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Journal of Virology, August 2000, p. 7485-7495, Vol. 74, No. 16
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Simian Immunodeficiency Virus (SIV) gag DNA-Vaccinated
Rhesus Monkeys Develop Secondary Cytotoxic T-Lymphocyte Responses
and Control Viral Replication after Pathogenic SIV Infection
Michael A.
Egan,1,*
William A.
Charini,1
Marcelo J.
Kuroda,1
Jörn E.
Schmitz,1
Paul
Racz,2
Klara
Tenner-Racz,2
Kelledy
Manson,3
Michael
Wyand,3
Michelle A.
Lifton,1
Christie E.
Nickerson,1
Tongming
Fu,4
John W.
Shiver,4 and
Norman L.
Letvin1
Division of Viral Pathogenesis, Department of Medicine,
Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, Massachusetts 022151; Department
of Pathology, Bernhard Nocht Institute for Tropical Medicine,
Hamburg, Germany2; Primedica-Mason
Laboratories, Worchester, Massachusetts 016083;
and Merck Research Laboratories, West Point, Pennsylvania
194864
Received 19 January 2000/Accepted 12 May 2000
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ABSTRACT |
The potential contribution of a plasmid DNA construct to
vaccine-elicited protective immunity was explored in the simian
immunodeficiency virus (SIV)/macaque model of AIDS. Making use of
soluble major histocompatibility class I/peptide tetramers and
peptide-specific killing assays to monitor CD8+
T-lymphocyte responses to a dominant SIV Gag epitope in genetically selected rhesus monkeys, a codon-optimized SIV gag DNA
vaccine construct was shown to elicit a high-frequency SIV-specific
cytotoxic T-lymphocyte (CTL) response. This CTL response was
demonstrable in both peripheral blood and lymph node lymphocytes.
Following an intravenous challenge with the highly pathogenic viral
isolate SIVsm E660, these vaccinated monkeys developed a secondary CTL response that arose with more rapid kinetics and reached a higher frequency than did the postchallenge CTL response in control
plasmid-vaccinated monkeys. While peak plasma SIV RNA levels were
comparable in the experimentally and control-vaccinated monkeys during
the period of primary infection, the gag plasmid
DNA-vaccinated monkeys demonstrated better containment of viral
replication by 50 days following SIV challenge. These findings indicate
that a plasmid DNA vaccine can elicit SIV-specific CTL responses in
rhesus monkeys, and this vaccine-elicited immunity can facilitate the
generation of secondary CTL responses and control of viral replication
following a pathogenic SIV challenge. These observations suggest that
plasmid DNA may prove a useful component of a human immunodeficiency
virus type 1 vaccine.
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INTRODUCTION |
There is an emerging consensus that
an effective human immunodeficiency virus type 1 (HIV-1) vaccine must
elicit both virus-specific neutralizing antibody and cytotoxic
T-lymphocyte (CTL) responses (21). The strategies that will
be needed to generate these different types of immune responses are
likely to be very different. Finding an approach for inducing an
antibody response that is capable of neutralizing a diversity of
primary patient HIV-1 isolates has proven elusive (17).
However, HIV-1-specific CTL can be generated in rodents and nonhuman
primates using a number of vaccine modalities, including live
recombinant vectors (10-12, 35, 36), peptides (3, 9,
13, 28, 34, 40), and plasmid DNA (2, 4-6, 22, 26, 38,
39). It will be important to characterize systematically the CTL
responses elicited by these various vaccine approaches to determine the
magnitude and durability of the responses, as well as the contribution
that such responses can make in containing HIV-1 replication. Such data
will be needed to select the optimal vaccine approaches to carry
forward into human testing.
DNA vaccination, the direct injection of plasmid DNA expressing genes
encoding the proteins of pathogens, has proven to be highly immunogenic
(7, 24, 27, 31, 32, 43, 44). Studies are currently ongoing
to examine the potential utility of plasmid DNA as a component of an
AIDS vaccine (2, 4-6, 26, 38, 39). Numerous reports have
demonstrated that DNA vaccines can elicit HIV-1 and simian
immunodeficiency virus (SIV)-specific CTL, T-helper cells, and
antibodies in mice and rhesus monkeys (19, 25, 37, 39,
45-48). Several trials of DNA vaccines, either alone or in
combination with other vaccine modalities, have shown promising results
in nonhuman primates (2, 19, 22, 25, 32, 38). Because they
are relatively stable, are inexpensive to produce, and do not carry
with them the pathogenic risks inherent in immunization with live
vectors, DNA vaccines represent an attractive solution to the HIV-1 pandemic.
The SIV/macaque model is useful in accessing HIV-1 vaccine strategies
(20). SIV isolates are lentiviruses that are similar in
nucleotide sequence to HIV-1. Moreover, certain SIV isolates induce an
AIDS-like disease in Asian macaque species. The power of this animal
model for exploring the efficacy of HIV-1 vaccine strategies has been
enhanced by the definition of a dominant SIV Gag CTL epitope in rhesus
monkeys expressing the major histocompatibility complex (MHC) class I
allele Mamu-A*01 (1, 29). The immune response to
this epitope can be monitored in infected and vaccinated animals using
both functional CTL assays and flow cytometric analysis of tetrameric
MHC class I/peptide-binding CD8+ T lymphocytes (8,
15-17, 35).
