2005, 79(14):8828. DOI:
Norman L. Letvin and Gary J. Nabel
Mascola, G. Kishko, Janelle C. Arthur, Ayako Miura, John R.
Michael Korioth-Schmitz, Shawn M. Sumida, Diana M. Truitt,
Dan H. Barouch, Zhi-yong Yang, Wing-pui Kong, Birgit
Vaccines in Mice and Nonhuman Primates
Immunodeficiency Virus Type 1 DNA
Immunogenicity of Human
Regulatory Element Enhances the
A Human T-Cell Leukemia Virus Type 1
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on August 30, 2012 by guest
JOURNAL OF VIROLOGY, July 2005, p. 8828–8834
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 14
A Human T-Cell Leukemia Virus Type 1 Regulatory Element
Enhances the Immunogenicity of Human Immunodeficiency
Virus Type 1 DNA Vaccines in Mice and
Dan H. Barouch,1† Zhi-yong Yang,2† Wing-pui Kong,2Birgit Korioth-Schmitz,1
Shawn M. Sumida,1Diana M. Truitt,1Michael G. Kishko,1Janelle C. Arthur,1
Ayako Miura,1John R. Mascola,2Norman L. Letvin,1
and Gary J. Nabel2*
Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston,
Massachusetts 02215,1and Vaccine Research Center, NIAID, National Institutes of
Health, Bethesda, Maryland 20892-30052
Received 18 January 2005/Accepted 18 April 2005
Plasmid DNA vaccines elicit potent and protective immune responses in numerous small-animal models of
infectious diseases. However, their immunogenicity in primates appears less potent. Here we investigate a novel
approach that optimizes regulatory elements in the plasmid backbone to improve the immunogenicity of DNA
vaccines. Among various regions analyzed, we found that the addition of a regulatory sequence from the R
region of the long terminal repeat from human T-cell leukemia virus type 1 (HTLV-1) to the cytomegalovirus
(CMV) enhancer/promoter increased transgene expression 5- to 10-fold and improved cellular immune re-
sponses to human immunodeficiency virus type 1 (HIV-1) antigens. In cynomolgus monkeys, DNA vaccines
containing the CMV enhancer/promoter with the HTLV-1 R region (CMV/R) induced markedly higher cellular
immune responses to HIV-1 Env from clades A, B, and C and to HIV-1 Gag-Pol-Nef compared with the parental
DNA vaccines. These data demonstrate that optimization of specific regulatory elements can substantially
improve the immunogenicity of DNA vaccines encoding multiple antigens in small animals and in nonhuman
primates. This strategy could therefore be explored as a potential method to enhance DNA vaccine immuno-
genicity in humans.
Plasmid DNA vaccines have shown promise as a novel vac-
cination modality based on their simplicity and versatility (31,
32, 36). In particular, DNA vaccines can elicit potent and
protective cellular and humoral immune responses in a variety
of small-animal models (10). However, they have proven sub-
stantially less immunogenic in nonhuman primate studies and
in clinical trials to date (8, 19, 33).
Several approaches have been explored to improve the im-
munogenicity of DNA vaccines. Our laboratories and others
have demonstrated that the addition of plasmids expressing
cytokines and immunomodulatory molecules can substantially
augment DNA vaccine-elicited immune responses in both mice
and nonhuman primates (3, 4, 15, 16, 21, 34, 37). However, the
practical requirements of manufacturing and establishing the
safety of the plasmid cytokines prior to the initiation of clinical
trials may prove a limitation of this strategy (7, 26). Other
approaches involve the addition of polymer adjuvants (29) and
the use of in vivo electroporation techniques (24, 35). These
strategies have similarly proven effective in animal models, but
their practical utility in clinical trials has yet to be demon-
In this study, we investigate a novel strategy involving opti-
mization of regulatory elements in the backbone of the plasmid
DNA vaccine. DNA vaccines often utilize a cytomegalovirus
(CMV) enhancer, promoter, and intron to drive high-level
expression of a transgene in mammalian cells (32, 38). Here,
we explore the effects of adding the regulatory R region from
the 5? long terminal repeat (LTR) of human T-cell leukemia
virus type 1 (HTLV-1), which acts as a transcriptional and
posttranscriptional enhancer (30). We find that these CMV/R
DNA vaccines elicit substantially higher human immunodefi-
ciency virus type 1 (HIV-1)-specific cellular immune responses
compared with the analogous parental DNA vaccines in both
mice and cynomolgus monkeys. Optimization of regulatory
elements thus represents a simple and effective strategy to
augment the immunogenicity of DNA vaccines in primates.
