key: cord-0999305-b0izt9hq
authors: Suschak, John J.; Williams, James A.; Schmaljohn, Connie S.
title: Advancements in DNA vaccine vectors, non-mechanical delivery methods, and molecular adjuvants to increase immunogenicity
date: 2017-06-12
journal: Hum Vaccin Immunother
DOI: 10.1080/21645515.2017.1330236
sha: 6d04e85d2c994d73c9a7e2692af91470f407ddeb
doc_id: 999305
cord_uid: b0izt9hq

A major advantage of DNA vaccination is the ability to induce both humoral and cellular immune responses. DNA vaccines are currently used in veterinary medicine, but have not achieved widespread acceptance for use in humans due to their low immunogenicity in early clinical studies. However, recent clinical data have re-established the value of DNA vaccines, particularly in priming high-level antigen-specific antibody responses. Several approaches have been investigated for improving DNA vaccine efficacy, including advancements in DNA vaccine vector design, the inclusion of genetically engineered cytokine adjuvants, and novel non-mechanical delivery methods. These strategies have shown promise, resulting in augmented adaptive immune responses in not only mice, but also in large animal models. Here, we review advancements in each of these areas that show promise for increasing the immunogenicity of DNA vaccines.

The constant emergence, and re-emergence, of known and novel pathogens challenges researchers to develop new vaccination technologies that allow for the rapid development of safe and effective vaccines. Nucleic acid (DNA and RNA) vaccines have characteristics that meet these challenges, including ease of production, scalability, consistency between lots, storage, and safety. DNA vaccine technology usually is based on bacterial plasmids that encode the polypeptide sequence of candidate antigens. The encoded antigen is expressed under a strong eukaryotic promoter, yielding high levels of transgene expression. 1 Inclusion of transcriptional enhancers, such as Intron A, enhance the rate of polyadenylation and nuclear transport of messenger RNA (mRNA). 2 The vaccine plasmids are generally produced in bacterial culture, purified, and then used to inoculate the host.

Modern DNA vaccine design generally relies on synthesis of the nucleic acid and possibly one-step cloning into the plasmid vector, reducing both the cost and the time to manufacture. Plasmid DNA is also extremely stable at room temperature, reducing the need for a cold chain during transportation. Vaccination with DNA plasmid removes the necessity for protein purification from infectious pathogens, improving safety. Furthermore, DNA vaccination has an excellent safety profile in the clinic, with the most common side effect being mild inflammation at the injection site. 3 Importantly, DNA vaccines provide a safe, non-live vaccine approach to inducing balanced immune responses, as the in vivo production of antigen allows for presentation on both class I and class II major histocompatibility complex (MHC) molecules (Fig. 1 ). This elicits antigen specific antibodies, 4 as well as cytotoxic T lymphocyte responses (CTL), 5 something that remains elusive in most non-live vaccines. DNA vaccines have also demonstrated the ability to generate follicular T helper populations, 6 which are critical for the induction of high quality antigen-specific B cell responses. 7 DNA vaccination has proven successful in several animal models for preventing or treating infectious diseases, allergies, cancer, and autoimmunity. [8] [9] [10] [11] [12] The early success of small animal studies led to several human clinical trials. However, the protective immunity observed in small animals and nonhuman primates was not observed in human studies when DNA vaccines were administered alone by needle delivery. Like the more conventional protein-based vaccines, DNA can be delivered by a variety of routes, including intramuscular (IM), intradermal (ID), mucosal, or transdermal delivery. Because DNA plasmids must enter host cell nuclei to be transcribed into mRNA, the early failure of DNA vaccines to elicit strong responses in humans was largely due to their delivery by needle injection, which deposits the DNA in intracellular spaces, rather than within cells. Improved delivery technologies, such as intramuscular or intradermal electroporation, have been used to facilitate transport of DNA into cells, resulting in much better immunogenicity in both clinical and non-clinical studies. [13] [14] [15] [16] [17] [18] [19] In one study, electroporation-enhanced DNA vaccination resulted in increased polyfunctional antigen-specific CD8 C T cells in patients receiving a HPV DNA vaccine expressing the E6 and E7 genes of HPV16 and HPV18 respectively. 20 The majority of DNA vaccinated patients displayed complete regression of their cervical lesions, as well as viral clearance, following DNA delivery. Other mechanical delivery approaches use physical force such as particle bombardment (gene gun) to deliver the DNA plasmids into targeted tissues or cells, with some clinical successes. [21] [22] [23] Delivery of a Hepatitis B DNA vaccine by particle bombardment resulted in sustained antibody titers in subjects who had previously failed to respond to a licensed subunit vaccine. 23 Needle-free pneumatic or jet injectors have also shown promise in both animal and human clinical trials, [24] [25] [26] [27] and function by injecting a high-pressure, narrow stream of injection liquid into the epidermis or muscles of test subjects. In addition to these improved mechanical delivery methods, several other approaches are being explored to increase the immunogenicity of DNA vaccines in humans. Here we review 3 of these approaches which show promise for advancing DNA vaccines: non-mechanical delivery, inclusion of molecular adjuvants, and improvements in DNA vaccine vectors.

As already mentioned, the greatest impediment to DNA vaccination is low immunogenicity due to difficulties in delivering DNA plasmid into the host cell. The transportation of DNA vaccine plasmids into cellular nuclei requires the crossing of several barriers. Vaccine plasmid must cross the phospholipid cellular membrane through endocytosis or pinocytosis, escape degradation in endosomes and lysosomes, survive cytosolic nucleases, and translocate across the nuclear envelope. In contrast to physical delivery systems, chemical delivery approaches use biopharmaceuticals to increase DNA vaccine transfection efficiency.

