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*
Wisconsin Regional Primate Research Center and
Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, WI 53715;
PowderJect, Inc., Madison, WI 53711;
§
Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom;
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Epimmune, Inc., San Diego, CA 92121;
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Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322; and
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Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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M
(CTPYDINQM)-specific CD8+ T lymphocyte responses in six of
six Mamu-A*01+ rhesus macaques. Tetramer
staining of fresh, unstimulated PBMC from two of the DNA-vaccinated
animals indicated that as much as 0.4% of all
CD3+/CD8
+ T lymphocytes were specific for
the SIVgag CTL epitope. Administration of MVA expressing the SIVgag CTL
epitope further boosted these responses, such that 0.8–20.0% of
CD3+/CD8+ T lymphocytes in fresh,
unstimulated PBMC were now Ag specific. Enzyme-linked immunospot assays
confirmed this high frequency of Ag-specific cells, and intracellular
IFN-
staining demonstrated that the majority of these cells produced
IFN- after peptide stimulation. Moreover, direct ex vivo
SIV-specific cytotoxic activity could be detected in PBMC from five of
the six DNA/MVA-vaccinated animals, indicating that this epitope-based
DNA prime/MVA boost regimen represents a potent method for inducing
high levels of functionally active, Ag-specific CD8+ T
lymphocytes in non-human primates. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD8+ T cell responses play a key role in the containment of lentivirus infections. Strong CTL responses have been found to correlate with reduced plasma RNA viral loads in HIV-infected individuals (3). It has also been demonstrated that CTL exert selective pressure on the AIDS virus populations, as evidenced by the eventual predominance of viruses with amino acid replacements in those regions of the virus to which CTL responses are directed (4, 5, 6, 7, 8, 9, 10). Adoptive transfer of autologous HIVgag-specific CD8+ CTL clones to three seropositive patients was capable of rapidly decreasing the percentage of productively infected CD4+ T cells (11). Similarly, SIV-infected macaques depleted of CD8+ T lymphocytes were subsequently unable to control virus replication (12, 13, 14). Together, these studies strongly support a role for CD8+ T cells in controlling AIDS virus infections and emphasize the importance of HIV vaccines to induce strong CD8+ T cell responses.
In comparing the various vaccine approaches designed to induce CD8+ T cell responses, epitope-based vaccines offer several advantages over vaccines encoding whole protein Ags. Not only are epitope-based vaccines capable of inducing more potent responses than whole protein vaccines (15), they confer the capacity to control qualitative aspects of the immune response by simultaneously targeting multiple dominant and subdominant epitopes (16, 17). This may be particularly important to the development of HIV vaccines, because the breadth of an immune response is likely to be crucial for controlling rapidly mutating pathogens such as HIV and hepatitis C virus (18, 19, 20, 21). The use of epitopes can also overcome any potential safety concerns associated with the vaccinating Ag, as exemplified in the case of the human papillomavirus E6 and E7 Ags, whose expression is clearly associated with cervical carcinoma (22, 23).
DNA vaccines are receiving considerable attention for their ability to induce cellular immune responses. They have induced immune responses to and in some cases even protected against various pathogens, such as influenza and malaria (24, 25, 26, 27). Although DNA vaccines encoding whole protein have effectively induced immunodeficiency virus-specific cellular immune responses in non-human primates (28, 29, 30, 31, 32, 33), they have been unable to reliably protect against vigorous AIDS virus challenges. This lack of protection, however, may be due to the inability of previous DNA vaccine regimens to induce potent enough cellular immune responses against HIV and SIV.
Current approaches to vaccine design have begun to take advantage of the synergistic effects of combining DNA vaccines with other approaches to induce stronger, more persistent cellular immune responses (34, 35, 36, 37, 38, 39). Recently, live pox viruses, such as modified vaccinia virus Ankara (MVA),4 have proven to be safe and effective vaccine vectors for inducing strong cellular immune responses (40, 41, 42, 43, 44). Combining a DNA prime with an MVA boost has generated strong protective CTL responses against malaria in mice (74). More importantly, these murine studies demonstrated that the combination of DNA and MVA induced significantly higher levels of epitope-specific immune responses than either method alone (38).
Extrapolation of the results of the immunogenicity of potential
vaccines in mice to primates is not always possible. Accordingly, in
our studies we have determined whether a combination DNA/MVA
vaccination regimen using a single CTL epitope can induce
virus-specific CTL in non-human primates. We have used the
Mamu-A*014-restricted SIV gag CTL epitope p11C,
CM (CTPYDINQM) (45, 46) as an immunogen to induce
AIDS virus-specific CTL responses. Using tetramers, ELISPOT,
intracellular IFN- staining, and fresh killing assays, we describe
the capability of an epitope-based DNA/MVA vaccine to induce in rhesus
macaques levels of functionally active epitope-specific
CD8+ T lymphocytes equivalent to those observed
during acute SIV infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rhesus macaques used in this study were identified as Mamu-A*01+ by PCR-sequence-specific priming and direct sequencing as previously described (47). Animals were maintained in accordance with the National Institutes of Health Guide to the Care and Use of Laboratory Animals and under the approval of the University of Wisconsin Research Animal Resource Center review committee.
DNA vectors pTH.HW and HC,CM
The pTH.HW vector was derived from the pTH.H vector
(48), which encoded for a polyepitope of 20 HIV and 3 SIV
CTL epitopes, including the Mamu-A*01-restricted CTL epitope
(TPYDINQML) (49). Following construction of the pTH.H
plasmid, the Mamu-A*01 CTL epitope was optimally defined and was found
to require an N-terminal cysteine residue (46), now termed
p11C, CM (50). The pTH.H vector was modified to include
the cysteine residue of this CTL epitope and was renamed pTH.HW
(51). The hepatitis B core Ag vector WRG7262 (PowderJect
Vaccines, Madison, WI) was modified to include the Mamu-A*01 CTL
epitope CTPYDINQM (HC,CM) within the immunodominant loop of the core
protein between aa 80 and 81.
