S1359644621002804

Drug Discovery Today d Volume xxx, Number xx d xxxx 2021 REVIEWS
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Methods to improve the immunogenicity
of plasmid DNA vaccines
Dalinda Eusébio a, Ana R. Neves a, Diana Costa a, Swati Biswas b, Gilberto Alves a,
Zhengrong Cui c, Ângela Sousa a,⇑
a
CICS-UBI – Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal
b
Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Jawahar Nagar, Shameerpet, Hyderabad 500078, Telangana,
India
c
The University of Texas at Austin, College of Pharmacy, Division of Molecular Pharmaceutics and Drug Delivery, Austin, TX 78712, USA
DNA vaccines have emerged as innovative approaches that have great potential to overcome the
limitations of current conventional vaccines. Plasmid DNA vaccines are often safer than other vaccines
because they carry only antigen genetic information, are more stable and easier to produce, and can
stimulate both humoral and cellular immune responses. Although the results of ongoing clinical trials
are very promising, some limitations compromise the immunogenicity of these vaccines. Thus, this
review describes different strategies that can be explored to improve the immunogenicity of plasmid
DNA vaccines, including the optimization of the plasmid vector backbone, the use of different methods
for vaccine delivery, the use of alternative administration routes and the inclusion of adjuvants. In
combination, these improvements could lead to the successful clinical use of plasmid DNA vaccines.
Keywords: Adjuvants; Administration routes; Delivery systems; Immunogenicity; Plasmid DNA vaccines
Introduction expensive costs and limited storage time. In addition, protein-

Vaccination is the most cost-effective method to fight and erad- based vaccines sometimes fail to offer an adequate immune
icate several pathogenic and infectious agents spread around the response, especially for pathogens that replicate intracellularly,
world, contributing to human and animal well-being. The pro- including viruses.
tection induced by vaccination is mediated through a complex Recently, DNA vaccines have emerged as alternatives that
interaction between innate and adaptative immune systems have great potential to overcome the limitations of current con-
[1]. At present, there are several different types of vaccines in ventional vaccines. DNA vaccines are considered to be third-
clinical use that can be categorized into two distinct generations. generation vaccines. They are normally based on bacterial DNA
First-generation vaccines comprise traditional live-attenuated plasmids and a strong eukaryotic promoter that enhances the
and inactivated vaccines, constituted of weakened or killed ver- expression of the antigenic protein, inducing immune responses
sions of the pathogen, respectively. The second generation com- against a wide range of pathologies and diseases, such as allergies
prises subunit vaccines, which contain selected fragments of the [4], cancers [5], and autoimmune and infectious diseases [6,7].
pathogen [2]. These fragments can be antigenic proteins, toxoids DNA vaccines offer numerous advantages over conventional vac-
(inactivated toxins), virus-like particles (VLPs), polysaccharides, cines, including the ability to stimulate both humoral and cellu-
or protein-polysaccharide conjugates [3]. These second- lar immune responses, which are beneficial in preventing
generation vaccines present several disadvantages including intracellular pathogen infections [8]. DNA vaccines that are
⇑ Corresponding author. Sousa, Â. (angela@fcsaude.ubi.pt)
1359-6446/Ó 2021 Elsevier Ltd. All rights reserved.

