mRNA Vaccines in Disease Prevention and Treatment

Signal Transduction and Targeted Therapy www.nature.
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REVIEW ARTICLE OPEN
mRNA vaccines in disease prevention and treatment

1,2,3,4,5 ✉ 1,2,3,4,5 ✉
Gang Zhang1,2,3,4,5, Tianyu Tang1,2,3,4,5, Yinfeng Chen1,2,3,4,5, Xing Huang and Tingbo Liang
mRNA vaccines have emerged as highly effective strategies in the prophylaxis and treatment of diseases, thanks largely although
not totally to their extraordinary performance in recent years against the worldwide plague COVID-19. The huge superiority of
mRNA vaccines regarding their efficacy, safety, and large-scale manufacture encourages pharmaceutical industries and
biotechnology companies to expand their application to a diverse array of diseases, despite the nonnegligible problems in design,
fabrication, and mode of administration. This review delves into the technical underpinnings of mRNA vaccines, covering mRNA
design, synthesis, delivery, and adjuvant technologies. Moreover, this review presents a systematic retrospective analysis in a logical
and well-organized manner, shedding light on representative mRNA vaccines employed in various diseases. The scope extends
across infectious diseases, cancers, immunological diseases, tissue damages, and rare diseases, showcasing the versatility and
potential of mRNA vaccines in diverse therapeutic areas. Furthermore, this review engages in a prospective discussion regarding
the current challenge and potential direction for the advancement and utilization of mRNA vaccines. Overall, this comprehensive
review serves as a valuable resource for researchers, clinicians, and industry professionals, providing a comprehensive
understanding of the technical aspects, historical context, and future prospects of mRNA vaccines in the fight against various
diseases.
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Signal Transduction and Targeted Therapy (2023)8:365 ; https://doi.org/10.1038/s41392-023-01579-1
INTRODUCTION possess multiple beneficial features over traditional vaccines.3,8

Vaccines have proven remarkable efficacy in preventing the spread Indeed, mRNA vaccines use the body cell as the core facility for a
of infectious diseases, causing the preservation of countless lives natural induction of both innate and adaptive immunity (Fig. 1),
annually.1,2 The extensive implementation of vaccines in recent enabling posttranslational modification and full functionality of
decades has led to the elimination of smallpox and an extremely protein products, allowing the correct translation folding and
low incidence of polio, measles, and other infectious diseases.3 The assembly in the host cells of multimeric and versatile proteins that
World Health Organization reports that vaccination prevents cannot be produced in bioreactors, and allowing the transfer of
approximately 2 million mortalities from measles, influenza, the produced intracellular and transmembrane proteins to their
pertussis, and tetanus every year.4 Moreover, the use of vaccines suitable cellular locations.9–14 mRNA vaccines can be designed to
in cancer management shows potent efficacy in preclinical trials, encode any antigen based on the unique attributes of diseases.
becoming one of the most promising treatments in the field of Moreover, compared with DNA vaccines, mRNA vaccines avoid the
immune oncology and gaining more attention than ever. potential risk of insertional mutagenesis in the host genome and
Conventional vaccines have several disadvantages that may limit cause adjustable expression of the selected antigen.15–17 From the
their application in disease prevention and treatment. For instance, commercial point of view, the mRNA vaccine allows rapid
the underlying procedure for the development of dendritic cell development and large-scale production through a cell-free
(DC) vaccines involves a labor-intensive and time-consuming process due to the highly productive transcription reaction
process that necessitates the preparation of patient-autologous in vitro, which is also extremely cost-effective.1,3,7,15–17 Notably,
cells. The engineering and fabrication of microorganism-based although mRNA itself also has several disadvantages compared
vaccines entail intricate and complex processes. Peptide vaccines with other vaccine modalities (e.g., poor stability and potent
exhibit MHC restriction, selectively activating monoclonal T cells, immunogenicity that limit the usage of mRNA vaccines in vivo),
thus having a high risk of immune escape.5 DNA vaccines have improvements in modifications and delivery largely address these
risks of genomic alteration, long-term expression, and generation obstacles, ensuring the maintenance of in vivo stability as well as
of anti-DNA autoantibodies that might impede their utilization in the balance between the initiation of a robust immune responses
humans.6,7 Therefore, it is essential to select a suitable vaccine and irreversible adverse reactions caused by lasting func-
format with promising value for disease prevention and treatment. tion.1,3,7,12,15–20 For all these reasons, mRNA vaccines have
Vaccines using messenger RNA (mRNA), a single nucleotide emerged as a promising modality in the prevention and treatment
sequence that functions as a template for protein translation, of a number of diseases.
1
Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, Zhejiang University School of Medicine, 310009 Hangzhou, Zhejiang, China; 2Department
of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, 310003 Hangzhou, Zhejiang, China; 3Zhejiang Clinical Research
Center of Hepatobiliary and Pancreatic Diseases, 310003 Hangzhou, Zhejiang, China; 4The Innovation Center for the Study of Pancreatic Diseases of Zhejiang Province, 310009
Hangzhou, Zhejiang, China and 5Cancer Center, Zhejiang University, 310058 Hangzhou, Zhejiang, China
Correspondence: Xing Huang (huangxing66@zju.edu.cn) or Tingbo Liang (liangtingbo@zju.edu.cn)
These authors contributed equally: Gang Zhang, Tianyu Tang
Received: 10 January 2023 Revised: 1 July 2023 Accepted: 30 July 2023
© The Author(s) 2023

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Fig. 1 Dual effects of mRNA vaccine on immune activation. mRNA vaccines induce both innate and adaptive immunity. Endocytosis of
exogeneous mRNA by antigen presenting cells is sensed by TLR3 and TLR7/8 in the endosomes as well as RIG-1, NOD2, LGP2, and MDA-5 in
the cytosol, inducing strong IFN-I responses, then triggering proinflammatory cytokine production, thereby activating innate immunity (left).
mRNA-encoded protein is released out of the cell to activate B cells, while mRNA-encoded or re-endocytosed proteins are degraded as
peptides in the proteasome to be presented on MHC-I or MHC-II molecules to activate CD4+ and CD8+ T cells, cocontributing to adaptive
immunity activation (right). This figure is created using Adobe Illustrator and is inspired by these two papers257,258
This comprehensive review provides an in-depth exploration of

the technical foundations of mRNA vaccine, encompassing
essential aspects such as mRNA design, synthesis, delivery, and
adjuvant technologies. This comprehensive review presents a
methodical and structured analysis of representative mRNA
vaccines used in a diverse array of medical conditions, including
infectious diseases, cancers, immunological diseases, tissue
damages, and rare diseases. Furthermore, this review includes a
forwards-looking discourse on the current obstacles and potential
possibilities in the development and implementation of mRNA
vaccines.
MRNA VACCINE DEVELOPMENT

The development of mRNA vaccines is the culmination of
extensive research spanning several decades. The discovery of
mRNA dates back to 1961, and its isolation for in vitro protein
expression was first achieved in 1969.21,22 In 1990, in vitro
transcribed mRNA was successfully validated as a template for
synthesizing proteins in mouse skeletal muscle cells in vivo,
marking a breakthrough in in vivo mRNA expression and laying
the groundwork for the development of mRNA vaccines.23 In
Fig. 2 Pipeline for the development of mRNA vaccines. The
1992, vasopressin mRNA was injected into the hypothalamus, development of mRNA vaccines includes a series of steps, including
successfully expressed, and yielded physiological responses.24 sequencing design, in vitro transcription, purification, nanoprecipi-
Subsequently, in 1993 and 1995, mRNA was found to elicit both tation, and filtration. This figure is created using Adobe Illustrator
innate and adaptive immunity.25–27 Despite these promising and refers to this paper4
findings, the development of mRNA vaccines initially faced limited
investment, mainly owing to concerns over their instability, mRNA design
inefficient in vivo transportation, and possible innate immuno- The advancement of mRNA vaccines faced a major obstacle
genicity. However, due to their safety, straightforward design, and owing to the instability of mRNAs and poor translational
simplicity of manufacturing, research on mRNA has persevered. efficiencies.4,28 In vitro, transcribed mRNA comprises five primary
Ultimately, this persistence paid off, as evidenced by the elements, namely, the 5ʹ cap, 5ʹ untranslated region (UTR), an
development of highly effective mRNA vaccines against COVID- open reading frame (ORF), 3ʹ UTR, and a poly(A) tail, all of which
19, which have played a pivotal role in the ongoing efforts to simulate the structure of an endogenous mRNA.4,28 To enhance
control the pandemic. To date, a comprehensive framework has mRNA translational efficacy, scientists have devised various
been established for the development of mRNA vaccines, techniques to modify each of these components and optimize
including design, synthesis, and delivery technologies (Fig. 2). mRNA design.
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The 5′ cap, a modified nucleotide structure situated at the 5′ against emerging infectious diseases. ORF sequences have been
end of the mature mRNA molecule, comprises a guanine optimized to enhance mRNA stability and translation efficiency.
nucleotide linked to the mRNA via a triphosphate linkage, with One such approach involves optimizing the codon usage of the
additional methylations at the 7th position of the guanine and/or ORF, thereby improving translation efficiency and reducing
the 2′ position of the first transcribed nucleotide.29,30 It has vital premature termination.4,28,40 Another strategy involves incorpor-
roles in several aspects of mRNA stability and functions, including ating specific RNA modifications, including a pseudouridine, to
protection against exonucleases, enhancement of mRNA transla- enhance the stability and accuracy of mRNA translation.4,16,28,41
tion efficiency, and facilitation of transport from the nucleus to the Additionally, the use of nonnatural amino acids in the ORF can
cytoplasm.4,31 In mRNA vaccines, the inclusion of a 5′ cap structure expand the epitope repertoire presented by the antigen, thereby
is critical for the stabilization of mRNA molecules and the potentially inducing a broader immune response. The develop-
promotion of efficient translation to the encoded protein. Notably, ment of efficient and effective ORF design strategies is vital for the
the 5′ cap modification, particularly the m7G cap, boosts mRNA success of mRNA vaccines. These endeavors are expected to result
translational efficiency by facilitating its recognition by the in the development of more potent and versatile mRNA vaccines
translation initiation complex.4,32 Furthermore, prior researches with broad application prospects for disease prevention and
have highlighted the capability of the m7G cap to protect mRNA treatment.
from nucleases, thereby enhancing its stability and Notably, modified nucleosides have gained widespread popu-
immunogenicity. larity within mRNA technology owing to their ability to enhance
The poly(A) tail is a critical posttranscriptional modification of the stability, translational efficiency, and immunogenicity of mRNA
mRNA that significantly contributes to its stability, export, and molecules.42–44 These nucleoside analogs can be integrated into
translation. In eukaryotic cells, the process of mRNA maturation the mRNA sequence in in vitro transcription, resulting in the
involves the addition of a long chain of adenine nucleotides at the formation of modified mRNA molecules with superior properties
3′ end of the mRNA molecule, with a typical length ranging from relative to their unmodified counterparts. Among the most
50–250 nucleotides.31,33 A key function of the poly(A) tail is to frequently employed modified nucleosides in mRNA are pseu-
safeguard mRNA from exonucleases, which are enzymes that can douridine, 5-methylcytidine, and 2-thiouridine.42–45 Pseudouridine
degrade RNA from its ends.29,31 Additionally, the poly(A) tail improves mRNA stability and translational efficiency while
facilitates the export of mRNA from the nucleus to the cytoplasm, reducing the activation of innate immune responses.45
wherein it can be translated into proteins.34–36 Furthermore, the 5-Methylcytidine elevates protein expression levels, while
poly(A) tail is involved in the initiation of protein synthesis.34–36 It 2-thiouridine enhances the precision of translation by increasing
interacts with poly(A)-binding protein, which recruits the ribo- the binding affinity between mRNA and ribosomes.45 Other
some to the mRNA, thus promoting efficient translation.34–36 The modifications, including N1-methylpseudouridine and 5-methox-
introduction of the poly(A) tail in mRNA vaccines serves two yuridine, have also been utilized to improve mRNA stability and
critical purposes. First, it stabilizes the mRNA molecule and translation efficiency.45 The incorporation of modified nucleosides
protects it from degradation by cellular enzymes. Second, it in mRNA technology holds considerable promise for the devel-
enhances the mRNA’s translation efficiency, leading to increased opment of more effective and safer mRNA-based therapeutics,
expression of the antigen and a more potent immune response. including vaccines and gene therapies for a wide range of human
The length of the poly(A) tail in mRNA vaccines is meticulously diseases.
optimized to balance mRNA stability and translation efficiency.
The UTRs of mRNA play a crucial role in the regulation of gene mRNA vaccine synthesis
expression.31,32 Located at the 5′ and 3′ ends, these regions are The production of mRNA by in vitro transcription involves the use
involved in the control of mRNA stability, translation efficiency, of RNA polymerase enzymes for synthesizing mRNA from a DNA
and subcellular localization, thereby regulating the production template outside a living cell. The upstream process entails using a
and function of the corresponding protein.4 The coding sequence plasmid as a template and transcribing it into primary mRNA using
of mRNA determines the protein sequence, while the UTRs T7, SP6, or T3 RNA polymerase.3 This reaction takes only a few
regulate its expression. Specifically, the 5′ UTR plays critical roles in hours and yields a few milligrams of primary mRNA per milliliter of
regulating mRNA stability and translational efficiency, with reaction. Subsequently, capping of primary mRNA occurs during
modifications to the 5′ cap structure and length of the 5′ UTR transcription using a Cap analog instead of the natural substrate
enhancing the two.28,37 Alternative splicing of the 5′ UTR can alter or via a two-step enzymatic reaction using RNA 2′-O-ribose
the translational efficiency of mRNA.37 Similarly, the 3′ UTR transferase, RNA methyltransferase, and a methyl donor sub-
regulates mRNA stability through the binding of regulatory strate.46–49 Although utilizing Cap analogs is a rapid and practical
proteins and microRNAs, which can either destabilize or stabilize approach to cap mRNA, its employment is impeded by the
mRNA. Modifying the 3′ UTR, for instance, by adding poly(A) tails, relatively high costs and the instability associated with the
can enhance mRNA stability and protein expression. In mRNA resultant m7GpppN cap structure.4,49 Conversely, the two-step
vaccine design, UTRs are meticulously engineered to optimize enzymatic reaction produces a more authentic and stable
protein expression and immune responses.29 The 5′ UTR can be m7GpppN cap structure, albeit requiring additional steps and
modified to enhance translation efficiency, while the 3′ UTR can enzyme reactions, as well as a meticulous selection of suitable
be modified to stabilize mRNA and prolong protein expression, enzymes and methyl donor substrates.28 To meet clinical quality
resulting in improved immunogenicity and efficacy of mRNA standards, the mRNA generated upstream needs to undergo
vaccines.37 multiple purification steps to separate and purify it from the
The ORF, beginning with a start codon and ending with a stop reaction mixture. Size exclusion chromatography is a commonly
codon, is a critical segment of mRNA translated into a protein by utilized method for separating mRNA molecules based on their
the ribosome.4,28 The length of the ORF can vary from a few sizes and shapes.50–52 This approach is both simple and gentle,
hundred to several thousand nucleotides.38,39 The sequence of the making it effective for removing impurities, including residual
ORF is responsible for determining the identity and structure of DNA, RNA, and proteins. Reverse-phase high-performance liquid
the protein synthesized, thus playing a pivotal role in the chromatography separates mRNA molecules based on their
effectiveness of mRNA.38,39 In the context of mRNA vaccines, the hydrophobicity, thus providing high resolution and purity, but it
importance of ORF design is paramount, as it directly affects the can be time-consuming and requires expensive equipment.
production of the target antigen. Advances in mRNA vaccine Affinity chromatography is another strategy for purifying mRNA
technology have facilitated their rapid design and production vaccines, whereby specific ligands are used to capture and purify