The use of liposomes as a carrier molecule has become a popular DNA vaccine delivery method as liposomes not only enhance transfection efficiency, but also have an adjuvant effect. Liposomes are spherical vesicles composed of phospholipids and cholesterol arranged into a lipid bilayer, allowing for fusion with cellular lipid membranes. 28 DNA plasmid can be either bound to the liposome surface, or encased within the hydrophobic core of the liposome. This facilitates delivery of the DNA vaccine plasmid into the cells. Importantly, lipid vesicles can be formulated as either unilamellar or multilamellar. Multilamellar vesicles allow for sustained delivery of vaccine over an extended period of time. While the use of liposomes for IM injection has resulted in some reactogenicity issues, 29, 30 liposome/DNA vaccine complexes have demonstrated an immunological benefit. IM injection of a liposome/ influenza nucleoprotein formulation increased antibody titers 20-fold compared with vaccine alone. 31, 32 Boosting of antibody titers did not diminish the cytotoxic T cell response. Likewise, inclusion of a liposome formulation in a P. falciparum vaccine enhanced the IFN-g production. 33, 34 An ensuing human trial involving DNA plasmids encoding the influenza H5 HA, nucleoprotein, and M2 genes reported cellular immune response rates and antibody titers comparable to that of the currently available inactivated protein-based H5 vaccines. 35 Additionally, liposomes have shown promise as a candidate for delivery of DNA vaccines to mucosal tissue. 36 A recent study demonstrated that vaccination with liposome encapsulated influenza A virus M1 induced both humoral and cellular immune responses that protected against respiratory infection. 36 Liposomes have also been shown to be an effective delivery method for intranasal DNA vaccination, conferring protective immune responses against infection. 37, 38 DNA vaccine delivery can also be accomplished through the use of biodegradable polymeric micro-and nanoparticles consisting of amphiphilic molecules between 0.5-10 mm in size. Similar to loading of DNA plasmid on liposomes, plasmid molecules can be either encapsulated or adsorbed onto the surface of the nanoparticles. [39] [40] [41] [42] These particles function as a carrier system, protecting the vaccine plasmid from degradation by extracellular deoxyribonucleases. In addition to shielding plasmid DNA from nucleases, micro-and nanoparticles promote the sustained release of vaccine instead of the bolus type of delivery characteristic of larger submicrometer complexes. 39, 43 High molecular weight cationic polymers have proven significantly more effective than cationic liposomes in aggregating DNA vaccine plasmid. Plasmid DNA immobilized within biodegradable chitosan-coated polymeric microspheres (ranging from 20 to 500 mm) can induce both mucosal and systemic immune responses. 44 Microspheres may be delivered either by the oral or intraperitoneal route, allowing for direct transfection of dendritic cells (DC), thereby increasing DC activation. The benefits of microsphere formulations have been shown in mice, non-human primates, and humans [45] [46] [47] [48] [49] against a wide range of diseases including hepatitis B, 50 tuberculosis, 51 and cancer. 52 These results suggest that microparticle-based delivery systems are capable of significantly improving DNA vaccine immunogenicity, and boosting cellular and humoral immune responses.

The use of liposomes or nanoparticles appears to be safe and well tolerated in clinical studies. Microparticle-based delivery Optimized gene sequences are inserted into a plasmid backbone and then delivered to the host via one of several delivery methods. Vaccine plasmid enters the nucleus of host myocytes and antigen presenting cells by using host cellular machinery. The plasmid components are transcribed and protein is produced. The cell provides endogenous post-translational modifications to antigens, producing native protein conformations. Vaccine-derived endogenous peptides are presented on MHC class I molecules. Engulfment of apoptotic or necrotic cells by APC also allows for cross-presentation of cell-associated exogenous antigens. Secreted antigen is captured and processed by antigen presenting cells, and presented on MHC class II. Antigen experienced APC migrate to the draining lymph node to stimulate CD4 C and CD8 C T cell populations. In addition, shed antigen can be captured by antigen-specific high affinity immunoglobulins on the B cell surface for presentation to CD4 C T cells, driving B cell responses.

systems can increase gene expression, as well as, DNA vaccine immunogenicity. Although many of the earliest carrier formulations did not show a significant clinical benefit, more recent studies highlighted herein yielded promising clinical data. As microparticles can be prepared with significant structural diversity (size, surface charge, lipid content), they offer considerable flexibility of vaccine formulation. This allows for optimization of the vaccine based on the specific needs of the clinician.

Another approach that has been effective in increasing DNA vaccine immunogenicity is the use of "vaccine cocktails" containing the DNA vaccine as well as plasmids encoding adjuvanting immunomodulatory proteins. Plasmid DNA contains unmethylated deoxycytidylate-phosphate-deoxyguanylate (CpG) motifs that function as a "built in" adjuvant. [53] [54] [55] [56] [57] [58] [59] Molecular adjuvant plasmids expressing cytokines, chemokines, or co-stimulatory molecules may be co-administered with the antigenic DNA vaccine plasmid. Cells transfected by molecular adjuvant plasmids secrete the adjuvant into the surrounding region, stimulating both local antigen presenting cells (APC) and cells in the draining lymph node. This results in durable, but low level, production of immune modulating cytokines that can tailor the immune response toward a more desirable outcome without the concerns of a systemic cytokine storm. While human data are limited, a wide range of inflammatory and helper T cell cytokines have been studied, in conjunction with DNA vaccination, in small animal models. 60, 61 In particular, we have highlighted a few of the most prominent molecular adjuvants with demonstrated ability to increase DNA vaccine immunogenicity. 62 A more comprehensive list of molecular adjuvants is included in Table 1 .