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Please cite this article in press as: D. Eusébio et al., Drug Discovery Today (2021), https://doi.org/10.1016/j.drudis.2021.06.008
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based on plasmids are unable to revert to virulent forms, and so resident cells. Thus, depending on the cells that are transfected
are very valuable in terms of safety. They possess the intrinsic at the immunization site, researchers have proposed three differ-
advantage of target flexibility as they are easy to synthesize by ent pathways that can lead to antigen presentation (Fig. 2). First,
changing the nucleic acid sequences. Furthermore, they intro- the DNA-encoded antigens can be expressed by transfected
duce nucleic acids encoding pathogenic antigens to the host cells somatic cells (such as myocytes and keratinocytes) and are pre-
using eukaryotic promoters, closely resembling live infections sented on MHC-I molecules to cytotoxic CD8 + T cells. However,
and thus inducing both cell and antibody-mediated immune as these somatic cells do not present antigenic proteins effi-
responses [9]. In addition, these vaccines allow reduced process- ciently, the other two pathways might be more important for
ing time, as it is possible to perform a rapid formulation and DNA vaccine immunogenicity [15,18].
large-scale manufacture and isolation, and so these vaccines are In the second pathway, the transfected somatic cells express
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highly cost-effective. Moreover, DNA vaccines are more ther- the encoded antigen and release derived peptides via exosomes
mostable than conventional vaccines, and thus their storage or apoptotic bodies, which are endocytosed by APCs, and the
and transport are relatively easy. captured antigens are processed and cross-presented through
Since publication of the initial evidence that nucleic acids MHC-II molecules to helper CD4 + T cells [18]. In this case, a
could be used for immunization, DNA vaccines have become humoral immune response is triggered when B cells recognize
progressively more sophisticated and improved. Five DNA vacci- the antigen through the presentation of activated antigen-
nes have already been licensed for veterinary applications. These specific CD4 + T cells. As a result, the B-cell immunity generates
include one against infectious hematopoietic necrosis virus in antibodies and memory B cells against the antigenic protein [19].
salmon [10], one against West Nile virus infection in horses Memory B cells are responsible for the induction of a strong
[11], one dedicated to the treatment of malignant melanoma in immune response when reinfection occurs, so this exogenous
dogs [12], one targeting growth hormone-releasing hormone pathway gives the DNA vaccine a preventive effect.
(GHRH) to enhance protection against pneumonia caused by The third, and perhaps the most significant molecular path-
Mycoplasma hyopneumoniae in swine [13], and the fifth against way induced by DNA vaccines is the direct transfection of APCs.
H5N1 avian influenza virus in chickens [14]. To date, however, This results in endogenous antigen processing and presenting
no DNA vaccine has been approved for human use. Several clin- through MHC-II molecules to helper CD4 + T cells, and through
ical trials that have been conducted in recent years showed only MHC-I molecules to cytotoxic CD8 + T cells. Following activa-
limited success because of poor immunogenicity. Therefore, tion, cytotoxic CD8 + T cells begin to proliferate and differentiate
more work on DNA vector design and delivery performance is into effector cells, named cytotoxic T lymphocytes (CTLs). Then,
still required to enhance the immunogenicity of DNA vaccines the effector cells are able to detect infected cells that present the
to the levels required for human regulatory approval and com- target antigen and cell death is triggered via different mecha-
mercial use [15]. nisms [20]. The effector mechanisms that CTLs use to kill target
Different methods can be applied for the delivery of DNA vac- cells comprise the secretion of death-inducing effector mole-
cines. These methods must overcome extra and intracellular bar- cules, including chemokines and effector cytokines such as
riers to transport DNA efficiently into the nucleus. DNA can be interferon-gamma (IFN-c) and tumor necrosis factor (TNF-a), as
administered through several routes, including intramuscular, well as the production and release of cytotoxic granules such as
transdermal, intradermal, subcutaneous, intravenous, oral or perforin and granzymes. In addition, CTLs can induce the apop-
pulmonary administration [16]. This review aims to summarize tosis of target cells via Fas–Fas-ligand interactions, resulting in
recent achievements in the development of methods for DNA the activation of the caspase cascade [21]. As cell death, and con-
vaccine delivery. In addition, developments in DNA vaccine sequently death of intracellular pathogens, is triggered by the
design and adjuvants will be analyzed, and ongoing human clin- activation of cytotoxic CD8 + T cells, this endogenous pathway
ical trials will be explored. can provide a therapeutic effect of the DNA vaccine. Thus, the
combination of both preventive and therapeutic effects
enhances a strong cellular and humoral immune response that
Mechanism of action of DNA vaccines is not seen with other types of vaccines [22].
DNA vaccines can induce not only humoral responses but also Before the adaptive immunity mentioned above is activated,
cell-mediated responses. After a DNA vaccine is administered, recombinant plasmids also induce the activation of innate
the recombinant plasmid needs to translocate to the nucleus immunity, which is dependent on the recognition of
for transcription, followed by translation to build the antigen pathogen-associated molecules that are not naturally present in
in the cytoplasm. Following expression of the encoded antigen the host. The innate immune response is activated by the recog-
by the host cellular machinery, specific immune responses are nition of the double-stranded DNA (dsDNA) of the bacterial plas-
induced by the expressed antigen [17]. In general, the immune mid backbone, owing to the presence of CpG dinucleotides
response requires antigen processing and presentation, in the motifs [20]. In the cytosol of transfected cells, some molecules
context of class I and class II major histocompatibility complex act as dsDNA sensors that activate innate immune responses
molecules (MHC-I or MHC-II), to CD8 + and CD4 + T cells, through the STING-TBK1 signaling cascade. This interaction
respectively [15]. This process is schematically represented in between the stimulator of interferon genes and the TANK-
Fig. 1. binding kinase 1 in the presence of intracellular DNA plasmids
The administration of a DNA vaccine might lead to the direct leads to the production of type I interferons (IFNs) and inflam-
transfection of antigen-presenting cells (APCs) or other tissue- matory cytokines [23,24]. In addition, the CpG dinucleotides
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FIGURE 1
Antigen processing and presentation in the context of major histocompatibility complex (MHC) molecules after administration of a DNA vaccine. APC,
antigen-presenting cell.
motifs that are common in bacterial DNA could also contribute the simplest method of vaccination: intramuscular injection of
to the immunogenicity of DNA vaccines. The Toll-like receptor naked plasmid DNA (pDNA). When vaccines are administered
9 (TLR9) was identified as a receptor for CpG motif DNA, which as naked pDNA, the immune responses are often weak due to
stimulates the expression of molecules that both initiate an poor cellular uptake and DNA degradation. Indeed, approxi-
inflammatory response and help to induce adaptive immune mately 95–99% of the naked DNA that is administered intramus-
responses [25]. Thus, understanding the mechanisms of activa- cularly remains in the extracellular space and it is rapidly
tion of both branches of immunity makes it possible to improve degraded by endonucleases. The use of delivery systems to pro-
the effectiveness of the immune responses that are induced by tect the pDNA and to help its cellular internalization represents
DNA vaccines. a promising strategy to overcome this constraint, and thus to
However, as previously mentioned, the DNA vaccines that improve the immunogenicity of DNA vaccines. Another consid-
have been licensed to date are only for veterinary use. Although eration in DNA vaccine immunogenicity is the route of adminis-
numerous clinical trials have been conducted, none of the DNA tration. Intramuscular administration of a DNA vaccine by
vaccine candidates has met the regulatory requirements needed needle-based injection is invasive and only induces weak
for approval and commercialization for human applications. immune responses because only a small fraction of the injected
The following steps need to be taken before any DNA vaccine DNA is taken up by cells [28]. This review describes and analyzes
can be submitted for approval to the regulatory agencies: labora- the different strategies that have been applied to enhance DNA
tory demonstration of the proof of concept; design and establish- vaccine immunogenicity, including optimization of the plasmid
ment of the manufacturing process; demonstration of adequate vector backbone, the use of alternative routes of administration,
quality and nonclinical safety; clinical trial approval; demonstra- the development of innovative delivery systems, and the use of
tion of clinical safety and efficacy; and marketing authorization adjuvants to potentiate the immune responses.
application [26]. Thus, the success achieved in preclinical studies
has not yet been translated into human clinics, mostly because
of the low immunogenicity of DNA vaccines observed in Optimization of the plasmid vector backbone
humans. Clinical trials using DNA vaccines have induced Plasmid DNA vectors have been used extensively in DNA-based