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the mRNA molecules.32,50,51 This method can provide high interactions, hydrophobic interactions, and covalent bonding.41,56
specificity and yield but may require additional steps for ligand These have lower immunogenicity and toxicity than polyplexes
immobilization and can be costly. Ion exchange chromatography and LNPs and can be engineered to enhance their stability and
is another common method for mRNA purification, which targeting specificity.56,57 However, their transfection efficiency
separates molecules based on their charge.29,50,51 Although this may be lower than that of LNPs, and their production can be more
method has high yield and purity, it may require multiple steps complex and costly.
and careful optimization to achieve optimal results. In addition to In general, the choice of delivery system depends on several
chromatography-based methods, precipitation-based approaches, factors, including the specific characteristics of the mRNA vaccine
including isopropanol or ethanol precipitation, can also be used to and the desired transfection efficiency, safety, stability, and target
purify mRNA vaccines.51 These methods are simple and cost- specificity.
effective but are less effective in removing impurities, and
additional steps for resuspension and quality control may be mRNA vaccine adjuvants
needed. Ultimately, the purification method chosen for mRNA Immunogenicity modulation is a nonnegligible issue in mRNA
vaccines depends on various factors, including the desired purity vaccine development. Although in vitro transcriptional mRNA has
level, scalability, cost, and downstream applications. shown some self-adjuvant potential, it is typically not enough to
elicit comprehensive protective immunity and requires intensified
mRNA vaccine delivery repeated/booster regimens for optimal effectiveness.58 Multiple
The delivery of mRNA vaccines into cells presents significant strategies have been applied for adjuvants of mRNA vaccines to
challenges due to the inherent instability of RNA and the need to regulate their immunogenicity. TriMix is a combination of mRNAs
protect it from degradation in the extracellular environment. Over that encode three distinct immune-stimulating proteins: CD40
the past few decades, researchers have explored various delivery ligand (CD40L), CD70, as well as constitutively active Toll-like
systems to overcome these challenges and enhance the efficacy receptor 4 (TLR4).59–61 Due to its ability to improve DC activation
of mRNA vaccines. and enhance the elicitation of CD8+ T-cell responses, TriMix has
One of the earliest approaches was the use of naked mRNA been incorporated into numerous vaccination studies. Moreover,
molecules, which were directly injected into cells or tissues.4,28,30 the utilization of cationic lipids is widely recognized for its ability
Herein, mRNA is delivered without a carrier, allowing it to be to improve RNA uptake and facilitate its endosomal escape,
translated into antigen proteins within cells. While naked mRNA resulting in increased adjuvant activity for mRNA vaccines.62,63
vaccines are relatively easy to produce and have shown promise in Furthermore, the incorporation of a synthetic mRNA sequence
preclinical studies, they are less stable and may elicit weaker with a polymeric carrier has been shown to enhance the
immune responses than mRNA vaccines delivered with carriers.38 adjuvanticity of various subunit vaccines.64 CureVac has devel-
Another early approach was the mRNA-DC vaccine, which oped RNActive® vaccines, which demonstrate inherent self-
involved the loading of DCs with mRNA encoding the desired adjuvant activity by incorporating naturally occurring nucleotides
antigen.4,28,30 The DCs then present the antigen to the immune complexed with protamine.65,66 The co-delivery of this mRNA
system, leading to a robust immune response. This approach has construct has been proven to significantly amplify B and T-cell
shown promise in preclinical studies for the treatment of cancers responses along with the amplification of subpopulations (e.g.,
and infectious diseases. In recent years, lipid-based nanoparticles Th1 and Th2 cells) and pre-germinal center B cells. However, the
(LNPs) and polyplexes/polymeric nanoparticles have been two of adjuvant properties of these strategies usually activate type I
the most commonly used mRNA vaccine delivery systems.38,41 interferon (IFN-I), which might cause the suppression of protein
LNPs are extensively utilized as delivery systems for mRNA translation as well as CD8+ T-cell activation.67,68 To overcome this
vaccines owing to their biocompatibility, stability, and ability to limitation, a hybrid nanoparticle system comprising a poly lactic-
protect mRNA from degradation.4,28,38,41,53,54 LNPs can be co-glycolic acid core and a lipid shell has been developed for
categorized based on the nature of their lipid components, simultaneous delivery of mRNA and a hydrophobic TLR7 adjuvant
surface charge, and surface modifications.41,55 One category is (gardiquimod). Poly lactic-co-glycolic acid facilitates the integra-
cationic LNPs, with positively charged lipid components interact- tion of the adjuvant within the nucleus, whereas the lipid shell
ing with the negatively charged phosphate backbone of mRNA, enables the loading of mRNA via electrostatic interactions. This
facilitating the latter’s delivery into target cells.38 Previous studies approach has demonstrated potent immune responses targeting
have provided evidence of the efficacy of ionizable LNP-based specific antigens and highly effective antitumour activities.69
vaccines against different infectious diseases.4,55 Ionizable LNPs
hold great potential as a delivery vehicle for mRNA-based
vaccines.4,38,41 These nanoparticles are composed of a central MRNA VACCINES IN INFECTIOUS DISEASES
core of mRNA enclosed by a lipid bilayer that incorporates mRNA vaccines are applied as prophylaxis against infectious
ionizable lipids, which allow effective mRNA encapsulation and diseases by encoding disease-specific antigens. To date, many
protection from degradation in the extracellular milieu. Moreover, preclinical and clinical trials using mRNA vaccines to induce
the ionizable lipids are instrumental in promoting the endosomal antiviral immunity have been performed in multiple infectious
release and cytoplasmic transport of the mRNA cargo, which is diseases, including severe acute respiratory syndrome coronavirus
pivotal for efficient protein expression. Polyethylene glycol (PEG)- 2, zika virus, human immunodeficiency virus, influenza virus,
ylated LNPs have a hydrophilic coating of PEG on their surface, cytomegalovirus, respiratory syncytial virus, varicella-zoster virus,
which enhances biocompatibility and reduces toxicity.28,38 and rabies virus (Table 1 and Fig. 3).
Polyplexes and polymeric nanoparticles are versatile delivery
systems that have been extensively studied for mRNA vaccines. mRNA vaccines against severe acute respiratory syndrome
Polyplexes are formed by electrostatic interactions between coronavirus 2
positively charged polymers, such as polyethyleneimine, and Since the beginning of 2021, severe acute respiratory syndrome
negatively charged mRNA molecules.14 These effectively protect coronavirus 2 (SARS-CoV-2) has infected countless people as well
mRNA from degradation, facilitate cellular uptake and enhance as caused millions of deaths worldwide. The majority of SARS-CoV-
immunogenicity due to their cationic charge.14 Polymeric 2 infections do not pose a life-threatening risk to individuals
nanoparticles can be formed from various polymers, including without preexisting diseases; however, in cases of severe infection,
poly lactic-co-glycolic acid and PEG, and mRNA can be encapsu- uncontrolled immune responses can be triggered in the lungs,
lated through multiple mechanisms, involving electrostatic destroying epithelial cells and alveoli, causing pulmonary edema,

Table 1. Clinical trials for mRNA vaccines in infectious diseases
Catalogue NCT number mRNA vaccine Encoded antigen Vehicle Phase Status
Severe acute respiratory syndrome NCT04283461 mRNA-1273 Full-length, prefusion stabilized spike protein Lipid Nanoparticle Phase I Completed
coronavirus 2 NCT04470427 mRNA-1273 Full-length, prefusion stabilized spike protein Lipid Nanoparticle Phase III Completed
NCT04368728 BNT162b1, BNT162b2 Spike glycoprotein receptor-binding domain Lipid Nanoparticle Phase II/III Completed
NCT04480957 LUNAR-COV19 Spike protein with two proline substitutions Lipid Nanoparticle Phase I/II Completed
NCT04860258 CVnCoV Spike protein with two proline substitutions Lipid Nanoparticle Phase III Terminated
NCT04847102 ARCoV Spike glycoprotein receptor-binding domain Lipid Nanoparticle Phase III Recruiting
NCT05364047 LVRNA009 Spike glycoprotein receptor-binding domain Lipid Nanoparticle Phase I Recruiting
Zika virus NCT03014089 mRNA-1325 Glycoproteins of Zika virus Unknown Phase I Completed
NCT04917861 mRNA-1893 Glycoproteins of Zika virus Lipid Nanoparticle Phase II Active, not
recruiting
Human immunodeficiency virus NCT02042248 AGS-004 Human immunodeficiency virus-1 antigen DC Phase I Completed
NCT00381212 AGS-004 Human immunodeficiency virus-1 antigen DC Phase II Completed

Influenza NCT03076385 VAL-506440 Membrane-bound form of the hemagglutinin Lipid Nanoparticle Phase I Completed
glycoprotein
NCT03345043 VAL-339851 Membrane-bound form of the hemagglutinin Lipid Nanoparticle Phase I Completed
glycoprotein
NCT05333289 mRNA-1030, mRNA-1020, Unknown Unknown Phase I/II Recruiting
mRNA-1010
Cytomegalovirus NCT03382405 mRNA-1647, mRNA-1443 Unknown Lipid Nanoparticle Phase I Completed
Respiratory syncytial virus NCT05127434 mRNA-1345 Unknown Unknown Phase II/III Recruiting
Rabies virus NCT03713086 CV7202 Glycoprotein of Rabies virus Lipid Nanoparticle Phase I Completed
NCT02241135 CV7202 Glycoprotein of Rabies virus Lipid Nanoparticle Phase I Completed
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Fig. 3 Landscape of mRNA vaccines in infectious diseases. mRNA vaccines have been developed against multiple infectious diseases to date,
including severe acute respiratory syndrome coronavirus 2, zika virus, human immunodeficiency virus, influenza virus, cytomegalovirus,
respiratory syncytial virus, varicella-zoster virus, and rabies virus. This figure is created using Adobe Illustrator and integrates the current
literature-based knowledge
a dangerous increase in vascular permeability and death.70,71 The induce the prefusion conformation. A study performed by Corbett
spike protein, which is found on the surface of SARS-CoV-2, et al. in 2020 exhibited the administration of mRNA-1273 triggered
facilitates the virus’s entry into host cells by binding to the potent humoral and cellular immunity against original and mutant
angiotensin-converting enzyme 2 receptors on the surface of the (D614G) SARS-CoV-2 in preclinical models.73,74 The administration
host cells.72 Therefore, the spike protein represents a prime target of the vaccine effectively provided protection to mice, preventing
for SARS-CoV-2 mRNA vaccines encoding either the receptor- SARS-CoV-2 infection in the nasal passages and lungs without
binding domain or the full-length spike protein. To date, two evident adverse effects or pathological changes in the respiratory
mRNA vaccines designed to target the spike protein of the system. The following phase I clinical trial conducted in July 2020
coronavirus disease 2019 (COVID-19) have gained approval and validated the safety and efficacy of mRNA-1273 in humans. The
widespread usage globally. These vaccines include mRNA-1273 geometric mean titers of anti-S-2P neutralizing antibodies after
developed by Moderna and BNT162b2 developed by BioNTech/ the second vaccination were 299,751, 782,719, and 1,192,154 in
Pfizer. Meanwhile, several other mRNA vaccines targeting the patients who received 25 μg, 100 μg, or 250 μg of mRNA-1273,
spike protein are currently undergoing clinical trials assessing their respectively, suggesting a robust humoral immune response in
safety and efficacy. participants. A robust T cell-mediated cytokine response was also
The initial phase I clinical study for COVID-19 vaccine was detected.75 The majority of the reported adverse events following
conducted on mRNA-1273, which was developed by Moderna. In vaccination were mild to moderate in nature. These included
the formulation of LNPs to encapsulate modified mRNA, the symptoms such as headache, chills, injection site pain, fatigue, and
ionizable lipid SM-102 was utilized. The mRNA sequence was myalgia, with more than half of the participants experiencing
modified with N1-methylpseudouridine encoding the spike these effects. Patients who received the 250 μg dose exhibited a
protein of SARS-CoV-2 with two proline substitutions (S-2P), which higher incidence (21%) of severe adverse events, particularly when

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the second vaccine was administered.75 In September 2020, study performed by Muik et al. in 2022, sera from 51 individuals
elderly participants were also involved in the trial, without any receiving two or three doses of BNT162b2 vaccine were tested
trial-limiting adverse effects observed.76 The phase III randomized, against original type, Beta, Delta, or Omicron pseudoviruses.83
placebo-controlled study was carried out at multiple medical After two doses, the neutralizing titers against the Omicron
centers in the United States from July to December 2020 and variant showed a reduction of more than 22-fold compared to
involved 30,420 volunteers, and the results showed that SARS- the titers against the wild-type. One month after receiving the
CoV-2 infection was diagnosed in 185 participants in the control third vaccine dose, the neutralizing titers against the Omicron
group, while this infection was diagnosed in only 11 patients in variant enhanced by 23-fold compared to the titers after two
the vaccinated group. mRNA-1273 demonstrated a 94.1% effec- doses, which were analogous to the levels of neutralizing titers
tiveness against SARS-CoV-2 infection and a 100% efficacy against against the original type observed after two doses. Together,
severe COVID-19 disease, with a transient and mild local and BNT162b2 is associated with superior safety and exhibits potent
systemic reaction induced by mRNA-1273.77 In 2022, Creech et al. efficiency against COVID-19, which is also effective in the context
evaluated mRNA-1273 in 6 to 11-year-old children in phase II/III of variants.
trial.78 In the first phase of the trial, 751 children were Multiple trials have compared the efficacy of mRNA-1273 and
administrated 50 μg or 100 μg doses of the mRNA-1273 vaccine. BNT162b2 vaccines. In a study performed by Wang et al. in 2022, a
On the basis of the results of safety and immunogenicity, the comparison was made between mRNA-1273 and BNT162b2
50 μg dose level was chosen for the second phase of the trial. The vaccines in terms of breakthrough SARS-CoV-2 infections,
second phase of the trial involved the random administration of hospitalizations, and deaths during the period when the delta
two injections of mRNA-1273 (50 μg each) or placebo to a group variant was predominant.84 The monthly incidence rate of
of 4,016 children, and these participants were then monitored for breakthrough infections showed a gradual increase from July to
a median duration of 82 days after the first injection. At this dose November 2021 in both the BNT162b2 cohort and the mRNA-1273
level, the observed adverse events were primarily mild and cohort. However, the incidence rate was elevated in the BNT162b2
temporary, with injection-site pain, headache, and fatigue being cohort compared with the mRNA-1273 cohort. Specifically, in
the most commonly reported. As of the data-cut-off date, no November, the incidence rate reached 2.8 cases per 1000 person-
severe side effects associated with the vaccine were reported, days in the BNT162b2 cohort as well as 1.6 cases per 1000 person-
such as multisystem inflammatory syndrome, myocarditis, or days in the mRNA-1273 cohort. After conducting matching
pericarditis. At 1 month following the second injection, children analysis, it was found that the mRNA-1273 cohort, consisting of
receiving mRNA-1273 at a 50 μg level exhibited a neutralizing 62,584 individuals, exhibited a markedly decreased hazard for
antibody titer of 1610, whereas young adults receiving the 100 μg breakthrough infections relative to BNT162b2 cohort, which also
level had a titer of 1300. Serologic responses were observed in a included 62,584 individuals. Among the patients who experienced
minimum 99.0% of participants within both age cohorts. At a breakthrough infections, it was observed that individuals receiving
point when the dominant circulating variant was Delta, the the mRNA-1273 vaccine were generally older compared to those
evaluated vaccine effectiveness against COVID-19 occurring receiving the BNT162b2 vaccine. There were also differences in
14 days or more after the initial injection was 88.0%. Overall, terms of sex, racial and ethnic composition, and the presence of
mRNA-1273 shows promising anti-COVID-19 efficacy, significantly comorbidities and adverse social determinants of health. After
protecting individuals from COVID-19. conducting the matching analysis, these differences were no
BNT162b1 and BNT162b2 are two COVID-19 mRNA vaccines longer found to be statistically significant. Among the individuals
developed by BioNTech and Pfizer. These vaccines are enclosed receiving the mRNA-1273 vaccine, the 60-day hospitalization risk
within LNPs and formulated utilizing Acuitas Therapeutics’ was 12.7%, with 392 out of 3,078 recipients requiring hospitaliza-
ionizable lipid ALC-0315. The mRNA in these vaccines is nucleo- tion. In comparison, among those receiving the BNT162b2
side-modified, with all uridines substituted by N1-methylpseu- vaccine, the 60-day hospitalization risk was slightly higher at
douridine, which enhances mRNA translation. BNT162b1 encoded 13.3%, with 2,489 out of 18,737 recipients requiring hospitaliza-
a secreted S glycoprotein receptor-binding domain (RBD) protein, tion. In terms of mortality, the 60-day mortality rate for mRNA-
while BNT162b2 encoded the S-2P protein. The relevant phase I 1273 recipients was 1.14%, with 35 out of 3,078 individuals
clinical study was performed in April 2020, and healthy experiencing mortality. For BNT162b2 recipients, the 60-day
participants in distinct groups were treated with either placebo mortality rate was 1.10%, with 207 out of 18,737 individuals
or two doses of one of the two vaccines mentioned above at experiencing mortality. Among the matched cohorts consisting of
differential doses (10 μg, 20 μg, 30 μg, and 100 μg) with a 21-day 3,054 individuals in each group, recipients of the mRNA-1273
interval. Both BNT162b1 and BNT162b2 resulted in a strong vaccine showed a decreased risk of 60-day hospitalizations
serologic response against the virus in a dose-dependent manner, compared to those of the BNT162b2 vaccine. Similarly, a study
especially following the second dose. The highest level of performed by Dickerman et al. examined the efficacy of the
neutralizing antibodies was detected on day 35, which was BNT162b2 and mRNA-1273 vaccines in a group of U.S. Veterans.85
14 days after the second dose.79,80 Although both BNT162b1 and Each vaccine group consisted of 219,842 individuals. During the
BNT162b2 elicited a potent and robust immune response, 24-week follow-up period, which was characterized by the
BNT162b2 was related to a lower risk of systematic adverse predominance of the alpha variant, the assessed risk of
effects than BNT162b1, especially in elderly participants, leading documented infection was 5.75 events per 1000 individuals in
to the selection of BNT162b2 to be used in a broader cohort the BNT162b2 group and 4.52 events per 1000 individuals in the
enrolled in phase III clinical study involving 43,448 participants mRNA-1273 group. The additional quantity of events per 1000
enrolled from April to December 2020.80 A total of 21,720 individuals for BNT162b2 relative to mRNA-1273 was 1.2 for
participants received BNT162b2, while 21,728 participants documented infection, 0.44 for symptomatic COVID-19, 0.55 for
received a placebo. The results revealed that eight patients in hospitalization for COVID-19, 0.10 for ICU admission for COVID-19,
the vaccinated group were diagnosed with SARS-CoV-2 infec- and 0.02 for mortality from COVID-19. The relative excess risk of
tion, whereas 162 patients in the placebo group were found to documented infection for BNT162b2 compared to mRNA-1273
be infected, suggesting that the efficacy of BNT162b2 was 95%. over a 12-week follow-up period, during which the delta variant
Among the infected patients, 10 were severely ill, with nine of was predominant, was 6.54 events per 1000 persons. Together,
them belonging to the placebo group and one to the vaccinated relative to people vaccinated with mRNA-1273, those with
group.81 In addition, BNT162b2 vaccination elicited a strong and BNT162b2 show lower rates of symptoms, hospitalization, ICU
enduring response of T follicular helper cells in humans.82 In a admission, and death, despite the higher infection rate.

