Hindawi Publishing Corporation

Journal of Biomedicine and Biotechnology

Volume 2010, Article ID 174378, 16 pages
doi:10.1155/2010/174378

Review Article
DNA Vaccines: Developing New Strategies against Cancer
Daniela Fioretti,1 Sandra Iurescia,1 Vito Michele Fazio,2, 3 and Monica Rinaldi1
1 Institute

of Neurobiology and Molecular Medicine, Department of Medicine, National Research Council (CNR),
Via Fosso del Cavaliere 100, 00133 Rome, Italy
2 Section of Molecular Medicine and Biotechnology, Interdisciplinary Center for Biomedical Research,

University Campus Bio-Medico, Via Alvaro

del Portillo 21, 00128 Rome, Italy
3 Unit`
a Operativa Oncologia, Istituto di Ricovero e Cura a Carattere Scientifico Casa Sollievo della Soerenza,
71013 San Giovanni Rotondo, Foggia, Italy
Correspondence should be addressed to Monica Rinaldi, monica.rinaldi@artov.inmm.cnr.it
Received 20 November 2009; Accepted 5 February 2010
Academic Editor: Soldano Ferrone
Copyright 2010 Daniela Fioretti et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Due to their rapid and widespread development, DNA vaccines have entered into a variety of human clinical trials for vaccines
against various diseases including cancer. Evidence that DNA vaccines are well tolerated and have an excellent safety profile proved
to be of advantage as many clinical trials combines the first phase with the second, saving both time and money. It is clear from the
results obtained in clinical trials that such DNA vaccines require much improvement in antigen expression and delivery methods
to make them suciently eective in the clinic. Similarly, it is clear that additional strategies are required to activate eective
immunity against poorly immunogenic tumor antigens. Engineering vaccine design for manipulating antigen presentation and
processing pathways is one of the most important aspects that can be easily handled in the DNA vaccine technology. Several
approaches have been investigated including DNA vaccine engineering, co-delivery of immunomodulatory molecules, safe routes
of administration, prime-boost regimen and strategies to break the immunosuppressive networks mechanisms adopted by
malignant cells to prevent immune cell function. Combined or single strategies to enhance the ecacy and immunogenicity
of DNA vaccines are applied in completed and ongoing clinical trials, where the safety and tolerability of the DNA platform are
substantiated. In this review on DNA vaccines, salient aspects on this topic going from basic research to the clinic are evaluated.
Some representative DNA cancer vaccine studies are also discussed.

1. Introduction
Spontaneous tumour regression has followed bacterial, fungal, viral, and protozoal infections. Intratumoral infections
may reactivate defensive functions, causing tumour regression.
These phenomena inspired the development of numerous rudimentary cancer immunotherapies, starting with
nonspecific immunostimulatory approaches first used by
William Coley [1] and leading to the concept of therapeutic
vaccination against cancer. The recent identification and
characterization of genes coding for tumour antigens (Ag)
has enabled the design of antigen-specific cancer vaccines
based on plasmid DNA and recombinant viral vectors. Gene
therapy can be used to manipulate the immune system to

help bodys natural defences to recognise and target cancer

cells.
In the last few years, it is estimated that in Europe there
were almost 3 000 000 cancer cases diagnosed (excluding non
melanoma skin cancers) and more than 1 500 000 deaths
from cancer each year [2]. Standard therapeutic procedures
currently in practice, including surgery, radiation, and
chemotherapy have not greatly impacted the spread and
recurrence of progressive malignancies [3], reducing the
ability of the immune system to provoke spontaneous
regressions. Newer strategies are needed to improve upon the
current treatment success rate.
Historically, Wol and colleagues [4] first demonstrated
that long-term gene expression in mouse skeletal muscle
could be achieved with direct intramuscular injection of

2
plasmid DNA. This and other early studies, demonstrating
the feasibility of direct intramuscular gene transfer for DNA
vaccination purpose, propelled the first vaccination studies
utilizing plasmid DNA in protection scenarios involving
influenza [5] and HIV-1 [6]. Cellular and humoral immune
responses have been demonstrated after the injection of
naked plasmid DNA vaccines into the dermis or muscle
tissue of mice [5, 7]. Such responses have induced protection
in preclinical models of infectious disease and malignancies
(for review, see Donnelly et al. [8]).
The DNA vaccine is a prime example of a modern
genetic vaccine. The use of naked plasmid DNA as vaccine to
elicit the immune system against disease provides a variety
of practical benefits for large-scale vaccine production that
are not as easily manageable with other forms of vaccines
including recombinant protein or whole tumor cells [9, 10].
The eectiveness to screen for antigens rapidly and to
design specific types of expression constructs has made the
study of DNA vaccines a valuable field for immunotherapy
of cancer.
New technologies including gene-expression profiling
has increased the list of candidate tumor antigens. Investigators have focused on targets which are either tumourspecific, including idiotypic antigens of B-cell tumours [11]
or tumour-associated antigens [12] that are also expressed by
the normal cell of origin [13] and that include the so-called
cancer-testis antigens [14].
Examples under intensive investigation are the antigens
of melanoma (http://www.cancer.gov/cancertopics/types/
melanoma), prostate cancer (http://www.cancer.gov/cancer
topics/types/prostate), and other epithelial cancers (http://
www.cancer.gov/cancertopics/types/skin).
DNA vaccines oer the opportunity to incorporate additional genes encoding molecules aimed at overcoming the
weak immunogenicity of tumor antigens and the patients
tolerized immune repertoire.
This paper briefly summarizes findings and key technologies that have contributed to the rapid progress of DNA
vaccines (mode of action, design, and optimization of DNA
vaccines) as well as the state of the art of some of the
more encouraging clinical studies using or against tumor
antigens.