DNA vaccinations
Frequencies of the DNA vaccinations using the Dermal PowderJect
XR gene gun (PowderJect Vaccines) are outlined in Fig. 1
. Each immunization delivered 32 µg of
DNA, precipitated on gold beads, unilaterally over a total of eight
skin sites in the abdominal and inguinal lymph node areas at 350 or 500
psi. The first vaccination delivered to each of the six animals
employed only the pTH.HW plasmid, while all subsequent booster
vaccinations involved codelivery of equal molar amounts of both the
pTH.HW and HC,CM vectors.
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The six DNA-vaccinated rhesus macaques were inoculated with
attenuated MVA encoding the same HIV/SIV polyepitope HW (MVA.HW) used
in the pTH.HW vector (37, 44, 48). Each vaccination
consisted of 5 x 108 PFU of MVA.HW in a
volume of 150 µl of PBS delivered intradermally and dispersed over
three chest sites on each animal. Animals 95045 and 95058 received
their MVA inoculations 9 and 13 wk after their last DNA vaccination,
animal 96031 received its inoculations 2 and 6 wk after its last DNA
vaccination, and animals 94004, 96123, and 96118 received their
inoculations 18 and 22 wk after their last DNA vaccination (Fig. 1
).
The MVA.HW was cultured on primary chick embryo fibroblasts derived
from eggs of a pathogen-free stock and prepared as previously described
(37). No lesions were found associated with the
inoculations.
Isolation of PBMC
PBMC were isolated from EDTA-treated whole blood using Ficoll/diatrioate gradient centrifugation. Cells were then washed twice in R10 medium (RPMI 1640 supplemented with penicillin (50 U/ml), streptomycin (50 µg/ml), L-glutamine (2 mM), and 10% FBS (BioCell, Carson, CA)).
Generation of in vitro cultured CTL effector cells
PBMC were isolated from EDTA blood drawn 1, 2, or 4 wk after
each DNA vaccination, and experiments were conducted using freshly
isolated PBMC unless otherwise stated. CTL cultures were initiated by
culturing 5 x 106 PBMC in R10 with peptide
(10 µM). On day 3 half of the medium was replaced with R10 medium
containing 20 U of rIL-2/ml (provided by M. Gately, Hoffmann-La
Roche, Nutley, NJ). Medium was supplemented every other day with rIL-2
until day 7, when cells were stimulated with 5 x
106 peptide-pulsed -irradiated (3000 rad)
autologous PBMC. Again, rIL-2 was added every 2 days until day 14 when
the CTL activity of the cultures was assessed in a standard
51Cr release assay. Peptides were obtained from
Biosynthesis (Lexisville, TX) as desalted products. Lyophilized
aliquots were resuspended in HBSS with 10% DMSO (Sigma) to a final
concentration of 1 mg/ml.
Cytotoxicity assays
The CTL activity of in vitro stimulated CTL cultures was assessed as previously described (46). Briefly, 5 x 105 Mamu-A*01-transfected 721.221 cells (46) or B-LCLs derived from a Mamu-A*01+ rhesus macaque were incubated for 1.5 h with 80 µCi of Na251CrO4 (New England Nuclear Life Sciences Products, Boston, MA) and 5 µg of corresponding peptide in 200 µl of R10 medium. These target cells were plated into duplicate wells of a 96-well U-bottom microtiter plate (5 x 103 cells/well) and incubated with effector CTL for 5 h. The reported percent specific lysis represents 51Cr release from CTPYDINQM peptide-pulsed targets minus 51Cr release from target cells pulsed with an irrelevant SIVnef peptide (NQGQYMNTPR). Spontaneous release was always <20% of maximal release. Data reported for in vitro stimulated CTL cultures are based on single CTL assays tested at an E:T cell ratio of 20:1 unless otherwise noted. No appreciable difference in lysis was observed between either Mamu-A*01+ B-LCL or Mamu-A*01-721.221 cells as targets in 51Cr release assays.
The CTL activity of freshly isolated PBMC was assessed in a similar manner as for in vitro stimulated CTL cultures with the following exceptions: 1) target cells were Mamu-A*01+ B-LCLs; 2) triplicate rather than duplicate wells were plated; 3) the irrelevant peptide (SNEGSYFF) used in these experiments was derived from the influenza virus nucleoprotein; and 4) data reported for fresh PBMCs were based on single CTL assays tested at E:T cell ratios of 150:1 and 50:1.
Mamu-A*01/CTPYDINQM tetramers
Soluble tetrameric Mamu-A*01 MHC class I/SIVgag CTPYDINQM peptide complexes were constructed as previously described (52) with the exception of the PCR primers required for amplification of the soluble MHC molecule. Primers Mamu-A*01–5p (5'-GGA ATT CCA TAT GGG ATC TCA TTC AAT GAA ATA TTT CTA CAC CTC CAT G-3') and Mamu-A*01–3p (5'-CGC GGA TCC GGA CTG GGA AAA CGG CTC-3') were designed to amplify the Mamu-A*01 heavy chain from a pKG5 vector containing a cDNA for the rhesus MHC class I molecule Mamu-A*01. Once the PCR products were cloned into an expression vector using NdeI and BamHI cloning sites (52), the rhesus MHC molecules were expressed and then folded with human ß2m and peptide.
Tetramer staining
Lymphocytes (2 x 105) from 2-wk in
vitro CTL cultures were stained in the dark for 30 min at room
temperature with the Mamu-A*01-PE tetramer (0.5 µg/100 µl for in
vitro cultures, 0.1 µg/100 µl for fresh PBMC) and an
anti-rhesus CD3-FITC-conjugated mAb (10 µl; BioSource, Camarillo,
CA) in a 100-µl volume of FACS buffer (PBS from Life Technologies
with 2% FCS from BioCell). To cool down the cells quickly to 4°C,
the plates were placed at -20°C for 5 min. An anti-CD8-PECy5
Ab (1 µl; Coulter, Hialeah, FL) was added for 10 min at 4°C, and
the cells were washed three times with FACS buffer and fixed with 450
µl of 2% paraformaldehyde (PFA).
