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Journal of Virology, July 1999, p. 5466-5472, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Use of Major Histocompatibility Complex Class I/Peptide/
2M
Tetramers To Quantitate CD8+ Cytotoxic T Lymphocytes
Specific for Dominant and Nondominant Viral Epitopes in
Simian-Human Immunodeficiency Virus-Infected Rhesus Monkeys
Michael A.
Egan,1,*
Marcelo J.
Kuroda,1
Gerald
Voss,1
Jörn E.
Schmitz,1
William A.
Charini,1
Carol I.
Lord,1
Meryl A.
Forman,2 and
Norman L.
Letvin1
Division of Viral Pathogenesis, Department of
Medicine, Beth Israel Deaconess Medical Center, Harvard Medical
School, Boston, Massachusetts 02215,1 and
Beckman Coulter, Inc., Miami, Florida 331162
Received 23 December 1998/Accepted 1 April 1999
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ABSTRACT |
To evaluate the impact of the diversity of antigen recognition by T
lymphocytes on disease pathogenesis, we must be able to identify and
analyze simultaneously cytotoxic T-lymphocyte (CTL) responses specific
for multiple viral epitopes. Many of the studies of the role of
CD8+ CTLs in AIDS pathogenesis have been done with simian
immunodeficiency virus (SIV)- and simian-human
immunodeficiency virus (SHIV)-infected rhesus monkeys. These studies
have frequently made use of the well-defined SIV Gag CTL
epitope p11C,C-M presented to CTL by the HLA-A homologue molecule
Mamu-A*01. In the present study we identified and fine mapped two novel
Mamu-A*01-restricted CTL epitopes: the SIVmac Pol-derived
epitope p68A (STPPLVRLV) and the human immunodeficiency
virus type 1 (HIV-1) Env-derived p41A epitope (YAPPISGQI).
The frequency of CD8+ CTLs specific for the p11C,C-M,
p68A, and p41A epitopes was quantitated in the same animals with a
panel of tetrameric Mamu-A*01/peptide/
2m complexes. All
SHIV-infected Mamu-A*01+ rhesus monkeys tested had a high
frequency of SIVmac Gag-specific CTLs to the p11C,C-M
epitope. In contrast, only a fraction of the monkeys tested had
detectable CTLs specific for the SIVmac Pol p68A and HIV-1
Env p41A epitopes, and these responses were detected at very low
frequencies. Thus, the p11C,C-M-specific CD8+ CTL response
is dominant and the p41A- and p68A-specific CD8+ CTL
responses are nondominant. These results indicate that CD8+
CTL responses to dominant CTL epitopes can be readily quantitated with the tetramer technology; however, CD8+ CTL responses
to nondominant epitopes, due to the low frequency of these
epitope-specific cells, may be difficult to detect and quantitate
by this approach.
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INTRODUCTION |
Although considerable data have
accrued in studies of AIDS pathogenesis supporting a role for
CD8+ cytotoxic T lymphocytes (CTL) in containing human
immunodeficiency virus type 1 (HIV-1) replication, the importance of
epitope specificity in antigen recognition by these cells remains
poorly defined. Containment of HIV-1 replication in vivo has been
correlated temporally with the generation of virus-specific CTL
(5, 10, 23). In addition, a potent CTL response in
chronically infected individuals is associated with low virus load and
a stable clinical status (18, 21). However, little is known
about the diversity of HIV-1 epitopes recognized by these CTL and
the ramifications of diverse antigen recognition for disease
pathogenesis. Some have suggested that the breadth of antigen
recognition by CD8+ CTL may have a significant impact on
success in controlling virus spread (23). To evaluate the
impact of the diversity of antigen recognition by T lymphocytes on
disease pathogenesis, we must be able to identify and analyze
simultaneously CTL responses specific for multiple viral epitopes.
Recently it has become possible to define with quantitative precision
distinct subpopulations of epitope-specific CD8+ CTL by
soluble major histocompatibility complex (MHC) class
I/peptide/beta-2-microglobulin (2m) tetrameric complexes and
flow cytometric analysis (2-4, 6, 8, 11, 17, 21). The
application of this technique has so far been used primarily for the
study of virus-specific CTL with specificity for dominant epitopes
presented to T cells by MHC class I molecules. The tetramer technique
for studying CTL should, however, be useful in evaluating CTL specific
for multiple epitopes of the same virus. This might be accomplished through the use of a panel of tetrameric MHC class I/peptide/2m complexes that define a variety of CTL epitopes restricted by either a single or multiple MHC class I molecules.
Simian immunodeficiency virus (SIVmac)- and simian-human
immunodeficiency virus (SHIV) infection-infected rhesus monkeys develop a disease with remarkable similarities to HIV-1-induced disease in
humans (13, 14, 24), providing powerful animal models in
which to study AIDS pathogenesis. The evaluation of rhesus monkey CTL
responses to SIVmac and SHIV has been facilitated by the use of
well-defined viral CTL epitopes and their restricting MHC class I
molecules. Specifically, SIVmac- and SHIV-specific CTL have been
evaluated with considerable sensitivity in these animal models through
the study of T-cell responses to the Gag epitope p11C,C-M presented
by the HLA-A homologue molecule Mamu-A*01 (1, 11, 15, 16).
The definition of additional CTL epitopes and the development of
tetrameric staining approaches for their evaluation would considerably
increase the utility of these animal models.
In the present study we have identified two additional
Mamu-A*01-restricted CTL epitopes in SHIV-infected rhesus monkeys. We have generated tetrameric Mamu-A*01/peptide/2m complexes that recognize CD8+ CTL specific for these epitopes and used
them to evaluate the breadth of the Mamu-A*01-restricted CTL response
in these monkeys.
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MATERIALS AND METHODS |
Animals.
Heparinized blood samples were obtained from rhesus
monkeys (Macaca mulatta) experimentally infected with
uncloned SIVmac strain 251, SHIV-89.6 (monkeys 206, 287, and
556), or SHIV-HXBc2 (monkeys L3 and L9). The experimental monkeys in
the present study were infected with SHIV or SIVmac 3 to 72 months
prior to their evaluation. The animals were maintained in accordance
with the guidelines of the Committee on Animals for the Harvard Medical School (Cambridge, Mass.) and the Guide for the Care and Use of Laboratory Animals (20).
Selection of Mamu-A*01+ rhesus monkeys.