2. Cancer Antigens
Scientists have identified a large number of cancer-associated
antigens, many of which are now being used to perform
cancer treatment vaccines both in basic research and in
clinical trials. The list of candidate tumor antigens grows
daily, largely because of expanding genetic technology
including human genome sequencing and gene-expression
profiling. Tumor antigens have been classified into two
broad categories: tumor-specific shared antigens and tumorspecific unique antigens [15]. Shared antigens or tumorassociated antigens (TAAs) are expressed by more than one
type of tumor cells. A number of TAA are also expressed
on normal tissues, albeit in dierent amounts. As reported
in the ocial National Cancer Institute website (NCI,
http://www.cancer.gov/), representative examples of such

Journal of Biomedicine and Biotechnology

shared antigens are the cancer-testis antigens [14], human
epidermal growth factor receptor 2 (HER2)/neu protein
(reviewed in [16]), and carcinoembryonic antigen (CEA)
[17]. Unique tumor antigens result from mutations induced
through physical or chemical carcinogens; they are therefore
expressed only by individual tumors. Tumor-specific unique
antigens encompass melanocyte/melanoma dierentiation
antigens, such as tyrosinase [18], MART1 [19] and gp100
[20], prostate-specific antigen (PSA) (reviewed in [21]) and
Idiotype (Id) antibodies [11]. Optimally designed cancer
vaccines should combine the best tumor antigens with the
most eective immunotherapy agents and delivery strategies
to achieve positive clinical results. An important dilemma for
vaccination against overexpressed tumor-associated antigens
is how to induce eective immunity against the chosen
target without leading to damaging autoimmunity. The
precision oered by DNA vaccines will induce focused
immunity against selected antigens, and, as they become
more powerful, targets will have to be selected carefully to
avoid autoimmunity. Recently, an NCI pilot prioritization
study produced a well-vetted, priority-ranked list of cancer
antigens [22]. Antigen prioritization involved developing
a list of ideal cancer antigen criteria/characteristics and
assigning relative weights to those criteria using pairwise
comparisons. The result of criteria weighting was as follows:
(a) therapeutic function, (b) immunogenicity, (c) role of the
antigen in oncogenicity, (d) specificity, (e) expression level
and percent of antigen-positive cells, (f) stem cell expression,
(g) number of patients with antigen-positive cancers, (h)
number of antigenic epitopes, and (i) cellular location of
antigen expression [22]. Such an eort to prioritize cancer
antigens represents the logical next step in attempting to
focus translational eorts on cancer vaccine regimens with
the highest potential for success.
A biological issue limiting the ecacy of cancer vaccines
is the low immunogenicity of cancer antigens. Strategies to
enhance antigen immunogenicity are discussed in a later
section.

3. Priming the Immune System

DNA vaccines are simple vehicles for in vivo transfection
and antigen production. A DNA vaccine is composed of a
plasmid DNA that encodes the antigen of interest under
the control of a mammalian promoter (i.e., CMV-intA,
CMV immediate/early promoter, and its adjacent intron A
sequence) and can be easily produced in the bacteria [23].
The optimized gene sequence of interest is delivered to
the skin (intradermally), subcutaneum or to the muscle by
one of several delivery methods. Using the host cellular
machinery, the plasmid enters the nucleus of transfected
local cells (such as myocytes or keratinocytes), including resident antigen presenting cells (APCs). Here, gene expression
from plasmid is followed by generation of foreign antigens.
Although the elucidation of all immunological components
involved following DNA immunization has not been entirely
achieved, the mode of action of plasmid DNA vaccines
appears twofold. DNA plasmids, which are derived from
bacteria, stimulate the innate immune system by interacting

Journal of Biomedicine and Biotechnology

with Toll-like receptor 9 (TLR9) [24]), a receptor found on
APCs, although the dierential expression of TLR9 in mice
and primate immune cells makes more complex their role
as adjuvants in primates. This nonspecific immune response
augments the antigen-specific immune response, where the
direct and indirect presentations of antigen to APCs are
involved. Two overarching models have been proposed. The
antigen encoded by the plasmid is produced in host cells,
either in professional APCs leading to direct priming of
immune responses or in nonprofessional cells from where
the antigen can be transferred to APCs leading to crosspriming.
A series of studies intended to determine how such vaccines could work investigated the source of Ag presentation,
the immunological properties of the DNA itself, and the role
of cytokines in eliciting the immune responses.
Early studies showed that DNA delivery method aected
the cell types that were transfected. Gene Gun (bombardment of the epidermis with plasmid coated onto gold
microbeads) tended to directly transfect epidermal keratinocytes and also Langerhans cells, which were shown to
migrate rapidly to regional lymph nodes [25]. In this case,
professional APCs were transfected directly and behave as the
source of Ag presentation.
Alternatively, intramuscular injection of plasmid predominantly led to transfection of myocytes. Myocytes lack
expression of major histocompatibility complex class (MHC)
II and costimulatory molecules and thus would not be
expected to prime T lymphocytes directly. Instead, immune
priming likely occurs by dendritic cells (DCs) [26, 27]
that presumably migrate to the site of DNA inoculation in
response to inflammatory or chemotactic signals following
vaccination [28, 29]. These DCs are thought to present
antigen by cross-presentation of extracellular antigen or
following direct transfection of plasmid DNA [26, 30].
Thus, in terms of induction of immunity, there is an
influence of the site and procedure used for injection, with
muscle and skin cells clearly able to act as antigen depots
but unable to prime the immune response. It is likely that
cross-presentation from these sites to APCs is the major
route to priming [26], but there is also evidence for direct
transfection of APCs, especially when delivery is to skin sites
through a gene gun [25]. The host-synthesized antigen is
then processed and presented by APCs in the context of both
MHC I and MHC II.
Antigen-loaded APCs travel to the draining lymph nodes,
where they present peptide antigens to nave T cells, thereby
eliciting both humoral and cellular immune responses.
Although plasmid DNA vaccines vectors can induce antibody
and CD4+ T cell helper responses, they are particularly
suited to induce CD8+ T cell responses because they express
antigens intracellularly, introducing them directly into the
MHC I antigen processing and presentation pathway [31].
Whatever, the process that conveys antigens to the APC
seems highly ecient since DNA vaccines, that produce only
very low levels of antigen, can induce all arms of the immune
response [32].
One lesson learned in the last years is that the development of plasmid DNA as cancer vaccine raises key issues

3
such as the need to break immunological tolerance, gradual
loss of MHC and antigen in tumour cells, regulatory T cells
that could negatively influence the induction of antitumour
responses, systemic defects in dendritic cells, secretion of
immunosuppressive cytokines, resistance to apoptosis [33,
34] as is discussed elsewhere.

