Methods 31 (2003) 207–216
www.elsevier.com/locate/ymeth
DNA vaccine strategies: candidates for immune modulation
and immunization regimens
Nicole A. Doria-Rose and Nancy L. Haigwood*
Departments of Microbiology and Pathobiology, University of Washington and Viral Vaccines Program, Seattle Biomedical Research Institute,
4 Nickerson Street, Seattle, WA 98109, USA
Accepted 25 April 2003
Abstract
DNA vaccine strategies can differ greatly, with significant effects on the outcome of immunization. In this article, we discuss
plasmid design strategies and vaccine regimens. Effectiveness against a pathogen can be affected by the choice of antigen and inclusion of multiple antigens. Gene expression and the resulting immune response can be improved by gene modification and choice
of promoters. In designing vaccine regimens, one must consider further dose, timing of doses, adjuvants, and routes of vaccination.
Many vaccines are enhanced by combining DNA with other vaccines in ‘‘prime-boost’’ regimens, in which the second vaccine is
often a recombinant viral vector or purified protein subunit. Prime-boost vaccines including DNA can elicit immune responses that
differ in magnitude, quality, and balance of cellular and humoral responses from those elicited by single components and thus
provide further enhancement for DNA immunizations.
Ó 2003 Elsevier Science (USA). All rights reserved.
Keywords: Antigen; Adjuvant; Plasmid; Promoter; Prime–boost
1. Introduction
When plasmid vectors expressing human influenza
virus proteins showed protection of mice from disease
after live influenza challenge [1,2], it was a milestone
event in the field of vaccinology. DNA vaccines offer
many advantages over conventional immunization approaches: they are simple to make and deliver, and they
elicit both humoral and cellular immunity. Intracellular
expression of native, oligomeric, surface-embedded antigens in the context of host cell surface proteins recapitulates the route that viral and parasitic pathogens
use. Finally, multiple antigens can be delivered by
combining several expression plasmids in the same immunization. Thus, DNA vaccines hold the promise of
delivering single or multiple native pathogen antigens
combined with safety, effectiveness, and ease of production.
*
Corresponding author. Fax: 1-206-284-0313.
E-mail address: [email protected] (N.L. Haigwood).
Many features must be considered in designing an
effective DNA immunization regimen. The choice of
antigen(s), vector, delivery route, dose, timing, adjuvants, and boosting agents will all influence the outcome
of vaccination because they affect the magnitude and
quality of immunity elicited. In this article, we address
issues of vaccine strategy pertinent to DNA vaccine
design. In Sections 2–8, we focus on issues of plasmid
design because gene expression is the sine qua non of
DNA vaccines. In Sections 9–13, we consider the effects
of regimen alterations, including ‘‘prime–boost’’ protocols that combine DNA with other vaccines.
2. Plasmid design
The first choice in designing a DNA vaccine is the
target antigen(s). One must select the pathogen genes and
the form of the gene, whether secreted, intracellular,
membrane bound, wild type, or mutated. Once the gene(s)
is chosen, various modifications are commonly used to
improve or alter the immunogenicity of the vaccines.
1046-2023/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S1046-2023(03)00135-X
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N.A. Doria-Rose, N.L. Haigwood / Methods 31 (2003) 207–216
3. Antigenic targets: choice of genes
A common starting point for vaccine antigen selection is the target of the immune responses of infected or
immune hosts. For example, patients infected with HIV
have neutralizing antibodies directed against the envelope protein, and long-term non-progressors may have
higher levels of this antibody. Therefore, most HIV
vaccine candidates include envelope antigens [3]. Recent
data indicating the importance of cytotoxic T lymphocytes (CTL) in control of HIV, especially in virus-exposed seronegative people [4], have led to studies in
which the gag gene, or major CTL epitopes encoded
therein, is included or is even used alone [5]. Likewise,
vaccines under development for Ebola virus are based
on the antigens to which survivors have a response [6].
An alternative approach is to examine other vaccines for
the same pathogen as a starting point. A hepatitis B
DNA vaccine uses the surface antigen HBsAg and thus
is directly comparable to the licensed recombinant protein vaccine [7]. A multi-gene malaria DNA vaccine
candidate includes antigens to which immune responses
are seen in volunteers who were immunized with irradiated Plasmodium parasites, as well as in partially immune individuals living in malaria-endemic areas [8].
When the antigens capable of eliciting a protective response are unknown, it is possible to use genome-based
or library approach, as reviewed in this issue (see Allan
and Wren).
4. Nature of the desired immune response: Th1 versus Th2
Another criterion for vaccine design is the ability of
the antigen to modulate the immune response, specifically the T helper (Th) subtype. Th1 immune responses
are primarily cellular, whereas Th2 immune responses
are mostly associated with antibody production. For
many pathogens, such as Leishmania, Th1 responses
correlate with a protective outcome, while Th2 responses are associated with non-protective or even
harmful results [9–11]. For other diseases, including
helminth worms, a Th2 response is desirable [12,13].
A Th1 or Th2 bias may be measured by cytokine
profiles produced by antigen-stimulated cells (see Sasaki
et al.). IL-4 and IL-10 are typically used as markers for
Th2, while IFNc and TNFa are considered Th1 cytokines [14–16]. Th types are best understood and defined
in the murine system. In mice, the bias may also be
measured by examining the ratio of antigen-specific
IgG2a–IgG1. A high proportion of IgG2a indicates a
Th1 response, while IgG1 indicates a Th2 response
[9,17–19].
In general, Th1 responses are considered better for
controlling intracellular pathogens, such as viruses;
however, many viruses, such as rabies, can be controlled
by antibody alone. Furthermore, assays to determine
Th1/Th2 bias are useful in comparing different regimens,
but they do not always reflect the extent of cellular or
humoral responses that have been elicited; one may find
both antibody and CTL in an animal with bias to Th1 or
Th2 [18]. When the protective response is poorly defined, caution should be exercised when choosing a route
or adjuvant to deliberately bias the response.