Cytokines are a class of immunoregulatory proteins that affect the behavior of other cells, and are critical for immune cell signaling. Cytokine-encoding genes can be delivered either as a separate plasmid, or as additional genes encoded within the antigen containing plasmid. The most extensively studied molecular adjuvant is Interleukin-2 (IL-2). IL-2 plays an essential role in the immune response by promoting the differentiation of na€ ıve T cells into effector T cells, as well as driving the generation of memory T cell pools. It is also required for the proliferation of Natural Killer (NK) cells. Inclusion of IL-2 has resulted in improved immunogenicity for HIV, [63] [64] [65] influenza, 66 and SARS-CoV 67 anti-viral DNA vaccines. Interestingly, a therapeutic vaccine encoding for the BCR/ABL-pIRES genes of myeloid leukemia and IL-2 also demonstrated enhanced immune responses, suggesting that IL-2 molcular adjuvants have the capability of alleviating the symptoms of chronic infection. 68 Similar to IL-2, IL-15 is a cytokine that induces NK and T cell proliferation. IL-15 is necessary for the generation of primary antigen-specific CD4 C and CD8 C T cell responses. It also plays a substantial role in establishment of memory CD8 C T cell populations. [69] [70] [71] [72] [73] Results of small animal studies suggest that the adjuvant effect of IL-15 is most potent when delivered in tandem with other cytokines. For example, a synergistic effect was seen when IL-15 and IL-21 were co-delivered with a DNA vaccine against Toxoplasma gondii infection. 74, 75 Additionally, sequential administration of IL-6, IL-7, and IL-15 genes augmented long-term CD4 C T cell memory responses to a foot and mouth disease DNA vaccine. 76 Therefore, depending on the antigen, it may be necessary to deliver IL-15 in combination with other molecular adjuvants. Notably, a study in rhesus macaques suggests that delivery of an IL-15 encoding DNA vaccine itself resulted in increased proliferation of NK and T cells, with no adverse effects. 77 Another recent study demonstrated that co-vaccination of rhesus macaques with SIV pol plasmid and HIV env plasmid plus IL-15 allowed for faster control of viremia than the group not formulated with IL-15. 78 Moreover, macaques vaccinated with IL-15 exhibited increased T cell proliferation compared with those receiving the antigen plasmid alone, suggesting that IL-15 has a robust effect on T cell memory responses.

IL-12 is another pro-inflammatory cytokine secreted by both dendritic cells and monocytes. IL-12 plays an integral role in shaping the innate and adaptive immune responses to infection. 79 of antigen-specific CD8 C T cells, and the expression of cytotoxic mediators such as interferon-g (IFN-g), granzyme B, and perforin. 82,83 IL-12 was the first cytokine to be evaluated for use as a molecular adjuvant, and several studies have shown that inclusion of IL-12 expression plasmids within the vaccine formulation enhances T h1 immune responses. [87] [88] [89] [90] [91] [92] [93] [94] [95] Vaccination of mice with a bicistronic plasmid expressing IL-12 and Yersinia pestis resulted in increased mucosal IgA and serum IgG, providing significantly higher levels of protection against challenge than antigen-only groups. 96 Studies in rhesus macaques have shown similar increases in DNA vaccine immunogenicity. Co-vaccination with SIV gag and IL-12 allowed for dose sparing, 97 as well as increased breadth of T cell responses. 89, 91, 98, 99 Additionally, multiple human clinical studies using vaccines adjuvanted with IL-12 have proven safe 100 and highly immunogenic, yielding high level CD4 C and CD8 C T cell responses. 87, 101, 102 Furthermore, inclusion of IL-12 expression plasmids can improve weakly immunogenic vaccines. A recent clinical study demonstrated that addition of IL-12 improved the immunogenicity of a Hepatitis B DNA vaccine, resulting in increased vaccine immunogenicity, as well as sustained memory T cell responses. 103 The final immunomodulatory cytokine that has received considerable focus as a molecular adjuvant is granulocytemacrophage colony stimulating factor (GM-CSF). GM-CSF recruits antigen presenting cells to the vaccination site and promotes DC maturation. 104 It has been successfully used in multiple DNA vaccines. [105] [106] [107] Plasmid-encoded GM-CSF, when co-delivered with a rabies virus DNA vaccine in mice, resulted in increased CD4 C T cell responses, antibody production, and protection from lethal viral challenge. 108 Likewise, a bicistronic DNA vaccine encoding HIV-1 gp120 and GM-CSF recruited inflammatory cellular infiltrates and elicited a potent CD4 C T cell response. 109 However, the benefit of GM-CSF molecular adjuvants remains unclear. Recent studies have shown that co-administration of GM-CSF plasmid with an antigen-encoding DNA vaccine can have deleterious effects. Co-delivery of GM-CSF suppressed the response to a DNA vaccine encoding Dengue virus type 1 and type 2, and also failed to improve the response elicited by a Hepatitis C vaccine. 110 Furthermore, inclusion of plasmid GM-CSF provided minimal adjuvant effect when co-administered with a malaria DNA vaccine in rhesus macaques. 111 Likewise, GM-CSF had no clear effect on T cell responses in patients receiving a melanoma DNA vaccine. 112 One possible explanation for these results is that high levels of GM-CSF can expand myeloid suppressor cell populations, and suppress the generation of adaptive immune responses. Alternatively, the lack of improved immunogenicity seen in clinical trials may be due to the relative lack of GM-CSF receptors on rhesus and human APC compared with murine cells. 113 While no specific adverse effects have been reported, the use of GM-CSF as an adjuvant may require some fine-tuning, particularly if GM-CSF expression levels must be considered with regards to immunosuppression.