4. Advantages and Disadvantages of

DNA Vaccines
The use of DNA vectors represents an important platform for
clinical applications, in which large-scale vaccine production
is not easily manageable with other forms of vaccine
including recombinant protein, whole tumor cells, or viral
vectors [35].
Although viral mediated gene transfer by genetically modified lentiviruses, adenoviruses, adeno-associated
viruses, and retroviruses is advantageous because of its high
transfection eciency and stability [36], the largest hurdles
using viral vectors are to overcome the immunogenicity of
the viral packaging proteins. Furthermore, viral methods are
disadvantageous because of their high expense, toxic side
eects, limits on transgene size, and potential for insertional
mutagenesis [37].
On the one hand, nonviral vectors are highly flexible, are capable of encoding a number of immunological
components, are associated with a lower cytotoxicity, are
relatively more stable, and are potentially more cost-eective
for manufacture and storage (Table 1).
Their safety in terms of adverse reactions after injection
has been demonstrated in animal models [38, 39] as well as
in human clinical trials.
The first clinical trail, initiated to monitor the safety
and ecacy of a DNA vaccine against HIV-1 infection [40],
demonstrated that DNA plasmid vaccines were safe and were
capable of inducing detectable immune cellular and antibody
responses [4042].
The simple plasmid backbone coupled with the technology of gene manipulation allows incorporation of genes,
which are then expressed by cells transfected in vivo.
Although the transfection process is inecient and varies
with the target tissue and means of delivery, sucient DNA
is generally taken up to prime the immune response [32].
DNA vaccines are free of the problems associated with
producing recombinant protein vaccines, and they are also
safer than live attenuated which can cause pathogenic
infection in vivo. Additionally, studies with DNA vaccines
have shown that even after multiple immunizations, antiDNA antibodies are not produced [43].
The ability to introduce antigen to the host immune
system, thus enabling it to elicit strong Th1 type CD4+ T
cells and CD8+ cytotoxic T cells, is a unique feature of
DNA vaccines which makes them distinct from conventional
protein or peptide vaccines. Because of this feature, they can
readily induce humoral as well cellular immune responses
[44].
Plasmid-based gene transfer can also deliver oligonucleotides that can alter gene splicing or gene expression, for
example, siRNA [35, 45].

Journal of Biomedicine and Biotechnology

Table 1: DNA vaccines main features.

Design
Manufacture
Safety
Stability

Advantage
Allowing the introduction of several immunological
components; synthetic and PCR methods for simple
modifications
Rapid production and formulation; easily engineered,
reproducible, large-scale production and isolation
No pathogenic infection in vivo; no significant adverse
events in any clinical trial; neutralizing immune
responses rarely observed; boost strategy is possible
Long shelf life; relative temperature insensitivity; lack
necessity of a cold chain stored on a large scale

Immunogenicity

5. Enhancing Efficacy and Immunogenicity of

DNA Vaccines
Despite immunogenicity of DNA vaccines has been well
established in animal models, low immunogenicity has
been the major deterrent towards the development of DNA
vaccines in large animal models and human. In order to overcome this hurdle, several approaches have been investigated
including plasmid design, immunomodulatory molecules,
delivery techniques, and prime-boost strategy (Table 2).
5.1. Plasmid Design. Early in the development of DNA vaccines, it became clear that maximizing the expression of the
encoded Ag was critical to the induction of potent immune
responses. Strong viral promoters, such as CMV-intA, are
generally favoured over regulated or endogenous eukaryotic
promoters [70]. Furthermore nuclear targeting sequence
(NTS) could be introduced to increase the eciency of
nuclear plasmid uptake from cytoplasm after intramuscular
injection [48, 71].
The utilization of codon-optimized sequences instead
of the wild-type coding sequences is a general and potent
method to improve vaccination. An optimal coding sequence
is determined back from the amino acid sequence of the
antigen by algorithms that take into account the abundance of specific tRNAs in the cytosol of human cells
and the predicted structure of the mRNA. Thereafter the
selected gene sequence is constructed in vitro using synthetic
oligonucleotides. Adverse rare codons are avoided and
secondary structures in the mRNA are minimized. Thereby,
the synthetic gene is optimal for expression and consequently
for the induction of an immune response [72].
The flexibility of plasmid design coupled with the
technology of gene manipulation allows also gene optimization. Indeed, the variable regions of the heavy (VH )
and light chain (VL ) of the tumor immunoglobulin, specific
for the B-cell malignancies, can be readily cloned and
combined into single-chain variable fragment (scFv) format,
encoding a single polypeptide consisting of VH and VL genes
linked together in frame by a short 15-amino acid linker
[73].

Drawback
Low transfection eciency

Lower immunogeni-city in larger animals and

human compared to mice

As already discussed, the backbone of bacterial DNA

includes cytosine-phosphate-guanine (CpG) unmethylated
regions as sequence motifs that stimulate innate immunity,
creating an inflammatory milieu for triggering the adaptive
immune response [74]. The role of CpG motifs as adjuvants
of immune response to DNA vaccines is well documented
in mice [75]. Preclinical studies showed that the addition
of CpG motifs in the plasmid can result in the induction
of proinflammatory cytokines, for example, IL-12 or IFNI [75]. CpGs are recognized by TLR9, a receptor found on
APCs, helping cytotoxic T-lymphocyte (CTL) dierentiation
and priming. The coadministration of genes encoding
ligands for Toll-like receptors (TLRs) or their signaling
molecules has been shown to improve the immunogenicity
of DNA vaccines [66, 76].
Engineering DNA vaccine design for maximizing
epitope-specific immunity has allowed epitope enhancement
by sequence modification. The recent molecular understanding of the immune response is leading to new strategies
to induce more eective immune responses. Self-tolerance
might lead to deletion of T cells specific for the most eective
epitopes, leaving only low-avidity T cells [77, 78]. Therefore,
not all sequences are optimal antigenic epitopes. A process
termed epitope enhancement is expected to make the
sequences of many epitopes of cancer more immunogenic
[79]. Epitope sequences can be modified to increase the
anity of the epitope peptide for the MHC molecule. The
knowledge of sequence motifs for peptide binding is the key
to improve the primary and/or secondary anchor residues
that provide much of the specificity of binding to the MHC
molecule [80, 81]. This strategy can greatly increase the
potency of a vaccine and can convert a subdominant epitope
into a dominant one by making it more competitive for
available MHC molecules, thereby increasing the level of
specific peptideMHC complexes on the antigen presenting
cell surface [82]. Epitope enhancement has been used to
increase the anity for both MHC class I and class II
molecules (reviewed in [83]).
To enhance the immunogenicity of DNA, vaccines encoding immunostimulatory RNA, such as double-stranded
RNA or replicon RNA, were also generated [84].

Journal of Biomedicine and Biotechnology

Table 2: DNA Vaccine Enhancing Strategies.

