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Vaccine 30 (2012) 4414–4418 Contents lists available at SciVerse ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Review RNA-based vaccines Jeffrey B. Ulmer ∗ , Peter W. Mason, Andrew Geall, Christian W. Mandl Novartis Vaccines, Cambridge, MA 02139, United States a r t i c l e i n f o Article history: Received 3 February 2012 Received in revised form 10 April 2012 Accepted 18 April 2012 Available online 28 April 2012 Keywords: Nucleic acid vaccine Viral vector a b s t r a c t Nucleic acid vaccines consisting of plasmid DNA, viral vectors or RNA may change the way the next generation vaccines are produced, as they have the potential to combine the benefits of live-attenuated vaccines, without the complications often associated with live-attenuated vaccine safety and manufacturing. Over the past two decades, numerous clinical trials of plasmid DNA and viral vector-based vaccines have shown them to be safe, well-tolerated and immunogenic. Yet, sufficient potency for general utility in humans has remained elusive for DNA vaccines and the feasibility of repeated use of viral vectors has been compromised by anti-vector immunity. RNA vaccines, including those based on mRNA and self-amplifying RNA replicons, have the potential to overcome the limitations of plasmid DNA and viral vectors. Possible drawbacks related to the cost and feasibility of manufacturing RNA vaccines are being addressed, increasing the likelihood that RNA-based vaccines will be commercially viable. Proof of concept for RNA vaccines has been demonstrated in humans and the prospects for further development into commercial products are very encouraging. © 2012 Elsevier Ltd. All rights reserved. Contents 1. 2. 3. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4414 Nucleic acid vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4414 2.1. DNA vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4415 2.2. Viral vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4415 2.3. RNA vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4415 Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4417 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4417 1. Background The discovery and development of new and improved vaccines have been greatly facilitated by the application of new technologies to identify protective antigens, to optimally present antigens to the immune system, and to manufacture vaccines using highly characterized, synthetic methods of production. This progress has been spurred by a need to move beyond empirical approaches to vaccines research and development, and has ushered in several new paradigms including reverse, structural and synthetic vaccinology approaches, respectively [1]. The use of nucleic acid-based vaccines to combine the benefits of in situ expression of antigens, with the safety of inactivated and subunit vaccines, has been a key advancement. Upon their discovery more than 20 years ago, nucleic acid vaccines promised to be a safe and effective means to mimic immunization with a live organism vaccine, particularly for induction of T cell immunity [2]. In addition, the manufacture of nucleic acid-based vaccines offered the potential to be relatively simple, inexpensive and generic. Since then, clinical trials have amply demonstrated 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 has led to extensive activity to identify enabling technologies. The main areas for improvement have been directed toward the nucleic acid vector, targeting the innate immune system to enhance immunogenicity, and delivery systems to overcome the barriers to efficient transfection of host cells in vivo. Significant progress has been made on all these fronts. This review paper will focus on the use of an alternative nucleic acid vector, namely RNA, as the basis of a new generation of vaccines. 2. Nucleic acid vaccines ∗ Corresponding author at: Novartis Vaccines, 350 Massachusetts Ave., Cambridge, MA 02139, United States. E-mail address: [email protected] (J.B. Ulmer). 0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2012.04.060 By definition, nucleic acid vaccines are based on DNA or RNA encoding the antigen(s) of interest. In their simplest form, they can J.B. Ulmer et al. / Vaccine 30 (2012) 4414–4418 consist of highly purified nucleic acids formulated in a buffer. Most often, however, specialized delivery systems are utilized to increase vaccine potency. Means to facilitate nucleic acid delivery involve (1) viral particles to take advantage of the efficiency of viral entry mechanisms, (2) non-viral formulations of DNA or RNA involving lipids, polymers, emulsions or other synthetic approaches to avoid the use of viral vectors, and (3) physical delivery technologies, such as electroporation in situ. The majority of the preclinical and clinical experiences with nucleic acid vaccines so far have been with DNA vaccines and DNA-based viral vectors. 