MATERIALS AND METHODS
Plasmid construction. The parental 1012 DNA vaccine plasmid contains the
human CMV immediate early (IE) enhancer, promoter, and intron. To construct
the CMV/R regulatory element, a SacII/HpaI fragment of the 1012 plasmid
containing the majority of the CMV IE intron was replaced with a 227-bp
EcoRV/HpaI fragment of the HTLV-1 R region (28). The resulting CMV/R
plasmid thus contains the human CMV IE enhancer/promoter, followed by the
HTLV-1 R region and a 123-bp fragment of CMV IE 3? intron. The splice donor
in the R region and the splice acceptor in the CMV IE 3? intron serve as the pair
of splicing signals. The RSV/R and mUB/R plasmids were similarly constructed
by replacing the CMV enhancer/promoter region of the CMV/R plasmid with a
381-bp AflIII/HindIII fragment of the Rous sarcoma virus (RSV) enhancer/
promoter or an 842-bp SpeI/EcoRV fragment of the mouse ubiquitin B (mUB)
* Corresponding author. Mailing address: Vaccine Research Center,
NIAID, National Institutes of Health, Room 4502, Bldg. 40, MSC-
3005, 40 Convent Drive, Bethesda, MD 20892-3005. Phone: 301-496-
1852. Fax: 301-480-0274. E-mail: firstname.lastname@example.org.
† These authors contributed equally to this work.
on August 30, 2012 by guest
enhancer/promoter, respectively. The mUB enhancer/promoter has been de-
scribed previously (40).
In vitro expression studies. Murine fibroblast 3T3 cells were transfected with
0.5 ?g or parental 1012 (CMV), CMV/R, RSV, RSV/R, mUB, mUB/R DNA
vaccines expressing HIV-1 Env gp145 ?CFI (9) in six-well plates using calcium
phosphate; 24 h after transfection, cells were harvested and lysed in 50 mM
HEPES, 150 mM NaCl, 1% NP-40 with protease inhibitors and 10 ?g total
protein was electrophoresed by sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (PAGE), and gp145 expression was assessed by Western blot
analysis. A 1:5,000 dilution of human HIV immunoglobulin G (IgG) was utilized
as the primary antibody, and a 1:5,000 dilution of horseradish peroxidase-con-
jugated goat anti-human IgG was utilized as the secondary antibody. The blots
were developed with the ECL Western blot developing system (Amersham
Biosciences, Piscataway, NJ).
Animals and immunizations. Six- to 8-week-old BALB/c mice (Charles River
Laboratories, Wilmington, MA) were immunized intramuscularly with 50 ?g
HIV-1 DNA vaccines expressing either clade B Env gp145?CFI (9) or a Gag-
Pol-Nef fusion protein (13) in 100 ?l sterile saline divided between the right and
left quadriceps muscles. Adult cynomolgus monkeys (Bioqual, Rockville, MD)
were immunized intramuscularly with 8 mg of a multivalent HIV-1 DNA vaccine
in 1 ml sterile saline delivered by Biojector inoculation into the right and left
quadriceps muscles. This multiclade, multigene DNA vaccine has been previ-
ously described (17) and includes a mixture of plasmids expressing HIV-1 Env
gp145?CFI from clades A, B, and C and a clade B Gag-Pol-Nef fusion protein
(four plasmids in a 1:1:1:3 ratio) or these antigens delivered on separate plasmids
as individual genes (six plasmids in a 1:1:1:1:1:1 ratio). All murine and primate
studies were approved by our Institutional Animal Care and Use Committees.