Strategy

Approach
intramuscular/EP

Routes of
administration

intradermal/EP
gene gun
tattoo perforating needle
intratumor
high-pressure liquid delivery

cytokine
Genetic
immunomodulators
as Adjuvants

chemokine
T cell helper epitopes
Toll-receptor ligands
heat shock proteins
plasmid DNA/plasmid DNA+EP

Prime-boost strategy

plasmid DNA/recombinant
protein
plasmid DNA/viral vector
viral vector/plasmid DNA

Engineering vaccine design for manipulating antigen

presentation and processing pathways is one of the most
important aspect that can be easily handled in the DNA
vaccine technology. If an antibody response is the goal, it
is clearly desirable to direct antigen expression to the endoplasmic reticulum (ER), in which folding and secretion can
occur. An appropriate leader (signal) sequence can achieve
this. (reviewed in [47]). For induction of CTLs, addition
of genes encoding molecules such as ubiquitin, aimed to
enhance degradation and peptide production in the proteasome, can be eective (reviewed in [85]). Similarly, targeting
expression to dierent subcellular pathways such as the
endosome or lysosome can amplify CD4+ T cell responses
[85]. Thus, DNA vaccines can be designed to induce an
appropriate eector pathway, including antibody against
cell-surface antigens, or CTL response against intracellular
antigens expressed only as MHC class I-associated peptides.
Since tumor antigens are often weakly immunogenic and the
immune repertoire in patients may have been tolerized, the
central question is whether DNA vaccines can activate and
maintain the high level of immunity required to suppress
cancer cell growth.
The pivotal position of CD4 T helper (Th) cells in helping
B cells to produce antibody and control induction and
maintenance of CD8 T cells [86] has led some investigators to
focus on their importance in responses to DNA vaccination.
By selecting genes encoding microbial proteins fused to

Indication
prostate cancer; B-cell
lymphoma
prostate cancer; colon
cancer
cervical cancer
melanoma
melanoma; renal
carcinoma
B-cell lymphoma;
colon cancer
liver cancer; prostate
cancer; melanoma; B-cell
lymphoma
B-cell lymphoma
prostate cancer; follicular
lymphoma; colon
carcinoma
lung carcinoma
cervical cancer
prostate cancer; colon
cancer
prostate carcinoma; breast
cancer
liver cancer; melanoma;
prostate carcinoma
prostate cancer

References
[4648]
[49, 50]
[51, 52]
[53]
[54, 55]
[50, 56]

[5662]
[63]
[46, 47, 64, 65]
[66]
[67]
[46, 47, 50]
[68] NCT00363012
[58, 60, 69]
[59]

the tumor antigen sequence, it was possible to activate

Th cells and to dramatically amplify immunity against
tumor cells [87]. As discussed in the following section,
DNA vaccines oer the opportunity to activate Th cells
and transform weak and ineective immunity to a powerful
antitumor attack [88].
5.2. Immunomodulatory and Immunoenhancing Molecules.
Even though specific antibody and CTL responses could be
induced in clinical trials with naked DNA vaccines, by the
intramuscular or intradermal route, high doses of DNA were
necessary to elicit detectable immune responses [89, 90].
Large quantities, that is, 510 mg, are required to induce only
modest immunogenicity [91].
Modifying the microenvironment of the vaccinated site
by coadministration of genetic, that is, DNA plasmids coding
for immunostimulatory molecules, protein, or chemical
adjuvants, improves the low immunogenicity of DNA vaccines [31].
Progress has been made in developing improved techniques for encapsulating plasmid DNA (liposomes, polymers, and microparticles) although few of these formulations
have been shown to elicit immune responses that are superior
to those elicited by simple intramuscular plasmid DNA, still
disappointing in human clinical trials [92].
Considering the ease in design and construction of
plasmid DNA used to target a particular neoplasm, biological

Journal of Biomedicine and Biotechnology

Table 3: Adjuvant molecules employed in cancer clinical trials to

enhance the immune response.
Adjuvant
cytokines
(GM-CSF, IL-12, IL-2)
Genetic bacterial toxins
(pDOM/tetanus toxin FrC)
immunomodulatory
molecules (HSP70)
Protein cytokines (GM-CSF, IL-2)

Phase
I/II-II

References
NCT00019448
[56, 62]

I/II

[46, 47, 64, 65]

I/II

[67]

I/II-II

[58, 59, 61];

adjuvants can be tailored and encoded within the same DNA

vector as well [35]. A vast array of molecules able to modulate
immune responses can be delivered (Table 3).
They include chemokines to attract APC [93], activating
cytokines [94, 95], costimulatory molecules, APC-targeting
antibodies, and molecules to manipulate antigen presentation and/or processing [96].
One of the common cytokines employed in plasmid DNA
vaccine is granulocytemacrophage colonystimulating factor (GM-CSF), a molecule able to enhance immune
responses by inducing proliferation, maturation, and migration of DCs as well as expansion and dierentiation of B and
T lymphocytes [62].
In addition to codelivery, DNA vaccines allow fusion of
genes encoding activating molecules to the antigen-encoding
sequence. This is an advantage, and fusion genes can create
single vaccines capable of multiple functions.
Biragyn and colleagues showed that the eciency of
DNA vaccination in vivo could be greatly increased by
encoding a fusion protein consisting of scFv fused to a
proinflammatory chemokine moiety that facilitates targeting
of APCs for chemokine receptor-mediated binding, uptake,
and processing of scFv antigen for subsequent presentation
to CD4+ or CD8+ T cells, or both [63]. In two independent
models, vaccination with DNA constructs encoding a fusion
protein consisting of scFv fused to the monocyte chemotactic
protein 3 (MCP-3) or the interferon inducible protein 10
(IP-10a) generated superior protection against a large tumor
challenge (20 times the minimum lethal dose), as compared
with the best available protein vaccines [63].
Additional strategies to activate eective immunity
against poorly immunogenic tumor antigens employ the
DNA fusion genes vaccines to activate T cell help for
antitumor responses. The CD4+ Th cell, as pivotal cell of the
immune response able to induce high levels of immunity and
the maintenance of the response, has been extensively studied
by Stevenson and coworkers [97]. The requirement for
foreign sequences to induce Th for the B-cell response and to
help the CTL response has been known for many years [98,
99]. Since Th cells control responses to vaccination, it is quite
obvious that self-antigens, which do not contain epitopes
likely to be recognized by available Th cells, are incapable
of inducing immunity. A strategy to activate Th cells for
inducing antitumor immunity is to engage a repertoire
against nontolerized antigens. The use of xenogeneic antigen

