Journal ofMaterials Chemistry B

FEATURE ARTICLE

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Biomaterials at t

CdlBNipEstddr

immunity. His academic interesbiomaterials and immunology, witinnovative vaccine strategies andnological pathways.

aDepartment of Chemical and Biological Eng

University of New York, Buffalo, NY 14260-4bDepartment of Microbiology and Immun

University of New York, Buffalo, NY 14260-4cThe Witebsky Center for Microbial Patho

Buffalo, The State University of New York, B

Cite this: J. Mater. Chem. B, 2014, 2,8053

Received 30th June 2014Accepted 12th September 2014

DOI: 10.1039/c4tb01058b

www.rsc.org/MaterialsB

This journal is © The Royal Society of C

he interface of nano- and micro-scale vector–cellular interactions in geneticvaccine design

Charles H. Jones,a Anders P. Hakanssonbc and Blaine A. Pfeifer*a

The development of safe and effective vaccines for the prevention of elusive infectious diseases remains a

public health priority. Immunization, characterized by adaptive immune responses to specific antigens, can

be raised by an array of delivery vectors. However, current commercial vaccination strategies are predicated

on the retooling of archaic technology. This review will discuss current and emerging strategies designed to

elicit immune responses in the context of genetic vaccination. Selected strategies at the biomaterial–

biological interface will be emphasized to illustrate the potential of coupling both fields towards a

common goal.

1. Introduction

Vaccines are undeniably one of the most signicant medicaladvancements of mankind, as the prevention of infectiousdiseases results in the sparing of more than 3 million lives per

harles H. Jones received B.S.egrees in Chemical & Biomo-ecular Engineering andiochemistry in 2011 fromorth Carolina State Universityn Raleigh, NC. He is currentlyursuing his Ph.D. in Chemicalngineering at the State Univer-ity of New York at Buffalo. Hishesis work focuses on theevelopment of novel geneelivery vectors and theiresulting impact upon adaptivets lie at the intersection ofh a special focus on developingelucidating underlying immu-

ineering, University at Buffalo, The State

200, USA. E-mail: blainepf@buffalo.edu

ology, University at Buffalo, The State

200, USA

genesis and Immunology, University at

uffalo, NY 14260-4200, USA

hemistry 2014

year and economic savings on the order of tens of billions of USdollars.1–3 Recent shortages of inuenza vaccines and theinability to overcome elusive diseases such as HIV-1 haveprovided an essential reminder of the tenuous nature of theworld's vaccine supply and the strategies for its delivery. Thedevelopment and assessment of new strategies is required toovercome such barriers, as opposed to the renement of currentapproaches, to create safer and more effective vaccines.

The principle of controlled immunization (vaccination) waspioneered by Edward Jenner and others in the early 19th centuryand has remained relatively unchanged in clinical development

Anders P. Hakansson is anAssistant Professor of Microbi-ology and Immunology at theState University of New York atBuffalo. He received degreesfrom Lund University in Medi-cine (B.S.) and Medical Micro-biology (Ph.D.). He was a part ofnumerous research fellowshipsincluding positions at David E.Briles' laboratory at the Univer-sity of Alabama, Birmingham;the Channing Laboratory at

Brigham and Women's Hospital (Harvard Medical School); andthe Division of Infectious Disease at Boston Children's Hospital(Harvard Medical School). His research seeks to understand howthe respiratory pathogen Streptococcus pneumoniae (pneumo-coccus) causes disease in humans and how human defense mech-anisms, including breast milk, can provide disease-protectiveproperties.

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and practice. These early endeavors led to the vaccine meth-odologies available today. Despite success, the underlyingprocesses required to produce immunity were largely unknown.Thus, research in the past quarter century has focused on theassembly of working models of adaptive immunity. Briey,adaptive immunity consists of a cellular response of T and Bcells upon exposure to antigens. The cascade is initiated by theinteraction of these cells with antigen-presenting cells (APCs;macrophages, dendritic cells, and neutrophils) displaying pro-cessed antigens (epitopes) on major histocompatibilitycomplexes (MHCs). T cells are stimulated by epitope presenta-tion in MHC-I or MHC-II and accompanying co-stimulatorymolecules (e.g., CD40, CD80, and CD86) through T cell receptors(TCRs), leading to activation and maturation of various T cellsubsets. MHC-II presentation is recognized by TH2 cells whichsubsequently activate B cells through the cross-linkage of B cellreceptors (BCRs), prompting maturation into long-lived anti-body-producing plasma cells or memory B cells.4 Similarly,MHC-I presentation is recognized by TH1 cells which thenactivate macrophages, CD8 T cells, and other cells responsiblefor cell-mediated immunity and phagocyte-dependent protec-tive mechanisms.

Generally, adaptive responses can be segmented into twodistinct protection mechanisms. First, antibody-mediated(humoral) responses are instigated by exposure to externallylocalized antigens such as bacterial and viral surface proteins orsecreted products such as tetanus toxoid (Fig. 1), resulting inMHC-II presentation and plasma cell antibody production.4

Antibody production facilitates the neutralization and clearanceof complimentary antigens. Conversely, cell-mediated immu-nity is characterized by responses that do not result in antibodyproduction but rather involve the activation of macrophages,cytotoxic T-lymphocytes, and antigen-specic cytotoxic T cells inconjunction with the release of proammatory cytokines(Fig. 1).4 Unlike humoral responses, cell-mediated immunity isdirected towards the eradication of intracellular infections thataffect both phagocytic and nonphagocytic cells by promotingdestruction of infected host cells. Examples of cell-mediatedresponses include any antigen of intracellular origin, producedor residing within the APC itself, such as viral infections (HIV,

Blaine Pfeifer is an AssociateProfessor of Chemical Engi-neering at the State University ofNew York at Buffalo. He receivedChemical Engineering degreesfrom Colorado State University(B.S.) and Stanford University(M.S. and Ph.D.) and completedhis postdoctoral work at theMassachusetts Institute of Tech-nology. His research seeks toapply cellular, metabolic, andprocess engineering in the context

of natural product biosynthesis and the development of therapiesfor infectious disease and cancer.

