08 transfectant was
also used in this study (7).
Mamu-B*08 peptide-binding motif from endogenous ligands
Transfected 721.221 cells producing soluble Mamu-B*08 were cultured in
a hollow fiber bioreactor (Toray) in a CP2500 unit (Biovest International),
and supernatant containing soluble Mamu-B*08 was collected. Approxi-
mately 25 mg of soluble Mamu-B*08 was affinity purified from superna-
tant with W6/32 Ab (30) coupled to Sepharose 4B matrix (GE Healthcare
Systems). Bound peptide was eluted from Mamu-B*08 H and L chains
with an acid boil and passed through an ultrafiltration stirred cell with a 3
kDa membrane (Millipore). Ten percent of the eluted peptide pool under-
went 14 cycles of N-terminal Edman degradation sequencing on an ABI
Procise 492 protein sequencer (Applied Biosystems). The relative domi-
nance of amino acids at each position in the peptide pool was determined
by calculating the fold increase in picomoles of an amino acid over the
previous cycle. Amino acids exhibiting a fold increase of 2.0–2.49, 2.5–
3.49, and ?3.5, over the previous round are considered weak, strong, and
dominant amino acids, respectively, at a given position in the peptide pool.
The remaining peptide pool was fractionated by reversed-phase HPLC and
sprayed via nanospray on an ESI-quadrupole time-of-flight Q-Star Elite
mass spectrometer (Applied Biosystems). Approximately 40 peptide-con-
taining fractions were generated. Individual peptide sequences were deter-
mined by MS/MS analysis. A peptide motif was compiled using resultant
Edman sequencing data and individual peptide sequences (31).
Positional scanning combinatorial library and peptide synthesis
The PSCL was synthesized as described previously (32). Each pool in the
library contains 9-mer peptides with one fixed residue at a single position.
With each of the 20 naturally occurring residues represented at each po-
sition along the 9-mer backbone, the entire library consisted of 180 peptide
the sequence of Mamu-B*08 are indicated by dots (.). Residues predicted to form the B and F binding pockets are indicated based on previous studies
Comparison of the ?1 and ?2 domains of the MHC class I alleles, Mamu-B*08, Mamu-B*03, and HLA-B*2705. Residues matching
7764SIMILAR MHC I BINDING PROFILES BETWEEN EC
Peptides used in screening studies were purchased as crude or purified
material from Mimotopes, Pepscan Systems, A and A Labs, Genescript, or
the Biotechnology Center at the University of Wisconsin Madison. Pep-
tides synthesized for use as radiolabeled ligands were synthesized by A and
A Labs and purified to ?95% homogeneity by reversed-phase HPLC. Pu-
rity of these peptides was determined using analytical reversed-phase
HPLC and amino acid analysis, sequencing, and/or mass spectrometry.
Peptides were radiolabeled with the chloramine T method (33). Lyophi-
lized peptides were resuspended at 4–20 mg/ml in 100% DMSO, then
diluted to required concentrations in PBS ? 0.05% (v/v) Nonidet P-40
SIV peptide sequences were derived from the SIVmac239 sequence,
(www.ncbi.nlm.nih.gov/GenBank/index.html) GenBank accession no.
MHC purification and peptide binding assays
MHC class I purification was performed using affinity chromatography as
described previously (33, 34). The Mamu-B*08 and Mamu-B*03 mole-
cules were purified from cell lysates of stable membrane-bound MHC class
I transfectants using the anti-HLA class I (A, B, and C) Ab W6/32. HLA-
B*2705 molecules were purified from the EBV-transformed homozygous
cell line LG2 using the HLA-B and -C Ab B123.2 (35–37). Protein purity,
concentration, and depletion efficiency steps were monitored by
Quantitative assays for peptide binding to detergent-solubilized MHC
class I molecules were based on the competitive inhibition of binding of a
high-affinity radiolabeled standard probe peptide and performed as detailed
in prior studies (33, 34, 37, 38). Peptides were tested at six different con-
centrations covering a 100,000-fold dose range in three or more indepen-
dent assays. The radiolabeled peptides used for the Mamu-B*08, Mamu-
B*03, and HLA-B*2705 assays were, respectively, peptide 3130.0006
(sequence RRDYRRGL, an N175to Y analog of the SIV Vif172–179RL8
epitope), the artificial ligand 3130.0012 (sequence RRAARAEYL), and the
human 60s rL28 38–46 peptide (sequence FRYNGLIHR). For each pep-
tide, the concentration of peptide yielding 50% inhibition of the binding of
the radiolabeled probe peptide (IC50) was calculated. Under the conditions
used, where [radiolabeled probe] ? [MHC] and IC50? [MHC], the mea-
sured IC50s are reasonable approximations of the true Kdvalues (39).
Control for MHC specificity is implicit by the fact that in each binding
assay, only one species of purified MHC class I molecule is utilized. Con-
trol wells to measure nonspecific (background) binding were also included.
Specificity is further implied because not all peptides bound all three mol-
ecules, several peptides bound none of the three molecules, and each mol-
ecule tested bound a unique set of peptides. In each experiment, a titration
of the unlabeled version of the radiolabeled probe was also tested as a
positive control for inhibition.
Analysis of the PSCL data was performed as described previously (40).
Briefly, IC50nM values for each mixture were standardized as a ratio to the
geometric mean IC50nM value of the entire set of 180 mixtures and then
normalized at each position so that the value associated with optimal bind-
ing at each position corresponds to 1. For each position, an average (geo-
metric) relative binding affinity (ARB) was calculated, and then the ratio of
the ARB for the entire library to the ARB for each position was derived.
We have denominated this ratio, which describes the factor by which the
normalized geometric average binding affinity associated with all 20 resi-
dues at a specified position differs from that of the average affinity of the
entire library, as the specificity factor (SF). As calculated, positions with
the highest specificity will have the highest SF value. Primary anchor po-
sitions were then defined as those with an SF ? 2.4. This criterion iden-
tifies positions where the majority of residues are associated with signifi-
cant decreases in binding capacity.
Secondary anchor designations were based on the SD of residue specific
values at each position. Dominant secondary anchor positions were defined
as those where the SD was ?3 and the SF ? 2.4, as well as positions
associated with an SD ? 2 if the SF is between 1.5 and 2.4. Weak sec-
ondary anchors have been defined as positions associated with a SD in the
2.5–3 range with an SF ? 1.5, or an SF in the 1.5–2.4 range with an
SD ? 2.
To identify predicted binders, all possible 9-mer peptides in SIV se-
quences were scored using the matrix values derived from the PSCL anal-
ysis as described previously (40). The final score for each peptide repre-
sents the product of the corresponding matrix values for each peptide
residue-position pair. Peptides of 8, 10, or 11 residues in length were also
selected using the combinatorial library as described previously (41).
Briefly, 8-mers were selected by scoring residues 1–7 with the correspond-
ing residues in the 9-mer matrix, and the C terminus was scored using the
corresponding 9-mer C-terminal value. Similarly, for 10- and 11-mer pep-
tides, residues 1–8 were scored using the corresponding residues of the
9-mer matrix, and the C terminus with the 9-mer C-terminal residues. Pep-
tides scoring among the top 2.5% (n ? 82) for each size were selected. An
additional set of 50 peptides representative of scores in each 10% range of
the lower 97.5% was selected to evaluate the efficacy of the prediction
IFN-? ELISPOT assay
We performed ELISPOT assays as described previously (42). Briefly,
PBMC were isolated from EDTA-anticoagulated blood using Ficoll-Paque
PLUS (GE Healthcare Systems) and density centrifugation. 1 ? 105PBMC
were used per well in precoated ELISpotPLUSkits (Mabtech) according to
the manufacturer’s instructions for the detection of IFN-?-secreting cells.
All tests were performed in duplicate or triplicate using individual peptides
at 10 ?M. The positive control, Con A (Sigma-Aldrich), was used at a final
concentration of 5 ?g/ml. The negative control wells were devoid of any
stimulation. The 96-well plates were incubated for 12–18 h at 37°C in
Wells were imaged and counted with an AID EliSpot reader version 4.0
(AID) and analyzed as described previously (38, 42). A response was con-
sidered positive if the mean number of spot-forming cells (SFC) from the
duplicate (or triplicate) sample wells exceeded background (mean of wells
without peptide stimulation) plus two SD. Background levels were sub-
tracted from each well, and assay results are shown as SFC per 1 ? 106
PBMC. Responses of ?50 SFC per 1 ? 106PBMC were not considered
Two of the 210 peptides binding Mamu-B*08 ? 500 nM were unable
to be tested in ELISPOT assays (supplemental Table I).5However, neither
the Rev nor Nef peptide would be considered a novel response as both
overlapped previously described Mamu-B*08-restricted epitopes by eight
of nine amino acids.
Defining CD8?T cell epitopes using peptide-specific CD8?
