Enhanced potency of plasmid DNA microparticle human immunodeficiency virus vaccines in rhesus macaques by using a priming-boosting regimen with recombinant proteins.
ABSTRACT DNA vaccines have been used widely in experimental primate models of human immunodeficiency virus (HIV), but their effectiveness has been limited. In this study, we evaluated three technologies for increasing the potency of DNA vaccines in rhesus macaques. These included DNA encoding Sindbis virus RNA replicons (pSINCP), cationic poly(lactide-co-glycolide) (PLG) microparticles for DNA delivery, and recombinant protein boosting. The DNA-based pSINCP replicon vaccines encoding HIV Gag and Env were approximately equal in potency to human cytomegalovirus (CMV) promoter-driven conventional DNA vaccines (pCMV). The PLG microparticle DNA delivery system was particularly effective at enhancing antibody responses induced by both pCMV and pSINCP vaccines and had less effect on T cells. Recombinant Gag and Env protein boosting elicited rapid and strong recall responses, in some cases to levels exceeding those seen after DNA or DNA/PLG priming. Of note, Env protein boosting induced serum-neutralizing antibodies and increased frequencies of gamma interferon-producing CD4 T cells severalfold. Thus, PLG microparticles are an effective means of delivering DNA vaccines in nonhuman primates, as demonstrated for two different types of DNA vaccines encoding two different antigens, and are compatible for use with DNA prime-protein boost regimens.
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ABSTRACT: The present study was undertaken to determine immune response and protection efficacy of a spike (S) protein fragment containing neutralizing epitopes (4F/4R) of turkey coronavirus (TCoV) by priming with DNA vaccine and boosting with the recombinant protein from the corresponding DNA vaccine gene segment. Turkeys were vaccinated by priming with either one dose (G1-750DP) or two doses (G3-750DDP) of 750μg DNA vaccine expressing 4F/4R S fragment and boosting with one dose of 200μg 4F/4R S fragment. One dose of 100μg DNA vaccine mixed with polyethyleneimine (PEI) and sodium hyaluronate (HA) followed by one dose of 750μg DNA vaccine and one dose of 200μg 4F/4R S fragment were given to the turkeys in group G2-100DPH. After infectious challenge by TCoV, clinical signs and TCoV detected by immunofluorescence antibody (IFA) assay were observed in less number of turkeys in vaccination groups than that in challenge control groups. TCoV viral RNA loads measured by quantitative real-time reverse transcription-PCR were lower in vaccinated turkeys than those in challenge control turkeys. The turkeys in G3-750DDP produced the highest level of TCoV S protein-specific antibody and virus neutralization (VN) titer. Comparing to the turkeys in G1-750DP, significantly less TCoV were detected by IFA in the turkeys in G2-100DPH receiving an extra dose of 100μg DNA mixed with PEI and HA. The results indicated that DNA-prime protein-boost DNA vaccination regimen targeting TCoV S fragment encompassing neutralizing epitopes induced humoral immune response and partially protected turkeys against infectious challenge by TCoV.Veterinary Immunology and Immunopathology 02/2013; · 1.88 Impact Factor
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ABSTRACT: The first clinical trial of an MF59(®)-adjuvanted influenza vaccine (Novartis) was conducted 20 years ago in 1992. The product that emerged (Fluad(®), Novartis) was licensed first in Italy in 1997 and is now licensed worldwide in 30 countries. US licensure is expected in the coming years. By contrast, many alternative adjuvanted vaccines have failed to progress. The key decisions that allowed MF59 to succeed in such a challenging environment are highlighted here and the lessons that were learned along the way are discussed. MF59 was connected to vaccines that did not succeed and was perceived as a 'failure' before it was a success. Importantly, it never failed for safety reasons and was always well tolerated. Even when safety issues have emerged for alternative adjuvants, careful analysis of the substantial safety database for MF59 have shown that there are no significant concerns with widespread use, even in more 'sensitive' populations.Expert Review of Vaccines 01/2013; 12(1):13-30. · 4.22 Impact Factor
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ABSTRACT: Identification of optimal antigen(s) and adjuvant combination(s) to elicit potent, protective, and long-lasting immunity has been a major challenge for the development of effective vaccines against chronic viral pathogens, such as HIV-1, for which there are not yet any licensed vaccines. Here we describe the use of a novel adjuvant approach employing Carbopol 971P(®) NF (hereafter referred to as Carbopol971P), a cross-linked polyanionic carbomer, in combination with the Novartis proprietary oil-in-water adjuvant, MF59, as a potentially safe and effective adjuvant to augment humoral immune responses to the HIV-1 envelope glycoprotein (Env). Intramuscular immunization of small animals with recombinant Env glycoprotein (gp140) formulated in Carbopol971P plus MF59 gave significantly higher titers of binding and virus neutralizing antibodies as compared to immunization using gp140 with either MF59 or Carbopol971P alone. In addition, the antibodies generated were of higher avidity. Importantly, the use of Carbopol971P plus MF59 did not cause any serious adverse reactions or any obvious health problems in animals upon intramuscular administration. Hence, the Carbopol971P plus MF59 adjuvant formulation may provide a benefit for future vaccine applications.Vaccine 02/2012; 30(17):2749-59. · 3.77 Impact Factor
JOURNAL OF VIROLOGY, July 2005, p. 8189–8200
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 13
Enhanced Potency of Plasmid DNA Microparticle Human
Immunodeficiency Virus Vaccines in Rhesus Macaques
by Using a Priming-Boosting Regimen with
