JOURNAL OF VIROLOGY, Feb. 2008, p. 1723–1738
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 4
Patterns of CD8?Immunodominance May Influence the Ability of
Mamu-B*08-Positive Macaques To Naturally Control Simian
Immunodeficiency Virus SIVmac239 Replication?†
John T. Loffredo,1Alex T. Bean,1Dominic R. Beal,1Enrique J. León,1Gemma E. May,1
Shari M. Piaskowski,2Jessica R. Furlott,1Jason Reed,1Solomon K. Musani,3Eva G. Rakasz,1
Thomas C. Friedrich,1Nancy A. Wilson,1David B. Allison,3and David I. Watkins1,2*
Wisconsin National Primate Research Center (WNPRC), University of Wisconsin—Madison, Madison, Wisconsin 537151;
Department of Pathology and Laboratory Medicine, University of Wisconsin—Madison, Madison, Wisconsin 537062; and
Section on Statistical Genetics, Department of Biostatistics, University of Alabama at Birmingham,
Birmingham, Alabama 352943
Received 19 September 2007/Accepted 21 November 2007
Certain major histocompatibility complex (MHC) class I alleles are strongly associated with control of
human immunodeficiency virus and simian immunodeficiency virus (SIV). CD8?T cells specific for epitopes
restricted by these molecules may be particularly effective. Understanding how CD8?T cells contribute to
control of viral replication should yield important insights for vaccine design. We have recently identified an
Indian rhesus macaque MHC class I allele, Mamu-B*08, associated with elite control and low plasma viremia
after infection with the pathogenic isolate SIVmac239. Here, we infected four Mamu-B*08-positive macaques
with SIVmac239 to investigate why some of these macaques control viral replication. Three of the four
macaques controlled SIVmac239 replication with plasma virus concentrations below 20,000 viral RNA cop-
ies/ml at 20 weeks postinfection; two of four macaques were elite controllers (ECs). Interestingly, two of the
four macaques preserved their CD4?memory T lymphocytes during peak viremia, and all four recovered their
CD4?memory T lymphocytes in the chronic phase of infection. Mamu-B*08-restricted CD8?T-cell responses
dominated the acute phase and accounted for 23.3% to 59.6% of the total SIV-specific immune responses.
Additionally, the ECs mounted strong and broad CD8?T-cell responses against several epitopes in Vif and
Nef. Mamu-B*08-specific CD8?T cells accounted for the majority of mutations in the virus at 18 weeks
postinfection. Interestingly, patterns of viral variation in Nef differed between the ECs and the other two
macaques. Natural containment of AIDS virus replication in Mamu-B*08-positive macaques may, therefore, be
related to a combination of immunodominance and viral escape from CD8?T-cell responses.
Understanding the immunological and genetic basis of the
natural control of AIDS virus replication should assist in hu-
man immunodeficiency virus (HIV) vaccine design. Of partic-
ular interest are human “elite controllers” (ECs), rare individ-
uals who spontaneously control HIV viremia to extremely low
levels (17). Similarly, a limited number of macaques sponta-
neously control simian immunodeficiency virus (SIV) replica-
tion and become ECs (49, 76).
Several lines of evidence suggest that CD8?T cells play a
key role in immune control of immunodeficiency virus repli-
cation. The transient in vivo depletion of circulating CD8?
lymphocytes in SIV-infected macaques, including EC ma-
caques, results in dramatic increases in plasma viremia (23, 32,
52, 69). CD8?T-cell responses also exert selective pressure on
replicating viruses, resulting in the emergence of variants that
escape immune detection in both HIV (5, 12, 20, 26, 36, 64, 65)
and SIV (3, 7, 19, 35, 51, 59, 60) infection. In addition, it is well
established that the expression of specific major histocompat-
ibility complex (MHC) class I alleles is associated with reduced
plasma viremia and/or slower disease progression in humans
(14, 15, 29, 34, 55) and macaques (49, 57, 61, 63, 76, 77). In
particular, human long-term nonprogressor and EC cohorts
are enriched for HLA-B27 and HLA-B57. Numerous studies
have implicated these molecules in the presentation of
epitopes that elicit effective HIV-specific CD8?T-lymphocyte
responses (6, 20, 26, 36, 38, 54, 55, 70). This assertion is further
supported by studies describing associations between viral
escape from the immunodominant HLA-B27-restricted
Gag263-272KK10 response and disease progression (8, 20, 26, 36).
Unfortunately, there are many difficulties inherent to study-
ing HIV-infected humans. Viral control appears to be medi-
ated soon after the resolution of acute-phase viremia with the
appearance of CD8?T-cell responses in HIV-infected individ-
uals (11, 41), yet HIV is infrequently diagnosed during primary
infection (42, 73). Hence, the study of immune responses ini-
tially involved in controlling HIV replication is extremely dif-
ficult. An additional complication arises due to the diversity of
HIV isolates with which individuals might be infected (24, 27).
Therefore, complete immunological monitoring would neces-
sitate the sequencing of acute-phase virus and the synthesis of
custom peptide sets matched to the infecting virus for every
AIDS research with nonhuman primates provides an animal
* Corresponding author. Mailing address: Department of Pathology
and Laboratory Medicine, University of Wisconsin—Madison, 555 Sci-
ence Dr., Madison, WI 53711. Phone: (608) 265-3380. Fax: (608)
265-8084. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 5 December 2007.
model to complement human studies. In particular, SIV-in-
fected ECs also provide examples of successful immune con-
tainment of pathogenic immunodeficiency virus replication.
However, unlike in human studies, researchers working with
macaques have direct control over key variables such as virus
strain, host genotype, and route of infection. Inoculum vari-
ability is eliminated because macaques can be infected with a
clonal viral stock, e.g., SIVmac239, enabling complete and
accurate tracking of early immune responses by use of corre-
sponding peptides in ex vivo immunological assays. Moreover,
the timing of immune responses after infection, the associated
viral sequence evolution, and plasma virus concentrations may
be closely monitored. Hence, the immunology and pathogen-
esis of acute infection can be more readily studied in macaques
than in humans and may aid in our understanding of the
correlates of immune protection.
Previously, we assembled a cohort of 192 Indian rhesus
macaques, all infected with SIVmac239 (49, 76). Fourteen of
these animals were considered ECs and controlled virus rep-
lication to fewer than 1,000 viral RNA (vRNA) copies/ml in
the chronic phase of SIV infection. Nine of the 14 ECs ex-
pressed Mamu-B*17 (76). In addition to Mamu-B*17, we re-
cently discovered that Mamu-B*08 is enriched in EC cohorts
and associated with reduced chronic-phase plasma virus con-
centrations (49). Over 50% of Mamu-B*08-positive macaques
become ECs. Interestingly, a preliminary binding motif for
Mamu-B*08 appears comparable to that of HLA-B27, an al-
lele associated with elite control in humans (48). The similarity
between Mamu-B*08 and HLA-B27 makes SIVmac239-in-
fected, Mamu-B*08-positive macaques an ideal system for
modeling human ECs.