Rhesus
monkeys were screened for the presence of the Mamu-A*01 allele by a
PCR-based technique as previously described (9). EDTA-preserved whole blood from rhesus macaques was subjected to Ficoll
diatrizoate density gradient centrifugation to isolate leukocytes, and
the washed cell pellets were resuspended in 200 µl of
phosphate-buffered saline. DNA extraction was then carried out with a
QIAmp blood kit (Qiagen Inc., Chatsworth, Calif.). PCR was performed on
200 to 500 µg of extracted DNA with allele-specific primers in a 50 µl reaction mixture consisting of 60 mM Tris (pH 8.5), 2 mM
MgCl2, 15 mM ammonium sulfate, 2 mM deoxynucleoside triphosphates (0.5 mM each), and 5 µl of Taq polymerase.
Primers A*01/F (5'-GAC AGC GAC GCC GCG AGC CAA-3') and A*01/R (5'-GCT GCA GCG TCT CCT TCC CC-3') were used at final concentrations of 800 nM
each. Two additional primers specific for a conserved MHC class II
sequence (based on the macaque homologue of HLA-DRB3) were included in
the reaction as internal positive controls. Primers 5' MDRB (5'-GCC TCG
AGT GTC CCC CCA GCA CGT TTC-3') and 3' MDRB (5'-GCA AGC TTT CAC CTC GCC
GCT G-3') were used at final concentrations of 680 nM each. PCR was
carried out with a GeneAmp System 9600 thermocycler (Perkin-Elmer Inc.,
Norwalk, Conn.). Samples were denatured at 96°C for 2 min followed by
5 cycles of 25 s at 96°C and 60 s at 72°C; 21 cycles of
25 s at 96°C, 50 s at 67°C, and 45 s at 72°C; and
4 cycles of 25 s at 96°C, 60 s at 55°C and 80 s at
72°C. The PCR products were analyzed by 1% agarose gel
electrophoresis. Ten microliters of each PCR reaction mixture was
loaded per lane.
Potential Mamu-A*01-positive animals were identified by the presence of
two bands, a 685-bp amplified product and a 260-bp band. DNA sequence
analysis was then performed on all potential positive samples to
confirm nucleotide sequence identity with the published Mamu-A*01
prototype sequence (16). Prior to being sequenced, the
amplified DNA was treated with 1 U per reaction of shrimp alkaline
phosphatase and 10 U of exonuclease I for 15 min at 37°C followed by
15 min at 80°C. The sequencing templates were then purified with a
QIAquick PCR purification kit (Qiagen, Inc.). For each template, 70 ng
of DNA was used for DNA sequencing together with 5 pmol of primer. Four
PCR primers were used for sequencing: A*01/F and A*01/R, whose
sequences are shown above, and B/1+ (5'-CTG CGC GGC TAC TAC AAC CA-3')
and G/1+ (5'-ATG TAA TCC TTG CCG TCG TA-3'). Sequencing was carried out
at a central core sequencing facility on an ABI-373 stretch
DNA-sequencing machine, using ABI AmpliTaq FS dye terminator chemistry
(Perkin-Elmer, Inc.). All animals used in this study were genotypically
Mamu-A*01 positive based on the above screening and were also Mamu-A*01 positive by functional CTL assay.
Peptide mapping of CTL epitopes.
The optimal
SIVmac Pol and HIV-1 Env peptides presented by
Mamu-A*01 to CTL were determined in functional effector cell assays. PBMC of Mamu-A*01+, infected rhesus monkeys were isolated
by centrifugation over Ficoll-Hypaque (Ficopaque; Pharmacia). The
PBMC were then specifically stimulated with antigen either by culture
with autologous B lymphoblastoid cell line (B-LCL) cells infected with
recombinant vaccinia virus or by the addition of 20- or 25-amino-acid
peptides. When using recombinant vaccinia virus-infected B-LCL cells as
stimulator cells, PBMC from infected, Mamu-A*01+ monkeys
were maintained at a density of 2 × 106/ml and
cocultured with an equal number of paraformaldehyde-fixed, autologous
B-LCL cells infected with a recombinant vaccinia virus expressing HIV-1
gp160 (v299). When long peptides were used as stimulating antigens,
PBMC from infected, Mamu-A*01+ monkeys were cultured with
50 µg of the indicated peptide (SIV Pol p68
[WQVTWIPEWDFISTPPLVRLVFNLV] or HIV-1 Env p41
[GKAMYAPPIEGQIRCSSNIT])/ml and maintained at a density of
5 × 106/ml. On day 3 of culture, the medium was
supplemented with human recombinant interleukin 2 (rIL-2) (20 U/ml;
provided by Hofmann-La Roche), and the cultures were maintained for an
additional 7 days. The cells were then centrifuged over Ficoll-Hypaque
and assessed as effectors in a standard 51Cr release assay
at an effector-to-target (E/T) ratio of 10:1 as described below. The
target cells were autologous B-LCL cells pulsed with decreasing
concentrations (10 to 0.01 ng/ml) of the indicated peptides.
Cytotoxicity assay.
PBMC from infected,
Mamu-A*01+ monkeys were cultured with 1.0 µg of the
indicated optimal peptide (p11C,C-M [CTPYDINQM], p68A [STPPLVRLV], p41A [YAPPISGQI]) and maintained
at a density of 2 × 106/ml. On day 3 of culture, the
medium was supplemented with human rIL-2 (20 U/ml; provided by
Hofmann-La Roche), and the cultures were maintained for an additional 7 days. The cells were then centrifuged over Ficoll-Hypaque and assessed
as effectors in a standard 51Cr release assay at an E/T
ratio of 10:1. The target cells were Mamu-A*01+ B-LCL cells
pulsed during overnight 51Cr labeling either with 1.0 µg
of the same peptide used to stimulate the cultured cells or with 1.0 µg of the control peptide p11B (ALSEGCTPYDIN) per ml. All
wells were assayed in quadruplicate. The plates were incubated for
5 h in a humidified incubator at 37°C. Specific release was
calculated as (experimental release 
spontaneous
release)/(maximum release spontaneous release) × 100.
Mamu-A*01/peptide/2m complex formation and staining of
peptide-specific CD8+ T lymphocytes.
Mamu-A*01/p11C,C-M/2m (SIVmac Gag),
Mamu-A*01/p68A/2m (SIVmac Pol), and Mamu-A*01/p41A/2m
(HIV-1 Env) complexes were prepared as previously described
(11). Phycoerythrin (PE)-labeled ExtrAvidin (Sigma) was
mixed stepwise with biotinylated Mamu-A*01/peptide complexes at a molar
ratio of 1:4 to produce the tetrameric complexes. All antibodies used
in this study were directly coupled to fluorescein isothiocyanate
(FITC), PE-Texas red (ECD), or allophycocyanin (APC). The following
monoclonal antibodies were used: anti-CD8
(Leu2a)-FITC (Becton
Dickinson), anti-CD8-ECD (Coulter), and anti-CD3-APC (FN18;
kindly provided by D. M. Neville, Jr., National Institutes of
Health, Bethesda, Md.).