5. Antigen combinations
Unlike attenuated, inactivated, or protein subunit
vaccines, DNA vaccines are usually in the same form
(purified plasmid); hence, there are no issues of incompatible buffers or stabilizers [20]. Many DNA vaccine
designs take advantage of this property and can encode
multiple antigens, with several benefits. For example,
different antigens are likely to be targets of humoral and
cellular responses. In HIV infection and related nonhuman primate models, only the envelope is targeted by
neutralizing antibody, whereas several other gene
products may be targets of CTL. In addition, infected
hosts develop unique individual CTL responses to viral
gene products [21,22]. This development may be due to
the variability of the MHC genes in these outbred
populations, such that different MHC haplotypes are
able to present epitopes from a range of genes. Therefore, it is appropriate to use more than one antigen in an
AIDS vaccine. Furthermore, HIV sequences vary tremendously; the inclusion of more antigens increases the
chance of providing at least one that is cross-reactive
with the circulating virus to which a given vaccinee may
be exposed. Such a strategy has been successful for influenza, which mutates rapidly and has several circulating forms [23]. Likewise, many pathogens undergo
antigenic variation; multiple versions of the same gene,
from divergent isolates, may be useful when combined in
a single vaccine. This strategy is analogous with the use
of several strains in a single inactivated vaccine, as is
commonly provided for influenza and polio. Another
advantage of using multiple genes is the ability to target
more than one stage of the life cycle of a pathogen. In
malaria, no single antigen has been shown to be protective, whereas a vaccine with four genes, two each
from the blood and liver stages of the parasite, protected
macaques from a lethal challenge with Plasmodium
knowlesi [8]. Multiple antigen DNA vaccines may even
be used to protect against several different pathogens.
Pediatric vaccines against multiple pathogens, such as
measles–mumps–rubella, are highly desirable because
they reduce the number of injections that must be given
[20]. A recent study showed that a combination DNA
vaccine protected mice from influenza, respiratory syncytial virus, and herpes simplex virus 1 [24]. Mixing
antigens may improve responses in unexpected ways: for
N.A. Doria-Rose, N.L. Haigwood / Methods 31 (2003) 207–216
example, a recent study showed that adding an anthrax
antigen to a Yersinia pestis DNA vaccine enhanced
protection from plague in a mouse model [25].
Some antigens bias the immune response to a Th1 or
Th2 type; in a combination, one antigen may predominate. Often, a Th2 bias will overcome a Th1 response.
Influenza virus hemagglutinin (HA) gene predominantly
elicits IgG2a in mice, but a mix of HA and the bovine
herpes virus gD gene elicits IgG1 to both antigens (Th2
type) [17]. A similar outcome is observed with measles
vaccines. The measles nucleoprotein gene alone elicits a
greater IgG2a response, whereas the HA gene alone
gives more IgG1. When mixed, both genes elicit more
IgG1 [18]. This effect can be seen in more direct measures of immune function. It has been shown that adding HIV vpr to a multi-gene DNA vaccine given to mice
reduced cellular responses to the other genes and altered
the IgG isotype ratios [26].
6. Modification of genes to influence immune responses
Genes may be modified to produce a protein that is
naturally secreted, membrane bound, cytosolic, or associated with an organelle. Altering the cellular localization can influence the immune response. For example,
Japanese encephalitis virus envelope (E) protein was
modified for secretion by deleting the transmembrane
portion of the protein; the secreted form elicited much
higher antibody titers and better protection than the
wild-type protein [27]. A study of various forms of
hepatitis B virus surface antigens showed that, while all
forms elicited comparable levels of CTL, the secreted
forms elicited higher antibody titers and a higher IgG1/
IgG2a ratio [28]. In addition, a study by Hasan et al. [29]
showed that immunization of mice with a plasmid expressing a truncated form of the Varicella zoster virus
gE antigen tended to promote a Th2-type response,
whereas a construct expressing a non-truncated form
induced a Th1 response. A naturally secreted protein
can be modified to enhance the rate of secretion by replacement of its signal sequence with that of a highly
expressed protein, such as tissue-type plasminogen activator (t-PA) [30–33]. In addition, t-PA signal sequences have been used to change an intracellular
protein into a secreted one, increasing the production of
antibodies in immunized subjects [34]. Likewise, a secreted protein can be retained in the cell if the signal
sequence is removed [27]. (See Ertl and Thomsen for
molecular engineering of constructs containing multiple
antigen-encoding genes.)
Targeting of gene products for degradation can also
improve immunogenicity, although with concomitant
reduction of protein expression. This paradoxical effect
is achieved because the proteins are degraded by the
proteasome complex and loaded onto MHC class I for
209
antigen presentation. Induction of CTL is enhanced,
although antibody induction may be eliminated. Targeting can be achieved by adding a ubiquitination signal
[35], or ubiquitin itself as a protein fusion [36].
Some pathogen genes are immunosuppressive, yet
they may be attractive vaccine components because they
are major targets of immune responses in natural infection. If the determinants of the immunosuppressive
function are known, and do not overlap with important
epitopes, it may be beneficial to mutate them. For example, HIV Nef downregulates expression of the signaling molecule CD4 on helper T-lymphocytes, making
it undesirable in a vaccine; however, it does contain
important CTL epitopes. A mutant nef gene that was
incapable of this immunosuppressive function, and
contained a t-PA fusion, elicited functional CTL in mice
[37].
Several recent studies have shown improved immunogenicity of DNA vaccines in which the antigen is
fused to an immune regulatory molecule. Fusion of the
model antigen human Ig [38] or the deltaPLD gene of
the sheep pathogen Corynebacterium pseudotuberculosis
[39] to the co-stimulatory molecule CTLA-4 resulted in
targeting of the antigen to lymphoid tissue and improved immune responses. Fusion of HIV gp120 or the
tumor antigen sFv to proinflammatory cytokines, including MCP-3, resulted in increased cellular responses
and neutralizing antibodies, or improved protection
from tumors, in vaccinated mice [40].
Polyepitope antigens are an alternative to the expression of whole genes. When immunodominant epitopes are known, such antigens may serve to focus the
immune response onto protective epitopes (and also
away from non-protective ones) (see Ertl and Thomsen).
7. Codon optimization
Additional modifications can be made to increase
protein production in transfected host cells. The most
effective of these is codon optimization. Many pathogens have a very different codon bias and/or genomic
GC content compared with mammals. This bias results
in low expression levels of their genes in transfected
mammalian cells and low immunogenicity of DNA
vaccines. For such organisms, it can be useful to synthesize the genes de novo, retaining the natural amino
acid sequences but using the human-preferred codons
for those amino acids. Codon optimization has been
performed for genes of the protozoan parasites Schistosoma mansoni [41], Entamoeba histolytica [42], and
Plasmodium species [8,43] and for the immunodeficiency
viruses HIV and SIV [44,45]. This procedure results in
increased protein production, accelerated seroconversion, increases of up to 100-fold in antibody titers
[43,45,46], and improved CTL responses [44,46].
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Several additional effects of codon optimization may
play a role in improved immunogenicity. When AT-rich
genes are recoded to the preferred human codons, multiple cytidine–phosphate–guanosine (CpG) sequences
may be introduced. Unmethylated CpG, as on plasmids
grown in bacteria, is known to be immunostimulatory
[47,48]. CpG-rich plasmids without inserts can also be
used as adjuvants [49]. Thus, a codon-optimized gene may
have an inherent adjuvant effect due to its CpG motifs,
although this is not always the case [46]. An additional
mechanism may operate in HIV: codon optimization
serves to remove inhibitory sequences, which reduce export of RNA from the nucleus as well as translation levels
[50].