This study was initiated to evaluate a plasmid DNA vaccine-elicited SIV
Gag-specific CTL response in rhesus monkeys and to determine the
contribution of that restricted immune response to containing a
highly pathogenic primary SIV infection.
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MATERIALS AND METHODS |
Animals.
All animals were maintained in accordance with the
guidelines of the Committee on Animals for the Harvard Medical School
and Guide for the Care and Use of Laboratory Animals
(6a).
Selection of Mamu-A*01+ rhesus
monkeys.
Rhesus monkeys were screened for the presence of the
Mamu-A*01 allele using a PCR-based technique as previously
described (15-17). EDTA-preserved whole blood from rhesus
monkeys was subjected to Ficoll diatrizoate density gradient
centrifugation to isolate leukocytes, and the washed cell pellets were
resuspended in 200 µl of phosphate-buffered saline (PBS). DNA
extraction was then carried out using a QIAmp blood kit (Qiagen Inc.,
Chatsworth, Calif.). PCR was performed on 200 to 500 µg of extracted
DNA using allele-specific primers in a 50-µl reaction consisting of
60 mM Tris (pH 8.5), 2 mM MgCl2, 15 mM ammonium sulfate, 2 mM deoxynucleoside triphosphates (0.5 mM each), and 5 µl of
Taq polymerase. Primers A*01/F (5'-GAC AGC GAC GCC GCG AGC
CAA-3') and A*01/R (5'-GCT GCA GCG TCT CCT TCC CC-3') were used at a
final concentration of 800 nM each. Two additional primers specific for
a conserved MHC class II sequence (based on the macaque homologue of
HLA-DRB3) were included in the reaction as internal positive controls.
Primers 5' MDRB (5'-GCC TCG AGT GTC CCC CCA GCA CGT TTC-3') and 3' MDRB (5'-GCA AGC TTT CAC CTC GCC GCT G-3') were used at a final
concentration of 680 nM each. PCR was carried out using a Perkin-Elmer
GeneAmp System 9600 thermocycler (Perkin-Elmer Inc., Norwalk, Conn.). Samples were denatured at 96°C for 2 min, followed by 5 cycles of
25 s at 96°C and 60 s at 72°C, followed by 21 cycles of
25 s at 96°C, 50 s at 67°C, and 45 s at 72°C,
followed by 4 cycles of 25 s at 96°C, 60 s at 55°C, and
80 s at 72°C. PCR products were analyzed by 1% agarose gel
electrophoresis. Ten microliters of each PCR product was loaded per lane.
Potential Mamu-A*01+ animals were identified by
the presence of two bands, a 685-bp amplified product and a 260-bp
band. DNA sequence analysis was then performed on all potential
positive samples to confirm nucleotide sequence identity with the
published Mamu-A*01 prototype sequence (14).
Prior to sequencing, amplified DNA was treated with 1 U of shrimp
alkaline phosphatase and 10 U of exonuclease I per reaction for 15 min
at 37°C followed by 15 min at 80°C. The sequencing templates were
then purified using a QIAquick PCR purification kit (Qiagen). For each
template, 70 ng of DNA was used for DNA sequencing together with 5 pmol
of primer. Four PCR primers were used for sequencing: A*01/F and A*01/R, whose sequences are shown above, and B/1+ (5'-CTG CGC GGC TAC
TAC AAC CA-3') and G/1+ (5'-ATG TAA TCC TTG CCG TCG TA-3'). Sequencing
was carried out at a central core sequencing facility on an ABI-373
stretch DNA sequencing machine, using ABI AmpliTaq FS dye terminator
chemistry (Perkin-Elmer). All animals used in this study were
genotypically Mamu-A*01+, based on the above
screening as well as functional CTL assay.
Immunization of rhesus monkeys.
Monkeys were immunized with
5 mg of plasmid DNA, 2.5 mg in each quadricep muscle by intramuscular
inoculation. Either an SIV gag-expressing plasmid
(n = 5) or an empty control vector (n = 4) was inoculated. Injections were performed using a Biojector needle-free injection system and the supplied Biojector no. 3 syringe
as instructed by the manufacturer (Bioject, Portland, Oreg.).
Recombinant vaccine constructs.
The vaccination plasmid,
V1R-SIV gag (Fig. 1), is a pUC-based vector that utilizes the human
cytomegalovirus immediate-early promoter with intron A and bovine
growth hormone transcription terminator/polyadenylation signal as
expression regulatory elements and expresses full-length SIV
gag (30). The SIV gag open reading frame is homologous to that of SIVmac239 and was synthesized using optimal codons for human gene expression (18).
Western blotting.