humoral and cellular immune responses, but the levels of these therapeutics. These vectors can be divided into a transcription
responses were not sufficient to generate significant clinical ben- unit, containing the eukaryotic promoter and the therapeutic
efits [16,27]. This inadequacy has been attributed to the presence gene, and the bacterial backbone required for its amplification,
of bacterial sequences in the DNA backbone, and to the route of which contains the origin of replication and selection markers
administration or delivery method used for these vaccine candi- (such as antibiotic resistance genes). Nevertheless, bacteria-
dates. All five licensed DNA vaccines are administered through derived sequences are not required for transgene expression
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FIGURE 2
Immune activation by DNA vaccines. Three different pathways can lead to antigen presentation after administration of a DNA vaccine, resulting in the
induction of both cellular and humoral immunity. APC, antigen-presenting cell; CTL, cytotoxic T lymphocytes; IFN, interferon; MHC, major histocompatibility
complex; TNF, tumor necrosis factor.
and are usually associated with serious safety concerns. For transgene expression [30]. As an example, the expression vector
instance, antibiotic resistance genes may disseminate into pcDNA3 was modified to generate the pVAX1 vector by replacing
human microbiota via horizontal gene transfer, increasing the the ampicillin selection marker with a kanamycin selection mar-
incidence of antibiotic-resistant infections worldwide [29]. More- ker, because of the potential for ampicillin to cause autoimmu-
over, bacterial sequences can cause severe side effects, such as nity. It was demonstrated that mice inoculated with pcDNA3–
inflammatory reactions in the host, and usually lead to the for- ANXB1 developed an autoimmunity response that did not occur
mation of repressive heterochromatin around these sequences in pVAX–ANXB1 inoculated mice [31]. Moreover, the sucralose
in the pDNA backbone, resulting in the silencing of episomal selection system, which incorporates transient expression
enhancers, has been developed to remove the need for an antibi- an efficient antigen-specific immune response. For example, sub-
otic selection marker. The SV40 enhancer was incorporated cutaneous injection mainly results in the transfection of fibrob-
upstream of the cytomegalovirus (CMV) promoter to improve lasts and keratinocytes, whereas dermal delivery primarily
extrachromosomal transgene expression, and the human T- transfects LCs. When compared to IM administration, ID admin-
lymphotropic virus type I (HTLV-I) R region was incorporated istration results in enhanced expression of the antigen, improv-
downstream of the CMV promoter to increase translation effi- ing the immunogenicity of DNA vaccines. These results may be
ciency. This antibiotic-free vector platform has increased trans- due to the presence of a higher density of APCs in the dermis
gene expression and improved HIV-1 gp120 DNA vaccine- than in other anatomical regions [38,39]. Thus, as APCs are more
induced neutralizing antibody titers in rabbits [32]. prevalent in the skin than in muscle, dermal immunization
A minicircle DNA (mcDNA) technology has been developed appears to be more suitable for DNA vaccines. Consequently,
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to remove bacterial elements completely. This innovative vector ID administration is a promising route for the administration
can be produced through an in vivo site-specific recombination of DNA vaccine.
system, in which a parental plasmid (PP) is recombined into Alternately, targeting DNA vaccine to secondary lymphoid
two molecules: a mcDNA and a miniplasmid (mP). The mcDNA organs is a suitable strategy to transfect as many APCs as possible
encodes the therapeutic expression cassette, whereas mP encodes using mucosal (oral, pulmonary, intranasal) or intravenous
the bacteria-derived sequences. Thus, the mcDNA technology routes. For example, oral administration of DNA vaccine allows
allows the complete removal of the undesired bacterial sequences the uptake of DNA by intestinal APCs, whereas pulmonary or
and decreases the size of the DNA vector, which reduces toxicity intranasal (IN) administration allows uptake by APCs in the res-
and improves transfection efficiency. In addition, the mcDNA piratory tract. Bioengineered bacteria have been used for the oral
vector can improve the level and duration of transgene expres- delivery of therapeutic proteins or antigens, because biomole-
sion [33,34]. The mcDNA technology has been applied success- cules on the surface of bacteria can easily mediate their entry into
fully in DNA vaccinations, and one study has demonstrated target cells [40]. Briefly, bacterial ghosts are non-living empty cell
that mcDNA is superior to pDNA in eliciting antigen-specific envelopes obtained by protein E-mediated lysis of Gram-negative
CD8 + T-cell responses in mice [35]. Another study demonstrated bacteria, which can be used as vaccine delivery systems due to
that a novel mcDNA vaccine induced enhanced HIV-1-specific their intrinsic adjuvant ability. In a recent study, a Neisseria gon-
immune responses in mice [36]. Recently, a novel Cre orrhoeae DNA vaccine was efficiently delivered by Salmonella
recombinase-mediated in vivo mcDNA (CRIM) platform was used enteritidis ghosts and the oral immunization of mice elicited
to produce mcDNA [37]. In this platform, the plasmid was able to greater CD4 + and CD8 + T cell responses [41]. Another approach
replicate as a complete parental plasmid in vitro and then trans- is to apply bacterial vector DNA vaccine delivery systems, such as
form into mcDNA in vivo by itself. The HN gene was used as an a recombinant attenuated Salmonella vector. For instance, a
antigen model to construct the traditional plasmid and the novel mcDNA vaccine was delivered orally to chickens via a
mcDNA, and the results revealed that the CRIM–HN vaccine sig- regulated-delayed-lysis Salmonella strain, which was genetically
nificantly improved the host immune response and provided engineered to lyse gradually in vivo after immunization [37]. IN
efficient protection against wild-type Newcastle disease virus is an interesting route of vaccine administration that offers some
challenge in chickens [37]. Thus, mcDNA vectors appears to be valuable opportunities. For instance, IN immunization with DNA
a promising approach for enhancing the immunogenicity of vaccine can elicit both systemic and mucosal immune responses,
DNA vaccines. and the latter is particularly important as mucosal immune
responses provide front-line protection against pathogen inva-
sion that initiates at the mucosal surface. Furthermore, IN vacci-
Methods for DNA vaccine delivery nation eliminates the pain, stresses and biohazards associated
The route by which DNA vaccine is delivered can significantly with needle-based injection and dismisses the need for trained
influence its immunogenicity and efficacy. As DNA delivery medical workers [42,43].
methods affect the types of host cell that are transfected, the
delivery method also plays a key role in determining the efficacy Physical methods
of DNA vaccines. Each method of delivery introduces DNA vac- Over recent years, several physical methods have been developed
cines to distinct areas of immune surveillance and consequently and clinically tested for the administration of DNA vaccines
primes the immune system in distinct ways [38]. through the skin, including needle-free approaches, such as the
The traditional intramuscular (IM) administration of vaccines gene gun, electroporation, jet injectors, and microneedle
via needle and syringe has low efficiency because only a small systems.
fraction of injected DNA is taken up by somatic cells and
expressed. Moreover, IM administration predominantly leads to Gene gun
the transfection of myocytes. For that reason, over the past dec- The gene gun, also called particle-mediated gene transfer, is a
ade, the focus in the DNA vaccination field has moved from IM delivery method developed for the physical introduction of
towards intradermal (ID) administration. As a natural barrier to DNA into cells. In brief, pDNA is coated onto particles of heavy
the entry of pathogens, the skin is an interesting target organ metals (such as gold, silver, or tungsten) and bombarded into tar-
for DNA vaccination. It is a very immune-active organ that con- geted tissues using a high-pressure instrument. As a result of the
tains a high frequency of APCs, such as the Langerhans cells high force, the DNA-coated particles penetrate the cell mem-
(LCs) and dermal dendritic cells, which are required to stimulate brane, increasing the cellular uptake [44] and consequently stim-
ulating immune responses that are greater than those produced of microneedle arrays, and different needle shapes and lengths
by standard vaccination. For instance, the administration of can be used [54]. Types of microneedle array that can be used
DNA vaccines using gene gun technology induced robust to deliver DNA include solid microneedles coated with DNA
immune responses against antigens in wild-type mice [45]. Fur- before skin penetration, solid microneedles coated with DNA
thermore, a particle-mediated epidermal delivery (PMED) gene that dissolve upon insertion, uncoated microneedles that dam-
gun device was successfully used in preclinical trials to deliver a age the epidermis before the application of a transdermal patch
DNA vaccine against dengue virus in non-human primates containing the DNA, and hollow microneedles for liquid delivery
[46]. Although carrying the disadvantage of expensive costs, into the skin [52–54]. Microneedles have been used successfully
the gene gun method has advantages such as good safety, high for DNA vaccine delivery in vivo. For instance, Ali et al. [55] devel-
efficiency, non-invasiveness, and (low) DNA dose. oped a microneedle array loaded with a functional HPV-16 E6/E7
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DNA vaccine encapsulated into peptide (RALA) nanoparticles for

