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Recent advances in mRNA vaccine technology

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 Available online at www.sciencedirect.com
ScienceDirect
Recent advances in mRNA vaccine technology
Norbert Pardi1, Michael J Hogan2 and Drew Weissman1
Messenger RNA (mRNA) vaccines represent a relatively new
vaccine class showing great promise for the future. This
optimism is built on recently published studies demonstrating
the efficacy of mRNA vaccines in combatting several types of
cancer and infectious pathogens where conventional vaccine
platforms may fail to induce protective immune responses.
These results would not have been possible without critical
recent innovations in the field, such as the development of safe
and efficient materials for in vivo mRNA delivery and advanced
protocols for the production of high quality mRNA. This review
summarizes the most important developments in mRNA
vaccines from the past few years and discusses the challenges
and future directions for the field.
Addresses
1
Department of Medicine, University of Pennsylvania, Philadelphia, PA,
19104, USA
2
Department of Pathology and Laboratory Medicine, Children’s Hospital
of Philadelphia, Philadelphia, PA, 19104, USA
Corresponding authors:
Pardi, Norbert (pnorbert@pennmedicine.upenn.edu), Weissman,
Drew (dreww@pennmedicine.upenn.edu)
Current Opinion in Immunology 2020, 65:14–20
This review comes from a themed issue on Vaccines
Edited by Bali Pulendran and Rino Rappuoli
https://doi.org/10.1016/j.coi.2020.01.008
0952-7915/ã 2020 Published by Elsevier Ltd.
Introduction: overcoming early challenges for
RNA vaccines
The concept of genetic (DNA and RNA) vaccines was
raised decades ago with the hope that a flexible, easy-to-
produce, safe and effective vaccine class could be gener-
ated. Until the late 2000s, the emphasis was on the
development of DNA-based approaches [1] due to the
hurdles stemming from RNA’s instability, inefficient in
vivo delivery, and its stimulation of excessive inflamma-
tory responses. Producing in vitro-transcribed (IVT) mes-
senger RNA (mRNA) is a fairly straightforward process
[2,3], but making high quality ‘therapeutic’ mRNA that is
highly translatable and does not induce serious inflam-
mation were major limitations in the field until very
recently. By the early 2010s, the latter problem was
largely resolved by a number of critical innovations,
Current Opinion in Immunology 2020, 65:14–20
including the incorporation of modified nucleosides
[4,5] (particularly modified uridine), optimization of cod-
ing sequences [6], and stringent purification of IVT
mRNA by high performance liquid chromatography
(HPLC) [7] to remove double-stranded RNA (dsRNA)
contaminants; all of these techniques serve to dampen the
innate sensing of synthetic mRNA, thus reducing toxicity
and improving translation of the mRNA [8]. The final
impediment to the viability of mRNA therapeutics has
been inefficient cytoplasmic delivery. Although several
approaches such as ex vivo-loaded dendritic cells (DCs),
intranodal delivery of mRNA, and mechanical methods
(gene gun, electroporation) were developed to deliver
naked mRNA for vaccination [9], these approaches are
either complicated and expensive (ex vivo loading of DCs)
or difficult to use in humans (intranodal delivery, elec-
troporation); thus, the ideal way to deliver mRNA would
be with a material that protects it from degradation and
facilitates efficient cellular uptake after simple injection.
The past few years have witnessed a surge in develop-
ment of highly efficacious delivery materials for nucleic
acids, with some remarkable results [10].
Recent innovations
The most important innovations in mRNA vaccine tech-
nology in recent years have been in the areas of: 1)
engineering of mRNA sequences, 2) development of
methods that enable simple, rapid and large-scale cGMP
production of mRNA; and 3) development of highly
efficient and safe mRNA vaccine delivery materials.
Engineering of mRNA sequences
Given that the 5’ and 3’ untranslated regions (UTRs) of
mRNA can significantly influence the rate of translation
and half-life of the transcript, optimization of the UTRs is
of interest to mRNA vaccine design [11–13]. A recent
study used a cell culture-based systematic selection pro-
cess to identify new UTRs that significantly increase
protein expression from IVT mRNA [14]. The authors
identified several 3’ UTR sequences that could induce
approximately threefold higher protein production from
the associated transcript compared to the benchmark of
the human b-globin 3’ UTR. The novel 3’ UTR motifs
were validated in mRNA vaccination and genetic repro-
gramming studies where they induced more potent ther-
apeutic effects compared to mRNA with the b-globin 3’
UTR.
As mentioned above, the vast majority of effective mRNA
vaccines use some kind of a delivery material, but two
recent publications reported on an interesting new vac-
cine format that showed great potency in the absence of a
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 Recent advances in mRNA vaccine technology Pardi, Hogan and Weissman 15