8054 | J. Mater. Chem. B, 2014, 2, 8053–8068

inuenza, and smallpox) and intracellular bacteria. Recently, ithas been recognized that TH17 responses can elicit protectiveimmunity against various bacterial and fungal pathogens.5 Inthis case, protection is mediated through the recruitment ofneutrophils, subsequent release of anti-microbial peptides, andIL-17-driven TH1 responses. Additionally, TH17 cells have beendocumented in regulating/augmenting B cell antibody genera-tion and germinal center and ectopic inducible bronchus-associated lymphoid tissue (iBALT) formation.6,7

Despite centuries of development, only 27 human diseasesare recognized by the CDC as preventable by vaccination.8

However, these examples do not account for rapidly emergingand immunologically evasive entities (malaria, HIV) or wherebasal regulatory mechanisms have gone awry (cancer). To thisend, some sub-optimal vaccines have been replaced withimproved alternatives and new strategies. Notably, more thanhalf of all new vaccines have been developed in the past 35years.2 However, infectious diseases still cause signicantmorbidity andmortality, especially in countries that do not haveaccess to modern medicine.9 Thus, new and broadly accessiblevaccines are needed to improve current rates of vaccinationagainst diseases amongst individuals who may be at riskbecause of their age, medical condition, occupation, orgeographical location.

This review will provide an overview of the main types ofvaccines as they pertain to current and emerging applicationsand production schemes. An emphasis will be placed on recentgenetic vaccination strategies. In particular, genetic vaccinedesign and vector-mediated delivery routes will be described inthe context of improving immune responses and therapeuticoutcomes. Finally, the types of genetic antigen delivery vectorsdiscussed and compared will include biological and biomaterialcarriers and the prospect of combining respective advantages ofeach.

2. Vaccine strategies and productionschemes

Vaccines can broadly be classied into four groups, each rep-resenting a different approach to eliciting adaptive immunity.These include: (1) live attenuated or (2) inactivated (killed)organisms; (3) puried components (toxoid, subunit, conju-gate); and (4) DNA-based vaccines.

2.1 Live attenuated organisms

Since Edward Jenner's use of bovine poxvirus (a viral entityclosely resembling smallpox but non-pathogenic in humans) toelicit protection against smallpox, the concept of live organismspromoting protective immunity has become the paradigm ofvaccine design (Fig. 1). Fundamentally, this approach is predi-cated on the identication and neutralization of virulencefactors (attenuation) while maintaining benecial immunoge-nicity. The strategy has been successfully utilized to combatpathogens and diseases such as measles, mumps, rubella,varicella zoster (chickenpox), inuenza, rotavirus, polio, yellowfever, and rabies.

This journal is © The Royal Society of Chemistry 2014

Fig. 1 General antigen processing. Upon entry into the body, a pathogenic organism encounters an antigen presenting cell (APC; dendritic cellsor macrophages) and is internalized for intracellular processing. The APCs will present the antigen to T helper cells via major histocompatibilitycomplexes (MHCs), which will then present either to naıve B cells or killer T cells. Subsequent activation steps result in either antibody production(humoral response) or cell mediated responses. After an ‘active’ phase of immunity, long-term (adaptive immunity) is established through theformation of memory B and T cells.

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Attenuated vaccines are successful due to their array ofantigens available for immune induction, innate adjuvantproperties, and evolutionarily optimized invasive properties.Thus, the use of live vaccines triggers immune responses thatare similar to what occurs during the natural progression ofinfection, prompting strong cell-mediated and humoralresponses as a result. In addition, outcomes typically includelong-term immunity with aminimal regimen of vaccine dosages(only one to two).

Once an infectious organism has been identied, there arenumerous methods of attenuation in preparation for dosagescale up. Some examples include the use of mutagenic agents(e.g., formaldehyde) or serial passages in cell culture or animalembryos (e.g., egg-based attenuation of viruses). Using egg-based attenuation as an example, a target virus is grown in aseries of eggs and becomes increasingly adapted with eachpassage. In this case, by using evolutionary adaptation as a tool,the virus becomes less etiologically t while still being recog-nizable by the human immune system. More generally, themanufacturing processes for live organism-based vaccines arestraight-forward as production processes are already in place orexisting unit operation-based infrastructure allows the accu-mulation of high concentrations of cultured microbial/viralorganisms.

Important limitations of live organism-based vaccinesinclude the potential for reversion to a form capable of causingdisease, delays associated with egg-based production schemesin the face of pandemic needs, and refrigeration requirements.Some organisms have the potential to revert back to a patho-genic state due to the failure to properly identify and removevirulence factors or when mutations occur during the produc-tion and immunization processes resulting in more pathogenicorganisms, exemplied by the oral polio vaccine (OPV), aningested live vaccine that has a propensity to mutate. Thesemutations have resulted in rare cases of paralytic polio, and forthese reasons, the process is no longer used in the US and has

This journal is © The Royal Society of Chemistry 2014

been replaced by a fully inactivated polio vaccine (IPV).10

Today's egg-based vaccine manufacturing has the capacity toannually produce up to 400 million dosages of a trivalent uvaccine.9 However, time delays, egg allergy, and scalabilityissues are causes for concern for many experts.9 To addressthese items, alternatives such as cell-culture and vaccine-strategy redesign have the potential to provide broader immunecoverage and improved public distribution as compared tocurrent practices. Another limitation is the standard require-ment of refrigeration to maintain live vaccine effectiveness.9

Thus, live vaccines may not be viable options in developingcountries, remote locations, or climates lacking widespreadrefrigeration capabilities.

2.2 Inactivated organisms

An alternative to live vaccines are fully inactive (dead) organ-isms incapacitated using chemicals (e.g., formaldehyde,formalin, or b-propiolactone), heat, or radiation. These atten-uation processes remove the pathogen's ability to replicate butkeep it physically intact, permitting immune modulation.Inactive vaccines are more stable and do not pose virulencereversion risks. The approach is most suitable when theimmune response is provoked by the organism itself (asopposed to a product that must be biosynthesized and secreted)and when inactivation does not reduce antigenimmunogenicity.

Due to the inability to replicate in vivo, inactivated vaccinesare oen administered concomitantly with an adjuvant(immune-potentiator; e.g., aluminium salts or an oil-in-wateremulsion of squalene [MF59]) to increase potency. In addition,these vaccines typically are not effective in eliciting cell-medi-ated immunity (due to the lack of endogenously producedantigens required for MHC-I presentation) and result in shorterlengths of protection, necessitating the use of boosters to createlong-term immunity. Manufacturing of inactivated vaccines issimilar to those of live, attenuated vaccines prior to applying the

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inactivation method of choice. Since the inactivated organismdoes not (usually) require delicate transport, these vaccinestypically do not require refrigeration and can be lyophilized,which increases accessibility.

2.3 Puried components or subunit and conjugate vaccines

When an immunogenic antigen is known to confer protectiveimmunity, it is safer andmore efficient to focus on the synthesisand/or isolation of this particular target for vaccine productionas compared to using either entire live (attenuated) or killedorganisms.