Electroporation the treatment of cervical cancer. The authors reported that, after
Electroporation is a physical transfection method that uses an microneedling, the concentration of antibodies was twice as high
electrical pulse to generate transient pores in the cell membrane, as that in control groups, and sera from mice vaccinated with
allowing efficient transfer of DNA into the cells. Once optimum microneedle/RALA-E6/E7 displayed greater T-cell-mediated cyto-
electroporation conditions are determined, this method can toxicity in response to HPV-16 oncogenic antigen-expressing
transfect a large number of cells in a short time [47]. Further- cells (TC-1) than in response to sera from mice that received
more, electroporation causes substantial cell death because of the vaccine intramuscularly. Moreover, microneedle/RALA-E6/
the high temperature induced by the high voltage application. E7 slowed tumor growth in a therapeutic model of vaccination
However, the local tissue damage is followed by inflammation and was more effective than intramuscular vaccination. Another
and recruits dendritic cells (DCs), macrophages, and lympho- recent study has successfully developed a charge reversal pH-
cytes, inducing significant antibody and T-cell immune sensitive copolymer as the release-layer for DNA delivery using
responses [48]. This method has been applied effectively in microneedles [56]. The DNA vaccine encoding an Alzheimer
humans to enhance naked DNA vaccine delivery. For instance, antigenic determinant induced robust antigen-specific immune
data from an HIV-1 DNA vaccine trial demonstrated that the responses in vivo. Therefore, microneedle arrays are promising
use of electroporation after intramuscular vaccination had a sig- devices that can efficiently deliver DNA into the skin.
nificant dose-sparing effect and provided superior immunogenic-
ity when compared with standard intramuscular delivery
Jet injection
without electroporation of the same vaccine. After three immu-
Jet injectors are needle-free devices that can be used to deliver
nizations, individuals developed a CD4 + or CD8 + T cell
drugs through the skin. This delivery method works by forcing
response to the HIV-1 DNA vaccine delivered via electroporation
the liquid that is loaded in the device through a small orifice,
that was greater than that induced by DNA vaccination without
which creates a high-pressure stream at a velocity sufficient to
electroporation [49]. In another phase I clinical trial, it was
penetrate the skin. Depending on the diameter of the orifice,
demonstrated that electroporation of an HPV DNA vaccine
the vaccine can be applied intradermally, subcutaneously, or
expressing E6/E7 antigens generated an enhanced polyfunc-
intramuscularly [57]. In a phase I clinical trial [58], the delivery
tional HPV-specific CD8 + T cell response, as shown by an
of an HIV-1 DNA vaccine using a Biojector device was compared
increase in cytolytic activity, proliferative capacity, and secretion
to delivery by needle and syringe, and it was concluded that the
of effector molecules. In addition, most DNA vaccinated patients
Biojector device improved the potency of priming for antibody
displayed both a complete regression of their cervical lesions and
and CD8 + T cells responses, suggesting that increasing transfec-
HPV viral clearance [50]. These and other clinical trials have
tion efficiency is key in improving the potency of DNA vaccines.
established that electroporation-based DNA delivery technology
These results demonstrate that needle-free injection devices
enhances cellular uptake and immune responses to DNA vacci-
show considerable promise for the delivery of DNA vaccines.
nes in humans [51]. Overall, electroporation is an effective
method to increase the efficiency of DNA uptake and to cause
the sterile inflammation necessary to augment the immunogenic Chemical methods
outcomes of DNA vaccination significantly. The administration of naked pDNA produces weak humoral and
cellular immune responses, necessitating the use of a suitable
Microneedles arrays DNA delivery platform for safe and effective target-specific intra-
Microneedle devices are composed of micro-sized needles that cellular delivery of DNA vaccines. The naked DNAs are degraded
can be arranged in small arrays to mediate localized delivery of rapidly by nucleases, whereas DNAs that are incorporated into
therapeutic molecules [52]. In microinjection, the skin is pene- delivery systems, including natural/synthetic cationic biopoly-
trated to a specific and reproducible depth by a microneedle mers, lipids or inorganic particles are protected from environ-
using simple mechanical force. This method effectively reaches mental degradation. The nano-sized, condensed DNA
the immune-cell-rich epidermis and dermis [53]. As microneedles constructs not only protect DNA but also promote cellular inter-
are very small (ranging from 25 to 2000 mm in length), delivery nalization. Multifunctional nanoparticles can often be created as
of the vaccine is painless and minimally invasive when com- delivery systems to target APCs by modifying the surface with
pared to conventional needle-based injection. Different materials targeting ligands. Nanoparticles are considered promising adju-
and manufacturing techniques can be applied for the production vants in vaccination as they possess a pathogen-like structure.
In addition, nanoparticles can also be surface-modified with response in chickens. The formulated DNA vaccine stimulated
immune-stimulatory molecules to enhance immune reactions. lymphocyte proliferation and upregulated the levels of IL-2, IL-
4 and IFN-c.
Liposomes
Liposomes are lipid bilayers with an aqueous core. These solid Increasing adjuvanticity by chemical modification
lipid nanoparticles are promising colloidal carrier systems, offer- The adjuvanticity of chitosan has been increased by functional-
ing appreciable stability, reduced toxicity, and desirable release izing trimethylchitosan with a TLR-7 agonist, 9-benzyl-8-
profiles for the entrapped bioactive. Liposomes can comprise hydroxyadenine (HA) [68]. HA was connected to trimethylchi-
anionic, cationic, or neutral (phospho)lipids, although anionic tosan via a polyethylene glycol (PEG) spacer through the hydro-
and neutral liposomes are considered to be less optimal for xyl group of chitosan. This decoration improved the interleukin-
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DNA vaccine delivery because of weak interactions between the 8-related immune-stimulatory capacity of chitosan in human
DNA and liposome [59]. In a recent study, cationic liposomes THP-1 macrophages. In another study, chitosan was modified
were used to deliver three malarial antigens encoded in DNA vac- with poly(d,l-lactide-co-glycolide) (PLGA), and subsequently, a
cines, and it was demonstrated that DNA-loaded cationic lipo- hydrogel nano-formulation of pDNA–chitosan–PLGA complex
somes provided strong humoral responses against antigens in was created using poloxamer 407 gel to minimize systemic pDNA
BALB/c mice [60]. In another recent study, two DNA–liposome degradation and to enhance the internalization of pDNA into
complexes (DDA and DMT) were prepared and their immuno- the APCs [69]. The results indicated that the nanoparticulated
genicity and protective efficacy against Mycobacterium tuberculosis vaccine released pDNA in a sustained manner and was taken
infection were compared with that of Bacillus Calmette–Guérin up by APCs efficiently.
(BCG) vaccination in C57BL/6 mice. When compared with
DDA vaccination, DMT vaccination elicited significantly more Mannosylation for APC targeting
IL2 + T cell responses and provided enhanced and persistent pro- To increase the stimulation of an immune response, nanoparti-
tection against M. tuberculosis infection in mice. DMT liposomes cles are often selectively delivered to APCs using mannose
also resulted in a slower and longer-lasting release of DNA, which ligands. These ligands bind to mannose receptors that are overex-
might be attributed to the improved protection of DMT adju- pressed on APCs and promote efficient internalization of the
vanted vaccines [61]. Liposomes can be used widely as an effi- nanocarrier. Recently, mannosylated chitosan (MCS) nanoparti-
cient delivery system in the preparation of DNA vaccines. cles have also been employed for DNA vaccine delivery to APCs
(Fig. 3) [70,71]. To achieve enhanced mucosal immune protec-
Naturally occurring polymers tion against pulmonary tuberculosis, Mycobacterium tuberculosis
Naturally occurring polysaccharides, in particular chitosan, have vaccine was formulated into nanoparticles of size ~400 nm using
been investigated for drug [62] and DNA vaccine delivery [63]. MCS [70]. The multi-T-epitope DNA vaccine loaded into MCS
Chitosan, especially the deacetylated form, which is extracted nanoparticles induced enhanced humoral and cellular immunity
from crustacean and insect shells is positively charged due to in the lung airways (as compared to chitosan DNA vaccines and
the presence of free primary amine groups. These groups interact BCG vaccines) when administered via the broncho-alveolar
electrostatically with the phosphate groups of DNA and allow route. The activation of the peptide-specific Th1 response and a
binding of DNA with the polymers. Polysaccharides are inexpen- polyfunctional CD4 + T response was much greater for the
sive, biocompatible, biodegradable materials that are amenable APC-targeted vaccine because of the increased accessibility of
to functionalization, which warrants their application as multi- APCs in the alveoli to the DNA vaccines, which provided
functional vaccine delivery vehicles. More interestingly, chitosan improved protection of the lung from airway Mycobacterium bovis
has drawn attention as an adjuvant for DNA vaccines as it helps BCG challenge. In another study, mannosylated phenylalanine
to induce type I interferons and DC activation, triggering innate grafted chitosan was utilized as a DNA delivery vector to increase
and adaptive immune reactions [64,65]. For example, chitosan the delivery of a hepatitis B DNA vaccine to APCs [71]. The man-
enhanced the immunogenicity of a multivalent chimeric gene nosylated polyplexes were taken up effectively by macrophages.
DNA vaccine against Trueperella pyrogens [66]. The chitosan- The intradermal immunization of BALB/c mice with the poly-
entrapped DNA vaccine was better than the free plasmid at pro- plexes, as compared to other tested formulations, including
ducing an immune response against the infection in mice. The FuGene HD, stimulated the proliferation of specific T-cells and
release of plasmid DNA was also prolonged when the encapsu- greatly enhanced the serum antibody titer.
lated form was used. One of the disadvantages of chitosan is its
low solubility in the physiological pH range of 6.8–7.2, which Synthetic polymers
could be overcome by chemical modification [63]. In one study, Synthetic cationic polymers such as poly(ethyleneimine) (PEI)
nanoparticles containing a combination of two water-soluble and dendrimers are used as DNA-complexing agents to deliver
chitosan derivatives, N-2-hydroxypropyl trimethylammonium genes of interest [72] and to prepare DNA vaccines [73–75]. PEI
chloride chitosan and N,O-carboxymethyl chitosan, were used is a polyamine polymer containing primary, secondary, and ter-