Zhang et al.
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Multiple studies have been conducted to explore the utilization individuals receiving the vaccine, as compared with those who
of mRNA-1273 or BNT162b2 in the context of SARS-CoV-2 variants. were unvaccinated, showed a progressive increase with each
Both mRNA-1273 and BNT162b2 vaccines demonstrated additional dose. Specifically, for a fourth dose, the effectiveness
enhanced potency and breadth in memory B-cell response, was observed to be 49% against overall infection, 69% against
effectively triggering neutralizing immunity against the SARS- symptomatic infection, and 86% against severe outcomes. Lauring
CoV-2 omicron variant.86–89 Fabiani et al. conducted a study to et al. assessed clinical severity and efficacy of BNT162b2 and
assess the effectiveness of mRNA vaccines and the waning mRNA-1273 vaccines against COVID-19 caused by the omicron,
protection against SARS-CoV-2 infection as well as severe COVID- delta, and alpha variants of the SARS-CoV-2 virus.95 The study
19 in a population of 33,250,344 individuals aged 16 years and involved a total of 5,728 individuals with COVID-19 and 5,962
above receiving their initial dose of either BNT162b2 or mRNA- individuals without COVID-19 in the United States. Among
1273 vaccine and showed no better diagnosis of SARS-CoV-2 individuals who received two vaccine doses, the rates were 85%
infection in Italy.90 In the period characterized by the prevalence for the alpha variant, 85% for the delta variant and 65% for the
of the delta variant, vaccine efficacy against SARS-CoV-2 infection omicron variant. For individuals who received three vaccine doses,
notably declined from 82% at 3–4 weeks to 33% at 27–30 weeks the rate was 94% against the delta variant and 86% against the
after the second dose. In the same time intervals, the effectiveness omicron variant. In-hospital mortality was 7.6% (81/1060) for
of the vaccines against severe COVID-19 also experienced a alpha, 12.2% (461/3788) for delta, and 7.1% (40/565) for omicron.
decline, although the decline was not as pronounced, from 96% to For unvaccinated patients with COVID-19 who were hospitalized,
80%. At 27–30 weeks after the second dose of the vaccine, high- the severity of illness, as measured by the WHO clinical
risk individuals, including those aged 80 years and older, as well as progression scale, was found to be elevated for the delta variant
those aged 60–79 years, did not appear to be adequately compared to the alpha variant, with an adjusted proportional
protected against infection. Abu-Raddad et al. investigated the odds ratio of 1.28. Conversely, the severity of illness was lower for
impact of mRNA vaccine boosters on SARS-CoV-2 omicron the omicron variant relative to the delta variant, with an adjusted
infection in 2,239,193 individuals administrated with a minimum proportional odds ratio of 0.61. Compared with unvaccinated
of two doses of the BNT162b2 or mRNA-1273 vaccine in Qatar.91,92 patients, vaccinated patients exhibited lower severity of illness for
After 35 days of follow-up, the cumulative incidence of sympto- each variant, including the alpha variant (adjusted proportional
matic omicron infection among individuals who received the odds ratio 0.33), the delta variant (0.44), and the omicron variant
BNT162b2 vaccine was 2.4% in the booster cohort and 4.5% in the (0.61). Together, mRNA-1273 and BNT162b2 also show protective
nonbooster cohort. The effectiveness of the booster dose in efficacy in the context of COVID-19, with BNT162b2 showing
protecting against symptomatic omicron infection, when com- superior efficacy against COVID-19 variants. Although this effect
pared to the initial primary series, was determined to be 49.4%. declines over time, further booster doses can partially reverse this
The efficacy of the booster dose in reducing COVID-19-related phenomenon, representing a strategy against COVID-19 variants.
hospitalization and mortality owing to omicron infection, com- On August 31, 2022, the U.S. Food and Drug Administration
pared to the initial vaccine series, was estimated to be 76.5%. The (FDA) has updated the emergency use authorizations for the
effectiveness of the BNT162b2 booster dose in decreasing Moderna COVID-19 Vaccine and the Pfizer-BioNTech COVID-19
symptomatic infection with the delta variant, relative to the initial Vaccine to allow for the use of bivalent formulations as a single
vaccine series, was estimated to be 86.1%. Among individuals who booster dose (derived from https://www.fda.gov/news-events/
received the mRNA-1273 vaccine, the cumulative incidence of press-announcements/coronavirus-covid-19-update-fda-
symptomatic omicron infection was 1.0% in the booster cohort authorizes-moderna-pfizer-biontech-bivalent-covid-19-vaccines-
and 1.9% in the nonbooster cohort after 35 days. The effectiveness use). The updated boosters include two mRNA elements derived
of the mRNA-1273 booster dose in reducing symptomatic omicron from the SARS-CoV-2 virus. These bivalent formulations consist of
infection, relative to the primary vaccine series, was estimated to one component from the initial type of the virus and another
be 47.3%. In addition, Accorsi et al. investigated the relation element owned by the BA.4 and BA.5 lineages of the omicron
between 3 doses of BNT162b2 or mRNA-1273 and symptomatic variant. The recommended interval for administering the booster
infection resulted from the SARS-CoV-2 omicron and delta dose is at least 2 months after the primary or previous booster
variants.93 Among the reported cases, 18.6% (n = 2,441) of vaccination. The Moderna COVID-19 Vaccine, Bivalent, has been
omicron cases, 6.6% (n = 679) of delta cases, and 39.7% approved as a standalone booster shot for individuals who are 18
(n = 18,587) of controls had received three doses of mRNA years old or older. The Pfizer-BioNTech COVID-19 Vaccine, Bivalent,
vaccines. Furthermore, 55.3% (n = 7245) of cases, 44.4% has been authorized as a single booster dose for people who are
(n = 4570) of delta cases, and 41.6% (n = 19,456) of controls had 12 years old and above. The FDA conducted an evaluation of
received two doses of mRNA vaccines. Lastly, 26.0% (n = 3412) of immune response data involving around 600 adults aged 18 and
cases, 49.0% (n = 5044) of delta cases, and 18.6% (n = 8721) of above who had already received two doses of the primary series
controls were reported to be unvaccinated. After adjusting for and an additional booster dose of the monovalent Moderna
relevant factors, the odds ratio for receiving three doses compared COVID-19 vaccine. These individuals were administered a second
to being unvaccinated was 0.33 for omicron cases and 0.065 for booster dose of the monovalent Moderna COVID-19 vaccine or
delta cases. Similarly, the odds ratio for three vaccine doses Moderna’s experimental bivalent COVID-19 vaccine, which
compared to two doses was 0.34 for omicron cases and 0.16 for includes the original strain and the BA.1 Omicron variant,
delta cases. Grewal et al. conducted a study to estimate the minimum 3 months after their initial booster shot. After a period
marginal efficacy of a fourth dose relative to a third dose, as well of 28 days, the group receiving the bivalent vaccine demonstrated
as the overall vaccine effectiveness of BNT162b2 and mRNA-1273 a superior immune response against the BA.1 Omicron variant
against any infection, symptomatic infection, and severe out- compared to the group receiving the monovalent Moderna
comes (hospital admission or death) associated with the omicron COVID-19 vaccine. Since the bivalent (original and omicron BA.1)
variant of SARS-CoV-2.94 When comparing a fourth dose of the and monovalent Moderna COVID-19 vaccines are manufactured
vaccine (with 95% of recipients receiving mRNA-1273) adminis- using the same process, the safety data obtained from the
tered seven days or more after vaccination to a third dose bivalent vaccine are relevant and applicable to the monovalent
received 84 or more days prior, the marginal effectiveness was Moderna COVID-19 vaccine. To assess the efficacy of a single
estimated to be 19% against any infection, 31% against booster shot of the Pfizer-BioNTech COVID-19 vaccine, Bivalent,
symptomatic infection, and 40% against severe outcomes for individuals aged 12 and above, the FDA examined immune
(hospital admission or death). The effectiveness of the vaccine in response data from around 600 individuals over the age of 55

Zhang et al.
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previously receiving a two-dose primary series and an additional CD4+ and CD8+ T cells. Further infection with SARS-CoV-2 in
booster dose using the monovalent Pfizer-BioNTech COVID-19 vaccinated mice showed that ARCoV protected mice from SARS-
vaccine. These individuals were administered a second booster CoV-2 infection with no measurable viral RNA in the lungs of the
dose of the monovalent Pfizer-BioNTech COVID-19 vaccine or vaccinated mice.99 Two doses of ARCoV in nonhuman primate
Pfizer-BioNTech’s experimental bivalent COVID-19 vaccine, which models triggered potent humoral responses characterized by
includes the original strain and the BA.1 Omicron variant, between elevated titers of neutralizing antibodies and strong cellular
4.7 and 13.1 months after their initial booster dose. responses against SARS-CoV-2 in cynomolgus macaques. The
After 1 month, the immune responses against BA.1 Omicron challenge assay revealed no detectable viral small guide RNAs in
variant in the group receiving the bivalent vaccine were found to the trachea and lung lobes of all the vaccinated cynomolgus
be superior to the immune responses observed in the group macaques, while robust viral replication was present in macaques
receiving the monovalent Pfizer-BioNTech COVID-19 vaccine. receiving a placebo treatment. These results suggested the ability
Because the bivalent vaccine and the monovalent vaccine are of ARCoV to prevent SARS-CoV-2 replication in the lower
manufactured using the same process, the safety data are relevant respiratory tract.100 A phase III clinical study was initiated in April
to the Pfizer-BioNTech COVID-19 vaccine. Following this approval, 2021 in multiple centers in Indonesia and Mexico (NCT04847102).
the FDA has revised the emergency use authorizations for the Further exposure of clinical results is required to assess the
Moderna COVID-19 vaccine and the Pfizer-BioNTech COVID-19 effectiveness of this mRNA vaccine.
Vaccine, eliminating the usage of the monovalent Moderna and LUNAR-COV19 is a self-replicating mRNA vaccine encoding an
Pfizer-BioNTech COVID-19 vaccines for booster doses in people 18 S-2P antigen developed by Arcturus in 2020, with the aim of
years and older and 12 years and older, respectively. These offering robust immunity with a single low-dose administration.101
monovalent vaccines are still authorized for application as a LUNAR-COV19 used in preclinical models induced a robust T-cell
primary series for individuals aged 6 months and above, as response with an expanded CD44+CD62L- effector/memory
outlined in their respective letters of authorization. The Pfizer- subset, enhanced the proportion of IFN-γ+ CD8+/CD4+ T cells,
BioNTech COVID-19 vaccine is presently authorized for a single as well as resulted in potent humoral responses with high titers of
booster shot for people who are 5 to 11 years old, minimum neutralizing antibodies. Eighty percent of mice treated with 10 mg
5 months after finishing a primary series of the Pfizer-BioNTech LUNAR-COV19 exhibited PRNT50 titers >320 at 30 days after
COVID-19 vaccine. Overall, the bivalent vaccine represents a new vaccination. The human ACE2 transgenic C57BL/6 mouse model
step in mRNA vaccines against COVID-19. was used for the challenge assay, revealing unchanged weight
One of the problems with mRNA vaccines is the requirement of and no clinical sign in the vaccinated mice after infection with
extremely low-temperature storage, which limits their application original type SARS-CoV-2, while mice that received placebo
in areas with poor conditions and low economic levels. CVnCoV is showed an increased clinical score and a significant decrease in
a chemically unmodified mRNA vaccine encoding S-2P developed weight after infection.101 The assessment of the viral load revealed
by CureVac AG, which is stable at +5 °C for at least 3 months and no detectable SARS-CoV-2 RNA in both lungs of the vaccinated
was first reported in April 2020. Preclinical models using CVnCoV mice compared to the mice treated with a placebo. LUNAR-COV19
revealed that this vaccine induced robust humoral responses as used in phase II clinical study (NCT04480957) was well tolerated,
well as strong T-cell responses with potent induction of IFN-γ+ and increased neutralizing antibody levels were observed in the
TNF+ T cells. In addition, the animals infected with SARS-CoV-2 enrolled patients. Further investigation is required for the broader
with the spike D614G substitution 4 weeks after vaccination application of this vaccine.
showed no detectable virus in the lower respiratory tract after a Together, the approvals of mRNA vaccines not only protect
dose of 10 μg. Moreover, CVnCoV decreased the histopathological numerous individuals from COVID-19 but also provide valuable
alterations in the lungs of mice infected with SARS-CoV-2.96 The experience for the development of mRNA vaccines against other
phase I clinical trial performed in June 2020 exhibited that two diseases. Of note, although various anti-SARS-CoV-2 mRNA
doses of CVnCoV administered to individuals were safe and well vaccines have been prepared and used in humans, there are still
tolerated. CVnCoV significantly increased the levels of IgG problems that have not been solved, and the mechanism of action
antibodies to S-protein, as well as RBD in a dose-dependent is still unclear. For example, the duration of the protection
manner, and the median antibody titers after two 12 μg doses of provided by the mRNA vaccine in humans against COVID-19, as
CVnCoV were similar to those in the serum from patients with well as how to increase the levels of IgA antibodies, which are
COVID-19.97 Therefore, a dose of 12 μg was chosen for the phase those that mainly protect the upper respiratory tract, are not yet
II/III trial. The randomized phase IIb/III clinical trial was conducted known. How to reduce the rate of adverse effects, as the incidence
in 47 centers all over the world from December 2020 to April 2021. of systemic adverse events induced by mRNA vaccines is still
After more than 40 days of observation, 83 patients among the higher compared to those triggered by inactivated virus vaccines
12,851 in the CVnCoV group were diagnosed with SAR-CoV-2 or protein subunits, as demonstrated in previous clinical trials.
infection, and 145 patients among the 12,211 in the placebo Long-term monitoring might provide more detailed and useful
group were diagnosed with SAR-CoV-2 infection; the overall information leading to the safe and extensive application of mRNA
vaccine efficacy of only 48.2% was partly owing to the presence of vaccines.
SARS-CoV-2 variants.97 The same year, CureVac AG announced its
second-generation mRNA vaccine CV2CoV, which possesses mRNA vaccines against Zika virus
optimized noncoding regions to improve the level of the targeted Zika virus (ZIKV) is an RNA virus with a positive sense, single-
antigen. CV2CoV induced higher titers of neutralizing antibodies stranded genome measuring 11 kilobases in length.102 People
and stronger T-cell responses in nonhuman primates than infected with ZIKV often develop fever, headache, rash, malaise,
CVnCoV. Moreover, the findings of the challenge assay displayed and conjunctivitis that last between two and seven days. However,
that CV2CoV induced stronger protection with lower viral loads in its tropism for progenitor neural cells causes neurodevelopmental
both the upper and lower respiratory tract. Clinical trials have birth defects and congenital malformation in a limited number of
been planned and will be performed soon in the future.98 instances.103 Preventive vaccination is the only option against the
ARCoV is an LNP mRNA vaccine encoding an RBD protein that complications of ZIKV infection, as no drug against this virus is
was developed by Abogen in 2020. ARCoV mRNA-LNP used in available.104 Membrane and envelope proteins are common
preclinical mouse models triggered high titers of neutralizing antigens for mRNA vaccines against ZIKV. To date, several ZIKV
antibodies and strong T-cell responses against SARS-CoV-2, with vaccines developed on the basis of the mRNA platform have been
significantly increased IFN-γ and TNF-α secreted by virus-specific tested in preclinical models. In 2017, Pardi et al. designed an