Sometimes a bacterial-based disease is not directly linked tothe physical makeup of a bacterium, but rather to an internallyproduced toxin that leads to disease in the infected host.Immunization with an inactivated variant of the toxin, called atoxoid, leads to a neutralizing antibody-mediated (humoral)response. For example, Clostridium tetani produces a neurotoxin(tetanospasmin) which causes tetanus. Puried toxin is inacti-vated using formaldehyde prior to immunization. This strategyresults in nearly 100% clinical efficacy against tetanus.Production of toxoid vaccines are simple, as in the example of C.tetani, where bacteria are cultured, the toxin puried, andinactivation completed prior to additional purication andsterilization. To boost immune responses, toxoids are typicallyformulated with adjuvants, such as aluminium or calcium salts.

Similarly, use of only part of a target antigen is designated asa subunit vaccine. These acellular forms of vaccines includethose for diphtheria and pertussis, which are commonly pack-aged together with C. tetani toxoid to create the ‘DTP’ vaccine.As with toxoid-based vaccines, subunit immunization resultsprimarily in a humoral response.

When poorly-immunogenic virulence factors are used astargets for immunization, conjugate vaccines are utilized tocreate more powerful combined immune responses. This isconducted by the conjugation of a molecule of interest to animmunogenic carrier protein. Conjugate vaccines such as theStreptococcus pneumoniae polysaccharide vaccine are composedof an assortment of pneumococcal polysaccharides attached tocarrier proteins such as CRM197 (diphtheria toxin) or the outermembrane protein complex from Neisseria meningitides.

Since many of the puried antigens presented above arebacterially derived, production can be adjusted to utilize anindustrial microbial host such as Escherichia coli via recombi-nant DNA strategies. Genetic transfer into well-characterizedhosts will facilitate the replacement of current pathogenically-derived vaccine targets with safer production routes and thepotential for more potent antigens. The new antigen route alsooffers the advantage of established process engineeringschemes associated with the new host that can be easily adaptedto vaccine production.

2.4 DNA vaccines

DNA-based vaccination is an emerging immunization strategyutilizing the administration of one or more genes encodingfunctionally active antigens from a targeted pathogen.11–13 Uponuptake by immune effector cells, host-mediated expression of

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antigenic targets results in the induction of both cellular andhumoral immunity. The ability to combine the simplicity andspecicity of the response from puried component vaccines(humoral) with cell-mediated immunity induction, the hall-mark of live vaccines, has established genetic vaccination as anattractive alternative to traditional vaccine strategies.11

DNA-based technology, unlike other antigenic immuniza-tion strategies, is highly malleable, as it combines the tools andpower of molecular biology with the increasing informationavailable through genome sequencing projects. This synergisticcombination allows for the rapid design and assembly ofpotential antigenic targets and eliminates the requirement ofisolating pathogen-derived antigens for vaccine production. Inaddition, manufacturing of plasmid DNA vaccines is conductedusing E. coli as a production host, thus, taking advantage ofstandard fermentation methodologies and production infra-structure. Since plasmids encoding different antigens areproduced and processed in the same manner, rapid alterationsin the production line can be implemented to accommodateemerging diseases or any unexpected shortages.9,14 In ademonstration of the potential scale and exibility of DNA-based vaccine production, previous studies have suggested thatusing established infrastructure will allow DNA vaccines tomeet global demands.15 DNA vaccines are also relatively stable,reducing and possibly removing the need for cold chain trans-port and storage.16

Despite production advantages, genetic antigens are them-selves not immunogenic, which necessitates the use of adju-vants and carriers, but allows for repeated homologousvaccination (i.e., successive treatment with the same antigen)without fear of declining efficacy.17 The remainder of this articlewill cover current strategies and vectors designed to improveand direct DNA vaccine outcomes.

3. Enhancing immune potency ofDNA vaccines

DNA vaccine technology is a highly exible platform that isroutinely applied to inuence in vivo responses. Approachesinclude optimizations of the antigen expression cassette,inclusion of adjuvants (traditional andmolecular), utilization ofalternative immunization strategies, and the development andformulation of next-generation delivery vectors (Table 1).

3.1 Expression cassette engineering

An ideal plasmid for DNA vaccination should be easily producedat the commercial scale, unable to integrate into the humangenome, and mediate high levels of transgene expression.Various steps have been taken to address these criteria,including optimization of the plasmid backbone (origin ofreplication, backbone size and content, and antibiotic resis-tance markers),18–24 engineering of the promoter and enhancerregions,25–29 development of improved DNA and mRNAsequences,13,30–32 and the incorporation of leader and poly-adenylation (polyA) sequences.33

This journal is © The Royal Society of Chemistry 2014

Table 1 DNA vaccination strategies to improve efficacy

Strategies Examples Purpose References

Plasmid modications� Backbone size reduction Minicircle and MIP plasmids Reduce in vivo silencing, enhance gene delivery efficiency 18 and 19� Antibiotic resistancemarker removal/replacement

RNA-out Reduce amount of bacterial backbone DNA 19

� Origin of replication pUC Provide high copy levels without being able to replicate in other hosts 34� Promoter choice CMV, CAGG, RSV Facilitate desired constitutive expression levels 27, 29 and

30� Enhancer elements SV40 enhancer Activate transcription of genes 29� Leader sequences Introns Positively augment promoter activity (increased transcription) and

enhance polyadenylation19

Sequence modications� Codon optimization Modication of GC/AT content Enhance translation 180� Strong translation startsequence

Kozak consensus sequence Enhance translation 44

� Multiple terminationsequences

Prevent read-through translation 34

� Splicing signal Splicing enhancers Enhance transcription 41–43� Polyadenylation Increase stability of mRNA product 34� Localization sequences LAMP1, TPA Bias antigenic processing towards desired response (Th1 vs. Th2) 46 and 47

Molecular adjuvants� Cytokines IL-1, IL-12, IL-15, GM-CSF Secreted signaling molecules that control the duration and strength of

an immune response58–63

� Chemokines (specializedtype of cytokine)

XCLs, CCLs, CXCLs, and CX3CLs Chemo-attractants for the induction of ‘immunocompetent’ localenvironment

55–57

� Signaling ligands TRIF Secreted ligands bind desired receptors to instigate desired immunepathways

67 and 68

� Transcription factors IRF1, IRF3, IRF7 Regulate immune responses at the genetic level 49 and69–72

� Co-stimulatory molecules CD40, CD80, CD86 Provide additional control over the activation of T cells 64–66

Immunization� Naked With or without general adjuvants Direct injection of pDNA 181� Delivery assisted Electroporation, gene gun, jet

injector, topical patchEnhanced delivery of pDNA delivery utilizing methods designed toincrease localized concentrations (pooled regions and intracellular)

181

� Carrier assisted Cationic polymers and lipids,biological carriers, inorganicmaterial