For staining of fresh, unstimulated PBMC, 1 x
106 PBMC were stained as described for in vitro
CTL cultures, except that cells were incubated with the
anti-CD3-FITC mAb, anti-CD8-PECy5 mAb, and Mamu-A*01-PE
tetramer simultaneously for 40 min at room temperature. Sample data
were acquired on a Becton Dickinson FACSCalibur instrument and analyzed
using CellQuest software (Becton Dickinson Immunocytometry Systems, San
Jose, CA). Background tetramer staining of in vitro stimulated cultures
from naive Mamu-A*01+ animals was routinely
<0.2% and <0.08% for fresh, unstimulated PBMC (data not shown).
IFN- ELISPOT assay
Ninety-six-well flat-bottom plates (U-Cytech, Utrecht, The
Netherlands) were coated with 5 µg/well of anti-IFN- mAb MD-1
(U-Cytech) overnight at 4°C. The plates were washed 10 times with
PBST (PBS containing 0.05% Tween 20) and blocked with PBS containing
2% BSA for 1 h at 37°C. PBS containing 2% BSA was discarded
from the plates, and freshly isolated PBMC in R5 medium were added. The
R5 contained 5 µg/ml Con A (Sigma, St. Louis, MO), 1 µM p11C, CM
peptide, 1 µM of an irrelevant SIVenv peptide (ELGDYKLV), or no
peptide. PBMC (5 x 104, 2.5 x
104, 1.2 x 104, and
6 x 103) were plated in triplicate (100
µl/well) and incubated for 14 h at 37°C in 5%
CO2. The cells were then removed, and 200
µl/well of ice-cold deionized water was added to lyse the remaining
PBMC. Plates were incubated on ice for 15 min and washed 20 times with
PBST. One microgram per well of rabbit polyclonal biotinylated
anti-IFN- detector Ab (U-Cytech) was added, and plates were
incubated for 1 h at 37°C before being washed 10 times with
PBST. Fifty microliters per well of a gold-labeled anti-biotin IgG
(U-Cytech) was added for 1 h at 37°C and washed 10 times
with PBST. Thirty microliters per well of activator mix (U-Cytech) was
then added and developed for 30 min to allow formation of silver salt
precipitate at the site of gold clusters. Wells were then washed with
distilled water and air-dried, and spots were counted
(53).
Intracellular IFN- staining
Freshly isolated PBMC (1 x 106) were
incubated at 37°C for 1 h with 50 ng/ml PMA and 1 µg/ml
ionomycin, 5 µM p11C, CM peptide-pulsed
Mamu-A*01+ B-LCL, or
Mamu-A*01+ B-LCL alone as a control. Cells were
treated with 10 µg/ml of brefeldin A (BFA) for 4–5 h at 37°C to
inhibit export of protein from the endoplasmic reticulum. Cells were
washed twice with FACS buffer (PBS and 2% FCS), stained with
CD8-PerCP (Becton Dickinson) and Mamu-A*01-PE tetramers as described
above, fixed with PFA overnight, and washed twice with FACS buffer. The
cells were then treated with 150 µl of permeablization buffer (0.1%
saponin in FACS buffer) for 5 min at room temperature, washed once with
0.1% saponin, and incubated in the dark with 1 µl of anti-human
IFN--FITC mAb (PharMingen, San Diego, CA) for 50 min. Cells were
washed three times with 0.1% saponin buffer and once with PBS before
the 100-µl cell suspension was fixed with 450 µl of 2%
PFA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To demonstrate that a CTL response can be induced in primates
using experimental vaccines encoding well-characterized CTL epitopes,
we used the PowderJect XR gene delivery system (PowderJect Vaccines) to
vaccinate Mamu-A*01+ rhesus macaques with the
pTH.HW vector encoding for the Mamu-A*01-restricted SIVgag CTL epitope
p11C, CM (46) (Fig. 1). Chest and inguinal lymph node
regions provided a sufficiently large region of skin to vaccinate. Two
and 4 wk following the first vaccination PBMC were isolated, cultured
for 2 wk in vitro with the p11C, CM peptide, and analyzed for CTL
responses using standard 51Cr release assays at
an E:T cell ratio of 20:1. A p11C, CM-specific CTL response was
detected in one of the six vaccinated animals (animal 94004; 17%
specific lysis) from PBMC taken 2 wk after the first vaccination (Fig. 2A), and in three animals
(animals 96031, 96123, and 94004; 13, 19, and 19% specific lysis,
respectively) from PBMC taken 4 wk after the first vaccination (data
not shown). Responses from cultured PBMC taken before DNA vaccination
were all <5% specific lysis (Fig. 2A).
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+ T
lymphocytes in each culture that expressed TCRs specific for the p11C,
CM peptide/Mamu-A*01 complex. Staining of the 2-wk in vitro cultures
with these tetramers revealed that four of the animals (95058, 96031,
96123, and 94004) were responding with levels of tetramer-positive
cells between 3 and 8% (Fig. 2B). These responses were well
above the levels of 0.2% or less detected in cultured PBMC derived
from each animal before DNA vaccination (Fig. 2B) or the
background responses of less than 0.2% detected in cultured PBMC from
three naive Mamu-A*01+ macaques (data not shown).