The PE-coupled tetrameric Mamu-A*01/peptide/
2m complexes were used
in combination with anti-CD8
-FITC, anti-CD8
-ECD, and
anti-CD3-APC to stain 100 µl of fresh blood or 2 × 10
5 lymphocytes isolated by Ficoll-Hypaque density gradient
centrifugation
following in vitro peptide stimulation as previously
described
(
11). Whole-blood samples were lysed with an
Immunoprep reagent
system and a Q-prep workstation (Beckman Coulter
Inc.). Ten thousand
gated events were collected, and samples were
analyzed on a Coulter
EPICS Elite ESP flow cytometer. Data analysis was
performed with
the EPICS Elite software (version 4.02; Beckman Coulter
Inc.).
Data presentation was performed by using WINMDI software version
2.7 (Joseph Trotter, La Jolla, Calif.) and PowerPoint 97 (Microsoft,
Redmond, Wash.).
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RESULTS |
Identification of novel Mamu-A*01-restricted SIVmac and
SHIV CTL epitopes.
To increase the power of the rhesus
monkey model for exploring the role of CTL in AIDS
immunopathogenesis, we sought to identify Mamu-A*01-restricted
SIVmac- and SHIV-derived CTL epitopes in addition to the
Gag p11C,C-M peptide. PBMC from Mamu-A*01+, SHIV-infected
rhesus monkeys were stimulated in vitro with pools of overlapping 20- or 25-amino-acid peptides spanning the entire SIVmac Pol and
HIV-1 Env proteins. The peptide-stimulated cultures were then assessed
for CTL activity specific for autologous B-LCL cells pulsed with each
of the individual peptides contained within the pool of stimulating
peptides (data not shown). In studies of PBMC from three
Mamu-A*01+, SHIV-infected monkeys, potential
Mamu-A*01-restricted CTL epitopes were detected in an HIV-1 Env
peptide (p41 [GKAMYAPPIEGQIRCSSNIT]; amino acids 393 to
412) (19) and in a SIVmac Pol peptide (p68 [WQVTWIPEWDFISTPPLVRLVFNLV]; amino
acids 876 to 900) (7).
Identification of the optimal HIV-1 Env-derived
Mamu-A*01-restricted CTL epitope.
Since the peptide
binding motif for Mamu-A*01 has recently been shown to have a proline
residue at position 3 (1), we focused the fine mapping of
these epitopes on 8- to 10-amino-acid peptides within these
sequences that contain a proline at that position. For
characterization of the HIV-1 Env-derived epitope, two different experimental approaches were used to generate HIV-1 Env-specific effector cells. In the first approach, paraformaldehyde-fixed, autologous B-LCL cells infected with a recombinant vaccinia virus (vv299) expressing the entire HIV-1 gp160 were used to stimulate PBMC
from a SHIV-infected monkey. In the second approach, the 20-amino-acid
peptide p41 was used to stimulate PBMC from a SHIV-infected monkey.
Neither strategy should bias the specificity of the expanded effector
cell population, since both depend upon intracellular antigen
processing to generate the Mamu-A*01-binding peptide. HIV-1
Env-specific effector T cells generated by each of these approaches
were used in a standard 51Cr release assay to assess lysis
of autologous B-LCL cells pulsed with each of the peptides shown in
Fig. 1C. Effector cells generated by both
approaches exhibited preferential lysis of autologous B-LCL cells
pulsed with the nine-amino-acid peptide p41A (YAPPISGQI) (Fig. 1A and B).
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FIG. 1.
Mapping of the optimal Mamu-A*01-restricted HIV-1 Env
CTL epitope. (A) PBMC from a SHIV-HXBc2-infected rhesus monkey
(L28) were cultured for 10 days with paraformaldehyde-fixed, autologous
B-LCL cells infected with a vaccinia virus (vv299) expressing HIV-1
gp160. The effector cells were assayed at an E/T ratio of 10:1 with
autologous B-LCL targets cultured overnight with the indicated
synthetic peptides. (B) PBMC from a SHIV-HXBc2-infected rhesus
monkey (L3) were cultured for 10 days with the HIV-1 gp160
20-amino-acid p41 peptide (GKAMYAPPIEGQIRCSSNIT) at a final
concentration of 50 µg/ml. The effector cells were assayed at an E/T
ratio of 10:1 with Mamu-A*01+ B-LCL targets cultured
overnight with the indicated synthetic peptides. (C) Sequences of
p41-derived peptides with a proline (P) at position 3.
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Identification of the optimal SIVmac Pol-derived
Mamu-A*01-restricted CTL epitope.
To map the SIVmac
Pol-derived Mamu-A*01-restricted CTL epitope, PBMC from an
SIVmac-infected monkey were stimulated in vitro with the
25-amino-acid p68 peptide. The resulting effector cells were then used
to assess lysis of autologous B-LCL cells pulsed with a number of 8-, 9-, and 10-amino-acid peptides within the SIVmac Pol sequence
containing a proline at position 3 (Fig.
2B). The nine-amino-acid peptide p68A
(STPPLVRLV) was preferentially recognized by the
Pol-specific effector cells (Fig. 2A).
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FIG. 2.
Mapping of the optimal Mamu-A*01-restricted
SIVmac Pol CTL epitope. (A) PBMC from an
SIVmac-infected rhesus monkey (GL9) were cultured for 10 days
with 10 µg of the SIVmac Pol 25-amino-acid p68 peptide
(WQVTWIPEWDFISTPPLVRLVFNLV) per ml. The effector cells were assayed at
an E/T Ratio of 10:1 with autologous B-LCL cells cultured overnight
with the indicated synthetic peptides. (B) Sequences of p68-derived
peptides with a proline (P) at position 3.
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We then sought to confirm that p68A was the optimal
Mamu-A*01-restricted SIVmac Pol epitope by assessing
the interaction of
selected peptides with the Mamu-A*01
molecule. Fluorescent dye-coupled
tetrameric MHC class
I/peptide/
2m complexes have recently been
shown to bind
subpopulations of epitope-specific CD8
+ T cells,
allowing for flow cytometric analysis of epitope-specific
CTL.
Critical to the development of these tetrameric staining
reagents is the efficient in vitro folding of the soluble MHC
class I
monomers around specific peptides. The efficiency of this
in vitro
folding reaction can be monitored by assessing the conversion
of the
31-kDa MHC class I heavy chain and the 12-kDa
2m to a
43-kDa MHC
class I/peptide/
2m monomer (
11). Peptides that bind
with
high affinity to the MHC class I heavy chain should induce
efficient
folding of the MHC class I/peptide/
2m monomers. Peptides
that fail
to bind efficiently to the MHC class I heavy chain should
fail to
produce high-molecular-weight MHC class I/peptide/
2m
monomers.