Enhanced delivery and expression of pDNA 181

� Prime boost Homologous/heterologous Alternative administration route designed to boost responses by rstpriming the immune system

182 and183

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Commonly overlooked, design of the plasmid backbone isthe rst and arguably most critical step in the development of apotent genetic vaccine. Since large quantities of plasmid DNA(pDNA) are required per dose, production is generally carriedout using high-copy bacterial plasmids, such as pUC-basedvectors. This family of vectors contains a modied pBR322origin of replication (mutated to remove regulatory constraintson plasmid number) allowing bacterial maintenance of over 500copies per bacterium, effectively increasing plasmid yields andlowering production cost.34 Equally important, plasmid size andbacterial components (bacterial plasmid elements such asorigins of replication and antibiotic resistance markers)signicantly inuence the magnitude and duration of trans-gene expression.35 Reduction in gene expression (silencing) isbelieved to occur at the nuclear stage by trimethylated-mediated

This journal is © The Royal Society of Chemistry 2014

chromatin-linked transcriptional blockage.23 Thus, expressionvectors have been designed to drastically improve transgeneexpression by the removal of any bacterial pDNA sequence.18–23

For example, minicircle (MC) pDNA vectors are small expres-sion vectors devoid of all bacterial DNA (including origin ofreplication and resistance markers). This is made possible bythe inclusion of several engineered sites on the plasmid back-bone that result in recombination-based removal of bacterialbackbone DNA but not the transcriptional unit. However,current MC vectors utilize temperature-sensitive lower copyorigins of replication, reducing the potential for large scaleproduction when compared to pUC-based vectors. Additionally,since recombination occurs within the bacterial productionhost, additional purication steps are also required to recoverthe nal MC plasmid. Similarly, transgene silencing is also

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initiated when greater than 1 kb of non-coding DNA is placedoutside the transcription unit (regardless of whether it isbacterial DNA).36 To simultaneously overcome both bacterialbackbone and non-coding DNA silencing, a new expressionsystem, termed mini-intronsic plasmid (MIP), was developed.19

This system shis the position of the bacterial replication originand selection marker from the backbone to an intron within thetransgene cassette. The same study demonstrated that trans-gene expression was stronger and extended when compared toeither plasmid or minicircle systems. Finally, to enable theability to rapidly customize vaccine antigens and retain opti-mized transgene expression, generalized cloning sites must beinserted to locations that will permit the ‘drop-in’ of all requiredexpression sequences.

Secondary to physical plasmid construction is the choice anddesign of the transcriptional unit. A typical (monovalent) tran-scriptional unit is arranged with a promoter upstream and apolyA signal downstream of a genetic antigen (Fig. 2). Theselection of a strong constitutive promoter, usually in the formof a viral promoter, to mediate high levels of transgeneexpression is required to achieve an effective immune response.Traditionally, transcriptional units have been driven by thehuman cytomegalovirus immediate-early (CMV) or the chicken-b actin with CMV early enhancer (CAGG) promoters.34,37,38 Ifreduced levels of transgene expression are desired, alternativeviral promoters such as the simian vacuolating virus 40 (SV40)or cellular-specic promoters such as the human elongationfactor 1-a (EF-1a) may be considered. Additional engineering ofthe CMV promoter (incorporation of a downstream HTLV-1 R-U5 region) has been demonstrated to drive higher levels oftransgene expression and ultimately improve cellular immuneresponses in mice and nonhuman primates.27 Aer thepromoter, intron sequences are typically included between the

Fig. 2 Generic design of a plasmid DNA vaccine.

8058 | J. Mater. Chem. B, 2014, 2, 8053–8068

promoter and gene construct as their presence has demon-strated elevated expression.33 It is thought that the inclusion ofintrons positively augments promoter activity39 and the rate ofpolyadenylation and/or nuclear transport associated with RNAsplicing.40 Transgene expression may be further elevated by theaddition of splicing enhancers internally and anking theintron.41–43 The spliced untranslated leader region (UTR) isdesigned to be less than 150 base-pairs long, contain no openreading frames (ORFs), and reduce occurrences of secondarystructures. Consequently, the translation initiation sequence(Kozak consensus sequence) is generally inserted at the start ofthe secondary exon and immediately prior to the ATG startcodon.44

A genetic vaccine's immune potency can be signicantlyelevated by codon optimization of the transgene sequence tomatch the target organism. This increase is related to the GCenrichment of nucleotides present in mammalian DNA ascompared to AT-rich sequences in bacterial DNA. Therefore, theavailability of tRNAs for translation and the variation in codonusage leads to distinct translation differences between theantigenic source and the transgene host cells. Additionally, dualstop codons are oen utilized to prevent read through trans-lation. Following an additional 30 UTR, a polyA signal is insertedto mediate mRNA processing and to limit enzymatic degrada-tion.33 Alternatively, targeting and signalling sequences can beincorporated to direct and enhance antigen processing towardseither MHC-I or MHC-II presentation.45 For example, endo-somal/lysosomal targeting promotes an MHC-II (predominatelyhumoral) response; thus, the addition of lysosomal-associatedmembrane protein 1 (LAMP1) or tissue plasminogen activator(TPA; to promote antigen secretion) signal sequences arecommonly used strategies.46,47 Conversely, N-terminal ubiquitintags are utilized to direct antigen entry into the proteasomaldegradation pathway which results in predominately MHC-Iresponses.48

When designing each of the previously described geneticsequences, optimization to promote and prolong transcribedmRNA is essential to further improve immune responses.Specically, modications of the mRNA sequence that do notinterfere with the antigen's nal protein structure and resultingpresentation are required. Alterations include removal ofunstable secondary structures (usually caused by GC-richregions), cis-acting motifs (e.g., TATA boxes), repeat sequences,cryptic splice sites, undesired ribosomal binding sites, andcruciforms (multiple hairpin loops which confer sensitivity tonucleases).33

A major advantage of the DNA platform is the exibility andease in customization of antigens to address disease-specicbarriers. Furthermore, although the text above describes thecreation of monovalent DNA platforms, the systems can easilybe adapted to deliver and express a broad coverage of antigens,which do not necessarily have to target the same disease. Withthis in mind, the development of an all-encompassing geneticvaccine platform that has the potential to provide coverage ofnumerous antigens in a single dose is an emerging trend in theeld of DNA vaccines.