Similar analysis of the 4-wk in vitro cultures revealed that all six
animals had responded to the vaccination (tetramer levels between 1 and
9%; data not shown). Taken together, the 51Cr
release assays and tetramer staining of in vitro cultures revealed that
all six DNA-vaccinated macaques were generating low, but detectable,
p11C, CM-specific responses after a single DNA vaccination. Booster DNA vaccinations enhanced CD8+ T cell responses
Two to four booster vaccinations codelivering the pTH.HW and an
additional plasmid (HC,CM) were then administered. The HC,CM
plasmid expresses a highly immunogenic hepatitis B core Ag (HBcAg)
shown to augment the cellular immune responses to inserted T cell
epitopes (54, 55, 56, 57). Initially, for the first two animals
(95045 and 95058) a schedule of booster vaccinations with substantial
resting periods was chosen based on previous studies in primates in
which Ab responses were significantly enhanced by lengthening the
resting periods between DNA vaccinations (58). When
vaccinations were begun in the second set of four animals, the resting
periods between vaccinations was shortened because the beneficial
effects of the resting periods was believed to be less critical to the
induction of a strong cellular immune response than to a strong humoral
response. PBMC were isolated 2 wk after each DNA vaccination, and in
vitro stimulated cultures were initiated. The administration of booster
DNA vaccinations effectively increased responses in each of the six
vaccinated macaques as measured by 51Cr release
assays (Fig. 2A) and tetramer staining (Fig. 2B).
Following the third or fourth booster vaccination, levels of p11C,
CM-specific lysis in in vitro stimulated cultures were elevated in
some animals to levels >30%. Levels of tetramer-positive
CD3+/CD8+ T lymphocytes
were also increased to >20% in some cultures. In the majority of
cultures tested these two independent measurements did roughly
correspond. Following the fourth and fifth vaccinations of some animals
(96118, 96123, and 94004), the responses appeared to plateau and even
decline. This may be a consequence of the reduced resting period
between DNA vaccinations in this group of three animals
(58). Nevertheless, booster DNA vaccinations were capable
of enhancing the levels of p11C, CM-specific
CD8+ T cell responses in all six vaccinated
macaques.
Tetramer-positive cells detected in fresh, unstimulated PBMC after DNA vaccination
Due to the strong p11C, CM-specific T cell responses observed
in in vitro stimulated cultures, we reasoned that the frequency of
p11C, CM-specific CD8+ T lymphocytes might be
high enough to detect low levels of tetramer-positive cells in fresh,
unstimulated PBMC. To follow the time course of Ag-specific
CD8+ T lymphocyte induction in DNA-vaccinated
animals, fresh unstimulated PBMC from animals 96118, 96123, and 94004
were stained 2, 4, 6, and 13 days following their fifth DNA
vaccination. Levels of tetramer-positive
CD3+/CD8+ T cells peaked
6 days after the DNA vaccination and declined by day 13 (data not
shown). When the percentages of tetramer-positive cells in unstimulated
PBMC were followed in all six macaques after their last two DNA
vaccinations, two of the six animals showed levels of p11C,
CM-specific CD3+/CD8+
T lymphocytes as high as 0.4% (Fig. 3
and Table I). This DNA vaccine regimen,
therefore, was able to induce levels of p11C, CM-specific
CD8+ T lymphocytes high enough to be detected in
fresh, unstimulated PBMC of some animals using tetramers.
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Because MVA has proven very effective at both inducing and
boosting CTL responses in mice and primates (37, 42, 51),
we were interested in assessing the effects of MVA boosting the six
DNA-vaccinated macaques. One week after administering MVA.HW fresh
unstimulated, Ficoll-separated PBMC were assessed for the percentage of
CD3+/CD8+ T lymphocytes
which were specific for the p11C, CM epitope. Tetramer analysis
revealed that 0.8, 18.0, 8.3, 1.2, 1.6, and 20.0% of the
CD3+/CD8+ T lymphocytes
from these six macaques were specific for the p11C, CM epitope (Fig. 4). These levels represented a
significant increase over the 0.4% levels detected weeks earlier in
some of the Mamu-A*01+ macaques after DNA
vaccination (Fig. 3 and Table I). Responses were then followed over the
subsequent 3 wk and were observed to decline to levels <1% in the
majority of animals. Interestingly, in animals 95058 and 94004, which
had demonstrated exceptionally high responses 1 wk post-MVA (18 and
20% respectively), levels were maintained above 8 and 2%,
respectively, after 2 wk. Responses in animal 95058 were maintained at
this high level up to 4 wk (Fig. 4).
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A second administration of MVA.HW was given to all six macaques 4
wk after receiving their first MVA.HW. As before, responses peaked 1 wk
after this second MVA.HW and increased over resting levels in the
majority of animals (Fig. 4). Only in animal 95045 were stronger
responses induced by this additional MVA.HW than had been induced by
the first MVA.HW. In the three macaques examined, 96118, 96123, and
94004, responses again declined by the second week. Therefore, while a
second administration of MVA.HW was capable of boosting levels of
Ag-specific responses, these levels did not generally exceed those
induced after the first MVA.HW. Longer resting periods between MVA
vaccinations, as used in other studies (42), may have
allowed for better induction by the second MVA.
Frequency of p11C, CM-specific IFN--producing T cells
determined by ELISPOT
The ELISPOT assay represents an effective method for measuring the
frequency of Ag-specific IFN--producing T cells in the circulation
(59, 60, 61). Fresh, unstimulated PBMC were tested at 1 and 2
wk following administration of their first MVA.HW with Con A, the p11C,
CM peptide, an irrelevant SIV envelope peptide (ELGDYKLV), or no
peptide. ELISPOT indicated that 1 wk following the first MVA.HW
vaccination, strong Ag-specific responses were observed in three of the
DNA/MVA-vaccinated animals tested. Animals 96118 and 96123 had 92 p11C,
CM-specific SFCs/50,000 input cells (effector frequency 1,
840/106 PBMC) and 44 p11C, CM-specific
SFCs/50,000 input cells (effector frequency,
880/106 PBMC), respectively (Fig. 5A). SFC responses in each of
the animals titrated according to the number of input cells per well
and were similar to values induced by Con A stimulation. Animal 94004,
with 20.0% tetramer-positive cells, was capable of demonstrating a
better response to the p11C, CM peptide, 206 SFCs/50,000 cells
(effector frequency, 4,120/106 PBMC), than to Con
A (effector frequency, 1,940/106 PBMC). None of
the animals demonstrated significant responses to the irrelevant
peptide or to wells without any peptide even at 50,000 cells/well (less
than five spots per well).