ICS assays. CD4?and CD8?T-lymphocyte responses were evaluated by
intracellular cytokine staining (ICS) for gamma interferon (IFN-?) and tumor
necrosis factor alpha (TNF-?) as previously described (17). Briefly, splenocytes
from immunized mice were harvested and incubated with pools of 15-amino-acid
peptides overlapping by 11 amino acids (2.5 ?g/ml each) covering the entire
HIV-1 Env protein, followed by treatment with 10 ?g/ml brefeldin A (Sigma, St.
Louis, MO). Cells were then fixed, permeabilized, and stained using rat anti-
mouse CD3, CD4, CD8, IFN-?, and TNF-? monoclonal antibodies (BD Pharm-
ingen, San Diego, CA). The IFN-?- and TNF-? positive cells in the CD4?and
CD8?cell populations were analyzed with the program FlowJo (Tree Star,
ELISPOT assays. ELISPOT assays were utilized to assess IFN-? production
by murine splenocytes or monkey peripheral blood mononuclear cells (PBMC)
as previously described (4, 5). For cell depletion studies, murine splenocytes were
incubated with magnetic microbeads coated with anti-CD4 (L3T4) or anti-CD8
(Ly-2) monoclonal antibodies (Miltenyi Biotec, Auburn, CA), and separation
using MiniMACS columns was performed according to the manufacturer’s in-
structions. Cell depletions were approximately 95 to 98% efficient. IFN-? secre-
tion was then measured in response to overlapping 15-amino-acid peptides
spanning the entire Env, Gag, Pol, or Nef protein.
Statistical analyses. Immunologic data are presented as means with standard
errors. Statistical analyses were performed with GraphPad Prism version 4.01
(GraphPad Software, Inc., 2004). Comparisons of mean cellular immune re-
sponses between groups of animals were performed by two-tailed nonparametric
Mann-Whitney tests. In all cases, P values of less than 0.05 were considered
Optimization of expression from plasmid DNA vaccines in
vitro. To develop improved plasmid DNA expression vectors,
alternative regulatory elements were constructed and assessed
for their ability to drive in vitro expression of a modified HIV-1
Env protein, gp145?CFI (9). The regulatory R sequence from
the 5? LTR of HTLV-1 (12, 30) was incorporated between the
CMV enhancer/promoter and splicing acceptor site in the pa-
rental 1012 plasmid backbone to construct CMV/R DNA vac-
cines (Fig. 1A). We also incorporated this R element down-
stream from the Rous sarcoma virus and mouse mUB
enhancer/promoter elements to create, respectively, RSV/R
and mUB/R DNA vaccines.
To assess antigen expression from plasmids containing these
transcriptional regulatory elements, we transfected 3T3 cells
with these expression vectors and measured gp145?CFI ex-
pression by Western blots. The expression of gp145?CFI from
the CMV/R plasmid was 5- to 10-fold higher than expression
from the parental 1012 plasmid (Fig. 1B, lanes 2 versus 3).
Thus, addition of the HTLV-1 R element substantially in-
creased antigen expression driven by the CMV promoter.
Baseline expression from the mUB plasmid was higher than
from the 1012 plasmid but was not further enhanced by addi-
tion of the R element (Fig. 1B, lanes 4 versus 5), suggesting
that the effects of adding the R element were promoter de-
pendent. An increase in expression was observed in the RSV/R
compared to the RSV plasmid (Fig. 1B, lanes 6 versus 7).
Expression from RSV plasmids is routinely lower than from
the 1012 plasmid (23).