as a carrier for Newcastle disease viral DNA vaccines [67]. The tiary amines, and is available as branched and linear forms. PEI
result from the study indicated that the DNA was released from establishes strong electrostatic interactions with DNA due to
the nanoparticles in a sustained manner following an initial the presence of a large number of primary amines in the poly-
burst release, and that intranasal administration of the meric chain. The PEI–DNA polyplex is spherical and is endocy-
nanoparticle-condensed DNA enhanced the mucosal immune tosed by living cells via a receptor-mediated endocytosis
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FIGURE 3
Mannosylated chitosan (MCS) nanoparticles loaded with plasmid DNA as a targeted system for vaccine delivery via mannose receptors. APC, antigen-
presenting cell.
process. The low pKa values of the amine groups give the PEI a pseudorabies virus (PrV) in pigs, which was administered intra-
proton sponge effect, which causes swelling and rupture of endo- nasally [79]. The results indicated that the polyplex was able to
somes and release of the PEI–DNA polyplex into the cytoplasm. induce systemic and mucosal antibodies. Interestingly, a similar
The DNA is then translocated to the nucleus. level of IgA antibodies against PrV was observed in mucosal sal-
Cell-penetrating peptides (CPPs) have been used to shuttle iva when this vaccine was administered either intranasally or
pDNA vectors [76] and antigens to modify the response of via the intramuscular route. In another recent study, a pDNA
immune cells and to improve vaccination efficacy. For example, vaccine encoding tyrosine hydroxylase as an antigen against
the CL22 peptide was employed to deliver a plasmid that neuroblastoma was conjugated with linear PEI (20 KDa). This
encoded tumor-associated antigens (TAAs) and to transfect this vaccine induced a cellular immune response and antitumor effi-
vector into DCs. This peptide showed a transfection efficiency cacy in neuroblastoma-engrafted mice [80]. The results indicated
equivalent to that of the best available non-viral agents for engi- higher production of immunostimulating IFN-c in DNA–PEI-
neering DCs and, consequently, for preparing vaccines for cellu- treated mice than in mice treated with free DNA. Moreover,
lar immunotherapy [77]. The MPG peptide was recently used for the rate of survival of tumor-bearing mice was much higher fol-
in vivo delivery of pDNA. This peptide can bind noncovalently to lowing the treatment with DNA-PEI vaccine compared to free
single-stranded (ss) and double-stranded (ds)-oligonucleotides DNA treatment.
and can protect them from DNase-mediated degradation [59].
Graphene oxide (GO) is widely employed for the delivery of Chemically modified dendrimers
biomolecules and has been used as the carrier vehicle to deliver Dendrimers, which are synthetic water-soluble polymers with
a vaccine antigen into APCs. GO is the oxidized form of gra- spherical architecture, have gained attention as DNA vaccine
phene and is a potential carrier platform for bioactive molecules vectors [81–84]. These polymers have primary amine groups on
because it has unique physicochemical properties, especially a their spherical surface, which increase with the number of gener-
surface that can be easily modified. GO functionalized with var- ations. They have been chemically modified and used in several
ious polymers has been shown to elicit strong immunological studies as DNA-condensing agents. In a recent study, a polyami-
responses [78]. doamine (PAMAM) dendrimer was conjugated to a CPP, HIV
transactivator of transcription (Tat), to increase the intracellular
Chemically modified PEIs penetration of a dendrimer–DNA polyplex [81]. The Tat-
Various chemical modifications have been performed on PEI to peptide-modified dendrimers were more efficient in delivering
engineer efficient vectors for DNA vaccine delivery [73,79,80]. DNA than were free dendrimers. Tat-conjugated dendrimers of
In a recent study, PEI was conjugated to PEG, condensed with generation 5 were also utilized in another study for transdermal
pDNA-encoding HIV proteins, and formulated into PLGA micro- administration of H5–DNA vaccine to provide protection against
spheres [73]. The PEG was used to stabilize the DNA in the water- H5N1 influenza virus challenge [82]. Other cell-targeting or
soluble core compartment. The release of DNA from PLGA micro- internalizing peptides have also been conjugated to dendrimers.
spheres was sustained after an initial 15% burst release. The intra- PAMAM dendrimer of generation 5 was conjugated to MHC class
muscular administration of the DNA vaccine polyplex in II-targeting peptide [83]; the modified dendrimer delivered
microsphere formulation in mice induced an adequate immune loaded DNA into DCS effectively and induced immune stimula-
response at a low dose. In another study, PEI was conjugated to tion under in vitro and in vivo assay conditions. Upon subcuta-
PLGA and the PEI–PLGA polymer was used as a vector for a neous administration in mice, the DNA–peptide–dendrimer
DNA vaccine encoding the immunogenic glycoprotein B against complex caused T-cell activation and rejection of tumor forma-
tion. An amine-terminated poly(ether imine) dendrimer of gen- carriers activate the immune system for a longer time by control-
eration 4 has also been used in a study [84]. This dendrimer deliv- ling cargo release at a specific site [78]. Native or synthetically
ered the pDNA rabies vaccine effectively and enhanced the optimized pathogen-associated molecular patterns (PAMPs) are
immunogenicity in mice when administered via intramuscular being used to activate specific pattern recognition receptors
injection [84]. (PRRs) and to enhance and elicit specific immune responses
against co-administered antigens without toxicity. Classes of
PAMPs, such as TLR agonists, nucleotide-binding oligomeriza-
Adjuvants for DNA vaccines tion domain (NOD)-like receptors (NOD/NLRs), stimulator of
Adjuvants are substances that can be incorporated into vaccines interferon genes (STING), retinoic acid-inducible gene I (RIG-I),
to enhance antigen-specific immunogenicity. They function and C-type lectin (CLR) agonists, have begun to be employed.
KEYNOTE (GREEN)
through a range of mechanisms that includes the activation of Targeting specific PRRs from different classes provides a wide
the innate immune system, enhancement of antigen uptake range of immune responses because each receptor activates a dis-
and presentation by APCs, the formation of a depot at the injec- tinct signaling pathway, thereby influencing innate and subse-
tion site for slow-release of the antigen, induction of chemotaxis, quent adaptive immune responses to produce defined cellular
and upregulation of co-stimulatory molecules without compro- and antibody responses [91]. In the past decade, researchers have
mising safety [15]. Owing to their ability to improve immune developed synergistic adjuvant combinations to augment their
responses, adjuvants make it possible to reduce the number of adjuvanticity and elicit the desired mix of immunological
doses needed or the amount of antigen needed in each dose. responses. Combination of adjuvants requires an understanding
Adjuvants must be adapted according to the nature of the anti- of both the nature of the immunogen and the type of immune
gen, immunization schedule, administration route, type and response required [85]. A more comprehensive list of genetic
duration of required immunity and pharmaceutical parameters adjuvants is included in Table 1, and a list of adjuvants tested
[85]. Adjuvants are divided into two types: immunostimulatory in vivo for their ability to enhance the immunogenicity of DNA
molecules and delivery systems. Immunostimulatory molecules vaccines is included in Table 2. Tables 3 and 4 summarize impor-
stimulate immune responses by interacting with specific recep- tant findings from some recent preclinical and clinical studies
tors [86]. Common vaccine adjuvants include aluminum salts with DNA vaccines, respectively.
[87], oil-in-water emulsions [88] and synthetic CpG In recent years, clinical trials have demonstrated the clinical
oligodeoxynucleotides (ODN) that are recognized by TLR9 [89]. activity of DNA vaccines and an improved induction of immune
As plasmids can be easily designed to encode immunomodu- response (Table 4). Some DNA vaccines have been licensed for
latory proteins, cytokines, chemokines, and co-stimulatory mole- veterinary use and some have undergone clinical trials in
cules have also been used extensively to enhance the humans, but to date, none are licensed for human use. Gene-
immunogenicity of DNA vaccines. These adjuvants can be based formulations require greater safety evaluation than do con-
encoded within the antigen-encoding plasmid, encoded in a sep- ventional vaccines, and the timeline for approving DNA vaccines
arate plasmid or used as proteins in combination with the vac- for humans is relatively lengthy. Most of the clinical trials under-
cine. However, plasmid-mediated administration of the genetic taken to date are investigating DNA vaccines for infectious dis-
adjuvant synchronizes the immune-stimulating effect with the eases and cancer. Adjuvants and other immunostimulants have
expression of the antigen, and also minimizes side effects by hav- been included, either as recombinant proteins or encoded by
ing these molecules expressed only locally [90]. Delivery systems, plasmid DNA and other formulations [92,93]. DNA delivery sys-
such as nanoparticles, can protect antigens from degradation tems [74] and strategies such as prime-boost combinations [93]
in vivo and at the same time can enhance the immune response have also been employed. In addition to simple intramuscular
in a way that is based on its specific characteristics [86]. The injection [94], approaches now include the use of pressurized
application of delivery carriers has overcome problems such as devices, electroporation, or noninvasive needle-free jet delivery
low immunogenicity, and the effects of carriers on the immune systems [95,96,97]. Optimizing codon usage and the use of
system have attracted considerable attention. Antigen-loaded DNA to prime an individual followed by a heterologous vaccina-
TABLE 1
Genetic adjuvants used in DNA vaccines and tested in vivo.
Genetic adjuvant Molecule nature Molecule function Animal model Reference
IL-2 Cytokine Stimulates the proliferation of both T and NK cells Chicken [98]
IL-12 Cytokine Induces the differentiation of naïve T cells into Th1 cells Mice [99]
IL-15 Cytokine Stimulates the proliferation of both NK and T cells Mice [100]
Flt3L Cytokine Specific growth factor that expands and mature dendritic cells (DCs) Mice [101]
GM-CSF Cytokine Recruits antigen-presenting cells (APCs) to the site of immunization Mice [102]
and stimulates DC maturation
RANTES (CCL5) Chemokine Promotes the activation of DCs and T cells Mice [103]
CD40L Co-stimulatory Induces the production of cytokines by DCs and the differentiation of T cells Mice [104]
CD54 (ICAM-1) Co-stimulatory Important in both T cell recirculation and activation Mice [105]
CD80 (B7-1) Co-stimulatory Enables T cell proliferation and activation Mice [106]
CD86 (B7-2) Co-stimulatory Enables T cell activation, differentiation, and function Mice [107]
KEYNOTE (GREEN)
TABLE 2
10
KEYNOTE (GREEN)
Types, mechanism and (dis)advantages of actual adjuvants. (Adapted from [86;108]).