T cell lines
Previous methods used to define Mamu-B*08-restricted SIV epitopes were
also utilized in this investigation (7). First, we generated peptide-specific
CD8?T cell lines. After several weeks of in vitro culture to increase
specificity, we examined these cell lines in IFN-?/TNF-? intracellular cy-
tokine staining (ICS) assays with Mamu-B*08 transfectants to verify that
immunogenic peptides were restricted by Mamu-B*08. These peptide-spe-
cific CD8?T cell lines were used in similar ICS assays to confirm the
minimal optimal epitope for each viral region. CD8?T cell lines were
cultured with media containing 100 U of IL-2/ml (National Institutes of
Health AIDS Research and Reference Reagent Program).
MHC class I tetramer and surface staining
MHC class I tetramers were constructed for the six novel SIV epitopes with
minor modifications as previously described (38, 43). We performed MHC
class I tetramer stains on PBMC and CD8?T cell lines to verify minimal
optimal epitopes as previously described (38, 42).
Sequencing of plasma viral RNA (vRNA)
SIVmac239 sequencing data was obtained from other studies involving
SIV-infected Mamu-B*08-positive macaques (7, 21). Viral sequencing was
performed based on methods described previously (7, 21, 28, 44).
Sequencing of endogenously bound Mamu-B*08 ligands
indicates peptide binding similarity to HLA-B27
With the emergence of SIV-infected Mamu-B*08-positive rhesus
macaques as a model to study elite control of HIV replication, we
decided to characterize a detailed peptide-binding motif of Mamu-
B*08. We first defined a preliminary motif for Mamu-B*08 using
an endogenous Mamu-B*08 peptide pool purified from Mamu-
B*08 transfectants. By subjecting ten percent of the eluted Mamu-
B*08 peptide pool to 14 cycles of N-terminal Edman degradation,
we determined the dominance of particular amino acids at each
5The online version of this article contains supplemental material.
7765The Journal of Immunology
position in the peptide pool. The pool sequencing analysis identi-
fied strong signals for R in position 2 and L at the C terminus (data
not shown). We then fractioned the remaining peptide pool by
reverse-phase HPLC into ?40 peptide-containing fractions. From
the various fractions spanning the HPLC chromatogram, 10
Mamu-B*08 ligands were selected and sequenced by MS/MS frag-
mentation (Table I). In agreement with the binding motif deter-
mined by Edman sequencing, all ten peptides contained an R at
position 2, and 8 of the 10 possessed L at the C terminus. The
majority (8 of 10) of the peptides sequenced were nonamers, while
the remaining two ligands were decamers.
We also tested the panel of sequenced ligands for their capacity
to bind to Mamu-B*08. The ten endogenous peptides bound
Mamu-B*08 with IC50values ?165 nM, including eight that
bound with much higher affinities of 17 nM or better (Table I). All
ten of these natural ligands also bound HLA-B*2705 with affinities
?500 nM, indicating that the binding similarities of Mamu-B*08
and HLA-B*2705 extend beyond epitope sequence similarity.
Determination of Mamu-B*08 peptide-binding motif using a
positional scanning combinatorial peptide library
To complement the pool sequencing analysis and derive a more
detailed quantitative motif for Mamu-B*08, we next tested the
capacity of a PSCL to bind purified Mamu-B*08 molecules. The
Mamu-B*08 binding capacity (IC50nM) for each mixture was
determined, and then normalized, as described in the Materials and
Methods, to generate the resulting Mamu-B*08 matrix shown in
Table II. Analysis of the matrix using previous established criteria
(40) confirmed position 2 and the C terminus as the main anchor
positions for Mamu-B*08 binding. The second position was espe-
cially critical for binding with a SF of 28.4.
In agreement with the pool sequencing derived motif, at position
2 only R was preferred (Table II). All other residues were associ-
ated with normalized average relative binding (ARB) values
?0.05. At the C terminus, a broader range of residues was per-
mitted. The PSCL analysis identified the aromatic residue F as the
most preferred followed by the hydrophobic aliphatic residues L
and I with ARB values of 0.673 and 0.375, respectively. The basic
residue K and the hydrophobic residue M were tolerated with ARB
values in the 0.1–0.12 range. All other residues were associated
with affinities ?10-fold lower than the optimal residue, F.
Influences on binding at other positions tended to be minor,
reflected by relatively low SF and SD. In fact, none of the positions
met the criterion we have utilized to define dominant secondary
anchors (see Ref. 40 and Materials and Methods), and only posi-
tion 1, associated with a SD of 2.7, appears to be a weak secondary
anchor. In position 1, the basic residues R, H, and K, as well as M
and S, were the most preferred, while the acidic residues D and E
were two of the most deleterious.
In summary, we used the PSCL to define a detailed motif for
Mamu-B*08. This motif was characterized by primary anchor
specificity for the basic residue R in position 2 and a preference for
hydrophobic (F, L, I, and M) and basic (K) residues at the C ter-
minus (Fig. 2).
Identification of SIV-derived Mamu-B*08 binding peptides
PSCL-based matrices offer an efficient means to identify MHC
class I binding peptides and the ability to identify nonmotif binders
(40, 41, 45–49). To identify SIV-derived Mamu-B*08 binders, we
used the Mamu-B*08 PSCL matrix to score all 9-mer peptides in
the SIVmac239 proteome. After synthesizing the 9-mer peptides
scoring in the upper 2.5% range (n ? 82), we tested them for
binding. An additional set of 50 lower scoring peptides, randomly
selected to provide five peptides to represent each successive 10%
scoring range, were also synthesized and tested. All of the SIV
peptides and their accompanying binding data can be found in
supplemental Table I.5We discovered that 49 (60%) of the 82
peptides selected bound Mamu-B*08 with an affinity of ?500 nM,
including 16 peptides that bound with affinities of 10 nM or better.
By contrast, none of the 50 peptides with scores beyond the upper
2.5% bound Mamu-B*08 with affinities ? 500 nM.
Both the endogenous ligands (Table I) as well as the previously
described Mamu-B*08-restricted SIV epitopes (7) demonstrated
that Mamu-B*08 is not restricted to binding only nonamers. Thus,
we applied the PSCL binding matrix, as described in Materials and
Methods, to predict SIV-derived 8-, 10-, and 11-mer binders. For
each size (8-, 10-, and 11-mer), the upper 2.5% scoring peptides
(n ? 82 for each size), plus an additional set of 50 peptides from
poorer scoring ranges, were synthesized and tested for binding. In
total 35 (42.7%), 58 (70.7%), and 37 (45.1%) of the 8-, 10-, and
11-mer peptide sets, respectively, bound with affinities of ?500
nM. As with the 9-mer peptide set, none of the peptides from the
lower scoring ranges bound with affinities ?500 nM. Overall, con-
sidering all four size sets together, 179 of the 328 (54.6%) peptides
scoring in the top 2.5% range were Mamu-B*08 binders. In the
course of subsequent analyses probing lower scoring ranges, we
found several additional Mamu-B*08 binders. In total, we identi-
fied 210 SIV-derived Mamu-B*08 binders (IC50? 500 nM) from
the 862 SIV-derived peptide candidates (supplemental Table I).5
Remarkable overlap in the peptide binding repertoires of
Mamu-B*08 in comparison with HLA-B*2705
Given the similarities between epitopes restricted by Mamu-B*08,
Mamu-B*03, and HLA-B*2705, we next sought to compare their
Table I. Representative Mamu-B*08 endogenous ligand sequences also bind Mamu-B*03 and HLA-B*2705
Source ProteinLength Sequencea
MHC Class I Binding Affinity (IC50nM)b
Vacuolar protein pump subunit SFD ? domain
Fibronectin receptor ? subunit
Proteosome subunit B2
Peroxisome biogenesis factor 1
Ribosomal protein S15a
Ribosomal protein S25
Non-lens ? ? crystallin-like protein
aDominant residues from pool sequencing are indicated in bold.
bIC50s ? 500 nM are not considered binders and are indicated in italics.
7766SIMILAR MHC I BINDING PROFILES BETWEEN EC
respective PSCL-derived motifs, as well as investigate the ex-
tent, if any, that their repertoires overlapped. For this compar-
ison, we created a PSCL matrix describing Mamu-B*03 binding
(supplemental Table II-A).5We also used the previously re-
ported PSCL matrix-based motif for HLA-B*2705 (40) (sup-
plemental Table II-B).5
The PSCL binding matrices indicated that Mamu-B*08, Mamu-
B*03, and HLA-B*2705 share very similar primary anchor spec-
ificities. Position 2 was identified as the dominant anchor position
for all three MHC class I molecules with an overwhelming pref-
erence for the positive residue R (Fig. 2). Unlike Mamu-B*08, a
second main anchor position was not identified for either Mamu-
B*03 or HLA-B*2705. Instead, the C terminus of Mamu-B*03
was defined as a dominant secondary anchor. In this position, the
most preferred residues were L, F, and I, representing a specificity
pattern very similar to that of Mamu-B*08. Position 1 was also
identified as a dominant secondary anchor and comparable to the
Mamu-B*08 specificity, a preference for basic residues was noted.