FIG. 1. In vitro expression of HIV-1 Env gp145 ?CFI from various
DNA expression vectors. (A). Schematic diagram of the alternative
cellular and viral regulatory elements in eukaryotic expression plas-
mids. (B). Western blot analysis of 3T3 cells transfected with empty
parental 1012 (CMV) (lane 1) or those expressing HIV Env gp145
?CFI with the CMV (lane 2), CMV/R (lane 3), mUB (lane 4), mUB/R
(lane 5), RSV (lane 6), or RSV/R (lane 7).
VOL. 79, 2005 ENHANCEMENT OF DNA VACCINES8829
on August 30, 2012 by guest
Immunogenicity of improved expression vectors in mice. We
reasoned that enhanced antigen expression may translate to
improved immunogenicity of these novel DNA vaccines in
vivo. To explore this possibility, we immunized BALB/c mice
(n ? 5/group) with 50 ?g of the parental 1012 DNA vaccine or
the CMV/R, RSV/R, mUB, or mUB/R DNA vaccines express-
ing HIV-1 Env gp145?CFI (9). Mice were immunized three
times at weeks 0, 2, and 6. On day 10 following the final
immunization, splenocytes were assessed for Env-specific cel-
lular immune responses by IFN-? and TNF-? intracellular
cytokine staining assays. The CMV/R DNA vaccine elicited
approximately twofold higher CD4?(P ? 0.15) and CD8?(P
? 0.043) T lymphocyte responses compared with the parental
1012 DNA vaccine expressing the same antigen (Fig. 2). In
contrast, the RSV/R, mUB, and mUB/R DNA vaccines did not
elicit enhanced CD8?immune responses, suggesting that the
HTLV-1 R element selectively improved immunogenicity in
the context of the CMV promoter.
We next compared the immunogenicity of the parental 1012
DNA vaccine and the CMV/R DNA vaccine expressing other
antigens. We immunized mice (n ? 8/group) with sham plas-
mids or with these DNA vaccines expressing the HIV-1 Gag-
Pol-Nef fusion protein (13). Mice were immunized twice at
weeks 0 and 6, and cellular immune responses were assessed by
IFN-? ELISPOT assays using splenocytes harvested 3 weeks
after the initial or boost immunization. Consistent with the
prior experiment, we observed approximately twofold higher
Gag- (P ? 0.038) and Pol-specific (P ? 0.020) responses elic-
ited by the CMV/R DNA vaccine compared to the parental
1012 DNA vaccine following the initial immunization (Fig.
3A). Nef-specific responses elicited by both plasmids remained
low. Following the boost immunization, responses elicited by
the CMV/R DNA vaccine remained approximately twofold
higher than responses elicited by the parental DNA vaccine
using both unfractionated splenocytes (Fig. 3B) and CD8-de-
pleted splenocytes (Fig. 3C).
Immunogenicity of CMV/R DNA vaccines in cynomolgus
monkeys. A number of strategies have improved the immuno-
genicity of DNA vaccines in inbred strains of mice, but few
have been assessed in outbred nonhuman primates (4, 24, 29).
FIG. 2. Immunogenicity of plasmid DNA vaccines expressing
HIV-1 Env gp145?CFI in mice. Mice (n ? 5/group) were immunized
with 50 ?g of the parental 1012 DNA vaccine or the CMV/R, RSV/R,
mUB, or mUB/R DNA vaccines expressing HIV-1 Env gp145 ?CFI at
weeks 0, 2, and 6. After the third immunization, splenocytes were
assessed for Env-specific cellular immune responses by IFN-? and
TNF-? ICS assays. (A) % CD3?CD4?IFN-?/TNF-??and (B) %
CD3?CD8?IFN-?/TNF-??splenocytes are shown.
FIG. 3. Immunogenicity of CMV/R plasmid DNA vaccines ex-
pressing HIV-1 Gag-Pol-Nef in mice. Mice (n ? 8/group) were immu-
nized with 50 ?g of the parental 1012 DNA vaccine or the CMV/R
DNA vaccine expressing HIV-1 Gag-Pol-Nef at weeks 0 and 6. Spleno-
cytes were assessed for Gag-, Pol-, and Nef-specific cellular immune
responses by IFN-? ELISPOT assays 3 weeks after the (A) initial and
(B) week 6 boost immunizations. (C) Splenocytes depleted of CD8?T
lymphocytes were also assessed in similar ELISPOT assays following
the boost immunization.