While not all Mamu-B*08- or Mamu-B*17-positive ma-
caques control viral replication (49, 75, 76), we and others have
previously shown that control of SIV replication is not due to
genes linked to Mamu-B*17 (75) or to polymorphisms in sev-
eral host genes, including CCR5, CXCR6, GPR15, RANTES,
interleukin-10 (IL-10), APOBEC3G, tumor necrosis factor al-
pha, and TSG101 (72). Rather, CD8?T cells appear to play
a critical role in the natural containment of SIV replication
Here, we investigated early immune responses and viral evo-
lution in four Mamu-B*08-positive Indian rhesus macaques
during primary SIVmac239 infection. From previous studies
(49), we estimated that two of the four macaques would con-
trol viral replication. We hypothesized that this control would
be a function of the Mamu-B*08-specific CD8?T-cell re-
sponses and viral escape. We found that containment of SIV
replication began before 10 weeks postinfection in three of the
four Mamu-B*08-positive macaques. During peak viremia, the
CD4?memory T-cell subset decreased in only two of the four
Mamu-B*08-positive macaques. However, the CD4?memory
T-cell numbers later rebounded in these two animals, and
CD4?memory T cells were then maintained in the chronic
phase of infection in all four macaques. While robust CD8?
immune responses were detected in all four animals, macaques
with the broadest Mamu-B*08-restricted immune responses
were the most successful at viral containment. Mutations within
Mamu-B*08 epitopes were detected in all four SIVmac239-
infected macaques. Interestingly, different patterns of viral varia-
tion in Nef distinguished the two ECs from the non-EC ma-
MATERIALS AND METHODS
Animals and viruses. Indian rhesus macaques (Macaca mulatta) were geno-
typed for the MHC class I alleles Mamu-A*01, -A*02, -A*08, -A*11, -B*01,
-B*03, -B*04, -B*08, -B*17, and -B*29 by using PCR amplification with se-
quence-specific primers as previously described (33, 49). While all four macaques
expressed Mamu-B*08, none of the four macaques expressed Mamu-B*17. Ma-
caques were infected intravenously with 100 50% tissue culture infective doses of
the pathogenic molecular clone SIVmac239 (37) (GenBank accession no.
M33262). SIV-infected animals were maintained at the National Primate Re-
search Center (University of Wisconsin—Madison, Madison, WI) and cared for
according to the regulations and guidelines of the University of Wisconsin In-
stitutional Animal Care and Use Committee.
Quantification of vRNA in plasma. The plasma virus concentrations of
SIVmac239-infected Indian rhesus macaques were monitored by quantitative
reverse transcription-PCR (QRT-PCR) as previously described (23, 47). Briefly,
vRNA was isolated from EDTA-anticoagulated plasma, reverse transcribed, and
detected using a one-step QRT-PCR kit (Invitrogen, Carlsbad, CA). The QRT-
PCR was performed under the previously described conditions on a LightCycler
1.2 (Roche, Indianapolis, IN). Each QRT-PCR assay was performed with an
internal standard curve prepared by 10-fold serial dilutions of a synthetic SIV gag
transcript. The copy number for each sample was then determined by interpo-
lation onto the standard curve, using LightCycler software version 4.0. Under
normal conditions, the threshold for detection in this assay is 30 vRNA eq/ml.
Quantification of circulating CD4?T lymphocytes. The absolute counts of
CD4?T cells and CD4?memory T cells/?l of blood were determined using a
two-platform method. Briefly, the frequency of CD3?CD4?(CD4?T cell) or
CD3?CD4?CD95?(CD4?memory T cell) cells within the lymphocyte popu-
lation was determined by flow cytometry using anti-human CD4 PerCP (clone
SK3; BD Biosciences, San Jose, CA), anti-human CD95 fluorescein isothiocya-
nate (FITC) (clone DX2; BD Biosciences), and anti-human CD3 allophycocya-
nin (clone SP34-2; BD Biosciences) monoclonal antibodies. Data were acquired
on a FACSCalibur (BD Biosciences) flow cytometer and analyzed by FlowJo
software version 8.4.5 (Tree Star, Inc., Ashland, OR). Lymphocyte counts per ?l
of blood were obtained from complete blood count analysis performed on a
Pentra 60C? Hematology analyzer (ABX Diagnostics, Irvine, CA). The CD4?
T-cell or CD4?memory T-cell count/?l of blood was then determined by mul-
tiplying the lymphocyte count/?l blood by the frequency of each cell subset
within the lymphocyte gate.
IFN-? ELISPOT assay. Enzyme-linked immunospot (ELISPOT) assays were
performed as previously described (47). Briefly, peripheral blood mononuclear
cells (PBMC) were isolated from EDTA-anticoagulated blood by using Ficoll-
Paque PLUS (GE Healthcare Bio-Sciences, Uppsala, Sweden) and density cen-
trifugation. A total of 1 ? 105PBMC were used per well in precoated ELISpotPLUS
kits (MABTECH Inc, Mariemont, OH) according to the manufacturer’s instruc-
tions for the detection of gamma interferon (IFN-?)-secreting cells. All tests
were performed in duplicate using individual peptides at 10 ?M or peptide pools
(10 15-mer peptides overlapping by 11 amino acids) at 1 ?M. Fifteen-mer
peptides were provided by the NIH AIDS Research and Reference Reagent
Program (Germantown, MD). Peptides fewer than 15 amino acids in length that
contained epitopes restricted by Mamu-A*01 (2, 4), Mamu-A*02 (46, 67, 71), and
Mamu-B*08 (48) were synthesized at the University of Wisconsin Biotechnology
Center (Madison, WI). The Mamu-B*08-restricted Env epitope Env573-581KL9
has not been previously described (J. T. Loffredo et al., unpublished data). The
positive control, concanavalin A (Sigma, St. Louis, MO), was used at a final
concentration of 5 ?g/ml. The negative-control wells were devoid of any stimu-
lation. The 96-well plates were incubated for 12 to 18 h at 37°C in 5% CO2.
CD8?cell-depleted ELISPOT assays were incorporated to identify CD4?
T-cell responses. In these cases, freshly isolated or cryopreserved PBMC were
depleted of CD8?cells by using a CD8 microbead kit for nonhuman primates
(Miltenyi, Auburn, CA) along with LS columns (Miltenyi) according to the
manufacturer’s protocols. Labeled cells were removed by magnetic separation,
and the remaining cells were used as described above in IFN-? ELISPOT assays.
Fluorescence-activated cell sorting analysis using CD3 FITC (clone SP34-2; BD
Biosciences), CD8 PerCP (clone SK1; BD Biosciences), and CD4 allophycocya-
nin (clone SK3; BD Biosciences) cell surface markers confirmed that the CD8?
cell depletions removed ?99% of the CD8?lymphocytes.
Wells were imaged and counted with AID EliSpot reader version 3.4.0 or 4.0
(AID, Strassberg, Germany) and analyzed as previously described (46, 47). A
response was considered positive if the mean number of spot-forming cells (SFC)
1724LOFFREDO ET AL. J. VIROL.
from the duplicate sample wells exceeded the background level (mean of wells
without peptide stimulation) plus 2 standard deviations. Background levels were
subtracted from each well, and assay results are shown as numbers of SFC per
1 ? 106PBMC. Responses of ?50 SFC per 1 ? 106PBMC were not considered
MHC class I tetramer and surface staining. Ex vivo MHC class I tetramer
stains were performed on freshly isolated or cryopreserved PBMC as previously
described (46). MHC class I tetramers were constructed with minor modifica-
tions as previously described (31, 46). Cryopreserved PBMC were thawed at 37°C
and washed twice in R10 (RPMI 1640 medium [HyClone, Logan, UT] supple-
mented with 10% fetal calf serum [HyClone], 2 mM L-glutamine [HyClone], and
1? antibiotic-antimycotic solution [HyClone]) before staining.
Briefly, ?5 ? 105cells were stained with 5 ?l of 0.1 mg/ml tetramer stocks, 3
?l of CD3 FITC (clone SP34-2; BD Biosciences), and 5 ?l of CD8 PerCP (clone
SK1; BD Biosciences) in ?100 ?l of R10. After the cells were fixed in 1%
paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), approximately
0.5 ? 105to 1 ? 105lymphocyte-gated events were acquired on a FACSCalibur
(BD Biosciences) and analyzed using FlowJo 8.4.5 (TreeStar, Inc.). Percentages
represent the numbers of lymphocyte-gated events that were CD3?CD8?and
MHC class I tetramer positive. The threshold of detection in these assays was
0.02% CD3?CD8?MHC class I tetramer-positive gated lymphocytes.
ICS assay. IL-2 and IFN-? intracellular cytokine staining (ICS) assays were
performed on freshly isolated PBMC as previously described (23, 71). Briefly,
each test contained ?5 ? 105PBMC. Peptide pools were used at a concentration
of 10 ?M. SIV peptide pools each contained 10 15-mer peptides overlapping by
11 amino acids. Fifteen-mer peptides were provided by the NIH AIDS Research
and Reference Reagent Program (Germantown, MD). Approximately 0.5 ? 105
to 1 ? 105lymphocyte-gated events were acquired on a FACSCalibur (BD
Biosciences) and analyzed using FlowJo 8.4.5 (TreeStar, Inc.). All values were
normalized by subtracting the background staining level (negative control of
PBMC in media without stimulation).