Gillis R. Otten,1* Mary Schaefer,1Barbara Doe,1Hong Liu,1Indresh Srivastava,1Jan zur Megede,1
Jina Kazzaz,1Ying Lian,1Manmohan Singh,1Mildred Ugozzoli,1David Montefiori,2
Mark Lewis,3† David A. Driver,1‡ Thomas Dubensky,1§ John M. Polo,1
John Donnelly,1Derek T. O’Hagan,1Susan Barnett,1
and Jeffrey B. Ulmer1
Chiron Corporation, Emeryville, California1; Duke University Medical Center, Durham, North Carolina2;
and Southern Research Institute, Frederick, Maryland3
Received 11 November 2004/Accepted 17 March 2005
DNA vaccines have been used widely in experimental primate models of human immunodeficiency virus
(HIV), but their effectiveness has been limited. In this study, we evaluated three technologies for increasing the
potency of DNA vaccines in rhesus macaques. These included DNA encoding Sindbis virus RNA replicons
(pSINCP), cationic poly(lactide-co-glycolide) (PLG) microparticles for DNA delivery, and recombinant protein
boosting. The DNA-based pSINCP replicon vaccines encoding HIV Gag and Env were approximately equal in
potency to human cytomegalovirus (CMV) promoter-driven conventional DNA vaccines (pCMV). The PLG
microparticle DNA delivery system was particularly effective at enhancing antibody responses induced by both
pCMV and pSINCP vaccines and had less effect on T cells. Recombinant Gag and Env protein boosting elicited
rapid and strong recall responses, in some cases to levels exceeding those seen after DNA or DNA/PLG
priming. Of note, Env protein boosting induced serum-neutralizing antibodies and increased frequencies of
gamma interferon-producing CD4 T cells severalfold. Thus, PLG microparticles are an effective means of
delivering DNA vaccines in nonhuman primates, as demonstrated for two different types of DNA vaccines
encoding two different antigens, and are compatible for use with DNA prime-protein boost regimens.
Both neutralizing antibodies and cell-mediated immunity
(CMI) likely will be required to protect against viruses that can
establish chronic infections, such as human immunodeficiency
virus (HIV) and hepatitis C virus (HCV). Moreover, in animal
models for HIV, both neutralizing antibodies against Env (9,
35–37) and cytotoxic T lymphocytes (CTL) that target multiple
viral antigens (3–5, 17, 18, 21, 26, 30, 32, 38, 39, 45, 48, 51–53,
59) can contribute to protection through prevention of infec-
tion and clearance of virus-infected cells, respectively. Vac-
cines consisting of inactivated pathogens or recombinant pro-
teins generally are not effective at inducing CTL and typically
are used to induce protective antibodies. In contrast, viruses
and intracellular bacteria can induce CTL responses, in part
due to neoexpression of the antigens during infection.
Plasmid DNA vaccines were born out of the need for induc-
ing both antibody and CMI responses, including CTL, without
the problems associated with live organism-based vaccines,
such as potential reversion to virulence and antivector immu-
nity that can limit boosting. Indeed, DNA vaccines that express
antigens from strong viral promoters have been used to elicit
protective antibodies and CMI in many animal models (14, 23).
However, naked DNA vaccines, i.e., plasmid DNA in saline,
have proven to be only modestly potent in humans, thereby
limiting their utility. Many approaches have been explored to
improve DNA vaccine potency, including better expression
vectors, DNA formulation and delivery systems, adjuvants, and
the use of booster vaccines.
We developed an alternative DNA vector that launches a
self-amplifying Sindbis virus (alphavirus) RNA replicon (15,
24). With this vector, once the RNA replicon is transcribed in
and exported from the nucleus, the RNA is replicated in the
cytoplasm by replicon-encoded enzymes and expresses the
gene of interest using a Sindbis subgenomic promoter. Plas-
mid-based replicons utilizing a Sindbis-derived RNA replicase
have been shown to be immunogenic in several murine models
at 10- to 1,000-fold lower doses than conventional plasmid
vectors (24, 33, 46). Similar DNA vaccines based on Semliki
Forest virus (SFV) have also been demonstrated to be effective
(1). So far, no data have been reported for alphavirus-based
DNA vaccines in primates.
We have addressed the inefficient delivery of DNA vaccines,
both within tissues and into cells (16), which has limited their
effectiveness. The vast majority of injected DNA is degraded
by nucleases in the extracellular spaces and within phagocytic
cells. One approach to improved delivery is to facilitate uptake
of DNA by antigen-presenting cells (APCs) using micropar-
ticles, such as chitosan (34, 49), polyethylenimine (31), and
* Corresponding author. Mailing address: Chiron Corporation, 4560
Horton St., Mail Stop 4.3, Emeryville, CA 94608. Phone: (510) 923-
2965. Fax: (510) 923-2586. E-mail: firstname.lastname@example.org.
† Present address: BioQual, Rockville, MD 20850.
‡ Present address: BioMedica Inc., San Diego, CA 92121.
§ Present address: Cerus Corporation, Concord, CA 94520.
poly(lactide-co-glycolide) (PLG) (25, 27, 28, 41, 54). We have
reported that DNA adsorbed to cationic PLG microparticles is
a very effective delivery system that markedly enhances im-
mune responses in small animals (6, 42, 54). PLG microparticle
adsorption captures DNA with high efficiency but still allows
for rapid release of the DNA from particles. The mode of
action of the PLG delivery system may involve uptake and
expression of DNA by APCs as well as prolonged expression of
antigen by protecting plasmid DNA from nuclease digestion
The studies presented here investigated the utility of two
pSINCP DNA vector (versus conventional cytomegalovirus
[CMV] promoter-driven) and the PLG microparticle DNA
delivery system, in rhesus macaques. Furthermore, we evalu-
ated these technologies both in the context of priming immune
responses and following boosting with recombinant proteins to
address compatibility and potential synergies.