To confirm the results of the functional SIVmac Pol peptide
fine-mapping experiment shown in Fig.
2, we initiated small-scale
in
vitro folding reactions to assess the ability of the two
nine-amino-acid
peptides p68A and p68B to induce folding of the
Mamu-A*01/
2m
complex. Formation of the folded 43-kDa
Mamu-A*01/peptide/
2m
complex was monitored by gel filtration. As
shown in Fig.
3A,
the peptide p68A bound
efficiently to Mamu-A*01, inducing the
formation of a 43-kDa
Mamu-A*01/peptide/
2m complex. In contrast
to this, the peptide
p68B failed to bind efficiently to Mamu-A*01
and failed to induce
folding of the 43-kDa Mamu-A*01/peptide/
2m
complex (Fig.
3B) above
that seen in the absence of exogenous
peptide (Fig.
3C).
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FIG. 3.
Folding of soluble Mamu-A*01 and human 2m around
SIVmac Pol-derived peptides in vitro. (A) Gel filtration
profile of soluble Mamu-A*01 monomers refolded with human 2m and the
SIVmac Pol-derived peptide p68A. (B) Gel filtration profile
of soluble Mamu-A*01 monomers refolded with human 2m and the
SIVmac Pol-derived peptide p68B. (C) Gel filtration profile
of soluble Mamu-A*01 monomers refolded with human 2m in the
absence of exogenous peptide.
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CTL specific for SIVmac Pol p68A and HIV-1 Env p41A
epitopes are detected in antigen-stimulated PBMC of some, but
not all, SIVmac- and SHIV-infected Mamu-A*01+ rhesus
monkeys.
To assess the utility of the newly defined epitopes
in monitoring CTL responses in SIVmac- and SHIV-infected,
Mamu-A*01+ rhesus monkeys, PBMC from chronically
infected animals were stimulated in vitro with 1.0 µg of the
optimal SIVmac Gag p11C,C-M, SIVmac Pol p68A, or HIV-1 Env p41A
peptide per ml for 10 days in the presence of rIL-2 and assessed for
antigen-specific CTL by two independent assays: the detection of MHC
class I/peptide/2m tetramers binding CD8+ T lymphocytes
and functional peptide-specific CTL activity (Table 1 and Fig.
4). In the PBMC of all SIVmac- and
SHIV-infected, Mamu-A*01+ monkeys tested, high numbers of
Mamu-A*01/p11C,C-M/2m tetramer-binding cells were detected following
in vitro peptide p11C,C-M stimulation; p11C,C-M-specific cells
constituted from 17.4 to 66.7% of the peptide-stimulated
CD8+ T cells. Consistent with the Mamu-A*01/p11C,C-M/2m
tetramer-binding data, high levels of functional p11C,C-M-specific CTL
activity were detected in PBMC of all SIVmac- and SHIV-infected,
Mamu-A*01+ monkeys tested.
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TABLE 1.
CTL specific for SIVmac Gag p11C,C-M,
SIVmac Pol p68A, and HIV-1 Env p41A epitopes detectable
in PBMC of SHIV- and SIVmac-infected, Mamu-A*01+
rhesus monkeys after in vitro culture
with peptidea
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FIG. 4.
Tetramer binding to peptide-stimulated PBMC from
SHIV- and SIVmac-infected, Mamu-A*01+ rhesus monkeys.
PBMC from SHIV-89.6-infected monkey 287 and
SIVmac-infected monkey 4DK were stimulated in vitro with 1.0 µg of the indicated optimal peptide (p11C, C-M [CTPYDINQM], p68A
[STPPLVRLV], or p41A [YAPPISGQI]) per ml for 10 days in
rIL-2-containing medium. The PE-coupled tetrameric
Mamu-A*01/peptide/2m complexes were used in combination with
anti-CD8-FITC, anti-CD8-ECD, and anti-CD3-APC to stain 2 × 105 lymphocytes isolated by Ficoll-Hypaque density
gradient centrifugation following this in vitro peptide stimulation.
The percentage of CD8+ T lymphocytes staining positively
with the Mamu-A*01/peptide/2m complex is indicated in the upper
right quadrant.
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Mamu-A*01/p41A/
2m tetramers binding CD8
+ T cells were
detected in all the SHIV-infected animals tested following in vitro
peptide p41A stimulation, with p41A-specific cells ranging from
0.7 to
6.6% of CD8
+ T cells. However, greater than 10%
functional Env-specific CTL
activity could only be detected in the PBMC
of three of five SHIV-infected
animals. Animals L3 and L9 were
chronically infected with SHIV-HXBc2,
which encodes the HIV-1 Env p41A
CTL epitope (YAPPISGQI) defined
in the studies shown in
Fig.
1. Animals 206, 287, and 556 were
chronically infected with
SHIV-89.6, which encodes a modified
HIV-1 Env p41A CTL
epitope (YAPPITGQI) containing an S-to-T amino
acid change at position 6. Despite this difference in the
HIV-1
Env CTL epitope, Mamu-A*01/p41A/
2m tetramer binding and
p41A-specific
CTL activity in the PBMC of these two groups of animals
did not
appear to differ
significantly.
Mamu-A*01/p68A/
2m tetramer-binding cells were detected in four
of five SHIV-infected animals and in all SIVmac-infected
animals
tested following in vitro peptide p68A stimulation.
In the SHIV-infected
animals, Mamu-A*01/p68A/
2m binding
cells ranged from 0.5 to 16.2%
of CD8
+ T cells, and in the
SIVmac-infected animals,
Mamu-A*01/p68A/
2m
binding cells ranged from 0.6 to
9.6% of CD8
+ T cells. Functional p68A-specific CTL were
detected in two of
four SHIV-infected animals staining positive with
the Mamu-A*01/p68A/
2m
tetramer and in three of five
SIVmac-infected
animals.
These results demonstrate that SIVmac Gag p11C,C-M-specific
CD8
+ CTL are readily detected in all infected
Mamu-A*01
+ monkeys, while CD8
+ CTL specific for
the SIVmac Pol and HIV-1 Env epitopes are present
at a
lower frequency and are detected in only a fraction of the
animals
tested.
SIVmac Pol p68A and HIV-1 Env p41A tetramer-binding cells
are only occasionally detected in freshly isolated PBMC of chronically
infected Mamu-A*01+ rhesus monkeys.