This journal is © The Royal Society of Chemistry 2014

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3.2 Adjuvants

Although rst-generation DNA vaccines were noted for theirpoor immunogenicity, recent molecular adjuvants (plasmidexpressed immune-potentiator molecules) have dramaticallyincreased the ability to induce a more robust and directedresponse.49 Historically, DNA vaccination was conducted usingadministration of naked DNA either with or without traditionaladjuvant addition. When adjuvants were included, aluminiumsalts (alum) or oil-based (MF59) adjuvants were commonoptions. These choices operate through the creation of‘immunocompetent’ local environments which leads to therecruitment of respective immune-effector cells (alum attractsmacrophages and monocytes; whereas, MF59 also attractsgranulocytes), accelerates monocyte differentiation, andaugments antigen uptake.50,51 However, these adjuvants serve asgeneral immunostimulatory elements that have little inuenceupon the direction and specicity of an immune response.

In general, DNA vaccines have a ‘built-in’ adjuvant in theform of the CpG motif (i.e., a cytosine triphosphate deoxy-nucleotide (C) linked to a guanine triphosphate deoxynucleo-tide (G) by a phosphodiester linkage (p)). Immunostimulatoryproperties arise due to aberrant lack of methylation, leading torecognition as a pathogen-associated molecular pattern (PAMP)by the pattern recognition receptor Toll-Like Receptor 9(TLR9).52 Addition of several CpG motifs in DNA vaccines hasresulted in improved immunogenicity, presumably by increasedinnate immune activation.53,54

Interestingly, due to the modularity of DNA vaccines, geneticadjuvants are oen included (either on the antigen-encodingplasmid or on a separate expression vector) to further increasethe resulting immune response. Upon vaccination, cells trans-fected with plasmids encoding molecular adjuvants will expressand secrete the desired product (generally a signalling mole-cule) into the surrounding region creating a focused immuno-competent local environment. Unlike general adjuvants, theresult is long-lasting and can be easily tailored to the specicvaccination strategy. These adjuvants operate by biasing thecellular immune response towards TH1 induction (effectiveagainst viruses, intracellular bacteria, and cancer), externalantigens (neutralizing antibody response), or by the recruitmentof immune effector cells. Since these adjuvants are derived fromknown immune-modulating agents, a higher level of controlcan be exercised in the direction of the desired response.Examples have included peptides (agellin and inuenza Avirus nucleoprotein), chemokines (XCLs, CCLs, CXCLs, andCX3CLs),55–57 cytokines (GM-CSF, interleukins),58–63 surfaceexpressed co-stimulatory molecules,64–66 receptor ligands,67,68

and transcription factors.49,69–72 Molecular adjuvants that biastowards intracellular antigenic trafficking (i.e., stimulating cell-mediated immunity) include the use of IL-2, IL-10, IL-12, IL-15,IL-18, TGFb, and IFNg.62,63,73 Alternatively, use of IL-4, IL-5, andIL-15 can bias a humoral-based response.58,63

As opposed to the direct expression of a dedicated molecularadjuvant, an alternative route towards a desired immunologicalresponse involves the use of transcription factors to shi geneexpression patterns. These adjuvants operate by controlling (or

This journal is © The Royal Society of Chemistry 2014

inducing) the production of immunogenic cytokines and che-mokines. There has been signicant commercial developmentof transcription factor-based vectors including TBK1 (TANK-binding kinase 1; general immunopotentiator;71 InvivoGen),IRF1 (interferon regulatory factor 1; increases humoralresponses,72 limited cell-mediated response;69 InvivoGen), IRF3(increases cell-mediated responses;72 InvivoGen), and chimericIRF7/3 (increases both cell-mediated and humoral responses;70

InvivoGen).Of the previously described adjuvants, GM-CSF (granulocyte-

macrophage colony-stimulating factor) was one of the rst co-delivery cytokine plasmids supporting DNA-induced immunity.Accordingly, GM-CSF has become a common and widely studiedmolecular adjuvant that transitioned to human use.74 Unfortu-nately, human application was unable to replicate the samelevel of efficacy that was present during animal trials. Thiscytokine molecular adjuvant operates by recruiting, activating,and/or enhancing the response of professional phagocytic cells.Nonetheless, other recruitment-oriented molecular adjuvantsare being developed and can potentially coordinate the trig-gered movement and functionality of adaptive immunity-mediated effector cells.75

Alternatively, immunogenicity can be enhanced by thedelivery of surface-expressed co-stimulatory molecules, such asCD80 (B7-1) and CD86 (B7-2),76 that are required as a secondarysignal for the induction of T cells. These adjuvants havesuccessfully been used to potentiate cytotoxic lymphocyte (CTL)activity in cancer,77,78 HIV,79 and tuberculosis studies.80 Simi-larly, expression of CD40, a surface receptor on B cells anddendritic cells, has demonstrated improved humoral immunitywith a switch to a TH1 response.81–83 Emphasizing co-stimula-tory molecules, blockage of the receptors through expression ofinhibitors or siRNA can assist in the regulation of immuneresponses that are present during chronic viral infection andcancer.84,85 In summary, the diversity and specicity of molec-ular adjuvants holds enormous potential and will likelycontinue to receive signicant attention on the basis ofencouraging preliminary results reported in the clinic.

3.3 Immunization strategies

Signicant effort has been devoted to patient administration ofpDNA for increased localized concentrations (i.e., a ‘depot’effect) and intracellular ux (using methods such as electro-poration).35 These strategies have resulted in elevatedresponses; however, even when administered with adjuvantsand carriers, the immune response is typically weaker than thatproduced by comparable viral vectors or proteinaceous/toxoidantigens.35 These limitations have driven the development of aconsecutive immunization strategy, known as ‘prime-boost’,which combines the small but focused response of the DNAplatform with the expansive immune activation of liverecombinant vaccines.86 The process involves immunizationwith a genetic antigen with adjuvant or carrier, followed by asecondary administration of the same antigen using either theoriginal vector or a new biological/synthetic vector. Thiscombination was heralded as an innovative administration

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strategy that promotes both humoral and cell-mediated vaccine-specic immunity. The underpinning of this successful strategyis based upon the powerful secondary response that is elicitedfollowing primary memory formation mediated by DNAimmunization.

Initial studies were predicated on the utilization of the samevaccination vector during re-administration (i.e., homologousprime-boost) which, though successful in generating andboosting the humoral response to an antigen, failed to signi-cantly strengthen cell-mediated immunity.87 Presumably thisoccurs due to the rapid clearance of homologous vaccinationagents.86 In contrast, heterologous prime-boost strategiesutilizing a different vector for the secondary administration ofthe antigen resulted in signicantly higher cell-mediatedresponses.88 Furthermore, heterologous prime-boost studieshave demonstrated that responses are fundamentally differentthan those induced by consecutive administration of eithervaccine modality89 – eliciting responses 10 times higher thaneither platform in isolation.90

4. Vector delivery options

The goal of DNA vaccination is the successful transfection ofimmune effector cells leading to the production of preventativeimmunity. Initial efforts prioritized injection of naked pDNAboosted with adjuvants (alum, complete Freund's adjuvant[CFA], and MF59), physical methods (gene gun, electroporation,ultrasound, magnetofection), and viral-encapsulation as unas-sisted DNA vaccination resulted in limited success.91 Howeverrecently, emerging delivery vectors have demonstrated potentialto elicit robust and better directed immune responses. An idealvector-based strategy is one that is easily modied and manu-factured (at a large scale) and sturdy during long-term storage.