In addition to cytokine-encoding plasmids, several other methods for increasing DNA vaccine immunogenicity exist. The increased understanding of immune signaling pathways has led to the development of adjuvant plasmids encoding adhesion molecules, chemokines, costimulatory molecules, and Toll-like receptor (TLR) ligands. These molecular adjuvants have had some success in small animal models. For example, the innate immune signaling molecule TRIF increased the antibody response generated by a swine fever virus DNA vaccine. 114 Moreover, TRIF increased the protective activity of an influenza HA-encoding DNA vaccine. 115 Similar results were seen in studies encoding the dsRNA receptors MDA5 and RIG-I. 116, 117 Additionally, antigen-fusion constructs, whereby the antigen of interest is linked to a "carrier protein," can increase the immune visibility of the vaccine, and enhance DNA vaccine potency. [118] [119] [120] A major advantage of DNA vaccination is the ability of multiple molecules such as molecular adjuvants to be inserted into the plasmid. Unlike the addition of recombinant cytokines, costimulatory molecules, and TLR ligands, which have a limited duration due to the short half-life of recombinant protein in vivo, molecular adjuvant-encoding plasmids will express protein for the same duration as the antigen, stimulating the immune system for a greater length of time. This can be done without fear of eliciting a cytokine storm, as generation of the adjuvanting signal will be localized to the site of vaccination. Of note, homologous recombination between plasmidencoded cytokines and the host gene sequence does not appear to be a significant concern, as multiple studies have shown that only extrachromosomal plasmid DNA has been identified following intramuscular injection. 121, 122 Furthermore, many current plasmids have been-codon optimized to improve gene expression in mammalian cells. This has resulted in changes to the cytokine gene sequence, limiting the possibility for homologous recombination and/or integration. Molecular adjuvants therefore show great promise for both increasing immunogenicity and extending the longevity of the immune response.

Plasmid DNA vectors contain functional elements, such as the origin of replication and selection markers, that are only required during the prokaryotic growth process in E. coli. These "bacterial region" elements ( Fig. 2 ) are no longer needed once cell culture is halted, and may have a negative effect on vaccine stability, uptake, and efficacy. Additionally, these elements can pose safety concerns, particularly if widely used antibiotic resistance markers are horizontally transmitted to host enteric bacteria populations. 123, 124 These concerns have been addressed by development of small bacterial RNA-based antibiotic free selection markers. 124, 125 Noncoding RNA markers are preferable to protein markers since proteins, like antibiotic resistance markers, can be expressed in the host organism after vector transfection, or horizontally transmitted to host bacteria. Noncoding RNA markers are also very small (< 200 basepairs) which decreases the overall vector size; this is advantageous since vector transfection efficiency is inversely related to vector size, [126] [127] [128] perhaps because smaller vectors are more resistant to delivery associated shear forces 129 and may have improved nuclear localization since they are more motile in the cytoplasm. 130 Additionally, some bacterial region protein marker genes have been shown to dramatically reduce vector expression. For example, the TN5 derived NPT-II kanamycin resistance marker (kanR) gene in the pVAX1 vector bacterial region significantly reduces transgene expression. Three groups have demonstrated that pVAX1 bacterial region mediated repression of transgene expression can be alleviated by replacement of the kanR gene with either a tRNA RNA selection marker, the RNA-OUT antisense RNA selection marker, or the endogenous pUC origin RNAI antisense RNA selection marker. [131] [132] [133] Consistent with this, removal of the pVAX1 bacterial region in a minicircle vector improved humoral and cellular immune responses up to 3fold compared with a pVAX1 vector control. 134 DNA vaccine vectors with dramatically higher transgene expression have recently been developed through identification of novel bacterial region and eukaryotic region vector configurations. Pioneering work by Mark Kay's laboratory at Stanford University demonstrated that bacterial regions larger than 1 kbase silenced transgene expression in quiescent tissue such as the liver, likely due to untranscribed bacterial region mediated heterochromatin formation that spreads to the eukaryotic region and inactivates the promoter. [135] [136] [137] Minicircle vectors, in which the bacterial region is removed by the action of a phage recombinase during production, alleviated this silencing. 135, 136, 138 However, production of minicircle vectors is low yield and poorly scalable due to the required in vivo or in vitro recombination during manufacture. 139 In an effort to create alternative short bacterial region vectors that could be efficiently manufactured, the Mini-Intronic Plasmid (MIP) and Nanoplasmid TM vector plasmid platforms were developed. MIP vectors incorporate a RNA-OUT selection marker-pUC origin bacterial region within a 3 0 UTR intron. In this configuration the bacterial region is within the transcription unit and the downstream polyA signal is linked to the eukaryotic promoter without an intervening selection marker or replication origin. Nanoplasmid TM vectors are RNA-OUT selection marker vectors in which the large pUC bacterial replication origin is replaced by a small R6K bacterial replication origin. In this configuration, the < 500 basepair (bp) bacterial region separates the polyA signal and the eukaryotic promoter. Unlike minicircles, both MIP and Nanoplasmid TM RNA-OUT selection vectors can be efficiently manufactured in gram/liter yields without antibiotic selection. 140 As expected, both vector platforms alleviate gene silencing in quiescent tissues similarly to minicircle vectors. 141, 142 However, unexpectedly both MIP and Nanoplasmid TM vectors dramatically improve overall gene expression up to 10-fold compared with plasmid and minicircle vectors in quiescent (liver) and non-quiescent tissues. 141, 142 The improved expression level after ID and IM delivery has application to improve DNA vaccination since increased expression level is correlative with improved humoral and cellular immune response. 62 Another approach to improve DNA vaccines is to engineer the vector to increase innate immune activation. DNA vaccines are potent triggers of innate immunity. Various studies have determined several innate immune pathways are activated by DNA vaccination (Fig. 2) . Most of the intrinsic adjuvant effect of DNA is mediated by cytoplasmic innate immune receptors that nonspecifically recognize B DNA and activate Sting or Inflammasome mediated signaling, 53,143 but unmethylated CpG sequences specific for TLR9 activation may also be important for priming CD8 T cell responses. 144, 145 Along these lines, DNA vaccine vectors may be sequence modified to introduce immunostimulatory xxCGxx TLR9 agonists into the vector to increase innate immune activation. This approach has been used to improve DNA vaccine immunogenicity, 58, 59, 146 but the results are variable. Some of the variability may be due to unintended inhibition of the eukaryotic promoter expression resulting from integration of CpG motifs into non-permissive sites in the vector. 125 As well, certain DNA delivery methods may not transfer DNA to the endosome as effectively as other deliveries (e.g. liposomes), preventing unmethylated CpG interaction with, and activation of, TLR9. Part of the complexity is that optimal TLR9 activating xxCGxx motifs are species-specific; different xxCGxx agonist motifs differentially modulate the immune response 147 and many xxCGxx motifs are immunosuppressive.