Sequencing of plasma vRNA. Viral sequencing was performed based on meth-
ods previously described (23, 60). Briefly, vRNA was extracted from plasma by
using a Qiagen MinElute kit (Valencia, CA). We used a Qiagen One Step
RT-PCR kit to amplify overlapping regions between approximately 300 and 800
nucleotides in length that spanned the entire SIVmac239 genome. The RT-PCR
conditions for all amplicons were as follows: 50°C for 30 min; 95°C for 15 min;
45 cycles of 94°C for 30 s, 53°C for 1 min, and 72°C for 150 s; and 68°C for 20 min.
Cycling ramp rates were 2°C per second. The amplified cDNA was purified using
a Qiagen PCR purification kit. Plasmids containing cloned sequences were pu-
rified using a QIAprep Spin Miniprep kit (Qiagen). Both strands of each am-
plicon were sequenced on a 3730 DNA analyzer (Applied Biosystems, Foster
City, CA). Sequences were assembled using Aligner version 1.6.3 (CodonCode,
Deadham, MA). DNA sequences were conceptually translated and aligned to the
wild-type SIVmac239 sequence with the MacVector 9.0 trial version (Accelrys,
Statistical analysis. Statistical analyses consisted of two sample t tests for
CD4?memory T-cell (CD3?CD4?CD95?gated lymphocytes) and total CD4?
T-cell (CD3?CD4?gated lymphocytes) counts in Mamu-B*08-positive and
Mamu-B*08-negative SIVmac239-infected macaques at seven different time
points. The time points were categorized into seven intervals, as 0, 2, 6, 8, 10, 12,
and 18 (or 28) weeks postinfection. Mean values for comparison between Mamu-
B*08-positive (n ? 4) and Mamu-B*08-negative (n ? 4) macaques at each of the
time points were obtained using the TTEST procedure in version 9.1 of SAS
The Z-score test was employed to compare the viral loads observed in each
of the four Mamu-B*08-positive macaques against the means of the progressor
(n ? 175) and EC (n ? 10) cohorts at 12 different time points. None of the
SIVmac239-infected macaques in the progressor and EC cohorts expressed
Mamu-B*08. The viral load data from the 187 animals within these two cohorts
were obtained from a prior investigation (49). The time points were defined here
as the numbers of weeks, between 0 and 20, after SIVmac239 infection, subdi-
vided into 12 intervals. The goal was to ascertain whether the viral loads in
Mamu-B*08-positive macaques were significantly different from the mean of the
EC cohort or significantly lower than the mean of the progressor cohort.
Before performing inferential testing, we checked for key underlying assump-
tions of the t tests and Z-score tests (i.e., normality of residuals and homosce-
dasticity). The failure of the data to support these assumptions led us to trans-
form the data for both CD4 counts and viral loads via the natural logarithm to
improve conformity to the assumptions. To circumvent the assumption of nor-
mality of residuals, we employed one-way nonparametric analysis using an exact
Wilcoxon rank sum test with the continuity correction for the CD4?T-cell counts
only to further verify our results. Because statistical significance and direction of
effects were the same across these analytic approaches, we based our interpre-
tations on results from t tests only.
Nucleotide sequence accession numbers. The SIV genome sequences from 18
weeks postinfection were given the following GenBank accession numbers:
EU280803 (r91003-SIVmm239), EU280805 (r01027-SIVmm239), EU280804
(r00032-SIVmm239), and EU280806 (r02019-SIVmm239).
Mamu-B*08-positive, SIV-infected macaques control SIVmac239
viral replication and recover their CD4?memory T lympho-
cytes after the acute phase of infection. We infected four
Mamu-B*08-positive Indian rhesus macaques in an attempt to
understand the dynamics of early infection and the way that
the majority of macaques expressing this protective allele con-
trol viral replication. After challenging the macaques intrave-
nously with the cloned viral isolate SIVmac239, we followed
plasma viral concentrations, peripheral blood CD4?counts
(including total and memory subsets), antigen-specific re-
sponses (CD8?and CD4?), and viral evolution during the first
20 weeks after SIV infection.
Three of the four macaques controlled their viral set points
of SIVmac239 replication below 20,000 vRNA copies/ml, and
two of these had viral set points of ?1,000 vRNA copies/ml
(Fig. 1). Only one macaque, animal r91003 (Mamu-A*01 pos-
itive and -B*08 positive), displayed plasma virus concentra-
tions no different from those seen in 175 SIVmac239-infected
macaques that progressed to AIDS (?500,000 vRNA copies/
ml) and was thus termed a progressor. In contrast, animal
r01027 (Mamu-A*01-positive and -B*08-positive) had a viral
set point of ?20,000 vRNA copies/ml and was termed a slow
progressor/controller. Controller macaques maintain viral set
points ?10-fold lower than our previously studied SIV-in-
fected macaque cohort (49). Macaques r00032 (Mamu-A*02
positive and -B*08 positive) and r02019 (Mamu-B*08 positive)
controlled replication of this pathogenic SIVmac239 isolate to
?1,000 vRNA copies/ml and were classified as ECs. When
comparing the plasma virus concentrations of the three Mamu-
B*08-positive macaques that controlled SIV viremia with those
of 175 macaques that progressed to AIDS, we found that the
EC macaques r00032 and r02019 had viral set points signifi-
cantly below those of 175 macaques that progressed to AIDS
(P ? 0.0066 and P ? 0.0076, respectively). The difference
between the viral set point of controller r01027 and those of
the progressor macaques was near statistical significance (P ?
0.0578). We also found that the plasma virus concentrations in
r01027, r00032, and r02019 first began to differentiate them-
selves from those in the progressors between weeks 6 and 8
postinfection and trended toward significance as early as 4
weeks postinfection (data not shown). These data further sug-
gest that the initial immunopathogenic events of immunodefi-
ciency virus infection are crucial in controlling viral replication.
We also measured concentrations of circulating CD4?T
cells in our four SIV-infected Mamu-B*08-positive macaques.
High levels of acute-phase immunodeficiency virus replication
leads to the destruction of the CD4?memory T-cell compart-
ment (44, 53), thereby crippling the immune system during the
critical stage of HIV/SIV infection. As expected, four Mamu-
B*08-negative macaques (r95107, r98059, r00041, and r01035)
that progressed to AIDS lost their CD4?memory T cells
(defined as CD3?CD4?CD95?lymphocytes) during the
VOL. 82, 2008Mamu-B*08 IMMUNODOMINANCE MAY INFLUENCE VIRAL CONTROL 1725
acute phase of infection and never recovered their CD4?
memory T lymphocytes (Fig. 2). Two Mamu-B*08-negative
animals, r98059 and r00041, were infected with SIVmac239 at
the same time as the four Mamu-B*08-positive macaques,
while r95107 and r01035 were two Mamu-B*08-negative his-
torical controls. By comparison, only two of the four Mamu-
B*08-positive macaques experienced an acute-phase loss in
their CD4?memory T-cell levels during peak viremia. CD4?
memory T-cell levels rebounded in all four macaques (Fig. 2).
For each time point between weeks 6 and 18 postinfection, the
Mamu-B*08-positive macaques preserved a significantly higher
number of CD4?memory T cells than the four Mamu-B*08-
negative macaques (P ? 0.014). Interestingly, the Mamu-B*08-
positive progressor macaque r91003 began to show a decline in
its CD4?memory T-cell count only at 18 weeks postinfection,
perhaps due to high plasma virus concentrations. The total
CD4?T-cell counts (defined as CD3?CD4?lymphocytes)
were not statistically different between the groups of Mamu-
B*08-positive and -B*08-negative macaques (data not shown).
