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of June 13, 2013.
This information is current as Vaccinia Virus Ankara Boost Regimen
Vaccinated with a DNA Prime/Modified
Blood Lymphocytes from Rhesus Macaques
Activity in Fresh, Unstimulated Peripheral
Induction of AIDS Virus-Specific CTL
Watkins
Altman, Bernard Moss, Andrew J. McMichael and David I.
Shipley, Jim Fuller, Tomas Hanke, Alessandro Sette, John D.
Bianca R. Mothé, Susan Steffen, Jon E. Boyson, Tim
Todd M. Allen, Thorsten U. Vogel, Deborah H. Fuller,
http://www.jimmunol.org/content/164/9/4968
2000; 164:4968-4978; ;J Immunol
References http://www.jimmunol.org/content/164/9/4968.full#ref-list-1
, 37 of which you can access for free at: cites 72 articlesThis article
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2000 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month byThe Journal of Immunology
by guest on June 13, 2013http://www.jimmunol.org/Downloaded from
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
1
Todd M. Allen,
2
* Thorsten U. Vogel,* Deborah H. Fuller,
‡
Bianca R. Mothe´,* Susan Steffen,*
Jon E. Boyson,
3
* Tim Shipley,
‡
Jim Fuller,
‡
Tomas Hanke,
§
Alessandro Sette,
¶
John D. Altman,
储
Bernard Moss,** Andrew J. McMichael,
§
and David I. Watkins*
†
The observed role of CTL in the containment of AIDS virus replication suggests that an effective HIV vaccine will be required to
generate strong CTL responses. Because epitope-based vaccines offer several potential advantages for inducing strong, multispe-
cific CTL responses, we tested the ability of an epitope-based DNA prime/modified vaccinia virus Ankara (MVA) boost vaccine
to induce CTL responses against a single SIVgag CTL epitope. As assessed using both
51
Cr release assays and tetramer staining
of in vitro stimulated PBMC, DNA vaccinations administered to the skin with the gene gun induced and progressively increased
p11C, C3M (CTPYDINQM)-specific CD8
ⴙ
T lymphocyte responses in six of six Mamu-A*01
ⴙ
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
ⴙ
/
CD8
␣
ⴙ
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
ⴙ
/CD8
␣
ⴙ
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
ⴙ
T
lymphocytes in non-human primates. The Journal of Immunology, 2000, 164: 4968–4978.
While antiretroviral therapies have been successful in
reducing viral loads in HIV-infected patients, these
drugs are having little impact on the global epidemic.
In 1998 alone, there were ⬎5.8 million new cases of HIV infection
worldwide (1). Reservoirs for HIV still persist in HIV-infected
patients undergoing antiretroviral therapy (2), and almost 90% of
the world’s HIV-infected population resides within countries un-
able to afford these drugs. Therefore, the development of vaccines
designed to prevent HIV infections, rather than treat the disease, is
the best hope.
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 se-
lective pressure on the AIDS virus populations, as evidenced by
the eventual predominance of viruses with amino acid replace-
ments in those regions of the virus to which CTL responses are
directed (4–10). 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
(12–14). 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 ad-
vantages 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 con-
trol 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 (18–21). The use of epitopes can also over-
come any potential safety concerns associated with the vaccinating
Ag, as exemplified in the case of the human papillomavirus E6 and
*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;
¶
Epimmune, Inc., San Diego, CA 92121;
储
Emory Vaccine
Center, Emory University School of Medicine, Atlanta, GA 30322; and **Laboratory
of Viral Diseases, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, MD 20892
Received for publication July 28, 1999. Accepted for publication February 23, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Grants R01AI41913 and RR0167 and a Cremer Schol-
arship from the Department of Pathology, University of Wisconsin, Madison (to
B.R.M.). D.I.W. is a recipient of an Elizabeth Glaser Foundation award. This paper
is WRPRC Publication No. 39-030.
2
Address correspondence and reprint requests to Dr. Todd M. Allen, Wisconsin
Regional Primate Research Center, University of Wisconsin, Madison, WI 53715.
E-mail address: tallen@primate.wisc.edu
3
Current address: Department of Molecular and Cellular Biology, Harvard Univer-
sity, Cambridge, MA 02138.
Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00
by guest on June 13, 2013http://www.jimmunol.org/Downloaded from
E7 Ags, whose expression is clearly associated with cervical car-
cinoma (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 (24–27). Al-
though DNA vaccines encoding whole protein have effectively in-
duced immunodeficiency virus-specific cellular immune responses
in non-human primates (28–33), they have been unable to reliably
protect against vigorous AIDS virus challenges. This lack of pro-
tection, however, may be due to the inability of previous DNA
vaccine regimens to induce potent enough cellular immune re-
sponses against HIV and SIV.
Current approaches to vaccine design have begun to take ad-
vantage of the synergistic effects of combining DNA vaccines with
other approaches to induce stronger, more persistent cellular im-
mune responses (34–39). Recently, live pox viruses, such as mod-
ified vaccinia virus Ankara (MVA),
4
have proven to be safe and
effective vaccine vectors for inducing strong cellular immune re-
sponses (40–44). 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*01
4
-restricted SIV gag CTL epitope p11C, C3M (CTP
YDINQM) (45, 46) as an immunogen to induce AIDS virus-spe-
cific 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 ma-
caques levels of functionally active epitope-specific CD8
⫹
T lym-
phocytes equivalent to those observed during acute SIV infection.
Materials and Methods
Animals
Rhesus macaques used in this study were identified as Mamu-A*01
⫹
by
PCR-sequence-specific priming and direct sequencing as previously de-
scribed (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 Re-
source Center review committee.
DNA vectors pTH.HW and HC,C3M
The pTH.HW vector was derived from the pTH.H vector (48), which en-
coded for a polyepitope of 20 HIV and 3 SIV CTL epitopes, including the
Mamu-A*01-restricted CTL epitope (TPYDINQML) (49). Following con-
struction of the pTH.H plasmid, the Mamu-A*01 CTL epitope was opti-
mally defined and was found to require an N-terminal cysteine residue (46),
now termed p11C, C3M (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,C3M) 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
g 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 vacci-
nations involved codelivery of equal molar amounts of both the pTH.HW
and HC,C3M vectors.
MVA inoculations
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 ⫻10
8
PFU of MVA.HW in a volume of 150
l 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/diatrio-
ate gradient centrifugation. Cells were then washed twice in R10 medium
(RPMI 1640 supplemented with penicillin (50 U/ml), streptomycin (50
g/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⫻10
6
PBMC in R10 with peptide (10
M). 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 ⫻
10
6
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
51
Cr 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 concen-
tration of 1 mg/ml.
Cytotoxicity assays
The CTL activity of in vitro stimulated CTL cultures was assessed as
previously described (46). Briefly, 5 ⫻10
5
Mamu-A*01-transfected
721.221 cells (46) or B-LCLs derived from a Mamu-A*01
⫹
rhesus ma-
caque were incubated for 1.5 h with 80
Ci of Na
251
CrO
4
(New England
Nuclear Life Sciences Products, Boston, MA) and 5
g of corresponding
peptide in 200
l of R10 medium. These target cells were plated into
duplicate wells of a 96-well U-bottom microtiter plate (5 ⫻10
3
cells/well)
and incubated with effector CTL for 5 h. The reported percent specific lysis
represents
51
Cr release from CTPYDINQM peptide-pulsed targets minus
51
Cr 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
51
Cr 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 excep-
tions: 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 pep-
tide 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
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.
4969The Journal of Immunology
by guest on June 13, 2013http://www.jimmunol.org/Downloaded from
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 ex-
pressed and then folded with human

2
m and peptide.
Tetramer staining
Lymphocytes (2 ⫻10
5
) 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
g/100
l for in vitro cultures, 0.1
g/100
l for fresh PBMC) and an
anti-rhesus CD3-FITC-conjugated mAb (10
l; BioSource, Camarillo,
CA) in a 100-
l 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
l; 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
l of 2% paraformaldehyde
(PFA).