MATERIALS AND METHODS
Plasmids. The plasmid pCMVKm2.GagMod.SF2, constructed from the ex-
pression vector pCMVKm2 (8) and containing a codon-optimized HIVSF2gag
sequence, has been described previously (61). A codon-optimized env sequence
encoding the ectodomain (gp140) of env from the SF162 HIV isolate (2) was
cloned into the expression vector pCMVLink (61) to produce the plasmid
The Sindbis virus-derived plasmid replicon pSINCP was constructed by mod-
ifying a previously described version of a Sindbis plasmid replicon, pSIN1.5 (24).
The entire nonstructural protein (nsP) gene coding sequences from the human
dendritic cell tropic strain of Sindbis virus (SINCR) (22) were substituted for the
existing nsP sequences in pSIN1.5. The vector was modified further by substitu-
tion of the CMV promoter-containing plasmid backbone with that from
pCMVLink (61). To prepare the pSINCP plasmid DNA vaccines, the gag and env
inserts were excised from the pCMVKm2 and pCMVLink constructs and were
inserted into pSINCP immediately downstream of the Sindbis subgenomic junc-
tion region promoter. Plasmid DNA was prepared using endotoxin-free kits
(QIAGEN, Valencia, CA).
Production and purification of o-gp140SF162?V2 protein. The sequence en-
coding a V2-deleted ectodomain of the HIV SF162 env gene protein from
HIV-1SF162 (gp140SF162?V2) was cloned as a 2.1-kb EcoRI-XbaI DNA frag-
ment containing two mutations in the primary and three mutations in secondary
cleavage sites (56). The resulting env sequence was cloned into the EcoRI and
XbaI sites of the pCMV3 expression vector, which was transfected into CHO
cells, and stable env-expressing cell lines were selected. The highest expressing
gp140SF162?V2-CHO cell clone was adapted to low serum medium (0.5% fetal
bovine serum) and adjusted to a cell density and perfusion rate that facilitated
the maximum expression of gp140SF162?V2 protein in its oligomeric confor-
mation (o-gp140SF162?V2) (55, 56). The bioreactor-adapted gp140?V2-CHO
cell clone was used to seed a 12.5-liter bioreactor for production runs. At the end
of the run, medium was concentrated 20-fold through a 100-kDa membrane and
stored at ?80°C in the presence of 1 mM EDTA and 1 mM EGTA. The
purification of o-gp140SF162?V2 was performed as described elsewhere (55,
Preparation of PLG microparticles for DNA adsorption. Cationic PLG-cetyl-
trimethylammonium bromide (CTAB) microparticles were prepared using a
modified solvent evaporation process as described previously (6, 54). The mi-
croparticles were prepared using an homogenizer (IKA, Wilmington, N.C.) at
high speed to emulsify 10 ml of a 5% (wt/vol) PLG polymer solution in meth-
ylene chloride with 1 ml of phosphate-buffered saline (PBS). The primary emul-
sion was then added to 50 ml of distilled water containing CTAB (0.5% [wt/vol]).
This resulted in the formation of a water-in-oil-in-water emulsion that was stirred
at 6,000 rpm for 12 h at room temperature, allowing the methylene chloride to
evaporate. The resulting microparticles were washed four times in distilled water
by centrifugation at 10,000 ? g and lyophilized. Plasmid DNA was adsorbed to
PLG-CTAB microparticles by incubating 1 mg of DNA in 1 ml of 1? Tris-EDTA
(TE) buffer with 100 mg of microparticles overnight at 4°C with gentle rocking.
The microparticles were then pelleted by centrifugation at 11,424 ? g for 10 min,
washed with 1? TE buffer, recentrifuged, and suspended in 5 ml of deionized
water and lyophilized. The size distribution of the microparticles was determined
using a particle size analyzer (Mastersizer; Malvern Instruments).
Preparation of HIV p55 gag protein adsorbed onto anionic PLG micropar-
ticles. As described previously (29), microparticles were prepared by homoge-
nizing 10 ml of 6% (wt/vol) PLG polymer solution in methylene chloride with 40
ml of distilled water containing SDS (1% [wt/vol]) at high speed using a 10-mm
probe. This resulted in an oil-in-water emulsion, which was stirred at 1,000 rpm
for 12 h at room temperature, and the methylene chloride was allowed to
evaporate. The resulting microparticles were filtered through 38-?m mesh,
washed three times in distilled water, and lyophilized. The size distribution of the
microparticles was determined using a Mastersizer (Malvern Instruments) par-
ticle size analyzer.
Fifty micrograms of lyophilized PLG-sodium dodecyl sulfate (SDS) particles
were incubated with 0.5 mg of p55Gag protein in 10 ml of 25 mM Borate buffer,
pH 9, with 6 M urea. Particles were left on a lab rocker at room temperature for
5 h. The microparticles were separated from the incubation medium by centrif-
ugation, washed once with borate buffer with 6 M urea, washed three times with
distilled water, and lyophilized.
The amount of protein adsorbed to microparticles was determined by dissolv-
ing 10 mg of the microparticles in 2 ml of 5% SDS–0.2 M NaOH solution at room
temperature. Protein concentration was measured by bicinchoninic acid protein
assay (Pierce, Rockford, IL). The Zeta potential for both blank and adsorbed
microparticles was measured using a Zeta analyzer (Malvern Instruments).