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B). Although the levels of tetramer-positive cells
had declined slightly in animals 96118 and 96123, the number of
IFN--producing cells detected in each of these animals at this 2 wk
point were now reduced by almost 90%. This may suggest that by 2 wk,
in the setting of declining levels of Ag, in addition to being deleted
some of these Ag-specific cells also lost their ability to produce
IFN- in response to the p11C,CM epitope. In 94004, the drop in
tetramer-positive cells by 2 wk post-MVA.HW was reflected by a more
comparable decline in IFN--producing cells. Nonetheless, detectable
levels of IFN--producing cells were still present 2 wk after
administration of MVA.HW. Further analysis conducted 21 wk after the
first MVA.HW indicated that positive ELISPOT responses were still
detectable in 96118, 96123, and 94004 at effector frequencies of
125/106, 35/106, and
230/106 PBMC, respectively (data not shown).
Tetramer-positive lymphocytes produce IFN- in response to
antigenic stimulation
Given the surprisingly high levels of Ag-specific lymphocytes
detected by the tetramer and ELISPOT assays, we used an additional
assay to confirm these results. Fresh PBMC from animals 95045, 95058,
and 96031 taken 1 wk after the second MVA.HW and fresh PBMC from a
naive Mamu-A*01+ animal (95084) were incubated
with BFA in the absence or the presence of mitogenic or Ag-specific
stimulation and stained for CD8, Mamu-A*01/p11C, CM tetramers,
and IFN-. In the absence of stimulation no IFN- was produced
(Fig. 6A). As a control,
treatment of cells with mitogens PMA/ionomycin induced the production
of IFN- in tetramer-positive as well as tetramer-negative cells
(Fig. 6B). Treatment of Ag-specific cells with the cognate
peptide has been shown to internalize the TCRs on the cell surface
(62). Because the cells have been treated with BFA, egress
of newly synthesized TCRs to the cell surface is prevented, abolishing
tetramer staining. Treatment of PBMC from each of the vaccinated
animals with the p11C, CM peptide resulted in a reduction in the
fraction of tetramer-positive cells as expected (Fig. 6C).
However, the fraction of cells that demonstrated the production of
intracellular IFN- after p11C, CM peptide stimulation was nearly
equivalent to the fraction of untreated cells that was previously
tetramer positive (Fig. 6A). Treatment of fresh PBMC from
these animals with an irrelevant peptide induced no internalization of
TCR and little or no production of IFN- (data not shown). These
results indicate that the majority of tetramer-positive
CD8+ cells in these DNA/MVA-vaccinated macaques
are functionally active and capable of responding specifically to the
p11C, CM peptide through the production of intracellular
IFN-.
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Because we induced high levels of peptide p11C, CM-specific
CD8+ T lymphocytes in our DNA/MVA-vaccinated
animals as detected by tetramer assays (Fig. 4), and those cells
produced IFN- upon activation (Figs. 5 and 6), we were interested in
determining whether we could detect direct ex vivo CTL activity from
fresh, unstimulated PBMC. Normally, CTL activity from PBMC of
vaccinated macaques, and even in chronically SIV infected animals, is
only detectable after in vitro stimulation with peptide. PBMC were
tested in 51Cr release assays at E:T cell ratios
of 150:1 and 50:1 against target B-LCLs pulsed with either peptide
p11C, CM or an irrelevant peptide. Although these experiments used
unconventionally high E:T ratios, similar ratios were required to
detect ex vivo CTL activity in intestinal intraepithelial lymphocytes
of SIV-infected macaques (63), as well as in PBMC of
HIV-infected patients (64, 65).
Thawed PBMC from animals 95045, 95058, and 96031 taken 1 wk after their
first MVA.HW, and fresh PBMC from a naive
Mamu-A*01+ macaque 96078, were initially tested.
The percent specific lysis from thawed PBMC of animals 95045 and 96031
at the higher E:T ratio were low, 5 and 10%, respectively, over
background levels in the control animal (Fig. 7). 95058, however, which had 18.0%
tetramer-positive
CD3+/CD8+ T cells at
this time point, demonstrated a more significant level of 15% specific
lysis. These levels of specific lysis were reproducible in a replicate
experiment (data not shown).
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). Nonetheless, significant responses were still
detectable in PBMC from animal 94004 (27% and 18%, respectively, at
E:T cell ratios of 150:1 and 50:1), compared with a background level of
<10% lysis in the control animal. Therefore, not only was this
epitope-based DNA/MVA vaccine capable of inducing high levels of
Ag-specific CD8+ T cells, the levels of induced
CTL were sufficient to exhibit cytolytic activity in fresh,
unstimulated PBMC in the majority of animals. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M-specific
CD8+ T cell responses in six
Mamu-A*01+ rhesus macaques. Tetramer staining of
fresh, unstimulated PBMC revealed that some DNA-vaccinated animals had
levels of epitope-specific
CD3+/CD8+ T lymphocytes
as high as 0.4%. Boosting DNA-vaccinated animals with recombinant MVA
expanded the percentage of epitope-specific
CD3+/CD8+ T lymphocytes
in fresh PBMC to levels between 1.2 and 20%. ELISPOT and intracellular
IFN- staining indicated that these CD8+ cells
were functionally active. Furthermore, in five of six
DNA/MVA-vaccinated animals the levels of Ag-specific T lymphocytes were
sufficiently high to detect, for the first time, direct ex vivo
vaccine-induced cytotoxic activity in non-human primates. The levels of
Ag-specific CD8+ T lymphocytes induced by this
epitope-based DNA/MVA vaccination regimen are equivalent to those
observed in acutely SIV-infected rhesus macaques (66).