4.1 Viral vectors

Viral vectors are oen heralded for their high transfection effi-ciency both in vitro and in vivo.35 Efficient delivery is attributedto the natural evolution of viral vectors towards improving genetransfer into the host for self-replication purposes. An array ofviral vectors for which both replicating and non-replicatingforms exist including lenti, adeno, pox, herpes, alpha, andmeasles viruses.92 However, vectors are primarily designed to benon-integrative (eliminating lentivirus) and non-replicative.Other vectors based on vaccine candidates have been underu-tilized (poliovirus and yellow fever) despite the formation of life-long protective immunity.92 To date, viral vectors are notcommonly utilized outside of providing vaccine targets. Thislimited interest in vaccine-associated use arises from smallgenetic cargo capacity, genetic instability, difficulty inmanufacturing and production, and prior vector-associatedimmunity.35 Although viral vectors are oen associated withdetrimental levels of immunogenic activations leading tosignicant cytotoxicity, well-constructed vaccine strategies canreduce off-target effects by modifying tropism and tissue/cell-specic promoter activity.93

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4.2 Bacterial vectors

Unlike viral vectors, innate biological properties of bacteriareduce ubiquitous delivery and bias delivery towards immuno-logical presentation mechanisms. The concept of utilizingbacteria to deliver genetic material was rst reported in the early1980s94 and has continually improved to produce vectors pos-sessing native or heterologous properties aimed to overcomespecic barriers associated with in vivo administration andsubsequent immune responses. This classication of vectorfalls within the ‘nonviral’ category of delivery systems but stillretains benecial biological traits of enhanced delivery.95

Strains of bacteria currently used include Listeria mono-cytogenes,96–99 Escherichia coli,94,100–109 Bidobacterium lon-gum,110–112 Salmonella spp.,113–117 Vibrio cholera,118–121 Shigellaspp.,122–124 Mycobacteria bovis,125 Yersinia enterocolitica,126 andLactococcus lacti.127 Although only a small fraction of the high-lighted vectors have been used in the context of DNA vaccines,most have engineering tools to facilitate the developmenttowards a gene delivery context. However, the reason for theusage of particular bacterial species and strains is predicated onsafety and economical concerns. For example, strains such as L.monocytogenes possess all the innate properties (ideal size forAPC uptake, adjuvant-like physical composition, and an endo-somal escape mechanism) to become a powerful gene deliveryvector. However, the organism is severely hindered by slowgrowth rates, under-developed microbiology techniques(compared to model organisms like E. coli), and the potentialreversion of pathogenicity.

Interestingly, an advantage of bacterial vectors is thepotential to protect against pathogens that enter through themucosa. This is accomplished through the use of strainscapable of replicating at mucosal membranes.103,117,128 In thiscase, the generation of a humoral response does not requireinclusion of a genetic vaccine but rather can be elicited bybacterially-expressed antigens presented with the vector.117

Considering the duality of bacterial vectors (i.e., the ability todeliver genetic cargo while simultaneously stimulating humoralimmunity through the presence or production of immunogenicmolecules), both humoral and cell-mediated protection can beobtained through administration of one vector.

Despite efforts to attenuate bacterial vectors and docu-mented successful cytotoxicity panels,129 lingering safetyconcerns have prevented widespread use. Biosafety issues havebeen addressed through various means including antibioticpre-treatment109 and auxotroph-conferring genetic manipula-tions.100,130–132 As a result, it is likely that additional engineeringwill allow the generation of safe and effective vectors. Further-more, if a safe alternative is obtained, bacteria offer unequivocalpotential in the individual or simultaneous delivery of all anti-genic types (DNA, RNA, protein, toxoid, and cellular compo-nents) without the need for additional adjuvants.

4.3 Biomaterial vectors

A myriad of biocompatible materials have been developed thatare capable of electrostatically binding to or encapsulatinggenetic cargo. Upon packaging genetic cargo, these synthetic

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carriers enter host cells through general or specialized endo-cytosis mechanisms before facilitating cytoplasmic release.Vectors include cationic lipids and polymers, polysaccharides,polypeptides, and inorganic particles. Historically, researchershave utilized poly(lactide-co-glycolic acid) (PLGA) and variantsthereof for antigen delivery.11 Use of PLGA was driven by a safebiocompatibility prole and a wealth of information availabledescribing delivery and degradation properties. However, asmore information was gained with respect to structure–func-tion relationships, a number of other polymer systems havebeen proposed and tested. Of these, liposomes have been widelystudied, but stability of these preparations limits clinicalapplication.133 Alternatively, polymers such as pH-sensitivepoly(beta-amino ester),134,135 acid-labile polyketals,136 andcationic polylactides (CPLAs)137,138 have been developed tofacilitate cargo release in the acidic endosomal compartment.Other naturally derived biomaterials have been adapted forvaccination purposes including polysaccharides,139 in additionto polypeptides in the context of synthetic peptide epitope-based polymers140 and self-adjuvating polymer–peptide conju-gates.141 Alternatively, inorganic molecules used as vaccinationagents include aluminium phosphate and aluminiumhydroxide, which operate by facilitating a depot effect forantigen release and activation of APCs. Analogously, emulsions(either oil-in-water or water-in-oil) utilizing Freund's adjuvantand MF59 are also capable of providing depot-type antigenrelease and triggering APC induction.

Biomaterial particle-based vaccine carriers can be readilytuned to fully engage desired immune effector cells. Further-more, there are three important compositional characteristicsof biomaterials that inuence the formation of adaptiveimmune responses and can be applied liberally to variouscircumstances (cancer, vaccines, wound healing, and tissueengineering). These include particle size, antigen selection andvalency, and the inclusion/coating of targeting ligands and/orimmune-potentiators.

