Vaccine 30 (2012) 4414 4418

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Vaccine

j ourna l ho me pag e: www.elsev ier .com

Review

RNA-based vaccines

Jeffrey B.Novartis Vaccin

a r t i c l

Article history:Received 3 FebReceived in reAccepted 18 AAvailable onlin

Keywords:Nucleic acid vaViral vector

Contents

1. Backg2. Nucleic acid vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4414

2.1. DNA vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44152.2. Viral vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44152.3. RNA vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4415

3. ProspRefer

1. Backgro

The dischave been ggies to idento the immcharacterizbeen spurrevaccines renew paradinology appvaccines towith the safadvancemeacid vaccine

Corresponbridge, MA 02

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0264-410X/$ http://dx.doi.oects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4417ences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4417

und

overy and development of new and improved vaccinesreatly facilitated by the application of new technolo-tify protective antigens, to optimally present antigensune system, and to manufacture vaccines using highlyed, synthetic methods of production. This progress hasd by a need to move beyond empirical approaches tosearch and development, and has ushered in severalgms including reverse, structural and synthetic vacci-roaches, respectively [1]. The use of nucleic acid-based

combine the benets of in situ expression of antigens,ety of inactivated and subunit vaccines, has been a keynt. Upon their discovery more than 20 years ago, nucleics promised to be a safe and effective means to mimic

ding author at: Novartis Vaccines, 350 Massachusetts Ave., Cam-139, United States.ress: jeffrey.ulmer@novartis.com (J.B. Ulmer).

immunization with a live organism vaccine, particularly for induc-tion of T cell immunity [2]. In addition, the manufacture of nucleicacid-based vaccines offered the potential to be relatively simple,inexpensive and generic. Since then, clinical trials have amplydemonstrated the safety and tolerability of nucleic acid vaccines[3], and robust manufacturing processes have been developed [4].However, potency in humans has been disappointing, which hasled to extensive activity to identify enabling technologies. The mainareas for improvement have been directed toward the nucleic acidvector, targeting the innate immune system to enhance immuno-genicity, and delivery systems to overcome the barriers to efcienttransfection of host cells in vivo. Signicant progress has been madeon all these fronts. This review paper will focus on the use of analternative nucleic acid vector, namely RNA, as the basis of a newgeneration of vaccines.

2. Nucleic acid vaccines

By denition, nucleic acid vaccines are based on DNA or RNAencoding the antigen(s) of interest. In their simplest form, they can

see front matter 2012 Elsevier Ltd. All rights reserved.rg/10.1016/j.vaccine.2012.04.060 Ulmer , Peter W. Mason, Andrew Geall, Christian W. Mandles, Cambridge, MA 02139, United States

e i n f o

ruary 2012vised form 10 April 2012pril 2012e 28 April 2012

ccine

a b s t r a c t

Nucleic acid vaccines consisting of plasmid DNA, viral vectors or RNA may change the way the nextgeneration vaccines are produced, as they have the potential to combine the benets of live-attenuatedvaccines, without the complications often associated with live-attenuated vaccine safety and manufac-turing. Over the past two decades, numerous clinical trials of plasmid DNA and viral vector-based vaccineshave shown them to be safe, well-tolerated and immunogenic. Yet, sufcient potency for general utilityin humans has remained elusive for DNA vaccines and the feasibility of repeated use of viral vectorshas been compromised by anti-vector immunity. RNA vaccines, including those based on mRNA andself-amplifying RNA replicons, have the potential to overcome the limitations of plasmid DNA and viralvectors. Possible drawbacks related to the cost and feasibility of manufacturing RNA vaccines are beingaddressed, increasing the likelihood that RNA-based vaccines will be commercially viable. Proof of con-cept for RNA vaccines has been demonstrated in humans and the prospects for further development intocommercial products are very encouraging.

2012 Elsevier Ltd. All rights reserved.

round. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4414/ locate /vacc ine

J.B. Ulmer et al. / Vaccine 30 (2012) 4414 4418 4415

consist of highly puried nucleic acids formulated in a buffer. Mostoften, however, specialized delivery systems are utilized to increasevaccine potency. Means to facilitate nucleic acid delivery involve(1) viral particles to take advantage of the efciency of viral entrymechanismlipids, polymthe use of vas electropoexperiencesvaccines an

2.1. DNA va

DNA vacels of infecsuccess at egens, whichpolyproteinbeen furthetion of plasa West Nileetic necrosifor dogs [7]apy for pigsantibody anindicationsthese immuconventionisms, or subfor this shodue, at leasand inadequcome theseand the moDNA delivesystem via immunolog[10]. Combinduction oliminary re