Dominance of Mamu-B*08-restricted CD8?T-cell responses
during the acute phase of SIVmac239 infection. We initially
examined the entire repertoire of SIV-specific immune re-
sponses by using ex vivo IFN-? ELISPOT with 15-mer peptides
that overlapped by 11 amino acids for the entire viral proteome
(Fig. 3). These assays also included eight minimal optimal
epitopes restricted by Mamu-B*08 (48) as well as known
epitopes and other peptides that bind to Mamu-A*01 (2, 4)
and Mamu-A*02 (46, 67, 71). In addition, we performed MHC
class I tetramer staining on the immunodominant epitopes
restricted by Mamu-A*01 (Tat28-35SL8 and Gag181-189CM9)
and Mamu-A*02 (Gag71-79GY9 and Nef159-167YY9) during
primary infection in macaques that expressed these alleles
(data not shown). Since CD8?T-cell responses usually peak at
3 weeks postinfection (66), we chose this time point to exten-
sively investigate the possible role of SIV-specific CD8?T cells
in disease protection or progression during the acute phase.
CD8?T-cell responses against the majority of the SIV pro-
teins were detected in all four SIV-infected macaques. While
these macaques all expressed Mamu-B*08, their immune re-
sponses should also be influenced by other known and un-
known MHC class I alleles that they expressed. As expected,
the progressor macaque r91003 (also Mamu-A*01 positive)
made robust immune responses against the immunodominant
Mamu-A*01-restricted CD8?T-cell epitopes Gag181-189CM9
and Tat28-35SL8 (Fig. 3 and data not shown). These CD8?
T-cell responses peaked at 3 weeks postinfection, with ?0.39%
(Gag181-189CM9) and ?2.24% (Tat28-35SL8) of CD3?CD8?
lymphocytes staining with the relevant tetramer. Aside from
these two Mamu-A*01-restricted responses, along with the
Mamu-A*01-restricted response against Gag254-262QI9, the
majority of the CD8?T-cell response generated by r91003
targeted a single Mamu-B*08-restricted epitope in Vif and an
epitope of unknown MHC class I restriction in Rev. Progressor
macaque r91003 made no substantial CD8?T-cell responses
against Nef, although three known Mamu-B*08-restricted
CD8?T-cell epitopes are located in this protein (48).
The controller macaque r01027 (also expressing Mamu-
A*01) mounted SIV-specific responses of a similar magnitude
against the Mamu-A*01-restricted CD8?T-cell epitopes
Tat28-35SL8 and Gag181-189CM9 (Fig. 3 and data not shown).
FIG. 1. Three of four Mamu-B*08-positive Indian rhesus macaques control pathogenic SIVmac239 viral replication. Longitudinal SIVmac239
plasma virus concentrations were plotted for four Mamu-B*08-positive macaques (blue lines) and the geometric mean of viral loads from 10
Mamu-B*08-negative ECs and 175 Mamu-B*08-negative animals that progressed to AIDS (red lines) from a previous study (49). SIV-infected
macaques r00032 (Mamu-A*02 positive and Mamu-B*08 positive) and r02019 (Mamu-B*08 positive) were considered ECs, with viral set points of
?1 ? 103vRNA copies/ml. These two macaques had significantly lower viral set points (weeks 10 to 20 postinfection) than the macaques that
progressed to AIDS (P ? 0.0066 for r00032 and P ? 0.0076 for r02019). Animal r01027 (Mamu-A*01 positive and Mamu-B*08 positive) is
considered a “controller,” with viremia ?1 log lower than typical SIV replication. The viral set point of r01027 was near statistical significance
compared to those of the macaques that progressed to AIDS (P ? 0.0578). None of these three macaques had P values significantly different from
that for the viral set point of the EC cohort. One of the four macaques did not control SIVmac239 replication to ?20,000 vRNA copies/ml after
10 weeks postinfection. Animal r91003 (Mamu-A*01 positive and Mamu-B*08 positive) exhibited typical viremia at ?10 weeks postinfection that
was not statistically different from that of the macaques that progressed to AIDS.
1726 LOFFREDO ET AL.J. VIROL.
MHC class I tetramer staining showed that the CD8?T-cell
response against Gag181-189CM9 peaked at 2 weeks postinfec-
tion (?0.77%), while the Tat28-35SL8-specific CD8?T cells
reached ?1.95% at 3 weeks postinfection. Along with a robust
CD8?T-cell response against Vif172-179RL8, several Mamu-
B*08-restricted responses of lower magnitude (?200 SFC/106
PBMC) were detected in r01027 (Fig. 3). A sizeable CD8?
T-cell response of unknown MHC restriction to Rev was also
identified in this macaque.
At 3 weeks postinfection, the two EC macaques r00032 and
r02019 mounted total SIV-specific immune responses near or
exceeding 10,000 SFC/106PBMC (Fig. 4). These included sev-
eral robust CD8?T-cell responses to both the Vif and Nef
epitopes restricted by Mamu-B*08 (Fig. 3 and 4). Additionally,
animal r00032 (also Mamu-A*02 positive) made a response
against the immunodominant Mamu-A*02-restricted CD8?T-
cell epitope Nef159-167YY9 (?2.4% at 2 and 3 weeks postin-
fection) (data not shown). Notably, we did not detect a re-
sponse to Gag71-79GY9, which is frequently codominant with
Nef159-167YY9 in Mamu-A*02-positive animals (46, 71). How-
ever, two responses, of ?500 SFC/106PBMC and of unknown
MHC class I restriction, were directed against Gag [Gag241-291(G)
and Gag361-411(J) peptide pools] in EC r00032 (Fig. 3). While
EC macaque r02019 did not express any other characterized
MHC class I alleles that restrict known minimal optimal
epitopes, a substantial response(s) of unknown restriction
against Vpr was identified.
Using the week 3 ELISPOT pool response data along with
responses to known minimal optimal epitopes and MHC class
I tetramer stains, we defined the proportion of the total SIV-
specific immune responses restricted by Mamu-B*08 and other
MHC class I molecules (Fig. 4). Mamu-A*01-positive ma-
caques mount two immunodominant, acute-phase CD8?T-
cell responses, Gag181-189CM9 and Tat28-35SL8 (3, 61), that
typically account for ?50% of the total SIV-specific CD8?
responses (56). However, the normally dominant Mamu-A*01-
restricted immune responses contributed only a slightly larger
percentage of the total SIV-specific immune response than the
Mamu-B*08-restricted immune responses in progressor ma-
caque r91003 and controller r01027. Mamu-A*01-restricted
CD8?T cells accounted for 44% of the total SIV-specific
immune response in r91003 and 26.5% of the total SIV-specific
FIG. 2. CD4?memory T cells are maintained in Mamu-B*08-positive macaques infected with SIVmac239. (A) Absolute counts of CD4?
memory T cells were obtained from the four SIVmac239-infected, Mamu-B*08-positive macaques (blue lines) in addition to the four SIVmac239-
infected, Mamu-B*08-negative macaques (red lines). Mamu-B*08-negative macaques r98059 and r00041 were infected along with the four
Mamu-B*08-positive macaques. CD4?memory T-cell counts from two additional Mamu-B*08-negative, -B*17-negative macaques (animals r95107
and r01035) were acquired from cryopreserved PBMC. Archived PBMC were not available at week 2 from r01035 or at week 18 from r01035 and
r00041. In place of the week 18 time point, absolute counts of CD4?memory T cells were acquired from the closest available time point (week
28), indicated in parentheses. (B) Geometric means of absolute counts of CD4?memory T cells. The blue line represents the four Mamu-B*08-
positive macaques, and the red line represents the four Mamu-B*08-negative macaques. By use of log-transformed arithmetic means, statistically
significant differences were found between the absolute CD4?memory T-cell counts of the Mamu-B*08-positive and -B*08-negative groups at the
following time points: week 6 (P ? 0.001), week 8 (P ? 0.001), week 10 (P ? 0.001), week 12 (P ? 0.014), and week 18 (or 28) (P ? 0.001).