For HLA-B*2705, positions 1 and 3 as well as the C terminus were
identified as weak secondary anchor positions. At the C terminus,
the most preferred residues for HLA-B*2705 were F, R, K, L, I, V,
and M, representing an overall pattern that resembles Mamu-B*08
(and Mamu-B*03) specificity, as well as previously published mo-
tifs for HLA-B*2705 (8–11). Comparison of the specificity at the
N terminus (P1) also revealed similarity to the preferences ob-
served for Mamu-B*08 and -B*03 (Fig. 2).
To determine whether these similar motifs would translate into
overlapping binding repertoires, we assessed the binding patterns
using 899 peptides. The 899 peptides tested included the SIV-
derived peptides (supplemental Table I),5the Mamu-B*08 endog-
enous ligands (Table I), and a number of known HLA-B27-re-
stricted T cell epitopes and endogenous ligands described in the
literature (15, 50–54) (Table III). We evaluated each peptide for its
capacity to bind Mamu-B*08, Mamu-B*03, and HLA-B*2705
(Table IV). Of the 899 peptides, 363 (40.4%) bound at least one of
the three MHC class I molecules with an IC50? 500 nM. The
positions were defined using the PSCL matrix data (Table II and supplemental Table II),5as described in Materials and Methods. Primary anchor positions
are identified by bold font and blue shading. Dominant secondary positions are indicated by bold font and green shading, whereas weak secondary positions
are highlighted by orange shading. Also shown are the most preferred residues at the corresponding position. In the case of the primary anchors, residues
associated with an ARB ? 0.1 are shown. At secondary positions, residues with an ARB ? 0.3 are shown.
Similarity of primary and secondary anchor positions among Mamu-B*08, Mamu-B*03, and HLA-B*2705. Primary and secondary anchor
Table II. PSCL-derived matrix describing 9-mer binding to Mamu-B*08a
aThe PSCL was tested for binding to Mamu-B*08, the data analyzed, and primary and secondary anchor positions defined, as described in Materials
and Methods. Values shown represent the average relative binding (ARB) of the corresponding library relative to other pools with the same fixed position.
SD indicates the SD between the ARB of pools at the same position. SF is the specificity factor, calculated as described in Materials and Methods,
representing the ratio of the average binding of the entire library to the average of pools at the indicated position. At the primary anchor positions (SF ?
2.4), the most preferred residues, associated with an ARB ? 0.1 are highlighted by bold font. The library average binding for Mamu-B*08 was 0.150.
7767The Journal of Immunology
degree of cross-reactivity between binders (IC50? 500 nM) was
exceptionally high, as 241 (66.4%) bound more than one of the
MHC class I molecules tested, including 167 (46%) that bound all
three molecules. We also discovered the cross-reactivity pattern to
be fairly uniform across the three MHC class I molecules. Specif-
ically, between 63 and 85% of the repertoire of any allele over-
lapped with the repertoire of another allele. These levels of overlap
are remarkable, and even higher than those typically seen among
alleles within the same HLA supertypes (34, 55–58). Correspond-
ingly, monospecificity was low with only 7% of the Mamu-B*08
binders, 14% of the Mamu-B*03 binders, and 25% of the HLA-
B*2705 binders failing to bind to another of the three MHC class
Of the previously reported HLA-B27 epitopes, we found that 19
of the 24 peptides that bound HLA-B*2705 with an affinity ? 500
nM also bound Mamu-B*08 (Table III). Fourteen of these epitopes
bound both Mamu-B*03 and -B*08, again demonstrating the sim-
ilar peptide binding specificity between Mamu-B*08, Mamu-
B*03, and HLA-B*2705. Overall, this shared specificity is re-
flected in an extraordinary degree of repertoire overlap between
the three MHC class I molecules.
Defining six novel SIV epitopes restricted by Mamu-B*08
The 500 nM threshold has been used to define candidate epitopes
in previous SIV epitope screens (38, 59–62) and has also been
shown to be associated with in vivo T cell recognition in human,
macaque, and murine systems (39, 63–65). On this basis, we ex-
amined whether the SIV-derived Mamu-B*08-binding peptides
identified in the binding studies above were recognized in vivo
using fresh PBMC from SIV-infected Mamu-B*08-positive ma-
caques. Using IFN-? ELISPOT assays, we tested seven macaques
at varying disease courses in the chronic phase of SIV infection
(?35 wk post-SIV infection). No Mamu-B*03-positive macaques
were available for SIV immunogenicity testing because of the low
Table IV. Mamu-B*08, Mamu-B*03, and HLA-B*2705 exhibit remarkable overlap in their peptide-binding repertoiresa
MHC Class I MoleculesBindersb
Percentage of Binders That Are Cross-Reactive with an IC50? 500 nM
Mamu-B*08 Mamu-B*03HLA-B*2705Both Neither
aBinding capacity to Mamu-B*08, Mamu-B*03, and HLA-B*2705 was tested for 899 peptides (10 Mamu-B*08 endogenous ligands from Table I,
27 HLA-B27-restricted T cell epitopes and endogenous ligands from Table III, and 862 SIV-derived peptides from supplemental Table I).
bBinders were defined as peptides with IC50values ?500 nM.
Table III. Known HLA-B27 T cell epitopes and endogenous ligands also bind to Mamu-B*08 and Mamu-B*03
Organism (source protein)Length Sequence
MHC Class I Binding Affinity (IC50nM)a
Bound Reference HLA-B*2705 Mamu-B*08Mamu-B*03
HIV (Gag p24)
HIV (Gag p24)
Endogenous HLA-B*2705 ligand (ATP-dependent RNA
Chlamydia trachomatis (efflux protein)
Endogenous HLA-B*2705 ligand (Histon H3.3)
EBV (EBNA 3C)
C. trachomatis (putative outer membrane protein)
Endogenous HLA-B*2705 ligand (26S proteasome
regulatory subunit S2)
C. trachomatis (hypothetical protein)
Endogenous HLA-B*2705 ligand (60S ribosomal protein
Endogenous HLA-B*2705 ligand (NADH-ubiquinone
C. trachomatis (invasin repeat-family-phosphatase)
Endogenous HLA-B*2705 ligand (Aggrecan)
HIV (Gag p17)
C. trachomatis (NADH-ubiquinone-oxidoreductase-?-chain)
C. trachomatis (hypothetical protein)
HIV (Env gp41)
Endogenous HLA-B*2705 ligand (60S ribosomal protein
C. trachomatis (hypothetical protein)
C. trachomatis (exodeoxyribonuclease-V ?-chain)
C. trachomatis (protease-ATPase)
Yersinia enterocolitica (Hsp 60-kDa)
C. trachomatis (hypothetical protein)
C. trachomatis (ATP-dependent zinc protein)
C. trachomatis (hypothetical protein)
21 403 1483 50
aIC50s ? 500 nM are not considered binders and are indicated in italics.
7768SIMILAR MHC I BINDING PROFILES BETWEEN EC
from seven Mamu-B*08-positive SIV-infected macaques were tested with Mamu-B*08 binding peptides in IFN-? ELISPOT assays. Animals were tested
during the chronic phase of SIV infection (?35 wk post-SIV infection). Results shown represent the 13 SIVmac239 epitopes known to be restricted by
Mamu-B*08. Mean values and SDs from triplicate wells were calculated for each assay. Background (the mean of wells without peptide) levels were
subtracted from each well. Mean responses ? 50 SFC per 1 ? 106PBMC were not considered positive. Assay results are shown as SFC per 1 ? 106PBMC.
Nef RL9* refers to the epitope at positions 8–16 in Nef, whereas Nef RL9 refers to the Nef epitope at positions 246–254. The SDs of Nef RL10 in r02019
(1522 SFC/106PBMC) and Nef RL10 in r99006 (1753 SFC/106PBMC) were 197 and 140, respectively.
Ex vivo whole PBMC IFN-? ELISPOT results of Mamu-B*08-restricted minimal optimal CD8?T cell epitopes. Freshly isolated PBMC
7769 The Journal of Immunology
frequency (?1%) of this allele in Indian rhesus macaque colonies
We evaluated the antigenicity of 208 SIV-derived peptides in
duplicate wells of two independent experiments. Seventy-five of
these peptides (36%) demonstrated functional reactivity in at least
one of the seven SIV-infected macaques (supplemental Table I).5
We also assessed two uninfected Mamu-B*08-positive macaques
in IFN-? ELISPOT assays. None of the peptides demonstrated
reactivity in either of these control animals (data not shown).