www.drugdiscoverytoday.com
Types Mechanism Advantages Disadvantages DNA vaccine Reference

Aluminum salt Induced cytokine network Considered the safest Adverse reactions pgp.LAMP-1 DNA vaccine [87]
Activate complement system adjuvant Inability to induce cyto-
Strong Th2 response Cheap price toxic T cell responses
Longer duration of
immune responses
Slow release of antigen
Oil-in-water MF59 Induces immunostimulatory effects at the injection For influenza virus Injection site pain – –
emulsion site Safe Reactions
Recruitment of cytokines Well tolerated Induced inflammatory
Th2-type response arthritis
AS01 Trigger specific innate signaling pathways For malaria – – –
Production of different cytokines and chemokines
H1-type functional immunity
AS03 Induces the production of protective antibodies For influenza virus Injection site pain – –
Reactions
Induced inflammatory
arthritis
Montanide™ Possesses immunostimulatory activity Slow release of antigens – BoHV-1 gD DNA vaccine [109]
Works by depot formation at the site of injection Local inflammation
Enhances cytotoxic T
lymphocyte (CTL)
activity
Delivery systems Liposomes Fusion with the phagocytic cell membrane, allow- Low toxicity and desir- High cost pCMFO/DMT DNA vaccine [61]
ing the antigen to enter the cytoplasm, participat- able release profile of Pain at the injection site
ing in the major histocompatibility complex-1 the bioactive
(MHC-I) pathway Appreciable stability
Polymeric Antigen is encapsulated in nanoparticles, consist- Biocompatibility, Stability of antigens in HPV-16 DNA vaccine [65]
nanoparticles ing of a degradable polymer biodegradability, non- microcapsules
Long-term repository effects toxic nature Optimal dose problem