Human RD cells in duplicate
25-cm2 tissue culture flasks were transfected with either
mock preparation (PBS) or 10 µg of V1R-SIV gag DNA construct using a
calcium phosphate-based transfection kit (Pharmacia Biotech,
Piscataway, N.J.). Twenty-four hours later, the cells were washed with
PBS three times and lysed in 0.5 ml of lysis buffer (50 mM Tris, 5 mM
EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl
fluoride, aprotinin [0.01 mg/ml; Sigma Chemical Co., St. Louis, Mo.])
at 4°C for 30 min. The aliquots of cell lysate (20 µl) were mixed
with an equal volume of 2× sample buffer (Novex, San Diego, Calif.)
and heated at 100°C for 5 min. The proteins were then resolved by
electrophoresis on 4 to 20% gradient sodium dodecyl sulfate
(SDS)-polyacrylamide gels (Novex). Following electrophoresis, proteins
were transferred onto a polyvinylidene difluoride membrane (Millipore)
under a constant current of 200 mA. The membrane was blocked with
BLOTTO solution (5% nonfat dry milk, 2% fetal calf serum, 0.2% Tween
20, and 10 mM Tris-Cl [pH 8.0] in PBS) overnight at room temperature
and then incubated with anti-p27 monoclonal antibody (1:1,000 dilution;
ABI, Columbia, Md.) for 2 h at room temperature. After washing
three times with BLOTTO solution, the membrane was incubated with
horseradish peroxidase-conjugated goat anti-mouse antibody for 1 h. The membrane was further washed with BLOTTO solution and developed
with an ECL (enhanced chemiluminescence reagent kit (Amersham,
Arlington Park, Ill.) for luminescent detection.
Vaccine preparations.
Inoculated cultures of terrific broth
(1.2% Bacto Tryptone, 2.4% Bacto Yeast Extract, 0.4% glycerol)
supplemented with 15 mM KH2PO4, 65 mM
K2HPO4, and 50 µg of kanamycin per ml were
grown overnight with shaking at 37°C. Minipreparations of plasmids
were made using a PERFECTprep plasmid DNA isolation kit (5 Prime-3 Prime, Inc., Boulder, Colo.). Maxipreparations of vaccine plasmids were
made by standard alkaline lysis followed by double CsCl gradient banding. A 1-liter overnight culture was centrifuged for 15 min at
7,500 rpm and resuspended in 14 ml of lysozyme buffer with lysozyme
(0.9% glucose, 10 mM EDTA, 25 mM Tris-Cl [pH 8], 4 mg of lysozyme
per ml). The suspension was then lysed with 28 ml of 0.2 M NaOH-1%
SDS, neutralized with 21 ml of sodium acetate 3 M (pH 4.8), and
centrifuged for 20 min at 17,000 rpm in a Sorvall centrifuge (Sorvall,
Braintree, Mass.). The supernatant was removed, centrifuged again for
20 min at 17,000 rpm, filtered, and ethanol precipitated with 3 volumes
of 95% ethanol. Following a 30-min incubation at 
20°C and
centrifugation at 6,500 rpm for 30 min in a Sorvall centrifuge, the
supernatants were discarded, and the ethanol-precipitated pellets were
air dried and resuspended in 11.5 ml of TSE buffer (10 mM Tris-Cl [pH
8], 1 mM EDTA, 10 mM NaCl). Optical-grade CsCl (13.2 g; Life
Technologies) and 2 ml of ethidium bromide (10 mg/ml) were added, and
the solution was ultracentrifuged at 60,000 rpm overnight at 20°C.
The CsCl-banded DNA was removed and then spun on a second CsCl
gradient. Following double CsCl banding, the ethidium was extracted
five times using CsCl-saturated isopropanol, and the DNA was
precipitated using 3 volumes of ethanol. The DNA was washed with 70%
ethanol, resuspended in TE buffer (10 mM Tris-Cl [pH 8], 1 mM EDTA),
extracted with phenol-chloroform, extracted with chloroform,
reprecipitated with ethanol, washed with 70% ethanol, and then
resuspended in sterile TE buffer. The DNA was quantitated and adjusted
to a concentration of 5 mg/ml with sterile isotonic saline.
Functional cytotoxicity assay.
Peripheral blood leukocytes
(PBL) from Mamu-A*01+ rhesus monkeys were
cultured with the optimal SIV peptide p11C (CTPYDINQM; 10 µg/ml)
and maintained at a density of 2 × 106 cells/ml. On
day 3 of culture, the medium was supplemented with recombinant human
interleukin-2 (IL-2; 20 U/ml; provided by Hoffmann-La Roche), and
cultures were maintained for an additional 7 days. Cells were then
centrifuged over Ficoll-Hypaque and assessed as effectors in a standard
51Cr release assay at an effector-to-target ratio ranging
from 20:1 to 0.6:1. Target cells were Mamu-A*01+
B-lymphoblastoid cell line (B-LCL) pulsed during overnight
51Cr labeling with 10 µg of either p11C or the control
peptide p11B (ALSEGCTPYDIN) per ml. All wells were assayed in
triplicate. Plates were incubated for 5 h in a humidified
incubator at 37°C. Specific release was calculated as [(experimental
release) (spontaneous release)/(maximum release) (spontaneous release)] × 100.
pCTL frequency determination by limiting dilution analysis
(LDA).
