Scientific background 2023
`Discoveries concerning nucleoside base modifications that enabled
`the development of effective mRNA vaccines against COVID-19
`
`When SARS-CoV-2 emerged in late 2019 and
`rapidly spread to all parts of the world, few thought
`that vaccines could be developed in time to help
`curb the increasing global disease burden. Yet,
`several vaccines were approved in record time,
`with two of the fastest approved and most effective
`vaccines produced with the new mRNA tech-
`nology. The concept of using mRNA for vacc-
`ination and in vivo delivery of therapeutic proteins
`was first proposed over 30 years ago, but several
`hurdles had to be overcome to make this a clinical
`reality. Early experiments demonstrated that in
`vitro transcribed mRNA stimulates undesired
`inflammatory responses and inefficient protein
`production in cells and tissues. A turning point was
`the discovery by Karikó and Weissman
`demonstrating that mRNA produced with modified
`nucleoside bases evades
`innate
`immune
`recognition and improves protein expression.
`These findings, combined with the development of
`efficient systems for in vivo mRNA delivery,
`stabilization of the SARS-CoV-2 spike antigen,
`and unparalleled investments by industry and
`governments, led to the approval of two highly
`successful mRNA-based COVID-19 vaccines in
`late 2020. The discovery by Karikó and Weissman
`was critical for making the mRNA vaccine platform
`suitable for clinical use at a time when it was most
`needed, making this an extraordinary contribution
`to medicine and paving the way for future mRNA
`applications.
`
`In today’s globally interconnected society the risk
`of new pandemics is greater than ever before.
`Pandemics are usually caused by zoonotic viruses
`that cross the species barrier into humans and
`spread
`through droplet- or aerosol-mediated
`transmission,
`causing
`airway
`infections.
`Developing and deploying vaccines
`rapidly
`enough to mitigate an ongoing pandemic is an
`enormous challenge that had never been met
`before the COVID-19 pandemic. The rapid sharing
`of the SARS-CoV-2 genome sequence, along with
`extensive prior developments in molecular bio-
`logy, vaccine research, and drug delivery over the
`past several decades spurred unprecedented
`activity among vaccine researchers during 2020.
`
`Scientists in academia and industry launched
`projects in record time, with financial and logistical
`backing from governments, industry, and non-
`profit organizations. The new mRNA vaccine plat-
`form represented one of the most interesting
`options, but how well it would work against this
`new virus was unknown. No mRNA-based vaccine
`had been approved for human use before.
`
`Virus vaccine platforms prior to COVID-19
`Most licensed anti-viral vaccines available today
`are produced with traditional techniques based on
`weakened or inactivated whole viruses (Figure 1).
`Live attenuated virus vaccines, such as the
`combined rubella-mumps-measles vaccine and
`the yellow fever virus vaccine, induce robust and
`long-lived antibody and T cell-mediated immunity.
`For the development of the yellow fever virus
`vaccine, Max Theiler was awarded the Nobel Prize
`in Physiology or Medicine in 1951. Vaccines
`based on inactivated viruses, such as the tick-
`borne encephalitis vaccine and the hepatitis A
`vaccine, induce effective but more transient
`immune responses, requiring repeated boosting.
`With the revolution of molecular biology and the
`development of technologies for recombinant
`protein production, opportunities for more targeted
`vaccine approaches arose. The first vaccine
`produced using this approach was the hepatitis B
`vaccine (HBV), approved in 1986, which was
`followed by the approval of the first human
`papillomavirus (HPV) vaccine in 2006. The HBV
`and HPV vaccines contain single protein
`components of the respective virus and are
`referred to as subunit vaccines. These vaccines
`protect against virus-induced cancers and are life-
`saving success stories [1]. Developments in
`molecular biology also allowed the engineering of
`carrier viruses encoding heterologous antigens of
`interest. Such viral vectors efficiently enter cells
`where the encoded antigens are produced by the
`endogenous protein synthesis machinery. The
`first example of a licensed viral vector vaccine was
`the Vesicular stomatitis virus-based vaccine
`against Ebola, approved in 2019, which was soon
`followed by an adenovirus-based Ebola vaccine
`[2].
`
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`
`
`Figure 1. Methods for vaccine production before the COVID-19 pandemic.
`Currently used vaccines are made from weakened or inactivated whole viruses, recombinant viral protein components
`(subunit vaccines), or viral vectors delivering antigens of interest (vector vaccines). The vaccination event stimulates
`antigen-specific immune responses, which provide protection if the vaccinated person is later exposed to the live
`pathogen.