For staining of fresh, unstimulated PBMC, 1 ⫻10
6
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
g/well of anti-IFN-
␥
mAb MD-1 (U-Cytech) over-
night at 4°C. The plates were washed 10 times with PBST (PBS containing
0.05% Tween 20) and blocked with PBS containing 2% BSA for1hat
37°C. PBS containing 2% BSA was discarded from the plates, and freshly
isolated PBMC in R5 medium were added. The R5 contained 5
g/ml Con
A (Sigma, St. Louis, MO), 1
M p11C, C3M peptide, 1
M of an irrel-
evant SIVenv peptide (ELGDYKLV), or no peptide. PBMC (5 ⫻10
4
,
2.5 ⫻10
4
, 1.2 ⫻10
4
, and 6 ⫻10
3
) were plated in triplicate (100
l/well)
and incubated for 14 h at 37°C in 5% CO
2
. The cells were then removed,
and 200
l/well of ice-cold deionized water was added to lyse the remain-
ing 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 ⫻10
6
) were incubated at 37°C for 1 h with 50
ng/ml PMA and 1
g/ml ionomycin, 5
M p11C, C3M peptide-pulsed
Mamu-A*01
⫹
B-LCL, or Mamu-A*01
⫹
B-LCL alone as a control. Cells
were treated with 10
g/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
l of permeablization buffer (0.1% sapo-
nin in FACS buffer) for 5 min at room temperature, washed once with 0.1%
saponin, and incubated in the dark with 1
l 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-
l cell
suspension was fixed with 450
l of 2% PFA.
Results
Induction of CTL responses following a single skin DNA
vaccination
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 (Pow-
derJect Vaccines) to vaccinate Mamu-A*01
⫹
rhesus macaques
with the pTH.HW vector encoding for the Mamu-A*01-restricted
SIVgag CTL epitope p11C, C3M (46) (Fig. 1). Chest and ingui-
nal 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, C3M
peptide, and analyzed for CTL responses using standard
51
Cr re-
lease assays at an E:T cell ratio of 20:1. A p11C, C3M-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, respec-
tively) from PBMC taken 4 wk after the first vaccination (data not
shown). Responses from cultured PBMC taken before DNA vac-
cination were all ⬍5% specific lysis (Fig. 2A).
In parallel, tetrameric complexes were used to determine the
percentage of CD3
⫹
/CD8
␣
⫹
T lymphocytes in each culture that
expressed TCRs specific for the p11C, C3M 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 re-
sponded to the vaccination (tetramer levels between 1 and 9%;
data not shown). Taken together, the
51
Cr release assays and tet-
ramer staining of in vitro cultures revealed that all six DNA-vac-
cinated macaques were generating low, but detectable, p11C,
C3M-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,C3M) were then administered. The
HC,C3M plasmid expresses a highly immunogenic hepatitis B
core Ag (HBcAg) shown to augment the cellular immune re-
sponses to inserted T cell epitopes (54–57). 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 en-
hanced by lengthening the resting periods between DNA vaccina-
tions (58). When vaccinations were begun in the second set of four
FIGURE 1. DNA and MVA vaccination schedule of six Mamu-A*01
⫹
macaques. Using the Dermal PowderJect XR gene gun six Mamu-A*01
⫹
rhesus macaques were vaccinated with DNA plasmids precipitated onto
gold particles. Two different DNA plasmids were employed, each encoding
the SIVgag CTL epitope CTPYDINQM. Three to five DNA vaccinations
were directed to skin tissues as indicated. Nine weeks (95045 and 95058),
2 wk (96031), and 18 wk (96118, 96123, and 94004) after their last DNA
vaccination these six macaques were boosted with two doses of 5 ⫻10
8
PFU of MVA.HW (MVA) spaced 4 wk apart.
4970 INDUCTION OF SIVgag-SPECIFIC CTL IN MACAQUES BY A DNA/MVA VACCINE
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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 re-
sponse 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 ma-
caques as measured by
51
Cr release assays (Fig. 2A) and tetramer
staining (Fig. 2B). Following the third or fourth booster vaccina-
tion, levels of p11C, C3M-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 in-
creased to ⬎20% in some cultures. In the majority of cultures
tested these two independent measurements did roughly corre-
spond. 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 en-
hancing the levels of p11C, C3M-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, C3M-specific T cell responses observed
in in vitro stimulated cultures, we reasoned that the frequency of
p11C, C3M-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 lym-
phocyte 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 vac-
cinations, two of the six animals showed levels of p11C, C3M-
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, C3M-specific CD8
⫹
T lymphocytes high enough
to be detected in fresh, unstimulated PBMC of some animals using
tetramers.