2.1. DNA vaccines DNA vaccines have been widely evaluated in many animal models of infectious and non-infectious diseases with generally good success at eliciting potent immune responses against encoded antigens, which have ranged from discrete T or B cell epitopes to large polyprotein complexes. The utility of DNA vaccines in animals has been further documented by the development and commercialization of plasmid DNA-based animal health products. These include a West Nile virus vaccine for horses [5], an infectious hematopoietic necrosis virus vaccine for fish [6], a melanoma cancer vaccine for dogs [7], and a growth hormone releasing hormone gene therapy for pigs [8]. In humans, proof of concept for induction of both antibody and T cell responses has been demonstrated for various indications in multiple clinical trials. However, the magnitude of these immune responses has been lower than those observed for conventional vaccines consisting of live or inactivated whole organisms, or subunit proteins formulated with adjuvants. The reasons for this shortcoming of DNA vaccines are not clear, but are likely due, at least in part, to inefficient delivery of DNA into human cells and inadequate stimulation of the human immune system. To overcome these limitations, various technologies have been evaluated and the most promising current approaches involve facilitation of DNA delivery by electroporation [9] and stimulation of the immune system via the use of genetic adjuvants (i.e., in situ expression of immunologically active molecules encoded by the DNA vaccine) [10]. Combinations of these approaches have resulted in potent induction of immunity in non-human primates [11,12] and preliminary results of human clinical trials are encouraging [3,9]. 2.2. Viral vectors In situ expression of antigens in a vaccinated host can be effectively achieved through the use of recombinant vectors, often DNA viruses, engineered to be safe and to encode the gene(s) of interest. Vectors based on adenoviruses and poxviruses have been studied extensively, although several other viral vectors are being evaluated at earlier stages of development. Both adenovirus and poxvirus vectors have demonstrated safety and immunogenicity in human clinical trials [13]. Notably, a poxvirus vector encoding HIV envelope protein used in a prime-boost regimen with recombinant envelope protein plus adjuvant elicited modest protection in a phase III efficacy trial [14]. One clear advantage of viral vectors over DNA vaccines is the efficiency with which the DNA payload is introduced into host cells, due to the natural invasiveness of the viral particle. Hence, the amount of plasmid DNA required for induction of immune responses is typically many orders of magnitude greater than the amount of DNA contained in a viral vector vaccine. Two potential limitations of viral vectors, though, are related to safety and the inherent immunogenicity of the vector itself. First, because viral vectors are usually originally derived from wild-type pathogenic viruses, there is at least a theoretical potential for reversion to a virulent state, just as there is for attenuated live virus-based vaccines. However, extensive safety testing of these vectors has demonstrated that this is likely not a major 4415 issue. Second, because viral vectors contain, and in some cases express, viral antigens in addition to the target antigen of interest, such vectors are usually quite immunogenic (i.e., elicit immune responses against the vectors themselves). Pre-existing anti-vector immunity (either due to prior infection with wild-type virus, vaccines or immunization with the vector) has been shown to blunt the ability of the vector to launch production of the target antigen and, hence, limits induction of immune responses against the antigen of interest. Strategies to circumvent this limitation have included use of certain adenovirus strains not commonly circulating in humans, to allow initial take of the viral vector vaccine, and heterologous prime-boost approaches involving different vectors, to allow effective boosting of immune responses against the target antigen. While these approaches can be effective, at least temporarily, they complicate the vaccination regimen and do not provide an optimal solution. 