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LNP-enclosed mRNA vaccine encoding the glycoproteins of the macaques. Rhesus macaques were initially primed with an mRNA
membrane and envelope of ZIKV.105 The administration of 30 μg vaccine containing a transmitted founder clade-B env protein
mRNA vaccine in C57BL/6 mice elicited a robust immune response lacking the N276 glycan. Multiple booster immunizations were
without any inflammation or other adverse events. The ZIKV administered to the rhesus macaques using autologous Envs that
reporter viral particle assay showed that the mean neutralizing IgG were repaired with the missing glycan and subsequently with
against the ZIKV virus peaked at 8 weeks after vaccination and was bivalent heterologous Envs from clades A and C. The vaccination
stable until 12 weeks after administration. Strong E-protein- regimen described was highly effective in inducing a strong
specific CD4+ T-cell responses were also observed as evidenced immune response, resulting in the production of neutralizing
by robust intracellular production of IL-2, TNF-α, and IFN-γ. antibodies against the most prevalent (tier-2) strains of HIV-1 and
Moreover, a challenge study showed that mice and nonhuman robust anti-Env CD4+ T cell responses. Upon conducting multiple
primates treated with the mRNA vaccine exhibited protection low-dose mucosal challenges with heterologous tier-2 simian-HIV
against ZIKV infection.105 The same year, Richner et al. developed AD8, the vaccinated animals demonstrated a 79% per-exposure risk
an LNP-enclosed mRNA vaccine encoding both original type and decrease. The findings suggest that the multiclade Env-Gag virus-
variant ZIKV membrane glycoproteins. Two doses of the mRNA like particle mRNA platform holds promise as a potential method for
vaccine potentiated the serum-neutralizing responses against developing an HIV-1 vaccine. Of note, a biotech firm, in collaboration
ZIKV and protected mice against ZIKV infection. The efficacy of the with the nonprofit partner IAVI (International AIDS Vaccine Initiative),
mRNA vaccine was also assessed in a mouse pregnancy model. has initiated a phase I clinical trial for an investigational mRNA HIV
The vaccinated mice were infected with ZIKV at embryo day six, vaccine (https://investors.modernatx.com/news/news-details/2022/
and the results exhibited two doses of mRNA vaccine significantly IAVI-and-Moderna-Launch-Trial-of-HIV-Vaccine-Antigens-Delivered-
reduced the levels of viral RNA in both fetal and placental Through-mRNA-Technology/default.aspx). The vaccine candidate in
tissues.106,107 Although the results of the mRNA ZIKV vaccine in question utilizes a prime and boost strategy to elicit targeted B-cell
preclinical studies are promising, further human clinical trials are responses with the objective of generating broadly neutralizing
needed. However, clinical trials for these vaccines in pregnant antibodies against HIV. The antigens employed in the vaccine were
women are undermined by ethical issues. developed as proteins by scientists at IAVI. They previously
investigated the prime antigen in an adjuvanted protein-based
mRNA vaccines against human immunodeficiency virus vaccine, inducing the desired B-cell response in 97% of trial
Human immunodeficiency virus (HIV) is a member of the participants. Notably, the development of the mRNA HIV vaccine is
Lentivirus genus of the retroviridae family and is divided into still in its initial stage. More research is needed to optimize this
two types: HIV-1 and HIV-2.108,109 HIV causes acquired immune treatment strategy for long-lasting immune responses. The studies
deficiency syndrome (AIDS), which infects 75 million people focus on the simultaneous administration of drugs that help
worldwide, causing more than 32 million AIDS-related deaths reactivate the HIV reservoir to make it visible to the immune system
(derived from Global HIV and AIDS statistics, 2019). No effective and may eventually improve the efficacy of the mRNA HIV vaccine.
preventive vaccine exists despite 30 years of research, primarily
because of the significant antigenic diversity of the protein found mRNA vaccines against influenza virus
in the HIV envelope and its dense "glycan shield that hides the Influenza viruses are members of the Orthomyxoviridae family
epitope of the crucial envelope protein. Multiple mRNA vaccines composed mainly of four types of influenza viruses: types A, B, C,
have been investigated in clinical studies to date. In 2016, Gandhi and D; among them, types A and B are clinically associated with
et al. used mRNA-transfected autologous DCs to stimulate the human diseases.114,115 The typical target of the mRNA vaccine
immune response against HIV-1.110 Fifteen patients were involved against influenza virus is the glycoprotein haemagglutinin (HA) on
in the trial and randomly assigned to two separate groups that the surface of the virus since it mediates viral entry. However,
received DC mRNA vaccines encoding HIV-1 antigen or placebo. owing to the rapid mutation of the influenza virus, which leads to
The proliferative response of CD4+ T cells to HIV-1 Gag was antigenic drift, the HA antigen component of the mRNA vaccine
significantly enhanced by DC mRNA vaccines, with a 3.4-fold requires annual review and modification. This feature makes the
increase compared to that in participants administrated with a mRNA vaccine the most suitable platform for preventing influenza
placebo. However, no significant release of IFN-γ was detected, virus infection and controlling the spread of the disease. In 2012,
and the increase in the CD8+ T cell proliferative response was Petsch et al. made a significant breakthrough by demonstrating
transient.110 In 2017, Jong et al. developed an HIV mRNA the effectiveness of an mRNA vaccine against influenza encoding
immunogen based on conserved targets of effective antiviral the full-length HA of influenza A/Puerto Rico/8/1934 (PR8HA).116
T-cell responses against HIV.111 The phase I trial using increasing The serum of the mRNA-vaccinated mice showed effective
doses of this vaccine showed that it was safe and well tolerated.111 seroconversion with an increased amount of virus-neutralizing
Despite these encouraging findings, the phase II clinical study in antibodies. Moreover, the CD8+ T cells from the vaccinated mice
the same year was stopped due to the production of insufficient had increased cytotoxic activity associated with viral clearance
immunogenicity by the vaccine. In 2020, Gay et al. combined AGS- and long-term immunological memory. The administration of
004, a DC mRNA vaccine, with the latency-reversing agent mRNA vaccines also induced long-term immunity and protected
vorinostat and evaluated the effect on the HIV reservoir. The animals (mice, ferrets, and pigs) from influenza A virus infection.116
aim of this combination therapy was to disrupt the virological Of note, the mRNA vaccine encoding HA from the PR8 H1N1 strain
latency by vorinostat and to deplete cells expressing HIV antigens triggered homologous and heterologous immune responses
and clear the HIV reservoir by the mRNA vaccine. However, against H1N1 and H5N1 strains, suggesting protection against
although the combination of AGS-004 and vorinostat was safe and heterogeneous viruses.116 In 2017, Lutz et al. developed an LNP-
well tolerated, no substantial impact on the immune response enclosed mRNA vaccine encoding the HA of the influenza virus
against HIV was observed, and the frequency of resting CD4+ strain H1N1pdm09.117 The use of an mRNA vaccine induced an
T-cell infection was stable throughout the entire treatment in all enhanced adaptive immune response represented by a transient
participants.112 A mRNA vaccine concurrently expressed local immunostimulatory milieu. The serum of the vaccinated mice
membrane-anchored HIV-1 envelope (Env) and simian immuno- showed an increased amount of multifunctional CD4+ and CD8+
deficiency virus (SIV) Gag proteins, was created to generate of T cells specifically against the influenza virus. The injection of the
virus-like particles.113 This vaccine formulation elicited the produc- mRNA vaccine induced a stable humoral response against the
tion of antibodies with broad neutralizing capabilities against HIV-1 influenza virus for at least one year, comparable with that of other
and demonstrated a reduction in the risk of infection in rhesus inactivated virus-based licensed vaccines, as demonstrated by a

Zhang et al.
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continuous follow-up of functional antibody and T-cell neutralization ability and superior whole-virion phagocytosis
responses.117 The same year, Bahl et al. developed mRNA vaccines activity compared with other vaccinated groups. The long-
encoding the HA proteins of H10N8 and H7N9,118 which induced lasting immune response encourages the use of this mRNA
robust humoral and cellular responses in preclinical mouse vaccine in future clinical studies.129 In 2021, Webster et al.
models, protecting mice from a lethal infection.118 Feldman administered an mRNA vaccine encoding CMV gB and PC by
et al. further performed the first randomized phase I clinical trial intramuscular injection to cynomolgus and rhesus macaques, and
utilizing two mRNA vaccines against H10N8 and H7N9.119 These an increased level of antigen-specific plasma antibody was
two vaccines were well tolerated without any serious vaccine- detected in both species. The elicited antibodies against PC were
related adverse events. The HA inhibition titers after the dose dependent, while the boosted antibodies against gB were
intradermal administration of 50 μg mRNA vaccine were ≥1:40 in similar in groups treated with 20 μg vaccine and 120 μg vaccine.
89.7% of patients. However, a significantly enhanced cellular However, mRNA had no significant influence on antibody-induced
response was not detected.119 In 2021, Chivukula et al. used cellular phagocytosis against CMV.130 Two phase I clinical trials are
unmodified and LNP-encapsulated mRNA encoding full-length HA active but not recruiting to assess the reactogenicity, safety, and
or full-length neuraminidase (NA).120 The HA and NA mRNA-LNP immunogenicity of the mRNA-1647 CMV vaccine (NCT05105048
formulations, whether administered as monovalent or multivalent and NCT05397223). A phase II clinical trial is recruited to assess the
vaccines, have demonstrated the ability to elicit robust functional efficacy, safety, and immunogenicity of the mRNA-1647 CMV
antibody and cellular immune responses in nonhuman primates. vaccine (NCT05683457). A phase I/II clinical study is also recruiting
The induced antigen-specific antibody responses have been found to assess the safety and immunogenicity of the mRNA-1647 CMV
to be correlated with protective effectiveness against viral vaccine in healthy individuals 9 to 15 years of age and individuals
challenge in mice. In 2022, McMahon et al. assessed immuno- 16 to 25 years of age (NCT05575492). A phase III clinical study is
genicity and protective efficacy of a quadrivalent nucleoside- recruiting healthy participants 16 to 40 years of age to assess the
modified mRNA vaccine against influenza in mice. This vaccine efficacy, safety, and immunogenicity of the mRNA-1647 CMV
formulation included four antigens from influenza A group 2 vaccine (NCT05085366). A phase I trial evaluating the safety,
viruses: HA stalk, NA, matrix protein 2, and nucleoprotein.121 The reactogenicity as well as immunogenicity of mRNA-1647 and
vaccination elicited antigen-specific cellular and humoral immu- mRNA-1443 CMV vaccines have been completed in healthy adults,
nity, protected mice from all challenge viruses, and provided but the findings are not disclosed (NCT03382405). A dose-finding
protection from morbidity at a dose of 125 ng per antigen. The study to assess the safety and immunogenicity of CMV vaccine
same year, Pardi et al. developed a pentavalent nucleoside- mRNA-1647 has also been completed in healthy adults, but the
modified mRNA vaccine that offered broad protection against results are not reported (NCT04232280). Together, no clinical data
influenza B viruses encoding antigens, B/Yamagata/16/1988-like have been reported regarding the safety, reactogenicity, safety,
lineage HA, B/Victoria/2/1987-like lineage HA, NA, matrix-2, and and immunogenicity of CMV mRNA vaccines to date. The
nucleoprotein.122 This vaccine provided protection from morbidity publication of these data has the potential to offer significant
at an impressively low dose of 50 ng per antigen. Additionally, insights for the advancement of anti-CMV mRNA vaccines.
Arevalo et al. developed a multivalent nucleoside-modified mRNA
vaccine targeting all known influenza virus subtypes.123 This mRNA vaccines against respiratory syncytial virus
multivalent vaccine, which encoded HA antigens from all 20 Respiratory syncytial virus (RSV) is an enveloped virus belonging to
known subtypes of influenza A/B virus lineages, elicited strong the Pneumovirus genus within the Paramyxoviridae family.131,132 It
antibody responses in mice and ferrets. These antibodies showed is the most common pathogen in infants and young children
reactivity against all 20 encoded antigens and provided protection causing acute lower respiratory infection. Older adults, especially
to mice and ferrets when challenged with both matched and those with deficient immunity, are also susceptible to RSV. The
mismatched viral strains. In general, mRNA vaccines with a rapid fusion protein (F protein) is targeted by the human immune
speed of production may become a critical treatment against system against RSV; thus, it is usually selected as the antigen for
influenza viruses. Further randomized studies are necessary to vaccine development. However, when RSV attaches to the
confirm the safety and effectiveness of mRNA influenza vaccines. targeted cell, the F protein is modified in a prefusion form, which
hides the potent neutralizing epitopes, leading to the immune
mRNA vaccines against cytomegalovirus evasion of RSV. In 2020, Espeseth et al. tested mRNA vaccines
Human cytomegalovirus (CMV) belongs to the Betaherpesvirinae encoding RSV F proteins with different conformations, and the
subfamily and possesses a genome size of 236 kilobases.124 results demonstrated that the native form of RSV F protein
Following primary infection, CMV typically establishes a latent generated high titers of neutralizing antibodies against both
state, persisting in the host without causing active disease. Virus prefusion- and postfusion-specific epitopes.133 The mRNA vaccine
reactivation in immunocompromised individuals can cause life- encoding the F protein with prefusion stabilizing mutations can
threatening complications involving the lung, gastrointestinal generate a humoral response toward prefusion-specific epitopes.
tract, liver, eye, or central nervous system. CMV is recognized as However, the stabilizing mutations do not generate higher titers
the most prevalent infectious cause of congenital malformations, of neutralizing antibody or enhanced T-cell response compared
with sensorineural hearing loss, developmental delay, and fetal with the effect of the mRNA vaccine encoding the native F
death in 10–15% of cases.124,125 The process of viral entry into protein.133 A phase I study is recruiting individuals aged 5 months
host cells is facilitated by the presence of viral envelope to <24 months to evaluate the safety and immunogenicity of
glycoproteins (g) gB and gH/gL (pentameric complex (PC)), and mRNA-1365 and mRNA-1345 (NCT05743881). A phase I trial is
cell−cell fusion events allow the dissemination of the virus.126,127 currently in progress, focusing on the tolerability and reactogeni-
In 2018, John et al. developed an mRNA vaccine encoding city of mRNA-1345 in various populations (NCT04528719). This
multiple CMV antigens, and the results using in vitro cell includes younger adults, women of child-bearing potential, older
experiments showed that the mRNA-transfected cells expressed adults, and RSV-seropositive children. The study involves different
high levels of the encoded antigens. The administration of mRNA dosing regimens, including ingle injections of up to 5 dose levels
CMV vaccines in mice generated long-lasting and high titers of in younger adults, 3 injections of the middle dose level
neutralizing antibodies against gB and PC. In addition, an administered with a 56-day interval in younger adults, a booster
enhanced proportion of IFN-γ-producing T cells was observed in injection around 12 months following the primary injection in
vaccinated mice.128 In 2020, Nelson et al. tested an mRNA vaccine older adults, and 3 injections of 1 of 2 dose levels given 56 days
encoding full-length gB in rabbits, which showed enhanced virus apart in RSV-seropositive children. Although infants and young

