Recent Advances in mRNA Vaccine Technology: Current Opinion in Immunology August 2020
Recent Advances in mRNA Vaccine Technology: Current Opinion in Immunology August 2020
Recent Advances in mRNA Vaccine Technology: Current Opinion in Immunology August 2020
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Messenger RNA (mRNA) vaccines represent a relatively new including the incorporation of modified nucleosides
vaccine class showing great promise for the future. This [4,5] (particularly modified uridine), optimization of cod-
optimism is built on recently published studies demonstrating ing sequences [6], and stringent purification of IVT
the efficacy of mRNA vaccines in combatting several types of mRNA by high performance liquid chromatography
cancer and infectious pathogens where conventional vaccine (HPLC) [7] to remove double-stranded RNA (dsRNA)
platforms may fail to induce protective immune responses. contaminants; all of these techniques serve to dampen the
These results would not have been possible without critical innate sensing of synthetic mRNA, thus reducing toxicity
recent innovations in the field, such as the development of safe and improving translation of the mRNA [8]. The final
and efficient materials for in vivo mRNA delivery and advanced impediment to the viability of mRNA therapeutics has
protocols for the production of high quality mRNA. This review been inefficient cytoplasmic delivery. Although several
summarizes the most important developments in mRNA approaches such as ex vivo-loaded dendritic cells (DCs),
vaccines from the past few years and discusses the challenges intranodal delivery of mRNA, and mechanical methods
and future directions for the field. (gene gun, electroporation) were developed to deliver
naked mRNA for vaccination [9], these approaches are
Addresses either complicated and expensive (ex vivo loading of DCs)
1
Department of Medicine, University of Pennsylvania, Philadelphia, PA, or difficult to use in humans (intranodal delivery, elec-
19104, USA
2
Department of Pathology and Laboratory Medicine, Children’s Hospital
troporation); thus, the ideal way to deliver mRNA would
of Philadelphia, Philadelphia, PA, 19104, USA be with a material that protects it from degradation and
facilitates efficient cellular uptake after simple injection.
Corresponding authors: The past few years have witnessed a surge in develop-
Pardi, Norbert (pnorbert@pennmedicine.upenn.edu), Weissman, ment of highly efficacious delivery materials for nucleic
Drew (dreww@pennmedicine.upenn.edu)
acids, with some remarkable results [10].
delivery in preclinical models [9] but those studies did not [42] to an influenza vaccine trial with inactivated influenza
reach clinical phase. Polymers used for mRNA delivery virus vaccines [43]. The magnitude and durability of
are often modified with fatty acid chains to improve the immune responses were relatively modest compared to
safety profile of the delivery material [21]. There has been preclinical studies [29] and will need to be improved. Of
significant progress in the field: Haabeth et al. and McKin- note,theionizable LNPsused intheabovestudiesappearto
lay et al. have recently developed novel lipid-containing contribute to effective immune responses through multiple
polymers called charge-altering releasable transporters mechanisms that are not fully appreciated, including effi-
(CARTs) that have efficiently targeted T cells and cient cellular uptake andtranslation of mRNA [27] as well as
resulted in clearance of established tumors in mice a recently reported adjuvant effect [34,44].
[22,23]. Manipulation of T cells is difficult and often
requires ex vivo operations (purification of T cells Of note, the above-mentioned preclinical and clinical
obtained from donors, electroporation with nucleic acid, trials were initiated years ago; thus, we presume that
expansion and reinfusion); thus, CARTs are very attrac- more effective LNP formulations will become available.
tive delivery materials with great potential in the areas of In this regard, similarly to the polymer-based CART
mRNA vaccines and gene therapy. Chahal et al. have platform, selective T cell targeting has also been achieved
described branched polyamine-based polymers called by mRNA-LNPs. Veiga et al. have used a novel platform,
dendrimers formulated with a lipid-anchored polyethyl- called ASSET (Anchored Secondary scFv Enabling Tar-
ene glycol (PEG) and an antigen-encoding, self-amplify- geting), in which a T cell-specific monoclonal antibody is
ing mRNA. Immunization with a single intramuscular linked to LNPs in order to target leukocytes [45]. This
dose of dendrimer-RNA nanoparticles elicited antigen- flexible platform holds great potential for mRNA vaccine
specific CD8+ T cell and neutralizing antibody responses and other applications as well.