Freshly isolated PBL were plated in twofold dilutions from
32,000 to 250 cells per well in 24-well replicates. The p11C-specific CTL were then expanded by the addition of 2 × 105
p11C-pulsed, irradiated, Mamu-A*01+ B-LCL.
Microcultures were fed on days 5 and 10 by the addition of culture
medium supplemented with recombinant IL-2 (10 U/ml). After 14 days of
culture, the effector cells were assessed for cytotoxicity against
51Cr-labeled p11C- and control peptide p11B-pulsed target
cells. Individual wells screened against p11C-pulsed targets that
demonstrated counts 3 standard deviations higher than p11B-pulsed
target cells were scored positive for p11C-specific CTL activity. By
plotting the fraction of negative wells against the number of cells
added per well, the p11C-specific precursor CTL (pCTL) frequency was extrapolated from the cell concentration at which 37% of the wells would be considered negative.
Mamu-A*01/peptide tetramer complex formation and staining of
peptide-specific CD8
+ T lymphocytes.
Mamu-A*01/p11C tetramer complexes were prepared as previously described
(15). Phycoerythrin (PE)-labeled streptavidin (Prozyme, San
Leondro, Calif.) was mixed stepwise with biotinylated Mamu-A*01/peptide complexes at a molar ratio of 1:4 to produce the tetrameric complexes. All antibodies used in this study were directly coupled to fluorescein isothiocyanate (FITC), phycoerythrin-Texas red (ECD), or
allophycocyanin (APC). The following monoclonal antibodies were used:
anti-CD8 (Leu2a)-FITC (Becton Dickinson), anti-CD8-ECD
(Coulter), and anti-CD3-APC (FN18; kindly provided by D. M. Neville, Jr., National Institutes of Health, Bethesda, Md.).
The PE-coupled tetrameric Mamu-A*01/p11C complexes were used in
combination with anti-CD8
-FITC, anti-CD8
-ECD, and anti-CD3-APC
to stain 100 µl of fresh blood or 2 × 10
5
lymphocytes isolated by Ficoll-Hypaque density gradient centrifugation
following in vitro peptide stimulation as previously described
(
15). The phenotyping of p11C-specific CD8
+ T
cells was done by staining 100 µl of whole blood with 1 µg
of
488-Neutravadin (Molecular Probes, Eugene, Oreg.)-coupled tetrameric
Mamu-A*01/p11C complexes in combination with anti-CD8
-ECD and
anti-CD3-APC along with one of the following: anti-CD11a-PE,
anti-CD28-PE,
anti-CD45RA-PE, anti-CD49d-PE, anti-CD62L-PE,
anti-CD95-PE, or
anti-HLA-DR-PE.
Whole-blood samples were lysed using a Coulter Immunoprep reagent
system and a Q-prep workstation (Beckman Coulter Inc.).
Ten thousand
gated events were collected, and samples were analyzed
on a Coulter
EPICS Elite ESP. The total number of Mamu-A*01/p11C
tetramer-binding
cells per microliter of whole blood was calculated
as the product of
(% Mamu-A*01/p11C tetramer binding) × (total
white blood cell
count per microliter of whole blood) × (% lymphocytes)
× (% CD8
+ cells). Total white blood cell counts and
percent lymphocytes
were determined by complete blood count analysis on
500 µl of
EDTA-anticoagulated blood using an automated hematology
analyzer
(T540; Coulter
Corporation).
Data analysis was performed using the EPICS Elite software (version
4.02; Beckman Coulter). Data presentation was performed
by using WinMDI
software version 2.7 (Joseph Trotter, La Jolla,
Calif.) and Microsoft
PowerPoint 97 (Microsoft, Redmond, Wash.).
SIVsm E660 challenge.
All animals were challenge
intravenously with 50 50% monkey infective doses (MID50)
of SIVsm E660 (kindly provided by V. Hirsch, National Institutes of Health).
In situ hybridization.
A 35S-labeled,
single-stranded, antisense RNA probe (Lofstrand Laboratories,
Gaithersburg, Md.) was used to detect viral mRNA. The hybridization was
done on frozen sections as previously described (42). The
sections were examined with a microscope equipped with epiluminescent
illumination (Axiophot; Carl Ziess Inc., Jena, Germany). Cells were
considered positive for viral gene expression if the grain count was
more than six times higher than the background count.
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RESULTS |
V1R-SIV gag DNA vaccine expression of the gag
gene.
To examine protein expression from the V1R-SIV gag DNA
vaccine (Fig. 1), we analyzed SIV Gag
protein expression by transient transfection of human RD cells followed
by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell
lysates (Fig. 2). Figure 2 shows that
V1R-SIV gag DNA-transfected cells express the SIV Gag protein to higher
levels (lanes 3 and 4) than cells transiently transfected with the
empty control vector (lanes 1 and 2).