It is difficult, time-consuming, and expensive to make
codon-optimized genes, but the effects can be very substantial, often making the investment worthwhile (see
Ertl and Thomsen).
8. Promoters
The vectors used for expression of the antigen can
have a large impact on immunogenicity. Promoters, enhancers, and introns can affect the level of expression of
the antigen (see Ertl and Thomsen). Most DNA vaccine
studies use plasmids carrying promoters that constitutively produce high levels of protein in most mammalian
tissues, notably the human cytomegalovirus (CMV) immediate–early 1 promoter [33]. However, there is some
concern about constitutive expression of antigens in inappropriate tissues, as pro-inflammatory antigens could
lead to organ damage. An attractive alternative, in
principle, is to use tissue-specific promoters. A musclespecific promoter, derived from the muscle creatine kinase gene, was used to confine expression of HIV-1 Gag
to muscle after intramuscular (i.m.) injection of plasmid
in mice. Vaccination elicited antibody and cellular responses, although they were at least 10-fold lower compared with a CMV construct [51,52]. Similarly, HIV-1
Nef was expressed in muscle using the desmin promoter.
In vaccinated mice, comparable levels of lymphoproliferation and IFNc production were elicited
by the muscle-targeted construct, compared with a
pcDNA3 CMV-driven vector, while antibody production was delayed but reached similar titers after four
immunizations [53].
modalities in ‘‘prime–boost’’ strategies. The second
vaccine modality is typically a recombinant protein or a
live vector.
10. Administration techniques
DNA may be administered by needle injection intradermally (i.d.) or i.m., to the skin or mucosa by biolistic means, intranasally or transcutaneously. Several
factors may influence the route of choice. Needle injection is easily performed and the DNA is prepared for
injection simply by resuspension in saline. The advantage of biolistic techniques, such as the Gene gun or
Biojector 2000, is in enhanced efficiency. It has been
shown in mice that approximately 100-fold less DNA is
required for a comparable antibody response than what
could be achieved with needle injection [1,54–57].
Biolistic and needle injections may produce different
types of immune responses. In many cases, the same
plasmid will elicit a Th1 response when given i.m. by
needle, but a Th2 or balanced Th1/Th2 response results
when delivered by gene gun [19,56,58]. The difference
may be due in part to the increased doses used for needle
injection. However, this finding is not universal; certain
antigens will bias the response one way or the other,
irrespective of the route used [18]. The gold carrier used
in gene gun delivery, though inert, can cause slight
damage to the skin when the gun is shot; the resulting
danger signal may create a Th2 bias [59].
Microencapsulation of DNA, or association of DNA
with microcapsules, has led to enhancement of CTL responses to encoded proteins. Biodegradable, non-antigenic poly-lactide polyglycolide (PLGA or PLG)
microspheres offer many advantages as a vaccine delivery
system (see Hobson et al.). Both cellular and humoral
immune responses can be elicited to antigens encapsulated in, or conjugated onto, PLG microspheres. Particles
used typically range in size from 1 to 10 lm in diameter, a
size that is readily phagocytosed by dendritic cells and
other antigen-presenting cells [60]. Microspheres elicit
both CD8+ and CD4+ T cell responses by releasing
antigen intracellularly [61]. Microsphere delivery enhances immune responses to DNA plasmids [62,63] (see
Sasaki et al. on use of adjuvants with DNA vaccines).
11. Number, size, and timing of doses
9. Immunization regimens
Once the vectors are constructed, the immunization
regimen must be considered. The timing, dose, route,
and use of adjuvants may all influence the immune response. In addition, many studies have shown an advantage of combining DNA vaccines with other
The number of doses affects the immune response. A
very immunogenic gene may require only a single dose,
as was found for the rabies G protein [55] and influenza
HA [64]. In most cases, more than one immunization is
required to provide a response strong enough to be
protective. For HIV, neutralizing antibody is usually
detected only after multiple doses [65]. In addition to
N.A. Doria-Rose, N.L. Haigwood / Methods 31 (2003) 207–216
changes in the magnitude of the immune response, more
doses of DNA may alter the type of response elicited. In
a study with the Powderject Gene Gun (Powderject,
Madison, WI) in macaques, antibodies to SIV were reduced after the sixth and seventh vaccinations, while
CTL responses increased [66].
For many antigens, 100 lg i.m. or 1 lg delivered by a
gene gun elicits vigorous and even protective responses
in mice. Larger animals, such as nonhuman primates,
require much higher doses, and doses for humans may
need to be higher still. For immunogenic proteins, an
effective dose is easy to achieve; for example, 4 lg of
hepatitis B vaccine from the Powderject device elicited
antibody titers in humans at protective levels [7]. However, for less immunogenic antigens, it may be difficult
to deliver a protective dose. The env genes of HIV and
related immunodeficiency viruses, SIV and the chimera
SHIV, are notoriously poor in stimulating neutralizing
antibody responses. Phase I clinical trials of HIV DNA
vaccines showed that very high doses were required.
Studies in infected [67] or uninfected [68] volunteers
suggested that 300 lg doses were superior to lower doses. In a study of pathogenic SHIV in macaques, it was
shown that 2.5 mg of a multi-gene plasmid gave better
protection than 250 lg when delivered i.m. or i.d. as part
of a prime–boost regimen [69]; however, recent studies
have used even higher doses (5 mg) of plasmid administered i.m. [70,71].
To address issues of dosing, we studied the effect of
the number of gene gun shots per dose on the immune
response of macaques to a DNA vaccine. Pig-tailed
macaques were inoculated with an expression plasmid
encoding the model antigen b-galactosidase, using the
Helios Gene Gun (Biorad, Hercules, CA). Groups of
four animals each received 5, 15, 30, or 45 shots, consisting of 1 lg plasmid carried on 0.5 mg of 1 lm gold
particles. The animals were immunized three times at
8-week intervals. Antibody responses were measured
three weeks after each immunization. We found that 30
and 45 shots were more effective doses than 5 or 15 shots
(Doria-Rose et al., submitted for publication). As shown
in Fig. 1, the higher doses resulted in earlier, more
consistent, and higher antibody titers against the
antigen.
The timing of doses also affects the outcome of vaccination. Studies of recombinant HIV-1 surface protein
gp120 in primates showed that a resting period of approximately 20 weeks between the second and third
immunizations resulted in significant, often 10-fold or
more increases in antibody and neutralizing antibody
titers [72]. Similarly, the time intervals between DNA
immunizations are also important. In a study using the
Powderject Gene Gun to deliver SIV vaccine to macaques, a three-dose regimen with intervals of 14–16
weeks induced higher antibody titers than a six-dose
regimen with 4–6-week intervals [73].