2.3. RNA vaccines Proof of concept for RNA vaccines was provided two decades ago, when intramuscular injection of mRNA in mice resulted in local production of an encoded reporter protein [15] and induction of immune responses against an encoded antigen [16]. In a direct comparison with a corresponding plasmid DNA vaccine, injection of similar doses of mRNA (on a mass basis) formulated in sucrose resulted in similar levels of reporter gene expression, suggesting equivalent efficiencies of cellular transfection in vivo by the two types of nucleic acid vaccines [15]. These initial publications were followed by many more demonstrating the general utility of eliciting immune responses by RNA vaccines (for review, see [17]). The variety of gene targets included reporter genes [15,18–20] viral antigens [16,21], tumor antigens [22–26], and allergens [27,28]. In these animal models, both antibody and T cell responses, including CD4+ and CD8+ , were elicited. Furthermore, functional immunity, as measured by protective efficacy against challenge with live pathogens or tumors, was achieved. In preclinical models of allergy, low doses of an RNA vaccine encoding allergens induce a Th1-biased immune response that provided resistance against subsequent allergic sensitization. Induction of antigen-specific immune responses can be achieved by administration of RNA vaccines via various routes, including intramuscular [15,19], intradermal [22], subcutaneous [16], intravenous [16], intrasplenic [21], and intranodal [24], as well as delivery into the skin by the gene gun [29,30]. In addition, a considerable amount of work has been done using mRNA vaccines to pulse dendritic cells in vitro, which are then administered as the immunizing agent (for review, see [31]). Hence, like DNA vaccines, RNA vaccines have demonstrated versatility in many animal models of infectious and non-infectious diseases. However, RNA vaccines have several attributes that provide potential advantages over DNA vaccines. First, there is a finite chance that plasmid DNA vaccines can integrate into the genome of the immunized host. Although there has been little evidence so far that integration occurs after DNA vaccination, use of RNA would eliminate this as an issue. Second, plasmid DNA vaccines must be delivered into and transcribed within the nucleus in order to transfect a cell, i.e. they must traverse two membrane barriers (plasma and nuclear membranes). This could be particularly problematic in non-dividing cells, such as mature myocytes, where the nuclear membrane remains intact. Several publications have demonstrated that microinjection of pDNA into the cytoplasm of non-dividing cells resulted in very low levels of gene expression, but direct intra-nuclear injection of the same number of pDNA copies led to efficient transfection [32–34]. In contrast, since RNA vaccines are translated directly in the cytoplasm, the need for delivery into the nucleus is obviated. Finally, the kinetics of antigen expression after RNA administration appears to peak 4416 J.B. Ulmer et al. / Vaccine 30 (2012) 4414–4418 Sub-genomic promoter Virus genome m7G 5' nonstructural proteins Capsid, E2/E1 glycoproteins An Sub-genomic promoter Replicon vector m7G 5' nonstructural proteins Antigen An Fig. 1. Schematic illustration of an RNA replicon vaccine. Example of an RNA replicon vector derived from a positive-strand alphavirus genome. All replicons encode genes (indicated here as nonstructural proteins) that drive their amplification within the cytoplasm of host cells. For use as vaccine vectors, replicons also encode antigen genes. For alphavirus vectors, antigen genes are most commonly inserted in place of the capsid and glycoprotein genes, which are not needed for genome replication. In this way, the vector amplifies its full-length genome when it is introduced into the cytoplasm, and then following genome amplification it initiates production of a sub-genomic mRNA encoding the target antigen. and decay rapidly, in contrast to DNA administration where antigen expression can persist for many weeks [35]. Hence, RNA vaccination better mimics antigen expression during an acute infection, which may be more conducive to induction of antigen-specific immune responses. The mechanisms of action for RNA vaccines have not been fully elucidated, but likely involve some of the same processes utilized by DNA vaccines for the expression and presentation of encoded antigens leading to induction of immune responses. After injection, the RNA is exposed to RNases in the tissue [36], which can degrade the vaccine and limit uptake of functional RNA by cells. In addition, the 2 -hydroyxl on the ribose sugar prevents the mRNA adopting a stable double -helix, due to steric hindrance, and makes the macromolecule more prone to hydrolysis. Nevertheless, various