Pulsed release of antigen Ability to be easily mod-
Targeting antigen-presenting cell (APC) ified into desired shapes
and sizes
Potential for single-dose
vaccines, reducing vacci-
nation costs
Graphene Derived from graphite by oxidation, retains the The surface can be easily Low solubility – –
oxide large planar structure and high surface area modified Poor stability
d
Effectively internalized Cytotoxic
Volume xx, Number xx

by APCs
Promotes the cross-pre-
sentation of antigen to
CD8 + T cells
d
xxx 2021
TABLE 2 (CONTINUED)

Types Mechanism Advantages Disadvantages DNA vaccine Reference

TLR agonist CpG-ODN TLR9 agonist For hepatitis B virus Large amount of systemic prM/E DNA vaccine [89]
Enhance antibody response (HBV), malaria, hepatitis pro-inflammatory
Induce a strong Th1 type cellular response B virus (HCV), cancer cytokines
Poly (I:C) TLR3 agonist For hepatitis Acutely toxic, can induce Connective tissue growth [110]
Direct activation of natural killer (NK) cells Without accumulation of autoimmune diseases factor (CTGF)/ mesothelin
Enhances antigen-specific CD8 + T cell responses toxicity (MSLN) DNA vaccine
d
Promotes antigen presentation by dendritic cells
Volume xx, Number xx

Bacterialghosts Contain well-known innate immune-stimulating Protection against sev- Induce potent proinflam- Neisseria gonorrhoeae DNA [41]
components eral intracellular matory cytokine vaccine
Stimulate cells through the TLR2 and TLR4 organisms responses in immune
pathways Effectively target DNA cells
vaccines to APCs at
mucosal surfaces
d
xxxx 2021
www.drugdiscoverytoday.com
KEYNOTE (GREEN)
11
KEYNOTE (GREEN)
TABLE 3
Example of DNA vaccine clinical trials in animal models.
Plasmid DNA vaccine Adjuvant Delivery system Animal Delivery Results References
(s) model method
Malarial DNA vaccine – DDAB + cholesterol + Mice Intramuscular Significant and multileveled antibody [60]
DSPE + PEG2000 and and cell-mediated response
intraperitoneal
injection
HIV-1 DNA vaccine – Poly(ethyleneimine) (PEI) Mice Intramuscular Increased immune responses at low [73]
in poly(d,l-lactide-co- injection dose
KEYNOTE (GREEN)
glycolide) (PLGA)
microspheres
Multi-antigen DNA pING PEI Mice Intramuscular Greater reduction in tumor volume [75]
vaccine injection and
intragastric
administration
PEI-DNA vaccine pING PEI Mice Intramuscular Anti-NB activity in the experimental [80]
injection model of the aggressive tumor growth
H5-DNA vaccine pBud-H5 Tat-PAMAM Mice Transdermal Higher hemagglutination inhibition [82]
inoculation titer, larger CD3+/CD4+ T cells, larger
CD3+/CD8+ T cell population, and
strong Th1-like cytokine responses
DNA vaccine – PPD PAMAM, HAPD Mice Subcutaneous Complexes in vivo preferentially [83]
PAMAM injection transfected dendritic cells in the
draining lymph nodes, promoted
generation of high affinity T cells, and
elicited rejection of established tumors
DNA vaccine pIRES-Rgp PETIM Mice Intramuscular Nanoformulation with PETIM dendrimer [84]
injection can produce an earlier onset of a high-
titered protective antibody response to
a plasmid-based rabies vaccine
pGJA-P/cDNA plasmid miR-9 – Mice Muscle Enhanced expression of antigen [111]
sponge injection proteins; first study indicated that
attenuating the effect of an
endogenous microRNA can enhance
the expression and immunogenicity of a
DNA vaccine
pWRG/ Venezuelan Interferon- – Mice Intramuscular Inclusion of innate immune agonists [112]
equine encephalitis ab (IFN-ab) electroporation can significantly boost protective
virus (VEEV) pWRG/ dsRNA and efficacy
Ebola virus (EBOV) CpG motifs
EBOV glycoprotein – – Macaque Intramuscular Individual vaccines elicited slightly [113]
(GP), Sudan virus electroporation higher IgG responses to EBOV or MARV
(SUDV) GP, Marburg than did the combination vaccines;
virus (MARV) GP, both the MARV and mixed vaccines
Ravn virus (RAVV) were able to protect macaques from
GP lethal MARV challenge;
vaccination of the mixed vaccines
would provide better protection against
EBOV if each vaccine were delivered to
a separate site rather than to a single
site
hetIL-15, plasmid IL-12 and – Macaque Intramuscular Sustained high levels of IL-15 in plasma, [114]
AG153 IL-15 injection with no significant toxicity;
hetIL-15-FC, plasmid followed by increased frequency of natural killer
AG256 in vivo (NK) and T cells undergoing
electroporation proliferation in peripheral blood;
high systemic levels of bioactive
cytokine, without the toxicity linked to
the high transient cytokine peak
associated with protein injection
Herpes simplex virus-2 IL-12 – Mice Electroporation Substantially greater protection against [115]
(HSV-2) vaccine a high-dose HSV-2 vaginal challenge
TABLE 3 (CONTINUED)
Plasmid DNA vaccine Adjuvant Delivery system Animal Delivery Results References
(s) model method
pcDNA3 CRT/E7; Gold nanoparticles Mice Gene gun Enhanced antigen specific CD8+ in both [116]
BPV1-L1 or CRT/E7 and ovalbumin antigenic
L2; systems;
HPV16-L1 generation of L1/L2-specific CD4+ T cell
or L2 immune responses and L1-specific
neutralizing antibodies;
therapeutic antitumor effects;
enhancement of CD8+ T cell immune
KEYNOTE (GREEN)
responses by DNA encoding L1 and L2
was also found to extend to an HPV-16
L1/L2 system
E1- and E3-deleted 3pRNA JetPEI transfection Mice Injection Vaccination with OVA in combination [117]
adenoviral vectors anti-GC reagent with 3pRNA protected mice from a
polyI:C subsequent OVA-encoding adenovirus
CpG infection in a CD8 + cell-dependent
manner;
3pRNA was superior to the other
adjuvants in antiviral vaccination
H1HA DNA vaccine Poly(dA:dT) – Mice Intramuscular DNA vaccine induced Irf7-dependent [118]
HA injection signaling, as part of the STING pathway,
critical for the generation of innate
cytokine signaling and antigen-specific
B and T cell responses
DNA vaccine pUMVC3/ Peptide PAMAM Mice Intrastromal Systemically administered non-small [119]
hIL-12 and cell lung cancer (NSCLC)-nanoprotein
pAAV- peritoneum (NP) selectively transfected lung cancer
eGFP-WPRE injection cells growing in mice
DDAB, xxx; DSPE, xxx; HA, 9-benzyl-8-hydroxyadenine; HAPD, xxx; NB, xxx; PAMAM, polyamidoamine; PETIM, xxx; pGJA-P, xxx; pIRES-Rgp, xxx; PPD, xxx;
pWRG, xxx; STING, stimulator of interferon genes; Tat, transactivator of transcription; WPRE, xxx. {AuQ: Please add missing explanations for
abbreviations.}
TABLE 4
DNA vaccines tested in clinical trials.
Plasmid DNA vaccine Adjuvant(s) Delivery method Model Clinical Results References
study
Idiotypic DNA vaccine Potato Intramuscular Patients with Phase I Ongoing [74]
virus X coat injection with B-cell non-
protein DNA/ poly Hodgkin’s
(PVXCP), (ethyleneimine) (PEI) lymphoma
Human complexes
chemokine
MIP3a
VGX-3100, synthetic – Electroporation Women with Phase Histopathological regression; [95]
plasmids targeting cervical IIb first therapeutic vaccine to show
Human papillomavirus- intraepithelial efficacy against CIN 2/3 associated
16 (HPV-16) and HPV-18 neoplasia (CIN) with HPV-16 and HPV-18
E6 and E7 proteins 2/3
Alpha fetoprotein (AFP) Sargramostim Intramuscular Patients with Phase I Ag-specific T cell responses were [92]
plasmid DNA (GM-CSF) injection hepatocellular detected;
plasmid DNA carcinoma more favorable progression-free
survival in one patient having higher
T-cell responses
Plasmid DNA expressing IL-2, IL-12, IL- Intramuscular Healthy Phase I CD4+ T-cell responses were [120]
HIV protein(s) 15 injection via needle volunteers demonstrated in 60–70% individuals
with or without in some trials, but CD8+ T-cell
electroporation, or responses were typically only
needleless device detected in < 25% of individuals;
minimal neutralizing and binding
antibody responses
(continued on next page)
TABLE 4 (CONTINUED)
Plasmid DNA vaccine Adjuvant(s) Delivery method Model Clinical Results References
study
GLS-6150, plasmids Intramuscular Patients with Phase I Decrease in Treg cell frequency and [93]
encoding hepatitis C IFNL3 injection with genotype 1a enhancement of HCV-specific T cell
virus (HCV) non- electroporation or 1b chronic responses without significant side
structural proteins HCV infection effects
Quadrivalent HPV – Intramuscular HIV-1 infected Phase II Vaccine antibody titers decreased [94]
Recombinant Vaccine injection women across all four HPV types;
(Types 6, 11, 16, 18) lower proportions of sustained
seropositivity were observed in
KEYNOTE (GREEN)
women with lower CD4 counts for all