VOL. 82, 2008Mamu-B*08 IMMUNODOMINANCE MAY INFLUENCE VIRAL CONTROL 1727
FIG. 3. Ex vivo whole PBMC IFN-? ELISPOT using peptides spanning the entire SIVmac239 proteome and the relevant MHC class
I-restricted minimal optimal CD8?T-cell epitopes at 3 weeks postinfection. Eighty-one peptide pools (10 15-mer peptides overlapping by 11 amino
acids) were tested in IFN-? ELISPOT assays spanning the complete SIVmac239 proteome. Total responses for each protein were calculated by
adding the mean values of the individual peptide pools for each SIV protein. Individual minimal optimal peptides were included to detect responses
1728 LOFFREDO ET AL.J. VIROL.
immune response in r01027, compared to 32.7% and 23.3%,
respectively, for Mamu-B*08-restricted CD8?T cells (Fig.
4A). Animal r00032 expressed both Mamu-A*02 and Mamu-
B*08. While Mamu-A*02-restricted responses accounted for
14.4% of the SIV-specific response, Mamu-B*08-restricted re-
sponses accounted for 48.7% of the total number of SIV-
specific CD8?T cells. Macaque r02019 did not express any
other characterized MHC class I alleles aside from Mamu-
B*08, and Mamu-B*08-restricted CD8?T cells accounted for
59.6% of its total SIV-specific response.
Despite the presence of as many as 12 MHC class I alleles
expressed in a given macaque (13, 16, 62), Mamu-B*08-re-
stricted CD8?T cells accounted for a large proportion (23.3 to
59.6%) of the total SIV-specific response. Furthermore, the
contribution of Mamu-B*08-restricted SIV-specific immune
responses is likely an underestimate because, unlike for
Mamu-A*01 (2, 4) and Mamu-A*02 (46), a comprehensive
peptide binding motif and epitope mapping study has not yet
been completed for Mamu-B*08.
Increased breadth of Mamu-B*08-restricted CD8?T-cell
responses against epitopes in Vif and Nef was associated with
elite control. Despite the fact that all four macaques made
strong Mamu-B*08-restricted CD8?T-cell responses, only two
animals were ECs, controlling viral replication to approxi-
mately 1,000 vRNA copies/ml (49, 76). We next analyzed the
breadth of the Mamu-B*08-restricted immune responses to
examine the possible role that multiple epitope-specific,
Mamu-B*08-restricted CD8?T-cell responses might play in
the control of SIV replication. At 3 weeks postinfection, the
progressor macaque r91003 made a narrowly focused immu-
nodominant response against Vif172-179RL8 that constituted
87% of the Mamu-B*08-restricted CD8?T cells detected in
this macaque (Fig. 4B). Controller macaque r01027 also made
an immunodominant response to this same epitope (53.7%,
Vif172-179RL8). However, three other Mamu-B*08-restricted
CD8?T-cell responses (Vif123-131RL9, Env573-581KL9, and
Nef137-146RL10) each accounted for ?10% of the Mamu-
B*08-restricted immune breadth in r01027. By contrast, the
EC macaques r00032 and r02019 made robust CD8?T-cell
responses to several of the eight Mamu-B*08-bound peptides,
none of which accounted for ?50% of the Mamu-B*08-re-
stricted CD8?T-cell response. At 3 weeks postinfection, five of
the eight Mamu-B*08-restricted responses were at ?1,000
SFC/106PBMC for the EC r00032, while the other EC r02019
had three responses exceeding this magnitude (Fig. 4B).
To study changes in the Mamu-B*08-restricted epitope
breadth over time, we performed MHC class I tetramer stains
on cryopreserved PBMC during the acute to early chronic
phase of SIV infection for all eight Mamu-B*08-restricted
CD8?T-cell epitopes (Fig. 5). We found that the MHC class
I tetramer stains largely agreed with the magnitude of the
IFN-? ELISPOT responses at 3 weeks postinfection (Fig. 4)
and subsequent time points (data not shown). At 3 weeks
postinfection, ECs r00032 and r02019 typically displayed the
largest percentages of SIV-specific CD8?T cells for each of
the eight Mamu-B*08-restricted CD8?T-cell responses. These
two ECs targeted three epitopes, two in Vif (RL9 and RL8)
and one in Nef (RL10), with massive CD8?T-cell responses
(0.76 to 7.31%) (Fig. 5). Controller macaque r01027 displayed
several low-level responses aside from its immunodominant
Vif172-179RL8 that were maintained over time. In contrast,
progressor r91003 mounted a Mamu-B*08-restricted immune
response focused primarily against the Vif172-179RL8 epitope
at 3 weeks postinfection, although a sizeable response against
Nef137-146RL10 was detected later after infection (Fig. 5). In-
terestingly, only r91003 failed to make a substantial response
against the Vif123-131RL9 epitope during any of the time
points, potentially implicating this response in Mamu-B*08-
restricted control. Not surprisingly, only low-level frequencies
for the majority of the Mamu-B*08-restricted CD8?T cells
were found in the three macaques controlling viremia at 18
weeks postinfection. This diminution of their robust acute-
phase responses was likely due to small amounts of antigen
stimulation due to low SIV plasma virus concentrations after
Stronger and broad CD4?T-cell responses in the Mamu-
B*08-positive EC macaques. Several previous studies have
shown an association between strong and broad CD4?T-cell
responses and control of HIV/SIV replication (10, 23, 25, 28,
50, 68). CD4?T cells may also play a role in the prevention of
CD8?T-cell exhaustion (45). Therefore, we investigated the
involvement of CD4-mediated immune responses in the four
Mamu-B*08-positive macaques. At 6 weeks postinfection, we
performed ex vivo IL-2 and IFN-? ICS assays to determine
whether responses to peptide pools detected in the PBMC
IFN-? ELISPOTs at 3 weeks postinfection were mediated by
CD4?or CD8?T cells. Progressor r91003 and controller
r01027 did not make any appreciable CD4?responses at this
time point (data not shown). In contrast, EC macaque r00032
made three responses directed against the Gag121-171(D),
Rev1-51(A), and Nef1-51(A) pools, while EC macaque r02019
responded to the Gag241-291(G) pool. All of these responses
were at or below 0.1% of the level for CD4?lymphocytes (data
We next determined whether CD4?T-cell responses broad-
ened after resolution of primary viremia, as a durable, long-
restricted by Mamu-A*01 and Mamu-A*02. While the Mamu-A*01-restricted Gag181-189CM9 epitope was not tested (NT) in r91003, we can verify
that a response to this epitope was detected at 3 weeks postinfection by the Gag161-211(E) peptide pool that contains the Gag181-189CM9 sequence
(692 SFC/106PBMC). MHC class I tetramer staining in r91003 confirms the presence of Gag181-189CM9-specific CD8?T cells at this time point
(see text). Two Mamu-A*02-restricted CD8?T-cell epitopes are labeled as Nef YY9. Nef YY9 refers to the epitope at positions 159 to 167
(YTSGPGIRY), while Nef YY92refers to the epitope at positions 221 to 229 (YTYEAYVRY). Minimal optimal peptides were used for the
following Mamu-B*08-restricted epitopes: Vif123-131RL9, Vif172-179RL8, Rev44-51RL8, Env573-581KL9, and Nef246-254RL9. Mamu-B*08 epitopes
annotated with an asterisk were represented with peptides slightly larger than the minimal optimal as these responses were in the process of being
fine mapped (Rev12-20KL9* represents two overlapping 15-mer peptides at positions 5 to 23, Nef8-16RL9* represents a 10-mer peptide at positions
7 to 16, and Nef137-146RL10* represents an 11-mer peptide at positions 136 to 146). Background levels were subtracted from each well. Mean
responses of ?50 SFC per 1 ? 106cells (white bars) were not considered positive.