We found a number of responses against previously character-
ized Mamu-B*08 epitopes in several macaques. Responses against
Vif123–131RL9, Vif172–179RL8, Rev12–20KL9, and Nef137–146RL10
were the most prevalent and detected in at least five of the seven
SIV-infected macaques (Fig. 3). The Nef137–146RL10-specific
CD8?T cell response was typically one of the strongest responses
and exceeded 800 SFC/106PBMC in three macaques in agreement
with previous data (7). In comparison, Rev44–51RL8, Nef8–16RL9,
and Nef246–254RL9 appeared subdominant during the chronic
phase of SIV infection. We detected these responses in less than
half of the macaques and at much lower levels, typically ?200
Several responses were also directed against viral regions that
did not contain epitopes known to be restricted by Mamu-B*08,
warranting further investigation. After excluding the peptides that
had considerable overlap (at least 6 aa) with the seven previously
defined Mamu-B*08-restricted epitopes, 14 immunogenic viral re-
gions, located in Gag, Pol, Vpx, Vpr, Env, and Nef, remained. We
generated CD8?T cell lines against 13 of the 14 novel regions. A
peptide-specific CD8?T cell line could not be generated against
Nef16–24LR9, likely because of its low frequency in a small num-
ber of macaques (data not shown).
Using the peptide-specific CD8?T cell lines in conjunction
with Mamu-B*08 transfectants in IFN-?/TNF-? ICS assays, we
established that 6 of the 14 regions with reactivity in ELISPOT
assays were CD8?T cell responses restricted by Mamu-B*08.
Given that Indian rhesus macaques express upward of 12 or more
MHC class I alleles (22, 66), it was not surprising that several of
the novel responses detected in ELISPOT assays were restricted by
other MHC class I molecules. We then used the Mamu-B*08-
restricted, peptide-specific CD8?T cell lines to identify the min-
imal optimal epitopes for cases where more than one peptide was
reactive for that region. Minimal confirmation occurred by test-
ing the functional avidity on peptide-specific CD8?T cell lines
and constructing MHC class I tetramers (data not shown). As
with the previous seven epitopes, all six novel responses con-
tained an R at position 2. Of the six novel epitopes, one was
located in Gag (Gag263–271YL9), one in Vpr (Vpr62–70IF9), and
four in Env (Env524–532KF9, Env573–581KL9, Env717–725LF9,
and Env868–876RL9). Like the Nef137–146RL10 epitope, the
Env573–581KL9 epitope was previously identified in Mamu-
B*03-positive SIV-infected macaques (23, 67).
Similar to the previously identified SIV epitopes, the newly
identified epitopes bound with extremely high affinity to Mamu-
B*08. Overall, 10 of the 13 Mamu-B*08-restricted epitopes bound
Mamu-B*08 with affinities ? 50 nM (Table V). Interestingly, all
13 Mamu B*08 epitopes identified to date were among the top
0.58% scoring peptides corresponding to their size in the PSCL
matrix-based predictions described above. For instance, all 10 of
the SIV 9-mer peptides restricted by Mamu-B*08 would have been
found by generating just 20 of the ?3330 possible 9-mers. Of the
13 Mamu-B*08-restricted epitopes, 9 and 10 peptides, respec-
tively, were found to bind HLA-B*2705 and Mamu-B*03 with
affinities ? 50 nM. Overall, only one peptide failed to bind all
three MHC class I molecules with an IC50? 500 nM.
In comparison to responses directed against Vif123–131RL9,
Vif172–179RL8, Rev12–20KL9, and Nef137–146RL10, the six novel
responses appeared to be subdominant during the chronic phase of
SIV infection (Fig. 3). We detected these responses in up to three
animals and typically at lower magnitudes. Interestingly, the SIV
epitope in Gag p27 (Gag263–271YL9) overlaps with an immuno-
dominant CD8?T cell epitope found in HLA-B*27-positive
humans infected with HIV-1 (Gag263–272KK10) and HIV-2
(Gag263–272RK10) (68–70). However, unlike the immunodomi-
nance of this Gag epitope observed in HIV-infected individuals
(13, 15, 18–20), we found Gag263–271YL9-specific CD8?T
cells in only two of the seven SIV-infected animals (r99006 and
r00078) at frequencies of 80 and 305 SFC/106PBMC, respec-
tively (Fig. 3).
Impact of viral variation in Mamu-B*08-restricted CD8?T cell
epitopes on Mamu-B*08 binding capacity
Previously, it has been shown that Mamu-B*08-specific CD8?T
cells selected for viral variation in several SIV epitopes (7, 21). We
therefore tested the Mamu-B*08 binding capacity of a number of
in vivo viral variants to determine whether they reduced the MHC
class I peptide binding to Mamu-B*08 (Table VI).
In general, the variant peptides studied were associated with
substantial decreases in Mamu-B*08 binding capacity, with only a
Table V. SIV epitopes restricted by Mamu-B*08 bind both macaque and human MHC class I molecules
Positions LengthSequence Short Namea
MHC Class I Binding Affinity (IC50nM)b
aNovel epitopes identified in this study are indicated in bold.
bIC50s ? 500 nM are not considered binders and are indicated in italics.
7770SIMILAR MHC I BINDING PROFILES BETWEEN EC
few having affinities within 3-fold of the WT epitope. Many of
these peptides did not exhibit variation at position 2 and the C
terminus, the primary anchor positions for Mamu-B*08 binding,
and bound with a biologically relevant affinity of ?500 nM (Table
VI). When variation was identified at the primary anchor positions,
these peptides typically bound weakly, if at all, with the majority
having affinities ? 500 nM. We tested a number of peptides with
variation in the primary anchor positions in IFN-? ELISPOT assays.
In comparison to the WT peptides, we found the variants to be greatly
reduced in magnitude, and in some instances, no reactivity was ob-
served (data not shown). However, ELISPOT is limited in its ability
more extensive testing will be necessary to thoroughly examine the
impact of these SIV variants on cellular recognition.
Although variation was found in the Nef137–146RL10 epitope,
these mutations did not greatly affect the binding capacity. In this
case, as previously reported (7), an alanine to proline substitution
at the residue immediately preceding the N terminus of the
Nef137–146RL10 epitope appears to affect peptide processing. A
similar alanine to proline substitution immediately preceding the
Table VI. Binding capacity of SIV variants within Mamu-B*08-restricted epitopes
Relative to WT
aPeptides with IC50s ? 500 nM are not considered binders and are indicated in italics. WT binding values are indicated in
bVariant not found in vivo at time of this submission.
cVariant found in vivo during the chronic phase of SIV infection (7).
dVariant found in vivo ?18 wk post-SIV infection (21).
ePreviously unpublished viral variant.
fPredominant viral variation is one amino acid upstream of this epitope, position 136: A to P (7, 21).
gPreviously published variant in Mamu-B*03-positive macaques escape (23).
7771The Journal of Immunology
N-terminal residue of the epitope has also been shown to inhibit
processing of an HLA-B57-restricted peptide in Gag of HIV (73).
The majority of the previously defined epitopes exhibited a wide
range of viral variation. However, at the time of this study, we
detected viral variation in only one of the newly discovered
epitopes, Env524–532KF9 (Table VI). In this case, an F to Y sub-
stitution at the C terminus of Env524–532KF9 reduced binding ?
40-fold from 7.2 to 321 nM. The lack of viral variation was par-
ticularly surprising for the Env573–581KL9 epitope. Viral escape
from Env573–581KL9-specific CD8?T lymphocytes was previ-
ously found in Mamu-B*03-positive SIV-infected macaques dur-
ing the chronic phase of SIV infection (23). However, we observed
no such variation in our SIV-infected Mamu-B*08-positive in-
fected macaques, despite detecting Env573–581KL9-specific CD8?
T cells in three of the seven macaques tested during the chronic
phase of infection. Two of these responses were ?600 SFC/106
PBMC (Fig. 3).
Characteristics of a successful immune response against HIV re-
main unknown. Investigating MHC class I alleles that are impli-
cated in slow disease progression may provide some insight. HLA-
B27 is one such allele that has been associated with elite control of
HIV infection (12–17). Recently, it has been established that
Mamu-B*08-positive macaques control SIVmac239 replication in
?50% of infected animals (6). Furthermore, Mamu-B*08-re-
stricted SIV epitopes fit the HLA-B27 peptide binding motif (7).
We therefore sought to investigate the binding similarities between
Mamu-B*08 and HLA-B27.
In this study, we found a remarkably high degree of overlap in
the peptide-binding repertoires of Mamu-B*08 and HLA-B*2705,
as well as Mamu-B*03. The considerable overlap between Mamu-
B*08 and Mamu-B*03 is not surprising given their high level of
sequence similarity (22). These two alleles differ by only two res-
idues in the ?1 and ?2 domains (Fig. 1). However, the extent to
which the binding specificity of the Mamu alleles cross-react with
HLA-B*2705 is very surprising even in light of previous obser-
vations (7, 24). HLA-B*2705 differs from Mamu-B*08 and Mamu-
B*03 at four of the seven key residues predicted to form the B
pocket, thought to determine the residue specificity at position 2 of
peptide ligands (74–76). In addition, differences are apparent in
key residues predicted to form the F pocket, believed to dictate the
C-terminal peptide specificity. HLA-B*2705 differs from Mamu-
B*08 at three of the four key F pocket residues. Overall, HLA-
B*2705 differs from Mamu-B*08 and Mamu-B*03 at 28 and 29
residues, respectively, throughout the ?1 and ?2 domains (Fig. 1).