cell types are capable of internalizing RNA by an active, saturable and specific process leading to local expression of antigen [35]. Uptake is mediated by membrane domains rich in caveolae and lipid rafts, and involves scavenger receptors [37]. Upon internalization, a portion of the RNA accumulates in the cytoplasm where it is translated into protein. This in situ production of antigen provides a means to mimic pathogen infections and expression of tumor antigens leading to efficient presentation of antigens by major histocompatibility complex (MHC) class I and II proteins, and induction of T cell responses in a manner analogous to that provided by DNA vaccines and viral vectors. Alternatively, RNA vaccines can be constructed for the efficient production and secretion (or cell-surface expression) of extracellular antigens to stimulate B cell responses and antigen-specific antibody production. The effectiveness of RNA vaccines may also be related to the fact that RNA is known to be a potent stimulator of innate immunity. In vitro, mRNA has been shown to activate dendritic cells and monocytes in a MyD88-dependent fashion involving signaling via Toll-like receptors (TLR) [38,39]. In vivo, it was recently demonstrated that an mRNA vaccine caused the upregulation of various genes involved in chemotaxis and cell activation [40] as well as induction of TLR7-dependent CD4+ and CD8+ T cell responses, and anti-tumor immunity [41]. Hence, the functionality of RNA vaccines involves at least two components: (1) local expression of antigen to facilitate presentation by MHC molecules and (2) engagement of pattern recognition receptors to stimulate innate immunity leading to potentiation of antigen-specific immune responses. Many of the above-referenced studies have used naked mRNA as the vaccine (i.e., simply formulated in a buffer). While this approach has been shown to elicit immune responses, the presence of degradative enzymes in tissues likely limits the amount of RNA that is available for internalization by cells in vivo. As a means to overcome this inherent drawback of using naked RNA, work has focused on delivery systems, adjuvants, and engineering of the RNA molecule. First, to protect RNA from degradation and enhance cellular uptake, encapsulation in liposomes [16,18,26] and complexation with cationic polymers [38,41] have proven effective. As an alternative delivery system, the gene gun has been used to directly introduce mRNA into the cytoplasm of cells [29]. Second, although RNA vaccines have a built-in adjuvant effect in the form of TLR engagement, mRNA vaccine potency has been enhanced by coadministration of recombinant GM-CSF [19] or Flt-3 ligand [42], or RNA encoding GM-CSF [43]. Finally, several approaches have been taken to improve the RNA molecule itself. Various modifications have been made to the 5 cap structure, the untranslated regions, and codon usage in the translated region (for review, see [44]), which have resulted in increased mRNA stability and expression. The feasibility of using RNA as the basis for a nucleic acid vaccine was initially regarded as questionable, due the inherent instability of mRNA in the presence of tissue fluids, the uncertainty of developing reasonable manufacturing processes yielding a stable formulation, and the anticipated high cost of the product. Each of these potential limitations is being addressed. Even naked mRNA is immunogenic in animals [19–21,25,45] and humans [46], indicating that RNA degradation in tissues after administration does not completely abrogate vaccine effectiveness. However, the efficiency of RNA delivery should be increased markedly through the use of enabling synthetic and viral delivery systems. For research purposes, in vitro transcribed mRNA can be obtained from plasmid DNA containing a bacteriophage promoter (T7, SP6 or T3) and over the past 10 years many technical refinements to the commercial kits have resulted in dramatic improvements in quality and yield [31,47]. More recently, RNA manufacturing by enzymatic transcription of appropriate DNA templates now seems attainable at reasonable cost and large scale. Long-term storage of lyophilized RNA vaccines have previously been studied and RNAse-free RNA vaccines were demonstrated to be no less stable than other conventional vaccines that require a cold chain to retain efficacy [48]. These advancements have enabled the development of process for the GMP production of mRNA vaccines in quantities sufficient for human clinical trials (for review, see [17]). While most of the published work has utilized mRNA as the vaccine, several publications have shown that RNA vaccines can also be derived from