To explore further
the relative frequency of CTL specific for the Mamu-A*01-restricted
SIVmac Pol and HIV-1 Env epitopes, we assessed
freshly isolated whole blood of SIVmac- and SHIV-infected, Mamu-A*01+ monkeys for binding of the
SIVmac Gag p11C,C-M, SIVmac Pol p68A, and HIV-1 Env
p41A/Mamu-A*01 tetramers to CD8+ T cells (Table
2 and Fig.
5). As shown in Table 2,
Mamu-A*01/p11C,C-M/2m tetramer-binding CD8+ T
cells were readily detected in freshly isolated peripheral blood of all
SIVmac- and SHIV-infected animals at a frequency of 0.2 to
14.7% of CD8+ T cells. Mamu-A*01/p41A/2m
tetramer-binding CD8+ T cells were detected in freshly
isolated peripheral blood of two of five SHIV-infected animals at a
frequency of 0.1 to 0.3% of CD8+ T cells. The
Mamu-A*01/p41A/2m tetramer failed to bind to PBMC of the
SIVmac-infected animals, confirming the specificity of the
reagent. Mamu-A*01/p68A/2m tetramer-binding CD8+ T cells
were detected in freshly isolated peripheral blood of one of five
SHIV-infected monkeys at a frequency of 0.1% of CD8+ T
cells and one of six SIVmac-infected monkeys at a
frequency of 0.4% of CD8+ T cells. It is interesting to
note that the animals with measurable Mamu-A*01-p68A tetramer (287 and
4DK) and Mamu-A*01/p41A/2m tetramer (L3 and 287) staining in freshly
isolated PBMC were the animals with the highest level of tetramer
staining following in vitro peptide stimulation.
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TABLE 2.
Binding of freshly isolated CD8+ peripheral
blood T cells from Mamu-A*01+, infected rhesus monkeys
to Mamu-A*01-Gag, -Env, and -Pol peptide tetramers
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FIG. 5.
Tetramer binding to freshly isolated PBMC from
SHIV- and SIVmac-infected, Mamu-A*01+ rhesus
monkeys. Fresh blood (100 µl) from SHIV-HXBc2-infected monkey
L3 and SIVmac-infected monkey 4DK were stained with the
indicated Mamu-A*01/peptide/2m complex in combination with
anti-CD8-FITC, anti-CD8-ECD, and anti-CD3-APC. The percentage
of CD8+ T lymphocytes staining positively with the
Mamu-A*01/peptide/2m complex is indicated in the upper right
quadrant.
|
|
| |
DISCUSSION |
The study of CTL is greatly facilitated by the elucidation of CTL
epitopes and the MHC class I molecules that present these peptides
to effector lymphocytes. Antigen-specific CTL populations are readily
expanded in vitro by stimulation with epitope peptides, and target
cells pulsed with these peptides can be used in 51Cr
release assays to avoid the high levels of background lysis associated
with the use of virus-infected target cells. To expand the utility of
the SIV-macaque model for studies of AIDS pathogenesis and
vaccine development, we sought to identify Mamu-A*01-restricted SIVmac and SHIV CTL epitopes in rhesus monkeys in
addition to the previously reported SIVmac Gag p11C,C-M
epitope (1, 16). Using PBMC from Mamu-A*01+,
SHIV-infected rhesus monkeys stimulated in vitro with pools of
overlapping peptides spanning the entire SIVmac Pol and HIV-1 Env proteins, we identified and fine mapped two novel
Mamu-A*01-restricted CTL epitopes: the SIVmac Pol-derived
epitope p68A (STPPLVRLV) and the HIV-1 Env-derived p41A
epitope (YAPPISGQI).
Allen et al. recently defined a consensus motif for peptides that bind
to the rhesus monkey HLA-A homologue molecule Mamu-A*01 (1).
Sequence analysis of peptides eluted from purified Mamu-A*01 molecules
revealed an enrichment of the signal for a proline residue in position
3, suggesting that proline at the third position of the CTL epitope
is the anchor residue critical for the binding of a peptide to
Mamu-A*01. They also identified threonine (T) at position 2, proline
(P) at position 4, isoleucine (I) at position 6, asparagine (N) at
position 7, and glutamine (Q) at position 8 as possible auxiliary
anchor residues or preferred residues of Mamu-A*01-restricted CTL
epitopes. The definition of the novel Mamu-A*01-restricted
SIVmac Pol and HIV-1 Env epitopes in the present study
confirms the predicted importance of proline as the position 3 anchor
residue. Of the six residues reported in the consensus sequence by
Allen et al. (1), p11C,C-M shows sequence identity with
five. The SIVmac Pol p68A (threonine at P2, proline at P3,
and proline at P4) and HIV-1 Env p41A (proline at P3, proline at P4,
and glutamine at P8) each contain three residues with sequence identity
to the consensus sequence (Table 3).
There is reason to suppose that further epitope-mapping studies should allow the definition of Mamu-A*01-restricted CTL epitopes derived from SIVmac Env and viral auxiliary proteins.
While all the Mamu-A*01+ rhesus monkeys evaluated had
high-frequency SIVmac Gag p11C,C-M-specific CTL
responses, only a fraction of these monkeys had detectable
functional CTL specific for SIVmac Pol p68A and HIV-1 Env
p41A, and these CTL were detected at relatively low frequencies by
tetramer binding. These findings suggest that Gag p11C,C-M is a
dominant epitope and both SIVmac Pol p68A and HIV-1 Env
p41A are nondominant CTL epitopes. The reason for the difference in
the frequency of CD8+ CTL specific for these epitopes
is unclear. The ability of a viral peptide to elicit CTL is likely to
be influenced by a number of factors: (i) the peptide-MHC class I
affinity and rate of peptide dissociation, (ii) the amount of viral
antigen expressed and the amount of antigen that can enter the MHC
class I antigen processing pathway, (iii) the rate at which viral
antigen is degraded, and (iv) the efficiency and selectivity of
peptide transport into the endoplasmic reticulum by the TAP
transporter (27). Some nondominant CTL epitopes appear
to be naturally processed and presented but remain poorly immunogenic
because of their low MHC class I binding affinities (22, 25,
26). It has been demonstrated that highly immunogenic CTL
epitopes invariably exhibit MHC class I antigen binding affinities
of 50 nM or less, while poorly immunogenic CTL epitopes often have
MHC class I antigen binding affinities of 500 nM or more
(25). The SIVmac Gag p11C,C-M epitope has been
shown to bind to Mamu-A*01 with a 50% inhibitory concentration (IC50) of 4.3 nM (1), which is consistent with
this peptide's immunodominance. The fact that SIVmac Pol
p68A- and HIV-1 Env p41A-specific CTL can be readily detected in some
chronically infected, Mamu-A*01+ rhesus monkeys suggests
that these epitopes can be naturally processed.