2.2. Viral ve

In situ etively achieviruses, engVectors basextensivelyated at earlivectors havclinical trialope proteienvelope pphase III efover DNA vis introducthe viral pfor inductiomagnitude vector vaccare related tor itself. Fifrom wild-tpotential fouated live vof these ve

issue. Second, because viral vectors contain, and in some casesexpress, viral antigens in addition to the target antigen of inter-est, such vectors are usually quite immunogenic (i.e., elicit immuneresponses against the vectors themselves). Pre-existing anti-vector

ity (r imility d, hen of ed ushumalogouw effn. Why coml sol

A va

of ofhen roduf immt comon ofose ting

twos wof el7]). 20] ns [2sponfunct chaical rgennce an-spe

of R], intlenic

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vitfor reemonn-intes there nto tttle ese ofaccins in raneticultes, ationtoplae expr of pince d fontiges, (2) non-viral formulations of DNA or RNA involvingers, emulsions or other synthetic approaches to avoid

iral vectors, and (3) physical delivery technologies, suchration in situ. The majority of the preclinical and clinical

with nucleic acid vaccines so far have been with DNAd DNA-based viral vectors.

ccines

cines have been widely evaluated in many animal mod-tious and non-infectious diseases with generally goodliciting potent immune responses against encoded anti-

have ranged from discrete T or B cell epitopes to large complexes. The utility of DNA vaccines in animals hasr documented by the development and commercializa-mid DNA-based animal health products. These include

virus vaccine for horses [5], an infectious hematopoi-s virus vaccine for sh [6], a melanoma cancer vaccine, and a growth hormone releasing hormone gene ther-

[8]. In humans, proof of concept for induction of bothd T cell responses has been demonstrated for various

in multiple clinical trials. However, the magnitude ofne responses has been lower than those observed foral vaccines consisting of live or inactivated whole organ-unit proteins formulated with adjuvants. The reasonsrtcoming of DNA vaccines are not clear, but are likelyt in part, to inefcient delivery of DNA into human cellsate stimulation of the human immune system. To over-

limitations, various technologies have been evaluatedst promising current approaches involve facilitation ofry by electroporation [9] and stimulation of the immunethe use of genetic adjuvants (i.e., in situ expression ofically active molecules encoded by the DNA vaccine)inations of these approaches have resulted in potentf immunity in non-human primates [11,12] and pre-sults of human clinical trials are encouraging [3,9].

ctors

xpression of antigens in a vaccinated host can be effec-ved through the use of recombinant vectors, often DNAineered to be safe and to encode the gene(s) of interest.ed on adenoviruses and poxviruses have been studied, although several other viral vectors are being evalu-er stages of development. Both adenovirus and poxviruse demonstrated safety and immunogenicity in humanls [13]. Notably, a poxvirus vector encoding HIV enve-n used in a prime-boost regimen with recombinantrotein plus adjuvant elicited modest protection in acacy trial [14]. One clear advantage of viral vectorsaccines is the efciency with which the DNA payloaded into host cells, due to the natural invasiveness ofarticle. Hence, the amount of plasmid DNA requiredn of immune responses is typically many orders ofgreater than the amount of DNA contained in a viraline. Two potential limitations of viral vectors, though,to safety and the inherent immunogenicity of the vec-rst, because viral vectors are usually originally derivedype pathogenic viruses, there is at least a theoreticalr reversion to a virulent state, just as there is for atten-irus-based vaccines. However, extensive safety testingctors has demonstrated that this is likely not a major

immuncines othe abgen anantigeincluding in heteroto alloantigeily, theoptima

2.3. RN

Proago, wlocal ption oa direcinjectiin sucrsuggesby thelicationutility see [1[15,18allergecell remore, againspreclining alleresistaantigetration[15,19intraspskin byof worcells inagent (have dand noattribuFirst, tgrate ibeen lition, uDNA vnucleumembbe parmyocypublicthe cyof gennumbetrast, sthe neeics of aeither due to prior infection with wild-type virus, vac-munization with the vector) has been shown to bluntof the vector to launch production of the target anti-nce, limits induction of immune responses against theinterest. Strategies to circumvent this limitation havee of certain adenovirus strains not commonly circulat-ns, to allow initial take of the viral vector vaccine, ands prime-boost approaches involving different vectors,ective boosting of immune responses against the targetile these approaches can be effective, at least temporar-plicate the vaccination regimen and do not provide an

ution.