Before the advent of tetramers, estimation of precursor CTL levels was
typically determined by limiting dilution analysis (LDA) assays. LDA
typically underestimates the levels of Ag-specific
CD8+ T cells by 10- to 100-fold compared with
tetramer or ELISPOT analysis (67, 68, 69). Corrected LDA
values for SIV-specific CD8+ T lymphocytes from
rhesus macaques immunized with a vaccinia virus-based subunit vaccine
(70) or immune-stimulating complexes (iscoms)
(71) suggest that these vaccines were capable of inducing
between 100 and 2000 protein-specific CD8+ T
lymphocytes/106 PBMC. In our study, tetramer
staining indicated that 0.4% of
CD3+/CD8+ T cells from
two of our DNA-vaccinated animals were specific for a single CTL
epitope, which would extrapolate to 1000/106
PBMC. Therefore, the levels of Ag-specific responses induced by the DNA
vaccinations in this study were equivalent to some previously published
levels of SIV-specific CTL induced in non-human primates.
The induction of p11C, CM-specific CD8+
responses in DNA/MVA-vaccinated macaques has also recently been
reported by Hanke et al. (38, 51). In these experiments,
tetramer staining of 1.3, 4.9, and 1.5% of
CD8+ T lymphocytes in frozen PBMC were induced
in three Mamu-A*01+ macaques following a
regimen of two DNA and two MVA vaccinations. In our
DNA/MVA-vaccinated animals, responses following the first MVA
(1.2–20.0%) were in most cases substantially higher. Hanke et al.
(51) also reported no significant lytic activity in 2-wk
in vitro stimulated cultures derived from PBMC of the DNA-vaccinated
macaques compared with this study in which responses were detected
after a single DNA vaccination. These differences were probably due to
the vaccine regimens, which in our experiments delivered greater
amounts of DNA and included a second DNA vector. This additional
hepatitis B core Ag vector was included to provide Th cell responses
during the DNA vaccination. In some animals, responses to the hepatitis
B core Ag appeared to correlate with the induction of good CTL
responses (data not shown). Therefore, the more effective priming of
CTL responses by our DNA vaccinations may account for the higher levels
of p11C, CM-specific CD8+ cells induced after
the MVA boost. The induction of p11C, CM-specific responses has also
recently been reported in Mamu-A*01+ macaques
vaccinated with MVA.HW alone (51) and MVA expressing
gag/pol of SIV (42). Tetramer staining levels in these
animals ranged between 1 and 5% of
CD3+/CD8ß+ cells. These
responses, however, were not detectable until after the second MVA
administration compared with only a single MVA in our study and those
of Hanke et al. (51), where a DNA prime was included,
illustrating the beneficial effects of priming with DNA.
Tetramer analysis of p11C, CM-specific T lymphocytes in
Mamu-A*01+ macaques chronically infected with SIV
indicates that between 0.7 and 10.3% of
CD3+/CD8+ T cells are
epitope specific (50, 66). The tetramer levels detected in
our DNA/MVA-vaccinated animals (1.2–20.0%) were equivalent to and in
some cases even greater than these levels in SIV-infected macaques.
In this study, the levels of Ag-specific cells measured in in vitro stimulated cultures using tetramers and 51Cr release assays were not always in complete agreement. Such discrepancies have been observed in other studies (50, 51, 72), although the reason for these differences is not understood. A similar discordance was also observed in this study when unstimulated PBMC were analyzed, suggesting that this phenomenon is not unique to in vitro stimulated cultures.
Although MHC class I tetramers are effective at determining the levels
of Ag-specific CD8+ T lymphocytes in fresh PBMC,
they do not reveal the functional state of the cells. In this study the
ability to measure p11C, CM-specific induction of IFN- in fresh
PBMC by intracellular IFN- staining was critical to assessing the
functionality of the vaccine-induced CD8+ T
lymphocytes. This analysis, which was not undertaken in other rhesus
macaque vaccination studies in which high levels of tetramer-positive
cells were induced (42, 51), revealed that the majority of
tetramer-positive cells were functionally active.
Comparisons of the number of p11C, CM-specific cells detected by
ELISPOT and tetramer staining revealed that ELISPOT generally
underestimated levels by between 1.3- and 10-fold. This discrepancy was
not due to these cells being nonfunctional as verified by intracellular
IFN- staining. Furthermore, because the tetramer staining of the
fresh, unstimulated PBMC was conducted at room temperature rather than
at 4°C, which has been observed to allow for binding of tetramers to
CTLs with minimal avidity (73), it is unlikely that this
discrepancy was due to an overestimation by the tetramers. Rather, the
1 µM concentration of peptide used in our ELISPOT experiments may
have been insufficient to induce the maximal number of
Ag-specific SFCs as has been observed in other studies (Ref.
60).5 It is noteworthy that the results from
the fresh killing assays in animals 96118, 96123, and 94004 examined 1
wk after the first MVA.HW corresponded much better with the ELISPOT
values than the tetramer values at this time point. This was especially
evident with animal 96118, which while possessing the lowest levels of
tetramer-positive cells (1.2%), possessed intermediate levels of SFCs
(92/50,000 cells) in ELISPOT and intermediate levels of specific lysis
(36% at an E:T cell ratio of 150:1) in fresh killing assays. While the
reasons for these discrepancies were not addressed by this study, these
results nonetheless suggest that analyses in addition to tetramer
staining, i.e., intracellular cytokine staining, ELISPOT, and fresh
killing assays, may prove crucial in our understanding of the role of
these vaccine-induced cells in viral containment. To this end, in a
similar study in which DNA/MVA-vaccinated animals were challenged with
SIV (51), these complementary assays were not undertaken,
and there was little or no correlation between the levels of
tetramer-positive cells and the containment of viral loads.