8830BAROUCH ET AL.J. VIROL.
on August 30, 2012 by guest
We therefore compared the immunogenicity of the parental
1012 DNA vaccine with CMV/R DNA vaccine expressing mul-
tiple HIV-1 antigens in cynomolgus monkeys. We immunized
two groups of adult cynomolgus monkeys (n ? 6/group) with
four-plasmid mixtures of 1012 or CMV/R DNA vaccines ex-
pressing HIV-1 Env gp145 ?CFI from clades A, B, and C and
the Gag-Pol-Nef fusion protein from clade B in a 1:1:1:3 ratio.
This multiclade, multivalent DNA vaccine has been previously
described (17) and is currently being evaluated in clinical trials.
A third group of monkeys was included to investigate
whether separating the Gag-Pol-Nef fusion protein into sepa-
rate genes encoded on separate plasmids would further in-
crease immune responses to these antigens. This third group of
monkeys received a six-plasmid mixture of CMV/R DNA vac-
cines encoding HIV-1 Env gp145 from clades A, B, and C and
separate Gag, Pol, and Nef proteins from clade B in a 1:1:1:
1:1:1 ratio. All monkeys received three immunizations of 8 mg
total DNA vaccine at weeks 0, 4, and 8.
We first compared cellular immune responses against Env
clade A, Env clade B, Env clade C, and Gag, Pol, and Nef from
clade B in monkeys that received the four-plasmid mixtures
under the control of CMV (1012) or CMV/R regulatory ele-
ments. Monkeys immunized with the parental 1012 DNA vac-
cines developed low and sporadic IFN-? ELISPOT responses
to Env 2 weeks following the second immunization at week 6,
and no clear responses above background were detected to
Gag, Pol, and Nef (Fig. 4A). In contrast, monkeys immunized
with the analogous CMV/R DNA vaccines exhibited signifi-
cantly higher responses to all antigens (Fig. 4B). Compared to
the parental 1012 DNA vaccines, the CMV/R DNA vaccines
elicited ?10-fold higher ELISPOT responses to Gag (P ?
0.0022), Pol (P ? 0.0043), and Nef (P ? 0.041) and 7- to 9-fold
higher responses to Env clade A (P ? 0.026), B (P ? 0.0087),
and C (P ? 0.030) at this time point. These results demonstrate
that the CMV/R DNA vaccines were markedly more immu-
nogenic than the parental 1012 DNA vaccines for multiple
HIV-1 antigens in nonhuman primates.
We also observed that separating the Gag-Pol-Nef fusion
protein into individual genes encoded on different plasmids
further improved these responses. In particular, monkeys that
received the 6-plasmid mixture of CMV/R DNA vaccines de-
veloped fourfold higher responses to Gag (P ? 0.0022), a trend
towards twofold higher responses to Pol (P ? 0.19), and four-
fold higher responses to Nef (P ? 0.049) (Fig. 4C), compared
to animals that received the four-plasmid mixture of CMV/R
DNA vaccines that included the Gag-Pol-Nef fusion protein
(Fig. 4B). As expected, Env-specific responses between these
two groups of monkeys that received the four-plasmid and
six-plasmid mixtures of CMV/R DNA vaccines were compara-
ble (P ? 0.48), since these groups received the same Env
The evolution of mean IFN-? ELISPOT responses in these
groups of monkeys was evaluated at weeks 0, 2, 6, 10, and 12.
Following the third DNA immunization at week 8, responses
increased in all groups of monkeys (Fig. 5). At week 10, the
parental 1012 DNA vaccines elicited Env- and Pol-specific
responses in the majority of animals, although Gag- and Nef-
specific responses remained low (Fig. 5A). In contrast, the
CMV/R DNA vaccines elicited potent and broad responses to
all antigens (Fig. 5B and C). At week 10, the four-plasmid
CMV/R DNA vaccines (Fig. 5B) elicited ?10-fold higher
ELISPOT responses to Gag (P ? 0.0022) and Nef (P ?