Despite these sequence differences, the overlapping binding mo-
tifs and peptide repertoires of these alleles are astounding. The
summary motifs exhibit the same narrow specificity at position 2,
and partially overlapping, although very similar, preferences at the
C terminus (Fig. 2). The fact that the motifs of each molecule are
not identical is reflected in the observation that while the overlaps
in the peptide-binding repertoires observed are unusually high,
they are not absolute. Each molecule is associated with its own
unique repertoire and specificity. However, when considered in the
context of other HLA- and Mamu-MHC class I alleles whose bind-
ing specificities have been characterized, the parallels between the
molecules studied herein remain remarkable. Mamu-B*08, Mamu-
B*03, and HLA-B*2705 share a preference for hydrophobic res-
idues at the C terminus. In addition, Mamu-B*08 and HLA-
B*2705 share a strong propensity for basic residues, which are also
well tolerated by Mamu-B*03. This pattern of specificity is rela-
tively rare among HLA class I alleles, being largely limited to
those in the HLA-B27-supertype. At position 2, all three MHC
class I molecules have an identical preference for R, a specificity
that has not been widely observed among alleles whose motifs
have been described in detail (8, 77). The rates of overlap in the
peptide-binding repertoires, ranging between 63 and 85% depend-
ing on pairing (Table IV), are higher than those typically observed
among alleles within an HLA supertype or even among very
closely related alleles of the same serological family (34, 55–58).
In a large fraction of cases, these molecules also bind the same
peptides with very similar IC50values (nM).
Binding cross-reactivity was evident not only in the case of
SIV-derived peptides, but also when comparing the binding of
known epitopes and ligands previously associated with HLA-B27
(15, 50–54) (Table III). Of the 24 selected sequences that bound
HLA-B*2705 with an IC50? 500 nM, 19 of them bound Mamu-
B*08. In addition, when searching the immune epitope database
(78) and SYFPEITHI databases (8), we found that two of the
Mamu-B*08 endogenous ligands, RRYNIIPVL and SRTPYH
VNL (Table I), were previously identified as HLA-B27 ligands.
Such functional overlap between Mamu-B*08 and HLA-B27, de-
spite substantial sequence diversity, may be best explained by con-
vergent evolution (79).
Using a systematic epitope screening process similar to that
used to investigate SIV epitopes restricted by Mamu-A*01, -A*02,
-A*11, -B*01, and -B*17 (36, 38, 59–62), we identified 210 SIV-
derived peptides that bound to Mamu-B*08 with an IC50of ?500
nM (supplemental Table I).5From these 210 peptides, 75 peptides,
spanning 21 viral regions, elicited responses in IFN-? ELISPOT
assays. Seven of the 21 regions contained Mamu-B*08-restricted
epitopes previously identified in SIV-infected EC macaques that
were depleted of their CD8?cells (28). An additional six regions
contained novel epitopes restricted by Mamu-B*08. Interestingly,
a majority of the 13 Mamu-B*08-restricted epitopes had a number
of overlapping peptides that bound Mamu-B*08 and were immu-
nogenic in IFN-? ELISPOT assays (supplemental Table I).5With
the strong preference for R at position 2 and a number of consec-
utive arginines within these viral regions, it is possible that over-
lapping minimal optimal epitopes may be present in these areas,
too. It should also be noted that our approach might fail to detect
CD8?T lymphocyte responses with low-binding affinities, as
those with an IC50? 500 nM were not tested in IFN-? ELISPOT
In each of the seven SIV-infected macaques, we identified be-
tween 4 and 12 Mamu-B*08-restricted responses in the chronic
phase of infection (Fig. 3). CD8?T cells directed against
Vif123–131RL9, Vif172–179RL8, Rev12–20KL9, and Nef137–146RL10
typically represented the strongest and most frequent immune re-
sponses. The six novel SIV-specific epitopes, located in Gag, Vpr,
and Env, appeared subdominant during the chronic phase of in-
fection (Fig. 3).
Gag263–271YL9 was the one of two regions containing an SIV
epitope in which an HLA-B27-restricted HIV epitope has also
been identified. The previously defined Mamu-B*08-restricted
epitope Nef137–146RL10 also appears to share homology with the
HIV epitope Nef105–114RI10 that is restricted by HLA-B*2705.
Interestingly, the region containing the Gag epitope is completely
conserved between SIVmac239 and HIV-2 and contains the im-
munodominant HLA-B*2705-restricted epitope Gag263–272KK10
in HIV-1 (13, 15, 18–20, 68–70). However, unlike in HIV-in-
fected HLA-B27-positive humans, the seven Mamu-B*08-positive
SIV-infected macaques rarely targeted this Gag epitope (Fig. 3).
Tetramer stains of both acute and chronic phase PBMC samples
confirmed this finding (data not shown). Therefore, although
Mamu-B*08 and HLA-B*2705 are functionally similar, the two
7772 SIMILAR MHC I BINDING PROFILES BETWEEN EC
MHC class I molecules largely target different epitopes in SIV and
This observation suggests that it may not be the epitopes them-
selves that contribute to the viral control associated with these
alleles, but rather other as yet unidentified characteristics that they
share. For instance, Mamu-B*08-restricted SIV epitopes, like
HLA-B27-restricted epitopes, commonly display a characteristic
dibasic peptide motif at the N terminus. It has previously been
shown that these peptides are relatively resistant to peptidase ac-
tivity, are more stable in the cytosol, and hence are more efficiently
presented by HLA-B27 (80). In the case of the 13 Mamu-B*08-
restricted SIV epitopes, 10 displayed this dibasic motif with either
R or K at the N terminus of the peptide. This suggests Mamu-
B*08, like HLA-B27, may be able to present peptides from Ags
when far fewer copies of the epitope are present and therefore may
eliminate virally infected cells more efficiently.
We also investigated the impact of viral escape variants on
Mamu-B*08 peptide binding. Overall, less than one-third of viral
variants identified in Mamu-B*08-restricted epitopes changed pri-
mary anchor residues, resulting in a drastic reduction in binding
(Table VI). Variation at other positions typically did not abrogate
binding to Mamu-B*08, suggesting that the observed viral varia-
tions more likely alters TCR engagement to disrupt recognition by
CD8?T cells. Interestingly, viral variation was evident at position
1 in five of the Mamu-B*08-restricted epitopes. Position 1 appears
to play some role in MHC class I binding (Table II and Fig. 2).
However, the effect of these mutations may also be in the disrup-
tion of the dibasic peptide motifs discussed above, resulting in the
alteration of peptide processing and/or stability.
The SIV-infected Indian rhesus macaque is the best animal
model available for HIV/AIDS research. Mamu-B*08-positive
macaques are especially important because of high frequency of
animals that become ECs (?50%) and the striking functional sim-
ilarity of this MHC class I molecule to the human EC allele, HLA-
B27. This study provided a detailed comparison of three alleles
associated with slow disease progression. In total, 13 Mamu-B*08-
restricted responses, located in Gag, Vpr, Vif, Rev, Env, and Nef
have now been identified, enabling more comprehensive patho-
genesis studies. Dissecting the mechanisms of elite control in
Mamu-B*08-positive macaques will be vitally important to guide
HIV vaccine design in the future.
We thank the MHC Genotyping Core at the Wisconsin National Primate
Research Center (WNPRC) (William Rehrauer, Chrystal Glidden, Gretta
Borchardt, and Debi Fisk) for genotyping our Indian rhesus macaques. We
also appreciate Jason Reed, Emma Gostick, and David A. Price for assis-
tance with the construction of MHC class I tetramers, Jessica Furlott for
immunological assay assistance, as well as Carrie Moore, Sandy Ngo, and
Amiyah Steen for performing MHC-peptide binding assays. We are grate-
ful to Clemencia Pinilla for providing us with the combinatorial peptide
library. Laura Valentine provided helpful discussions. We also thank the
Virology, Genetics, Immunology, and Animal Core Laboratories as well as
Research Support Services at the National Primate Research Center, Uni-
versity of Wisconsin Madison (WNPRC) for technical assistance. The fol-
lowing reagent was obtained through the National Institutes of Health
AIDS Research and Reference Reagent Program, Division of AIDS, Na-
tional Institute of Allergy and Infectious Diseases, National Institutes of
Health: IL-2, human (item no. 136) from Hoffman-La Roche.
The authors have no financial conflict of interest.