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FIG. 1.
SIV gag DNA expression vector V1R-SIV gag.
Kan(R), kanamycin resistance gene; CMV, cytomegalovirus; ori, origin of
replication; bGH, bovine growth hormone.
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FIG. 2.
In vitro expression of the V1R-SIV gag DNA vaccine
construct in transiently transfected human RD cells. Human RD cells
were transfected with either mock preparation (PBS; lanes 1 and 2) or
10 µg of V1R-SIV gag DNA construct (lanes 3 and 4) using calcium
phosphate. Twenty-four hours later, the cells were washed and lysed,
and the proteins were then resolved by SDS-PAGE. Sizes are indicated in
kilodaltons.
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SIV Gag-specific peripheral blood CD8+ T-lymphocyte
responses after DNA immunization.
To evaluate the immunogenicity
of a plasmid SIV gag DNA vaccine,
Mamu-A*01+ rhesus monkeys were immunized by
intramuscular inoculation with 5 mg of either an SIVmac 239 gag-expressing plasmid or a control empty plasmid. The
immunization schedule included inoculations at 0, 1, 2, and 6 months.
These monkeys were then monitored for the development of SIV
Gag-specific CD8+ T-lymphocyte responses. PBL were assessed
in whole blood without in vitro stimulation for Mamu-A*01/p11C tetramer
binding to CD8+ T cells. In addition, CD8+ T
lymphocytes from these animals were assessed for Mamu-A*01/p11C tetramer binding as well as for functional p11C-specific lytic activity
after in vitro p11C stimulation. Finally, p11C-specific CTL precursor
frequencies were determined by functional LDA. By week 4 following the
first plasmid DNA immunization, four of the five SIV gag
DNA-immunized monkeys had detectable Gag epitope-specific functional
CTL activity. The Gag epitope-specific CTL responses in these four
animals increased following subsequent DNA immunizations (Table
1). The control plasmid-immunized monkeys
remained negative for Gag epitope-specific functional CTL activity
throughout the course of the experiment.
Mamu-A*01/p11C tetramer binding to unstimulated
CD8
+ T cells in whole blood was also monitored
in the monkeys (Table
1;
Fig.
3). By week
6, 2 weeks after the second immunization, four
of the five SIV
gag DNA-vaccinated animals had detectable Mamu-A*01/p11C
tetramer-binding CD8
+ T cells in whole blood,
comprising 0.1 to 0.6% of all circulating
CD8
+ T
lymphocytes. Peak levels of Mamu-A*01/p11C tetramer-binding
CD8
+ T cells in whole blood were seen 2 weeks after
the fourth vaccination,
following immunizations at week 24 (Fig.
4A). The control plasmid
inoculated
monkeys never developed Mamu-A*01/p11C tetramer-binding
CD8
+ T lymphocytes that could be detected in whole blood
during the
course of the experiment.
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FIG. 3.
SIV Gag p11C tetramer binding to peripheral blood
CD8+ lymphocytes of SIV gag DNA-immunized
Mamu-A*-01+ rhesus monkeys. Arrows indicate the
weeks at which the monkeys were immunized. Percent p11C tetramer
binding represents Mamu-A*01/p11C tetramer-binding
CD8+ T cells in unstimulated whole blood samples at
each time point tested.
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FIG. 4.
SIV Gag p11C tetramer binding-CD8+ T
cells in whole blood of SIV gag DNA-immunized
Mamu-A*-01+ rhesus monkeys 2 weeks after the
fourth immunization. Cells were gated on CD8+
CD3+ lymphocytes. (A) Staining of unstimulated whole blood;
(B) staining of lymphocytes after in vitro expansion with p11C.
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PBL from the vaccinated monkeys were also stimulated in vitro with the
Gag peptide p11C in IL-2-containing medium. These cells
were assessed
for Mamu-A*01/p11C tetramer binding and functional
Gag epitope-specific
CTL activity. The peptide-stimulated PBL
from four of the five
gag DNA-vaccinated animals demonstrated
persistent high
levels of Mamu-A*01/p11C tetramer-binding CD8
+ T cells
(Table
1; Fig.
4B). Moreover, after in vitro peptide
stimulation, the
levels of Mamu-A*01/p11C tetramer-binding CD8
+ T cells in
these monkeys mirrored the levels of functional Gag
epitope-specific
CTL activity (Table
1; Fig.
5). The
tetramer-binding
CD8
+ T lymphocytes in the PBL of the
single monkey that developed
only a low level of tetramer-binding cells
in whole blood, N529
(Fig.
4A), failed to expand to a significant level
(Fig.
4B).
None of the control DNA-immunized animals developed
T-lymphocyte
populations that demonstrated significant levels of
Mamu-A*01/p11C
tetramer-binding CD8
+ T cells after in
vitro peptide stimulation. Using LDA, we were
able to detect
vaccine-induced p11C-specific CD8
+ pCTL in only one of the
four
gag DNA-vaccinated monkeys tested,
animal P091 (Table
1). As has been shown by others, the p11C-specific
pCTL frequency
calculated by LDA was typically 10- to 100-fold
lower than the number
of p11C-specific CD8
+ CTL measured by direct tetramer
staining in fresh blood.