An alternative strategy is to encode immunostimulatory RNA within the plasmid to increase innate immune activation. This approach has the potential advantage that additional innate immune pathways not normally stimulated by DNA alone are activated, resulting in polyvalent activation of multiple innate immune pathways to enhance immune activation. 148, 149 Like TLR9 for DNA, several innate immune TLRs for RNA are endosomal. 150 Activation of these receptors requires motif introduction into an expressed RNA, as well as cytoplasmic RNA shuttling into the endosome by autophagy. For example, 3 0 UTR , each of which can activate the STINGI TBK1I IRF3 pathway to induce type 1 interferon production. 143 An additional cytoplasmic innate immune pathway activated nonspecifically by transfected dsDNA is the cytoplasmic AIM2 inflammasome. 157 Other dsDNA receptors and innate immune activation pathways exist, 143 including a recently identified STING/IRF7 signaling pathway required for DNA vaccine immunogenicity. 158 By contrast, the endosomal innate immune receptor TLR9 recognizes specific unmethylated CpG DNA motifs in DNA vaccines. To improve innate immune activation, addition of optimized immunostimulatory CpG motifs in the vector backbone may be used to increase TLR9 activation. Immunostimulatory RNA expressed from the vector may be used to activate alternative RNA sensing innate immune receptors such as RIG-I using an additional RNA Polymerase III RNA expression cassette 117 (plasmid backbone adjuvant) or incorporation of RNA recognizing TLR agonist motifs such as CpG RNA into the 3 0 UTR. 152 Due to limited transgene expression after DNA vaccination in large animals, vector modifications (e.g., <500 bp bacterial region Nanoplasmid TM vectors; intronic bacterial region MIP vectors) and deliveries (e.g., Electroporation) that improve transgene expression also improve adaptive immunity. 62, 125, 159 Adapted under a Creative Commons Attribution license from Williams, 2013. 160 incorporation of a 20 bp immunostimulatory ssRNA encoding D type CpG upstream of a 28 bp hairpin dsRNA resulted in a 4-fold increase in antigen reactive IgG titers, 151 and a 2-fold increase in IFN-g secreting CD4 C and CD8 C T cells. 152 Moreover, several RNA-sensing innate immune receptors such as RIG-I, MDA5 and DDX3 are cytoplasmic. 143 DNA vaccine expressed RNA can be used to target these receptors directly, without autophagy. Of these, RIG-I is of particular interest since RIG-I agonists have demonstrated adjuvant properties to improve the humoral response, 153 humoral and CD4 C T cell response, 154, 155 and CD8 C T cell response 153 to co-administered antigens. 156 In addition, RIG-I is ubiquitiously expressed in most tissues (expression of TLRs typically is restricted to immune cell subtypes) and certain RIG-I agonists that can be expressed in DNA vaccines (e.g., a blunt dsRNA with a 3 0 triphosphate) are structurally conserved between humans and mice. A DNA vaccine vector that co-expresses with antigen a RIG-I dsRNA agonist in a vector backbone encoded RNA Polymerase III transcription unit (Fig. 2 ) enhanced the humoral and CD8 C T cell response after DNA vaccination. 117 DNA vaccines encoding immunostimulatory sequences that selectively improve CTL responses to encoded antigen may have niche application in vaccines for intracellular pathogens or cancer. Innovations that increase transgene expression may be used to improve the performance of immunomodulatory molecular adjuvant plasmids, in addition to traditional antigen expressing DNA vaccine plasmids. Collectively, vector design innovations that improve transgene expression level and innate immune activation are complementary to improved mechanical and nonmechanical DNA vaccine delivery platforms. Combining improved vectors with liposome or polymeric particle nonmechanical delivery, or with needle free injector device delivery, has the potential to increase immunogenicity with these well tolerated, safe, delivery platforms.

While DNA vaccination provides several advantages over more conventional vaccination strategies, further optimization is necessary before it becomes the predominant strategy in human patients. Despite initial setbacks, significant progress has been made in overcoming the problem of low immunogenicity in humans. A clearer understanding of the immune mechanisms governing DNA vaccine immunogenicity has illuminated several pathways that may be useful in further improving DNA vaccine efficacy. A large catalog of cytokines, chemokines, adhesion molecules, and transcription factors are in the process of being tested as molecular adjuvants, although it is likely that each will need to be carefully assessed for safety and tolerability. Likewise, continued development of vaccine delivery methods appears promising. New formulations exploiting sustained vaccine delivery methods, such as slow-releasing micropatches or multilamellar vesicles, are on the horizon. The strong appeal of needle-free injection and mucosal delivery, the ease of design, and the recent clinical successes with DNA vaccines suggests that this approach is on the precipice of redefining the field of vaccinology.

Disclosure of potential conflicts of interest James A. Williams has an equity interest in Nature Technology Corporation. Due to this relationship with Nature Technology Corporation, the author acknowledges that there is a potential conflict of interest inherent in the publication of this manuscript and assert that an effort to reduce or eliminate that conflict has been made where possible.

Effect of different promoters on immune responses elicited by HIV-1 gag/env multigenic DNA vaccine in Macaca mulatta and Macaca nemestrina

Intervening sequences increase efficiency of RNA 3 0 processing and accumulation of cytoplasmic RNA

Molecular mechanisms for enhanced DNA vaccine immunogenicity

Anti-HIV cellular immunity: recent advances towards vaccine design

DNA vaccines against human immunodeficiency virus type 1 in the past decade

Induction of CD4(C) T cell-dependent CD8(C) type 1 responses in humans by a malaria DNA vaccine

The role of follicular helper T cells and the germinal center in HIV-1 gp120 DNA prime and gp120 protein boost vaccination

Direct gene transfer into mouse muscle in vivo

Heterologous protection against influenza by injection of DNA encoding a viral protein

Cross-subtype antibody and cellular immune responses induced by a polyvalent DNA prime-protein boost HIV-1 vaccine in healthy human volunteers