ccines

concept for RNA vaccines was provided two decadesintramuscular injection of mRNA in mice resulted inction of an encoded reporter protein [15] and induc-une responses against an encoded antigen [16]. Inparison with a corresponding plasmid DNA vaccine,

similar doses of mRNA (on a mass basis) formulatedresulted in similar levels of reporter gene expression,equivalent efciencies of cellular transfection in vivo

types of nucleic acid vaccines [15]. These initial pub-ere followed by many more demonstrating the generaliciting immune responses by RNA vaccines (for review,The variety of gene targets included reporter genesviral antigens [16,21], tumor antigens [2226], and7,28]. In these animal models, both antibody and Tses, including CD4+ and CD8+, were elicited. Further-tional immunity, as measured by protective efcacyllenge with live pathogens or tumors, was achieved. Inmodels of allergy, low doses of an RNA vaccine encod-s induce a Th1-biased immune response that providedgainst subsequent allergic sensitization. Induction ofcic immune responses can be achieved by adminis-NA vaccines via various routes, including intramuscularradermal [22], subcutaneous [16], intravenous [16],

[21], and intranodal [24], as well as delivery into the gene gun [29,30]. In addition, a considerable amounts been done using mRNA vaccines to pulse dendriticro, which are then administered as the immunizingview, see [31]). Hence, like DNA vaccines, RNA vaccinesstrated versatility in many animal models of infectious

fectious diseases. However, RNA vaccines have severalhat provide potential advantages over DNA vaccines.is a nite chance that plasmid DNA vaccines can inte-he genome of the immunized host. Although there hasvidence so far that integration occurs after DNA vaccina-

RNA would eliminate this as an issue. Second, plasmides must be delivered into and transcribed within theorder to transfect a cell, i.e. they must traverse two

barriers (plasma and nuclear membranes). This couldarly problematic in non-dividing cells, such as maturewhere the nuclear membrane remains intact. Severals have demonstrated that microinjection of pDNA intosm of non-dividing cells resulted in very low levelsression, but direct intra-nuclear injection of the sameDNA copies led to efcient transfection [3234]. In con-RNA vaccines are translated directly in the cytoplasm,r delivery into the nucleus is obviated. Finally, the kinet-n expression after RNA administration appears to peak

4416 J.B. Ulmer et al. / Vaccine 30 (2012) 4414 4418

Capsid, E2/E1 gl ycopro teins Anm7G

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tic illustration of an RNA replicon vaccine. Example of an RNA replicon vector dere as nonstructural proteins) that drive their amplication within the cytoplasm of

vectors, antigen genes are most commonly inserted in place of the capsid and glyclies its full-length genome when it is introduced into the cytoplasm, and then folloarget antigen.

apidly, in contrast to DNA administration where antigencan persist for many weeks [35]. Hence, RNA vaccinationics antigen expression during an acute infection, whichre conducive to induction of antigen-specic immune

hanisms of action for RNA vaccines have not beenated, but likely involve some of the same processesDNA vaccines for the expression and presentation oftigens leading to induction of immune responses. Aftere RNA is exposed to RNases in the tissue [36], whiche the vaccine and limit uptake of functional RNA byition, the 2-hydroyxl on the ribose sugar prevents theting a stable double -helix, due to steric hindrance,the macromolecule more prone to hydrolysis. Never-ious cell types are capable of internalizing RNA by anrable and specic process leading to local expression[35]. Uptake is mediated by membrane domains rich

and lipid rafts, and involves scavenger receptors [37].alization, a portion of the RNA accumulates in the cyto-re it is translated into protein. This in situ productionprovides a means to mimic pathogen infections andof tumor antigens leading to efcient presentation of

major histocompatibility complex (MHC) class I and IId induction of T cell responses in a manner analogousided by DNA vaccines and viral vectors. Alternatively,es can be constructed for the efcient production andr cell-surface expression) of extracellular antigens to

cell responses and antigen-specic antibody produc-fectiveness of RNA vaccines may also be related to theA is known to be a potent stimulator of innate immu-, mRNA has been shown to activate dendritic cells andin a MyD88-dependent fashion involving signaling viaceptors (TLR) [38,39]. In vivo, it was recently demon-

an mRNA vaccine caused the upregulation of variousved in chemotaxis and cell activation [40] as well asf TLR7-dependent CD4+ and CD8+ T cell responses, andimmunity [41]. Hence, the functionality of RNA vaccinesleast two components: (1) local expression of antigen

presentation by MHC molecules and (2) engagement ofgnition receptors to stimulate innate immunity leading

As an adirectlalthouof TLR coadmor RNAbeen tcationsregionsee [44expres

Thewas inity of develoformulthese pis immcating not cociencyuse of purposmid DNover thmerciaand yietranscrat reasRNA vvaccinventioThese the GMhuman

Whvaccinalso betural pfor a vaion of antigen-specic immune responses. the above-referenced studies have used naked mRNAine (i.e., simply formulated in a buffer). While thisas been shown to elicit immune responses, the pres-radative enzymes in tissues likely limits the amountt is available for internalization by cells in vivo. As avercome this inherent drawback of using naked RNA,cused on delivery systems, adjuvants, and engineering

molecule. First, to protect RNA from degradation andlular uptake, encapsulation in liposomes [16,18,26] andon with cationic polymers [38,41] have proven effective.