`
`Both traditional whole virus-based vaccines and
`viral vector-based vaccines require cell culture-
`based manufacturing
`facilities.
`Vaccine
`researchers have therefore long been interested
`in the development of subunit vaccines that
`circumvent the need for large scale cell cultures by
`delivering nucleic acid (DNA or mRNA) directly to
`vaccine recipients, exploiting the body’s own
`capacity to produce proteins. There was a strong
`sentiment that the availability of such platforms
`would not only increase the world’s capacity to
`make vaccines, but also facilitate more rapid and
`less costly vaccine production in response to
`pandemics.
`
`Early work on nucleic acid- and viral vector-
`based vaccines
`The first demonstrations that nucleic acid-based
`immunizations could work date back to the early
`1990´s when DNA vaccines [3] and mRNA
`vaccines [4] were first tested in mice. There were
`several
`potential
`advantages with
`these
`approaches. Not only are nucleic acid-based
`vaccines easy to manufacture; they are also
`flexible since the sequence can be easily changed
`to encode different antigens. Together with the
`ease of production, this makes iterative testing of
`new candidate vaccines and the generation of
`updated vaccines rapid and efficient. A biological
`advantage is that in addition to antibody and major
`histocompatibility complex
`(MHC) class
`II-
`restricted CD4+ T cell responses, which are also
`induced by other vaccine types, viral vector- and
`nucleic acid-based vaccines have the potential to
`stimulate cytotoxic CD8+ T cell responses since
`they allow presentation of endogenously produced
`
`antigenic peptides on MHC class I molecules.
`Induction of CD8+ T cells is particularly interesting
`in the context of cancer vaccines where the aim is
`to kill targeted tumor cells, and for anti-viral
`vaccines aimed
`to eliminate
`infected cells.
`However, despite the potential advantages of
`nucleic acid-based vaccines, whether they would
`be well-tolerated and stimulate sufficiently robust
`immune response in humans to represent a viable
`path forward for clinical vaccine development was
`unclear.
`
`Initially, DNA vaccines were considered more
`promising than mRNA vaccines since DNA is
`more stable. However, progress was slow and
`early encouraging results with DNA vaccines in
`small animals did not translate to humans [5]. A
`likely reason is that injected DNA must cross two
`barriers, the plasma membrane and the nuclear
`membrane, to reach the cellular compartment
`where transcription takes place (DNA conversion
`to mRNA). In contrast, mRNA-based vaccines
`only need to gain access the cell cytoplasm where
`translation takes place (mRNA conversion to
`protein), making delivery easier. An additional
`advantage with mRNA vaccines is that the
`delivered nucleic acid cannot integrate into the
`host genome, adding an important safety aspect
`to
`this platform. Despite
`these advantages,
`skepticism about the usefulness of the approach
`remained high since mRNA was considered too
`unstable for medical applications.
`
`Against this background, the vaccine field turned
`to the use of engineered viral vectors as these
`have their own intrinsic mechanisms to enter cells
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`and deliver genetic cargo. Since the 1990s, many
`different types of viral vector-based vaccines
`against a variety of pathogens have been tested
`preclinically, demonstrating both promising results
`and setbacks [6]. A drawback of viral vector-based
`vaccines
`is
`that
`in addition
`to
`the desired
`responses elicited against the antigen of interest,
`antibodies against the structural proteins used to
`package the vector may be induced, compro-
`mising booster responses if the same platform is
`used again. Nevertheless, effective viral vector-
`based vaccines using different types of engi-
`neered adenoviruses were developed during the
`COVID-19 pandemic and administered at scale,
`demonstrating their usefulness, especially in the
`early phase of a pandemic [7, 8].
`
`the 1990s, a small community of
`During
`investigators continued to explore the use of
`mRNA as a potential vaccine platform. Early
`studies had demonstrated that mRNA purified
`from cells was translated into protein when
`reintroduced into oocytes [9]. Delivery into tissue
`of a living organism was the next challenge. The
`first study to demonstrate that injection of naked
`mRNA into skeletal muscle resulted in protein
`production in vivo was published by Philip Felgner
`and colleagues in 1990 [10]. Soon thereafter,
`Martinon et al. demonstrated the induction of
`antigen-specific cytotoxic T lymphocyte responses
`in mice injected with liposome-formulated mRNA
`encoding the influenza virus nucleoprotein [4].