Protection against papillomavirus with a polynucleotide vaccine

A qualitative progression in HIV type 1 glycoprotein 120-specific cytotoxic cellular and humoral immune responses in mice receiving a DNA-based glycoprotein 120 vaccine

A DNA vaccine for venezuelan equine encephalitis virus delivered by intramuscular electroporation elicits high levels of neutralizing antibodies in multiple animal models and provides protective immunity to mice and nonhuman primates

Immunogenicity and protective efficacy of a DNA vaccine against Venezuelan equine encephalitis virus aerosol challenge in nonhuman primates

Codon-optimized filovirus DNA vaccines delivered by intramuscular electroporation protect cynomolgus macaques from lethal Ebola and Marburg virus challenges

A multiagent filovirus DNA vaccine delivered by intramuscular electroporation completely protects mice from ebola and Marburg virus challenge

Immunotherapy against HPV16/18 generates potent TH1 and cytotoxic cellular immune responses

In vivo electroporation enhances the immunogenicity of an HIV-1 DNA vaccine candidate in healthy volunteers

DNA vaccination with electroporation induces increased antibody responses in patients with prostate cancer

Clearance of persistent HPV infection and cervical lesion by therapeutic DNA vaccine in CIN3 patients

Genetic immunization is a simple method for eliciting an immune response

Induction of antigenspecific CD8C T cells, T helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis B virus DNA vaccine

Hepatitis B DNA vaccine induces protective antibody responses in human non-responders to conventional vaccination

Protection of mice against rotavirus challenge following intradermal DNA immunization by Biojector needle-free injection

Influenza virus h5 DNA vaccination is immunogenic by intramuscular and intradermal routes in humans

Enhancement of the immune response in rabbits to a malaria DNA vaccine by immunization with a needle-free jet device

Comparison of the CD8C T cell responses and antitumor effects generated by DNA vaccine administered through gene gun, biojector, and syringe

A liposome-based platform, VacciMax, and its modified water-free platform DepoVax enhance efficacy of in vivo nucleic acid delivery

Liposomal malaria vaccine in humans: a safe and potent adjuvant strategy

Phase I study of a Neisseria meningitidis liposomal vaccine containing purified outer membrane proteins and detoxified lipooligosaccharide

Vaxfectin enhances the humoral immune response to plasmid DNA-encoded antigens

Vaxfectin enhances antigen specific antibody titers and maintains Th1 type immune responses to plasmid DNA immunization

Vaxfectin enhances immunogenicity and protective efficacy of P. yoelii circumsporozoite DNA vaccines

Vaxfectin enhances both antibody and in vitro T cell responses to each component of a 5 gene Plasmodium falciparum plasmid DNA vaccine mixture administered at low doses

Phase 1 clinical trials of the safety and immunogenicity of adjuvanted plasmid DNA vaccines encoding influenza A virus H5 hemagglutinin

Oral vaccination with a liposome-encapsulated influenza DNA vaccine protects mice against respiratory challenge infection

CpG/Poly (I:C) mixed adjuvant priming enhances the immunogenicity of a DNA vaccine against eastern equine encephalitis virus in mice

Enhanced nasal mucosal delivery and immunogenicity of anti-caries DNA vaccine through incorporation of anionic liposomes in chitosan/ DNA complexes

Preparation, characterization, cytotoxicity and transfection efficiency of poly(DL-lactide-co-glycolide) and poly(DL-lactic acid) cationic nanoparticles for controlled delivery of plasmid DNA

Direct plasmid DNA encapsulation within PLGA nanospheres by single oil-in-water emulsion method

Biological potency of microsphere encapsulated plasmid DNA

Cationic microparticles: A potent delivery system for DNA vaccines

Preparation and in vitro transfection efficiency of chitosan microspheres containing plasmid DNA:poly(L-lysine) complexes

Microencapsulation of DNA within alginate microspheres and crosslinked chitosan membranes for in vivo application

Immune responses and protection obtained by oral immunization with rotavirus VP4 and VP7 DNA vaccines encapsulated in microparticles

Immunogenicity in mice of a cationic microparticle-adsorbed plasmid DNA encoding Japanese encephalitis virus envelope protein

Enhanced potency of plasmid DNA microparticle human immunodeficiency virus vaccines in rhesus macaques by using a priming-boosting regimen with recombinant proteins

Enhanced immunogenicity of microencapsulated multiepitope DNA vaccine encoding T and B cell epitopes of foot-and-mouth disease virus in mice

Poly-L-lysine-coated nanoparticles: a potent delivery system to enhance DNA vaccine efficacy

Augmented humoral and cellular immune responses to hepatitis B DNA vaccine adsorbed onto cationic microparticles

Enhanced protective efficacy of a tuberculosis DNA vaccine by adsorption onto cationic PLG microparticles

Plasmid DNA encoding human carcinoembryonic antigen (CEA) adsorbed onto cationic microparticles induces protective immunity against colon cancer in CEA-transgenic mice

TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA vaccines

Toll-like receptor-independent gene induction program activated by mammalian DNA escaped from apoptotic DNA degradation

Recognition of cytosolic DNA activates an IRF3-dependent innate immune response

Vaccination with plasmid DNA activates dendritic cells via Toll-like receptor 9 (TLR9) but functions in TLR9-deficient mice

TLR9¡/¡ and TLR9C/ C mice display similar immune responses to a DNA vaccine

Adjuvant effect of multi-CpG motifs on an HIV-1 DNA vaccine

Effect of plasmid backbone modification by different human CpG motifs on the immunogenicity of DNA vaccine vectors

DNA vaccines: developing new strategies to enhance immune responses

From plasmids to protection: a review of DNA vaccines against infectious diseases

Synthetic DNA vaccines: improved vaccine potency by electroporation and co-delivered genetic adjuvants

Engineering DNA vaccines via co-delivery of co-stimulatory molecule genes

Cytokine molecular adjuvants modulate immune responses induced by DNA vaccine constructs for HIV-1 and SIV