Vaccination. Rhesus macaques were housed at Southern Research Institute
(Frederick, MD) in accordance with Institutional Animal Care and Use Com-
mittee guidelines. Animals were immunized by intramuscular injection on weeks
0, 4, and 14 with DNA vaccines encoding HIVSF2p55Gag (0.5 mg) and HIVSF162
gp140Env (1.0 mg), with or without adsorption to PLG microparticles. Rhesus
were boosted with yeast-derived p55Gag protein (0.2 mg) adsorbed to anionic
PLG microparticles (Gag/PLG) (44) at week 29. Finally, the animals were
boosted with 0.1 mg CHO cell-derived o-gp140SF162?V2 (oligomeric, V2 loop-
deleted gp140Env protein) administered with the oil-in-water MF59 adjuvant
(Env/MF59) (47) at weeks 38 and 75.
Antibody assays. Antibodies against Env and Gag proteins were measured by
an enzyme-linked immunosorbent assay (ELISA). MaxiSorp plates (Nalge Nunc,
Rochester, NY) were coated overnight at 4°C with 50 ?l of 5 ?g/ml of Env
protein or Gag protein in PBS, pH 7.0. The coated wells were blocked for 1 h at
37°C with 150 ?l of 5% goat serum (Gibco BRL, Grand Island, NY) in phos-
phate-buffered saline (PBS). Serum samples were diluted initially 1:25 or 1:100
in the blocking buffer followed by threefold serial dilutions. The bound antibod-
ies were detected with horseradish peroxidase-conjugated goat anti-monkey im-
munoglobulin G (IgG) (Southern Biotechnology Associates, Birmingham, AL)
diluted 1:5,000 with the blocking buffer and incubated for 1 h at 37°C. For
development, 3,3?,5,5?-tetramethylbenzidine was incubated for 15 min, and the
reaction was stopped by adding 2 N HCl. The assay plates were then read on an
ELISA plate reader at an absorbance wavelength of 450 nm. A standard was
included on each plate, and a reference value of the standard was used for the
normalization of the sample ELISA titers. The titers represent the dilution
factors producing an optical density of 0.5.
Virus-neutralizing antibodies were assessed against human peripheral blood
mononuclear cells (PBMC)-grown, homologous HIV-1SF162virus, as previously
described (7). Titers were calculated as the greatest serum dilution that reduced
virus growth in humans by 80%, as measured by p24Gag antigen production.
Preimmune sera were used as negative controls.
Purification of Rhesus peripheral blood mononuclear cells (PBMC) and der-
ivation of B-lymphoblastoid cell lines (B-LCL). Rhesus PBMC were separated
from heparinized whole blood on Ficoll-Hypaque gradients. To derive rhesus
B-lymphoblastoid cell lines, PBMC were exposed to herpesvirus papio-contain-
ing culture supernatant from the 594S cell line in the presence of 0.5 ?g/ml
Cyclosporine A (Sigma). Rhesus PBMC were cultured at 2 ? 106to 3 ? 106per
well in 1.5 ml in 24-well plates for 8 days in AIM-V:RPMI 1640 (50:50) culture
medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum
(AR10). Antigen-specific cells were stimulated by the addition of a pool of either
Gag or Env peptides (20-mers, overlapping by 10; 10.7 ?g/ml total peptide).
Recombinant human IL-7 (15 ng/ml; R&D Systems, Minneapolis, MN) was
added at the initiation of culture. Recombinant human IL-2 (Proleukin; 20
IU/ml; Chiron) was added on days 1, 3, and 6.
51Cr-release assay for CTL activity. Autologous B-LCL were infected with
recombinant vaccinia viruses (rVV) expressing gag (rVVgag-polSF2) or env
(rVVgp160envSF162) and then labeled overnight with Na2[51Cr]O4(NEN, Bos-
ton, MA; 10 ?Ci per 2.5 ? 105B-LCL) and washed. rVV-infected,51Cr-labeled
B-LCL were added (2,500 per round-bottom well) to duplicate wells containing
threefold serial dilutions of cultured PBMC. Unlabeled B-LCL (1 ? 105per
8190OTTEN ET AL.J. VIROL.
well) were added to inhibit nonspecific cytolysis. After 4 h, 50 ?l of supernatant
from each culture was harvested, added to Lumaplates (Packard, Meriden, CT),
and counted with a Wallac Microbeta TriLux liquid scintillation counter (Perkin
Elmer Life Sciences, Boston, MA).51Cr released from lysed targets was normal-
ized by the formula percent specific51Cr release ? 100 ? (mean experimental
release ? spontaneous release)/(maximum release ? spontaneous release),
where spontaneous release is mean counts per minute (cpm) released from
target cells in the absence of PBMC and maximum release is mean cpm released
from target cells in the presence of 0.1% Triton X-100. For each dilution of
PBMC culture, a response toward a given antigen (Gag or Env) was scored as
positive if, for PBMC stimulated in culture with that particular antigen, lysis of
antigen-bearing targets exceeded lysis of irrelevant targets by at least 10% and if,
for targets bearing that antigen, lysis by PBMC stimulated with that antigen
exceeded lysis by PBMC stimulated with the irrelevant antigen by at least 10%.
Cultures were scored as positive if the above criteria were satisfied for at least
two consecutive dilutions of culture.
Lymphoproliferation assay (LPA). PBMC (2 ? 105) were incubated in flat-
bottom microtiter wells in a volume of 0.2 ml AR10 in the absence or presence
of p55 Gag protein (3 ?g/ml) or a pool of Env peptides (16 ?g/ml). Six replicate
cultures were established. After 4 days of incubation, [3H]thymidine ([3H]TdR;
Amersham, Piscataway, NJ) was added (1 ?Ci/well). Following overnight incu-
bation, cultures were harvested onto glass microfiber filters. Cellular uptake of
[3H]TdR was measured with a Microbeta TriLux liquid scintillation counter
(Perkin Elmer). Stimulation indices (SI) were calculated using the formula SI ?