`
`investigators developed
`In parallel, several
`alphavirus replicon vaccines, which have the
`added advantage that a higher copy number of
`antigen-encoding transcripts are produced in
`each cell, resulting in the induction of robust
`antigen-specific immune responses following in
`vivo delivery of naked mRNA [11, 12]. These early
`studies stimulated the field and led to the demon-
`stration of promising results in animal models, but
`it would take more than two decades until the first
`mRNA-based vaccine against an infection was
`tested in human clinical trials.
`
`The discovery of mRNA and systems for in
`vitro transcription
`To explore
`the potential of mRNA-based
`applications, an efficient system for mRNA pro-
`duction and manipulation was needed. For this,
`the field relied on a series of fundamental research
`discoveries starting
`in
`the 1950s. After
`the
`landmark discoveries of DNA as the inherited
`genetic material, the search started for the
`intermediate molecule that was transcribed from
`nuclear DNA and transported to the ribosomes in
`the cytoplasm
`to specify protein synthesis.
`Experiments on cells
`infected with
`the T2
`bacteriophage identified a metabolically active
`
`RNA fraction constituting approximately 1% of the
`total cellular RNA [13] that had proper base ratios
`[14]. This unstable form of RNA, or messenger
`RNA (mRNA), was proposed to be the missing
`intermediate carrier of information [15], and the
`hypothesis soon gained experimental support
`through pulse-labeling experiments in bacteria
`[16, 17]. Around the same time, insight into how
`cells produce RNA from DNA was gained through
`the discovery of RNA polymerase [18-20]. In the
`following decades, several RNA polymerases
`were identified in bacteria and eukaryotic cells,
`including single-subunit RNA polymerases from
`the T7 [21] and SP6 [22] bacteriophages.
`
`Building on the discovery of the more versatile
`bacteriophage RNA polymerases , Paul Krieg and
`Douglas Melton demonstrated
`that synthetic
`mRNA could be produced in large quantities in
`vitro by using the SP6 RNA polymerase and cDNA
`clones containing the SP6 promoter [23, 24].
`Furthermore, the in vitro produced SP6 mRNA
`was efficiently
`translated
`into protein when
`injected into frog oocytes [23]. Around this time,
`the T7 RNA polymerase was cloned by William
`Studier’s lab [25] and developed into an efficient
`and inducible in vitro transcription system with a
`patent filed in 1984 [26]. The T7 RNA polymerase
`had several advantageous features, including
`highly specific binding to the T7 promoter (a
`conserved stretch of nucleotides -17 to +6 relative
`to the transcriptional start site) and an ability to
`transcribe RNA at a high speed. Similar efforts to
`harness the in vitro transcription capacity of T7
`RNA polymerase were pursued [27]. The T7 in
`vitro transcription system became further opti-
`mized into a highly efficient cell-free system for
`large-scale production of any mRNA of interest,
`with major impact on science and biotechnology.
`
`Delivering in vitro transcribed mRNA to cells
`and tissues
`Another important research area focused on how
`to deliver nucleic acids into cells. An early strategy
`was to use liposomes, small cell membrane-like
`vesicles
`composed of phospholipids and
`cholesterol. Already in 1978, researchers had
`described successful attempts at delivering
`purified globin mRNA into mouse lymphocytes
`and human epithelial cells using liposomes [28,
`29] simply by trapping the mRNA inside the
`liposome vesicles. The field of nucleic acid de-
`livery improved thanks to the pioneering work by
`Philip Felgner while at Syntex Research. Felgner
`synthesized the first cationic lipid (DOTMA) and
`showed that it could form stable liposomes with
`nucleic acids [30]. The positively charged lipids
`improved both the entrapment of negatively
`charged nucleic acids (through electrostatic inter-
`actions) and fusion to the negatively charged cell
`
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`Bethesda, she set up her own group at the
`Department of Neurosurgery at the University of
`Pennsylvania in 1997. Karikó had a strong drive to
`advance
`the mRNA
`platform
`and
`she
`systematically investigated different components
`of in vitro transcribed mRNA to identify require-
`ments for optimal protein expression in cells and
`tissues [34]. Among several findings, she demon-
`strated that lipofectin-complexed mRNA encoding
`luciferase, a reporter protein, could be delivered to
`the rat brain and she showed that expression was
`improved when a longer poly(A) tail was added to
`the mRNA 3' end [35]. Encouraged by these
`results, Karikó continued her quest to make the
`mRNA platform suitable for clinical use.
