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Vaccine 30 (2012) 4414–4418
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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
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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. Therefore, the RNA vaccine approach holds promise as an effective means of eliciting
functional, protective immune responses in humans, but success
will likely require enabling delivery technologies. Next generation
replicon RNA vaccines will be formulated with synthetic delivery
technologies and will aspire to combine the effectiveness of live
attenuated vaccines, an equal or better safety profile than plasmid
DNA vaccines, and completely synthetic methods of manufacture.
Such a vaccine would possess the desired attributes of an ideal
vaccine.
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