Zhang et al.
12
children are frequently infected by RSV, few clinical trials have fatality.139 The rabies virus binds to its cellular target through the
been performed at this stage to date, but they have been surface glycoprotein RABV-G, gaining access to the peripheral
launched for adults. Moderna developed an mRNA vaccine named nerves and the central nervous system. In 2016, Schnee et al.
mRNA-1777 that encodes RSV F protein stabilized in the prefusion tested a vaccine composed of mRNA encoding RABV-G in mice
conformation, which became the first RSV mRNA vaccine entering and domestic pigs and discovered 2 doses of this vaccine-induced
a phase I clinical study for assessing its safety, tolerability, and virus-specific neutralizing titers ≥0.5 IU/ml and an increased
immunogenicity.134 A total of 72 healthy young adults from 18 to proportion of virus-specific CD4+ T cells.65 Antibody titers in mice
49 years old and 107 healthy old adults from 60 to 79 years old vaccinated with 20 μg and 80 μg mRNA vaccine remained stable
were enrolled in this study, randomly divided into two groups and throughout one year of measurement once a month, with mean
treated with mRNA-1777 or placebo. The safety profile of mRNA- titers of approximately 40 IU/ml. The vaccinated mice were
1777 was favorable, with no reports of serious adverse events and protected against intracerebral rabies virus infection, suggesting
good tolerability observed. mRNA-1777 induced geometric mean the satisfying immunogenicity of the mRNA vaccine.65 In 2017,
titers of neutralizing antibody peaking from day 29 to 60 Alberer et al. performed the first phase I clinical study in Germany
postinjection and declining over time. Intracellular cytokine using the mRNA rabies vaccine CV7201.140 A total of 101
staining of IFN-γ, IL-2, and TNF-α also showed enhanced CD4+ participants aged 18 to 40 were enrolled and vaccinated, and
T-cell responses in both young and old participants. These results the results demonstrated that CV7201 was generally safe and well
are promising for use in large randomized, placebo-controlled tolerated, with only one vaccine-related serious side effects
trials involving vulnerable adult populations in the future.134 In (moderate Bell’s palsy). RABV-G-specific IgM and IgG titers peaked
addition, multiple clinical trials have been performed. A phase I at days 21 and 42 postinjection. A significant enhancement in
study is recruiting adults 50 to 75 years old for assessing the serum IgG was found after the 1-year boost, suggesting the
safety, reactogenicity, and immunogenicity of the mRNA-1045 RSV establishment of an immune memory response in participants.
vaccine (NCT05585632). An observational study is currently RABV-G-specific CD4+ T cells transiently enhanced after vaccina-
recruiting participants to assess the real-world efficacy of the tion and declined to baseline levels 3 months after injection.140
Moderna mRNA-1345 vaccine in preventing lower respiratory tract Since the phase I clinical trial using mRNA rabies vaccine showed
disease caused by RSV, as well as to investigate additional health satisfying outcomes, future studies should focus on increasing
and economic outcomes (NCT05572658). A phase I/II study is antibody titers inducing a longer immune response to potentially
currently underway to evaluate the safety and immunogenicity of help the production of cheaper and more available rabies vaccines
a single intramuscular injection of 3 dose levels of an RSV mRNA to meet the needs of public health.
vaccine candidate formulated with two different LNPs (i.e., LNP
containing CL-0059 or CL-0137) in healthy adults aged 18–50
years and 60 years and older (NCT05639894). A phase II/III study is MRNA VACCINES IN CANCERS
recruiting adults aged 60 years and older to assess the safety and mRNA vaccines in cancers are usually applied in a therapeutic
tolerability of the mRNA-1345 vaccine and the vaccine’s ability to setting instead of a prophylactic approach in infectious dis-
prevent the first episode of RSV-associated lower respiratory tract eases.141 Indeed, it is typically designed to encode tumor-
disease in this population (NCT05127434). Although multiple associated antigens (TAAs) or neoantigens to activate antitumour
clinical studies have been launched, almost all are still at an early immune responses.142 To date, numerous clinical trials investigat-
stage, and the prophylactic effects of the mRNA vaccine against ing the effect of the mRNA vaccine against various cancers have
acute infection of the lower respiratory tract remain to be defined. been registered in the U.S. National Library of Medicine
(ClinicalTrials.gov), including melanoma, brain cancer, non-small
mRNA vaccines against varicella-zoster virus cell lung cancer (NSCLC), ovarian cancer, prostate cancer, blood
Varicella zoster virus (VZV), also referred to as human herpesvirus system cancer, digestive system cancer, and breast cancer (Table 2
3, is an alphaherpesvirus with a double-stranded DNA genome and Fig. 4).
that is widely distributed in the human population.102 Primary VZV
infection leads to varicella (chickenpox), and it becomes latent in mRNA vaccines against melanoma
ganglionic neurons. Latent VZV is reactivated in severe cases due Melanoma arises from the malignant conversion of melanocytes
to decreased cellular immunity against VZV, causing postherpetic that are widely distributed in the body (e.g., skin, mucosa, uvea,
neuralgia, which may lead to unbearable pain lasting for months inner ear, and rectum). Cutaneous melanoma is the most common
and affect the quality of life of patients. VZV encodes 10 type that accounts for ~1.7% of all newly diagnosed primary
glycoproteins: ORFS/L, gK, gN, gC, gB, gH, gM, gL, gI and malignancies, responsible for ~0.7% of all cancer mortality
gE.135–137 In 2020, Monslow et al. developed an LNP-enclosed worldwide.143 DC-based mRNA vaccines are mostly used to
mRNA vaccine encoding the VZV gE antigen, and its efficacy was combat melanoma. As early as 1996, Boczkowski et al. performed
compared with that of two other vaccines approved on the adoptive mRNA-pulsed DC transfer, discovering that a DC-pulsed
market, including one with a live attenuated virus and one with a mRNA vaccine encoding ovalbumin (OVA) protected mice from
subunit protein. Rhesus monkeys were divided into five groups OVA-expressing tumor cells and significantly reduced lung
and treated with VZV gE subunit protein, live-attenuated VZV, and metastases in a B16/F10.9 tumor model.144 In recent years, diverse
mRNA VZV vaccine at different doses. The results revealed the DC-based mRNA vaccines have been tested in melanoma patients.
safety of the two 50 μg mRNA VZV vaccines and the ability to Gaudernack et al. isolated autologous total mRNA from biopsied
trigger a potent humoral and cellular immunity comparable to melanoma tissue and introduced it into DCs via electropora-
that of the 50 μg subunit protein vaccine, indicating that the tion.145,146 Then, melanoma patients were vaccinated with
mRNA vaccine is a suitable platform for future production of the autologously derived tumor mRNA-transfected DCs, causing the
VZV vaccine.138 Although the translatability of the results was induction of a wide range of T-cell responses. Several antigens
promising, more clinical and preclinical investigations focused on have been used as targets for mRNA vaccine development, such
the effectiveness and safety of the vaccine are still urgently as MAGE-A3, MAGE-A2, gp100, and tyrosinase. MAGE-A3 and
needed. MAGE-C2 are exclusively expressed in germ cells and tumor cells
(including melanoma cells), while tyrosinase and gp100 are widely
mRNA vaccines against rabies virus expressed in both tumor and normal tissue. Aarntzen et al. utilized
Rabies virus is a negative-stranded RNA virus of the Rhabdoviridae mRNA to electroporate monocyte-derived DCs to encode gp100
family causing rabies, a zoonotic viral disease with nearly 100% and tyrosinase.147,148 These monocyte-derived DCs were then

Table 2. Clinical trials for mRNA vaccines in cancers
Disease NCT Number Disease condition Encoded antigen Vehicle Combination Phase Status
Melanoma NCT00204516 Stage III/IV Melan-A, MAGE-A1, MAGE-A3, Unknown GM-CSF Phase I/II Completed
survivin, gp100, tyrosinase,
personalized antigens
NCT00204607 Stage III/IV Melan-A, MAGE-A1, MAGE-A3, Protamine GM-CSF Phase I/II Completed
survivin, gp100, tyrosinase
NCT01278940 Advanced Tumor mRNA-encoded antigens DC IL-2 Phase I/II Completed
NCT01530698 Stage III/IV GP100, tyrosinase DC No Phase I/II Completed
NCT01676779 Stage III/IV Unknown DC No Phase II Completed
NCT00243529 Stage III/IV GP100 and tyrosinase DC No Phase I/II Completed
NCT01066390 Stage III/IV MAGE-A3, MAGE-C2, tyrosinase, DC No Phase I Completed
gp100
NCT00978913 Unknown Survivin, hTERT, p53 DC Cyclophosphamid Phase I Completed
NCT00940004 Stage III/IV GP100, tyrosinase DC No Phase I/II Completed

NCT02285413 Stage III/IV GP100, tyrosinase DC Cisplatinum Phase II Completed
NCT00672542 Metastatic Melan-A, tyrosinase, gp100, DC Proteasome siRNA- Phase I Completed
MAGE-3 tranfected DC
NCT01278940 Advanced Autologous tumor mRNA- DC IL-2 Phase I/II Completed
encoded antigens
NCT01684241 Advanced NY-ESO-1, tyrosinase No No Phase I Completed
NCT04526899 Anti-PD-1-refractory/relapsed, unresectable stage MAGE-A3, NY-ESO-1, TPTE, Liposome Cemiplimab Phase II Recruiting
III or IV tyrosinase
NCT01983748 Uveal Autologous tumor RNA- DC No Phase III Recruiting
encoded antigens
NCT03739931 Advanced OX40L, IL-23, IL-36γ Lipid Durvalumab Phase I Recruiting
Zhang et al.
Nanoparticle
NCT03897881 Unknown Personalized antigens Lipid Pembrolizumab Phase II Active, not
Nanoparticle recruiting
NCT01456104 Stage II/III/IV Murine tyrosinase-related DC No Phase I Active, not
peptide 2 recruiting
NCT02410733 Stage III/IV NY-ESO-1, MAGE-A3, TPTE, Liposome Pembrolizumab Phase I Active, not
tyrosinase recruiting
NCT04335890 Metastatic uveal Autologous tumor-RNA- DC No Phase I Active, not
encoded antigens recruiting
NCT00126685 Stage IV Autologous polymerase chain DC No Phase I/II Unknown
reaction-amplified tumor RNA-
encoded antigens
NCT00929019 Unknown GP100, tyrosinase DC No Phase I/II Terminated
NCT00961844 Stage II/III/IV hTERT, survivin, tumor cell- DC Temozolomid Phase I/II Terminated
derived mRNA-encoded
antigens
NCT03394937 Unknown 5 TAAS No Pembrolizumab Phase I Terminated
NCT03480152 Metastatic Autologous cancer cell-derived Unknown No Phase I/II Terminated
neoantigens
13
14
Table 2. continued
Brain Cancer NCT00846456 Glioblastoma Glioblastomas stem cell-derived DC No Phase I/II Completed
mRNA-encoded antigens
NCT00890032 Recurrent glioblastoma multiforme Autologous brain tumor stem DC No Phase I Completed
cell mRNA-encoded antigens
NCT00626483 Glioblastoma multiforme CMV pp65 LAMP DC Basiliximab, Phase I Completed
GM-CSF
NCT02529072 Recurrent astrocytoma, malignant glioma, and CMV pp65 LAMP DC Nivolumab Phase I Completed
glioblastoma
NCT03615404 Glioblastoma, malignant glioma, CMV pp65 LAMP, GM-CSF DC Tetanus toxoid Phase I Completed
medulloblastoma recurrent, pediatric
glioblastoma multiforme, pediatric brain tumor,
recurrent pediatric brain tumor
NCT04963413 Glioblastoma CMV pp65 LAMP, GM-CSF DC No Phase I Recruiting
NCT04573140 Adult glioblastoma, pediatric high-grade gliomas Autologous total tumor mRNA, DOTAP No Phase I Recruiting
CMV pp65 LAMP liposome
NCT03548571 IDH wild-type, MGMT-promotor methylated Survivin, hTERT, autologous DC Temozolomide Phase II/III Recruiting
glioblastoma tumor stem cell mRNA-encoded
antigens
NCT02649582 Glioblastoma multiforme WT1 DC Temozolomide Phase I/II Recruiting
NCT02465268 Glioblastoma multiforme CMV pp65 LAMP DC Saline, Td, GM-CSF Phase II Recruiting
Zhang et al.
NCT03688178 Glioblastoma CMV pp65 LAMP DC Varlilumab Phase II Recruiting

NCT00639639 Glioblastoma multiforme CMV pp65 LAMP DC Tetanus-Diphtheria, Toxoid Phase I Active, not
recruiting
NCT03927222 Glioblastoma CMV pp65 LAMP DC Temozolomide, Phase II Suspended
Tetanus-Diphtheria Toxoid,
GM-CSF
NCT04741984 Glioblastoma CMV pp65 LAMP Monocytes No Phase I Not yet
recruiting
NCT02808364 Recurrent glioblastoma Personalized antigens DC No Phase I Unknown
NCT02709616 Glioblastoma Personalized antigens DC Temozolomide, Phase I Unknown
Radiotherapy
NCT02366728 Glioblastoma CMV pp65 LAMP DC Basiliximab, Phase II Unknown
Temozolomide, Saline
NCT01291420 Glioblastoma WT1 DC No Phase I/II Unknown
Non-Small Cell NCT03164772 Metastatic NY-ESO-1, MAGE-C1, MAGE-C2, No Durvalumab, Phase I/II Completed
Cancer 5T4, survivin, muclin-1 Tremelimumab
NCT00923312 Stage IIIB/IV NY-ESO-1, MAGE-C1, MAGE-C2, No No Phase I/II Completed
5T4, survivin
NCT03948763 KRAS Mutant advanced or metastatic KRAS Lipid Pembrolizumab Phase I Recruiting
Nanoparticle

NCT03908671 Stage IIIB/IV Personalized neoantigens Unknown No Not applicable Not yet
recruiting
NCT03908671 Unresectable or metastatic Personalized neoantigens Unknown No Not applicable Unknown
Table 2. continued
Ovarian Cancer NCT04163094 Primary 3 OC TAAs Liposome Carboplatin/Paclitaxel, Phase I Active, not
Surgery recruiting
NCT03323398 Advanced or metastatic OX40L Lipid Durvalumab Phase I/II Active, not
NCT01334047 Relapsed hTERT, survivin, stem cell DC No Phase I/II Terminated
mRNA-encoded antigens
NCT01456065 Stage III hTERT DC Survivin Phase I Unknown
Prostate Cancer NCT01278914 Androgen resistant metastatic Unknown DC Unknown Phase I/II Completed
NCT01446731 Castration-resistant metastatic PSA, PAP, survivin, hTERT DC Docetaxel Phase II Completed
NCT00831467 Hormone refractory PSA, PSMA, PSCA, STEAP Unknown No Phase I/II Completed
NCT00004211 Metastatic PSA DC No Phase I/II Completed
NCT00010127 Metastatic Autologous tumor mRNA- DC No Phase I Terminated
encoded antigens

NCT04382898 Metastatic castration-resistant or high-risk RBL038, RBL039, RBL-040, RBL- Liposome Cemiplimab Phase I/II Active, not
localized 041, RBL-045 recruiting
NCT01197625 High Gleason score (9–10) or micrometastatic Primary prostate cancer tissue DC hTERT, Phase I/II Active, not
mRNA-encoded antigens Survivin recruiting
NCT02140138 Intermediate to high risk PSA, PSMA, PSCA, STEAP, PAP, DC No Phase II Terminated
mucin-1
NCT01153113 Metastatic hTERT DC No Phase I/II Withdrawn
NCT00006430 Metastatic Autologous prostate tumor DC No Phase I Unknown
tissue mRNA-encoded antigens
Blood System NCT00834002 AML WT1 DC No Phase I Completed
Cancer NCT01734304 AML WT1, PRAME, CMV pp65 LAMP DC No Phase I/II Completed
Zhang et al.
NCT00510133 AML hTERT, LAMP DC No Phase II Completed

NCT01686334 AML WT1 DC No Phase I/II Recruiting
NCT03083054 AML, high risk myelodysplastic syndromes WT1 DC No Phase I/II Active, not
recruiting
NCT00514189 AML AML mRNA-encoded antigens DC No Phase I Terminated
NCT00965224 AML, CML, myeloma WT1 DC No Phase II Unknown
NCT02315118 CD20 + CLL, B-cell non-Hodgkin’s lymphoma CD16-41BB-CD3zeta T cell Rituximab, Phase I/II Unknown
IL-2
NCT03739931 Lymphoma OX40L, IL-23, IL-36γ Lipid Durvalumab Phase I Recruiting
Nanoparticle
NCT03323398 Lymphoma OX40L Lipid Durvalumab Phase I/II Active, not
NCT01995708 Multiple myeloma CT7, MAGE-A3, WT1 DC Autologous stem cell Phase I Active, not
transplantation recruiting
Digestive NCT00228189 CRC with liver metastases CEA DC No Phase I/II Completed
System Cancer NCT03480152 Metastatic gastrointestinal cancer Personalized neoantigens Lipid No Phase I/II Completed
nanoparticle
NCT00003433 Metastatic CRC CEA DC No Phase I/II Completed
NCT03468244 Advanced ESC, GA, PAAD, CRC Personalized neoantigens Unknown No Not applicable Recruiting
15
Zhang et al.
16
administered to 45 patients with stage III and IV melanoma. The
AML acute myelocytic leukemia, CML chronic myeloid leukemia, CLL chronic lymphocytic leukemia, CRC colorectal cancer, HCC hepatocellular carcinoma, ESC esophageal squamous carcinoma, GA gastric
study demonstrated notable CD4+ and CD8+ T-cell responses
Terminated
Terminated
Terminated
Completed
Active, not
Recruiting
Recruiting
Recruiting
Not applicable Unknown
Unknown
Unknown
specific to tumor antigens, suggesting the potential effectiveness
recruiting
recruiting
Not applicable Not yet
of mRNA-electroporated DC vaccines for treating melanoma.
Status
Immunological adjuvants are usually used to stimulate and

amplify the immune responses to the targeted antigens to
regulate the in vivo immunogenicity of the mRNA vaccine. TriMix
has been mostly used as a DC-based mRNA vaccine against
Phase I/II
Phase I/II
Phase I/II
Phase I/II
Phase I/II
melanoma because it encodes the activation stimulator CD40L
Phase I
Phase I
Phase I
Phase I
Phase I
(CD4+ T-cell activator), the costimulatory molecule CD70 (CD8+
Phase
T-cell activator), and the constitutively active TLR 4 (DC antigen

presentation promotor). Wilgenhof et al. described a phase IB trial
that enrolled 15 advanced melanoma patients who were
Cytokine-induced killer
subjected to vaccination with autologous monocyte-derived DCs

electroporated with synthetic mRNA (TriMixDC-MEL).149 The
Cyclophosphamide
report revealed that the mRNA encoded CD40L, TLR4, CD70,

Pembrolizumab
HLA-II targeting signal, and a TAA (MAGE-A3, MAGE-C2, tyrosi-

Combination
Durvalumab
nase, or gp100). After vaccination, two patients exhibited a

complete response, two patients exhibited a partial response, and
Placebo
adenocarcinoma, PAAD pancreatic adenocarcinoma, TAAs tumor-associated antigens, TriMix mRNA encoding TLR4, CD40L, and CD70, DC dendritic cell
all patients showing an objective response had a progression-free
cells
No
No
No
No
No
No
No disease > two years. In addition, Jansen et al. showed a phase II

trial using TriMixDC-MEL as an adjuvant treatment in stage III/IV
melanoma.150 The findings of the study arm displayed 71% of
Nanoparticle
Nanoparticle
patients were free of disease as compared to 35% in the control

Unknown
Unknown
Unknown
Unknown
arm one year later. Given that the expression of PD-1/PD-L1 can
Vehicle
compromise the efficacy activated by mRNA vaccines, one study

T cell
Lipid
Lipid
DC
DC
DC
DC
No
investigated the effect of combined treatment of TriMixDC-MEL

and an immune checkpoint inhibitor (ipilimumab) in stage III/IV
melanoma (NCT01302496).151 The treatment elicited objective
Personalized tumor antigens
long-term clinical responses, with an overall survival of 28% (11/

Personalized neoantigens
39) and progression-free survival of 18% (7/39) after 5 years of

TGF-β receptor type II
follow-up. Eighty percent (12/15) of patients with immune

Survivin, hTERT, p53
OX40L, IL-23, IL-36γ
monitoring were vaccine responders, and among them,

Encoded antigen
Mucin-1, survivin
10 showed T-cell responses against at least two antigens.