Expression kinetics of the interleukin-2/immunoglobulin (IL-2/Ig) plasmid cytokine adjuvant

Co-expression of interleukin-2 by a bicistronic plasmid increases the efficacy of DNA immunization to prevent influenza virus infections

Enhancing immune responses against SARS-CoV nucleocapsid DNA vaccine by co-inoculating interleukin-2 expressing vector in mice

A BCR/ABL-hIL-2 DNA vaccine enhances the immune responses in BALB/c mice

IL-15 trans-presentation by pulmonary dendritic cells promotes effector CD8 T cell survival during influenza virus infection

Cutting edge: requirement for IL-15 in the generation of primary and memory antigen-specific CD8 T cells

IL-15 regulates CD8C T cell contraction during primary infection

Lack of IL-15 results in the suboptimal priming of CD4C T cell response against an intracellular parasite

Dendritic cell-derived IL-15 controls the induction of CD8 T cell immune responses

Protective efficacy of Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) adjuvated with recombinant IL-15 and IL-21 against experimental toxoplasmosis in mice

Synergy of mIL-21 and mIL-15 in enhancing DNA vaccine efficacy against acute and chronic Toxoplasma gondii infection in mice

Sequential administration of cytokine genes to enhance cellular immune responses and CD4 (C) T memory cells during DNA vaccination

Intramuscular delivery of heterodimeric IL-15 DNA in macaques produces systemic levels of bioactive cytokine inducing proliferation of NK and T cells

Protection against simian/human immunodeficiency virus (SHIV) 89.6P in macaques after coimmunization with SHIV antigen and IL-15 plasmid

Development of TH1 CD4C T cells through IL-12 produced by Listeria-induced macrophages

Resolution of cutaneous leishmaniasis: interleukin 12 initiates a protective T helper type 1 immune response

Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4C T cells

Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes

Interleukin-12: a cytokine at the interface of inflammation and immunity

IL-1 beta is required for IL-12 to induce production of IFN-gamma by NK cells. A role for IL-1 beta in the T cell-independent mechanism of resistance against intracellular pathogens

Mechanisms of IFN-gamma induction by natural killer cell stimulatory factor (NKSF/IL-12). Role of transcription and mRNA stability in the synergistic interaction between NKSF and IL-2

Interleukin 12 induces stable priming for interferon gamma (IFNgamma) production during differentiation of human T helper (Th) cells and transient IFN-gamma production in established Th2 cell clones

Safety and comparative immunogenicity of an HIV-1 DNA vaccine in combination with plasmid interleukin 12 and impact of intramuscular electroporation for delivery

Development of a multicomponent candidate vaccine for HIV-1

Combined effects of IL-12 and electroporation enhances the potency of DNA vaccination in macaques

SIV DNA vaccine co-administered with IL-12 expression plasmid enhances CD8 SIV cellular immune responses in cynomolgus macaques

Comparative ability of plasmid IL-12 and IL-15 to enhance cellular and humoral immune responses elicited by a SIVgag plasmid DNA vaccine and alter disease progression following SHIV(89.6P) challenge in rhesus macaques

Macaques co-immunized with SIVgag/pol-HIVenv and IL-12 plasmid have increased cellular responses

An interleukin 12 adjuvanted herpes simplex virus 2 DNA vaccine is more protective than a glycoprotein D subunit vaccine in a high-dose murine challenge model

Unique Th1/Th2 phenotypes induced during priming and memory phases by use of interleukin-12 (IL-12) or IL-28B vaccine adjuvants in rhesus macaques

KMP-11 DNA immunization significantly protects against L. donovani infection but requires exogenous IL-12 as an adjuvant for comparable protection against L. major

A nasal interleukin-12 DNA vaccine coexpressing Yersinia pestis F1-V fusion protein confers protection against pneumonic plague

A dose sparing effect by plasmid encoded IL-12 adjuvant on a SIVgag-plasmid DNA vaccine in rhesus macaques

Therapeutic vaccination with simian immunodeficiency virus (SIV)-DNA C IL-12 or IL-15 induces distinct CD8 memory subsets in SIV-infected macaques

HIV/SIV DNA vaccine combined with protein in a co-immunization protocol elicits highest humoral responses to envelope in mice and macaques

Plasmid IL-12 electroporation in melanoma

Levels of human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte effector and memory responses decline after suppression of viremia with highly active antiretroviral therapy

Safety and immunogenicity of an HIV-1 gag DNA vaccine with or without IL-12 and/or IL-15 plasmid cytokine adjuvant in healthy, HIV-1 uninfected adults

Correlation of antiviral Tcell responses with suppression of viral rebound in chronic hepatitis B carriers: a proof-of-concept study

Plasmid vaccine expressing granulocyte-macrophage colony-stimulating factor attracts infiltrates including immature dendritic cells into injected muscles

A plasmid encoding murine granulocyte-macrophage colony-stimulating factor increases protection conferred by a malaria DNA vaccine

A push-pull approach to maximize vaccine efficacy: abrogating suppression with an IL-13 inhibitor while augmenting help with granulocyte/macrophage colony-stimulating factor and CD40L

Cytokine GM-CSF genetic adjuvant facilitates prophylactic DNA vaccine against pseudorabies virus through enhanced immune responses

Manipulation of the immune response to a plasmid-encoded viral antigen by coinoculation with plasmids expressing cytokines

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4C T-cell responses

Variable effects of the co-administration of a GM-CSF-expressing plasmid on the immune response to flavivirus DNA vaccines in mice

A DNA vaccine encoding the 42 kDa C-terminus of merozoite surface protein 1 of Plasmodium falciparum induces antibody, interferon-gamma and cytotoxic T cell responses in rhesus monkeys: immuno-stimulatory effects of granulocyte macrophage-colony stimulating factor

A phase I study of immunization using particle-mediated epidermal delivery of genes for gp100 and GM-CSF into uninvolved skin of melanoma patients