Sindbis viru[51,52], Kunvaccines hato launch a potency of of RNA vaccdoses [51]. viral-particogous antigare producthat permitAntigen An

nomic promoter

om a positive-strand alphavirus genome. All replicons encode genesells. For use as vaccine vectors, replicons also encode antigen genes.ein genes, which are not needed for genome replication. In this way,

genome amplication it initiates production of a sub-genomic mRNA

ative delivery system, the gene gun has been used toroduce mRNA into the cytoplasm of cells [29]. Second,A vaccines have a built-in adjuvant effect in the formgement, mRNA vaccine potency has been enhanced byration of recombinant GM-CSF [19] or Flt-3 ligand [42],oding GM-CSF [43]. Finally, several approaches have

to improve the RNA molecule itself. Various modi-e been made to the 5 cap structure, the untranslatedd codon usage in the translated region (for review,hich have resulted in increased mRNA stability and

ibility of using RNA as the basis for a nucleic acid vacciney regarded as questionable, due the inherent instabil-A in the presence of tissue uids, the uncertainty of

reasonable manufacturing processes yielding a stable, and the anticipated high cost of the product. Each oftial limitations is being addressed. Even naked mRNAenic in animals [1921,25,45] and humans [46], indi-

RNA degradation in tissues after administration doestely abrogate vaccine effectiveness. However, the ef-A delivery should be increased markedly through theling synthetic and viral delivery systems. For researchn vitro transcribed mRNA can be obtained from plas-ntaining a bacteriophage promoter (T7, SP6 or T3) andst 10 years many technical renements to the com-s have resulted in dramatic improvements in quality1,47]. More recently, RNA manufacturing by enzymaticn of appropriate DNA templates now seems attainablele cost and large scale. Long-term storage of lyophilizedes have previously been studied and RNAse-free RNAere demonstrated to be no less stable than other con-accines that require a cold chain to retain efcacy [48].ncements have enabled the development of process foroduction of mRNA vaccines in quantities sufcient forical trials (for review, see [17]).ost of the published work has utilized mRNA as theeral publications have shown that RNA vaccines canived from sub-genomic replicons that lack viral struc-ns. Replicon RNA-based vaccines have been generated

of RNA viruses including, Semliki Forest virus [21,25],

s [20], poliovirus [49,50], tick-borne encephalitis virusjin virus [53], and bovine viral diarrhea [54]. RNA-basedve also been described in which the RNA vaccine is usedlive-attenuated virus infection. In this case, the inherentthe encoded live viral vaccine has permitted this typeine to elicit protective immunity at very low (ng) RNAMore commonly, experimental RNA-based vaccines arele delivered products engineered to express a heterol-en in place of the viral structural genes. These vaccinesed under special conditions (e.g., packaging cell lines)

production of single-round infectious particles carrying

J.B. Ulmer et al. / Vaccine 30 (2012) 4414 4418 4417

Table 1Superior attributes of RNA vaccines.

Parameter Vaccine type

Live Subunit Viral vector DNA mRNA Replicon RNA

Synthetic Generic manSafety Antibody indCTL inductioIn vivo exprControl of exAbsence of eIn vivo self aPotency in h

RNAs encodhigh levels oa live, sprRNA virusegous gene efor vaccine RNA vaccinThe RNA amcopies of anates, which[61]. Thus, oto be more direct compsignicantlafter replico

These repackaged in[52,62,63] Vefciency, amanufacturhas the potitated delivthose evaluwithout thetors.

3. Prospec

RNA vacpotential toviral delive(see Table tive approatheir advancines have bactive immpreclinical p[31]). In themelanoma tegy [46]. Santigens, sumetastatic In these exantigen-spedemonstrathumans. Clivaccines paof repliconsteins was shCMV seronpolyfunctio

lizinse reer typpronal, ely rn RNlogieated accin

vace.

nces

puolitury ser JBlogoutein. Sraro Blicatio1;53(a AR, thodsis BSe virullenge

be u1;75(ver KAN) virka salsenbaety anadjuncal exci1;72(ghia-rowthure Bdesai

succei F, W-DNA6;28(isle SEg of ufacturing

+/ + +/ uction + + +n + + ession + + pression + ukaryotic contaminants +/ mplication + + umans + + +/

ing the vaccine antigens [5559]. In this way, transient,f antigen production can be achieved without the use ofeading viral infection. Replicons derived from differents differ with regard to levels and duration of heterolo-xpression allowing the generation of a versatile toolboxor gene therapy applications [60]. An illustration of ane based on an alphavirus replicon is depicted in Fig. 1.plication process in the cytoplasm produces multipletigen-encoding mRNA and creates dsRNA intermedi-