(mean cpm in the presence of antigen)/(mean cpm in the absence of antigen). SI
values of ?5.0 were considered positive.
Intracellular cytokine staining (ICS) and flow cytometry. Rhesus PBMC were
incubated overnight at 37°C in the absence or presence of antigen (Gag peptide
pool, 30 ?g/ml, or Env peptide pool, 30 ?g/ml). Anti-CD28 (1 ?g/ml; Pharmin-
gen, San Diego, CA) was added as a source of costimulation, and Brefeldin A
(1:1,000; Pharmingen) was added to prevent cytokine secretion. Duplicate cul-
tures were prepared for each stimulation condition. After overnight incubation,
PBMC were stained for cell surface CD4 (anti-CD4 allophycocyanin conjugate,
clone SK3; Becton Dickinson, San Jose, CA) and CD8 (anti-CD8? peridinin
chlorophyll protein conjugate, clone SK1; Becton Dickinson), permeabilized
with Cytofix/Cytoperm (Pharmingen), and then stained for intracellular gamma
interferon (IFN-?) (monoclonal antibody 4S.B3, phycoerythrin conjugate;
Pharmingen) and tumor necrosis factor alpha (TNF-?) (MAb11, fluorescein
isothiocyanate [FITC] conjugate; Pharmingen). Stained cells were analyzed with
a FACSCalibur flow cytometer (Becton Dickinson). For each culture condition,
the mean frequency of cytokine-positive cells was calculated for the duplicate
cultures. To estimate the antigen-specific frequency, the unstimulated mean
frequency was subtracted from the stimulated mean frequency.
Antibody responses to pCMV and pSINCP DNA vaccines
adsorbed to cationic PLG microparticles. When administered
in saline, both pCMV and pSINCP vectors induced low to
intermediate titers of gag- and env-specific antibodies (Fig. 1).
At no time was there a significant difference in the gag anti-
body titers induced by the two vectors; however, pCMVenv-
vaccinated animals had higher titers of env-specific antibodies
(peak geometric mean titer, 2,460) than those vaccinated with
pSINCPenv (peak geometric mean titer, 70). When pCMV
and pSINCP vectors were adsorbed to PLG microparticles,
high titers of gag- and env-specific antibodies (peak geometric
mean titers, 5,000 to 20,000) were induced with just two doses.
The enhancing effects of PLG formulation were statistically
significant (P ? 0.05, analysis of variance [ANOVA] on log-
transformed titers), with the exception of pCMVenv (Fig. 1C),
where env titers 2 weeks after vaccination with pCMVenv/PLG
FIG. 1. Gag and Env-specific antibodies induced by DNA or DNA/PLG priming and protein boosting. Groups of 5 rhesus macaques were
vaccinated at weeks 0, 4, and 14 with pCMVgag and pCMVenv DNA/saline or DNA/PLG (A and C) or pSINCPgag and pSINCPenv DNA or
DNA/PLG (B and D). Rhesus were boosted at week 29 with recombinant Gag protein adsorbed onto anionic PLG microparticles and at weeks
38 and 75 with oligomeric gp140env protein formulated with MF59 adjuvant. Anti-Gag or -Env antibodies are plotted as group geometric mean
ELISA titers for DNA/saline (open symbols) and DNA/PLG (closed symbols), and error bars extend to 95% confidence upper limits.
VOL. 79, 2005 DNA MICROPARTICLE VACCINES 8191
were nonetheless fivefold greater than pCMVenv-induced ti-
ters. Env-specific antibody titers induced by PLG-adsorbed
pSINCP and pCMV were indistinguishable. Thus, PLG-ad-
sorption of pCMV and pSINCP vectors expressing either env
or gag led to substantial increases in antibody titers. Despite
high env-specific antibody titers in rhesus vaccinated with
PLG-adsorbed pCMVenv or pSINCPenv, no HIV neutralizing
activity was detected (Fig. 2).
CD4 T-cell responses to DNA vaccines adsorbed to cationic
PLG microparticles. CD4 T-cell responses to DNA vaccina-
tion were measured both by LPA and by cytokine flow cytom-
etry. Gag-specific proliferation was detected 2 weeks after the
first DNA vaccination and reached peak levels 2 weeks after
the second DNA vaccination (Fig. 3). A third DNA vaccination
did not increase stimulation indices. Considering stimulation
indices (SI) of ?5 as indicating significant proliferation, we
noted that the numbers of vaccinees with SI ? 5 2 weeks after
the first vaccination were 0 (pCMVgag), 2 (pCMVgag/PLG), 3
(pSINCPgag), and 4 (pSINCPgag/PLG), suggesting that PLG-
formulated DNA vaccines were better than unformulated
DNA and that the pSINCP vector was more effective than
pCMV. However, these differences between the vaccination
groups did not achieve statistical significance (P ? 0.08,
Kruskal-Wallis test). Rhesus #40 (pCMVgag/PLG) and rhesus
#37 (pSINCP) had much higher gag-specific proliferative re-
sponses than the others in their respective groups. There were
no obvious factors that contributed to the high responses of
these two individuals. When these outliers were excluded from
the analysis, no statistically significant differences in the mag-
nitudes of the stimulation indices between four groups were
observed (Fig. 3E and F), although the pSINCPgag/PLG-
vaccinated rhesus had higher responses on average than the
For env-specific proliferation assays, we used a pool of over-
lapping env peptides that stimulated higher responses than
recombinant env protein, which may modulate T-cell prolifer-
ation through a direct interaction with CD4. We were unable
to perform env LPA until 7 weeks after the second DNA
vaccination and therefore may not have observed maximal
responses, which were likely to have occurred 2 weeks after the
second DNA vaccination, as observed for Gag (Fig. 3). The
pCMVenv and pSINCPenv DNA vaccines and their PLG-
formulated versions induced env-specific T cells capable of
proliferating in LPA assays (Fig. 4). As observed for Gag,
rhesus #37 and #40 had much higher stimulation indices than
the others in their respective groups, indicating that these two
animals were unusually responsive to vaccination. Overall,
stimulation indices of the rhesus vaccinated with PLG-formu-
lated env DNA were not greater than those of the rhesus
vaccinated with unformulated env DNA (Fig. 4E and F).