F. van Griensven, D. Hu, J. W. Tappero, and K. Choopanya. 2006. Randomized,
P.,P. Gilbert,M. Gurwith,W. Heyward,M. Martin,
double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycop-
rotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand.
J. Infect. Dis. 194: 1661–1671.
2. Buchbinder, S. P., D. V. Mehrotra, A. Duerr, D. W. Fitzgerald, R. Mogg, D. Li,
P. B. Gilbert, J. R. Lama, M. Marmor, C. Del Rio, et al. 2008. Efficacy assessment
of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind,
randomised, placebo-controlled, test-of-concept trial. Lancet 372: 1881–1893.
3. McElrath, M. J., S. C. De Rosa, Z. Moodie, S. Dubey, L. Kierstead, H. Janes,
O. D. Defawe, D. K. Carter, J. Hural, R. Akondy, et al. 2008. HIV-1 vaccine-
induced immunity in the test-of-concept Step Study: a case-cohort analysis. Lan-
cet 372: 1894–1905.
4. Deeks, S. G., and B. D. Walker. 2007. Human immunodeficiency virus control-
lers: mechanisms of durable virus control in the absence of antiretroviral therapy.
Immunity 27: 406–416.
5. Yant, L. J., T. C. Friedrich, R. C. Johnson, G. E. May, N. J. Maness, A. M. Enz,
J. D. Lifson, D. H. O’Connor, M. Carrington, and D. I. Watkins. 2006. The
high-frequency major histocompatibility complex class I allele Mamu-B*17 is
associated with control of simian immunodeficiency virus SIVmac239 replica-
tion. J. Virol. 80: 5074–5077.
6. Loffredo, J. T., J. Maxwell, Y. Qi, C. E. Glidden, G. J. Borchardt, T. Soma,
A. T. Bean, D. R. Beal, N. A. Wilson, W. M. Rehrauer, et al. 2007. Mamu-
B*08-positive macaques control simian immunodeficiency virus replication.
J. Virol. 81: 8827–8832.
7. Loffredo, J. T., T. C. Friedrich, E. J. Leon, J. J. Stephany, D. S. Rodrigues,
S. P. Spencer, A. T. Bean, D. R. Beal, B. J. Burwitz, R. A. Rudersdorf, et al. 2007.
CD8?T cells from SIV elite controller macaques recognize Mamu-B*08-bound
epitopes and select for widespread viral variation. PLoS ONE 2: e1152.
8. Rammensee, H., J. Bachmann, N. P. Emmerich, O. A. Bachor, and S. Stevanovic.
1999. SYFPEITHI: database for MHC ligands and peptide motifs. Immunoge-
netics 50: 213–219.
9. Jardetzky, T. S., W. S. Lane, R. A. Robinson, D. R. Madden, and D. C. Wiley.
1991. Identification of self-peptides bound to purified HLA-B27. Nature 353:
10. Rotzschke, O., K. Falk, S. Stevanovic, V. Gnau, G. Jung, and H. G. Rammensee.
1994. Dominant aromatic/aliphatic C-terminal anchor in HLA-B*2702 and
B*2705 peptide motifs. Immunogenetics 39: 74–77.
11. Lopez de Castro, J. A., I. Alvarez, M. Marcilla, A. Paradela, M. Ramos,
L. Sesma, and M. Vazquez. 2004. HLA-B27: a registry of constitutive peptide
ligands. Tissue Antigens 63: 424–445.
12. Kaslow, R. A., C. Rivers, J. Tang, T. J. Bender, P. A. Goepfert, R. El Habib,
K. Weinhold, and M. J. Mulligan. 2001. Polymorphisms in HLA class I genes
associated with both favorable prognosis of human immunodeficiency virus
(HIV) type 1 infection and positive cytotoxic T lymphocyte responses to
ALVAC-HIV recombinant canarypox vaccines. J. Virol. 75: 8681–8689.
13. Goulder, P. J., R. E. Phillips, R. A. Colbert, S. McAdam, G. Ogg, M. A. Nowak,
P. Giangrande, G. Luzzi, B. Morgan, A. Edwards, et al. 1997. Late escape from
an immunodominant cytotoxic T lymphocyte response associated with progres-
sion to AIDS. Nat. Med. 3: 212–217.
14. Hendel, H., S. Caillat-Zucman, H. Lebuanec, M. Carrington, S. O’Brien,
J. M. Andrieu, F. Schachter, D. Zagury, J. Rappaport, C. Winkler, et al. 1999.
New class I and II HLA alleles strongly associated with opposite patterns of
progression to AIDS. J. Immunol. 162: 6942–6946.
15. Altfeld, M., E. T. Kalife, Y. Qi, H. Streeck, M. Lichterfeld, M. N. Johnston,
N. Burgett, M. E. Swartz, A. Yang, G. Alter, et al. 2006. HLA Alleles associated
with delayed progression to AIDS contribute strongly to the initial CD8?T cell
response against HIV-1. PLoS Med. 3: e403.
16. Gao, X., A. Bashirova, A. K. Iversen, J. Phair, J. J. Goedert, S. Buchbinder,
K. Hoots, D. Vlahov, M. Altfeld, S. J. O’Brien, and M. Carrington. 2005. AIDS
restriction HLA allotypes target distinct intervals of HIV-1 pathogenesis. Nat.
Med. 11: 1290–1292.
17. Carrington, M., and S. J. O’Brien. 2003. The influence of HLA genotype on
AIDS. Annu. Rev. Med. 54: 535–551.
18. Feeney, M. E., Y. Tang, K. A. Roosevelt, A. J. Leslie, K. McIntosh, N. Karthas,
B. D. Walker, and P. J. Goulder. 2004. Immune escape precedes breakthrough
human immunodeficiency virus type 1 viremia and broadening of the cytotoxic T
lymphocyte response in an HLA-B27-positive long-term-nonprogressing child.
J. Virol. 78: 8927–8930.
19. Betts, M. R., B. Exley, D. A. Price, A. Bansal, Z. T. Camacho, V. Teaberry,
S. M. West, D. R. Ambrozak, G. Tomaras, M. Roederer, et al. 2005. Character-
ization of functional and phenotypic changes in anti-Gag vaccine-induced T cell
responses and their role in protection after HIV-1 infection. Proc. Natl. Acad. Sci.
USA 102: 4512–4517.
20. Streeck, H., M. Lichterfeld, G. Alter, A. Meier, N. Teigen, B. Yassine-Diab,
H. K. Sidhu, S. Little, A. Kelleher, J. P. Routy, et al. 2007. Recognition of a
defined region within p24 gag by CD8?T cells during primary human immu-
nodeficiency virus type 1 infection in individuals expressing protective HLA
class I alleles. J. Virol. 81: 7725–7731.
21. Loffredo, J. T., A. T. Bean, D. R. Beal, E. J. Leon, G. E. May, S. M. Piaskowski,
J. R. Furlott, J. Reed, S. K. Musani, E. G. Rakasz, et al. 2008. Patterns of CD8?
immunodominance may influence the ability of Mamu-B*08-positive macaques
to naturally control simian immunodeficiency virus SIVmac239 replication. J. Vi-
rol. 82: 1723–1738.
22. Boyson, J. E., C. Shufflebotham, L. F. Cadavid, J. A. Urvater, L. A. Knapp,
A. L. Hughes, and D. I. Watkins. 1996. The MHC class I genes of the rhesus
monkey: different evolutionary histories of MHC class I and II genes in primates.
J. Immunol. 156: 4656–4665.
7773 The Journal of Immunology
23. Evans, D. T., D. H. O’Connor, P. Jing, J. L. Dzuris, J. Sidney, J. da Silva,
T. M. Allen, H. Horton, J. E. Venham, R. A. Rudersdorf, et al. 1999. Virus-
specific cytotoxic T lymphocyte responses select for amino-acid variation in sim-
ian immunodeficiency virus Env and Nef. Nat. Med. 5: 1270–1276.
24. Dzuris, J. L., J. Sidney, E. Appella, R. W. Chesnut, D. I. Watkins, and A. Sette.
2000. Conserved MHC class I peptide binding motif between humans and rhesus
macaques. J. Immunol. 164: 283–291.
25. Kaizu, M., G. J. Borchardt, C. E. Glidden, D. L. Fisk, J. T. Loffredo,
D. I. Watkins, and W. M. Rehrauer. 2007. Molecular typing of major histocom-
patibility complex class I alleles in the Indian rhesus macaque which restrict SIV
CD8?T cell epitopes. Immunogenetics 59: 693–703.
26. Kestler, H., T. Kodama, D. Ringler, M. Marthas, N. Pedersen, A. Lackner,
D. Regier, P. Sehgal, M. Daniel, N. King, and R. Desrosiers. 1990. Induction of
AIDS in rhesus monkeys by molecularly cloned simian immunodeficiency virus.
Science 248: 1109–1112.