`
`mRNA delivery to dendritic cells and the role
`of innate sensing
`In the late 1990s, Karikó teamed up with Drew
`Weissman, a physician scientist with an interest in
`basic immunology and vaccine development, who
`had joined the University of Pennsylvania in 1997.
`Weissman had received his MD and PhD degrees
`from Boston University
`in
`immunology and
`microbiology in 1987. After a residency period at
`Beth Israel Deaconess Medical Center at Harvard
`Medical School in Boston, he joined Anthony
`Fauci’s group at the National Institutes of Health
`(NIH) for a post-doctoral fellowship to investigate
`how the human immunodeficiency virus type 1
`(HIV-1) interacts with target receptors on different
`types of immune cells. Having established his own
`group at the University of Pennsylvania, he
`focused increasingly on vaccine research and the
`use of dendritic cells to prime immune responses.
`Ralph Steinman was awarded a Nobel Prize in
`Physiology or Medicine for the discovery of
`dendritic cells in 2011. With Weissman’s back-
`ground in immunology and Karikó’s expertise in
`RNA biochemistry, the two scientists comple-
`mented each other well and shared a passion for
`exploiting the use of mRNA in medical applica-
`tions.
`
`Together, Karikó and Weissman tested whether in
`vitro transcribed mRNA could be delivered to
`dendritic cells to exploit their antigen-presentation
`potential. A major goal of Weissman was to
`develop a vaccine against HIV-1, a virus that
`causes chronic
`infections. This was an
`exceptional challenge given the extensive immune
`evasion properties of this virus, setting it apart
`from viruses
`that cause acute
`infections.
`Weissman was interested in using dendritic cells
`to prime antigen-specific T cells and had
`developed systems to culture dendritic cells and
`assess their activation and antigen presenting
`capacities. Dendritic cells have exquisite abilities
`to both sense pathogens and prime naïve T cells
`and thus they bridge the innate and adaptive
`
`membranes, resulting in improved delivery into
`cells. Cationic lipid-based liposomes (lipofectin)
`opened the door to the field of engineered DNA
`and RNA delivery into cells. Lipofectin was soon
`used to deliver in vitro transcribed mRNA into
`cultured cells to demonstrate protein production
`[31], encouraging future therapeutic applications.
`However, in vivo applications of lipofectin showed
`unwanted side effects and researchers continued
`the search for improved delivery systems.
`
` A
`
` second major improvement was made in the lab
`of Pieter Cullis at
`the University of British
`Columbia with the development of ionizable
`cationic lipids. These lipids could be maintained in
`a positively charged or neutral form depending on
`the pH of the environment. Forming these lipid
`nanoparticles (LNPs) at low pH had the benefits of
`cationic lipids in efficiently entrapping negatively
`charged mRNA within the vesicles. However,
`when delivered in vivo and exposed to physio-
`logical pH, the lipids lost their charge, which had
`several benefits including lower in vivo toxicity.
`The important discoveries by Cullis team spurred
`large industrial interest in the development of
`ionizable lipids. Notable, the delivery of nucleic
`acids was further optimized through the T-
`connector that could generate dense lipid nano-
`particles made of four components: an i) ionizable
`cationic lipid, ii) a helper lipid, iii) cholesterol and
`iv) polyethenylene glycol (PEG)
`[32]. More
`efficient ionizable cationic lipids were identified in
`large-scale screening programs in several biotech
`companies. Consequently, lipid nanoparticles now
`enable safe and efficient in vivo delivery of nucleic
`acids, including mRNA, into human cells. This
`advance is of great importance for clinical appli-
`cations of nucleic acid-based technologies.
`
` vision to use mRNA for the delivery of
`therapeutic proteins
`The potential of using the new molecular biology
`techniques to create mRNA-based vaccines or to
`treat human diseases by delivering mRNA to
`replace defective genes with functional ones, or by
`overexpressing a therapeutic protein, stimulated
`an enormous interest. In 1992, Jirikowski et al.