Somewhat higher stimulation indices were seen in the
pSINCP-vaccinated animals compared to the pCMV-vacci-
nated animals, but the differences were not statistically signif-
To assess antigen-specific T-cell function and differentiation,
PBMC were stimulated overnight with pools of synthetic pep-
tides, stained for cell surface CD4 and CD8 and for intracel-
lular IFN-? and TNF-?, and analyzed by flow cytometry for
cytokine-positive cells. We focused our analysis on IFN-?/
TNF-?-double positive cells, which were the most prevalent
antigen-specific cells. Antigen-specific IFN-?/TNF-?-double
positive CD4 T-cell frequencies were near background 2 weeks
after the first vaccination (Fig. 5A and B). This was in contrast
to proliferative responses that were detectable at that time
(Fig. 3). Peak levels of antigen-specific IFN-?/TNF-?-double
positive CD4 T cells were observed 2 weeks after the second
DNA vaccination (Fig. 5C and D) and decreased thereafter
(Fig. 5E and F), with no apparent increase after the third DNA
vaccination (data not shown). Frequencies of antigen-specific
CD4 T cells were higher to some extent for the groups vacci-
nated with PLG-formulated DNA, but the differences were not
FIG. 2. Serum-neutralizing antibody titers in vaccinated rhesus.
HIV-1SF162neutralizing titers of sera obtained 2 weeks after the third
DNA vaccination and after each Env protein boost are shown for all
vaccines (A). Neutralizing titers of sera obtained 2 weeks after the
second Env protein boost are grouped by env DNA and formulation
used in the vaccine priming phase (B). Horizontal bars indicate group
geometric mean titers.
8192 OTTEN ET AL.J. VIROL.
FIG. 3. Gag-specific lymphoproliferation of rhesus PBMC. Stimulation indices for individual animals vaccinated with pCMVgag/saline (A),
pCMVgag/PLG (B), pSINCPgag/saline (C), or pSINCPgag/PLG (D). Figure legends show animal numbers. Vertical dotted lines denote DNA
vaccinations at weeks 0, 4, and 14 and Gag protein boost at week 29. Group geometric mean stimulation indices and 95% confidence upper limits
are shown for pCMVgag (E)- and for pSINCPgag (F)-vaccinated animals. Outliers (#40 and #37) were excluded.
VOL. 79, 2005 DNA MICROPARTICLE VACCINES8193
FIG. 4. Env-specific lymphoproliferation of rhesus PBMC. Stimulation indices for individual animals vaccinated with pCMVenv/saline (A),
pCMVenv/PLG (B), pSINCPenv/saline (C), or pSINCPenv/PLG (D). Figure legends show animal numbers. Vertical dotted lines denote DNA
vaccinations at weeks 0, 4, and 14 and Env protein boosts at weeks 38 and 75. Group geometric mean stimulation indices and 95% confidence upper
limits are shown for pCMV (E)- and for pSINCP (F)-vaccinated animals. Outliers (#40 and #37) were excluded.
8194OTTEN ET AL. J. VIROL.
FIG. 5. Cytokine-producing antigen-specific CD4 T cells induced by DNA or DNA/PLG vaccination. Antigen-specific CD4 T cells that
produced both IFN-? and TNF-? were enumerated by flow cytometry. Frequencies of cytokine-positive CD4 T cells are plotted for individual
animals. Horizontal bars indicate group mean frequencies. Gag-specific (A, C, E) and Env-specific (B, D, F) responses are shown for 2 weeks after
the first vaccination (week 2) (A and B) and for 2 weeks (C and D) and 7 weeks (E and F) after the second DNA vaccination (weeks 6 and 11,
VOL. 79, 2005 DNA MICROPARTICLE VACCINES8195
statistically significant, in part due to animal-to-animal varia-
CD8 T-cell responses to DNA vaccines adsorbed to cationic
PLG microparticles. Because the PBMC were stained for both
CD4 and CD8, we could also study the CD8?CD4?T-cell
population; however, antigen-specific, IFN-?/TNF-?-double
positive CD8 T cells were generally at background levels.
Therefore, PBMC were cultured with antigens and growth
factors under conditions that stimulated the production of
CTL. Assays for cytolytic activity were performed using as
targets51Cr-labeled autologous B-LCL that had been infected
with recombinant vaccinia virus vectors for gag (rVVgagpolSF2)
or env (rVVgp160envSF162). Figure 6 illustrates antigen-spe-
cific cytolytic activity from individual animals that were ob-
served 2 weeks after the second vaccination. Levels of cytolysis
were heterogeneous and ranged from undetectable to high.