The role of cytokine DNAs as vaccine adjuvants for optimizing cellular immune responses

The Toll-like receptor adaptor molecule TRIF enhances DNA vaccination against classical swine fever

Toll-like receptor adaptor molecules enhance DNA-raised adaptive immune responses against influenza and tumors through activation of innate immunity

MDA5 can be exploited as efficacious genetic adjuvant for DNA vaccination against lethal H5N1 influenza virus infection in chickens

Coexpressed RIG-I agonist enhances humoral immune response to influenza virus DNA vaccine

Coadministration with DNA encoding papillomavirus capsid proteins enhances the antitumor effects generated by therapeutic HPV DNA vaccination

Mutants of plant genes for developing cancer vaccines

Comparison of HPV DNA vaccines employing intracellular targeting strategies

Plasmid DNA vaccines: tissue distribution and effects of DNA sequence, adjuvants and delivery method on integration into host DNA

Potential DNA vaccine integration into host cell genome

Studying plasmid horizontal transfer in situ: a critical review

Marker-free plasmids for biotechnological applications -implications and perspectives

Improving DNA vaccine performance through vector design

Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency

Investigations of the effect of DNA size in transient transfection assay using dual luciferase system

Effects of circular DNA length on transfection efficiency by electroporation into HeLa Cells

Micro-minicircle Gene therapy: Implications of size on fermentation, complexation, shearing resistance, and expression

Size-dependent DNA mobility in cytoplasm and nucleus

plasmids free of antibiotic resistance markers, display high-level transgene expression in muscle, skin and tumour cells

Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields

Plasmid DNA size does affect nonviral gene delivery efficiency in stem cells

In vivo electroporation of minicircle DNA as a novel method of vaccine delivery to enhance HIV-1-specific immune responses

The extragenic spacer length between the 5 0 and 3 0 ends of the transgene expression cassette affects transgene silencing from plasmid-based vectors

Minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcriptional level

Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo

Towards effective non-viral gene delivery vector

Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo

Antibiotic-free production of a herpes simplex virus 2 DNA vaccine in a high yield cGMP process

A mini-intronic plasmid (MIP): a novel robust transgene expression vector in vivo and in vitro

Replicative minicircle vectors with improved expression

Microbial sensing by Toll-like receptors and intracellular nucleic acid sensors

Priming of CD8C T-cell responses after DNA immunization is impaired in TLR9-and MyD88-deficient mice

Essential role for TLR9 in prime but not prime-boost plasmid DNA vaccination to activate dendritic cells and protect from lethal viral infection

The combination of TLR-9 adjuvantation and electroporation-mediated delivery enhances in vivo antitumor responses after vaccination with HPV-16 E7 encoding DNA

Three types of human CpG motifs differentially modulate and augment immunogenicity of nonviral and viral replicon DNA vaccines as built-in adjuvants

Combined CpG and poly I:C stimulation of monocytes results in unique signaling activation not observed with the individual ligands

Triggering intracellular receptors for vaccine adjuvantation

Innate immune sensing and signaling of cytosolic nucleic acids

CpG RNA: identification of novel single-stranded RNA that stimulates human CD14CCD11cC monocytes

Incorporation of immunostimulatory motifs in the transcribed region of a plasmid DNA vaccine enhances Th1 immune responses and therapeutic effect against Mycobacterium tuberculosis in mice

Cutting edge: The RIG-I ligand 3pRNA potently improves CTL cross-priming and facilitates antiviral vaccination

Activation of the RIG-I pathway during influenza vaccination enhances the germinal center reaction, promotes T follicular helper cell induction, and provides a dose-sparing effect and protective immunity

Enhanced influenza virus-like particle vaccination with a structurally optimized RIG-I agonist as adjuvant

Crosstalk between cytoplasmic RIG-I and STING sensing pathways

Identification of aim2 as a sensor for DNA vaccines

A cGAS-Independent STING/IRF7 Pathway Mediates the Immunogenicity of DNA Vaccines

Vector optimization and needle-free intradermal application of a broadly protective polyvalent influenza A DNA vaccine for pigs and humans

Vector design for improved DNA vaccine efficacy, safety and production

Modulation of cellular responses by plasmid CD40L: CD40L plasmid vectors enhance antigen-specific helper T cell type 1 CD4C T cell-mediated protective immunity against herpes simplex virus type 2 in vivo

Costimulatory molecule immune enhancement in a plasmid vaccine model is regulated in part through the Ig constant-like domain of CD80/86

Macrophage colony-stimulating factor can modulate immune responses and attract dendritic cells in vivo

Intracellular adhesion molecule-1 modulates beta-chemokines and directly costimulates T cells in vivo

Coimmunization with IFN-gamma or IL-2, but not IL-13 or IL-4 cDNA can enhance Th1-type DNA vaccine-induced immune responses in vivo

Modulation of amplitude and direction of in vivo immune responses by co-administration of cytokine gene expression cassettes with DNA immunogens

IL-4 increases Simian immunodeficiency virus replication despite enhanced SIV immune responses in infected rhesus macaques

Interleukin 7 can enhance antigen-specific cytotoxic-T-lymphocyte and/or Th2-type immune responses in vivo

CD8 positive T cells influence antigen-specific immune responses through the expression of chemokines

DNA vaccines encoding interleukin-8 and RANTES enhance antigen-specific Th1-type CD4(C) T-cell-mediated protective immunity against herpes simplex virus type 2 in vivo

Co-immunization with plasmid IL-12 generates a strong T-cell memory response in mice

Coimmunization with an optimized IL-15 plasmid results in enhanced function and longevity of CD8 T cells that are partially independent of CD4 T cell help

Antigen-specific humoral and cellular immune responses can be modulated in rhesus macaques through the use of IFN-gamma, IL-12, or IL-18 gene adjuvants

John J. Suschak and Connie S. Schmaljohn would like to acknowledge funding from the Joint Science and Technology Office for Chemical and Biological Defense of the Defense Threat and Reduction Agency. The opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.