to break tolerance is likely due to the presence of some

foreign sequences in the xenogeneic antigen that are able
to activate Th cells [100]. Focusing on the antimicrobial
repertoire, the principle has been applied to realize the DNA
fusion gene vaccines encoding the tumor antigen linked
to an antigen derived from tetanus toxin. Fusion of the
Fragment C (FrC) of tetanus toxin amplified the immune
response against a range of tumor antigens, leading to
suppression of tumor growth [87]. Clinical trials by using
these approaches to breaking self-tolerance for therapeutic
purposes in patients with lymphoma and prostate carcinoma
are discussed elsewhere.
5.3. Route of Administration. It is increasingly apparent that
the immunogenicity of DNA vaccines greatly depends upon
the delivery methods used for immunization [101].
In a melanoma mouse model, DNA vaccination was
administered together with intratumoral delivery of antiangiogenic plasmids, encoding angiostatin, and endostatin.
Combined melanoma vaccination resulted in 57% tumorfree survival over 90 days after challenge [54]. In a modest
proportion of patients with malignant disease, intratumoral
injection of DNA led to regression of tumor at distant sites
[102].
The recent studies have confirmed that physical methods
are superior over other delivery methods that administer
DNA in various chemical solutions [103, 104].
Biolistic gene gun delivery involves adhering naked DNA
to gold beads and shooting the particles through a highpressured instrument. This system delivers DNA directly
into skin and Langerhans cells in a highly ecient process.
Gene gun immunization has been shown to induce a greater
CD8+ T cell response as well as to require less vaccine to
achieve tumor immunity [51].
A promising strategy is electroporation (EP), which in
primates increases not only the level but also the breadth of
response [105], overcoming the diculty in translating the
eectiveness of DNA vaccination from preclinical rodents to
large animals, including human subjects [106].
Electroporation-based DNA delivery technology dramatically enhances cellular uptake of DNA vaccines. EP itself
works as an adjuvant to enhance the necessary danger
signals that become detectable by the immune system.
The tissue damage caused by the application of EP causes
inflammation and recruits DCs, macrophages, and lymphocytes to the injection site [107, 108] inducing significant
immune responses, including antibody and T-cell responses.
Moreover, it is tolerable without anesthetic and does not
induce unwanted immune responses against the delivery
mechanism, therefore it can be used for repeat administrations.
A newly developed intradermal DNA delivery is the
tattoo technology. The tattoo device has a cartridge of nine
fine metal perforating needle that oscillate at a constant high
frequency and puncture the skin, leading to DNA transfer
to skin-associated cells. The expression of reporter genes
results in robust T-cell responses [109]. Recently tattooimmunization was applied in a phase I study to assess

Journal of Biomedicine and Biotechnology

the toxicity and ecacy of inducing tumor-specific T-cell
immunity against melanoma [53].
5.4. Prime-Boost Strategies. Vaccination schedules based on
combined prime-boost regimens using dierent vector systems to deliver the desired antigen (i.e., heterologous primeboost immunization regimen) appear to be a successful
improvement in DNA vaccine platform.
Actually, prime-boost regimens have shown promise in
eliciting greater immune response in humans compared with
DNA vaccination alone [101].
The DNA-prime-viral vector-boost approach focuses on
the induction of T-cell immune responses. In this approach,
homologous boost immunization carries the equivalent
antigen than the previous immunization. Viral vectors that
have been tested as booster vaccine include adenovirus,
vaccinia virus, fowlpox [110, 111] as well as recombinant
vesicular stomatitis virus [112].
Likewise, the DNA-prime-protein-boost approach employs recombinant protein antigens that match with the
antigens used in DNA prime immunization [68, 113, 114].
This strategy aims to develop balanced humoral and cellmediated immune responses with a focus on eliciting high
quality protective antibody responses.
The heterologous prime-boost vaccination regimen
exploits the ability of the immune system to generate a
large number of secondary antigen-specific T cells. Following
a priming immunization, a proportion of the antigenspecific T cell population transforms into antigen-specific
memory T cells, which have the ability to expand rapidly
upon encounter with the same antigen a second-time
round.
Since the priming and boosting vectors are dierent,
this strategy allows for greater expansion of the disease
antigen-specific T cell populations [115]. To date, heterologous prime-boost regimens are among the most potent
strategies to induce cellular immune responses. Compared
to homologous prime-boost approach with the same DNA
vaccine, boosting a primary response with a heterologous
vector will result in 410-fold higher T cell responses [116
118].
On the one end, a combination of DNA vaccines with
EP in a homologous prime-boost approach could generate
antibody responses comparable to those that are induced
by protein in Complete Freund Adjuvant, and also amplified CTL responses [46]. EP may provide a prime-boost
combination equivalent to that observed using viral vectors,
and it is now undergoing testing in the clinic using a DNA
vaccine for patients with prostate cancer. Repeated EP has
been accepted by patients without the need for general or
local anaesthesia and with no apparent long-term ill eects
[47].
5.5. Strategies to Break the Immunosuppressive Networks.
Immune suppression is a feature of the tumor microenvironment and a barrier to tumor immune therapy. The
microenvironment of tumors is established through the
activity of both myeloid and lymphoid regulatory cells,