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FIG. 5.
Plasmid DNA vaccine-induced Gag epitope-specific
functional CTL activity in PBL of vaccinated monkeys 2 weeks after the
fourth immunization. PBL of the vaccinated monkeys were activated in
vitro with p11C in IL-2-containing medium and assessed for lysis of
p11C-pulsed ( ) and control peptide p11B-pulsed ( ) B-LCL target
cells.
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DNA vaccine-induced SIV Gag-specific CD8+ T-lymphocyte
responses in lymph nodes.
Since HIV-1 replication occurs
predominantly in lymph nodes of infected individuals, it is important
that an HIV-1 vaccine elicit virus-specific CD8+ CTL that
are resident in secondary lymphoid organs. To determine whether DNA
vaccination elicited such a response, inguinal lymph nodes were
obtained from the immunized animals 1 week after the third immunization
and analyzed for SIV Gag epitope-specific CD8+ T
lymphocytes. Lymph node cell suspensions from two of the five plasmid
SIV gag DNA-vaccinated animals (P091 and 9298) showed detectable levels of Mamu-A*01/p11C tetramer-binding
CD8+ T cells (Table 2).
After p11C stimulation, lymph node cells from these same two animals,
as well as a third vaccinated animal (T258), showed high levels of
Mamu-A*01/p11C tetramer binding as well as functional p11C-specific
lytic activity. No SIV Gag epitope-specific CD8+ T
lymphocytes were detected in the lymph nodes of the control plasmid
DNA-immunized monkeys.
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TABLE 2.
Lymph node T lymphocytes from SIV gag plasmid
DNA-vaccinated monkeys demonstrate Gag epitope-specific CTL responses
10 weeks following the initial immunization
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Secondary CTL responses in DNA-vaccinated monkeys after SIV
challenge.
We then assessed the protection afforded these monkeys
by plasmid DNA vaccine-elicited immunity against SIV challenge as well as the durability of this protection. A subset of the animals were
challenged 4 weeks after the final DNA immunization (SIV gag
DNA-vaccinated animals P091 and N529; control DNA-vaccinated animals
R468, P967, and V299); the remaining animals were challenged 9 weeks
after the final DNA immunization. All animals were challenged with an
intravenous inoculation of 50 MID50 of the highly
pathogenic SIVsm E660. The challenged monkeys were monitored for the
development of SIV Gag epitope-specific CTL responses by Mamu-A*01/p11C
tetramer binding in whole blood, as well as by tetramer binding and
functional CTL activity after in vitro p11C stimulation of PBL. All SIV
gag plasmid DNA-vaccinated animals, regardless of the time
interval between the final immunization and viral challenge, developed strong secondary CTL responses to the challenge virus. The SIV gag DNA-vaccinated monkeys developed higher levels of
Mamu-A*01/p11C tetramer-binding cells in whole blood, and these cells
appeared earlier after virus challenge than did the control
DNA-vaccinated monkeys (Table 3). After
the initial burst of virus replication, the levels of circulating
Mamu-A*01/p11C tetramer-binding CD8+ T cells in the
SIV gag DNA-vaccinated monkeys and those of the control
DNA-vaccinated monkeys did not differ significantly (Fig. 6).
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TABLE 3.
SIV Gag epitope p11C-specific CD8+
T-lymphocyte responses of vaccinated animals after intravenous
challenge with SIVsm E660
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FIG. 6.
SIV Gag p11C tetramer binding CD8+ T
cells in whole blood after SIVsm E660 challenge in control DNA- and SIV
gag DNA-immunized monkeys. Percent p11C tetramer binding
represents Mamu-A*01/p11C tetramer-binding CD8+ T
cells in unstimulated whole blood samples at each time point tested.
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The phenotypes of the Mamu-A*01/p11C tetramer-binding and nonbinding
CD8
+ T cells in the vaccinated monkeys after virus
challenge were
analyzed by four-color flow cytometry.
CD8
+ T cells were analyzed for binding of the
Mamu-A*01/p11C 01 tetrameric
complex and CD11a, CD28, CD45RA, and MHC
class II DR expression
(data not shown). The phenotypes of the
Mamu-A*01/p11C tetramer-binding
and nonbinding CD8
+ T
cells were similar. Moreover, the phenotypic characteristics
were
similar to those of Mamu-A*01/p11C tetramer-binding and nonbinding
CD8
+ T cells seen in monkeys chronically infected
with SIVmac (
15).
The Mamu-A*01/p11C tetramer-binding T
cells showed a high level
of CD11a expression, a relatively low level
of CD45RA expression,
and heterogeneous CD28 and MHC class II
expression.
DNA-vaccinated monkeys develop secondary CTL responses in lymph
nodes after challenge.