delivery material [15,16]. This method utilizes the co- Figure 1

delivery of an mRNA encoding an alphavirus RNA-
dependent RNA polymerase plus a second mRNA that Linearized DNA template
encodes the antigen of interest and alphavirus genomic (a) 2017 methods (b) 2019 methods
features enabling its replication in the cytoplasm. This so-
In vitro transcription
called ‘transreplicon’ or ‘splitzicon’ system builds on the In vitro transcription
with CleanCap®
dose-sparing properties of self-amplifying mRNA [9], as a
very low dose (50 ng) was effective in inducing protective
immune responses in mice, even when delivered as AAA AAA
ppp cap1
unformulated mRNA. These findings are particularly
attractive as the utilization of low doses decreases the
Enzymatic capping
cost of the vaccine production. The absence of a delivery
material further decreases the cost, simplifies manufactur- cap1
AAA

ing and raises the possibility of vaccine lyophilization and

Cellulose
storage at ambient temperature. adsorption
HPLC
Optimization of mRNA production purification
Development of methods that enable rapid, simple, large-
scale and inexpensive production of high-quality mRNA
is a critical requirement for the future implementation of AAA AAA
cap1 cap1
mRNA vaccines. Although production of ‘therapeutic’
quality mRNA is not particularly challenging at present
Formulation of mRNA
[2,3], current protocols may not be ideal for mass produc-
tion. Most current mRNA production protocols (summa-
rized in Figure 1) entail separate enzymatic reactions for Vaccination
DNA template preparation, IVT, and 5’ capping, with Current Opinion in Immunology

nucleic acid precipitation after each step to remove other

reaction components. This iterative process involves
sample loss at each step and increases the time and cost
of production. Additionally, some current mRNA purifi-
cation methods — notably, HPLC purification — are not
easily scalable, further bottlenecking mass production.
While current approaches work well at small scale, they
are suboptimal for large-scale manufacturing. Ideally, the
final mRNA product would be prepared in a ‘one-pot’
reaction with a highly scalable purification method. Two
recently described innovations have made progress
towards this end. The first pertains to a co-transcriptional
capping strategy called CleanCap1, which adds a natural
5’ cap1 structure to a specific transcription start sequence
during IVT [17]. This development is significant
because previous protocols used either enzymatic cap-
ping [2] — adding additional reaction components and
purification steps to manufacturing — or co-transcrip-
tional capping with cap0 resulting innate immune activa-
tion when the IVT mRNA preparation contains dsRNA
contaminant [18,19]. The second methodological innova-
tion has offered an attractive alternative to HPLC purifi-
cation that is useful at laboratory and industrial scales
[20]: Baiersdorfer et al. described a simple method for
mRNA purification via adsorption of double-stranded
RNA contaminants to cellulose, a cheap and abundant
polysaccharide. The authors demonstrated that this
highly scalable and inexpensive method works just as
well as HPLC to remove dsRNA contaminants from IVT
mRNA samples. Taken together, research in the past
couple of years has greatly facilitated the large-scale
Schematic of recent innovations in mRNA preparation technology.
Shown are (a) methods used by some groups to prepare non-
inflammatory mRNA circa 2017 (e.g. [35]) and (b) simplified methods
to prepare the same, available as of the writing of this manuscript in
2019.
manufacture of therapeutic mRNA, and it is likely that
additional research in this vein will further improve the
simplicity and cost-efficiency of mRNA production.
Development of materials for efficient in vivo mRNA
delivery
As discussed above, the vast majority of mRNA vaccines
are designed to be injected with a carrier molecule that
protects mRNA from rapid degradation and delivers it to
the cytoplasm without significant toxicity. Recent work
has resulted in the development of multiple types of
lipid-based carriers, polymers, and peptides that have
all showed promise in preclinical and some clinical stud-
ies [10]. It is worth emphasizing that no efficient broadly
applicable in vivo mRNA delivery materials were avail-
able until very recently; thus, we believe that the most
significant recent progress in the mRNA vaccine field has
occurred in this area and that it has played a critical role in
advancing mRNA vaccines through several landmark
efficacy studies published within the last three years.
Polymers
Some early studies successfully used non-lipid polymers
such as polyethylenimine (PEI) for mRNA vaccine