four vaccine types;
durable titer response above the
seroconversion cut-off levels in HIV
PENNVAX-B, synthetic – Intramuscular HIV-infected Phase I Increased frequency of HIV-specific T- [96]
plasmids targeting the injection with subjects cells producing IFN-c and expansion
Gag, Pol and Env electroporation of activated HIV-specific CD8 + T-cells
proteins of HIV-1 capable of lytic protein upregulation;
robust increases in HIV-specific
cytokine production that are
statistically significant against all three
components of the immunotherapy;
HIV-specific cellular activation and
regulation of GrzB and Prf were
observed in a majority of treated
individuals
EP-1300 DNA vaccine – Electroporation Healthy Phase I Little or no specific immune responses [97]
volunteers to malarial antigens; immunogenicity
was poor
COVID-eVax, plasmid – Intramuscular Healthy adult Phase I/ Ongoing [121]
encoding SARS-CoV-2 injection followed by volunteers II
Receptor Binding electroporation
Domain (RBD) of the
spike protein
AG0302-COVID19, plasmid – Intramuscular Healthy adult Phase II Ongoing [122]
DNA encoding SARS- injection volunteers /III
CoV-2 spike protein
INO-4800, plasmid DNA – Intradermal injection Participants at Phase II/ Ongoing [123]
encoding SARS-CoV-2 followed by high risk of III
spike protein electroporation exposure to
SARS-CoV-2
ZyCoV-D, plasmid DNA – Intradermal Healthy Phase III Ongoing [124]
encoding SARS-CoV-2 administration with a human
spike protein needle-free injection volunteers
system
tion with the same antigen in an alternate format, for example, Conclusion and future perspectives
in a viral vector [92], are other promising strategies. The results In comparison with conventional vaccines, DNA vaccines are
of the clinical trials are very promising, but there are some limi- safer because they carry antigen genetic information, are more
tations that need to be addressed in future studies. A key issue in stable and easier to produce, and are considered a more economic
DNA vaccination is how to deliver the DNA plasmid into the biopharmaceutical product to distribute. Although DNA vaccines
desired cells optimally. Research has focused on methods to have the advantage of inducing efficient humoral and cellular
enhance the immune response because of inefficient uptake of immune responses, the application of naked DNA by traditional
naked plasmid DNA by cells, low immunogenicity of plasmid- IM injection results in low immunogenicity, resulting in limited
encoded antigens, and poor expression of antigen in human tis- ability to elicit significant clinical benefits. This limited immuno-
sues after immunization [97]. Thus, there is a need to optimize genicity can be strongly related to the composition of the pDNA
and improve the design of DNA vaccines, to improve the DNA backbone, the route of administration or the delivery methods
vector backbone and to consider the co-delivery of molecular used. To overcome this limitation, several strategies should be
adjuvants by exploring adequate delivery systems and adminis- considered. First, the DNA vector backbone can be improved
tration routes. In combination, these improvements may lead by exploring the use of the innovative mcDNA vector to elimi-
to the successful clinical use of these vaccines. nate the presence of prokaryotic sequences, and to avoid inflam-
matory reactions, antibiotic resistance or the formation of repres- charge. These characters assure the cell internalization of the tar-
sive heterochromatin that is responsible for the silencing of epi- get and facilitate its functionalization with specific ligands that
somal transgene expression. Second, alternative routes of allow delivery of the target, together with any immune-
administration, such as intranasal vaccination, are more efficient stimulatory molecules, to APCs, thereby improving the immune
than IM administration since they have the potential to facilitate response.
systemic and mucosal immunity, providing front-line protection
against pathogen invasions that initiate at the mucosal surface.
Such alternative routes of administration may also be more
attractive vaccination modalities, reducing the pain, stresses Acknowledgements
and biohazards associated with needle-based injection. Third, This work was supported by FEDER funds through the POCI -
KEYNOTE (GREEN)
the use of a suitable and biocompatible biomaterial is fundamen- COMPETE 2020 - Operational Programme Competitiveness and
tal to protecting the DNA vector from environmental degrada- Internationalization in Axis I - Strengthening research, techno-
tion, to compacting and carrying the genetic material and to logical development and innovation program (Project POCI-01-
delivering it specifically into target cells. To maximize the activa- 0145-FEDER-007491) and by National Funds from the FCT
tion of the desired immune response, and consequently the effi- (Foundation for Science and Technology) (Project UID/
cacy of DNA vaccines, the delivery system should present Multi/00709/2019). Dalinda Eusébio acknowledges a doctoral
nanometric size, uniform morphology and a positive surface fellowship from the FCT (Ref: 2020.10201.BD).
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Drug Discovery Today d Volume xxx, Number xx d xxxx 2021 REVIEWS

Introduction expensive costs and limited storage time. In addition, protein-

⇑ Corresponding author. Sousa, Â. (angela@fcsaude.ubi.pt)

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DNA vaccine encapsulated into peptide (RALA) nanoparticles for

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Types, mechanism and (dis)advantages of actual adjuvants. (Adapted from [86;108]).

Types Mechanism Advantages Disadvantages DNA vaccine Reference

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Volume xx, Number xx

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Types Mechanism Advantages Disadvantages DNA vaccine Reference

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women with lower CD4 counts for all

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