Although HIV- and SIV-specific CTL activity has been detected in fresh, unstimulated PBMC of seropositive humans (64, 65), and small intestine intraepithelial lymphocytes of chronically SIV-infected macaques (63), such responses have never been detected in the PBMC of vaccinated or SIV-infected macaques. Detectable levels of fresh killing were present in five of our six DNA/MVA-vaccinated animals, which serves to illustrate the potency of this vaccine regimen.
Whether vaccine-induced CD8+ T cell responses can control HIV and SIV infections remains unknown. Careful attention to the strength and breadth of a vaccine-induced CD8+ T cell response and proper assessment of these responses using assays that measure the levels of functionally active lymphocytes will be critical to the development of an effective HIV vaccine. The ability of this epitope-based DNA/MVA regimen to induce high levels of functionally active Ag-specific CTL against a single CTL epitope in non-human primates represents a first step toward addressing this issue. These findings now facilitate the immunizing of rhesus macaques with multiple CTL epitopes (both immunodominant and subdominant) to explore the role of vaccine-induced CD8+ T cell responses in controlling virus replication after SIV challenge.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Todd M. Allen, Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, WI 53715.
3 Current address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138.
4 Abbreviations used in this paper: MVA, modified vaccinia virus Ankara; Mamu, Macaca mulatta; ELISPOT, enzyme-linked immunospot; B-LCL, B lymphoblastoid cell line; PFA, paraformaldehyde; LDA, limiting dilution analysis; BFA, brefeldin A.
5 T. M. Allen, B. R. Mothe, J. Sidney, P. Jing, J. L. Dzuris, M. E. Liebl, T. U. Vogel, D. H. O’Connor, X. Wang, M. C. Wussow, J. A. Thomson, J. D. Altman, D. I. Watkins, and A. Sette, Submitted for publication.
Received for publication July 28, 1999. Accepted for publication February 23, 2000.
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production using an ELISPOT assay. J. Immunol. Methods 144:203.[Medline]
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|
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|
|
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|
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V. Novitsky, H. Cao, N. Rybak, P. Gilbert, M. F. McLane, S. Gaolekwe, T. Peter, I. Thior, T. Ndung'u, R. Marlink, et al. Magnitude and Frequency of Cytotoxic T-Lymphocyte Responses: Identification of Immunodominant Regions of Human Immunodeficiency Virus Type 1 Subtype C J. Virol., September 11, 2002; 76(20): 10155 - 10168. [Abstract] [Full Text] [PDF]
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|
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|
|
T. M. Allen, P. Jing, B. Calore, H. Horton, D. H. O'Connor, T. Hanke, M. Piekarczyk, R. Ruddersdorf, B. R. Mothe, C. Emerson, et al. Effects of Cytotoxic T Lymphocytes (CTL) Directed against a Single Simian Immunodeficiency Virus (SIV) Gag CTL Epitope on the Course of SIVmac239 Infection J. Virol., September 11, 2002; 76(20): 10507 - 10511. [Abstract] [Full Text] [PDF]
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|
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|
|
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|
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H. Horton, T. U. Vogel, D. K. Carter, K. Vielhuber, D. H. Fuller, T. Shipley, J. T. Fuller, K. J. Kunstman, G. Sutter, D. C. Montefiori, et al. Immunization of Rhesus Macaques with a DNA Prime/Modified Vaccinia Virus Ankara Boost Regimen Induces Broad Simian Immunodeficiency Virus (SIV)-Specific T-Cell Responses and Reduces Initial Viral Replication but Does Not Prevent Disease Progression following Challenge with Pathogenic SIVmac239 J. Virol., June 14, 2002; 76(14): 7187 - 7202. [Abstract] [Full Text] [PDF]
|
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|
|
R. R. Amara, J. M. Smith, S. I. Staprans, D. C. Montefiori, F. Villinger, J. D. Altman, S. P. O'Neil, N. L. Kozyr, Y. Xu, L. S. Wyatt, et al. Critical Role for Env as well as Gag-Pol in Control of a Simian-Human Immunodeficiency Virus 89.6P Challenge by a DNA Prime/Recombinant Modified Vaccinia Virus Ankara Vaccine J. Virol., May 13, 2002; 76(12): 6138 - 6146. [Abstract] [Full Text] [PDF]
|
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S. Santra, D. H. Barouch, M. J. Kuroda, J. E. Schmitz, G. R. Krivulka, K. Beaudry, C. I. Lord, M. A. Lifton, L. S. Wyatt, B. Moss, et al. Prior Vaccination Increases the Epitopic Breadth of the Cytotoxic T-Lymphocyte Response That Evolves in Rhesus Monkeys following a Simian-Human Immunodeficiency Virus Infection J. Virol., May 13, 2002; 76(12): 6376 - 6381. [Abstract] [Full Text] [PDF]
|
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T. M. Allen, L. Mortara, B. R. Mothe, M. Liebl, P. Jing, B. Calore, M. Piekarczyk, R. Ruddersdorf, D. H. O'Connor, X. Wang, et al. Tat-Vaccinated Macaques Do Not Control Simian Immunodeficiency Virus SIVmac239 Replication J. Virol., March 19, 2002; 76(8): 4108 - 4112. [Abstract] [Full Text] [PDF]
|
|
|
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E. G.-T. Wee, S. Patel, A. J. McMichael, and T. Hanke A DNA/MVA-based candidate human immunodeficiency virus vaccine for Kenya induces multi-specific T cell responses in rhesus macaques J. Gen. Virol., January 1, 2002; 83(1): 75 - 80. [Abstract] [Full Text] [PDF]
|