are known to be potent stimulators of innate immunityn a mass basis, replicon RNA vaccines have the potentialeffective than corresponding mRNA vaccines. Indeed, aarison of the two types of RNA vaccines demonstratedy higher and more persistent expression levels in vivon RNA administration [20].plicon vaccines have been administered as naked RNA

viral particles, or delivered by electroporation in situiral particle delivery of replicons has the advantage ofs previously described for viral vectors, but complicatesing, introduces theoretical safety considerations, andential limitation of anti-vector immunity. Hence, facil-ery of RNA replicons using synthetic systems, such asated for mRNA or DNA vaccines may increase potency

added complications commonly seen with viral vec-

ts

cines, particularly self-amplifying replicons, have the capture the advantages of both DNA vaccines and

ry while overcoming the drawbacks of each technology1). The prospect of RNA vaccines being a more effec-ch than other types of nucleic acid vaccines has led tocement into human clinical trials. So far, mRNA vac-een administered to cancer patients in several trials asunotherapeutic immunization protocols, supported byroof of concept in animal tumor models (for review, see

rst trial, mRNA encoding genes cloned from metastaticumors were used as an autologous immunization strat-ubsequent trials used combinations of known tumor

neutrain thefor othcine afunctiowill likreplicotechnoattenuDNA vSuch avaccin

Refere

[1] Rapcen

[2] Ulmeropro

[3] Ferapp201

[4] LarMe

[5] DavNilchacan200

[6] Gar(IHner

[7] GroSafas gic201

[8] Draof gNat

[9] Sarfor

[10] LorHIV200

[11] Belminch as MUC1, CEA, telomerase, MAGE-1, tyrosinase, inmelanoma [64] and renal cell carcinoma [65] patients.ploratory clinical trials, the mRNA vaccines elicitedcic immune responses (both antibodies and T cells),ing proof of concept that mRNA vaccines are active innical trials have also been performed with RNA repliconckaged in viral particles. A bivalent vaccine consisting

encoding cytomegalovirus (CMV) gB and pp65/IE1 pro-own to be well tolerated and immunogenic in healthyegative volunteers [66]. All 40 individuals generatednal CD4+ and CD8+ T cell responses, as well as virus

PBMC of2011;6(6

[12] Hirao LA,lation thragainst S

[13] Rollier CSas vaccin2011;23(

[14] Vaccari Ma step t2010;9(9

[15] Wolff JA,gene tra14658.+/ + ++ + ++ + ++/ + ++ + ++ + ++ + ++ + + ++/ +/ TBD

g antibodies. However, the magnitude of the responsescent trials was similar to those previously observedpes of nucleic acid vaccines. Therefore, the RNA vac-ach holds promise as an effective means of elicitingprotective immune responses in humans, but successequire enabling delivery technologies. Next generationA vaccines will be formulated with synthetic deliverys and will aspire to combine the effectiveness of livevaccines, an equal or better safety prole than plasmides, and completely synthetic methods of manufacture.cine would possess the desired attributes of an ideal

R, Mandl CW, Black S, De Gregorio E. Vaccines for the twenty-rstociety. Nature Reviews Immunology 2011;11(12):86572., Donnelly JJ, Parker SE, Rhodes GH, Felgner PL, Dwarki VJ, et al. Het-s protection against inuenza by injection of DNA encoding a viralcience 1993;259(5102):17459., Morrow MP, Hutnick NA, Shin TH, Lucke CE, Weiner DB. Clinicalns of DNA vaccines: current progress. Clinical Infectious Diseases3):296302.Ramirez OT. Plasmid DNA production for therapeutic applications.

in Molecular Biology 2012;824:271303., Chang GJ, Cropp B, Roehrig JT, Martin DA, Mitchell CJ, et al. Wests recombinant DNA vaccine protects mouse and horse from virus

and expresses in vitro a noninfectious recombinant antigen thatsed in enzyme-linked immunosorbent assays. Journal of Virology9):40407., LaPatra SE, Kurath G. Efcacy of an infectious hematopoietic necrosis

us DNA vaccine in Chinook Oncorhynchus tshawytscha and sockeye O.mon. Diseases of Aquatic Organisms 2005;64(1):1322.ugh DA, Leard AT, Bergman PJ, Klein MK, Meleo K, Susaneck S, et al.d efcacy of a xenogeneic DNA vaccine encoding for human tyrosinasetive treatment for oral malignant melanoma in dogs following sur-sion of the primary tumor. American Journal of Veterinary Research12):16318.Akli R, Li X, Schwartz RJ. Enhanced growth by ectopic expression

hormone releasing hormone using an injectable myogenic vector.iotechnology 1997;15(12):12859.NY, Weiner DB. Electroporation delivery of DNA vaccines: prospectsss. Current Opinion in Immunology 2011;23(3):4219.einer DB, Calarota SA, Kelly LM, Lisziewicz J. Cytokine-adjuvanted

vaccination strategies. Springer Seminars in Immunopathology3):2318., Yin J, Shedlock DJ, Dai A, Yan J, Hirao L, et al. Long-term program-antigen-specic immunity from gene expression signatures in the rhesus macaques immunized with an SIV DNA vaccine. PLoS One):e19681.