Inguinal lymph nodes were obtained from the
monkeys at the time of peak viral replication on day 15 postchallenge,
as well as 28 and 42 days postchallenge. Lymphocytes from these lymph nodes were analyzed for secondary Gag epitope-specific CTL responses by
tetramer staining and functional assays. At the time of peak virus
replication during primary infection, as was seen in the peripheral
blood, the SIV gag DNA-vaccinated animals developed higher
levels of Mamu-A*01/p11C tetramer-binding CD8+ T
cells than did the control-vaccinated animals as measured in freshly
isolated lymph nodes cells (Table 4).
This difference between the experimentally and control-vaccinated
groups of monkeys was not apparent in the Gag peptide-stimulated
lymphocytes when assessed for tetramer binding or functional CTL
activity. As was the case in the peripheral blood, the differences in
the levels of SIV Gag epitope-specific CD8+ T-cell
responses in the lymph nodes of the SIV gag DNA-vaccinated animals compared to the control DNA-vaccinated animals were not apparent in the specimens obtained 28 and 42 days after challenge.
View this table:
[in this window]
[in a new window]
|
TABLE 4.
Lymph node T lymphocytes from SIV gag plasmid
DNA-vaccinated monkeys demonstrate secondary Gag epitope-specific CTL
responses after SIVsm E660 challenge
|
|
Secondary CTL responses do not alter the level of virus replication
in lymph nodes immediately after challenge.
Inguinal lymph nodes
harvested on days 15 and 42 after viral challenge were analyzed for
viral RNA levels by in situ hybridization. As shown in Table
5, no differences were apparent between
the gag DNA- and control DNA-vaccinated monkeys in the SIV
RNA detected in these lymph nodes. Therefore, the potent secondary CTL
responses seen in the SIV gag DNA-vaccinated animals failed
to affect the level of SIV replication in lymph nodes during the first
weeks following infection.
View this table:
[in this window]
[in a new window]
|
TABLE 5.
In situ hybridization studies of SIV RNA in lymph node
sections from plasmid DNA-vaccinated monkeys after SIVsm E660
virus challenge
|
|
Peripheral blood CD4+ T lymphocyte counts after
challenge.
Clinical sequelae of infection were assessed in the
weeks immediately after challenge by monitoring peripheral blood
CD4+ T-lymphocyte counts in the SIV gag DNA- and
control DNA-vaccinated monkeys (Fig. 7).
By day 50 after challenge, two of the control (V299 and T720) and one
of the experimentally vaccinated (P091) monkeys demonstrated a
significant loss of circulating CD4+ T lymphocytes. Thus,
there was no apparent effect of vaccination on CD4+
T-lymphocyte preservation in the early weeks following viral challenge.
However, one control DNA-vaccinated animal (R468), the monkey with the
highest level of virus replication, had to be euthanized on day 64 postchallenge with clinical signs of SIV disease (weight loss,
lethargy, decreasing platelet counts, and anemia). All of the animals
after day 50 have maintained their peripheral blood CD4+
T-lymphocyte counts and have remained healthy.
View larger version (29K):
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[in a new window]
|
FIG. 7.
Peripheral blood CD4+ T-lymphocyte counts
after intravenous SIVsm E660 challenge in
Mamu-A*01+ rhesus monkeys immunized with the
control (A) or the SIV gag (B) DNA vaccine.
|
|
SIV gag DNA-vaccinated monkeys have lower viral loads
at set point after viral challenge than control DNA-vaccinated
monkeys.
The best predictor of eventual clinical course in SIVsm
E660-infected macaques is plasma viral load at set point, 8 weeks after
infection (23). We therefore assessed plasma viral RNA levels through day 80 of infection in these challenged monkeys. The
control DNA-vaccinated animals all had high levels of virus replication
which peaked at day 15; these levels stabilized at set point, between
104.3 and 107.5 viral RNA copies per ml (Fig.
8). The SIV gag DNA-vaccinated animals had comparable levels of virus replication during primary infection, peaking at day 15. However, in contrast to the control DNA-vaccinated monkeys, the SIV gag DNA-vaccinated animals
were better able to control the viral infection, with three of four vaccinated animals having undetectable plasma levels of virus (<1,500
viral RNA copies/ml) at day 50 postchallenge (Fig. 8). Day 50 plasma
samples from the three SIV gag-vaccinated animals with
undetectable levels of viral RNA were then reanalyzed using a more
sensitive assay (41). Two of the three animals (T258 and
9298) maintained undetectable levels of virus (<300 viral RNA
copies/ml). The other animal (N529) showed a low level of viral
replication (6,000 viral RNA copies/ml).
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 8.