7
as well as through the production of immune-suppressive
factors by malignant cells themselves.
Many tumor-infiltrating macrophages, referred to
myeloid-derived suppressor cells (MDSCs), have an immune-suppressive phenotype [119]. These macrophages
are abundant in many tumors arising in both humans and
mice and can exert powerful anti-inflammatory eects. In
addition to MDSCs, regulatory T cells (Tregs) also heavily
infiltrate many tumors [120]. These cells, characterized by
the expression of the transcription factor FoxP3 as well as
CD4 and CD25, play a key role in the regulation of adaptive
immunity. Tregs can suppress immune responses through
the secretion of suppressive cytokines like TGF- and IL35 [120, 121]. Tregs are a potential barrier to developing
productive immune therapies for cancer, and they represent
an attractive target for enhancing antitumor immunity.
Cancer immunotherapy is designed to specifically target
cancer types using components of the immune system.
Therefore DNA vaccines are also faced many obstacles that
include breaking peripheral T cell tolerance against tumor
self-antigens, to elicit appropriate immune reactions, as well
as overcoming tumor-derived immunosuppressive networks
and evasion tactics. Evasive mechanisms adopted by malignant cells to prevent immune cell function are numerous
and lead to the clonal expansion of non-immunogenic
tumor cells, by loss of tumor antigen, and to the apoptosis
prevention [35].
Tumour cells can downregulate expression of MHC
and target antigens and often secrete immunosuppressive molecules to defend themselves against attack [122].
Tumours can create a tolerogenic environment which spreads
to draining lymph nodes and can enhance regulatory Tcell activity. The hurdles to successful reversal of tolerance
and induction of eective immunity are becoming clear
and vaccines must incorporate elements to overcome them
[123].
Furthermore cancer cells secrete soluble factors in the
tumor microenviroment, for example, VEGF, IL-10, and
TGF-, that aect the maturation, dierentiation, and
activity of APCs as DCs [124], interfering with immune cells
maturation and eector properties. The tumour microenvironment may drive tumour growth and even selectively
support a subset of tumour cells, the cancer stem cells
(CSCs).
The DNA vaccination platform can be capable of
suppressing the progression of already established tumor
by targeting those secreted soluble factors in the tumor
microenvironment [125], reversing immunological attenuation mechanisms and improving DNA vaccine potency.
The concept of combining cancer vaccination with
angiogenesis inhibition is appealing, due to favorable safety
profile of both approaches, as well as possible biological
synergies [54]. DNA vaccination in mice against the VEGF
receptor, FLK-1, abrogated the tumor vasculature and
protected DNA vaccinated animals from tumor challenge
in prophylactic approach [126]. Expression of the plateletderived growth factor receptor (PDGFR) in stromal cells
directly correlates with advanced stage disease in human
colorectal cancer. DNA vaccine against PDGFR suppressed

Journal of Biomedicine and Biotechnology

growth and dissemination of human colorectal cancer cells

injected into mice [127].
In vivo coadministration of plasmids encoding the
chemokine macrophage inflammatory protein-1alpha (MIP1alpha) and the DC-specific growth factor fms-like tyrosine
kinase 3 ligand (Flt3L) with the plasmid DNA augments the
immunogenicity of the vaccine, mobilizing and activating
large numbers of DCs at the site of inoculation [128].
Consistent with the concept that most eective cancer
therapies are multimodal, combining Treg depletion with
active cancer immunotherapeutic interventions is an attractive prospect, supported by abundant data in mice [129
132] and by preliminary human trials [133135]. Lastly,
additional strategies aimed to altering regulatory T cell
function in cancer immunotherapy, including blocking Treg
tracking, dierentiation, and/or function and reducing
eector cell susceptibility to suppression, have already proven
successful in preliminary studies [136138].

The inclusion of FrC sequence, or other nonself antigens,

activates T-cell help to reverse tolerance and induces high
levels of immunity [47, 64, 65]. To further increase immunogenicity of DNA vaccine, the use of molecular adjuvants such
as cytokines and immunomodulatory molecules has been
extensively employed in clinical trials [56, 5863, 67].
Clinical trials conducted over the last few years have
led promising results, particularly when DNA vaccines
were used in combination with other form of vaccines, as
demonstrated in prostate and liver cancer clinical trials [58
60, 68]. Delivery of gene-based vaccines by physical methods,
that is, electroporation and gene gun, has demonstrated
to amplify the immune responses induced by therapeutic
vaccines against cancer [46, 47, 64].
In the following section, an overview of various types of
clinical trials will be given to highlight the issue for usage of
plasmid DNA in humans. Table 4 provides a brief summary
of clinical trials discussed in this review.

6. Human Clinical Trials

6.1. Lymphoma. DNA vaccination is an attractive and eective approach for active therapeutic vaccination against Bcell malignancies given the ease of production compared to
Id protein vaccines.
Patient-specific DNA vaccines for therapy of B cell
lymphomas and multiple myelomas based on scFv encoding
a chimeric immunoglobulin molecule consisting of VH and
VL genes derived from each patientss tumor were shown to
be eective in animal models [47, 73].
The first phase I/II trial of idiotypic vaccination for follicular B-cell lymphoma using a genetic approach [142] was
conducted by Hawkins and colleagues. Vaccines encoding
individual DNA idiotypic scFv fused to TTFrC were delivered
as naked DNA by i.m. injection in patients with follicular
lymphoma in clinical remission following chemotherapy,
and plasmid DNA were able to develop cellular or/and
humoral antiidiotype immune responses in 38% of patients
over a period of several months [47].
In a second study of vaccine therapy for B-cell lymphoma, the patients tumor scFv was linked to the IgG2a and
mouse immunoglobulin heavy- and light-chain constant
regions chains, respectively. In this phase, I/II trial patients
in remission after chemotherapy received two series of i.m.
DNA vaccinations and at the end of the second vaccination,
50% of patients exhibited humoral and/or T-cell anti-Id
responses; yet, these were cross-reactive with Id proteins
from other patients tumors. Subsequently, a third series of
vaccinations was carried out using human GM-CSF DNA
mixed with Id DNA: humoral or T-cell responses were
boosted in some cases [56].

The goals of the various clinical trials were to demonstrate

the safety and tolerability of the candidate vaccines, and to
explore the ecacy of DNA vaccines in humans. Injection
of the plasmid DNA construct is tolerated well in terms
of safety in the patient population and rarely involves
systemic toxicities. DNA vaccines that are currently being
tested do not show relevant levels of integration into host
cellular DNA [139, 140]. Besides, preclinical studies in
nonhuman primates as well as early studies in humans did
not detect increases in antinuclear or anti-DNA antibodies.
Participants in human trials of DNA vaccines are followed
for possible signs and symptoms of autoimmunity induced
by DNA vaccination giving no convincing evidence of
autoimmunity developing in association with a DNA vaccine
[40, 42, 141].
The earliest Phase I clinical trial for a DNA vaccine was
of an HIV-1 candidate tested in individuals infected by HIV1, followed by studies in volunteers who were not infected
by HIV-1 [40]. Other prophylactic and therapeutic DNA
vaccine trials followed, including trials that tested DNA vaccines against cancer influenza, malaria, hepatitis B, and other
HIV-1 candidates [24, 41, 42]. These trials demonstrated
that the DNA vaccine platform is well tolerated and safe,
as no adverse events were reported and all studies went to
completion.
The evident safety of DNA vaccines has led to a relaxation
of the requirements for approval by both the United States
Food and Drug Administration and the national competent
regulatory authorities in Europe. This is why many clinical
studies tend to melt the first phase with the second phase.
Then the issue has become the ecacy rather than toxicity.
Since tumour antigens are generally weakly immunogenic, they often induce a low level of spontaneous immunity
or, in other cases, the spontaneous response can lead to
tolerance [47]. The molecular precision oered by genebased vaccines, together with the facility to include additional genes to direct and amplify immunity, could lead to an
ecient methods to use the immune system against cancer.