`used mRNA injection for in vivo expression of
`vasopressin to treat diabetes insipidus in a rodent
`model
`[33]. Around
`this
`time, a Hungarian
`research scientist at the University of Penn-
`sylvania, Katalin Karikó, experimented with
`different forms of RNA with the ambition to opti-
`mize expression of therapeutic proteins. Karikó
`completed her PhD at the Biological Research
`Center in Szeged in 1982. Following post-doctoral
`work at the Hungarian Academy of Sciences and
`subsequent
`research positions at Temple
`University in Philadelphia and at the Uniformed
`Services University of the Health Sciences in
`
` A
`
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`

`immune systems [36]. Karikó and Weissman
`showed that dendritic cells pulsed with in vitro
`transcribed mRNA encoding the HIV-1 structural
`protein, Gag, stimulated primary CD4+ and CD8+
`T cell responses in vitro [37]. The team also found
`that the process of mRNA loading resulted in DC
`activation and maturation [38], which initially was
`interpreted as a positive effect since activated
`dendritic cells are superior in T cell priming. The
`negative consequences of
`innate
`immune
`activation by in vitro transcribed mRNA were not
`fully appreciated at this point. Interestingly, and
`somewhat counterintuitively, this would turn out to
`be a critical factor for advancing mRNA-based
`vaccines.
`
`The observation that dendritic cells were activated
`following uptake of in vitro transcribed mRNA led
`to critical questions about which signaling
`pathways were engaged? Dendritic cells express
`both surface and endosomal Toll-like receptors
`(TLRs), which recognize distinct molecular struc-
`tures referred to as pathogen-associated mole-
`cular patterns (PAMPs) [39]. TLR binding to
`PAMPs results in intracellular signaling and pro-
`duction of anti-viral cytokines including type 1
`interferons, an effective warning system to detect
`incoming pathogens. Studies of how TLRs
`distinguish different forms of nucleic acid had
`gained traction after Hemmi et al. showed that
`unmethylated CpG motifs, abundant in microbial
`but rare in mammalian DNA, activate TLR9 [40].
`
`Within a few years, the ligands for most nucleic
`acid sensing TLRs had been identified, including
`TLR3 that senses double-stranded RNA (dsRNA),
`a viral replication intermediate, and TLR7 and
`TLR8 that sense single-stranded viral RNA and
`some forms of synthetic RNA [41, 42]. In 2004,
`Karikó and Weissman reported that in vitro
`transcribed mRNA contains dsRNA contaminants
`that can activate TLR3, leading to a cytokine
`response
`[43]. Another
`important clue was
`obtained when Koski, Karikó and Weissman
`together with Brian Czerniecki and colleagues
`demonstrated that transfection of dendritic cells
`with in vitro transcribed mRNA stimulated a
`cytokine response similar to that observed with
`prokaryotic RNA. Experimental manipulations to
`increase the poly(A) length of in vitro transcribed
`mRNA
`led
`to significantly
`reduced
`IL-12
`production. However,
`this was not
`the
`full
`explanation for the observed effects. When four
`homopolynucleotides, polyuridylic acid
`(pU),
`
`
`polyguanylic acid (pG), polycytidylic acid (pC),
`polyadenylic acid (pA), were tested using IL-12 as
`a read-out for DC activation, only pU induced a
`response, suggesting that the nucleotide content
`also played a role [44]. A similar finding, using
`interferon alpha as a readout, was reported the
`same year from Reis e Sousa’s group in their
`studies of RNA recognition by TLR7 [41].
`
`The Kariko, Weissman breakthrough
`Karikó and Weissman continued their careful
`studies of different types of RNA and the work
`resulted in a breakthrough publication in 2005.
`The study described the influence of mRNA base
`modifications on
`the cytokine
`response by
`dendritic cells [45]. They showed that eukaryotic
`mRNA and tRNA, in which base modifications are
`abundant, did not stimulate a cytokine response
`while prokaryotic and in vitro-transcribed mRNA
`did. They further showed that the incorporation of
`pseudouridine (Ψ), 5-methylcytidine (m5C), N6-
`methyladenosine (m6A), 5-methyluridine (m5U) or
`2-thiouridine (s2U) into in vitro transcribed mRNA
`abrogated activation of inflammatory responses
`when these mRNAs were added to dendritic cells
`[45]. The incorporation of m6A and s2U almost
`completely abrogated recognition by TLR3, while
`TLR7 and TLR8 activation could be evaded using
`m6A, s2U, m5C, m5U and Ψ. Importantly, only
`modifications of uridines (m5U, s2U and Ψ)
`abolished DC activation (Figure 2).