Although in vitro transcription-based forms are less common
than DC-based forms, the development in molecular biotechnol-
ogy has made this form feasible for cancer treatment. BioNTech,
TriMix
NCT03948763 KRAS mutant advanced or metastatic CRC, PAAD KRAS
WT1
GenenTech, and Moderna are leading companies in the

CEA
pharmaceutical industry, and as such, they have announced

clinical updates regarding in vitro transcription-based vaccines
against melanoma. In 2004, Weide et al. performed a phase I/II
study to assess the safety and efficacy of a protamine-mRNA
NCT03480152 HCC, colon cancer, gastrointestinal cancer
vaccine encoding TAAs (gp100, Melan-A, Tyrosinase, MAGE-A1,

NCT02693236 Middle and advanced esophagus cancer
MAGE-A3, and survivin) in a group of 21 patients with metastatic

melanoma.152 After vaccination, one patient among 7 with
measurable disease experienced a complete response. Foxp3+/
NCT01291420 Metastatic or locally advanced
CD4+ regulatory T cells or myeloid suppressor cells were

NCT03908671 Advanced esophageal cancer
NCT03908671 Advanced esophagus cancer
significantly reduced in the peripheral blood of vaccinated

patients with or without keyhole limpet hemocyanin, respectively.
Two of 4 immunologically evaluable patients displayed a
NCT Number Disease condition
reproductive elevation in vaccine-specific T cells. BNT111 is a

NCT03431311 Metastatic CRC
liposomal RNA vaccine encoding four TAAs, MAGE-A3, NY-ESO-1,

NCT02316457 Triple negative
NCT03739931 Triple negative
PTEN, and tyrosinase, and its safety and effectiveness were

NCT00978913 Metastatic
NCT00003432 Metastatic
evaluated in 2015 after intravenous administration in a phase I

trial (Lipo-MERIT, NCT02410733).153 This study recruited 89
NCT03788083 Early
patients, with 42 suffering from measurable stage III/IV melanoma.

Three patients in the BNT111 monotherapy group exhibited a
partial response, seven exhibited stable disease, and one
exhibited complete metabolic remission of metastatic lesions, as
revealed by PET/CT imaging. The combination of BNT111 with PD-
1 blockade revealed that six of 17 patients experienced a partial
Table 2. continued
response. The disease was controlled for a long time in most of

the patients with partial response or stable disease in both groups
Breast Cancer
during a follow-up period of up to two years. The observed clinical

response was accompanied by the activation of CD4+ and CD8+
Disease
T-cell immune responses specifically targeting the vaccine

antigens. Additionally, the therapeutic adverse events experi-
enced by patients were predominantly mild to moderate flu-like

Zhang et al.
17
Fig. 4 Landscape of mRNA vaccines in cancers. mRNA vaccines have been developed against multiple cancers to date, including melanoma,
brain cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, blood system cancer, digestive system cancer, and breast cancer. This
figure is created using Adobe Illustrator and integrates the current literature-based knowledge
symptoms such as fever and chills. These symptoms were mostly leading to a delay in tumor growth. Additionally, in 2017,
observed early on, of short duration, easily manageable, and Fernandez et al. launched a phase I trial to evaluate the
typically resolved within 24 h. At present, BNT111 is being used in immunogenicity and safety of the ECI006 vaccine in melanoma
an ongoing phase II trial for the treatment of PD-1 inhibitor (a combination of TriMix and TAA-encoding mRNA)
refractory/recurrent and unresectable stage III/IV melanoma (NCT03394937). Nevertheless, the abovementioned mRNA vac-
(NCT04526899). On November 19, 2021, BioNTech was granted cines are designed to target TAAs, and central tolerance is
priority eligibility for the treatment of melanoma with BNT111 by inevitable. Therefore, personalized mRNA vaccines are warranted
the FDA. In 2022, Sittplangkoon et al. studied the immunogenicity to overcome this challenge.
and antitumour responses of mRNA that encodes tumor antigens The initial application of personalized RNA mutanome vaccines
with varying levels of N1-methylpseudouridine modification in a in human melanoma was reported in 2017.155 The authors
B16 melanoma model.154 The mRNA vaccine encoding OVA- identified nonsynonymous mutations in 13 melanoma patients
induced significant production of IFN-I and the maturation of DCs, by RNA and exome sequencing, and among them, ten per patient
with a negative correlation observed with elevating percentages were selected to construct two synthetic RNAs according to the
of N1-methylpseudouridin modification. Unmodified OVA-LNPs affinity to HLA class I/II. All patients were treated with a minimum
significantly reduced tumor growth, prolonged survival, and of eight and a maximum of 20 neoepitope vaccine injections.
increased intratumoural CD40+ DCs and the frequency of Increased responses were observed in one-third of patients who
granzyme B+/IFN-g+/TNF-a+ polyfunctional OVA peptide-specific previously showed weak responses against neoepitopes, while de
CD8+ T cells in a B16-OVA murine melanoma model. The robust novo responses were observed in the remaining patients. Eight
antitumour effects of unmodified OVA-LNPs were also found in patients with no radiologically detectable lesions at the beginning
the lung metastatic tumor model. In addition, the mRNA vaccine of the vaccination generated a vigorous immune response and
was also evaluated using B16 melanoma neoantigens (Pbk-Actn4), showed progression-free disease for 12–23 months. Moreover,

Zhang et al.
18
vaccination induced a significant decrease in the cumulative rate DC migration study involved 100 patients with resected, grade IV
of metastatic events and sustained progression-free survival. glioblastoma, but the results were not provided (NCT02366728).
When these RNA mutanome vaccines were used in combination Two clinical trials are recruiting patients to investigate the
with PD-1 blockade, a third of patients experienced a complete effectiveness of human CMV pp65-LAMP in glioblastoma, and
response to the vaccination. The study revealed that the the results are not disclosed (NCT02465268, NCT03688178). In
vaccination was well tolerated, with seven patients showing addition, a pp65-LAMP mRNA-loaded 1,2-dioleoyl-3-trimethylam-
vaccine-related immune responses. Apart from this trial, another monium-propane (DOTAP) liposome vaccine is being tested in
phase I multicenter study tested mRNA-4157 (a lipid-encapsulated high-grade glioma and glioblastoma in a phase I study, and the
personalized vaccine that encodes neoantigens selected based on results are not published (NCT04573140). Of note, mRNA vaccines
a proprietary algorithm) monotherapy or combined with pem- in malignant glioma mainly encode TAAs, and whether
brolizumab in resected solid tumors (including melanoma).147 neoantigen-based mRNA vaccines together with immune check-
Among the 13 patients in the monotherapy arm that included point inhibitors could show superior efficacy in glioma remains to
three suffering from melanoma, 12 remained disease-free after a be investigated.
median follow-up of 8 months, and no drug-related adverse
events of more than grade two were observed. Moreover, mRNA vaccines against non-small cell lung cancer
GenenTech and BioNTech started a series of phase I and II trials Lung cancer is the second most frequent cancer and the leading
for personalized lipid-encapsulated mRNA vaccines combined cause of cancer mortality worldwide,156 with NSCLC representing
with atezolizumab or pembrolizumab (e.g., NCT03289962 and 85% of all lung cancers.164 The 5-year survival rate is about 60% in
NCT03815058).147 All these pieces of evidence demonstrate that cases of resectable diseases, approximately 33% in cases of
personalized mRNA vaccines in combination with other anticancer unresectable regional disease, and 6.3% in cases of extended
approaches may pave the way for the treatment of melanoma. disease with metastasis.164,165 CV9201 and BI1361849 (CV9202) are
Diverse mRNA vaccines have been developed for the treatment two mRNA-based vaccines that were clinically tested in NSCLC.166
of melanoma, displaying potential therapeutic efficacy in clinical CV9201 is composed of a protamine-formulated sequence-
studies. However, no mRNA vaccine has been officially approved optimized mRNA that encodes five NSCLC-associated antigens:
for the treatment of melanoma. The combination of mRNA MAGE-C1, MAGE-C2, NY-ESO-1, 5T4 and survivin. CV9202 has the
vaccines with other therapeutic strategies may further enhance same composition as CV9201 with the addition of mucin-1.
their effectiveness and promote their potential for approval. Multiple clinical studies have been initiated to investigate their
efficacy in NSCLC. In 2019, Sebastian et al. reported a phase I/IIA
mRNA vaccines against brain cancer study using CV9201 in stage IIIB/IV NSCLC.167 A total of 46 locally
Primary brain cancer is less frequent in adults, representing 1–2% advanced (n = 7) or metastatic (n = 39) NSCLC patients with stable
of all cancer types worldwide.156,157 Malignant glioma is the most disease after first-line treatment were recruited and subjected to
common subtype in brain cancer, with glioblastoma being the five intradermal CV9201 injections (400–1600 µg of mRNA). The
most aggressive subtype.158 The 5-year survival of brain cancer maximum dose was recommended in phase IIA, all doses were
depends on its malignancy, with an approximate value of 32% in well tolerated, and most adverse events were mild-to-moderate
malignant glioma and approximately 5% in glioblastoma in the reactions in the injection site and flu-like symptoms. An antigen-
United States.159 DC-pulsed tumor mRNA vaccine is one of the first specific immunity was observed in 63% of assessable patients in
mRNA forms applied in human malignant glioma.160 Two studies phase IIA, and 60% (18/30) showed more than twofold activated
involved the application of autologous tumor mRNA-loaded DCs. IgD+CD38hi B cells. A total of 31% (9/29) and 69% (20/29) of
The first is a clinical study that recruited five patients who patients showed stable and progressive disease, respectively. The
underwent subtotal removal of malignant glioma without receiv- median overall and progression-free survival rates were 5 months
ing other therapy.161 All patients exhibited a specific CD8+ and 10.8 months, respectively, and the 2- and 3-year survival rates
cytotoxic T-cell response after treatment with autologous tumor were 26.7% and 20.7%, respectively. In the same year, Papachris-
mRNA-loaded DCs, and among them, three showed potent tofilou et al. reported a phase IB trial evaluating the effectiveness
cytolytic activity against autologous glioma cells. The other study of CV9202 in combination with local radiation treatment in 26
recruited seven glioblastoma patients in a phase I/II study for patients suffering from stage IV NSCLC with stable disease or
evaluating the efficacy of DC-pulsed cancer stem cell mRNA.162 partial response after standard first-line treatment.168 These
Two vaccinations were performed in all patients in the first week patients were classified into three strata: 1: no nonsquamous
after the end of the standard chemoradiotherapy, followed by one NSCLC, partial response/stable disease after treatment with four or
weekly vaccination for 3 weeks and then one vaccination or more cycles of pemetrexed- and platinum-based therapy and no
temozolomide every 2 weeks. Although tumor recurrence was mutation of EGFR (n = 16); 2: squamous NSCLC, partial response/
observed in five patients (at 10, 15, 17, 22, and 29 months after the stable disease after four or more cycles of nonplatinum compound
treatment), six patients in the control group died before the first and platinum-based treatment (n = 8); and 3: nonsquamous
patient experiencing recurrence in the vaccinated group, and NSCLC, stable disease/partial response after treatment for
three of the seven survived for more than 1000 days. To exert 3–6 months with EGFR-tyrosine kinase inhibitor, EGFR mutation
more specific antitumour effects, a randomized and blinded (n = 2). Patients received two injections of CV9202, followed by
clinical study on glioblastoma used a DC-pulsed mRNA vaccine radiation therapy (4 × 5 Gy). Patients in strata 1 and 3 subsequently
encoding CMV pp65 since this protein is expressed in >90% of were administrated with three further treatments with CV9202,
glioblastomas but not in the surrounding normal tissue.163 The while those in stratum 2 received four, after which all patients
authors assessed the impact of vaccine site preconditioning on DC were vaccinated with CV9202 at 3-week intervals for the first
migration. Twelve patients were randomly classified into two 6 months and then every 6 weeks thereafter. Vaccination of
groups and subjected to unilateral vaccine site preconditioning CV9202 was continued until disease progression required systemic
with tetanus/diphtheria toxoid or unpulsed autologous DCs. second-line treatment or in cases of patients encountering
Treatment with tetanus/diphtheria and mRNA vaccines signifi- unacceptable toxicity. Patients in strata 1 and 3 received
cantly prolonged both overall and progression-free survival, and maintenance pemetrexed or continued EGFR-tyrosine kinase
50% of patients were alive for more than 36.6 months. A pp65- inhibitor therapy, respectively. An antigen-specific immune
specific immune response was detected for several months in all response was detected in all three strata (a total of 25 evaluable
the long-term survivors, and the increased pp65-specific inter- patients), and at least a twofold increase in the magnitude of the
feron-γ levels were correlated with overall survival. A subsequent immune response against one or more of the CV9202 antigens