MVA significantly boosts levels of tetramer-positive cells in
fresh, unstimulated PBMC
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 as-
sessed for the percentage of CD3
⫹
/CD8
␣
⫹
T lymphocytes which
were specific for the p11C, C3M epitope. Tetramer analysis re-
vealed 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, C3M epitope (Fig. 4). These levels represented a signifi-
cant 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
FIGURE 2. DNA vaccinations prime and boost
the levels of epitope-specific CD8
⫹
T lymphocytes
in Mamu-A*01 macaques. Fresh PBMC taken 2 wk
after each DNA vaccination were used to derive 2
wk in vitro stimulated cultures from each animal,
with the exception of the cultures indicated (Œ) that
were initiated using thawed PBMC. A, Standard
51
Cr release assays (E:T cell ratio, 20:1) were initi-
ated 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-trans-
fected cells. Background peptide-specific lysis from
naive Mamu-A*01
⫹
animals tested in each assay
was consistently ⬍10% (dotted line). B, In vitro cul-
tured PBMC were also were stained with p11C,
C3M-specific Mamu-A*01 tetramers. Background
tetramer staining of in vitro stimulated cultures from
naive Mamu-A*01 animals was always ⬍0.2% (dot-
ted line). NT, time points when in vitro stimulated
cultures were not tested.
4971The Journal of Immunology
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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 main-
tained at this high level up to 4 wk (Fig. 4).
A second administration of MVA boosted responses moderately
A second administration of MVA.HW was given to all six ma-
caques 4 wk after receiving their first MVA.HW. As before, re-
sponses 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 in-
duced after the first MVA.HW. Longer resting periods between
MVA vaccinations, as used in other studies (42), may have al-
lowed for better induction by the second MVA.
Frequency of p11C, C3M-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 cir-
culation (59–61). Fresh, unstimulated PBMC were tested at 1 and
2 wk following administration of their first MVA.HW with Con A,
the p11C, C3M peptide, an irrelevant SIV envelope peptide
(ELGDYKLV), or no peptide. ELISPOT indicated that 1 wk fol-
lowing the first MVA.HW vaccination, strong Ag-specific re-
sponses were observed in three of the DNA/MVA-vaccinated an-
imals tested. Animals 96118 and 96123 had 92 p11C, C3M-
specific SFCs/50,000 input cells (effector frequency 1, 840/10
6
FIGURE 3. Tetramer-positive CD3
⫹
/
CD8
␣
⫹
T lymphocytes are detectable in
fresh PBMC of some macaques after mul-
tiple DNA vaccinations. Fresh, unstimu-
lated PBMC from six DNA-vaccinated
macaques were stained 6 or 14 days after
receiving their fourth or fifth DNA vacci-
nation. The percentage of CD3
⫹
/CD8
␣
⫹
,
p11C, C3M tetramer-positive T lympho-
cytes is indicated in the top right corner of
each plot. Significant responses ⬎0.10%
are in boldface. The background level from
fresh unstimulated PBMC of a naive
Mamu-A*01-positive animal was ⬍0.08%
(data not shown).
Table I. Percentage of tetramer-positive CD3
⫹
/CD8
␣
⫹
T lymphocytes detected in fresh, unstimulated PBMC
from DNA-vaccinated macaques
a
No. of DNA
Vaccinations Days Post-
DNA 95045
b
95058
b
96031
b
96118 96123 94004
0
c
0.02 0.04 0.01 0.01 0.00 0.02
4 14 –––0.21
e
0.08 0.53
28 0.03 0.05 – – – –
5 6 –––0.48 0.05 0.45
13 0.02 – – 0.21 0.08 0.34
28 0.01 0.07 0.03
d
–––
a
Tetramers typically stain ⬍0.08% of fresh, unstimulated CD3
⫹
/CD8
␣
⫹
T lymphocytes from PBL of naive Mamu-A*01
⫹
animals.
b
Thawed PBMC were used for samples from animals 95045, 95058, and 96031.
c
Thawed PBMC from each animal were used to test levels of pre-DNA tetramer-positive cells.
d
PBMC isolated 4 wk following animal 96031’s last (third, not fifth) DNA vaccination were tested.
e
Significant responses ⬎0.10% are in boldface.