6.2. Prostate Carcinoma. After the important insights provided in preclinical studies [49], the department of Oncology
of the University Hospital of Uppsala is recruiting participants for a phase I/II trial, where intradermal EP (DERMA
VAX) will be used as a delivery system. This study will assess
the feasibility and safety of vaccination with increasing doses
of xenogenic DNA coding for the Rhesus Prostate Specific
Antigen (rhPSA), a protein that is 89% homologous to

Journal of Biomedicine and Biotechnology

human PSA, administered in patients with relapsed prostate
cancer.
A phase I/II, dose escalation, DNA vaccination trial with
plasmid DNA, which carries prostate-specific membrane
antigen (PSMA), fused to a domain (DOM1) of Fragment
C of tetanus toxin, delivered either by i.m. or by i.m.
followed by EP, was performed in patients with recurrent
prostate cancer. The epitope used in this study, PSMA27
is a short stretch of 9 amino acids tumor-derived epitope
belonging to the PSMA. Preliminary analysis of CD8+ Tcell reactivity against the prostate-specific membrane antigen
target peptide indicated significant responses in 3 out of
3 patients and CD4+ T-cell responses against the DOM1.
These data validated EP as a potent method for stimulating
humoral responses induced by DNA vaccination in humans
[46, 47, 64].
Results of a phase I/II trial, conducted with DNA vaccine
encoding human prostatic acid phosphatase (PAP) coadministered intradermally with GM-CSF, in prostate cancer
patients (stage D0) are associated with an increased PSA
doubling time (PSADT), 6.5 months pretreatment versus 9.3
months in the 1 year posttreatment [61]. A longer PSADT is
associated with an extremely low risk of death from prostate
cancer. Besides, 14% of patients developed PAP-specific
IFN gamma-secreting CD8+ T-cells immediately after the
treatment course, and 41% of patients developed PAPspecific CD4+ and/or CD8+ T-cell proliferation, confirming
the preclinical studies [143].
Todorova and colleagues [58] enhanced the DNA vaccine
ecacy by heterologous prime-boost regimen in a Phase
I/II study. Prostate cancer patients were prime-boosted
with alternate injections of recombinant adenoviral vector
expressing PSMA and plasmid DNA encoding PSMA and
CD86 alongside receiving GM-CSF proteins as adjuvants.
After 36-month observation period from the first vaccine
injection, 86% of participants developed anti-PSMA antibody.
6.3. Melanoma. DNA vaccine platform is a promising
therapeutic approach also for the treatment of malignant
melanoma, as demonstrated by already completed and ongoing clinical trials.
In stage IV melanoma patients, a phase I/II pilot study
of intranodal delivery of Synchrotope MA2M plasmid DNA
vaccine induced both humoral and CTL responses against
cells expressing tumor two melanoma-associated antigens
[144]. Synchrotope MA2M plasmid is a bivalent DNA
vaccine encoding epitopes for both Melan-A (MART-1) and
tyrosinase with potential antineoplastic activity.
The same approach was used in a improved trial conducted with the Synchrovax SEM plasmid DNA vaccine
containing a plasmid pSEM that encodes 4 peptide epitope
sequences, Melan-A (2635), Melan-A (3196), tyrosinase
(19), and tyrosinase (369377), resulting in antigen-specific
immunity even though not induce regression of established
disease [145].
DNA plasmids encoding the gp100 nonmutated melanoma-melanocyte antigen alone were administered in patients

9
with metastatic melanoma. Rosenberg et al. showed that
neither intramuscular nor intradermal injection was capable of raising cellular immune reactivity or a significant
incidence of antitumor eects [57]. Increasing results were
obtained in a phase II study with interleukin-2 cytokines
as adjuvant used in combination with same vaccination
protocols (ClinicalTrials.gov Identifier: NCT00019448) and
in a phase II trial with human GM-CSF plasmid DNA in
conjunction with a multipeptide vaccine encoding gp100 and
tyrosinase peptidse [62].
6.4. Cervical Cancer. Current vaccination strategies are based
on the induction of neutralizing antibodies against the
major and minor capsid proteins, L1 and L2, of human
papillomavirus, and Gardasil is only eective against a subset
of HPV genotypes [146]. Further therapeutic interventions
for early-stage and late-stage cervical cancers or HPV-related
disease are uneective.
DNA plasmid platform could represent an ideal vaccine
against HPV infections since it could generate both humoral
immune response to prevent new infections as well as cellmediated immunity to eliminate established infection [146].
A recent phase I/II clinical trial in patients with
high-grade squamous intraepithelial lesion associated with
HPV16 provided DNA plasmid expressing a mutated nonfunctional E7 incapable of binding retinoblastoma protein,
with no transforming activity, linked to HSP70. A signal
sequence was also attached to the hybrid antigen which
results in secretion of the linked E7 antigen [67]. E7 HPV
antigen as well as E6 antigen are essential for transformation
and are coexpressed in HPV-associated lesions hence they
represent ideal targets for the development of HPV therapeutic vaccines.
6.5. Liver Cancer. In preclinical studies, mice were successfully DNA-based immunized [147]. In a prime-boost
approach, coadministration of plasmids DNA encoding
murine alpha fetoprotein (AFP) and murine GM-CSF was
followed by boosting with an AFP-expressing nonreplicating
adenoviral vector [60] leading to tumor protective immunity.
The early studies were applied in a phase I/II clinical
trial in patients with HLA-A 0201-expressing stage IIIVA
hepatocellular carcinoma. Vaccine therapy, comprising AFP
and sargramostim (GM-CSF) plasmids DNA, followed by
AFP adenoviral vector boost determined the dose-limiting
toxicity and maximum tolerated dose of adjuvant vaccination (ClinicalTrials.gov Identifier: NCT00093548).
6.6. Breast Cancer. Since HER-2/neu (HER2) oncogenic protein is a tumor antigen in patients with breast and ovarian
cancer, several vaccine strategies have been developed and
are being evaluated for safety and immunogenicity in phases
I and II clinical trials (ClinicalTrials.gov). Patients whose
tumors overexpress the antigen have both detectable antibody and T-cell immunity directed against HER2. Likewise
preclinical studies suggest that the HER2 protein, particularly the intracellular domain (ICD), is a tumor rejection
antigen [148].