www.sciencedirect.com Current Opinion in Immunology 2020, 65:14–20

16 Vaccines
delivery in preclinical models [9] but those studies did not
reach clinical phase. Polymers used for mRNA delivery
are often modified with fatty acid chains to improve the
safety profile of the delivery material [21]. There has been
significant progress in the field: Haabeth et al. and McKin-
lay et al. have recently developed novel lipid-containing
polymers called charge-altering releasable transporters
(CARTs) that have efficiently targeted T cells and
resulted in clearance of established tumors in mice
[22,23]. Manipulation of T cells is difficult and often
requires ex vivo operations (purification of T cells
obtained from donors, electroporation with nucleic acid,
expansion and reinfusion); thus, CARTs are very attrac-
tive delivery materials with great potential in the areas of
mRNA vaccines and gene therapy. Chahal et al. have
described branched polyamine-based polymers called
dendrimers formulated with a lipid-anchored polyethyl-
ene glycol (PEG) and an antigen-encoding, self-amplify-
ing mRNA. Immunization with a single intramuscular
dose of dendrimer-RNA nanoparticles elicited antigen-
specific CD8+ T cell and neutralizing antibody responses
against Zika, Ebola and influenza viruses and Toxoplasma
gondii in mice [24].
Peptides
Cell-penetrating peptides (CPPs) are rarely used for
mRNA vaccines but there has been some recent progress
in the field: Udhayakumar et al. developed CPPs contain-
ing amphipathic Arg-Ala-Leu-Ala motifs to condense
mRNA into particles that can disrupt and penetrate
membranes and demonstrated potent cytolytic T cell
responses after immunizing mice with CPP-complexed
mRNA [25]. It will be important for future studies to
explore whether this platform can induce potent antibody
responses or protection from pathogen infection.
Lipid nanoparticles
Ionizable lipid-containing nanoparticles (LNPs), initially
developed for siRNA delivery, are the most widely used
in vivo mRNA delivery materials at present [26]. After a
proof-of-concept for the in vivo translation of mRNA-LNPs
was demonstrated in 2015 [27], multiple vaccine studies
have used LNPs with unmodified or nucleoside-modified
mRNA that induced durable, protective immune responses
against multiple infectious pathogens, often after a single
dose [28–34,35,36–40], and have been effective in com-
batting cancer as well [41]. Several clinical trials using
mRNA-LNPs are underway, and some published data from
two Phase I influenza virus vaccine trials (NCT03076385
and NCT03345043) are available [29,42]. In these trials,
healthy adults were immunized twice, three weeks apart,
with placebo or nucleoside-modified mRNA-LNPs encod-
ing full-length influenza virus H10 and H7 hemagglutinin
(HA), and safety and immunogenicity were evaluated.
Nucleoside-modified mRNA-LNP influenza vaccines
induced humoral immune responses in the vaccine recip-
ients and the safety profile of the vaccines was comparable
Current Opinion in Immunology 2020, 65:14–20
[42] to an influenza vaccine trial with inactivated influenza
virus vaccines [43]. The magnitude and durability of
immune responses were relatively modest compared to
preclinical studies [29] and will need to be improved. Of
note,theionizable LNPsused intheabovestudiesappearto
contribute to effective immune responses through multiple
mechanisms that are not fully appreciated, including effi-
cient cellular uptake andtranslation of mRNA [27] as well as
a recently reported adjuvant effect [34,44].
Of note, the above-mentioned preclinical and clinical
trials were initiated years ago; thus, we presume that
more effective LNP formulations will become available.
In this regard, similarly to the polymer-based CART
platform, selective T cell targeting has also been achieved
by mRNA-LNPs. Veiga et al. have used a novel platform,
called ASSET (Anchored Secondary scFv Enabling Tar-
geting), in which a T cell-specific monoclonal antibody is
linked to LNPs in order to target leukocytes [45]. This
flexible platform holds great potential for mRNA vaccine
and other applications as well.
Kranz et al. have reported on lipoplexes that preferentially
target DCs after systemic delivery [46]. Selective DC
targeting with mRNA vaccines to induce strong immune
responses is a potentially critical finding, and this platform
has already demonstrated promise in clinical trials and has
been actively investigated in the context of personalized
cancer vaccines [47,48].
Future directions and outstanding questions
regarding mRNA vaccines
While the past several years have witnessed a rapid pace
of innovation in mRNA manufacturing, in vivo delivery,
and immunogenicity, there remains much room for
improvement and investigation. Here, we briefly high-
light three interrelated topics that, if better understood,
could propel the field further: (1) differences in mRNA
preparation, (2) differences between animal models and
humans, and (3) mechanisms of immunogenicity of
mRNA vaccines.
mRNA preparation
It is currently unknown whether certain formats of
mRNA vaccines are more effective for certain types of
immune responses or certain disease applications. This
question is prompted by the fact that the inflammatory
profile of synthetic mRNA can be dramatically altered by
multiple variables, including modified nucleosides [4,5],
purification to remove dsRNA species [7,20], sequence
engineering [6] intracellular RNA replication [49], and
delivery material [32,44,49]. The resulting cytokine and
chemokine milieu could be reasonably expected to sig-
nificantly impact the magnitude and/or quality of T and B
cell responses in ways that may be more useful for some
disease applications than others. Relevant mechanisms
may include the impact of type I IFNs on the inhibition of
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 Recent advances in mRNA vaccine technology Pardi, Hogan and Weissman 17