0.0022), fourfold higher ELISPOT responses to Pol (P ?
0.043), and trends toward 1.5- to 4-fold higher responses to
FIG. 4. Immunogenicity of multivalent CMV/R DNA vaccines ex-
pressing HIV-1 antigens in cynomolgus monkeys. Cynomolgus mon-
keys (n ? 6/group) were immunized with the four-plasmid multivalent
(A) 1012 DNA vaccines or (B) CMV/R DNA vaccines expressing
HIV-1 Env gp145 ?CFI from clades A, B, and C and a Gag-Pol-Nef
fusion protein. (C) A third group of monkeys was immunized with a
six-plasmid multivalent CMV/R DNA vaccine expressing HIV-1 Env
gp145 ?CFI from clades A, B, and C and separate gag, pol, and nef
genes. All monkeys received 8 mg total DNA at weeks 0, 4, and 8.
IFN-? ELISPOT responses were assessed 2 weeks after the second
immunization at week 6.
VOL. 79, 2005ENHANCEMENT OF DNA VACCINES 8831
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Env clades A, B, and C (Fig. 5B), compared with the four-
plasmid parental 1012 DNA vaccines (Fig. 5A). Gag-, Pol- and
Nef-specific responses remained highest in the animals that
received the six-plasmid CMV/R DNA vaccines with these
genes encoded on separate plasmids (Fig. 5C). These studies
confirm that the CMV/R DNA vaccines elicited substantially
higher-magnitude and broader cellular immune responses to
multiple antigens compared with the parental 1012 DNA vac-
cines. Thus, including the HTLV-1 R element and separating
the gag, pol, and nef genes significantly enhanced the immuno-
genicity of HIV-1 DNA vaccines in nonhuman primates.
The relatively low immunogenicity of DNA vaccines in clin-
ical trials to date has led to the development of various strat-
egies to improve the immunogenicity of these vaccines. Ap-
proaches that have proven effective in nonhuman primates
include the use of plasmid cytokines, polymer adjuvants, and in
vivo electroporation techniques (4, 24, 29). A disadvantage of
these approaches is that they require additional materials or
devices that add manufacturing, regulatory, and practical com-
plexities that may limit their large-scale clinical application. In
this study, we investigated the potential utility of a strategy
aimed at optimizing regulatory elements in the plasmid DNA
vaccine. We demonstrated that the addition of the HTLV-1 R
element to a CMV expression cassette markedly increased
DNA vaccine immunogenicity in both mice and cynomolgus
monkeys. Importantly, such a modification does not involve
any additional components or devices and thus does not in-
crease vaccine complexity. Optimizing immunogenicity by en-
gineering novel regulatory elements into the plasmid backbone
therefore represents a simple, practical, and effective approach
that could be advanced into clinical trials.
A previous study demonstrated that adding the HTLV-1 R
element downstream of an SV40 early promoter increased
transgene expression by approximately 10- to 40-fold in vitro
(30). The present study extends this observation by demon-
strating that the R element also potentiates expression from a
strong CMV promoter/enhancer. More importantly, we
showed that this enhanced expression translates into signifi-
cantly augmented immune responses in both rodents and non-
human primates. We speculate that the beneficial effects of the
R region likely involve recruiting key cellular transcription
factors that enhance transgene transcription (12, 39), although
the precise mechanism of action remains to be determined.
The R region may also function as an internal ribosome entry
site, suggesting that it may act at a posttranscriptional step as
well (2). Alternatively, it is possible that the combination of
CMV enhancer and HTLV-1 R regions stimulate optimal ex-
pression in professional antigen-presenting cells, which would
best facilitate the antigen-specific T-cell response. Interest-
ingly, the RSV/R and mUB/R DNA vaccines exhibited com-
parable transgene expression in vitro compared with CMV/R
DNA vaccines (Fig. 1) but nevertheless failed to result in
improved immunogenicity (Fig. 2). Thus, enhanced expression
in vitro is not the sole determinant of enhanced immunoge-
nicity in vivo.