Zhang et al.
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compared to baseline was observed in 20 patients. Ten patients currently recruiting ovarian cancer patients for a liposome-
showed at least a twofold increase in functional CD8+/CD4+ formulated mRNA vaccine together with (neo)-adjuvant che-
T cells compared to the value at baseline. Twelve patients (12/26) motherapy (NCT04163094). Regrettably, the phase I/II trial that
exhibited stable disease, and one showed a partial response and utilized autologous DCs loaded with amplified ovarian cancer
was also treated with pemetrexed maintenance. The most stem cell mRNA, hTERT, and survivin in recurrent platinum-
common CV9202-related side effects were flu-like symptoms sensitive epithelial ovarian cancer patients was terminated in 2021
and reactions at the injection site, with three patients developing (NCT01334047), with no results disclosed. The utilization of mRNA
grade 3 (fatigue and pyrexia). Recently, a phase I/II study vaccines in ovarian cancer is still in its early stages, and the
(NCT03164772) completed the assessment of the safety and number of patients enrolled in clinical trials remains limited. The
effectiveness of CV9202 in combination with the immune efficacy of mRNA vaccines in ovarian cancer should be further
checkpoint inhibitor durvalumab targeting PD-L1 and tremelimu- explored in a larger number of patients for a better evaluation of
mab targeting CTLA-4 for the treatment of NSCLC, but the results their efficacy.
are not published. In addition, a clinical study (NCT03908671)
involving patients with NSCLC and advanced esophageal cancer mRNA vaccines against prostate cancer
for the use of a personalized mRNA vaccine that encodes tumor- Prostate cancer is the second most common cancer and the fifth
specific antigens has been registered. Although a fraction of leading cause of cancer-related mortality among men globally,
patients with NSCLC experience beneficial effects from treatment with approximately 10 million men diagnosed.156,171 It causes over
with the mRNA vaccine, the overall survival is still limited, as 400,000 mortalities annually worldwide, which is projected to
reported in published studies. Further optimization of the mRNA reach over 800,000 deaths annually by 2040.156,171–173 Islam et al.
vaccine and the selection of suitable combination therapy is developed an adjuvant-pulsed mRNA vaccine nanoparticle con-
required to enhance its efficacy. In addition, only a few studies taining an OVA-coded mRNA and a palmitic acid-modified TLR7/8
have been completed with published findings, and more clinical agonist R848 (C16-R848) encapsulated with a lipid-polyethylene
trials are needed for the future application of mRNA vaccines glycol shell.174 This vaccine successfully preserved the adjuvant
in NSCLC. activity of the encapsulated C16-R848, and exhibited a notable
improvement in mRNA transfection efficacy, with a rate exceeding
mRNA vaccines against ovarian cancer 95%. This high transfection efficacy led to enhanced presentation
Ovarian cancer is one of the most dangerous gynecological of the OVA mRNA-derived antigen on MHC class I molecules in
cancers, with around 314 000 new cases and 207 000 mortalities in antigen-presenting cells. Vaccination elicited potent adaptive
2020 worldwide.156 It accounts for ~5% of female cancer-related immune responses by improving the extension and infiltration
death and has become the fifth leading cause of female cancer- of OVA-specific CD8+ T cells in OVA-expressing syngeneic allograft
related death global. A DC-pulsed mRNA vaccine encoding folate- mouse models of prostate cancer and suppressed tumor growth
receptor-α (FR-α) was used in 2004 for treating relapsed metastatic when offered postengraftment (60% reduction vs. control).
ovarian cancer.169 The patient involved in this study was a 62-year- CV9103 encodes four TAAs in prostate cancer: PSA, PSMA, PSCA,
old woman diagnosed with advanced serous papillary ovarian and STEAP, and it is the first-in-human tested mRNA vaccine.175
cancer IIIc with widespread peritoneal carcinomatosis and Two clinical trials on the use of CV1903 in prostate cancer have
increased CA-125. The patient was subjected to two tumor been conducted. One (NCT00831467) investigated the effect of
debulking procedures and experienced two tumor relapses. The three increasing doses (256 mg, 640 mg, and 1280 mg total mRNA)
vaccine treatment with autologous DCs engineered with mRNA in cohorts of three to six patients with prostate cancer, but the
encoding FR-α started at the moment of the second relapse, with results are not published. Phase I of another open, phase I/II,
a total of ten vaccinations administered at 4-week intervals. The uncontrolled, prospective study (NCT00906243) confirmed the
CT showed a partial response when the tumor volume was safety of the dose of 320 µg RNA per antigen, providing a
compared before the treatment and 3 months after the last recommended dose for phase IIa to explore the immunological
vaccination. The CT at 16 months of follow-up revealed a activity of that dose. Forty-four patients with increased PSA and
regression of over 50% of the lymph-node metastases, and mostly existing metastases (>80%) were recruited, and the results
consistently, the vaccinations induced an FR-α-specific immune showed a superior immunogenicity rate induced by the vaccine in
response. After six vaccinations, the IFN-γ produced by FR-α- prostate cancer patients; antigen-specific T cells were observed in
stimulated CD8+ cells and FR-α-stimulated CD4+ cells increased approximately 80% of patients independently of their HLA
30-fold and 15-fold compared to the amount in the prevaccination background, and approximately 58% reacted against multiple
samples, respectively. Similarly, granzyme B was increased after antigens. The PSA levels were stabilized in individual patients and
vaccination compared to the amount in the prevaccination dropped by more than 85% in one patient. One dose-limiting
samples. No systemic or local side reactions associated with the toxicity, urinary retention, was observed in six patients after the
therapy were observed, indicating that the vaccination was well use of the highest dose. The most frequent adverse events were a
tolerated. Another publication reported the application of DC- reaction at the injection site or flu-like symptoms (e.g., chills and
pulsed mRNA encoding WT1 in ovarian carcinoma and carcino- fever).
sarcoma.170 Two patients, one with serous ovarian cancer and the A subsequent clinical evaluation was performed due to the
other with ovarian carcinosarcoma, received four weekly vaccina- favorable safety profile and strong antigen-specific immune
tions, which induced increased CD137+ antigen-specific T cells, IL- responses of CV9103 to assess two additional antigens, PAP and
2, and IFN-γ in ovarian carcinoma and CD137+ antigen-specific mucin-1, developing a new vaccine termed CV9104 that was used
T cells, IL-2, and TNF-α in ovarian carcinosarcoma. Unfortunately, in two clinical studies.175 The first trial (NCT01817738) enrolled
the disease progressed after four vaccinations, and the patient patients with castrate refractory metastatic prostate cancer who
with ovarian carcinoma survived for 19 months, while the patient were subjected to surgery or androgen suppression therapy (by
with ovarian carcinosarcoma survived for 12 months after the end GNRH agonist or antagonist). The vaccination started at a dose of
of vaccine administration. In that same year, a phase I study was 1920 µg in weeks 1, 2, and 3, continued in weeks 5, 7, 9, 12, 15, 18,
conducted to assess the safety of active immunotherapy using and 24, then every 6 weeks for 12 months and every 3 months
fully mature, TERT-mRNA, and survivin-peptide double-loaded DCs thereafter until treatment discontinuation. The overall survival
in 15 patients with advanced epithelial ovarian cancer. However, from the time of randomization was up to 3.5–4 years. The second
the results were not published despite the completion of the trial using CV9104 (NCT02140138) was in an open-label rando-
study (NCT01456065). A first-in-human, open-label phase I study is mized trial involving 35 high-risk and intermediate-risk patients

Zhang et al.
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with prostate cancer. Patients received four doses of CV9104 et al. performed the first-in-human phase I trial involving 10 AML
vaccine in weeks 1, 2, 3, and 5, and then, these patients patients on TLR7/8-matured DC-pulsed mRNA vaccines encoding
underwent radical prostatectomy over one but within 2 weeks. WT1 and PRAME (two AML-associated antigens) as well as CMV
The primary outcome was the evaluation of the antigen-specific pp65.184 Seven patients were subjected to the complete regular
cellular and humoral immune response to the vaccine, while the 10 vaccinations, resulting in an increase in WT1 (2/10)-, PRAME (4/
secondary outcome was the measurement of the incidence and 10)-, and CMV pp65 (9/10)-specific CD8+ T cells, and CMV pp65-
severity of the adverse effects and the changes in PSA serum induced CD4+ T cells (4/7) in the peripheral blood. The median
levels. However, no clinical therapeutic results have been provided relapse-free survival was 1084 days, while the median overall
thus far. Together, the mRNA vaccines against prostate cancer are survival was not reached after 1057 days, with five patients (50%)
mostly at an attempted stage, and the potential values in survival relapse-free at the end of the observation.
require more support from clinical results. Some studies have focused on chronic lymphocytic leukemia
and lymphoma. The studies were mostly performed in preclinical
mRNA vaccines against blood system cancer trials. Kokhaei et al. showed that a DC-pulsed mRNA vaccine did
Hematological malignancies encompass a range of diseases not elicit a marked enhancement in IFN-γ-producing T cells
involving the abnormal proliferation of hematopoietic stem cells, compared with unpulsed DCs against B-cell chronic lymphocytic
including leukemia, myeloma, and lymphoma.176 Leukemia ranks leukemia.185 In contrast, mRNA vaccines have exhibited effective-
as the first leading cause of cancer death among blood diseases ness in lymphoma. In 2011, Fotin-Mleczek et al. reported the use
and the tenth leading cause of cancer deaths overall worldwide of a two-component mRNA vaccine (protamine-complexed)
(www.iarc.fr) according to the global cancer statistics for 2020 encoding TLR7 and tumor antigen Gallus gallus OVA, HsPSMA,
released by the International Agency for Research on Cancer. The or HsSTEAP for treating T-cell lymphoma in an E. G7-OVA-based
common leukemia types include acute myeloid leukemia (AML), mouse model, in which E. G7-OVA is a mouse T-cell lymphoma cell
chronic myeloid leukemia, chronic myelomonocytic leukemia, line stably expressing Gallus gallus OVA.186 The vaccine triggered
chronic neutrophilic leukemia, and atypical chronic myeloid antigen-specific CD4+ and CD8+ T-cell responses, and sustained
leukemia.177 mRNA vaccines have mainly been applied to AML immune memory, and the vaccination mediated a strong
in blood system cancer thus far. In 2005, Jarnjak-Jankovic showed antitumour response in both prophylactic and therapeutic
that a DC-pulsed tumor mRNA vaccine triggered specific T-cell contexts. In 2021, Tusup et al. conducted an assessment on the
responses against leukemia cells in vitro.178 Subsequently, efficacy of an mRNA vaccine in inducing immune response against
Driessche et al. reported a phase I clinical study with dose TCR CDR3 regions using a murine model based on EL4
escalation of an autologous DC-pulsed mRNA vaccine encoding T-lymphoma cell line, resulting in a feasible approach in
WT1 in 10 patients with AML.179 Patients were administered protection against T-lymphoma.187 In 2022, Slam et al. developed
intradermal injections every 2 weeks, receiving 5, 10, or 20 × 106 an mRNA vaccine consisting of an OVA-coded mRNA and a
DC in the ventromedial region of the thigh or upper arm. The four palmitic acid-modified TLR7/8 agonist R848 (C16-R848) together
doses were well tolerated by all patients, and no autoimmune or with a lipid-polyethylene glycol shell.174 Vaccination significantly
acute toxicities were observed throughout the entire trial. This increased the amplification and infiltration of OVA-specific CD8+
team further showed the results of the use of the above vaccine in T cells in OVA-expressing syngeneic allograft mouse models of
phase I/II study on AML patients.180 Patients with hematological lymphoma and prevented tumor growth when the vaccine was
remission after chemotherapy were enrolled 1 month after given before tumor engraftment (84% reduction vs. control). At
polychemotherapy for four biweekly vaccinations. Five (50%) present, a phase I study is ongoing for the evaluation of the effects
patients (two of them refractory to chemotherapy) showed of mRNA-2752 (a lipid nanoparticle encapsulating mRNAs encod-
complete disease remission (absence of blasts in blood and less ing human OX40L, IL-23, and IL-36γ) alone and combined with an
than 5% blasts in the bone marrow) after intradermal vaccination, immune checkpoint blockade after intratumoural injection in solid
with the myeloblast percentage decreasing to a normal level. Out tumors and lymphoma (NCT03739931).
of the five individuals, three exhibited long-term responses with Overall, the mRNA vaccine showed promising effects in AML in
complete remission that endured for over three years. A clinical trials, although none has been approved for its standard
significantly positive correlation was found between the long- therapy. Except for AML, mRNA vaccines in other human blood
term response and WT1-specific CD8+ T-cell number. This study system cancers are principally in the preclinical phase, and more
was performed again two years later on more patients, 17 in total, clinical trials are warranted to investigate their efficacy.
and among them, eight showed a complete response with a
median relapse-free survival of 47 months.181 On this basis, this mRNA vaccines against digestive system cancer
group reported a phase II trial about a DC-pulsed mRNA vaccine Cancer can arise in any tissue of the gastrointestinal tract,
encoding WT1 as postremission treatment in 30 AML patients at including the colon, stomach, esophagus, liver, and pancreas.188
high risk of relapse.182 Thirteen patients showed an antileukemic Digestive system cancer is a leading cause of cancer morbidity
response, with five-year overall and relapse-free survival rates of and death worldwide, and three million new cases and two million
53.8% and 50%, respectively (7.7% and 30.8% in nonresponders, deaths from gastrointestinal cancers occur every year.156 Ghola-
respectively). Patients aged ≤65 who had complete remission min et al. used a DC-pulsed tumor mRNA vaccine in esophageal
showed a longer 5-year survival (69.2%) than those aged > 65 squamous cell carcinoma in vitro, and the results showed a
years who experienced the same remission (30.8%), which is more significant induction of cytotoxicity (median >18.7% compared
than 51.7% in those aged ≤65 and 18% in those aged > 65 years with the control) and INF-γ secretion (> twofold compared with
present in the Swedish Acute Leukemia Registry. The same year, the control).189 Mahdi Forghanifard et al. also used a DC-pulsed
Khoury et al. also investigated a DC-pulsed mRNA vaccine mRNA vaccine encoding MAGE-A4, NY-ESO1, and LAGE1, which
encoding hTERT in 21 adult patients with AML: 16 in the first also promoted the activation of CTLs against esophageal cells
complete remission, three in the second complete remission, and in vitro.190 Peng et al. used a DC-pulsed mRNA vaccine derived
two with early disease recurrence.183 Among those in complete from HepG-2 cells or samples from hepatocellular carcinoma
remission, 11 (58%) developed a specific T-cell response and were (HCC) patients, and the results showed an increase in the number
free of disease at a median follow-up of 52 months. Four (57%) of CD8+ T cells in cytotoxic T lymphocytes (CTLs) and a promotion
patients older than 60 years were free of disease recurrence at a of cytotoxic activity in HCC in vitro.191 A preclinical study using
median follow-up of 54 months. To improve the effectiveness mRNA 5671 evaluated its therapeutic efficacy in colorectal cancer.
exerted by mRNA vaccines targeting monoantigens, Lichtenegger The vaccine described encodes the four frequently observed KRAS