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PBMC) and 44 p11C, C3M-specific SFCs/50,000 input cells (ef-
fector frequency, 880/10
6
PBMC), respectively (Fig. 5A). SFC re-
sponses 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, C3M
peptide, 206 SFCs/50,000 cells (effector frequency, 4,120/10
6
PBMC), than to Con A (effector frequency, 1,940/10
6
PBMC).
None of the animals demonstrated significant responses to the ir-
relevant peptide or to wells without any peptide even at 50,000
cells/well (less than five spots per well).
Samples derived 2 wk following the first MVA.HW were also
analyzed (Fig. 5B). 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,C3M 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 anal-
ysis conducted 21 wk after the first MVA.HW indicated that pos-
itive ELISPOT responses were still detectable in 96118, 96123,
and 94004 at effector frequencies of 125/10
6
, 35/10
6
, and 230/10
6
PBMC, respectively (data not shown).
Tetramer-positive lymphocytes produce IFN-
␥
in response to
antigenic stimulation
Given the surprisingly high levels of Ag-specific lymphocytes de-
tected 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,
C3M 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 tet-
ramer-positive as well as tetramer-negative cells (Fig. 6B). Treat-
ment 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. Treat-
ment of PBMC from each of the vaccinated animals with the p11C,
C3M 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,
C3M 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 produc-
tion of IFN-
␥
(data not shown). These results indicate that the
majority of tetramer-positive CD8
⫹
cells in these DNA/MVA-vac-
cinated macaques are functionally active and capable of respond-
ing specifically to the p11C, C3M peptide through the production
of intracellular IFN-
␥
.
Direct ex vivo cytotoxic activity in fresh PBMC from
DNA/MVA-vaccinated macaques
Because we induced high levels of peptide p11C, C3M-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 de-
termining 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 in-
fected animals, is only detectable after in vitro stimulation with
peptide. PBMC were tested in
51
Cr release assays at E:T cell ratios
of 150:1 and 50:1 against target B-LCLs pulsed with either peptide
p11C, C3M 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 lympho-
cytes 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
FIGURE 4. MVA.HW vaccination of DNA-primed macaques signifi-
cantly boosts levels of epitope-specific CD3
⫹
/CD8
␣
⫹
T lymphocytes in
fresh, unstimulated PBMC. Nine weeks (95045 and 95058), 2 wk (96031),
and 18 wk (96118, 96123, and 94004) after their last DNA vaccination six
macaques were boosted with two doses of 5 ⫻10
8
PFU of MVA.HW
spaced 4 wk apart. Fresh PBMC from DNA/MVA-vaccinated macaques
were isolated and tetramer stained to determine the percentage of p11C,
C3M-specific CD8
⫹
T cells. Background staining of fresh, unstimulated
PBMC taken from a naive Mamu-A*01
⫹
macaque (animal 96078) was
⬍0.08%. Levels detected in fresh PBMC taken 1–4 wk before the
MVA.HW vaccination were ⬍0.2% (data not shown).
4973The Journal of Immunology
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the higher E:T ratio were low, 5 and 10%, respectively, over back-
ground 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).
We next assessed for ex vivo CTL activity in fresh, unstimulated
PBMC from animals 96118, 96123, and 94004 1 wk after receiv-
ing their first MVA.HW, a point at which levels of tetramer stain-
ing were between 1.2 and 20.0%. More dramatic levels of fresh
killing of 36, 13, and 77% were detected in these animals at the
highest E:T cell ratio over background levels in the control animal.
Samples from animals 96118, 96123, and 94004 taken 1 wk after
the second MVA.HW vaccination were also assessed, although the
levels of tetramer-positive cells were lower at this time point.
These lower levels of tetramer-positive cells were paralleled by a
comparable reduction in the levels of specific lysis (Fig. 7). None-
theless, 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
FIGURE 5. Frequency of p11C, C3M-specific CD8
⫹
T cells as measured by an IFN-
␥
ELISPOT assay. Freshly isolated PBMC were assessed for their
ability to produce IFN-
␥
in response to Con A, peptide p11C, C3M(1
M), an irrelevant SIVenv 9 mer (ELGDYKLV; 1
M), or no peptide. PBMC
taken 1 wk (A)and2wk(B) following the first MVA.HW were plated at varying dilutions of input cells.