sub-genomic replicons that lack viral structural proteins. Replicon RNA-based vaccines have been generated for a variety of RNA viruses including, Semliki Forest virus [21,25], Sindbis virus [20], poliovirus [49,50], tick-borne encephalitis virus [51,52], Kunjin virus [53], and bovine viral diarrhea [54]. RNA-based vaccines have also been described in which the RNA vaccine is used to launch a live-attenuated virus infection. In this case, the inherent potency of the encoded live viral vaccine has permitted this type of RNA vaccine to elicit protective immunity at very low (ng) RNA doses [51]. More commonly, experimental RNA-based vaccines are viral-particle delivered products engineered to express a heterologous antigen in place of the viral structural genes. These vaccines are produced under special conditions (e.g., packaging cell lines) that permit production of single-round infectious particles carrying J.B. Ulmer et al. / Vaccine 30 (2012) 4414–4418 4417 Table 1 Superior attributes of RNA vaccines. Parameter Synthetic Generic manufacturing Safety Antibody induction CTL induction In vivo expression Control of expression Absence of eukaryotic contaminants In vivo self amplification Potency in humans Vaccine type Live Subunit Viral vector DNA mRNA Replicon RNA − − +/− + + + − − + + − − + + − − − +/− − + − − +/− + + + + − + +/− +/− + + +/− + + + + − +/− + + + + + + + + − +/− + + + + + + + + + TBD RNAs encoding the vaccine antigens [55–59]. In this way, transient, high levels of antigen production can be achieved without the use of a “live”, spreading viral infection. Replicons derived from different RNA viruses differ with regard to levels and duration of heterologous gene expression allowing the generation of a versatile toolbox for vaccine or gene therapy applications [60]. An illustration of an RNA vaccine based on an alphavirus replicon is depicted in Fig. 1. The RNA amplification process in the cytoplasm produces multiple copies of antigen-encoding mRNA and creates dsRNA intermediates, which are known to be potent stimulators of innate immunity [61]. Thus, on a mass basis, replicon RNA vaccines have the potential to be more effective than corresponding mRNA vaccines. Indeed, a direct comparison of the two types of RNA vaccines demonstrated significantly higher and more persistent expression levels in vivo after replicon RNA administration [20]. These replicon vaccines have been administered as naked RNA packaged in viral particles, or delivered by electroporation in situ [52,62,63] Viral particle delivery of replicons has the advantage of efficiency, as previously described for viral vectors, but complicates manufacturing, introduces theoretical safety considerations, and has the potential limitation of anti-vector immunity. Hence, facilitated delivery of RNA replicons using synthetic systems, such as those evaluated for mRNA or DNA vaccines may increase potency without the added complications commonly seen with viral vectors. 3. Prospects RNA vaccines, particularly self-amplifying replicons, have the potential to capture the advantages of both DNA vaccines and viral delivery while overcoming the drawbacks of each technology (see Table 1). The prospect of RNA vaccines being a more effective approach than other types of nucleic acid vaccines has led to their advancement into human clinical trials. So far, mRNA vaccines have been administered to cancer patients in several trials as active immunotherapeutic immunization protocols, supported by preclinical proof of concept in animal tumor models (for review, see [31]). In the first trial, mRNA encoding genes cloned from metastatic melanoma tumors were used as an autologous immunization strategy [46]. Subsequent trials used combinations of known tumor antigens, such as MUC1, CEA, telomerase, MAGE-1, tyrosinase, in metastatic melanoma [64] and renal cell carcinoma [65] patients. In these exploratory clinical trials, the mRNA vaccines elicited antigen-specific immune responses (both antibodies and T cells), demonstrating proof of concept that mRNA vaccines are active in humans. Clinical trials have also been performed with RNA replicon vaccines packaged in viral particles. A bivalent vaccine consisting of replicons encoding cytomegalovirus (CMV) gB and pp65/IE1 proteins was shown to be well tolerated and immunogenic in healthy CMV seronegative volunteers [66]. All 40 individuals generated polyfunctional CD4+ and CD8+ T cell responses, as well as virus neutralizing antibodies. However, the magnitude of the responses in these recent trials was similar to those previously observed for other types of nucleic acid vaccines. 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