VOL. 82, 2008 Mamu-B*08 IMMUNODOMINANCE MAY INFLUENCE VIRAL CONTROL1729
FIG. 4. Involvement of Mamu-B*08-restricted CD8?T-cell responses in the overall CD8?T-cell-mediated immune response against
SIVmac239 at 3 weeks postinfection. (A) Mamu-B*08-restricted CD8?T cells make a major contribution to the total SIV-specific immune
response during the acute phase of infection. The contributions of known responses are shown for Mamu-A*01, Mamu-A*02, and Mamu-B*08
by attributing peptide pools to particular MHC class I molecules based on the locations of known SIV epitopes in these pools. (B) Mamu-B*08-
restricted CD8?T cells recognize a broader epitope repertoire in EC macaques early in SIV infection. CD8?T-cell responses accounting for
?10% of the total Mamu-B*08-restricted immune response are indicated above the appropriate bar. Minimal optimal peptides were used for the
1730 LOFFREDO ET AL.J. VIROL.
lasting control may also be aided by CD4?T-cell responses
(45). IFN-? ELISPOT assays were performed on PBMC de-
pleted of CD8?cells at 20 weeks after SIV infection (Fig. 6).
Controller r01027 made only one detectable CD4?T-cell re-
sponse to a peptide pool in Gag. Surprisingly, despite high
levels of SIV viremia, the number of CD4?T-cell responses
made by progressor r91003 was comparable to those made by
the EC macaques r00032 and r02019. However, all of the
following Mamu-B*08-restricted epitopes: Vif123-131RL9, Vif172-179RL8, Rev44-51RL8, Env573-581KL9, and Nef246-254RL9. Mamu-B*08 epitopes
annotated with an asterisk were represented with peptides slightly larger than the minimal optimal as these responses were in the process of being
fine mapped (Rev12-20KL9* represents two overlapping 15-mer peptides positions 5 to 23, Nef8-16RL9* represents a 10-mer peptide at positions
7 to 16, and Nef137-146RL10* represents an 11-mer peptide at positions 136 to 146). Data for both panel A and panel B were derived from ex vivo
IFN-? ELISPOT assays at 3 weeks postinfection (Fig. 3). Background levels were subtracted from each well. Mean responses of ?50 SFC per 1 ?
106cells (white bars) were not considered positive.
FIG. 5. Comparison of Mamu-B*08-restricted CD8?T-cell responses detected by MHC class I tetramers during the first 18 weeks postinfection.
Cryopreserved PBMC from the four Mamu-B*08-positive macaques (black, r91003; green, r01027; blue, r00032; and red, r02019) were thawed and
stained at the indicated time points for the eight Mamu-B*08-restricted epitopes. Results of MHC class I tetramer stains are shown here as percentages
of CD3?CD8?MHC class I tetramer-positive gated lymphocytes. The threshold of detection in these assays was 0.02% CD3?CD8?MHC class I
tetramer-positive gated lymphocytes.
VOL. 82, 2008 Mamu-B*08 IMMUNODOMINANCE MAY INFLUENCE VIRAL CONTROL1731
CD4?T-cell responses detected in r91003 were low frequency,
with between 50 and 150 SFC/106CD8?cell-depleted PBMC.
In comparison, the ECs exhibited stronger CD4?immune
responses. EC r00032 mounted three CD4?T-cell re-
sponses, while EC r02019 had six CD4?T-cell responses at
?150 SFC/106CD8?cell-depleted PBMC. Almost half of
the CD4?T-cell responses generated by r91003, r00032, and
r02019 were against Gag, a protein that is frequently recog-
nized by CD4?T cells in successful vaccinees and ECs (10,
23, 25, 28, 68, 74). A number of the low-level responses
(?100 SFC/106CD8?cell-depleted PBMC) from these
three macaques were also found in frozen IFN-? CD8?
cell-depleted PBMC ELISPOT assays between weeks 14 and
26 postinfection (data not shown).
FIG. 6. Detection of CD4?T-cell responses at 20 weeks postinfection by ex vivo CD8?cell-depleted IFN-? ELISPOT. Thirty-five peptide
pools, with 10 15-mer peptides overlapping by 11 amino acids, spanning the entire SIVmac239 proteome (except for Pol and Env due to limited
availability of PBMC) were tested in IFN-? ELISPOT assays on PBMC depleted of CD8?cells. Each column represents the number of SFC per
106CD8?cell-depleted PBMC directed against a single peptide pool at 20 weeks postinfection. Only positive responses are indicated. Background
levels were subtracted from each well. Mean responses of ?50 SFC per 1 ? 106cells (dashed line) were not considered positive.
1732LOFFREDO ET AL.J. VIROL.
Early viral variation in several Mamu-B*08-restricted
CD8?T-cell epitopes. It has been shown that CD8?T cells
exert enormous selective pressures and can lead immunodefi-
ciency virus escape as early as 4 weeks postinfection, (3, 5, 7, 8,
12, 19, 20, 26, 35, 36, 51, 59, 60, 64, 65). We therefore inves-
tigated whether viral escape was a factor in determining
whether a Mamu-B*08-positive macaque develops high viral
loads or controls SIV replication. Sequencing the entire
SIVmac239 genome from plasma virus at 18 weeks postinfection
revealed only a few substitutions in the clonal SIVmac239 virus
(see Fig. S1 in the supplemental material). In total, mutations
resulting in 33 amino acid substitutions (17 complete amino
acid substitutions and 16 incomplete replacements) were found
in plasma virus from the four SIV-infected macaques (Table
1). A deletion in the RNA that encodes the gp120 Env protein
was also found in progressor macaque r91003 (positions 418 to
424) (see Fig. S1 in the supplemental material). Of these 33
amino acid substitutions, 7 were previously documented to be
the result of suboptimal nucleotides that routinely mutate in
the majority of Indian rhesus macaques in vivo, likely increas-
ing the fitness of SIVmac239 (1). An additional two substitu-
tions located in Env also do not appear to be associated with
MHC class I expression (Table 1). Of the remaining 24 sub-
stitutions, 16 were within defined MHC class I epitopes, while
8 cannot be accounted for at this time.
Mamu-B*08-restricted CD8?T cells were responsible for
selecting the majority of the viral variation in the replicating
plasma virus at 18 weeks postinfection. We found 10 amino
acid replacements in or near Mamu-B*08-restricted epitopes
(Table 1; also see Fig. S1 in the supplemental material). Six of
these were the result of complete nucleotide substitutions,
while four were identified as mixed-base substitutions. Surpris-
ingly, viral variation was observed in four of the eight known
Mamu-B*08-restricted CD8?T-cell epitopes (Vif123-131RL9,
Vif172-179RL8, Nef136-147RL10, and Nef246-254RL9), with at
least one mutation detected in each of the four macaques at 18
Selective pressures exerted by Mamu-A*01-restricted CD8?
T cells accounted for six amino acid replacements found at 18
weeks postinfection (Table 1; also see Fig. S1 in the supple-
mental material). Escape mutations were detected in both
Mamu-A*01-positive macaques (r91003 and r01027) in the
Tat28-35SL8 epitope, consistent with the rapid evolution of this
sequence (3, 59). Three concomitant changes in the overlap-
ping open reading frame for Vpr were also identified. The only
other substitution that was likely due to Mamu-A*01-restricted
CD8?T cells was in the Env726-735ST10 epitope in controller
macaque r01027. No variation was identified in Mamu-A*02-
restricted epitopes in EC macaque r00032.
We further defined the viral ontogeny in these four
Mamu-B*08-restricted epitopes (Vif123-131RL9, Vif172-179RL8,
Nef136-147RL10, and Nef246-254RL9) by population sequencing
of replicating plasma virus at seven time points between weeks
4 and 18 postinfection (Fig. 7). Epitope mutations were detected
as early as 10 weeks after SIV infection in Nef136-147RL10 in the
EC r02019. The majority of the viral variants detected at 18
weeks postinfection appeared between weeks 12 and 13 postin-
fection. Nonetheless, we saw viral variation in four of the eight
Mamu-B*08-restricted epitopes, indicating selective pressure
mediated by Mamu-B*08-restricted CD8?T cells on multiple
epitopes. A majority of these mutations were observed previ-
ously in the chronic phase, and selection was correlated with
expression of Mamu-B*08 (48).