`
`To date, researchers have uncovered more than
`one hundred different post-transcriptional modi-
`fications in RNA and shown that modifications are
`more extensive in RNA of eukaryotes than pro-
`karyotes [46, 47]. Pseudouridine (Ψ) was dis-
`covered already in 1951 [48] and is one of the
`most abundant RNA modifications, initially found
`in tRNAs and small nuclear RNAs (snRNAs) and
`more recently in other types of RNA. Cells modify
`RNA through enzymatic reactions, for example
`pseudouridine
`is catalyzed by pseudouridine
`synthase enzymes, or using small ribonucleo-
`protein (snoRNPs) complexes. RNA modifications
`contribute to RNA stability, base-pairing specifi-
`city, folding and other functional properties. Of the
`over one hundred RNA modifications known [49],
`limited functional data exists on most modifi-
`cations. Understanding the physiological implica-
`tions of these modifications therefore remains an
`active research field.
`
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`
`
`Figure 2. Evaluation of in vitro transcribed mRNA with or without nucleoside base modifications and
`transfection into primary dendritic cells.
`(a) The T7 in vitro transcription system was used to produce mRNA with canonical RNA bases (A, U, G and C) or
`modified bases. (b) The bases used for in vitro transcription of RNA-1571 are shown, with those that did not result in
`TNF-alpha secretion indicated in orange (modified from Karikó et al. Immunity 2005).
`
`The Karikó and Weissman discovery explained an
`observation made over 40 years earlier by Isaacs
`and colleagues demonstrating that delivery of
`deaminated RNA into cells resulted in a stronger
`type 1 interferon response than control RNA [50].
`Deamination increases the proportion of uridines
`in the RNA, which Kariko and Weissman had
`demonstrated was critical for DC activation. Later
`work showed that the use of N1-methylpseudo-
`uridine (m1Ψ), alone or in combination with m5C,
`further improved the mRNA platform both in terms
`of reducing recognition of innate immune recep-
`tors and increasing protein expression [51], the
`latter was in part explained by an increased
`ribosome occupancy on m1Ψ-containing mRNA
`[52]. Today, m1Ψ is the most common modified
`base used in mRNA vaccine production, including
`in the two COVID-19 vaccines approved in late
`2020, as discussed below.
`
`that
`their breakthrough discovery
`Following
`incorporation of modified bases evades undesired
`immune activation by in vitro transcribed mRNA,
`
`
`that
`Karikó and Weissman demonstrated
`pseudouridine-containing mRNA was also more
`efficiently translated, resulting in higher protein
`production in cells that have taken up the mRNA
`[53] (Figure 3). In the same study, they showed
`that delivery of modified mRNA into the spleen of
`mice led to increased protein production and
`decreased
`immune activation, an
`important
`demonstration for future therapeutic applications.
`Karikó, Weissman and colleagues further demon-
`strated that in vitro transcribed mRNA activates
`protein kinase R (PKR), an anti-viral protein that
`protects cells
`from
`invading pathogens by
`recognizing dsRNA by phosphorylating
`the
`eukaryotic translation initiation factor 2 alpha
`(eIF2a), blocking protein translation. The team
`showed that the use of modified bases reduced
`activation of PKR and improved protein production
`[54]. Recognition of in vitro transcribed mRNA by
`the 2’5’ oligoadenylate synthetase (OAS) and
`degradation by the OAS-induced Rnase L enzyme
`were also decreased with RNA containing
`modified bases [55].
`
`
`
`Figure 3. Higher protein
`expression from base-modified
`in vitro-transcribed mRNA.
`Base-modified in vitro transcribed
`mRNA was produced where
`uridines (U) were substituted with
`pseudouridine (Ψ). When base-
`modified mRNA was introduced
`into cells, an increased protein
`production compared
`to
`that
`achieved with unmodified mRNA
`was observed.
`
`
`
`
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`

`Furthermore, Karikó and colleagues showed that
`dsRNA contaminants produced during in vitro
`transcription could be removed through an HPLC
`purification step [56], or as later reported together
`with Uğur Şahin and colleagues at BioNTech, by
`using a cellulose-based purification step [57],
`further improving the expression of protein from in
`vitro transcribed mRNA.