211
Fig. 1. Dose–response experiment. Macaques received b-galactosidase
plasmid by Gene Gun and their antibody titers were measured three
weeks later. (A) Number of animals responding after each dose. (B)
Antibody titers to b-galactosidase. The geometric mean of each group
is shown. Vertical bars ¼ standard error of the mean.
12. Adjuvants
Adjuvants for DNA vaccines can be chemical in
form, or genetic, that is, co-immunizing with a stimulatory gene. Chemical adjuvants range from the traditional adjuvant alum [74] to PLG microparticles (see
above). Although CpG-unmethylated plasmids are immunostimulatory, CpG oligonucleotides may [75] or
may not [76] have an adjuvant effect for DNA vaccines.
Genetic adjuvants are usually cytokine genes, which
provide general immune stimulation and can also bias
the immune response toward a Th1 or Th2 type [77].
Genes for cholera toxin and heat-labile enterotoxin have
also been used as genetic adjuvants [78]. Genes that
stimulate apoptosis, such as caspases, may also improve
immunogenicity [79]. (See Sasaki et al. for further review
of adjuvants used for DNA immunization.)
In choosing an adjuvant that provides a Th1 or Th2
bias, it is important to know which type of response will
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N.A. Doria-Rose, N.L. Haigwood / Methods 31 (2003) 207–216
be protective (see above). For example, Leishmania
major requires a Th1-type response for effective immunity. Adding the Th1 cytokine IL-12 to a DNA vaccine
improved the potency and durability of a protective
response in mice [80]. Conversely, inappropriate cytokines may be harmful. For example, a DNA vaccine to
Japanese encephalitis virus was protective when given
alone, but co-administration of an IL-12 plasmid reduced antibody production and abrogated the protective response [13]. Some cytokines, such as IL-2 and
GM-CSF, often provide a balanced response and may
be more appropriate when the type of response that will
be protective is unknown, or when both humoral and
cellular responses are beneficial [15,81].
For cytokine adjuvants, the timing of administration
may also be important. Injection of mice with an IL-2
plasmid five days before that of an HIV gp120 plasmid
resulted in reduced antibody and CTL responses, while
administration on the same day or two to five days after
gp120 improved both responses [15]. Similar results were
seen with IL-2 and vaccination against hepatitis B [28].
In mice vaccinated and challenged with Leishmania,
administration of an IL-12 plasmid four days after
vaccination resulted in fewer lesions and more IFNc
production, whereas early or simultaneous administration enhanced infection [82]. A GM-CSF plasmid given
three days before, on the day of, or three days after
vaccination elicited responses that were Th2 biased,
balanced, and Th1 biased, respectively [83].
13. Prime–boost or combination immunization
Studies in a number of systems have demonstrated
the advantage of combining two or more vaccine modalities. The immune responses can be qualitatively and
quantitatively different when compared with one vaccine
type given alone. Generally, the regimens begin with one
or more doses of the first vaccine—‘‘prime’’—followed
by one or more doses of the second modality—‘‘boost.’’
The first major study to use this approach for SIV
showed sterilizing immunity elicited by priming with a
recombinant vaccinia virus that encoded SIV Envelope
protein and boosting with purified Envelope protein
[84]. In many cases, a combination of two modalities
elicits better immune responses than either vaccine alone
[57,85–88]. Priming with DNA and boosting with recombinant protein improve binding and neutralizing
antibody titers [89,90] whereas priming with DNA and
boosting with poxvirus increased cellular responses in
other studies [91,92].
Priming with DNA and boosting with another
agent, or vice versa, have conferred at least partial
protection in animal models of such diverse pathogens
as the protozoan parasites Leishmania [93] and malaria [94], the rickettsial bacteria Cowdria ruminantium
(heartwater) [95], and immunodeficiency viruses [5,69–
71,96], as well as models for human cancers [97–99].
Several such regimens are in Phase I clinical trials for
HIV [100] and malaria [101]. Table 1 lists notable
recent successes in animal models of pathogens. This
list is not exhaustive but meant to show the breadth
of use of prime–boost strategies. It is important to
note that the DNA vaccines in many of the vaccine
challenge models listed in Table 1 have demonstrated
protection from disease (but not necessarily from infection).
The order of immunization can affect the outcome of
prime–boost regimens. In a study of the herpes simplex
virus gB vaccine in mice, priming i.m. with DNA and
boosting i.m. with recombinant vaccinia encoding HSV
antigen elicited the best systemic responses, whereas
mucosal immunization elicited better mucosal and systemic responses when vaccinia was used for priming
[102].
We studied several prime–boost regimens for the
SHIV89.6P model of AIDS in macaques, including a
pair of regimens with opposite orders of vaccination.
Table 1
Examples of successful DNA prime–boost vaccines in animal models
Pathogen
Animal model
Prime
Boost
Outcome
Reference
Plasmodium
Mouse
Mouse
Macaque
Mouse
Mouse
Mouse
Macaque
Macaque
Macaque
Macaque
Macaque
Macaque
DNA, IM
DNA, ID, or IM
DNA, IM
DNA
DNA
DNA, IM, or GG
DNA
DNA, IM, and ID
DNA, IM
DNA, IM
DNA, GG
Vaccinia
Vaccinia, IP
Modified Vaccinia Ankara
Attenuated vaccinia
Vaccinia
Protein
Modified Vaccinia Ankara
Adenovirus
Protein, IM
Modified Vaccinia Ankara
Adenovirus
Vaccinia
DNA, GG
Protection from infection
Protection from infection
Protection from disease
Reduced lesions
Partial protection
Protection from infection
Protection from infection
Reduced virus load
Protection from disease
Protection from disease
Protection from disease
Protection from disease
[93]
[56]
[6]
[92]
[94]
[56]
[109]
[95]
[65]
[67]
Leishmania
Cowdria
Influenza virus
Ebola virus
SIV
SHIV
SHIV
SHIV
SHIV
IM, intramuscular injection; ID, intradermal injection; IP, intraperitoneal injection; GG, Gene Gun.
Doria-Rose et al., submitted for publication.
*
N.A. Doria-Rose, N.L. Haigwood / Methods 31 (2003) 207–216
213
virus, but the latter regimen elicited a higher cellular
response [71].
The mechanisms responsible for these improved responses have not been fully explained in the literature.