In a few instances Mamu-A*01/p41A/2m and Mamu-A*01/p68A/2m
tetramer staining of CD8+ T cells was detected in the
absence of demonstrable functional peptide-specific CTL activity. In
other instances functional peptide-specific CTL were detected in PBMC
that demonstrated very low Mamu-A*01/peptide/2m tetramer binding. We
have seen a quantitative correlation between functional CTL activity
and the level of Mamu-A*01/peptide/2m tetramer-binding
CD8+ T cells only when greater than 5% of all
CD8+ T cells bind the tetramer. A correlation between
functional CTL activity and the level of Mamu-A*01/peptide/2m
tetramer binding has not been demonstrated when the tetramer positivity
is as low as has been seen for the nondominant HIV-1 Env p41A and
SIVmac Pol p68A epitopes. A failure to detect HIV-1 Env
p41A- and SIVmac Pol p68A-specific functional CTL activity
may be due to the inconsistency of functional CTL assays when low
numbers of specific effector cells are used. It is also possible that
an underlying abnormality in T-cell help in the infected monkeys
results in a loss of detectable functional activity when low
epitope-specific effector numbers are accessed.
Circulating Mamu-A*01/p11C,C-M/2m tetramer-binding CD8+
T cells in the chronically infected monkeys ranged from 0.2 to 3.8% of
all CD8+ CD3+ cells, with one monkey as high as
14.7%. The level of Mamu-A*01/p11C,C-M/2m tetramer-binding
CD8+ T cells in these chronically infected animals remained
relatively stable during the course of their evaluation (data not
shown). In the setting of primary SIVmac infection, we have
recently demonstrated that tetramer-binding CD8+
CD3+ cells specific for the dominant p11C,C-M epitope
appeared as early as day 11 postinfection, peaked on day 13 at 1.3 to 8.3%, and declined coincident with the fall in virus load
(12). We do not yet know whether the kinetics of the
emergence of the CTL response or its anatomic compartmentalization
differs for CTL that recognize the dominant and nondominant epitopes.
Through the use of soluble MHC class I/peptide/2m tetrameric
complexes and flow cytometric analysis, it has become possible to
define with quantitative precision distinct subpopulations of
epitope-specific CD8+ CTL (2-4, 6, 8, 11, 17,
21). In the rhesus monkey model, the application of this
technique has so far been restricted to the study of CTL with
specificity for a single dominant epitope. In the current study,
soluble MHC class I/peptide/2m complexes were developed for multiple
epitopes of the same virus presented to CTL by the same MHC class I
molecule. This has allowed us to analyze the CD8+ CTL
responses to both dominant and nondominant epitopes. The results indicate that CD8+ CTL responses to dominant CTL
epitopes can be easily quantitated with this technology. However,
CD8+ CTL responses to nondominant epitopes, due to the
low frequency of these epitope-specific cells, may be difficult to
quantitate with the tetramer technology.
| |
ACKNOWLEDGMENT |
This work was supported by National Institutes of Health grants
AI42301, AI35166, and AI20729.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess
Medical Center, P.O. Box 15732, Boston, Massachusetts 02215. Phone: (617) 667-0526. Fax: (617) 667-8210. E-mail:
michael_egan{at}caregroup.harvard.edu.
| |
REFERENCES |
| 1.
|
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 simian immunodeficiency virus.
J. Immunol.
160:6062-6071[Abstract/Free Full Text].
|
| 2.
|
Altman, J. D.,
P. A. H. Moss,
P. J. R. Goulder,
D. H. Barouch,
M. G. McHeyzer-Williams,
J. I. Bell,
A. J. McMichael, and M. M. Davis.
1996.
Phenotypic analysis of antigen-specific T lymphocytes.
Science
274:94-96[Abstract/Free Full Text].
|
| 3.
|
Busch, D. H.,
I. M. Pilip,
S. Vijh, and E. G. Pamer.
1998.
Coordinate regulation of complex T cell populations responding to bacterial infection.
Immunity
8:353-362[Medline].
|
| 4.
|
Callan, M. F.,
L. Tan,
N. Annels,
G. S. Ogg,
J. D. Wilson,
C. A. O'Callaghan,
N. Steven,
A. J. McMichael, and A. B. Rickinson.
1998.
Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus in vivo.
J. Exp. Med.
187:1395-1402[Abstract/Free Full Text].
|
| 5.
|
Chen, Z. W.,
Z. C. Kou,
C. Lekutis,
L. Shen,
D. Zhou,
M. Halloran,
J. Li,
J. Sodroski,
D. Lee-Parritz, and N. L. Letvin.
1995.
T cell receptor V beta repertoire in an acute infection of rhesus monkeys with simian immunodeficiency viruses and a chimeric simian-human immunodeficiency virus.
J. Exp. Med.
182:21-31[Abstract/Free Full Text].
|
| 6.
|
Flynn, K. J.,
G. T. Belz,
J. D. Altman,
R. Ahmed,
D. L. Woodland, and P. C. Doherty.
1998.
Virus-specific CD8+ T cells in primary and secondary influenza pneumonia.
Immunity
8:683-691[Medline].
|
| 7.
|
Franchini, G.,
C. Gurgo,
H. G. Guo,
R. C. Gallo,
E. Collalti,
K. A. Fargnoli,
L. F. Hall,
F. Wong-Staal, and M. S. Reitz, Jr.
1987.
Sequence of simian immunodeficiency virus and its relationship to the human immunodeficiency viruses.
Nature
328:539-543[Medline].
|
| 8.
|
Gallimore, A.,
A. Glithero,
A. Godkin,
A. C. Tissot,
A. Pluckthun,
T. Elliott,
H. Hengartner, and R. Zinkernagel.
1998.
Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes.
J. Exp. Med.
187:1383-1393[Abstract/Free Full Text].
|
| 9.
|
Knapp, L. A.,
E. Lehmann,
M. S. Piekarczyk,
J. A. Urvater, and D. I. Watkins.
1997.
A high frequency of Mamu-A*01 in the rhesus macaque detected by polymerase chain reaction with sequence-specific primers and direct sequencing.
Tissue Antigens.
50:657-661[Medline].
|
| 10.
|
Koup, R. A.,
J. T. Safrit,
Y. Cao,
C. A. Andrews,
G. McLeod,
W. Borkowsky,
C. Farthing, and D. D. Ho.
1994.
Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome.
J. Virol.
68:4650-4655[Abstract/Free Full Text].
|
| 11.
|
Kuroda, M. J.,
J. E. Schmitz,
D. H. Barouch,
A. Craiu,
T. M. Allen,
A. Sette,
D. I. Watkins,
M. A. Forman, and N. L. Letvin.
1998.
Analysis of Gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex.