27. Friedrich, T. C., E. J. Dodds, L. J. Yant, L. Vojnov, R. Rudersdorf, C. Cullen,
D. T. Evans, R. C. Desrosiers, B. R. Mothe, J. Sidney, et al. 2004. Reversion of
CTL escape-variant immunodeficiency viruses in vivo. Nat. Med. 10: 275–281.
28. Friedrich, T. C., L. E. Valentine, L. J. Yant, E. G. Rakasz, S. M. Piaskowski,
J. R. Furlott, K. L. Weisgrau, B. Burwitz, G. E. May, E. J. Leon, et al. 2007.
Subdominant CD8?T cell responses are involved in durable control of AIDS
virus replication. J. Virol. 81: 3465–3476.
29. Prilliman, K., M. Lindsey, Y. Zuo, K. W. Jackson, Y. Zhang, and W. Hildebrand.
1997. Large-scale production of class I bound peptides: assigning a signature to
HLA-B*1501. Immunogenetics 45: 379–385.
30. Barnstable, C. J., W. F. Bodmer, G. Brown, G. Galfre, C. Milstein,
A. F. Williams, and A. Ziegler. 1978. Production of monoclonal antibodies to
group A erythrocytes, HLA and other human cell surface antigens-new tools for
genetic analysis. Cell 14: 9–20.
31. Wahl, A., and W. H. Hildebrand. Changes in MHC Class I Peptide Epitopes
Following Viral Infection. In: Methods in Molecular Biology: Modulation of
Antigen Presentation During Infection. Humana Press, Totowa, NJ. In press.
32. Pinilla, C., J. R. Appel, P. Blanc, and R. A. Houghten. 1992. Rapid identification
of high affinity peptide ligands using positional scanning synthetic peptide com-
binatorial libraries. BioTechniques 13: 901–905.
33. Sidney, J., S. Southwood, C. Oseroff, M. F. del Guercio, A. Sette, and
H. M. Grey. 2001. Measurement of MHC/peptide interactions by gel filtration.
Curr. Protoc. Immunol. Chapter 18: Unit 18.3.
34. Sidney, J., S. Southwood, and A. Sette. 2005. Classification of A1- and A24-
supertype molecules by analysis of their MHC-peptide binding repertoires. Im-
munogenetics 57: 393–408.
35. Sidney, J., M. F. del Guercio, S. Southwood, V. H. Engelhard, E. Appella,
H. G. Rammensee, K. Falk, O. Rotzschke, M. Takiguchi, R. T. Kubo, et al. 1995.
Several HLA alleles share overlapping peptide specificities. J. Immunol. 154:
36. Allen, T. M., J. Sidney, M. F. del Guercio, R. L. Glickman, G. L. Lensmeyer,
D. A. Wiebe, R. DeMars, C. D. Pauza, R. P. Johnson, A. Sette, and D. I. Watkins.
1998. Characterization of the peptide binding motif of a rhesus MHC class I
molecule (Mamu-A*01) that binds an immunodominant CTL epitope from sim-
ian immunodeficiency virus. J. Immunol. 160: 6062–6071.
37. Schneidewind, A., M. A. Brockman, J. Sidney, Y. E. Wang, H. Chen,
T. J. Suscovich, B. Li, R. I. Adam, R. L. Allgaier, B. R. Mothe, et al. 2008.
Structural and functional constraints limit options for cytotoxic T lymphocyte
escape in the immunodominant HLA-B27-restricted epitope in human immuno-
deficiency virus type 1 capsid. J. Virol. 82: 5594–5605.
38. Loffredo, J. T., J. Sidney, C. Wojewoda, E. Dodds, M. R. Reynolds, G. Napoe,
B. R. Mothe, D. H. O’Connor, N. A. Wilson, D. I. Watkins, and A. Sette. 2004.
Identification of seventeen new simian immunodeficiency virus-derived CD8?T
cell epitopes restricted by the high frequency molecule, Mamu-A*02, and po-
tential escape from CTL recognition. J. Immunol. 173: 5064–5076.
39. Sette, A., J. Sidney, M. F. del Guercio, S. Southwood, J. Ruppert, C. Dahlberg,
H. M. Grey, and R. T. Kubo. 1994. Peptide binding to the most frequent HLA-A
class I alleles measured by quantitative molecular binding assays. Mol. Immunol.
40. Sidney, J., E. Assarsson, C. Moore, S. Ngo, C. Pinilla, A. Sette, and B. Peters.
2008. Quantitative peptide binding motifs for 19 human and mouse MHC class
I molecules derived using positional scanning combinatorial peptide libraries.
Immunome Res. 4: 2.
41. Sidney, J., B. Peters, C. Moore, T. J. Pencille, S. Ngo, K. A. Masterman,
S. Asabe, C. Pinilla, F. V. Chisari, and A. Sette. 2007. Characterization of the
peptide-binding specificity of the chimpanzee class I alleles A*0301 and A*0401
using a combinatorial peptide library. Immunogenetics 59: 745–751.
42. Loffredo, J. T., B. J. Burwitz, E. G. Rakasz, S. P. Spencer, J. J. Stephany,
J. P. Vela, S. R. Martin, J. Reed, S. M. Piaskowski, J. Furlott, et al. 2007. The
antiviral efficacy of simian immunodeficiency virus-specific CD8?T cells is
unrelated to epitope specificity and is abrogated by viral escape. J. Virol. 81:
43. Hutchinson, S. L., L. Wooldridge, S. Tafuro, B. Laugel, M. Glick, J. M. Boulter,
B. K. Jakobsen, D. A. Price, and A. K. Sewell. 2003. The CD8 T cell coreceptor
exhibits disproportionate biological activity at extremely low binding affinities.
J. Biol. Chem. 278: 24285–24293.
44. O’Connor, D. H., A. B. McDermott, K. C. Krebs, E. J. Dodds, J. E. Miller,
E. J. Gonzalez, T. J. Jacoby, L. Yant, H. Piontkivska, R. Pantophlet, et al. 2004.
A dominant role for CD8?-T lymphocyte selection in simian immunodeficiency
virus sequence variation. J. Virol. 78: 14012–14022.
45. Udaka, K., K. H. Wiesmuller, S. Kienle, G. Jung, and P. Walden. 1995. Decrypt-
ing the structure of major histocompatibility complex class I-restricted cytotoxic
T lymphocyte epitopes with complex peptide libraries. J. Exp. Med. 181:
46. Stryhn, A., L. O. Pedersen, T. Romme, C. B. Holm, A. Holm, and S. Buus. 1996.
Peptide binding specificity of major histocompatibility complex class I resolved
into an array of apparently independent subspecificities: quantitation by peptide
libraries and improved prediction of binding. Eur. J. Immunol. 26: 1911–1918.
47. Udaka, K., K. H. Wiesmuller, S. Kienle, G. Jung, H. Tamamura, H. Yamagishi,
K. Okumura, P. Walden, T. Suto, and T. Kawasaki. 2000. An automated predic-
tion of MHC class I-binding peptides based on positional scanning with peptide
libraries. Immunogenetics 51: 816–828.
48. Lauemoller, S. L., A. Holm, J. Hilden, S. Brunak, M. Holst Nissen, A. Stryhn,
L. Ostergaard Pedersen, and S. Buus. 2001. Quantitative predictions of peptide
binding to MHC class I molecules using specificity matrices and anchor-stratified
calibrations. Tissue Antigens 57: 405–414.
49. Peters, B., H. H. Bui, S. Frankild, M. Nielson, C. Lundegaard, E. Kostem,
D. Basch, K. Lamberth, M. Harndahl, W. Fleri, et al. 2006. A community re-
source benchmarking predictions of peptide binding to MHC-I molecules. PLoS
Comput. Biol. 2: e65.
50. Peruzzi, M., N. Wagtmann, and E. O. Long. 1996. A p70 killer cell inhibitory
receptor specific for several HLA-B allotypes discriminates among peptides
bound to HLA-B*2705. J. Exp. Med. 184: 1585–1590.
51. Ugrinovic, S., A. Mertz, P. Wu, J. Braun, and J. Sieper. 1997. A single nonamer
from the Yersinia 60-kDa heat shock protein is the target of HLA-B27-restricted
CTL response in Yersinia-induced reactive arthritis. J. Immunol. 159: 5715–5723.
52. Kuon, W., H. G. Holzhutter, H. Appel, M. Grolms, S. Kollnberger, A. Traeder,
P. Henklein, E. Weiss, A. Thiel, R. Lauster, et al. 2001. Identification of HLA-
B27-restricted peptides from the Chlamydia trachomatis proteome with possible
relevance to HLA-B27-associated diseases. J. Immunol. 167: 4738–4746.
53. Alvarez, I., M. Marti,J.Vazquez,
J. A. Lopez de Castro. 2001. The Cys-67 residue of HLA-B27 influences cell
surface stability, peptide specificity, and T cell antigen presentation. J. Biol.
Chem. 276: 48740–48747.