against Zika, Ebola and influenza viruses and Toxoplasma
gondii in mice [24]. Kranz et al. have reported on lipoplexes that preferentially
target DCs after systemic delivery [46]. Selective DC
Peptides targeting with mRNA vaccines to induce strong immune
Cell-penetrating peptides (CPPs) are rarely used for responses is a potentially critical finding, and this platform
mRNA vaccines but there has been some recent progress has already demonstrated promise in clinical trials and has
in the field: Udhayakumar et al. developed CPPs contain- been actively investigated in the context of personalized
ing amphipathic Arg-Ala-Leu-Ala motifs to condense cancer vaccines [47,48].
mRNA into particles that can disrupt and penetrate
membranes and demonstrated potent cytolytic T cell Future directions and outstanding questions
responses after immunizing mice with CPP-complexed regarding mRNA vaccines
mRNA [25]. It will be important for future studies to While the past several years have witnessed a rapid pace
explore whether this platform can induce potent antibody of innovation in mRNA manufacturing, in vivo delivery,
responses or protection from pathogen infection. and immunogenicity, there remains much room for
improvement and investigation. Here, we briefly high-
Lipid nanoparticles light three interrelated topics that, if better understood,
Ionizable lipid-containing nanoparticles (LNPs), initially could propel the field further: (1) differences in mRNA
developed for siRNA delivery, are the most widely used preparation, (2) differences between animal models and
in vivo mRNA delivery materials at present [26]. After a humans, and (3) mechanisms of immunogenicity of
proof-of-concept for the in vivo translation of mRNA-LNPs mRNA vaccines.
was demonstrated in 2015 [27], multiple vaccine studies
have used LNPs with unmodified or nucleoside-modified mRNA preparation
mRNA that induced durable, protective immune responses It is currently unknown whether certain formats of
against multiple infectious pathogens, often after a single mRNA vaccines are more effective for certain types of
dose [28–34,35,36–40], and have been effective in com- immune responses or certain disease applications. This
batting cancer as well [41]. Several clinical trials using question is prompted by the fact that the inflammatory
mRNA-LNPs are underway, and some published data from profile of synthetic mRNA can be dramatically altered by
two Phase I influenza virus vaccine trials (NCT03076385 multiple variables, including modified nucleosides [4,5],
and NCT03345043) are available [29,42]. In these trials, purification to remove dsRNA species [7,20], sequence
healthy adults were immunized twice, three weeks apart, engineering [6] intracellular RNA replication [49], and
with placebo or nucleoside-modified mRNA-LNPs encod- delivery material [32,44,49]. The resulting cytokine and
ing full-length influenza virus H10 and H7 hemagglutinin chemokine milieu could be reasonably expected to sig-
(HA), and safety and immunogenicity were evaluated. nificantly impact the magnitude and/or quality of T and B
Nucleoside-modified mRNA-LNP influenza vaccines cell responses in ways that may be more useful for some
induced humoral immune responses in the vaccine recip- disease applications than others. Relevant mechanisms
ients and the safety profile of the vaccines was comparable may include the impact of type I IFNs on the inhibition of
mRNA translation and the chemoattraction or functional of Tfh, GC, and neutralizing antibody responses to these
modulation (activation, differentiation, etc.) of antigen- vaccines [55]. It remains to be seen whether these various
presenting cells, lymphocytes, or other immune cells at vaccine formats have truly distinct immunogenic proper-
the site of vaccination or draining lymphoid tissues [50]. ties. We would like to particularly emphasize that very
In recent reports, nucleoside-modified mRNA-LNP vac- few studies have directly compared the in vivo efficacy of
cines have been particularly associated with potent T different mRNA vaccine formats [34,56], and these stud-
follicular helper (Tfh) cell, germinal center (GC) B cell, ies in general have not compared the protective or thera-
and neutralizing antibody responses [34,51]. However, it peutic efficacy of the various vaccines; therefore, this will
is currently unknown whether nucleoside modification is be a critical area of future research.
explicitly required for strong Tfh and GC responses to
mRNA vaccines or if other methods might be able to Human versus animal studies
achieve a similar outcome. Less clear is what impact A major hurdle in bringing mRNA vaccines to the clinic is
nucleoside modification has on cytotoxic CD8+ T cell to translate the often remarkable immunogenicity seen in
responses. Unmodified non-replicating mRNA, on the preclinical studies to human vaccine trials. This has been
other hand, has been shown to generate robust CD8+ illustrated by two mRNA vaccines made by different
T cell responses against tumor antigens, in which case methods: a rabies vaccine made using unmodified mRNA
antibody responses are generally not relevant to measure with a protamine/mRNA adjuvant [57,58], and two influ-
[46,52]. Self-amplifying mRNA vaccines, meanwhile, enza vaccines made with nucleoside-modified mRNA-
appear to stimulate balanced T and B cell responses LNPs encoding hemagglutinin from H7N9 or H10N8
[53,54], but there is limited information about the quality viruses [29,42]. In both cases, the preclinical data were
Figure 2
ssRNA
B cell help
dsRNA
Differentiation signals
sequence
(Th1,Tfh,etc.)
engineering, UTRs
5’ 3’
CD8+T
cell help
Species differences in
immune sensing(?)