For particulate delivery systems, size determines bio-distribution and residence time,142,143 cellular uptake mecha-nisms,35,142 and passive targeting of cell types.144,145 In thecontext of vaccines, it is important for vectors to reach immu-nologically-important anatomical regions such as the lymphnodes in order to interact with APCs.143 This region can beaccessed through direct intranodal injections146 and traffickingthrough the lympathic vessels.143 Furthermore, 45 nm is theparticle cut-off size for effective drainage into the lymphnodes,145 which is greater than the 5.5 nm and less than the 1mm size limits for rapid renal elimination and reticuloendo-thelial system (RES) clearance, respectively.147,148 However,particles >100 nm are less effective at passive transport throughthe lymphatic system by interstitial ow.143 In other studies,while investigating size-dependent lymph node targeting, uo-rescently labelled particles less than 30 nmwere detected within2 hours; whereas, larger particles (100, 500, 1000 nm) weresparingly detected in lymph tissue and were observed to bepooled near the injection site.144,149,150

Aside from biodistribution, size largely determines themanner a particle will enter the cell. General endocytosis occurs

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on the order of 200 nm or less (smaller particles are taken upmore frequently), macropinocytosis on the order of 500 nm (upto 10 mm), and phagocytosis accommodating sizes up to 10mm.35 The last mechanism provides an alternative means ofdirecting a biomaterial carrier to the immune system. Namely,particles designed to only be engulfed by professional phago-cytes offer a targeted means of carriage and subsequentpresentation by tissue APCs present at the administrationsite.144,145 Thus, in the design of biomaterial vectors for vaccinepurposes, small particles appear to be more effective at target-ing and pooling at desired sites but larger particles offer theprospect of targeted APC delivery.

Considerations for antigen selection and valency are perhapsthe most critical parameters for biomaterials-driven vaccinestrategies. As before, the selection of antigen will largelydetermine the response elicited, but given the basis of geneticvaccines, most immune responses will be mixed (TH1, TH2, andTH17). However, using polymer chemistry tools, it is possible tostructurally gra and/or encapsulate different forms (pDNA,protein, subunit, toxoid) of the antigen of choice to synergisti-cally harness the duality of different vaccine classes.151 Analo-gously, high-density surface graing of antigenic material (notDNA) can result in increased crosslinking of antigen-specicTCRs and BCRs, which leads to lower thresholds for activa-tions.152,153 For example, Irvine and co-workers synthesizedlayered lipid particles composed of entrapped malarial antigensand multivalent surface coatings.154 This strategy resulted inincreased antibody production (over an order of magnitude) ascompared to non-surface-coated particles.

Similarly, the inclusion of targeting moieties or immune-potentiator molecules can improve and/or direct an immuneresponse. For example, delivery specicity to APCs can beimproved by inclusion of mannose, an antagonist ofCD206.155,156 Similarly, inclusion of different surface ligands,such as MHC-II ligand and CCR1/3/5 molecules, have demon-strated the ability to polarize immunological responses towardsthe development of tailor-made vaccines.157 In addition to thesummary provided above, comprehensive discussions ofbiomaterials in the context of gene delivery and vaccinationhave been covered in previously reviews.11,35

4.4 Biomaterial-interfaced vectors

4.4.1 Virus-mimicking strategies. Virus-like particles(VLPs) are self-assembled particles of viral-derived components(capsid and envelope proteins) that are promising alternativesto traditional vaccine strategies. Although closely mimickingthe natural structure of viruses, VLPs are unable to replicate orrevert to a prior virulent form (due to the lack of genomicDNA).158 In addition, VLPs are easily modied, produced, andscaled at a low cost. VLPs have the ability to deliver variouscargo including genetic material, peptides, proteins, smallmolecules, adjuvants, and other antigenic material.159 However,VLPs do not usually contain all the needed molecules to fullymediate an effective gene delivery response, and thus, requirethe inclusion of polymeric components to elevate gene delivery.VLPs oen contain the capsid that recognizes and binds viral-

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associated cellular receptors and a biological and/or syntheticendolysosomal escape mediator. Escape from the lysosome hasbeen accomplished through the addition of biomaterials toVLPs. Specically, viral particles have been complexed withcationic polymers and lipids to improve endolysosomal release(via proton sponge and lipid mixing escape mechanisms,respectively).160–162 Analogously, virosomes (reconstitutedvirion-like phospholipid bilayer spherical vesicles) contain allthe glycoproteins derived from viruses but are devoid of bothcapsid proteins and genetic material. They have gained atten-tion as potential gene delivery carriers due to their ease ofproduction and modication, coupled with their low cytotox-icity prole.163,164 However, their in vivo applications are limitedby the lingering risk of unwanted innate immunogenicity. Thus,numerous biomaterial-mediated modications, includingadditions of polyethylene glycol (PEG) and targeting mole-cules,165 have been conducted to overcome this obstacle andreduce off-target effects.

Alternatively, rather than simply modifying viral-like parti-cles with biomaterials, entirely biomaterial-based devices havebeen used to mimic biophysical properties (size, shape, andsurface antigens) of viruses. Of interest, self-assembled lipo-somes that mimic viral structures have been synthesizedthrough the assembly of lipids, transferrin, and DNA.166 Thisstrategy is marked by multicentre lamellar nanostructures witha transferrin coating which closely resembles the inuenza andherpes viruses.167 As of yet, this strategy has not been applied inthe context of genetic vaccination. Similarly, pH-sensitivenanogel systems that structurally and functionally resembletraits of viruses have also been developed.168 These gels mimic aviral structure through the use of a hydrophobic core and twolayers of hydrophilic shells. Additionally, serum albumin-linkedPEG was graed onto the surface to create a capsid-like struc-ture.168 Unlike traditional viral particles, these biomimeticsdemonstrated pH-sensitive swelling from physiological (pH 7.4)to the endosomal (pH 6.4) level, which in turn facilitatedendosomal escape and release of cargo. Nanogels have beensynthesized utilizing several natural and synthetic US Food andDrug Administration (FDA)-approved biopolymers including,poly(methyl methacrylate), poly(D,L-lactic acid), poly(glycolicacid), PLGA, poly(3-caprolactone), chitosan, poly-L-lysine,poly(g-glutamic acid), dextran, dextrin, mannan, pullulan,heparin, hyaluronic acid, and alginate.169

To date, most studies of biomaterial-interfaced virus-mimicking strategies have been performed in vitro, and their invivo efficacy has not been well-established. Combining theinnate properties of viral particles with the tool-set available forbiomaterial synthesis allows for limitless opportunities in thedesign and modication of vaccine strategies.