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Z. Hel, W.-P. Tsai, A. Thornton, J. Nacsa, L. Giuliani, E. Tryniszewska, M. Poudyal, D. Venzon, X. Wang, J. Altman, et al. Potentiation of Simian Immunodeficiency Virus (SIV)-Specific CD4+ and CD8+ T Cell Responses by a DNA-SIV and NYVAC-SIV Prime/Boost Regimen J. Immunol., December 15, 2001; 167(12): 7180 - 7191. [Abstract] [Full Text] [PDF]
|
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 |
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 W. S. Meng, L. H. Butterfield, A. Ribas, V. B. Dissette, J. B. Heller, G. A. Miranda, J. A. Glaspy, W. H. McBride, and J. S. Economou {alpha}-Fetoprotein-specific Tumor Immunity Induced by Plasmid Prime-Adenovirus Boost Genetic Vaccination Cancer Res., December 1, 2001; 61(24): 8782 - 8786. [Abstract] [Full Text] [PDF]
|
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R. S. Veazey, M.-C. Gauduin, K. G. Mansfield, I. C. Tham, J. D. Altman, J. D. Lifson, A. A. Lackner, and R. P. Johnson Emergence and Kinetics of Simian Immunodeficiency Virus-Specific CD8+ T Cells in the Intestines of Macaques during Primary Infection J. Virol., November 1, 2001; 75(21): 10515 - 10519. [Abstract] [Full Text] [PDF]
|
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 G. Ada Vaccines and Vaccination N. Engl. J. Med., October 4, 2001; 345(14): 1042 - 1053. [Full Text] [PDF]
|
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R. Maile, B. Wang, W. Schooler, A. Meyer, E. J. Collins, and J. A. Frelinger Antigen-Specific Modulation of an Immune Response by In Vivo Administration of Soluble MHC Class I Tetramers J. Immunol., October 1, 2001; 167(7): 3708 - 3714. [Abstract] [Full Text] [PDF]
|
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|
 |
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 T Hanke Prospect of a prophylactic vaccine for HIV Br. Med. Bull., September 1, 2001; 58(1): 205 - 218. [Abstract] [Full Text] [PDF]
|
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D. N. Forthal, G. Landucci, and E. S. Daar Antibody from Patients with Acute Human Immunodeficiency Virus (HIV) Infection Inhibits Primary Strains of HIV Type 1 in the Presence of Natural-Killer Effector Cells J. Virol., August 1, 2001; 75(15): 6953 - 6961. [Abstract] [Full Text]
|
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D. H. Barouch, S. Santra, M. J. Kuroda, J. E. Schmitz, R. Plishka, A. Buckler-White, A. E. Gaitan, R. Zin, J.-H. Nam, L. S. Wyatt, et al. Reduction of Simian-Human Immunodeficiency Virus 89.6P Viremia in Rhesus Monkeys by Recombinant Modified Vaccinia Virus Ankara Vaccination J. Virol., June 1, 2001; 75(11): 5151 - 5158. [Abstract] [Full Text]
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M. K. Slifka, R. Pagarigan, I. Mena, R. Feuer, and J. L. Whitton Using Recombinant Coxsackievirus B3 To Evaluate the Induction and Protective Efficacy of CD8+ T Cells during Picornavirus Infection J. Virol., March 1, 2001; 75(5): 2377 - 2387. [Abstract] [Full Text]
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T. U. Vogel, T. M. Allen, J. D. Altman, and D. I. Watkins Functional Impairment of Simian Immunodeficiency Virus-Specific CD8+ T Cells during the Chronic Phase of Infection J. Virol., March 1, 2001; 75(5): 2458 - 2461. [Abstract] [Full Text]
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T. M. Allen, B. R. Mothé, J. Sidney, P. Jing, J. L. Dzuris, M. E. Liebl, T. U. Vogel, D. H. O'Connor, X. Wang, M. C. Wussow, et al. CD8+ Lymphocytes from 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., January 15, 2001; 75(2): 738 - 749. [Abstract] [Full Text]
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 D. H. Barouch, S. Santra, J. E. Schmitz, M. J. Kuroda, T.-M. Fu, W. Wagner, M. Bilska, A. Craiu, X. X. Zheng, G. R. Krivulka, et al. Control of Viremia and Prevention of Clinical AIDS in Rhesus Monkeys by Cytokine-Augmented DNA Vaccination Science, October 20, 2000; 290(5491): 486 - 492. [Abstract] [Full Text]
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M. A. Cromwell, R. S. Veazey, J. D. Altman, K. G. Mansfield, R. Glickman, T. M. Allen, D. I. Watkins, A. A. Lackner, and R. P. Johnson Induction of Mucosal Homing Virus-Specific CD8+ T Lymphocytes by Attenuated Simian Immunodeficiency Virus J. Virol., September 15, 2000; 74(18): 8762 - 8766. [Abstract] [Full Text]
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D. T. Evans, P. Jing, T. M. Allen, D. H. O'Connor, H. Horton, J. E. Venham, M. Piekarczyk, J. Dzuris, M. Dykhuzen, J. Mitchen, et al. 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., August 15, 2000; 74(16): 7400 - 7410. [Abstract] [Full Text]
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) that were initiated using thawed PBMC.
A, Standard 51Cr release assays (E:T cell
ratio, 20:1) were initiated from PBMC derived pre- and postvaccination.
Mamu-A*01+ B-LCLs were used as target cells, with the
exception of samples from animal 95058 in which target cells were
Mamu-A*01-721.221-transfected cells. Background
peptide-specific lysis from naive Mamu-A*01+ animals tested
in each assay was consistently <10% (dotted line). B,
In vitro cultured PBMC were also were stained with p11C, C
). For comparison, levels of specific
lysis detected from a naive Mamu-A*01+ animal (96078)
tested concurrently are reproduced with each graph (
). Note that the
y-axis for the animal 94004 graph at 1 wk post-MVA
extends to 100% specific lysis. Responses 10% above those detected in
fresh PBMC from a naive Mamu-A*01+ macaque in the same
assay were considered significant.