Hokey DA, Morrow MP, Jure-Kunkel MN, Weiner DB. Immune modu-ough 4-1BB enhances SIV vaccine protection in non-human primatesIVmac251 challenge. PLoS One 2011;6(9):e24250., Reyes-Sandoval A, Cottingham MG, Ewer K, Hill AV. Viral vectorse platforms: deployment in sight. Current Opinion in Immunology3):37782., Poonam P, Franchini G. Phase III HIV vaccine trial in Thailand:

oward a protective vaccine for HIV. Expert Review of Vaccines):9971005.

Malone RW, Williams P, Chong W, Acsadi G, Jani A, et al. Directnsfer into mouse muscle in vivo. Science 1990;247(4949 Pt 1):

4418 J.B. Ulmer et al. / Vaccine 30 (2012) 4414 4418

[16] Martinon F, Krishnan S, Lenzen G, Magne R, Gomard E, Guillet JG, et al. Inductionof virus-specic cytotoxic T lymphocytes in vivo by liposome-entrapped mRNA.European Journal of Immunology 1993;23(7):171922.

[17] Pascolo S. Messenger RNA-based vaccines. Expert Opinion on Biological Ther-apy 2004;4(8):128594.

[18] Hoerr I, Obst R, Rammensee HG, Jung G. In vivo application of RNA leads toinduction of specic cytotoxic T lymphocytes and antibodies. European Journalof Immunology 2000;30(1):17.

[19] Carralot Jimmunitycines. Cel

[20] Johanninet al. A Shigh leve1995;23(

[21] Zhou X, BSemliki Fo

[22] Gransteindermal ceRNA. Jour

[23] Conry RMterization1995;55(

[24] Kreiter S,vaccinatitherapeu

[25] Ying H, ZCancer t1999;5(7

[26] Zhou WZmelanom100 mRN

[27] Roesler ES, et al. Ia panel 2009;124

[28] Weiss R, tion as a sof Vaccin

[29] Qiu P, Ziein efcie1996;3(3

[30] Aberle JHresponseborne enc

[31] Kreiter S,senger RN2011;23(

[32] Mirzayanforeign gemicroinje

[33] Thorburnneedle m

[34] Zabner J,moleculaChemistr

[35] Probst J, taneous cspecic, s

[36] Probst J, terizationTherapy 2

[37] Lorenz Cet al. Prmediated2011;8(4

[38] Scheel B,receptor-condense

[39] Kariko Kendogeno2004;279

[40] Fotin Mleet al. Highform for cJournal o

[41] Fotin-Mleet al. Mesdependenof Immun

[42] Kreiter S,and enhaResearch

[43] Hess PR, Boczkowski D, Nair SK, Snyder D, Gilboa E. Vaccination with mRNAsencoding tumor-associated antigens and granulocyte-macrophage colony-stimulating factor efciently primes CTL responses, but is insufcient toovercome tolerance to a model tumor/self antigen. Cancer Immunology andImmunotherapy 2006;55(6):67283.

[44] Pascolo S. Vaccination with messenger RNA (mRNA). Handbook of Experimen-tal Pharmacology 2008;183:22135.

[45] Conry RM, LoBuglio AF, Loechel F, Moore SE, Sumerel LA, Barlow DL, et al. A car-oembre Theide B,t phasmmunamo

NA ge9;71(es KL,cine sson C

the mon of 61.nuzzi ation omesuenza747ndl CWthesizure Mer RM

avis of t4;101vey Ts repnt. Jouich B, 2 antiinst tuO, Zhtor de

exprhoro Nis viruuenzadman single8;72:ri S, G

partises is3;77(ey IJ,al. Huunodine e18.rke Rctiouct comenerang Y,

dend1;243gott JMNA stdicineanssoation 2;7(1ide B,l. Dircinati9;32(ig SMadermune rapy 2nsteindomie for 9;28(P, Probst J, Hoerr I, Scheel B, Teufel R, Jung G, et al. Polarization of induced by direct injection of naked sequence-stabilized mRNA vac-lular and Molecular Life Sciences 2004;61(18):241824.g FW, Conry RM, LoBuglio AF, Wright M, Sumerel LA, Pike MJ,indbis virus mRNA polynucleotide vector achieves prolonged andl heterologous gene expression in vivo. Nucleic Acids Research9):1495501.erglund P, Rhodes G, Parker SE, Jondal M, Liljestrom P. Self-replicatingrest virus RNA as recombinant vaccine. Vaccine 1994;12(16):15104.