J. Exp. Med.
187:1373-1381[Abstract/Free Full Text].
|
| 12.
|
Kuroda, M. J.,
J. E. Schmitz,
W. A. Charini,
C. E. Nickerson,
M. A. Lifton,
C. I. Lord,
M. A. Forman, and N. L. Letvin.
1999.
Emergence of CTL coincides with clearance of virus during primary simian immunodeficiency virus infection in rhesus monkeys.
J. Immunol.
162:5127-5133[Abstract/Free Full Text].
|
| 13.
|
Letvin, N. L.,
M. D. Daniel,
P. K. Sehgal,
R. C. Desrosiers,
R. D. Hunt,
L. M. Waldron,
J. J. MacKey,
D. K. Schmidt,
L. V. Chalifoux, and N. W. King.
1985.
Induction of AIDS-like disease in macaque monkeys with T-cell tropic retrovirus STLV-III.
Science
230:71-73[Abstract/Free Full Text].
|
| 14.
|
Letvin, N. L., and N. W. King.
1990.
Immunologic and pathologic manifestations of the infection of rhesus monkeys with simian immunodeficiency virus of macaques.
J. Acquired Immune Defic. Syndr.
3:1023-1040.
|
| 15.
|
Miller, M. D.,
C. I. Lord,
V. Stallard,
G. P. Mazzara, and N. L. Letvin.
1990.
The gag-specific cytotoxic T lymphocytes in rhesus monkeys infected with the simian immunodeficiency virus of macaques.
J. Immunol.
144:122-128[Abstract].
|
| 16.
|
Miller, M. D.,
H. Yamamoto,
A. L. Hughes,
D. I. Watkins, and N. L. Letvin.
1991.
Definition of an epitope and MHC class I molecule recognized by gag-specific cytotoxic T lymphocytes in SIVmac-infected rhesus monkeys.
J. Immunol.
147:320-329[Abstract].
|
| 17.
|
Murali-Krishna, K.,
J. D. Altman,
M. Suresh,
D. J. Sourdive,
A. J. Zajac,
J. D. Miller,
J. Slansky, and R. Ahmed.
1998.
Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection.
Immunity
8:177-187[Medline].
|
| 18.
|
Musey, L.,
J. Hughes,
T. Schacker,
T. Shea,
L. Corey, and M. J. McElrath.
1997.
Cytotoxic-T-cell responses, viral load, and disease progression in early human immunodeficiency virus type 1 infection.
N. Engl. J. Med.
337:1267-1274[Abstract/Free Full Text].
|
| 19.
|
Myers, G.,
B. Korber,
S. Wain-Hobson,
R. F. Smith, and G. N. Pavlakis.
1993.
Human retroviruses and AIDS 1993: a compilation and analysis of nucleic acid and amino acid sequences.
Los Alamos National Laboratory, Los Alamos, N. Mex.
|
| 20.
|
National Research Council.
1996.
Guide for the care and use of laboratory animals.
National Academic Press, Washington, D.C.
|
| 21.
|
Ogg, G. S.,
X. Jin,
S. Bonhoeffer,
P. R. Dunbar,
M. A. Nowak,
S. Monard,
J. P. Segal,
Y. Cao,
S. L. Rowland-Jones,
V. Cerundolo,
A. Hurley,
M. Markowitz,
D. D. Ho,
D. F. Nixon, and A. J. McMichael.
1998.
Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA.
Science
279:2103-2106[Abstract/Free Full Text].
|
| 22.
|
Oukka, M.,
N. Riche, and K. Kosmatopoulos.
1994.
A nonimmunodominant nucleoprotein-derived peptide is presented by influenza A virus-infected H-2b cells.
J. Immunol.
152:4843-4851[Abstract].
|
| 23.
|
Pantaleo, G.,
J. F. Demarest,
H. Soudeyns,
C. Graziosi,
F. Denis,
J. W. Adelsberger,
P. Borrow,
M. S. Saag,
G. M. Shaw,
R. P. Sekaly, et al.
1994.
Major expansion of CD8+ T cells with a predominant V beta usage during the primary immune response to HIV.
Nature
370:463-467[Medline].
|
| 24.
|
Reimann, K. A.,
K. Tenner-Racz,
P. Racz,
D. C. Montefiori,
Y. Yasutomi,
W. Lin,
B. J. Ransil, and N. L. Letvin.
1994.
Immunopathogenic events in acute infection of rhesus monkeys with simian immunodeficiency virus of macaques.
J. Virol.
68:2362-2370[Abstract/Free Full Text].
|
| 25.
|
Sette, A.,
A. Vitiello,
B. Reherman,
P. Fowler,
R. Nayersina,
W. M. Kast,
C. J. Melief,
C. Oseroff,
L. Yuan,
J. Ruppert,
J. Sidney,
M. F. del Guerico,
S. Southwood,
R. T. Kubo,
R. W. Chesnut,
H. M. Grey, and F. V. Chisari.
1994.
The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes.
J. Immunol.
153:5586-5592[Abstract].
|
| 26.
|
van der Burg, S. H.,
M. J. Visseren,
R. M. Brandt,
W. M. Kast, and C. J. Melief.
1996.
Immunogenicity of peptides bound to MHC class I molecules depends on the MHC-peptide complex stability.
J. Immunol.
156:3308-3314[Abstract].
|
| 27.
|
York, I. A., and K. L. Rock.
1996.
Antigen processing and presentation by the class I major histocompatibility complex.
Annu. Rev. Immunol.
14:369-396[Medline].
|
Journal of Virology, July 1999, p. 5466-5472, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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-
Santra, S., Schmitz, J. E., Kuroda, M. J., Lifton, M. A., Nickerson, C. E., Lord, C. I., Pal, R., Franchini, G., Letvin, N. L.
(2002). Recombinant Canarypox Vaccine-Elicited CTL Specific for Dominant and Subdominant Simian Immunodeficiency Virus Epitopes in Rhesus Monkeys. J. Immunol.
168: 1847-1853
[Abstract]
[Full Text]
-
Dzuris, J. L., Sidney, J., Horton, H., Correa, R., Carter, D., Chesnut, R. W., Watkins, D. I., Sette, A.
(2001). Molecular Determinants of Peptide Binding to Two Common Rhesus Macaque Major Histocompatibility Complex Class II Molecules. J. Virol.
75: 10958-10968
[Abstract]
[Full Text]
-
Veazey, R. S., Gauduin, M.-C., Mansfield, K. G., Tham, I. C., Altman, J. D., Lifson, J. D., Lackner, A. A., Johnson, R. P.
(2001). Emergence and Kinetics of Simian Immunodeficiency Virus-Specific CD8+ T Cells in the Intestines of Macaques during Primary Infection. J. Virol.