Differences in immune responses to DNA vaccine versus
live viral vector vaccine delivery may reflect alternate
pathways of antigen presentation through dendritic cells
and other antigen-presenting cells. In the case of
boosting with poxviruses, it has been suggested that
DNA primes a population of memory cells that then
react quickly to the boosting antigen in an environment
of inflammatory signals elicited by the poxvirus [91]. In
general, recombinant viral vector infection often results
in a strong inflammatory response that can enhance the
onset of immunity to encoded antigens but makes it
impossible to boost with another dose of the same agent,
as memory responses result in rapid elimination of the
vector and lower immunity to the recombinant product.
Priming or boosting with DNA circumvents this response.
Each regimen included a DNA vaccine composed of six
plasmids encoding all SHIV genes. We used the gene
gun to deliver 30 shots of 2 lg of each plasmid per dose.
Three doses of DNA were given as prime or boost; the
other vaccines included vaccinia recombinants carrying
SHIV gag-pol and env genes and inactivated virus particles. We also compared priming with DNA and
boosting with vaccinia, to the reverse regimen as outlined in Table 2. We measured antiviral immune responses prior to infection and found that DNA
vaccination increased antibody titers when given as a
prime or as a boost. Upon infection of vaccinated and
control animals, we found that the regimens of vaccinia
recombinants were much more effective than DNA
alone or with particles. As summarized in Table 3, both
regimens with vaccinia provided protection from acute
CD4þ T cell loss caused by the virus. However, priming
with vaccinia and boosting with DNA were more effective than the reverse regimen in lowering the setpoint
(post-acute) viral load, a predictor of survival time in
macaques as well as human patients (Doria-Rose et al.,
submitted for publication).
Some studies have shown qualitatively different responses to single vaccines versus combinations. For
example, monkeys immunized with recombinant Modified Vaccinia Virus Ankara (MVA), or primed with
DNA and boosted with MVA, all had good control of a
pathogenic SHIV challenge; however, the combination
elicited different responses. The combination elicited
higher levels of IFNc-producing T cells but lower antibody titers relative to rMVA alone [103]. Similarly, an
adenovirus-based vaccine alone, or as a boost after
DNA vaccination, protected macaques from the same
14. Conclusion
DNA vaccines have demonstrated effectiveness in
stimulating immune responses, leading us to speculate
about the future. Most importantly, how will DNA
expression vectors be used in the future to prime or
boost responses, and will stand-alone DNA vaccines be
possible? The answers to these questions rely substantially on what advances are seen in plasmid design and
delivery. The ‘‘gold standard’’ of the human CMV
promoter will certainly be replaced as we find methods
Table 2
Example of vaccine experiment in macaques: comparison of prime–boost regimens followed by infection with SHIV89.6P
Group
Number animals
Dose 1 Week 0
Dose 2 Week 4/8
Dose 3 Week 25
Dose 4 Week 50
Dose 5 Week 73
Week 76
Control
1
2
3
4
6
6
6
6
6
Sham
DNA
DNA
DNA
Vaccinia-SHIV
Sham
DNA
DNA
DNA
Vaccinia-SHIV
Sham
DNA
DNA
DNA
DNA
Sham
DNA
SHIV particles
Vaccinia-SHIV
DNA
Sham
DNA
SHIV particles
Vaccinia-SHIV
DNA
Challenge
Challenge
Challenge
Challenge
Challenge
Table 3
Outcome of experiment outlined in Table 2
Group
Control
1
2
3
4
*
Prime
Boost
—
—
DNA
DNA
DNA
Vaccinia
DNA
Particle
Vaccinia
DNA
Significant difference from controls, p < 0:05.
Outcome of infection
CD4þ T cells
Viremia: Peak
Viremia: Setpoint
Acute loss
Acute loss
Acute loss
Slow or no loss
Slow or no loss
High
Medium
High
Low
Low
High
High
High
Medium
Low
214
N.A. Doria-Rose, N.L. Haigwood / Methods 31 (2003) 207–216
to identify or design synthetic promoters that are tightly
regulated and tissue-specific as well as more highly expressed. The in vivo transfection efficiency of antigenpresenting cells, such as dendritic cells, can certainly be
enhanced by effective cytokine- or ligand-induced recruitment of these cells to the site(s) of immunization of
the DNA vectors. Delivery of DNA to mucosal surfaces
(e.g., intravaginally or intranasally) and directly to
lymph nodes [104] are examples of promising approaches that are likely to undergo expanded testing in
coming years. If DNA can be effectively delivered transcutaneously [105], via bacterial carriers [106], or boosted
orally through food ingestion [107,108], much of the
current research on regimens may need to be revisited.
At this time, DNA vaccines are still in early development. The last 10 years of intensive DNA vaccine research in animal models have provided sufficient success
to act as an incentive for further development of this
powerful tool.
References
[1] E.F. Fynan, R.G. Webster, D.H. Fuller, J.R. Haynes, J.C.
Santoro, H.L. Robinson, Proc. Natl. Acad. Sci. USA 90 (1993)
11478–11482.
[2] J.B. Ulmer, J.J. Donnelly, S.E. Parker, G.H. Rhodes, P.L.
Felgner, V.J. Dwarki, S.H. Gromkowski, R.R. Deck, C.M.
DeWitt, A. Friedman, et al., Science 5102 (1993) 1745–1749.
[3] L. Stamatatos, D. Davis, Aids 15 (Suppl. 5) (2001) S105–S115.
[4] S.L. Rowland-Jones, T. Dong, K.R. Fowke, J. Kimani, P.
Krausa, H. Newell, T. Blanchard, K. Ariyoshi, J. Oyugi, E.
Ngugi, et al., J. Clin. Invest. 102 (1998) 1758–1765.
[5] E.G. Wee, S. Patel, A.J. McMichael, T. Hanke, J. Gen. Virol. 83
(2002) 75–80.
[6] T. Maruyama, L.L. Rodriguez, P.B. Jahrling, A. Sanchez, A.S.
Khan, S.T. Nichol, C.J. Peters, P.W. Parren, D.R. Burton, J.
Virol. 73 (1999) 6024–6030.
[7] M.J. Roy, M.S. Wu, L.J. Barr, J.T. Fuller, L.G. Tussey, S.
Speller, J. Culp, J.K. Burkholder, W.F. Swain, R.M. Dixon,
et al., Vaccine 19 (2000) 764–778.
[8] W.O. Rogers, W.R. Weiss, A. Kumar, J.C. Aguiar, J.A. Tine, R.
Gwadz, J.G. Harre, K. Gowda, D. Rathore, S. Kumar, S.L.
Hoffman, Infect. Immun. 70 (2002) 4329–4335.
[9] S.L. Reiner, R.M. Locksley, Annu. Rev. Immunol. 13 (1995)
151–177.