RD, Ding W, Ozawa H. Induction of anti-tumor immunity with epi-lls pulsed with tumor-derived RNA or intradermal administration ofnal of Investigative Dermatology 2000;114(4):6326., LoBuglio AF, Wright M, Sumerel L, Pike MJ, Johanning F, et al. Charac-

of a messenger RNA polynucleotide vaccine vector. Cancer Research7):1397400.

Selmi A, Diken M, Koslowski M, Britten CM, Huber C, et al. Intranodalon with naked antigen-encoding RNA elicits potent prophylactic andtic antitumoral immunity. Cancer Research 2010;70(22):903140.aks TZ, Wang RF, Irvine KR, Kammula US, Marincola FM, et al.herapy using a self-replicating RNA vaccine. Nature Medicine):8237., Hoon DS, Huang SK, Fujii S, Hashimoto K, Morishita R, et al. RNAa vaccine: induction of antitumor immunity by human glycoproteinA immunization. Human Gene Therapy 1999;10(16):271924., Weiss R, Weinberger EE, Fruehwirth A, Stoecklinger A, Mostbockmmunize and disappear-safety-optimized mRNA vaccination withof 29 allergens. The Journal of Allergy and Clinical Immunology(5), 1070-7 e111.Scheiblhofer S, Roesler E, Weinberger E, Thalhamer J. mRNA vaccina-afe approach for specic protection from type I allergy. Expert Reviewes 2012;11(1):5567.gelhoffer P, Sun J, Yang NS. Gene gun delivery of mRNA in situ resultsnt transgene expression and genetic immunization. Gene Therapy):2628., Aberle SW, Koer RM, Mandl CW. Humoral and cellular immune

to RNA immunization with avivirus replicons derived from tick-ephalitis virus. Journal of Virology 2005;79(24):1510713.

Diken M, Selmi A, Tureci O, Sahin U. Tumor vaccination using mes-A: prospects of a future therapy. Current Opinion in Immunology

3):399406.s R, Aubin RA, Paterson MC. Differential expression and stability ofnes introduced into human broblasts by nuclear versus cytoplasmicction. Mutation Research 1992;281(2):11522.

AM, Alberts AS. Efcient expression of miniprep plasmid DNA aftericro-injection into somatic cells. Biotechniques 1993;14(3):3568.

Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ. Cellular andr barriers to gene transfer by a cationic lipid. Journal of Biologicaly 1995;270(32):189979007.Weide B, Scheel B, Pichler BJ, Hoerr I, Rammensee HG, et al. Spon-ellular uptake of exogenous messenger RNA in vivo is nucleic acid-aturable and ion dependent. Gene Therapy 2007;14(15):117580.Brechtel S, Scheel B, Hoerr I, Jung G, Rammensee HG, et al. Charac-

of the ribonuclease activity on the skin surface. Genetics Vaccines006;4:4., Fotin-Mleczek M, Roth G, Becker C, Dam TC, Verdurmen WP,otein expression from exogenous mRNA: uptake by receptor-

endocytosis and trafcking via the lysosomal pathway. RNA Biology):62736.

Teufel R, Probst J, Carralot JP, Geginat J, Radsak M, et al. Toll-likedependent activation of several human blood cell types by protamine-d mRNA. European Journal of Immunology 2005;35(5):155766., Ni H, Capodici J, Lamphier M, Weissman D. mRNA is anus ligand for Toll-like receptor 3. Journal of Biological Chemistry(13):1254250.czek M, Zanzinger K, Heidenreich R, Lorenz C, Thess A, Duchardt KM,ly potent mRNA based cancer vaccines represent an attractive plat-ombination therapies supporting an improved therapeutic effect. Thef Gene Medicine; in press.czek M, Duchardt KM, Lorenz C, Pfeiffer R, Ojkic-Zrna S, Probst J,senger RNA-based vaccines with dual activity induce balanced TLR-7t adaptive immune responses and provide antitumor activity. Journalotherapy 2011;34(1):115.

Diken M, Selmi A, Diekmann J, Attig S, Husemann Y, et al. FLT3 lig-nces the cancer therapeutic potency of naked RNA vaccines. Cancer

2011;71(19):613242.

cinGen

[46] Wersof I

[47] YammR200

[48] Jonvac

[49] Jackin lati45

[50] Vignizgenin173

[51] MasynNat

[52] Koliveing200

[53] Harvirume

[54] EdlHeraga

[55] Li Hvecand

[56] Kalatitin

[57] Wion 200

[58] Perconviru200

[59] Calet immequ303

[60] Gehinfedireof G

[61] Watoid201

[62] Pigof RMe

[63] Johpor201

[64] Weet avac200

[65] RittIntrimmThe

[66] BerRancin200yonic antigen polynucleotide vaccine for human clinical use. Cancerrapy 1995;2(1):338.