In both mice and cynomolgus monkeys, CMV/R DNA vac-
cines expressing HIV-1 antigens elicited higher cellular im-
mune responses than the parental 1012 DNA vaccines express-
ing the same antigens. However, the magnitude of the
observed effects differed substantially between the two species.
While the CMV/R DNA vaccines elicited only twofold higher
responses in mice (Fig. 3), the CMV/R DNA vaccines elicited
?10-fold higher cellular immune responses to Gag, Pol, and
Nef and seven- to ninefold higher responses to Env after two
FIG. 5. Mean responses to multivalent CMV/R DNA vaccines ex-
pressing HIV-1 antigens in cynomolgus monkeys. (A to C) Cynomol-
gus monkeys were vaccinated as described in the legend to Fig. 4.
Mean and standard errors of IFN-? ELISPOT responses in these
groups of animals were assessed at weeks 0, 2, 6, 10, and 12 following
8832BAROUCH ET AL.J. VIROL.
on August 30, 2012 by guest
immunizations in cynomolgus monkeys (Fig. 4 and 5). We
suspect that this difference may reflect the lower baseline im-
munogenicity of the parental 1012 DNA vaccines in nonhuman
primates and that the beneficial effects of the R element may
appear more striking in limiting situations. Consistent with this
observation, the R element had the greatest effect at enhancing
the weakest responses elicited by the parental 1012 DNA vac-
cine against Gag and Nef. However, Env- and Pol-specific
cellular immune responses were also significantly higher when
induced by CMV/R DNA vaccines compared with the parental
1012 DNA vaccines.
We also observed that the six-plasmid mixture of CMV/R
DNA vaccines that included Gag, Pol, and Nef on separate
plasmids elicited significantly higher cellular immune re-
sponses to these antigens compared to the four-plasmid mix-
ture of CMV/R DNA vaccines that included the Gag-Pol-Nef
fusion protein. These effects are particularly notable since the
separate gag, pol, and nef plasmids were each utilized at one-
third the dose of the plasmid encoding the Gag-Pol-Nef fusion
protein. We speculate that this may reflect enhanced transla-
tion or mRNA stability of the shorter genes compared with the
fusion gene, which might potentially affect antigen processing
A limitation of the current study is that we were not able to
assess the protective efficacy afforded by the CMV/R DNA
vaccines. Since HIV-1 does not infect cynomolgus monkeys, we
could not perform viral challenges in these animals. However,
accumulating data have confirmed the importance of cellular
immune responses in controlling HIV-1 replication in humans
and simian immunodeficiency virus replication in rhesus mon-
keys (14, 22, 27). Moreover, vaccines aimed at eliciting virus-
specific cellular immune responses have afforded partial con-
trol of simian-human immunodeficiency virus and simian
immunodeficiency virus challenges in rhesus monkeys (1, 4, 11,
18, 20, 25, 29). Thus, we believe that the markedly increased
magnitude and breadth of HIV-1-specific cellular immune re-
sponses afforded by the CMV/R DNA vaccines in nonhuman
primates in the present study will likely prove beneficial in the
development of second-generation DNA vaccines for HIV-1
and other pathogens. In particular, incorporating the HTLV-1
R element and utilizing separate genes in place of fusion genes
represent simple and practical strategies to improve DNA vac-
cines. These strategies could be advanced readily into clinical
We thank Srinivas Rao, Vi Dang, Kristin Beaudry, Faye Yu, Kristi
Martin, Darci Gorgone, and Michelle Lifton for generous advice,
assistance, and reagents.
We acknowledge support from the NIH Vaccine Research Center
(G.J.N. and N.L.L.) and NIH grant AI-58727 (D.H.B.).
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