[10] A.K. Abbas, K.M. Murphy, A. Sher, Nature 383 (1996) 787–793.
[11] P.J. Openshaw, F.J. Culley, W. Olszewska, Vaccine 20 (Suppl. 1)
(2001) S27–S31.
[12] F.D. Finkelman, J.F. Urban Jr., J. Allergy Clin. Immunol. 107
(2001) 772–780.
[13] H.W. Chen, C.H. Pan, H.W. Huan, M.Y. Liau, J.R. Chiang,
M.H. Tao, J. Immunol. 166 (2001) 7419–7426.
[14] G.J. Gorse, M.J. McElrath, T.J. Matthews, R.H. Hsieh, R.B.
Belshe, L. Corey, S.E. Frey, D.J. Kennedy, M.C. Walker, M.M.
Eibl, Vaccine 16 (1998) 493–506.
[15] D.H. Barouch, S. Santra, T.D. Steenbeke, X.X. Zheng, H.C.
Perry, M.E. Davies, D.C. Freed, A. Craiu, T.B. Strom, J.W.
Shiver, N.L. Letvin, J. Immunol. 161 (1998) 1875–1882.
[16] N.L. Letvin, D.C. Montefiori, Y. Yasutomi, H.C. Perry, M.E.
Davies, C. Lekutis, M. Alroy, D.C. Freed, C.I. Lord, L.K.
Handt, et al., Proc. Natl. Acad. Sci. USA 94 (1997) 9378–9383.
[17] R. Braun, L.A. Babiuk, S. Littel-van den Hurk, J. Gen. Virol. 79
(1998) 2965–2970.
[18] A.I. Cardoso, N. Sixt, A. Vallier, J. Fayolle, R. Buckland, T.F.
Wild, J. Virol. 72 (1998) 2516–2518.
[19] D.M. Feltquate, S. Heaney, R.G. Webster, H.L. Robinson, J.
Immunol. 158 (1997) 2278–2284.
[20] N.A. Halsey, Pediatr. Infect. Dis. J. 20 (2001) S40–S44.
[21] M.R. Betts, D.R. Ambrozak, D.C. Douek, S. Bonhoeffer, J.M.
Brenchley, J.P. Casazza, R.A. Koup, L.J. Picker, J. Virol. 75
(2001) 11983–11991.
[22] D.H. OÕConnor, T.M. Allen, T.U. Vogel, P. Jing, I.P. DeSouza,
E. Dodds, E.J. Dunphy, C. Melsaether, B. Mothe, H. Yamamoto, et al., Nat. Med. 8 (2002) 493–499.
[23] J.J. Donnelly, A. Friedman, J.B. Ulmer, M.A. Liu, Vaccine 15
(1997) 865–868.
[24] A.M. Talaat, R. Lyons, S.A. Johnston, Vaccine 20 (2001) 538–
544.
[25] E.D. Williamson, A.M. Bennett, S.D. Perkins, R.J. Beedham, J.
Miller, L.W. Baillie, Vaccine 20 (2002) 2933–2941.
[26] V. Ayyavoo, S. Kudchodkar, M.P. Ramanathan, P. Le, K.
Muthumani, N.M. Megalai, T. Dentchev, L. Santiago-Barrios,
C. Mrinalini, D.B. Weiner, Aids 14 (2000) 1–9.
[27] M.S. Ashok, P.N. Rangarajan, Vaccine 20 (2002) 1563–1570.
[28] M. Geissler, V. Bruss, S. Michalak, B. Hockenjos, D. Ortmann,
W.B. Offensperger, J.R. Wands, H.E. Blum, J. Virol. 73 (1999)
4284–4292.
[29] U.A. Hasan, D.R. Harper, S. Argent, G. Layton, B.W. Wren,
W.J. Morrow, Vaccine 18 (2000) 1506–1514.
[30] Z. Li, A. Howard, C. Kelley, G. Delogu, F. Collins, S. Morris,
Infect. Immun. 67 (1999) 4780–4786.
[31] W.W. Leitner, M.C. Seguin, W.R. Ballou, J.P. Seitz, A.M.
Schultz, M.J. Sheehy, J.A. Lyon, J. Immunol. 159 (1997) 6112–
6119.
[32] R.L. Burke, C. Pachl, M. Quiroga, S. Rosenberg, N. Haigwood,
O. Nordfang, M. Ezban, J. Biol. Chem. 261 (1986) 12574–
12578.
[33] B.S. Chapman, R.M. Thayer, K.A. Vincent, N.L. Haigwood,
Nucleic Acids Res. 19 (1991) 3979–3986.
[34] D. Haddad, S. Liljeqvist, S. Stahl, I. Andersson, P. Perlmann, K.
Berzins, N. Ahlborg, FEMS Immunol. Med. Microbiol. 18
(1997) 193–202.
[35] Y. Wu, T.J. Kipps, J. Immunol. 159 (1997) 6037–6043.
[36] F. Rodriguez, J. Zhang, J.L. Whitton, J. Virol. 71 (1997) 8497–
8503.
[37] X. Liang, T. Fu, H. Xie, E. Emini, J. Shiver, Vaccine 20 (2002)
3413.
[38] J.S. Boyle, J.L. Brady, A.M. Lew, Nature 392 (1998) 408–
411.
[39] P.J. Chaplin, R. De Rose, J.S. Boyle, P. McWaters, J. Kelly,
J.M. Tennent, A.M. Lew, J.P. Scheerlinck, Infect. Immun. 67
(1999) 6434–6438.
[40] A. Biragyn, I.M. Belyakov, Y.H. Chow, D.S. Dimitrov, J.A.
Berzofsky, L.W. Kwak, Blood 100 (2002) 1153–1159.
[41] F.F. Hamdan, A. Mousa, P. Ribeiro, Parasitol. Res. 88 (2002)
583–586.
[42] D. Gaucher, K. Chadee, Vaccine 20 (2002) 3244.
[43] D.L. Narum, S. Kumar, W.O. Rogers, S.R. Fuhrmann, H.
Liang, M. Oakley, A. Taye, B.K. Sim, S.L. Hoffman, Infect.
Immun. 69 (2001) 7250–7253.
[44] S. Andre, B. Seed, J. Eberle, W. Schraut, A. Bultmann, J. Haas,
J. Virol. 72 (1998) 1497–1503.
[45] L. Vinner, H.V. Nielsen, K. Bryder, S. Corbet, C. Nielsen, A.
Fomsgaard, Vaccine 17 (1999) 2166–2175.
[46] L. Deml, A. Bojak, S. Steck, M. Graf, J. Wild, R. Schirmbeck,
H. Wolf, R. Wagner, J. Virol. 75 (2001) 10991–11001.
[47] D.M. Klinman, A.K. Yi, S.L. Beaucage, J. Conover, A.M.