Because of this heterogeneity, we defined criteria for positivity
(Materials and Methods) and scored each culture against those
criteria (Table 1). CTL response rates for pCMVgag/saline and
pCMVgag/PLG were similar, although CTL appeared earlier
with pCMVgag/PLG vaccination. The pCMVgag vaccines (sa-
line or PLG) appeared more effective at inducing Gag CTL
responses than pSINCPgag (saline or PLG), with a higher
frequency of responders (Table 1). However, neither pCMV
nor pSINCP was effective at inducing Env CTL, with or with-
out PLG. The differences between Gag and Env CTL response
rates could reflect vector-related differences, i.e., the gag vac-
cines were superior to the env vaccines, or differences in the
assays, e.g., inhibitory peptides in the env pool but not in the
gag pool, inefficient processing and presentation of env by the
rVVenv-infected B-LCL targets, or more epitopes for CTL
recognition in gag than in env.
p55Gag protein boosting. As noted above, the third DNA
vaccination did not lead to increases in either antibodies or T
cells beyond what was observed after the second vaccination.
Because recombinant proteins have been shown in other sys-
tems to boost immune responses in DNA-primed rhesus all
animals were given intramuscular recombinant gag protein
that was adsorbed to anionic PLG microparticles. This formu-
lation has been reported to be immunogenic in rhesus (44). As
shown in Fig. 1A and B, after boosting with gag protein, the
anti-gag antibody titers were approximately 10-fold higher in
the animals primed with PLG/CTAB-DNA than those primed
with naked DNA. Thus, more effective priming by PLG-for-
mulated DNA was associated with higher antibody titers after
gag protein boosting.
Most animals showed increased gag-specific lymphoprolif-
eration after boosting with Gag protein/PLG (Fig. 3A to D);
however, postboosting stimulation indices mostly did not ex-
ceed those induced by DNA priming. When we compared
rhesus primed with pCMVgag/PLG to those primed with
pCMVgag/saline (Fig. 3E) or pSINCPgag/PLG-primed to
pSINCPgag/saline rhesus (Fig. 3F), there were no differences
in their postboosting proliferative responses. Consistent with
the proliferation data, no increases in IFN-?/TNF-? double-
positive gag-specific CD4 T cells were seen in the cytokine flow
cytometry assays performed after the Gag protein boosting
(data not shown). Finally, recombinant Gag protein did not
restore Gag-specific CD8 CTL to the levels seen 2 weeks after
the second DNA priming vaccination. Thus, in this DNA
prime-protein boost regimen, PLG-adsorbed Gag protein was
more effective at boosting antibodies than T cells.
Env protein boosting. Two weeks after boosting with oligo-
meric Env protein (at week 38), antibody titers increased to
high levels (geometric mean ? 27,000) in all animals (Fig. 1).
Unlike Gag, there were no significant differences in Env anti-
body titers between the DNA/PLG-primed and DNA/saline-
primed animals. Over the next 34 weeks, Env antibody titers
declined but remained above preboost levels. The second Env
protein boost at week 75 restored antibody titers to levels seen
after the first Env protein boost. Env protein boosting was
sufficient to induce neutralizing antibodies: 50% of the animals
had measurable neutralizing antibodies after the first protein
boost and 100% had neutralizing antibodies after the second
protein boost (Fig. 2A). Furthermore, neutralizing titers in-
creased from a geometric mean of 5 after the first Env boost to
a geometric mean of 64 after the second boost (P ? 0.001, t test
on log-transformed titers). The neutralizing titers were not
statistically different between the different DNA-primed
groups (Fig. 2B, ANOVA on log-transformed titers).
Two weeks after Env protein boosting, Env-specific lympho-
proliferation SIs had increased 4- to 12-fold over preboost
levels (Fig. 4) and were 2- to 5-fold higher than after env DNA
priming (7 weeks after second DNA prime). Despite strongly
increased env-specific proliferation after the first env protein
boost, frequencies of IFN-?-positive CD4 T cells were rela-
tively low (?0.1%; Fig. 7). However, after the second Env
protein boost, these frequencies increased substantially and
significantly (P ? 0.001, Mann-Whitney) to a mean of 0.6%.
Four animals had env-specific frequencies of ?1%, and the
highest was 3.8%. As expected, CTL responses were not
boosted by Env protein boosts (Table 1).
In summary, boosting DNA-primed macaques with Env pro-
tein resulted in neutralizing antibodies and IFN-?-producing,
proliferation-competent CD4 T cells that were markedly
higher than achieved by DNA priming.
This study evaluated DNA vaccine expression and formula-
tion technologies for plasmids encoding HIVSF2p55Gag and
HIVSF162gp140 Env that were sequence modified for high
levels of expression. First, two fundamentally different types of
DNA vectors were compared: a conventional plasmid vector,
pCMV, containing genes driven by a highly active CMV pro-
moter, and a plasmid-based Sindbis replicon vector, pSINCP.
The present study demonstrates that in rhesus macaques the
pSINCP Sindbis-based DNA replicon vaccines were similar in
potency to pCMV, at least at the dose used. Thus, pSINCP
may provide an alternative to pCMV for clinical testing. It
remains to be determined whether pSINCP is more potent
than pCMV at limiting doses, as shown previously for rodents
(24, 33, 46, 50).
We have developed DNA-adsorbed PLG microparticles as a
means of increasing the delivery of DNA into cells, particularly
APCs (16). DNA/PLG vaccines are very potent in small ani-
mals (42, 54), and the results presented here demonstrate that
DNA/PLG led, in particular, to a marked enhancement of
antibody responses, with a lesser effect on CD4 T cells and
CTL. Why antibody titers were more greatly increased than
8196 OTTEN ET AL.J. VIROL.
FIG. 6. CTL activity from cultured PBMC obtained 2 weeks after the second DNA or DNA/PLG vaccination. Shown are CTL from individual
animals vaccinated with pCMV/saline (A), pCMV/PLG (B), pSIN/saline (C), pSIN/PLG (D), or not vaccinated (E). Gag- and Env-stimulated
(stim) cultures were tested against both rVVgag-infected targets and rVVenv-infected targets. Culture dilutions meeting the criteria for positivity
(see Materials and Methods) are indicated (*).