`
`Research leading up to the mRNA vaccines
`against COVID-19
`By 2010, three main companies with programs
`focusing on the emerging mRNA technology had
`been established: CureVac, founded in 2000
`aimed to develop vaccines against infections and
`cancer; BioNTech founded in 2008 had the
`objective
`to develop personalized cancer
`vaccines; and Moderna, founded in 2010 planned
`to use the mRNA platform to reprogram somatic
`cells to pluripotent cells and to deliver therapeutic
`proteins, for example to repair damaged tissue. All
`three companies collaborated closely with acade-
`mic researchers to improve the technology and
`evaluate their respective platforms in disease
`areas of interest.
`
`The team behind Curevac, including Ingmar
`Hoerr, Günter Jung, Steve Pascolo and Hans-
`Georg Rammensee, had realized the potential of
`the mRNA technology early on. They developed
`approaches to improve the efficiency of protein
`production through optimizations of the mRNA 5’
`and 3' untranslated
`regions and codon
`optimization, without using modified bases. In
`2000, they reported that administration of RNA,
`either naked or liposome-complexed, induced
`antigen-specific adaptive immune responses in
`mice (antibody and CD8+ T cell responses) with
`the liposome-encapsulated RNA giving higher
`responses [58]. They evaluated their first mRNA
`vaccine in humans approximately eight years later
`when genetic material from tumors of melanoma
`patients was extracted and used to generate
`mRNA that was administered as an autologous
`vaccine with granulocyte-macrophage colony-
`stimulating factor (GM-CSF) as an adjuvant. The
`approach was shown to be safe and to increase
`anti-tumor immune responses in some patients
`[59]. In 2012, the Curevac team reported elicitation
`of protective immune responses against influenza
`virus infection in several animal models [60] and in
`2017, the first mRNA-based vaccine against an
`infectious disease, rabies, was tested in clinical
`trials.
`
`Activities in the mRNA vaccine field now expanded
`rapidly. In 2017, promising pre-clinical results of
`
`that used
`mRNA-based Zika virus vaccines
`modified bases were reported by Norbert Pardi
`and Weissman [61] and by Michael Diamond and
`colleagues at Washington University School of
`Medicine [62]. The latter study, which described
`vaccination of pregnant females, demonstrated
`protection against viral transmission to the fetus, a
`major concern with Zika virus infections. In 2017,
`Moderna announced the start of a clinical trial with
`an mRNA-based vaccine against Zika virus
`(ClinicalTrials.gov: NCT03014089). Moderna also
`initiated two phase I clinical trials to evaluate the
`safety and immunogenicity of their mRNA vaccine
`candidates against influenza virus H10N8 and
`H7N9, two avian influenza strains with pandemic
`potential
`[63,
`64]
`(ClinicalTrials.gov
`NCT03076385 and NCT03345043).
`
`Around the time of the Zika vaccine trial, Moderna
`also initiated collaborations with Barney Graham
`and his team at the Vaccine Research Center at
`the NIH to develop an mRNA-based vaccine
`against Middle East Respiratory Syndrome
`coronavirus (MERS-CoV). The vaccine encoded a
`prefusion-stabilized form of the MERS spike
`where, among other modifications, prolines were
`introduced in the S2 domain to prevent the
`metastable prefusion form transitioning into the
`post-fusion form [65]. Early work by Qiao et al. had
`showed that the introduction of prolines in the
`influenza virus hemagglutinin 2 domain (HA2),
`which undergoes a loop to helix transition at low
`pH, interferes with the ability of the influenza virus
`to fuse with host membranes [66]. Based on this
`finding, and the knowledge that viruses from
`different families have evolved similar solutions for
`fusing with target cells, appropriately positioned
`prolines have been substituted into the spike
`glycoproteins of several viruses to stabilize them
`in their respective prefusion forms, including but
`not limited to HIV-1 [67], Respiratory syncytial
`virus [68] and SARS-CoV-2 [69]. The high-
`resolution structure of the SARS-CoV-2 spike
`published in record time by Jason McLellan’s
`group in early 2020 proved invaluable for several
`of the successful COVID-19 vaccines, as well as
`for the definition of neutralizing antibody epitopes
`and antibody escape mutations in later emerging
`SARS-CoV-2 variants, information that is of great
`importance for our understanding of vaccine-
`induced
`immune protection. The prefusion-
`stabilized form of the SARS-CoV-2 spike was
`used
`in
`the mRNA vaccines developed by
`Pfizer/BioNTech and Moderna (Figure 4), as well
`as in the vector vaccine by Janssen and the
`protein-based vaccine developed by Nov