Carralot JP, Reese A, Scheel B, Eigentler TK, Hoerr I, et al. Results of thee I/II clinical vaccination trial with direct injection of mRNA. Journalotherapy 2008;31(2):1808.to A, Kormann M, Rosenecker J, Rudolph C. Current prospects forne delivery. European Journal of Pharmaceutics and Biopharmaceutics3):4849.

Drane D, Gowans EJ. Long-term storage of DNA-free RNA for use intudies. Biotechniques 2007;43(5):67581.A, Messinger J, Palmer MT, Peduzzi JD, Morrow CD. Gene expressionuscle and central nervous system following intramuscular inocu-encapsidated or naked poliovirus replicons. Virology 2003;314(1):

M, Gerbaud S, van der Werf S, Escriou N. Naked RNA immu-with replicons derived from poliovirus and Semliki Forest virus

for the generation of a cytotoxic T cell response against the A virus nucleoprotein. Journal of General Virology 2001;82(Pt 7):., Aberle JH, Aberle SW, Holzmann H, Allison SL, Heinz FX. In vitro-

ed infectious RNA as an attenuated live vaccine in a avivirus model.edicine 1998;4(12):143840., Aberle JH, Aberle SW, Allison SL, Heinz FX, Mandl CW. Mimickingvirus immunization with a noninfectious RNA vaccine. Proceed-he National Academy of Sciences of the United States of America(7):19516.J, Anraku I, Linedale R, Harrich D, Mackenzie J, Suhrbier A, et al. Kunjinlicon vectors for human immunodeciency virus vaccine develop-rnal of Virology 2003;77(14):7796803.Hogdal LJ, Rehermann B, Behrens SE. Dendritic cells transfected withgen-encoding RNA replicons cross-prime CD8 T cells and protect micemor challenge. Vaccine 2010;28(49):776473.u YF, Asakawa M, Kuma H, Hirata T, Ueda Y, et al. A cytoplasmic RNArived from nontransmissible Sendai virus with efcient gene transferession. Journal of Virology 2000;74(14):65649.H, Veits J, Rautenschlein S, Zimmer G. A recombinant vesicular stom-s replicon vaccine protects chickens from highly pathogenic avian

virus (H7N1). Vaccine 2009;27(8):117483.DG, Frolov I, Mason PW. Third-generation avivirus vaccines based-cycle, encapsidation-defective viruses. Advances in Virus Research77126.reer CE, Thudium K, Doe B, Legg H, Liu H, et al. An alphavirus repli-cle chimera derived from venezuelan equine encephalitis and sindbis

a potent gene-based vaccine delivery vector. Journal of Virology19):10394403.

Betts MR, Irlbeck DM, Davis NL, Swanstrom R, Frelinger JA,moral, mucosal, and cellular immunity in response to a humaneciency virus type 1 immunogen expressed by a Venezuelanncephalitis virus vaccine vector. Journal of Virology 1997;71(4):

, Heinz FX, Davis NL, Mandl CW. Heterologous gene expression bys and replicon vectors derived from tick-borne encephalitis virus andparison of this avivirus system with an alphavirus replicon. Journall Virology 2005;86(Pt 4):104553.Swiecki M, McCartney SA, Colonna M. dsRNA sensors and plasmacy-ritic cells in host defense and autoimmunity. Immunological Review(1):7490., Sheahan BJ, Soden DM, OSullivan GC, Atkins GJ. Electroporation

imulates immunity to an encoded reporter gene in mice. Molecular Report 2009;2(5):7536.n DX, Ljungberg K, Kakoulidou M, Liljestrom P. Intradermal electro-of naked replicon RNA elicits strong immune responses. PLoS One):e29732.

Pascolo S, Scheel B, Derhovanessian E, Pugfelder A, Eigentler TK,ect injection of protamine-protected mRNA: results of a phase 1/2on trial in metastatic melanoma patients. Journal of Immunotherapy5):498507., Haentschel M, Weimer KJ, Heine A, Muller MR, Brugger W, et al.al vaccinations with RNA coding for TAA generate CD8+ and CD4+

responses and induce clinical benet in vaccinated patients. Molecular011;19(5):9909.

DI, Reap EA, Katen K, Watson A, Smith K, Norberg P, et al.zed, double-blind, phase 1 trial of an alphavirus replicon vac-cytomegalovirus in CMV seronegative adult volunteers. Vaccine2):48493.

RNA-based vaccines1 Background2 Nucleic acid vaccines2.1 DNA vaccines2.2 Viral vectors2.3 RNA vaccines

3 ProspectsReferences