mRNA translation and the chemoattraction or functional
modulation (activation, differentiation, etc.) of antigen-
presenting cells, lymphocytes, or other immune cells at
the site of vaccination or draining lymphoid tissues [50].
In recent reports, nucleoside-modified mRNA-LNP vac-
cines have been particularly associated with potent T
follicular helper (Tfh) cell, germinal center (GC) B cell,
and neutralizing antibody responses [34,51]. However, it
is currently unknown whether nucleoside modification is
explicitly required for strong Tfh and GC responses to
mRNA vaccines or if other methods might be able to
achieve a similar outcome. Less clear is what impact
nucleoside modification has on cytotoxic CD8+ T cell
responses. Unmodified non-replicating mRNA, on the
other hand, has been shown to generate robust CD8+
T cell responses against tumor antigens, in which case
antibody responses are generally not relevant to measure
[46,52]. Self-amplifying mRNA vaccines, meanwhile,
appear to stimulate balanced T and B cell responses
[53,54], but there is limited information about the quality
of Tfh, GC, and neutralizing antibody responses to these
vaccines [55]. It remains to be seen whether these various
vaccine formats have truly distinct immunogenic proper-
ties. We would like to particularly emphasize that very
few studies have directly compared the in vivo efficacy of
different mRNA vaccine formats [34,56], and these stud-
ies in general have not compared the protective or thera-
peutic efficacy of the various vaccines; therefore, this will
be a critical area of future research.
Human versus animal studies
A major hurdle in bringing mRNA vaccines to the clinic is
to translate the often remarkable immunogenicity seen in
preclinical studies to human vaccine trials. This has been
illustrated by two mRNA vaccines made by different
methods: a rabies vaccine made using unmodified mRNA
with a protamine/mRNA adjuvant [57,58], and two influ-
enza vaccines made with nucleoside-modified mRNA-
LNPs encoding hemagglutinin from H7N9 or H10N8
viruses [29,42]. In both cases, the preclinical data were

Figure 2

(a) Input variables (b) Output variables

modified vs. unmodified

ribonucleosides Cytokine and
chemokine milieu
delivery
materials B cell activation

ssRNA
B cell help

dsRNA

Differentiation signals
sequence
(Th1,Tfh,etc.)
engineering, UTRs
5’ 3’

CD8+T
cell help
Species differences in
immune sensing(?)