4.4.2 Microbial-like particles. Although recombinantbacteria have been actively investigated for gene delivery andvaccination, a novel approach has been demonstrated with theuse of bacteria-mediated delivery of nanoparticle systems,termed ‘microbots’.170 This study utilized an attenuated strainof L. monocytogenes and surface conjugated nanoparticles thatwere loaded with pDNA. Surface conjugation was mediatedthrough biotin–streptavidin interactions.170 Transfection with

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these hybrid vectors resulted in high levels of gene delivery andproduction of target proteins. Separately, our group has devel-oped a hybrid gene delivery vector containing bacterial andbiomaterial components. A bacterial core, E. coli, was surface-modied using a cationic polymer (poly(beta-amino ester)) inan effort to combine normally disparate vector-associatedproperties and tools sets to systematically overcome genedelivery barriers.171 This approach facilitated tailored APCcellular responses to levels signicantly exceeding individualvector components or commercially-available transfectionagents in vitro. In addition, in vivo ovalbumin gene deliverymouse models demonstrated that the hybrid device canmediate signicant antibody responses without the need ofadjuvants. Furthermore, the system is primed for an expansivecombinatorial effort to vary both the biomaterial and biologicalcomponents of the hybrid design. Here, the variety of cationicbiomaterials commonly used in gene delivery offer new capa-bilities in the context of hybrid vector construction. As oneexample, we have recently tested the use of CPLA as an alter-native biomaterial component (data submitted). Analogously, E.coli has been used in conjunction with commercial reagents(lipofectamine) to improve gene delivery.172

Outer membrane vesicles (OMVs) are naturally occurringproteoliposomes that bud from Gram-negative bacteria.173

These single lipid bilayer particles range in size from 50–250nm. Aer discovery of OMVs, it was quickly realized that in vivoadministration could prompt protective humoral and mucosalimmune responses without the need of adjuvants.173 Presum-ably this arises from the presence of lingering immunoactivevirulence factors on the vesicle surface.174 Using molecularbiology tools, targeting ligands and additional immune-poten-tiator molecules have been attached to the OMV surface toimprove resulting responses.174 Similar to synthetic vectors,OMVs are acellular, making them promising alternatives to liveor attenuated pathogen-based vaccines. Most notably, Neisseriameningitides-derived OMVs have been commercialized byNovartis for the synthesis of European-approved vaccine Bex-ero.175 Similarly, non-denatured cell envelopes from Gram-negative bacteria (bacterial ghosts [BGs]) have demonstratedthe ability to mediate gene delivery.167 BGs are producedthrough the heterologous expression of the bacteriophage-derived lysis gene E. These vectors contain all intrinsic surfacecomponents (polysaccharides, agella, and mbriae) but aresubstantially larger than OMVs (retaining size characteristics ofthe host bacterium). Additionally, as with OMVs, BGs retainintrinsic adjuvant properties derived from residual lipopoly-saccharides (LPSs). Although this set of vectors has documentedsuccess in vivo and possesses a robust tool-set for vector engi-neering, biomaterial-functionalization has yet to occur but is apromising potential future avenue.

5. Current and emerging trends

Genetic vaccination has the potential for combating and even-tually overcoming a variety of diseases that have overwhelmedcurrent standard-of-cares. Yet challenges remain, and despitedecades of research, the elicitation of a signicant DNA-

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mediated immune response in a safe and scalable manner isstill elusive. However, these barriers are beginning to fall. As theuse of biological and biomaterial vectors, in the wake ofadvancements in adaptive immunity understanding, evolvespast initial design strategies towards tailoring and functionali-zation that more effectively instigates and directs immuneresponses, DNA vaccination will continue to grow in terms ofimpact and potential.

Specically, tradition vector studies utilized simple antigenencapsulation (through innate, physical, or electrostaticcapture) and systemic administration methodologies. Use ofthese systems has resulted in some success, but due to systemiceffects and/or low efficacy, they do not transition well to non-model-based applications. Thus, current research is investi-gating the following topics: (1) targeted delivery; (2) cellularspecicity; (3) vector combination and interfacial design; (4)response-modulation; (5) designed antigen release and activa-tion kinetics. Each of these examples are classical researchareas in gene and drug delivery but have not been readilyapplied in the eld of immunology. Ultimately, investigatingthe underlying biological mechanisms of DNA vaccines arisingfrom chemical- and structure-properties of individual deliveryagents must be the focus of future studies. Doing so will facil-itate the transition from empirical experimentation to an era ofrational design.

Fig. 3 Global gene therapy clinical trial information. Breakdown of trials bclinical data (http://www.wiley.com/legacy/wileychi/genmed/clinical/).

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6. Clinical trials and commercialproducts

Although substantial progress has been made in the preclinicaldevelopment of gene-based vaccines, the ultimate goal is totranslate observed animal model efficacy to human studies.However, given the early stage of the eld of gene therapy, nonaked DNA vaccines have been used in a randomised clinicaltrial.176 As such, since genetic vaccine clinical data is limited,the remainder of the review will emphasize the development ofgeneral gene-based therapies that have advanced to clinicaltrials and corresponding commercial entities. Numerous trialsof various phases are being conducted using a myriad of strat-egies (Fig. 3). Despite their limitations, viral vectors still remainthe primary vector of choice. In addition, the overall number oftrials has grown over the years (Fig. 3C).

It is probable that the approval and subsequent commer-cialization of these gene-based trials remains several yearsaway. This is evident by the small number of approved gene-based products. These include Gendicine™ (SiBiono Gene TechCo.; rst globally approved clinical gene therapy), Oncorine™(Sunway Biotech Co. Ltd), Cerepro® (Ark Therapeutics Group;rst GMP certication in the EU; rst and only adenoviral vectorthat has completed a phase III clinical trial), and Glybera(UniQure). However, no gene-based therapies are approved bythe FDA, though three gene-based products are available for

y phase (A), vector (B), and year (C). Information gathered from pooled

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veterinary use.177–179 Interestingly, all of these products arevaccines – demonstrating the potential of genetic vaccination.We surmise that the insights provided by on-going andapproved gene-based therapies will help shape improvedstudies that will result in a ‘burst’ of successful trials (includingthose in vaccination) in the future.

7. Conclusions

Despite initial challenges, emerging technological advance-ments have generated renewed interest in the development ofrationally-designed genetic vaccination strategies. Specically,the efficacy of genetic vaccination strategies can be improved bysystematically engineering and optimizing the expressioncassette (plasmid), delivery vector, and immunization/admin-istration method. These new strategies coupled with the variousinherent advantages of DNA vaccines – their ease of design andmanufacturing, strong safety record, and stability – provide theframework for immunologically-effective and economically-palatable vaccine production schemes. However, delivery vectorselection will require a thorough understanding of the biologyof the vaccine target and the resultant disease pathology.Through the continual renement of DNA vaccine technology,the prospects for prophylactic treatment of human and animaldisease will help shape the future of vaccinology.

Acknowledgements

The authors recognize support from NIH awards AI088485(BAP) and DC013554 (APH) and a SUNY-Buffalo Schomburgfellowship (CHJ).

Notes and references

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