VOL. 79, 2005DNA MICROPARTICLE VACCINES8197
were T-cell responses is not known. Our T-cell assays mea-
sured proliferative capacity, cytokines (IFN-?, TNF-?), and
CTL, and it is possible that we may have seen a greater PLG
effect had we directly measured T-cell help for B cells. On the
other hand, relatively greater effect of PLG delivery on anti-
body response may have implications for understanding the
mode of action of DNA vaccines in primates. Substantially
increased antibody titers may be a result of prolonged expres-
sion of antigen in situ, as was seen in mice (54). In vitro studies
have shown that PLG microparticles protect plasmid DNA
from nuclease digestion (12), thereby potentially prolonging
the availability of the plasmid in vivo. This may be important,
since the amount of antigen produced in situ by a DNA vaccine
is very small (60). For T-cell responses, the relatively lower
increase in responses suggests that expression of antigen in
APCs is not a limiting step in primates, assuming PLG func-
tioned similarly here as it may in mice. It has been shown that
expression of DNA vaccine antigens in APCs is not required
for induction of T-cell responses, including CTL (57). In fact,
in mice it appears that cross-priming of antigens produced in
non-APCs such as muscle cells is the predominant means of
eliciting CTL responses after needle injection (10, 11, 13, 19).
These results suggest that means to targeting DNA vaccines
for expression in non-APCs may also be useful, particularly for
T-cell responses. Indeed, electroporation, which is a physical
delivery system that results in increased uptake of DNA by
myocytes (40, 58), was relatively more effective for enhance-
ment of T-cell responses in rhesus macaques than it was for
To determine the optimal number of DNA or DNA/PLG
vaccinations, we monitored immune responses after each vac-
cination. Two vaccinations were required to obtain maximal
antibody (Fig. 1) and T-cell responses (Fig. 2, 3, 4, Table 1).
Interestingly, while Gag-specific lymphoproliferative responses
were detectable after the first vaccination, IFN-?- and TNF-
?-producing CD4 T cells were detected only after the second
DNA vaccination, suggesting that two vaccinations were re-
quired for effector cell differentiation. We noted that a third
DNA vaccination at week 14 did not result in further increases
in antibody titers or antigen-specific T cells, particularly for the
DNA/PLG groups. The vaccination regimen may not have
allowed sufficient time between immunizations, which has been
suggested previously to be important in primates (20). Another
contributing factor may have been the antibody levels at the
time of the third immunization. In the DNA/PLG groups, the
anti-Gag and anti-Env antibody titers were still high (Fig. 1),
which may have prevented effective boosting by neutralizing
the small amount of antigen produced in situ.
Despite the apparent ineffectiveness of the third DNA vac-
cination, the effectiveness of boosting DNA-primed macaques
with recombinant protein was demonstrated here, as has been
reported previously (9). In this case, we sought to determine if
a strong DNA prime could affect the quantity and quality of
responses after the protein boost. Interestingly, in only some
cases did a stronger DNA prime result in higher levels of
immune responses after the protein boost, such as for anti-Gag
antibodies in the DNA/PLG groups versus naked DNA groups.
In contrast, anti-Env antibodies (both ELISA and neutralizing)
were not different after the protein boost, despite significantly
higher responses in the DNA/PLG groups after the DNA
prime. Reasons for these differences are not known but may be
related to the nature of the recombinant antigens (Gag versus
Env) or the adjuvant/delivery systems used (anionic PLG mi-
croparticles versus MF59 oil-in-water emulsion). Importantly
though, the quality of the anti-Env T-cell responses was influ-
enced by DNA vaccine priming. Strong Th1-type cytokine
(IFN-?) production from CD4 T cells was observed after Env
protein boosting, despite the use of MF59, a Th2-promoting
adjuvant. In fact, in all animals the CD4 IFN-? response was
higher after the second Env protein boost compared to the
DNA prime. In contrast, priming of macaques with Env pro-
tein vaccines in other studies did not induce this type of Th1
response (unpublished observations). Therefore, strong DNA
vaccine priming induces a Th1 type of T-cell response that can
be substantially boosted with recombinant Env protein.
In summary, we have demonstrated that two fundamentally
different DNA vectors (pCMV, pSINCP) are effective in ma-
caques. The potencies of both DNA vaccines were enhanced
by delivery via PLG microparticles, and boosting with recom-
binant proteins further increased antibodies and T cells. The
combined use of alternative DNA vectors, improved formula-
tion and delivery systems, and adjunct technologies such as
FIG. 7. IFN-?-producing antigen-specific CD4 T cells induced by
Env protein boosting of DNA (saline or PLG)-primed rhesus. PBMC
were assayed by CFC 2 weeks after Env protein boosts. Frequencies of
env-specific IFN-?-positive CD4 T cells are plotted for individual an-
imals. Horizontal bars indicate group mean frequencies: 73 ? 10?5
after first Env protein boost, 601 ? 10?5after second Env protein
TABLE 1. Gag and Env CTL responses of monkeys vaccinated
with DNA and boosted with proteina
No. of Gag- or Env-CTL-positive rhesus at wk:
26 1116 2024 31 4077
aAnti-Gag and anti-Env CTL responses are shown as the number of respond-
ing animals per group of 5, based on criteria described in Materials and Methods.
bND, not determined.
8198OTTEN ET AL.J. VIROL.
booster vaccines will likely be vital to effective use of DNA
vaccines in humans.
We gratefully acknowledge contract support from NIH-NIAID-
We thank Nancy Miller (NIAID) for helpful suggestions.
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