54. Appel, H., W. Kuon, M. Kuhne, M. Hulsmeyer, S. Kollnberger, S. Kuhlmann,
E. Weiss, M. Zeitz, K. Wucherpfennig, P. Bowness, and J. Sieper. 2004. The
solvent-inaccessible Cys67 residue of HLA-B27 contributes to T cell recognition
of HLA-B27/peptide complexes. J. Immunol. 173: 6564–6573.
55. Sidney, J., S. Southwood, V. Pasquetto, and A. Sette. 2003. Simultaneous pre-
diction of binding capacity for multiple molecules of the HLA B44 supertype.
J. Immunol. 171: 5964–5974.
56. Sidney, J., S. Southwood, D. L. Mann, M. A. Fernandez-Vina, M. J. Newman,
and A. Sette. 2001. Majority of peptides binding HLA-A*0201 with high affinity
cross-react with other A2-supertype molecules. Hum. Immunol. 62: 1200–1216.
57. Lamberth, K., G. Roder, M. Harndahl, M. Nielsen, C. Lundegaard,
C. Schafer-Nielsen, O. Lund, and S. Buus. 2008. The peptide-binding specificity
of HLA-A*3001 demonstrates membership of the HLA-A3 supertype. Immuno-
genetics 60: 633–643.
58. Sylvester-Hvid, C., M. Nielsen, K. Lamberth, G. Roder, S. Justesen,
C. Lundegaard, P. Worning, H. Thomadsen, O. Lund, S. Brunak, and S. Buus.
2004. SARS CTL vaccine candidates; HLA supertype-, genome-wide scanning
and biochemical validation. Tissue Antigens 63: 395–400.
59. Allen, T. M., B. R. Mothe, J. Sidney, P. Jing, J. L. Dzuris, M. E. Liebl,
T. U. Vogel, D. H. O’Connor, X. Wang, M. C. Wussow, et al. 2001. CD8?
lymphocytes from simian immunodeficiency virus-infected rhesus macaques rec-
ognize 14 different epitopes bound by the major histocompatibility complex class
I molecule mamu-A*01: implications for vaccine design and testing. J. Virol. 75:
60. Mothe, B. R., J. Sidney, J. L. Dzuris, M. E. Liebl, S. Fuenger, D. I. Watkins, and
A. Sette. 2002. Characterization of the peptide-binding specificity of Mamu-B*17
and identification of Mamu-B*17-restricted epitopes derived from simian immu-
nodeficiency virus proteins. J. Immunol. 169: 210–219.
61. Sette, A., J. Sidney, H. H. Bui, M. F. del Guercio, J. Alexander, J. Loffredo,
D. I. Watkins, and B. R. Mothe. 2005. Characterization of the peptide-binding
specificity of Mamu-A*11 results in the identification of SIV-derived epitopes
and interspecies cross-reactivity. Immunogenetics 57: 53–68.
62. Loffredo, J. T., J. Sidney, S. Piaskowski, A. Szymanski, J. Furlott, R. Rudersdorf,
J. Reed, B. Peters, H. D. Hickman-Miller, W. Bardet, et al. 2005. The high
frequency Indian rhesus macaque MHC class I molecule, Mamu-B*01, does not
appear to be involved in CD8? T lymphocyte responses to SIVmac239. J. Im-
munol. 175: 5986–5997.
63. Sette, A., A. Vitiello, B. Reherman, P. Fowler, R. Nayersina, W. M. Kast,
C. J. Melief, C. Oseroff, L. Yuan, J. Ruppert, et al. 1994. The relationship be-
tween class I binding affinity and immunogenicity of potential cytotoxic T cell
epitopes. J. Immunol. 153: 5586–5592.
64. Vitiello, A., L. Yuan, R. W. Chesnut, J. Sidney, S. Southwood, P. Farness,
M. R. Jackson, P. A. Peterson, and A. Sette. 1996. Immunodominance analysis of
CTL responses to influenza PR8 virus reveals two new dominant and subdomi-
nant Kb-restricted epitopes. J. Immunol. 157: 5555–5562.
65. van der Most, R. G., K. Murali-Krishna, J. L. Whitton, C. Oseroff, J. Alexander,
S. Southwood, J. Sidney, R. W. Chesnut, A. Sette, and R. Ahmed. 1998. Iden-
tification of Db- and Kb-restricted subdominant cytotoxic T cell responses in
lymphocytic choriomeningitis virus-infected mice. Virology 240: 158–167.
66. Daza-Vamenta, R., G. Glusman, L. Rowen, B. Guthrie, and D. E. Geraghty.
2004. Genetic divergence of the rhesus macaque major histocompatibility com-
plex. Genome Res. 14: 1501–1515.
67. Evans, D. T., P. Jing, T. M. Allen, D. H. O’Connor, H. Horton, J. E. Venham,
M. Piekarczyk, J. Dzuris, M. Dykhuzen, J. Mitchen, et al. 2000. Definition of five
new simian immunodeficiency virus cytotoxic T lymphocyte epitopes and their
7774SIMILAR MHC I BINDING PROFILES BETWEEN EC
restricting major histocompatibility complex class I molecules: evidence for an
influence on disease progression. J. Virol. 74: 7400–7410.
68. Nixon, D. F., A. R. Townsend, J. G. Elvin, C. R. Rizza, J. Gallwey, and
A. J. McMichael. 1988. HIV-1 gag-specific cytotoxic T lymphocytes defined with
recombinant vaccinia virus and synthetic peptides. Nature 336: 484–487.
69. Nixon, D. F., S. Huet, J. Rothbard, M. P. Kieny, M. Delchambre, C. Thiriart,
C. R. Rizza, F. M. Gotch, and A. J. McMichael. 1990. An HIV-1 and HIV-2
cross-reactive cytotoxic T cell epitope. AIDS 4: 841–845.
70. Rowland-Jones, S., R. A. Colbert, T. Dong, S. McAdam, M. Brown, K. Ariyoshi,
S. Sabally, H. Whittle, and A. McMichael. 1998. Distinct recognition of closely-
related HIV-1 and HIV-2 cytotoxic T cell epitopes presented by HLA-B*2703
and B*2705. AIDS 12: 1391–1393.
71. Valentine, L. E., S. M. Piaskowski, E. G. Rakasz, N. L. Henry, N. A. Wilson, and
D. I. Watkins. 2008. Recognition of escape variants in ELISPOT does not always
predict CD8?T cell recognition of simian immunodeficiency virus-infected cells
expressing the same variant sequences. J. Virol. 82: 575–581.
72. Bennett, M. S., H. L. Ng, A. Ali, and O. O. Yang. 2008. Cross-clade detection of
HIV-1-specific cytotoxic T lymphocytes does not reflect cross-clade antiviral
activity. J. Infect. Dis. 197: 390–397.
73. Draenert, R., C. L. Verrill, Y. Tang, T. M. Allen, A. G. Wurcel, M. Boczanowski,
A. Lechner, A. Y. Kim, T. Suscovich, N. V. Brown, et al. 2004. Persistent rec-
ognition of autologous virus by high-avidity CD8 T cells in chronic, progressive
human immunodeficiency virus type 1 infection. J. Virol. 78: 630–641.
74. Madden, D. R., D. N. Garboczi, and D. C. Wiley. 1993. The antigenic identity of
peptide-MHC complexes: a comparison of the conformations of five viral pep-
tides presented by HLA-A2. Cell 75: 693–708.
75. Madden, D. R. 1995. The three-dimensional structure of peptide-MHC com-
plexes. Annu. Rev. Immunol. 13: 587–622.
76. Saper, M. A., P. J. Bjorkman, and D. C. Wiley. 1991. Refined structure of the
human histocompatibility antigen HLA-A2 at 2.6 A resolution. J. Mol. Biol. 219:
77. Rapin, N., I. Hoof, O. Lund, and M. Nielsen. 2008. MHC motif viewer. Immu-
nogenetics 60: 759–765.
78. Peters, B., J. Sidney, P. Bourne, H. H. Bui, S. Buus, G. Doh, W. Fleri,
M. Kronenberg, R. Kubo, O. Lund, et al. 2005. The immune epitope database and
analysis resource: from vision to blueprint. PLoS Biol. 3: e91.
79. Sette, A., J. Sidney, B. D. Livingston, J. L. Dzuris, C. Crimi, C. M. Walker,
S. Southwood, E. J. Collins, and A. L. Hughes. 2003. Class I molecules with
similar peptide-binding specificities are the result of both common ancestry and
convergent evolution. Immunogenetics 54: 830–841.
80. Herberts, C. A., J. J. Neijssen, J. de Haan, L. Janssen, J. W. Drijfhout, E. A. Reits,
and J. J. Neefjes. 2006. Cutting edge: HLA-B27 acquires many N-terminal di-
basic peptides: coupling cytosolic peptide stability to antigen presentation. J. Im-
munol. 176: 2697–2701.
7775The Journal of Immunology