Krieg, Proc. Natl. Acad. Sci. USA 93 (1996) 2879–2883.
N.A. Doria-Rose, N.L. Haigwood / Methods 31 (2003) 207–216
[48] A.M. Krieg, A.K. Yi, S. Matson, T.J. Waldschmidt, G.A.
Bishop, R. Teasdale, G.A. Koretzky, D.M. Klinman, Nature 374
(1995) 546–549.
[49] Y. Kojima, K.Q. Xin, T. Ooki, K. Hamajima, T. Oikawa, K.
Shinoda, T. Ozaki, Y. Hoshino, N. Jounai, M. Nakazawa, et al.,
Vaccine 20 (2002) 2857–2865.
[50] R. Schneider, M. Campbell, G. Nasioulas, B.K. Felber, G.N.
Pavlakis, J. Virol. 71 (1997) 4892–4903.
[51] A. Bojak, D. Hammer, H. Wolf, R. Wagner, Vaccine 20 (2002)
1975–1979.
[52] A. Bojak, J. Wild, H. Wolf, R. Wagner, Vaccine 20 (2002) 1980–
1984.
[53] N. Cazeaux, Y. Bennasser, P. Vidal, Z. Li, D. Paulin, E.
Bahraoui, Vaccine 20 (2002) 3322.
[54] A.M. Bennett, R.J. Phillpotts, S.D. Perkins, S.C. Jacobs, E.D.
Williamson, Vaccine 18 (1999) 588–596.
[55] D.L. Lodmell, M.J. Parnell, J.R. Bailey, L.C. Ewalt, C.A.
Hanlon, Vaccine 20 (2001) 838–844.
[56] S.C. Oliveira, G.M. Rosinha, C.F. de-Brito, C.T. Fonseca, R.R.
Afonso, M.C. Costa, A.M. Goes, E.L. Rech, V. Azevedo, Braz.
J. Med. Biol. Res. 32 (1999) 207–214.
[57] P. Degano, J. Schneider, C.M. Hannan, S.C. Gilbert, A.V. Hill,
Vaccine 18 (1999) 623–632.
[58] A.A. Belperron, D. Feltquate, B.A. Fox, T. Horii, D.J. Bzik,
Infect. Immun. 67 (1999) 5163–5169.
[59] R. Weiss, S. Scheiblhofer, J. Freund, F. Ferreira, I. Livey, J.
Thalhamer, Vaccine 20 (2002) 3148–3154.
[60] Y. Tabata, Y. Ikada, Biomaterials 9 (1988) 356–362.
[61] L.D. Falo Jr., M. Kovacsovics-Bankowski, K. Thompson, K.L.
Rock, Nat. Med. 1 (1995) 649–653.
[62] M.L. Hedley, J. Curley, R. Urban, Nat. Med. 4 (1998) 365–368.
[63] D. OÕHagan, M. Singh, M. Ugozzoli, C. Wild, S. Barnett, M.
Chen, M. Schaefer, B. Doe, G.R. Otten, J.B. Ulmer, J. Virol. 75
(2001) 9037–9043.
[64] H.L. Robinson, C.A. Boyle, D.M. Feltquate, M.J. Morin, J.C.
Santoro, R.G. Webster, J. Infect. Dis. 176 (Suppl. 1) (1997) S50–
S55.
[65] S.W. Barnett, S. Lu, I. Srivastava, S. Cherpelis, A. Gettie, J.
Blanchard, S. Wang, I. Mboudjeka, L. Leung, Y. Lian, et al., J.
Virol. 75 (2001) 5526–5540.
[66] D.H. Fuller, P.A. Rajakumar, L.A. Wilson, A.M. Trichel, J.T.
Fuller, T. Shipley, M.S. Wu, K. Weis, C.R. Rinaldo, J.R.
Haynes, M. Murphey-Corb, J. Virol. 76 (2002) 3309–3317.
[67] R.R. MacGregor, J.D. Boyer, K.E. Ugen, K.E. Lacy, S.J.
Gluckman, M.L. Bagarazzi, M.A. Chattergoon, Y. Baine, T.J.
Higgins, R.B. Ciccarelli, et al., J. Infect. Dis. 178 (1998) 92–
100.
[68] J.D. Boyer, A.D. Cohen, S. Vogt, K. Schumann, B. Nath, L.
Ahn, K. Lacy, M.L. Bagarazzi, T.J. Higgins, Y. Baine, et al., J.
Infect. Dis. 181 (2000) 476–483.
[69] R.R. Amara, F. Villinger, J.D. Altman, S.L. Lydy, S.P. OÕNeil,
S.I. Staprans, D.C. Montefiori, Y. Xu, J.G. Herndon, L.S.
Wyatt, et al., Science 292 (2001) 69–74.
[70] D.H. Barouch, S. Santra, J.E. Schmitz, M.J. Kuroda, T.M. Fu,
W. Wagner, M. Bilska, A. Craiu, X.X. Zheng, G.R. Krivulka,
et al., Science 290 (2000) 486–492.
[71] J.W. Shiver, T.M. Fu, L. Chen, D.R. Casimiro, M.E. Davies,
R.K. Evans, Z.Q. Zhang, A.J. Simon, W.L. Trigona, S.A.
Dubey, et al., Nature 415 (2002) 331–335.
[72] K.P. Anderson, C. Lucas, C.V. Hanson, H.F. Londe, A. Izu, T.
Gregory, A. Ammann, P.W. Berman, J.W. Eichberg, J. Infect.
Dis. 160 (1989) 960–969.
[73] D.H. Fuller, M.M. Corb, S. Barnett, K. Steimer, J.R. Haynes,
Vaccine 15 (1997) 924–926.
[74] J.B. Ulmer, C.M. DeWitt, M. Chastain, A. Friedman, J.J.
Donnelly, W.L. McClements, M.J. Caulfield, K.E. Bohannon,
D.B. Volkin, R.K. Evans, Vaccine 18 (1999) 18–28.
215
[75] D.M. Klinman, K.M. Barnhart, J. Conover, Vaccine 17 (1999)
19–25.
[76] R. Weeratna, C.L. Brazolot Millan, A.M. Krieg, H.L. Davis,
Antisense Nucleic Acid Drug Dev. 8 (1998) 351–356.
[77] J.J. Kim, N.N. Trivedi, L.K. Nottingham, L. Morrison, A. Tsai,
Y. Hu, S. Mahalingam, K. Dang, L. Ahn, N.K. Doyle, et al.,
Eur. J. Immunol. 28 (1998) 1089–1103.