Viral variation in the Mamu-B*08-restricted CD8?T-cell
epitopes appeared to be more pronounced in the progressor
(r91003) and controller (r01027) macaques (Fig. 7). The
Vif123-131RL9 epitope showed the same pattern of viral varia-
tion (alanine to valine) in all four macaques. By contrast,
mutations in the second Vif epitope, Vif172-179RL8, were de-
tected only in the controller r01027. The Nef136-146RL10
epitopes displayed two patterns of viral variation. The first
pattern was seen in the two ECs and involved an isoleucine
(I)-to-threonine (T) change at position eight of the epitope.
The second pattern was found in the other two animals and
involved a change of alanine (A) to proline (P) in the amino
acid N-terminal to Nef136-146RL10 that may alter antigen pro-
cessing (48). Interestingly, a similar A-to-P substitution imme-
diately N-terminal of an HLA-B57-restricted HIV epitope in
Gag is known to alter antigen processing and to abrogate
recognition (18). The second Nef epitope, Nef246-254RL9, ac-
cumulated substitutions only in progressor r91003 and control-
ler r01027. In both cases, there was a leucine (L)-to-proline (P)
substitution at the C terminus of the epitope.
Previous investigations have linked particular MHC class I
alleles to control of HIV replication (14, 15, 29, 34, 55), yet
understanding how CD8?T cells restricted by these protective
alleles contribute to viral control remains a mystery. Recently,
we identified an association between the Indian rhesus ma-
caque MHC class I allele Mamu-B*08 and control of SIVmac239
replication (49). While identifying CD8?T-cell responses re-
stricted by this allele in EC macaques, we also discovered that
the preliminary peptide binding motif of Mamu-B*08 appears
TABLE 1. Amino acid replacements in the circulating plasma virus
of the four Mamu-B*08-positive macaques at 18 weeks after
Type of substitution
No. of amino acid replacements
CompleteMixed base Total
MHC class I associated
Not MHC associated
aExpressed by r91003 (progressor), r01027 (controller), r00032 (EC), and
bExpressed by r91003 (progressor) and r01027 (controller).
cExpressed by r00032 (EC).
dSubstitutions at position 67 in Env were previously detected in 33 of 35
SIV-infected macaques regardless of MHC class I genotype (60).
eThis number includes concomitant changes in the overlapping open reading
frame for Vpr (see Fig. S1C in the supplemental material).
VOL. 82, 2008 Mamu-B*08 IMMUNODOMINANCE MAY INFLUENCE VIRAL CONTROL1733
to be similar to the peptide binding motif of HLA-B27 (48), an
MHC class I allele associated with control of HIV replication
in humans. The high percentage of Mamu-B*08-positive ma-
caques that become ECs (?50%) and the functional similarity
of Mamu-B*08 to HLA-B27 make these MHC class I-defined
macaques ideal for modeling human ECs. We therefore stud-
ied the immunopathogenic events of acute-phase SIV infection
in four Mamu-B*08-positive macaques in an attempt to further
understand CD8-mediated viral control in ECs.
In this observational study, we show that three of four
Mamu-B*08-positive macaques controlled replication of the
pathogenic SIVmac239 isolate (Fig. 1). Two of these macaques
(r00032 and r02019) were ECs with plasma viral concentra-
tions of approximately 1,000 vRNA copies/ml at 20 weeks
postinfection. The third macaque (r01027) showed some mea-
sure of control of this highly pathogenic virus, with a plasma
viral concentration of ?20,000 vRNA copies/ml at 20 weeks
postinfection. Only macaque r91003 had plasma viremia sim-
ilar to the viral set points in the majority of animals that
progressed to AIDS (?5 ? 105vRNA copies/ml).
We then investigated how the majority of Mamu-B*08-pos-
itive macaques controlled viral replication. Recent experi-
ments have demonstrated that the rapid depletion of the
CD4?memory T cells during the acute phase of HIV/SIV
infection might be an important factor contributing to disease
progression (44, 53). Interestingly, while the CD4?memory T
cells in the PBMC were depleted during primary SIV infection
in two of the four Mamu-B*08-positive macaques, all four of
FIG. 7. Mutations in Mamu-B*08-restricted CD8?T-cell epitopes occurred as early as 10 weeks postinfection. The ontogeny of substitutions
in Vif123-131RL9, Vif172-179RL8, Nef137-146RL10, and Nef245-254RL9 was followed by sequencing of plasma virus between 4 and 18 weeks after
SIVmac239 infection. The Nef137-146RL10 epitope is annotated with an asterisk to signify that the amino acid residue immediately N-terminal to
the epitope, alanine (A), is also displayed. Viral variation at this position is associated with Mamu-B*08 expression (48). Amino acids identical to
the wild-type sequence are shown as dots. Complete amino acid replacements are shown in uppercase; sites of mixed-base heterogeneity are shown
1734 LOFFREDO ET AL. J. VIROL.
the Mamu-B*08-positive macaques recovered their CD4?
memory T cells by the chronic phase of infection (Fig. 2). By
contrast, four Mamu-B*08-negative macaques infected with
SIVmac239 experienced acute-phase loss of their CD4?mem-
ory T-cell subset and none of the Mamu-B*08-negative ma-
caques recovered this important CD4?T-cell subset. At 18
weeks postinfection, the progressor macaque (r91003) started
to show signs of CD4?memory T-cell depletion, likely related
to the high plasma virus concentrations in this animal.
Mamu-B*08-restricted CD8?T-cell responses contributed
substantially to the acute phase of SIVmac239 infection (Fig.
4). However, the pattern of immunodominance varied from
animal to animal, and breadth seemed to correlate with suc-
cessful control of immunodeficiency virus replication. At 3
weeks postinfection, progressor r91003 focused its Mamu-
B*08-restricted CD8?T-cell responses primarily on only one
of the eight mapped Mamu-B*08 CD8?T-cell epitopes,
Vif172-179RL8. Vif172-179RL8-specific cells accounted for 87%
of the Mamu-B*08-restricted CD8?T cells detected in this
animal. By contrast, the two ECs (r00032 and r02019) divided
their robust CD8?T-cell responses among several Mamu-
B*08-restricted epitopes (Fig. 3 to 5). Controller macaque
r01027 also made several Mamu-B*08-restricted CD8?T-cell
responses at 3 weeks postinfection, but these were of a much
lower magnitude than those of the ECs. Indeed, both EC ma-
caques targeted at least the two epitopes in Vif (Vif123-131RL9
and Vif172-179RL8) and one epitope in Nef (Nef137-146RL10) with
high-frequency CD8?T-cell responses. Thus, high-frequency
CD8?T-cell responses against epitopes in Vif and Nef ap-
peared to correlate with a successful disease outcome.
The Vif123-131RL9 response may be especially crucial in the
control of viral replication. Vif123-131RL9-specific CD8?T
cells were generated to a sizeable frequency only in the three
macaques that controlled viral replication (Fig. 3 to 5). Inter-
estingly, we have recently shown that two SIVmac239?nef-
vaccinated Mamu-B*08-positive macaques controlled replica-
tion of a pathogenic heterologous challenge with SIVsmE660
(M. R. Reynolds et al., unpublished data). No replication of
the challenge virus was seen in one of the two Mamu-B*08-
positive macaques, whereas the other had a peak of 14,000
vRNA copies/ml at 2 weeks postchallenge. In the macaque that
experienced replication of the SIVsmmE660 challenge virus,
we detected only two vaccine-induced anamnestic CD8?T-cell
responses. Both of these were restricted by Mamu-B*08 re-
sponses (Vif123-131RL9 and Env573-581KL9). During the acute
phase after SIVsmE660 challenge, the Env573-581KL9-spe-
cific CD8?T cells expanded to a frequency of ?1%. How-
ever, the Vif123-131RL9-specific CD8?T-cell response was
immunodominant, peaking at 4.84% of the level for CD3?