Current Opinion in Immunology

Representation of unknown factors in mRNA vaccine design. Shown are (a) a variety of potential features in mRNA vaccines that may influence
the immune response, and (b) a number of relevant immune pathways that might be affected. A transfected antigen-presenting cell is depicted as
an important mediator of many of these effects. Abbreviations: APC, antigen-presenting cell; ssRNA, single-stranded RNA; dsRNA, double-
stranded RNA; UTRs, untranslated regions; Th1, type 1 helper cell; Tfh, T follicular helper cell.

www.sciencedirect.com Current Opinion in Immunology 2020, 65:14–20

18 Vaccines
extremely promising in both mice and larger animals (pigs
for rabies and ferrets and cynomolgus macaques for
influenza), and two doses of vaccine were sufficient to
stimulate strong and sustained titers of neutralizing anti-
bodies. In contrast, when the same vaccines were tested
in human volunteers, two doses of vaccine stimulated
unexpectedly low neutralizing antibody titers and sero-
conversion frequencies. The discrepancies between the
animal models and human studies prompt a number of
important questions: are mRNA vaccines or their adju-
vants sensed or translated differently in humans versus
other animals? Are there different biological require-
ments for potent antibody responses in humans compared
to these animals? And does pre-existing immunity, for
example, against prior influenza virus exposures in
humans, impact the immunogenicity of a nucleoside-
modified mRNA-LNP vaccine? Animal models for influ-
enza virus vaccine studies may potentially be improved
by including a prior inoculation with live and/or inacti-
vated virus to better recapitulate the immune landscape
in human adults receiving an mRNA vaccine. Future
studies should also examine the issue of ‘original anti-
genic sin;’ in this phenomenon, sequential exposure to
divergent influenza virus antigens tends to boost antibody
responses that preferentially recognize the first antigen, at
the expense of those that recognize the second with high
affinity [59].
Mechanisms of immunogenicity of mRNA vaccines
Given the many variables in mRNA vaccine formulation
outlined above and shown on Figure 2, it will be impor-
tant to determine which features promote protective
immunity so these vaccines may be improved in the
future. One major outstanding question in mRNA vac-
cine design is whether type I IFNs enhance or inhibit
protective immunity; in fact, there is good evidence for
both. A detrimental effect of type I IFN has been
explicitly demonstrated in the context of self-amplifying
mRNA vaccines. Pepini et al. showed that blocking the
activity of the type I IFN receptor improved both the
expression of mRNA-encoded protein as well as antigen-
specific antibody and CD8+ T cell responses [49]. In
contrast, a report by Kranz et al. showed that type I
IFN signaling was required for optimal antitumor immu-
nity induced by an unmodified mRNA-LNP vaccine [46].
Taken together, it seems likely that type I IFNs may be
detrimental for some types of mRNA vaccine and useful
for others. Our current knowledge about the topic is
discussed in detail in a recent publication [50].
Other mechanisms that may influence the B or T cell
response against all mRNA vaccines include the half-life
of antigen availability, the kinetics or magnitude of anti-
gen presentation on MHC class I and II, contributions
from other components of the innate immune system
(NK cells, neutrophils, macrophages, etc.), and the cyto-
kine milieu induced by the mRNA and/or the delivery
Current Opinion in Immunology 2020, 65:14–20
material. Further research into all of these areas will
contribute both to the further improvement of mRNA
vaccines and to our basic understanding of immunology.
Conclusions
The past several years yielded critically important
advancements in the field of mRNA vaccines and pro-
vided evidence for the viability of this novel vaccine
modality. New manufacturing methods and delivery
materials will facilitate the rapid, inexpensive mass pro-
duction of next-generation mRNA vaccines. Data from
human trials for both cancer and infectious disease
mRNA vaccines are encouraging, but further improve-
ments of the delivery materials and a more complete
understanding of the mechanisms of action of various
mRNA vaccine types are needed to rationally manipulate
these formulations in order to increase efficacy while
minimizing adverse events after vaccine administration.
Conflict of interest statement
In accordance with the University of Pennsylvania poli-
cies and procedures and our ethical obligations as
researchers, we report that Drew Weissman is named
on patents that describe the use of nucleoside-modified
mRNA as a platform to deliver therapeutic proteins.
Drew Weissman and Norbert Pardi are also named on
a patent describing the use of modified mRNA in lipid
nanoparticles as a vaccine platform. We have disclosed
those interests fully to the University of Pennsylvania,
and we have in place an approved plan for managing any
potential conflicts arising from licensing of our patents.
Acknowledgements
N.P. was supported by the National Institute of Allergy and Infectious
Diseases (1R01AI146101). D.W. was supported by the National Institute of
Allergy and Infectious Diseases (R01-AI050484, R01-AI124429 and R01-
AI084860). M.J.H. is a Cancer Research Institute Irvington Fellow
supported by the Cancer Research Institute. The authors apologize to all
colleagues whose great studies could not be cited here owing to space
limitations.
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