[78] J. Arrington, R.P. Braun, L. Dong, D.H. Fuller, M.D. Macklin,
S.W. Umlauf, S.J. Wagner, M.S. Wu, L.G. Payne, J.R. Haynes,
J. Virol. 76 (2002) 4536–4546.
[79] S. Sasaki, R.R. Amara, A.E. Oran, J.M. Smith, H.L. Robinson,
Nat. Biotechnol. 19 (2001) 543–547.
[80] S. Gurunathan, C. Prussin, D.L. Sacks, R.A. Seder, Nat. Med. 4
(1998) 1409–1415.
[81] J.I. Sin, J.J. Kim, K.E. Ugen, R.B. Ciccarelli, T.J. Higgins, D.B.
Weiner, Eur. J. Immunol. 28 (1998) 3530–3540.
[82] A.H. Noormohammadi, H. Hochrein, J.M. Curtis, T.M. Baldwin, E. Handman, Vaccine 19 (2001) 4043–4052.
[83] K. Kusakabe, K.Q. Xin, H. Katoh, K. Sumino, E. Hagiwara, S.
Kawamoto, K. Okuda, Y. Miyagi, I. Aoki, K. Nishioka, D.
Klinman, J. Immunol. 164 (2000) 3102–3111.
[84] S.L. Hu, K. Abrams, G.N. Barber, P. Moran, J.M. Zarling, A.J.
Langlois, L. Kuller, W.R. Morton, R.E. Benveniste, Science 255
(1992) 456–459.
[85] H.L. Robinson, D.C. Montefiori, R.P. Johnson, K.H. Manson,
M.L. Kalish, J.D. Lifson, T.A. Rizvi, S. Lu, S.L. Hu, G.P.
Mazzara, et al., Nat. Med. 5 (1999) 526–534.
[86] M.J. Estcourt, A.J. Ramsay, A. Brooks, S.A. Thomson, C.J.
Medveckzy, I.A. Ramshaw, Int. Immunol. 14 (2002) 31–37.
[87] D.H. Fuller, L. Simpson, K.S. Cole, J.E. Clements, D.L.
Panicali, R.C. Montelaro, M. Murphey-Corb, J.R. Haynes,
Immunol. Cell Biol. 75 (1997) 389–396.
[88] T. Matano, M. Kano, H. Nakamura, A. Takeda, Y. Nagai, J.
Virol. 75 (2001) 11891–11896.
[89] S.W. Barnett, S. Rajasekar, H. Legg, B. Doe, D.H. Fuller, J.R.
Haynes, C.M. Walker, K.S. Steimer, Vaccine 15 (1997) 869–873.
[90] S.P. Mossman, C.C. Pierce, M.N. Robertson, A.J. Watson, D.C.
Montefiori, M. Rabin, L. Kuller, J. Thompson, J.B. Lynch,
W.R. Morton, et al., J. Med. Primatol. 28 (1999) 206–213.
[91] J. Schneider, S.C. Gilbert, C.M. Hannan, P. Degano, E. Prieur,
E.G. Sheu, M. Plebanski, A.V. Hill, Immunol. Rev. 170 (1999)
29–38.
[92] T. Hanke, R.V. Samuel, T.J. Blanchard, V.C. Neumann, T.M.
Allen, J.E. Boyson, S.A. Sharpe, N. Cook, G.L. Smith, D.I.
Watkins, et al., J. Virol. 73 (1999) 7524–7532.
[93] R.M. Gonzalo, G. del Real, J.R. Rodriguez, D. Rodriguez, R.
Heljasvaara, P. Lucas, V. Larraga, M. Esteban, Vaccine 20
(2002) 1226–1231.
[94] M. Sedegah, G.T. Brice, W.O. Rogers, D.L. Doolan, Y.
Charoenvit, T.R. Jones, V.F. Majam, A. Belmonte, M. Lu, M.
Belmonte, D.J. Carucci, S.L. Hoffman, Infect. Immun. 70 (2002)
3493–3499.
[95] A. Nyika, A.F. Barbet, M.J. Burridge, S.M. Mahan, Vaccine 20
(2002) 1215–1225.
[96] N.L. Haigwood, C.C. Pierce, M.N. Robertson, A.J. Watson,
D.C. Montefiori, M. Rabin, J.B. Lynch, L. Kuller, J. Thompson,
W.R. Morton, et al., Immunol. Lett. 66 (1999) 183–188.
[97] K.R. Irvine, R.S. Chamberlain, E.P. Shulman, D.R. Surman,
S.A. Rosenberg, N.P. Restifo, J. Natl. Cancer Inst. 89 (1997)
1595–1601.
[98] W.S. Meng, L.H. Butterfield, A. Ribas, V.B. Dissette, J.B.
Heller, G.A. Miranda, J.A. Glaspy, W.H. McBride, J.S. Economou, Cancer Res. 61 (2001) 8782–8786.
[99] S. Pasquini, S. Peralta, E. Missiaglia, L. Carta, N.R. Lemoine,
Gene Ther. 9 (2002) 503–510.
[100] B.S. Graham, Annu. Rev. Med. 53 (2002) 207–221.
[101] M. Habeck, Lancet Infect. Dis. 2 (2002) 584.
216
N.A. Doria-Rose, N.L. Haigwood / Methods 31 (2003) 207–216
[102] S.K. Eo, M. Gierynska, A.A. Kamar, B.T. Rouse, J. Immunol.
166 (2001) 5473–5479.
[103] R.R. Amara, F. Villinger, S.I. Staprans, J.D. Altman, D.C.
Montefiori, N.L. Kozyr, Y. Xu, L.S. Wyatt, P.L. Earl, J.G.
Herndon, et al., J. Virol. 76 (2002) 7625–7631.
[104] K.J. Maloy, I. Erdmann, V. Basch, S. Sierro, T.A. Kramps,
R.M. Zinkernagel, S. Oehen, T.M. Kundig, Proc. Natl. Acad.
Sci. USA 98 (2001) 3299–3303.
[105] Z. Cui, R.J. Mumper, J. Control Release 81 (2002) 173–184.
[106] H. Mollenkopf, G. Dietrich, S.H. Kaufmann, Biol. Chem. 382
(2001) 521–532.
[107] A.M. Walmsley, C.J. Arntzen, Curr. Opin. Biotechnol. 11 (2000)
126–129.
[108] D.E. Webster, M.L. Cooney, Z. Huang, D.R. Drew, I.A.
Ramshaw, I.B. Dry, R.A. Strugnell, J.L. Martin, S.L. Wesselingh, J. Virol. 76 (2002) 7910–7912.
[109] N.J. Sullivan, A. Sanchez, P.E. Rollin, Z.Y. Yang, G.J. Nabel,
Nature 408 (2000) 605–609.