10

Journal of Biomedicine and Biotechnology

Table 4: Phases I/II-II clinical trials: key summary.

Tumor

Study ID

Patients no.

Lymphoma

UK-007

25

NCT00859729

18

UK-112

20

NCT00582140

22

Bulgarian
Drug
Agency Register

52

NCT00033228

618

Objectives
Determine the safety,
dose, immunogenicity
Determine the feasibility
and safety
Determine the safety and
functionality of DNA
vaccine delivery system
Determine the
feasibility and safety
Determine the
immunological responses

Status
Completed

Melanoma

NCT00085137

327

Cervical cancer

NCT00121173

150

Liver cancer

NCT00093548

325

Breast cancer

NCT00363012

56

Characterize the humoral

immune response
against PSMA
Determine the safety
and tolerability
Determine the
immunological and
clinical responses
Determine the safety
and tolerability
Determine any
antitumor response
Determine the feasibility
and toxicity
Determine the eect
Determine changes
in lesion size and
HPV viral load
Determine the immune
responses
Correlate measures of
immune response with
clinical response
Determine the doselimiting toxicity and
maximum tolerated dose
Determine the optimal
biological dose
Determine disease-free
survival of patients treated
Determine and
characterize
the immunologic
memory
to the HER2-ICD

Side eects

Absence of toxicity
CD8+ T-cell
reactivity against the
target peptide
PAP-specific IFN
gamma-secreting
CD8+ T-cells
PAP-specific
CD4+ and/or CD8+
T-cell proliferation
Specific humoral
immune response
against PSMA
Antigen-specific
Immunity
No regression of
established disease

Brief and
acceptable
pain at the
injection site

Open
recruiting

Open

Prostate cancer
Determine the safety

Response
Absence of toxicity
Cellular and/or
humoral responses

Completed
(Aug 2009)

Completed

Completed
(July 2009)
Open
Not
Recruiting

Open
Not
Recruiting

Immunological
ecacy
in terms of
T-cell response
Absence of
toxicity
In the highest-dose
cohort the number
of patients with
complete histologic
regression is
higher than the
unvaccinated cohort,
but not significant.

Completed
(Feb 2009)

Absence of
toxicity

Open
recruiting

Absence of
toxicity

Grade I/II toxicity

Grade I toxicity

Transient
injection-site
discomfort

Journal of Biomedicine and Biotechnology

Salazar and colleagues are studying the immune response
in patients overexpressessing HER2 epitope who have
undergone vaccine therapy in a heterologous prime-boost
regimen (ClinicalTrials.gov identifier: NCT00363012). After
vaccination with a plasmid encoding HER2 ICD in patients
with advanced stage HER2 overexpressing breast and ovarian
cancers patients receive HER2 ICD protein treatment intradermally at 6 months postvaccination with the pNGVL3hICD vaccine. The injection site is biopsied and examined
for infiltrating T-cell and antigen-presenting cell populations
and blood samples are examined for the presence of memory
markers to demonstrate the development of HER2 ICD
memory immunity.

7. Conclusions and Future Directions

Plasmid DNA is a new generation biotechnology product
that is just beginning to enter the marketplace. Progress
in the application of DNA vaccines as an immunization
protocol is evident from the increasing number of such
vaccines under evaluation in clinical trials and by the recent
approval of several DNA vaccine products for veterinary
applications.
The goal of DNA vaccination will be the development
of eective immunization strategies against previously established tumors. Because of tolerance to tumour antigens,
eorts are ongoing to optimize the DNA vaccine technology
platform. Strategies to improve antigen expression, inclusion
of adjuvants in the formulation, or as immune modulators to
improve the immunogenicity, and the use of next-generation
delivery methods are under intensive investigation. Current
eort to prioritize cancer antigens represents the logical
next step in attempting to focus translational eorts on the
most promising cancer antigens into vaccines for cancer
treatment or prevention. It is likely that these vaccines
will have to be combined with other treatment modalities.
It has become appreciated that vaccine approaches may
enhance subsequent responses to radiotherapy and that
certain chemotherapies actually enhance responses to vaccines. Accordingly, several late-stage clinical trials are already
evaluating the benefit of vaccination in addition to conventional chemotherapy. One attractive setting is in patients
during complete remission after standard adjuvant treatment
(chemotherapy, radiotherapy, etc., or a combination) to
whom vaccination can be given after immunological recovery [149]. Combining immunotherapy with conventional
chemotherapy, antiangiogenic therapy, and other approaches
could yield synergistic or additive therapeutic results.
There is still much to do in terms of optimizing vaccine
design, activation and selecting appropriate target antigens,
improving immune recruitment, and delivery technology.
Nevertheless, in the next years an increasing number of
DNA vaccines will enter more advanced phases of human
studies, aimed to establish their ecacy as real clinical products. Therapeutic regimens composed of optimal vaccine
formulations with combinations of immunotherapy agents
and delivery strategies could oer hope to patients suering
from incurable cancer that current standard therapies cannot
provide alone.

11

Acknowledgments
This work was supported by MUR Grant FIRB 2006
(RBIP0695BB), and by the Italian Banca Marche. D. Fioretti
and S. Iurescia. have been supported by MUR Grant FIRB
2006 (RBIP0695BB). Both of them contributed equally to
the preparation of this paper.

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Diabetes Research
Volume 2014

Hindawi Publishing Corporation

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Volume 2014

Hindawi Publishing Corporation

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Volume 2014

International Journal of

Journal of

Endocrinology

Immunology Research
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Disease Markers

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Volume 2014

Volume 2014

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BioMed
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PPAR Research
Hindawi Publishing Corporation
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Hindawi Publishing Corporation

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Volume 2014

Volume 2014

Journal of

Obesity

Journal of

Ophthalmology
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Volume 2014

Evidence-Based
Complementary and
Alternative Medicine

Stem Cells
International
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Volume 2014

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Volume 2014

Journal of

Oncology
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Volume 2014

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Volume 2014

Parkinsons
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Computational and
Mathematical Methods
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Volume 2014

AIDS

Behavioural
Neurology
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Research and Treatment

Volume 2014

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Volume 2014

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Volume 2014

Oxidative Medicine and

Cellular Longevity
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Volume 2014