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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
We have demonstrated the ability of an epitope-based DNA/MVA
vaccine to induce the highest reported levels of Ag-specific CD8
⫹
T lymphocyte responses detected in a mammalian species. Gene
gun DNA vaccinations targeted to the skin proved effective at
priming p11C, C3M-specific CD8
⫹
T cell responses in six
Mamu-A*01
⫹
rhesus macaques. Tetramer staining of fresh, un-
stimulated 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 recombi-
nant 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 equiv-
alent 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 (67–69). Corrected LDA values for SIV-
specific CD8
⫹
T lymphocytes from rhesus macaques immunized
with a vaccinia virus-based subunit vaccine (70) or immune-stim-
ulating complexes (iscoms) (71) suggest that these vaccines were
capable of inducing between 100 and 2000 protein-specific CD8
⫹
T lymphocytes/10
6
PBMC. In our study, tetramer staining indi-
cated 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 1000/10
6
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, C3M-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 de-
tected 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
FIGURE 6. The majority of tetramer-positive cells
isolated 1 wk following the second MVA.HW produce
IFN-
␥
in response to stimulation with the p11C,
C3M peptide. Fresh Ficoll-purified PBMC purified 1
wk after the second MVA.HW were stained for CD8
␣
,
intracellular IFN-
␥
production, and tetramers to deter-
mine the percentage of tetramer-positive cells capable
of producing IFN-
␥
in response to the p11C, C3M
peptide. A, Unstimulated PBMC. B, PMA/ionomycin-
stimulated cells. C, p11C, C3M peptide (5
M)-
stimulated cells, which in response to the peptide have
internalized their TCR (therefore tetramer negative)
and produced intracellular IFN-
␥
. The percentage of
positively staining cells is indicated in each quadrant.
The levels of tetramer-positive cells illustrated in Fig.
6 are slightly lower than those reported in Fig. 4, since
the percentage of CD8
␣
⫹
(not CD3
⫹
/CD8
␣
⫹
) lym-
phocytes is reported.
4975The Journal of Immunology
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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,
C3M-specific CD8
⫹
cells induced after the MVA boost. The
induction of p11C, C3M-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
⫹
/CD8

⫹
cells. These responses, however, were not de-
tectable 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, C3M-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
51
Cr 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 ob-
served in this study when unstimulated PBMC were analyzed, sug-
gesting 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, C3M-specific induction of IFN-
␥
in
fresh PBMC by intracellular IFN-
␥
staining was critical to assess-
ing 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-pos-
itive cells were functionally active.
Comparisons of the number of p11C, C3M-specific cells de-
tected 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 ver-
ified by intracellular IFN-
␥
staining. Furthermore, because the tet-
ramer 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
M 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 in-
termediate levels of SFCs (92/50,000 cells) in ELISPOT and in-
termediate levels of specific lysis (36% at an E:T cell ratio of
150:1) in fresh killing assays. While the reasons for these discrep-
ancies were not addressed by this study, these results nonetheless
suggest that analyses in addition to tetramer staining, i.e., intra-
cellular 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-in-
fected macaques (63), such responses have never been detected in
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.
FIGURE 7. Direct ex vivo cytotoxic activity detected in fresh PBMC
from DNA/MVA-vaccinated macaques. Freshly isolated PBMC were
tested in a
51
Cr release assay using peptide-pulsed B-LCL from a Mamu-
A*01
⫹
rhesus macaque. PBMC were tested at E:T cell ratios of 150:1 and
50:1 (⽧). 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.
4976 INDUCTION OF SIVgag-SPECIFIC CTL IN MACAQUES BY A DNA/MVA VACCINE
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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 mea-
sure 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 func-
tionally 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 ma-
caques 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.
Acknowledgments
We thank Maurice Gately for providing rIL-2. We also thank Carol
Emerson and the animal care staff at the Wisconsin Regional Primate Re-
search Center for their assistance in carrying out these experiments.
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