75: 10515-10519
[Abstract]
[Full Text]
-
Maile, R., Wang, B., Schooler, W., Meyer, A., Collins, E. J., Frelinger, J. A.
(2001). Antigen-Specific Modulation of an Immune Response by In Vivo Administration of Soluble MHC Class I Tetramers. J. Immunol.
167: 3708-3714
[Abstract]
[Full Text]
-
Millrain, M., Chandler, P., Dazzi, F., Scott, D., Simpson, E., Dyson, P. J.
(2001). Examination of HY Response: T Cell Expansion, Immunodominance, and Cross-Priming Revealed by HY Tetramer Analysis. J. Immunol.
167: 3756-3764
[Abstract]
[Full Text]
-
Barouch, D. H., Santra, S., Kuroda, M. J., Schmitz, J. E., Plishka, R., Buckler-White, A., Gaitan, A. E., Zin, R., Nam, J.-H., Wyatt, L. S., Lifton, M. A., Nickerson, C. E., Moss, B., Montefiori, D. C., Hirsch, V. M., Letvin, N. L.
(2001). Reduction of Simian-Human Immunodeficiency Virus 89.6P Viremia in Rhesus Monkeys by Recombinant Modified Vaccinia Virus Ankara Vaccination. J. Virol.
75: 5151-5158
[Abstract]
[Full Text]
-
Barouch, D. H., Craiu, A., Santra, S., Egan, M. A., Schmitz, J. E., Kuroda, M. J., Fu, T.-M., Nam, J.-H., Wyatt, L. S., Lifton, M. A., Krivulka, G. R., Nickerson, C. E., Lord, C. I., Moss, B., Lewis, M. G., Hirsch, V. M., Shiver, J. W., Letvin, N. L.
(2001). Elicitation of High-Frequency Cytotoxic T-Lymphocyte Responses against both Dominant and Subdominant Simian-Human Immunodeficiency Virus Epitopes by DNA Vaccination of Rhesus Monkeys. J. Virol.
75: 2462-2467
[Abstract]
[Full Text]
-
Allen, T. M., Mothé, B. R., Sidney, J., Jing, P., Dzuris, J. L., Liebl, M. E., Vogel, T. U., O'Connor, D. H., Wang, X., Wussow, M. C., Thomson, J. A., Altman, J. D., Watkins, D. I., Sette, A.
(2001). CD8+ Lymphocytes from Simian Immunodeficiency Virus-Infected Rhesus Macaques Recognize 14 Different Epitopes Bound by the Major Histocompatibility Complex Class I Molecule Mamu-A*01: Implications for Vaccine Design and Testing. J. Virol.
75: 738-749
[Abstract]
[Full Text]
-
Barouch, D. H., Santra, S., Schmitz, J. E., Kuroda, M. J., Fu, T.-M., Wagner, W., Bilska, M., Craiu, A., Zheng, X. X., Krivulka, G. R., Beaudry, K., Lifton, M. A., Nickerson, C. E., Trigona, W. L., Punt, K., Freed, D. C., Guan, L., Dubey, S., Casimiro, D., Simon, A., Davies, M.-E., Chastain, M., Strom, T. B., Gelman, R. S., Montefiori, D. C., Lewis, M. G., Emini, E. A., Shiver, J. W., Letvin, N. L.
(2000). Control of Viremia and Prevention of Clinical AIDS in Rhesus Monkeys by Cytokine-Augmented DNA Vaccination. Science
290: 486-492
[Abstract]
[Full Text]
-
Kuroda, M. J., Schmitz, J. E., Lekutis, C., Nickerson, C. E., Lifton, M. A., Franchini, G., Harouse, J. M., Cheng-Mayer, C., Letvin, N. L.
(2000). Human Immunodeficiency Virus Type 1 Envelope Epitope-Specific CD4+ T Lymphocytes in Simian/Human Immunodeficiency Virus-Infected and Vaccinated Rhesus Monkeys Detected Using a Peptide-Major Histocompatibility Complex Class II Tetramer. J. Virol.
74: 8751-8756
[Abstract]
[Full Text]
-
Evans, D. T., Jing, P., Allen, T. M., O'Connor, D. H., Horton, H., Venham, J. E., Piekarczyk, M., Dzuris, J., Dykhuzen, M., Mitchen, J., Rudersdorf, R. A., Pauza, C. D., Sette, A., Bontrop, R. E., DeMars, R., Watkins, D. I.
(2000). Definition of Five New Simian Immunodeficiency Virus Cytotoxic T-Lymphocyte Epitopes and Their Restricting Major Histocompatibility Complex Class I Molecules: Evidence for an Influence on Disease Progression. J. Virol.
74: 7400-7410
[Abstract]
[Full Text]
-
Egan, M. A., Charini, W. A., Kuroda, M. J., Schmitz, J. E., Racz, P., Tenner-Racz, K., Manson, K., Wyand, M., Lifton, M. A., Nickerson, C. E., Fu, T., Shiver, J. W., Letvin, N. L.
(2000). Simian Immunodeficiency Virus (SIV) gag DNA-Vaccinated Rhesus Monkeys Develop Secondary Cytotoxic T-Lymphocyte Responses and Control Viral Replication after Pathogenic SIV Infection. J. Virol.
74: 7485-7495
[Abstract]
[Full Text]
-
Skinner, P. J., Daniels, M. A., Schmidt, C. S., Jameson, S. C., Haase, A. T.
(2000). Cutting Edge: In Situ Tetramer Staining of Antigen-Specific T Cells in Tissues. J. Immunol.
165: 613-617
[Abstract]
[Full Text]
-
Chen, Z. W., Craiu, A., Shen, L., Kuroda, M. J., Iroku, U. C., Watkins, D. I., Voss, G., Letvin, N. L.
(2000). Simian Immunodeficiency Virus Evades a Dominant Epitope-Specific Cytotoxic T Lymphocyte Response Through a Mutation Resulting in the Accelerated Dissociation of Viral Peptide and MHC Class I. J. Immunol.
164: 6474-6479
[Abstract]
[Full Text]
-
Allen, T. M., Vogel, T. U., Fuller, D. H., Mothe, B. R., Steffen, S., Boyson, J. E., Shipley, T., Fuller, J., Hanke, T., Sette, A., Altman, J. D., Moss, B., McMichael, A. J., Watkins, D. I.
(2000). Induction of AIDS Virus-Specific CTL Activity in Fresh, Unstimulated Peripheral Blood Lymphocytes from Rhesus Macaques Vaccinated with a DNA Prime/Modified Vaccinia Virus Ankara Boost Regimen. J. Immunol.
164: 4968-4978
[Abstract]
[Full Text]
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Abstract
Introduction