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.
extremely promising in both mice and larger animals (pigs material. Further research into all of these areas will
for rabies and ferrets and cynomolgus macaques for contribute both to the further improvement of mRNA
influenza), and two doses of vaccine were sufficient to vaccines and to our basic understanding of immunology.
stimulate strong and sustained titers of neutralizing anti-
bodies. In contrast, when the same vaccines were tested Conclusions
in human volunteers, two doses of vaccine stimulated The past several years yielded critically important
unexpectedly low neutralizing antibody titers and sero- advancements in the field of mRNA vaccines and pro-
conversion frequencies. The discrepancies between the vided evidence for the viability of this novel vaccine
animal models and human studies prompt a number of modality. New manufacturing methods and delivery
important questions: are mRNA vaccines or their adju- materials will facilitate the rapid, inexpensive mass pro-
vants sensed or translated differently in humans versus duction of next-generation mRNA vaccines. Data from
other animals? Are there different biological require- human trials for both cancer and infectious disease
ments for potent antibody responses in humans compared mRNA vaccines are encouraging, but further improve-
to these animals? And does pre-existing immunity, for ments of the delivery materials and a more complete
example, against prior influenza virus exposures in understanding of the mechanisms of action of various
humans, impact the immunogenicity of a nucleoside- mRNA vaccine types are needed to rationally manipulate
modified mRNA-LNP vaccine? Animal models for influ- these formulations in order to increase efficacy while
enza virus vaccine studies may potentially be improved minimizing adverse events after vaccine administration.
by including a prior inoculation with live and/or inacti-
vated virus to better recapitulate the immune landscape
Conflict of interest statement
in human adults receiving an mRNA vaccine. Future
In accordance with the University of Pennsylvania poli-
studies should also examine the issue of ‘original anti-
cies and procedures and our ethical obligations as
genic sin;’ in this phenomenon, sequential exposure to
researchers, we report that Drew Weissman is named
divergent influenza virus antigens tends to boost antibody
on patents that describe the use of nucleoside-modified
responses that preferentially recognize the first antigen, at
mRNA as a platform to deliver therapeutic proteins.
the expense of those that recognize the second with high
Drew Weissman and Norbert Pardi are also named on
affinity [59].
a patent describing the use of modified mRNA in lipid
nanoparticles as a vaccine platform. We have disclosed
Mechanisms of immunogenicity of mRNA vaccines
those interests fully to the University of Pennsylvania,
Given the many variables in mRNA vaccine formulation
and we have in place an approved plan for managing any
outlined above and shown on Figure 2, it will be impor-
potential conflicts arising from licensing of our patents.
tant to determine which features promote protective
immunity so these vaccines may be improved in the
future. One major outstanding question in mRNA vac- Acknowledgements
N.P. was supported by the National Institute of Allergy and Infectious
cine design is whether type I IFNs enhance or inhibit Diseases (1R01AI146101). D.W. was supported by the National Institute of
protective immunity; in fact, there is good evidence for Allergy and Infectious Diseases (R01-AI050484, R01-AI124429 and R01-
both. A detrimental effect of type I IFN has been AI084860). M.J.H. is a Cancer Research Institute Irvington Fellow
supported by the Cancer Research Institute. The authors apologize to all
explicitly demonstrated in the context of self-amplifying colleagues whose great studies could not be cited here owing to space
mRNA vaccines. Pepini et al. showed that blocking the limitations.
activity of the type I IFN receptor improved both the
expression of mRNA-encoded protein as well as antigen- References and recommended reading
specific antibody and CD8+ T cell responses [49]. In Papers of particular interest, published within the period of review,
have been highlighted as:
contrast, a report by Kranz et al. showed that type I
IFN signaling was required for optimal antitumor immu- of special interest
nity induced by an unmodified mRNA-LNP vaccine [46]. of outstanding interest
Taken together, it seems likely that type I IFNs may be
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