Plasma viral load analysis after intravenous SIVsm E660
challenge in Mamu-A*01+ rhesus monkeys immunized
with the control (A) or the SIV gag (B) DNA vaccine.
|
|
| |
DISCUSSION |
While a number of recent studies in nonhuman primates have
suggested that plasmid DNA immunization may prove useful as a component of an HIV vaccine, caveats associated with each of these studies have
made it difficult to generalize from these findings with any sense of
certainty. Although plasmid DNA immunization protected chimpanzees from
infection with HIV-1 (5), the poor replicative ability and
lack of pathogenicity in chimpanzees of the SF2 challenge isolate used
in that study have raised questions as to the significance of that
observation. We have previously reported that plasmid DNA priming and
recombinant envelope protein boosting protected macaques from infection
with a chimeric SIV/HIV (SHIV) challenge (22). However, the
lack of pathogenicity in macaques of the SHIV isolate used in that
study makes it difficult to conclude anything from that observation
concerning the eventual utility of such a vaccine approach in humans.
Finally, a DNA priming-recombinant poxvirus boosting approach has
recently shown some protective efficacy in macaques against a
pathogenic SHIV challenge (33). In this study, however, a
prior nonpathogenic SHIV challenge in the same monkeys obscures the
contribution of the immunizations on the protection that was seen. The
present study was designed to assess gag DNA immunization
alone in eliciting Gag-specific CTL and protection against a highly
pathogenic SIV challenge.
It has been difficult to generalize from HIV vaccine studies in
nonhuman primates not only because of the diversity of immunization regimens employed but also because of differences in the challenge viruses used. In the various studies that have been reported, the
plasmid DNA constructs used have differed in construction and in the
methods by which they have been delivered as immunogens. It is clear
now that the immunogenicity of a plasmid DNA vaccine in a higher
primate is tied to the promoters that are employed in their
construction, whether or not codon optimization has been used in their
construction, the quality and quantity of DNA used in each inoculation,
the route by which the plasmid DNA is administered, and how the plasmid
is formulated. The results of the present experiments indicate that
when at least some of these variables are optimized, a plasmid DNA
vaccine can elicit a CTL response of high enough frequency to be
detected in unstimulated whole blood using the soluble MHC class I
tetramer technology.
The central goal of this study was to assess CTL immediately following
virus challenge to determine whether the CTL that were generated
following infection arose earlier and reached a higher frequency in the
experimentally vaccinated monkeys than in those animals that received
the control vaccinations. It has been well documented that immunization
with a number of effective vaccines does not elicit sterilizing
immunity. Rather, vaccine-elicited immunity facilitates the generation
of secondary immune responses that contain the spread of replicating
virus during the first days following virus infection, limiting the
pathogenicity of the virus. Priming for the induction of such a
secondary immune response is certainly a realistic goal for a plasmid
DNA immunogen. In fact, the present study clearly indicates that
high-frequency secondary CTL responses are observed in the
gag DNA-vaccinated monkeys following infection with SIV.
Moreover, the statistically significant containment of SIV replication
at the time of set point in the gag DNA-vaccinated monkeys
suggests that those secondary CTL responses may play a useful role in
containing SIV replication.
The present study includes a clinical evaluation of the SIV-challenged
monkeys for more than 300 days following infection. It has been clearly
demonstrated that viral set point postinfection in SIVsm E660-infected
macaques predicts the clinical outcome of the infection
(23). Thus a higher virus load by 8 weeks postinfection likely indicates a rapid clinical course of disease. Therefore, there
is reason to suppose that the gag DNA-vaccinated monkeys will have a more benign clinical course than the control DNA-vaccinated monkeys. The control DNA-vaccinated monkey (R468) with the highest level of virus replication had to be euthanized on day 64 postchallenge with clinical signs of SIV disease. Interestingly, the one SIV gag DNA-vaccinated monkey that failed to control virus
replication (P091), despite a measurable decline in CD4 counts, has
remained healthy.
The use of the soluble MHC class I tetramer technology for monitoring
vaccine-elicited CTL in these monkeys is of enormous value. This
technical approach allows us to quantitate with ease and precision Gag
epitope-specific CTL in lymph nodes and peripheral blood without
relying on in vitro stimulation of the antigen-specific lymphocyte
populations. It also allows us to compare the relative efficacy of DNA
vaccination with that of other previously evaluated vaccine
technologies. Interestingly, the immunongenicity of the gag
DNA plasmid vaccine construct used in the present study does not appear
to be quite as great as the immunogenicity of a recombinant MVA
construct in rhesus monkeys (35). It will be important to assess the relative protection afforded by these different immunogens against SIV challenge. It is, however, likely that the immune responses
elicited by this DNA immunogen might be augmented by formulating the
DNA with a variety of novel adjuvants or delivering the DNA with
cytokines. Finally, there is also reason to assume that combining DNA
immunogens with live vectors may also further augment the
vaccine-elicited CTL responses.
| |
ACKNOWLEDGMENTS |
This work was supported by National Institutes of Health grants
CA50139 and AI85343.
| |
FOOTNOTES |
*
Corresponding author. Present address: Wyeth Lederle
Vaccines and Pediatrics, Bldg. 180, Rm. 216-37, 401 N. Middletown Rd., Pearl River, NY 10965. Phone: (914) 732-3036. Fax: (914) 732-4941. E-mail: eganm{at}WAR.Wyeth.com.
| |
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Journal of Virology, August 2000, p. 7485-7495, Vol. 74, No. 16
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