CD8?lymphocytes in the peripheral blood at 3 weeks post-
challenge, implicating this response in the control of viral
We then examined whether escape from Mamu-B*08-re-
stricted responses could account for progression or control of
SIVmac239 replication. It has been previously shown that
escape mutations could lead to loss of viral control in SIV-
infected macaques (7, 35). Also, viral escape from the immu-
nodominant HIV-specific response against the HLA-B27-re-
stricted epitope Gag263-272KK10 has been associated with loss
of control of viral replication (8, 20, 26, 36). We sequenced
replicating plasma virus at 18 weeks postinfection and found
that the majority of the amino acid replacements at this time
were selected for by Mamu-B*08-restricted CD8?T-cell re-
sponses (Table 1). By comparison, viral variation was not de-
tected in any of the Mamu-A*02-restricted epitopes, and
amino acid replacements were present in only two Mamu-
A*01-restricted epitopes (Tat28-35SL8 and Env726-735ST10)
(see Fig. S1 in the supplemental material). Four of the eight
Mamu-B*08-restricted epitopes (Vif123-131RL9, Vif172-179RL8,
Nef136-147RL10, and Nef246-254RL9) exhibited viral variation
consistent with mutations identified in a previous study (48).
These results may suggest that Mamu-B*08-restricted CD8?T
cells exert more selective pressure than other MHC class I-
restricted responses during the critical early phase of infection.
Alternatively, the mutations observed in these epitope se-
quences could occur in regions of the viral genome that are not
under strong evolutionary constraints. Our group and others
have previously shown that fitness costs may play a role in
determining the rate at which escape mutations accumulate
and revert in vivo (21, 22, 43).
We also followed the ontogeny of mutations by population
sequencing these four Mamu-B*08-restricted epitopes at various
time points. We discovered the first evidence of viral variation in
circulating plasma virus at 10 weeks postinfection and mutations
within several Mamu-B*08-restricted epitopes by 13 weeks
postinfection (Fig. 7). Interestingly, the patterns of amino acid
substitutions in the Nef137-146RL10 and Nef246-254RL9 epitopes
differentiated the four Mamu-B*08-positive macaques into two
separate groups, the ECs and the non-ECs. It will be intriguing
to see if viral control is lost through the accumulation of ad-
ditional mutations as the three macaques that control SIV
replication progress further into the chronic phase of infection.
Similar to those in HLA-B27 and HLA-B57-positive individ-
uals (6, 9, 38), the acute-phase CD8?T-cell responses in
Mamu-B*08-positive macaques were dominated by Mamu-
B*08-specific CD8?T-cell responses (Fig. 3 and Fig. 4). Sur-
prisingly, Mamu-B*08 appeared to reduce the dominant influ-
ence of Mamu-A*01 on CD8?T-cell responses. Previously,
such immunodomination was described to occur in macaques
expressing both Mamu-A*01 and Mamu-A*02 during the
acute phase of SIVmac251 infection (58). Typically, in Mamu-
A*01-positive macaques, the Mamu-A*01-restricted Tat28-35SL8-
and Gag181-189CM9-specific CD8?T cells account for ?50%
of the acute-phase SIV-specific immune responses (56). How-
ever, for both r91003 and r01027, the Mamu-A*01-restricted
contribution was below 50%. Responses restricted by Mamu-
A*02 in r00032 also appeared to be diminished in this Mamu-
B*08-positive macaque compared to those in Mamu-A*02-pos-
itive macaques that do not express Mamu-B*08. It should be
emphasized, however, that these data are derived from only
three animals and that larger cohorts of Mamu-B*08-positive
macaques expressing other MHC class I molecules should be
studied to clarify this issue.
HIV disease progression in HLA-B27-positive humans ap-
pears to be associated with viral escape from the immunodom-
inant Gag263-272KK10 response during the chronic phase of
infection (8, 20, 26, 36). However, additional HIV-specific
CD8?T-cell responses may be involved in the initial control of
viremia. Because immune responses in HIV-infected individ-
uals are normally characterized using IFN-? ELISPOT with
VOL. 82, 2008 Mamu-B*08 IMMUNODOMINANCE MAY INFLUENCE VIRAL CONTROL1735
consensus peptides, often after primary infection, definition of
the entire breadth of the HLA-B27-restricted CD8?T-cell
responses may be incomplete. While preliminary, our current
findings appear to indicate that the breadth of CD8?T-cell
responses restricted by protective MHC class I alleles, rather
than a single immunodominant response, may be important in
determining the control of replication of the AIDS virus.
Surprisingly, while Mamu-B*08 appears to bind similar pep-
tides to HLA-B27, an immunodominant CD8?T-cell response
directed against Gag has not been identified for Mamu-B*08.
Rather, Mamu-B*08 restricts robust CD8?T-cell responses
largely from Vif and Nef. Moreover, we are currently attempt-
ing to identify all of the SIV epitopes restricted by Mamu-
B*08. At this time, we have identified only a single Gag-specific
CD8?T-cell response, and it is both low frequency and rec-
ognized in only a few Mamu-B*08-positive macaques (J. T.
Loffredo et al., unpublished data). Hence, while CD8?T cells
targeting Gag have been shown to be extremely effective in
controlling immunodeficiency virus replication (7, 8, 20, 26, 35,
36, 39, 51, 70), it may be possible for individuals to control
viremia by directing responses against other proteins as well.
Mamu-B*08-positive, SIV-infected macaques may offer an in-
triguing model for studying such a mechanism. Interestingly,
both HLA-B27 and Mamu-B*08 present many epitopes con-
taining two N-terminal basic amino acids. These peptides are
relatively resistant to peptidase activity, and thus, those pep-
tides may be more stable, and this may result in more efficient
MHC class I antigen presentation (30).
Approximately 50% of Mamu-B*08-positive macaques
become ECs, controlling replication of the pathogenic
SIVmac239 isolate to ?1,000 vRNA copies/ml (49). Given that
the chronic-phase viral set points in 175 macaques that pro-
gressed to AIDS were ?500,000 vRNA copies/ml, and approx-
imately two-thirds of macaques die by 1 year postinfection
(40), this level of control is remarkable. However, not all
Mamu-B*08-positive macaques become ECs after SIVmac239
infection, and the immune system’s role in successful viral
containment remains difficult to define. Understanding how
some of these macaques become progressors or controllers
might give us key insights into how to make an effective HIV
vaccine. Here, we provide evidence from a small yet provoca-
tive study that the immunodominance and breadth of CD8?
T-cell responses restricted by protective MHC class I alleles
may facilitate the development of elite control. Further exper-
iments are needed to explore this intriguing idea, especially
given the fundamental implications that this might have for
We thank the MHC Genotyping Core at the WNPRC (William
Rehrauer, Chrystal Glidden, Gretta Borchardt, and Debi Fisk) for
genotyping our Indian rhesus macaques. We also gratefully acknowl-
edge Gnankang Napoe ´, Emma Gostick, and David A. Price for assis-
tance with the construction of MHC class I tetramers, Taeko Soma for
QPCR, Kim Weisgrau for technical assistance, and Alex Blasky for
assistance with DNA sequencing. Laura Valentine and Matthew Reyn-
olds provided helpful discussions. We also thank the Virology, Genet-
ics, Immunology, and Animal core laboratories as well as Research
Support Services at the National Primate Research Center, University
of Wisconsin—Madison (WNPRC) for technical assistance. The fol-
lowing reagents were obtained through the NIH AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID, NIH: com-
plete SIVmac239 peptide sets (15-mer peptides overlapping by 11
amino acids) of Gag (item no. 6204), Vif (item no. 6205), Tat (item no.
6207), Pol (item no. 6443), Rev (item no. 6448), Vpr (item no. 6449),
Vpx (item no. 6450), Env (item no. 6883), and full-length Nef (item no.
This research was supported by National Institutes of Health (NIH)
contract HHSN266200400088C and NIH grants R01 AI049120, R01
AI052056, R24 RR015371, and R24 RR016038 to D.I.W. as well as
R21 AI068586 to T.C.F. Additionally, this publication was made pos-
sible in part by grant number P51 RR000167 from the National Center
for Research Resources (NCRR), a component of the NIH, awarded
to the WNPRC. This project has also been funded in part by a grant
from the Japan Health Sciences Foundation to D.I.W. This work was
conducted in part at a facility constructed with support from Research
Facilities Improvement grant numbers RR15459-01 and RR020141-01
This publication’s contents are solely the responsibility of the au-
thors and do not necessarily represent the official views of the NCRR
or the NIH.
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