Zhang et al.
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mutations (G12C, G12D, G12V, and G13D).192 When used as on mRNA vaccines against NSCLC, a clinical study of personalized
monotherapy or in combination with pembrolizumab, it promotes mRNA vaccines that encode tumor-specific antigens in patients
an augmentation in CD8+ T-cell responses in mice. Similarly, Kim with NSCLC and advanced esophageal cancer has been registered
et al. showed that a DC-pulsed CEA mRNA vaccine with (NCT03908671), and the results in esophageal cancer are still
modification of calreticulin and the TAT protein transduction unknown.
domain induced a potent CD4+ and CD8+ T-cell response and Altogether, although clinical trials using mRNA vaccines to
antitumour effects in mice with colon cancer.193 Clinically, Wan combat digestive system cancer are limited, some effectiveness was
et al. used a CD40-B-cell-pulsed mRNA vaccine encoding alpha- shown in a fraction of patients, providing a foundation for further
fetoprotein for the treatment of HCC since their hypothesis was development of efficient treatments for digestive system cancer.
that the vaccine may boost a robust and prime naïve T-cell
response;194 however, they did not report any preclinical or clinical mRNA vaccines against breast cancer
results to date. Maeda reported a phase I clinical trial on a DC- Breast cancer is the most frequently diagnosed cancer in women
pulsed heat-shock protein 70-encoded mRNA vaccine used at and the leading cause of cancer-related death globally.156 Global
increasing doses in hepatitis C virus-related HCC.195 Twelve Cancer Statistics 2020 reports that female breast cancer has
patients were enrolled, divided into three cohorts, and treated surpassed lung cancer and has become the most frequently
with three vaccinations every three weeks (1 × 107, 2 × 107, and diagnosed cancer.156 Breast cancer includes three major subtypes:
3 × 107 DCs). The dose of 3 × 107 DCs was the recommended dose ER+, HER2+, and triple-negative breast cancer (TNBC).203 Conven-
according to the outcome of the pretreatment. Two patients tional endocrine or targeted drugs are not effective against TNBC
experienced complete response without recurrence, five patients compared with other subtypes, and TNBC has the worst prognosis,
experienced disease progression, and five experienced stable with over 50% of patients experiencing relapse within the initial 3
disease. The two patients with complete response showed no to 5 years following diagnosis and a median overall survival of
disease recurrence for 44 and 33 months, respectively. Lesterhuis 10.2 months.203,204 In 2013, five partners in academia and industry
et al. conducted a comparison between the effects of DC-pulsed led by BioNTech AG launched The Mutanome Engineered RNA
CEA peptide and DC-pulsed CEA mRNA vaccines in patients Immuno-Therapy project (NCT02316457) to validate a pioneering
diagnosed with resectable liver metastases of colorectal cancer.196 mRNA vaccine concept targeting individually expressed tumor
All patients received three intravenous and intradermal vaccina- antigens and tumor neo-antigens in patients with TNBC from
tions every week. However, anti-CEA-specific antibodies were clinical and industrial perspectives.205,206 This project developed a
detected in eight (8/11) patients in the peptide group, but no computational medicine platform to identify tumor neoantigens
antibodies were found in the five patients in the mRNA group. In and TAAs in patients with TNBC, set up an mRNA vaccine
addition, an mRNA vaccine encoding neoantigens induced warehouse for shared tumor antigens solving >95% of TNBC
specific T-cell immune responses in patients with gastrointestinal patients as well as a manufacturing process for producing a
cancer.197 The mRNA-based vaccine mRNA 4650 was clinically personalized mRNA vaccine. In addition, this platform evaluated
evaluated for the treatment of various digestive system cancers, the associated biomarkers identifying molecular and immunolo-
including gastrointestinal cancer and liver cancer.198,199 Patients gical signatures correlated with clinical events following vaccina-
with gastrointestinal cancer treated with an intramuscular tion and identified synergistic agents and optimized protocols of
administration of mRNA 4650 developed CD4+ and CD8+ T-cell personalized vaccines. The vaccine consists of “off-the-shelf”
responses against tumor neoantigens. mRNA 4157 was designed mRNA selected from a presynthesized mRNA and a vaccine
to encode 34 unique neoantigens, and a phase I clinical study is warehouse encoding neoantigens expressed in individual patient
ongoing in patients with MSI-high colorectal cancer and other tumors as well as an mRNA engineered on-demand encoding
solid tumors.200 It induces antigen-specific T cells and is well patient-specific sequence stretches that incorporate nonsynon-
tolerated when used as monotherapy or in combination with ymous mutations. Every tumor is profiled before treatment to
pembrolizumab, leading to complete or partial responses. Suso select the proper shared tumor antigens and detect mutations by
et al. published a case report of a pancreatic cancer patient exome sequencing. A cutting-edge platform is used for the
treated with a DC-pulsed telomerase-encoded mRNA vaccine.201 design, manufacture, and release of tailored mRNA vaccines based
The patient was a 62-year-old woman who was treated with on the output of the profiling. In 2019, Schmidt reported phase I/II
standard gemcitabine chemotherapy after developing multiple trials assessing the feasibility, safety, and biological effectiveness
metastatic lymph node lesions after surgery. Chemotherapy was of this personalized mRNA vaccine in Germany and Sweden.206
stopped because of the occurrence of severe neutropenia, and it Patients were allocated to one of two study arms at the end of the
was replaced with vaccination. The patient experienced a standard of care therapy. Patients in arm 1 were subjected to eight
remarkable decrease in lymph node metastases after 32 months vaccination cycles with a vaccine encoding shared TAAs selected
of vaccination without any increase in metabolic activity in the according to the tumor antigen expression profile (mRNA WARE-
lesions compared with other lymph nodes. Furthermore, no HOUSE vaccine). Patients in arm 2 were subjected to treatment
serious treatment-related adverse events were observed during with the mRNA WAREHOUSE vaccine followed by eight vaccina-
the 3-year vaccination. In 2013, Chen et al. compared the efficacy tion cycles with a vaccine encoding personalized 20 unique
of DC-pulsed mRNA encoding mucin-4 and/or survivin in neoepitopes identified by next-generation sequencing (mRNA
pancreatic cancer in vitro.202 All three cohorts induced a CTL MUTATION vaccine). Preliminary immune response results from
response, which was stronger for DCs cotransfected with both patients in arm2 have been disclosed at the Annual Meeting of the
antigens. A phase I clinical trial has been completed and evaluated European Society of Medical Oncology.206 Vaccine-triggered CD4+
the efficacy, safety, and tolerability in multiple cancers, including and/or CD8+ T-cell responses against 1-10 neoepitopes, as well as
colorectal cancer, although the findings were not published a great number of neoepitope-specific T-cell responses (10.3% of
(NCT03948763). In 2020, a phase I/II trial assessed the safety and peripheral CD8+ T cells), were found in all 14 patients vaccinated
immunogenicity of an mRNA-based, personalized vaccine against with the mRNA MUTATION vaccine. Moreover, approximately 30%
neoantigens in autologous gastrointestinal cancer (NCT03480152). of peripheral CD8+ T cells exhibited a diversified CD8+ T-cell
Specific immunogenic mutations as targets for the mRNA vaccine response, characterized by a high number of poly-epitopic TCR-
were identified in tumor-infiltrating lymphocytes. The vaccination clonotypes, which lasted for at least 6 months at high levels after
elicited a mutation-specific T-cell response against the predicted the last vaccination. Although vaccination induced specific T-cell
neoepitopes, but no objective clinical responses were found in the responses, the survival data are still unpublished, and the efficacy
four treated patients in this trial. As mentioned in the paragraph of the mRNA vaccine is not yet clear. Moreover, only one study

Zhang et al.
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investigated the efficacy of personalized anti-breast cancer mRNA MRNA VACCINES IN TISSUE DAMAGE
vaccines, and more trials are needed to promote them in clinical Tissue damage refers to any physical injury or harm that occurs to
practice. the body’s tissues. This can be resulted from a variety of factors,
such as trauma, infection, inflammation, and exposure to harmful
substances or radiation. Tissue damage can affect any part of the
MRNA VACCINES IN IMMUNOLOGICAL DISEASES body, including the skin, muscles, bones, organs, and nerves.
Autoimmune diseases are characterized by chronic inflammation Cardiovascular damage is the leading threat to human health
due to a dysregulated immune response to self-antigens.207 Many worldwide.173 They include but are not limited to coronary heart
clinical studies using mRNA vaccines against cancers or infectious disease, hypertension, heart failure, vascular calcification, and
diseases have exhibited their potential to trigger autoimmune cardiac fibrosis.139 Cardiovascular damage is mostly irreversible
diseases.8 However, mouse models have revealed their ability to and can only be controlled. In 2016, AstraZeneca developed
treat autoimmune diseases, although no clinical applications have AZD8601, an mRNA vaccine encoding VEGF-A165 with a minimal
yet been performed.208 The physiological induction and main- innate immune response.213–215 AZD8601 used in preclinical
tenance of peripheral tolerance are primarily determined by the models induced more blood vessels in local tissue and
presentation of self-antigens by antigen-presenting cells (APCs) significantly accelerated the healing of chronic wounds in a
with diminished surface expression of costimulatory molecules, dose-dependent manner.213–215 A clinical trial was subsequently
such as DC86. Conventional U-composed mRNA vaccines often started in 2017 in patients with coronary artery disease under-
elicit strong type I T helper cell responses driven by TLR signaling. going coronary artery bypass grafting surgery (NCT03370887).
Krienke et al. introduced a liposomal formulation that systemically Patients were randomly and equally divided into three groups and
delivers antigens encoded by the mRNA vaccine into lymphoid further treated with AZD8601 at different doses or placebo, with
tissue-resident CD11c+ APCs and replaced uridine (U) by the the evaluation of the safety of AZD8601 as the primary endpoint.
incorporation of N1-methylpseudouridine. This method avoids the The results were reported at the American Heart Association’s
significant activation of CD8+ T cells, CD4+ T cells, CD11+ APCs, Scientific Sessions 2021, showing the safety and tolerability of
natural killer cells, and B cells, as well as the secretion of IFN-α or AZD8601 as well as the positive trends in exploratory efficacy
other inflammatory cytokines in mice, suggesting that objectives. Rurik et al. developed an antifibrotic treatment strategy
nanoparticle-formulated N1-methylpseudouridin-modified mRNA based on chimeric antigen receptor T cells using CD5-targeted
is appropriate for the noninflammatory delivery of proteins into LNPs-mRNA. Ten micrograms of CD5/LNP-mRNA encoding FAP-
splenic CD11c+ APCs. In an experimental autoimmune encepha- CAR were intravenously injected into mice with cardiac injury
lomyelitis mouse model of multiple sclerosis induced by the induced by the delivery of AngII/PE. Echocardiography showed
selective expression of MOG (the epitope of myelin oligoden- remarkable functional improvement in the injured mice 2 weeks
drocyte glycoprotein in DCs), mice were vaccinated with MOG- after the initial treatment. Of note, left ventricular diastolic
encoding N1-methylpseudouridine mRNA after immunization with function was significantly improved and returned to the original
MOG, and the results showed that they were protected from healthy level during the follow-up period. The improvement in the
disease development. Vaccination also prevented further disease extracellular matrix burden was more evident in the mice treated
progression in mice with an established disease and even reverted with LNP-mRNA than in those treated with saline. Altogether,
pathology in some cases. The treatment suppressed disease- these findings were encouraging and provide possibilities for the
promoting TH1, TH17, and TH1/TH17 cells by inducing FOXP3+ treatment of irreversible cardiovascular diseases.
regulatory T cells and increasing the expression of T-cell Apart from cardiovascular diseases, mRNA vaccines have shown
exhaustion markers (e.g., PD-1, CTLA4, TIGIT, TIM-3, and LGA-3). effectiveness in multiple soft tissue damages.216 The administra-
Vaccination did not influence the immune responses to unrelated tion of mRNA-LNPs containing nucleoside-modified mRNA that
antigens, and this approach was effective in models induced by encodes HGF and EGF was found to stimulate liver regeneration in
different antigens (PLP, MBP, and MOBP), suggesting important mice with chronic choline-deficient ethionine-mediated liver
aspects of this approach, such as the possibility of optimizing the injury and acute acetaminophen-induced liver toxicity.217 In the
mRNA vaccine to elicit protective immune responses against same year, another study utilized mRNA that encodes VEGF-C to
specific pathologies and maintaining antigen-specific immune induce the growth of lymphatic vessels in mice.218 By adminis-
tolerance to treat autoimmune diseases. tering low dose of VEGF-C mRNA-loaded lipid nanoparticles
Allergy is a hypersensitivity reaction of the immune system to a (mRNA-LNPs), targeted lymphatic growth was induced, leading to
foreign substance that is typically harmless to most individuals. the remarkable reversal of lymphedema and restoration of
This foreign substance, known as an allergen, triggers an immune lymphatic function in an experimental mouse model. In a mouse
response that results in various symptoms, such as itching, model of diabetes, the delivery of nucleoside-modified mRNA
sneezing, watery eyes, and skin rash. Common allergens include encoding FGF-2 through mineral-coated microparticles improved
pollen, dust mites, certain foods, medications, and insect venom. the healing of dermal wounds by hastening the process of
Allergies can range from mild to severe and, in some cases, can be complete wound closure.219
life-threatening. mRNA vaccines also offer a safer approach to In 2015, Elangovan et al. showcased the promising potential of
preventing allergic conditions by encoding the allergen and mRNA-based therapeutic strategies in the field of bone regenera-
providing a purer immunizing antigen compared to traditional tion.220 They employed pseudouridine and 5-methylcytidine-
allergen extracts.209 In mice, mRNA vaccines that encode allergens modified mRNA encoding BMP-2, which was combined with
have been found to be effective in preventing type I allergies by polyethylenimine (PEI) and incorporated into collagen scaffolds
activating a Th1 cell response.210 After immunization, the mice prior to the implantation into rat calvarial defects. After a duration
were exposed to the corresponding allergen, and the resulting of 4 weeks, the PEI-BMP-2 mRNA-activated matrices exhibited a
inflammatory signatures (e.g., eosinophils, IL-4 and IL-5) were significant improvement in bone regeneration when compared
reduced, while anti-inflammatory responses were enhanced (e.g., with the PEI-complexed BMP-2 pDNA-activated matrices. Micro-
the induction of IFN-γ-producing cells).211 More importantly, computed tomography analysis revealed a significant increase in
mRNA vaccines have been exhibited to generate long-term both the amount of bone volume and total volume of regenerated
memory responses in mice, leading to potent anti-inflammatory bone in defects treated with scaffolds embedded with PEI-mRNA
responses upon re-exposure to allergens.212 These findings and PEI-pDNA complexes. Specifically, the defects treated with
illustrate the potential of mRNA vaccines for targeting allergies PEI-mRNA exhibited a 3.9-fold higher bone volume, while the total
without the need for booster vaccinations. volume of regenerated bone was 1.9-fold higher compared to the

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23
negative control group. Balmayor et al. also confirmed the treatment. mRNA vaccines have been reported to have the
osteogenic potential of nucleoside-modified BMP-2 mRNA treat- potential to treat multiple rare diseases. Cystic fibrosis is a
ment in a rat femur bone defect model.221 Furthermore, the hereditary condition, predominantly impacting the lungs, pan-
administration of a low dose (2.5 µg/defect) of nucleoside- creas, and other organs.226,227 It is caused by a mutation in the
modified mRNA within a fibrin gel matrix demonstrated speeded CFTR gene, causing the generation of thick, sticky mucus in the
up bone healing compared to the fibrin control group, as lungs and other organs. This mucus can clog airways and make it
evidenced by significant improvements observed just 2 weeks difficult to breathe, leading to chronic lung infections, lung
after application. A study was undertaken with the goal of damage, and respiratory failure. Cystic fibrosis can also affect the
augmenting long-lasting mRNA delivery to specific cells and pancreas, causing digestive problems and malnutrition, and it can
creating a convenient ready-to-use product. To achieve this, the lead to other complications, such as liver disease, diabetes, and
researchers developed a vacuum-dried construct known as infertility. Cystic fibrosis is a lifelong condition that currently has
transcript-activated matrices (TAMs). In a noncritical femoral bone no cure, while treatment helps symptom management as well as
defect rat model, collagen sponges were preloaded with improves quality of life. In 2018, Robinson et al. reported that a
nucleoside-modified BMP-2 mRNA-loaded lipid nanoparticles clinically relevant lipid nanoparticle-packed chemically modified
(mRNA-LNPs), resulting in a remarkable enhancement of bone mRNA encoding CFTR increased membrane-localized CFTR and
generation when compared to empty collagen sponges. exhibited rescued its role as a chloride channel in patient-derived bronchial
exceptional stability at room temperature for a minimum of epithelial cells; its nasal application restored CFTR-mediated
6 months, and facilitated prolonged protein generation for up to chloride secretion to conductive airway epithelia in CFTR-deficient
6 days. This seminal study showed BMP-2-encoding TAMs were mice, representing a promising platform for the correction of
effective in delivering sustained mRNA to target cells. In a cystic fibrosis.228 Preclinical evaluation of MRT5005 (an mRNA
subsequent investigation, the researchers explored the dose- encoding the CFTR protein) administered by nebulization
dependent impact of nucleoside-modified BMP-2-encoding TAMs validated cystic fibrosis correction in mice and nonhuman
on the promotion of new bone formation in a critical femoral primates.229 A phase I/II clinical study is currently in progress,
defect rat model. Micro-CT and histological analyses revealed that seeking participants for a randomized, double-blinded, placebo-
the higher dose of the product (15 µg/defect) exhibited approxi- controlled study. The trial aims to assess the safety, tolerability,
mately double the amount of newly formed bone compared to and biological activity of MRT5005 when administered via
the lower dose (3.75 µg/defect).222 A study conducted a compar- nebulization to adults diagnosed with cystic fibrosis
ison of BMP-9-PEI-activated matrix (collagen scaffold) and BMP-2- (NCT03375047).
PEI-activated matrix in terms of their ability to promote bone Inherited metabolic disorders are significant contributors to
regeneration. The results unveiled a superior capacity of BMP-9 illness and death in children.230 These disorders, which affect
mRNA transfection in enhancing the in vitro osteogenic differ- approximately 1 in 800 live births, often stem from mutations in a
entiation of human bone marrow mesenchymal stem cells single gene inherited in an autosomal recessive pattern.231
compared to the administration of BMP-2 mRNA. Furthermore, Inherited metabolic diseases are responsible for 10–15% of
when implanted in rat calvarial bone defects, BMP-9 mRNA pediatric acute liver failure cases, with mortality rates ranging
exhibited a remarkable 2-fold increase in the connectivity density from 22–65%.231 mRNA vaccines have been tested in several rare
of the regenerated bone compared to BMP-2 mRNA.223 To genetic disorders, such as hereditary tyrosinemia type 1, phenylk-
enhance the gene-activated collagen membrane, an additional etonuria (PKU), methylmalonic acidemia (MMA), propionic acid-
improvement was made by immersing the perforated collagen emia (PA), glycogen storage disease type 1a (GSD1a), and ornithine
membrane in a solution containing BMP-9 mRNA-PEI complexes, transcarbamylase (OTC) deficiency. PKU is a genetic metabolic
followed by a freeze-drying process. Upon application of this disorder resulting from insufficient functional phenylalanine
product to rat calvarial defects, a notable and significant formation hydroxylase (PAH) activity, causing the buildup of phenylalanine
of new bone was observed after a 4-week period of treatment.224 (Phe) in the blood and organs of those affected.232,233 Without
A combination therapy involving mRNA, stem cell transplantation, treatment, patients experience significant neurological damage.
and scaffolds has recently been investigated for bone regenera- Administering mouse Pah mRNA packaged in LNPs through
tion. In a rat model of calvarial bone defects, the implantation of repeated intravenous injection into a PKU (Pahenu2) mouse model
nucleoside-modified BMP-2 and VEGF-A mRNA-transfected bone produced therapeutic PAH protein, reduced Phe levels in the liver,
marrow mesenchymal stem cells within a collagen scaffold serum, and brain, and reversed the progression of the dis-
resulted in a significant augmentation of bone regeneration. The ease.234,235 These findings suggest Pah mRNA formulated in LNPs
simultaneous delivery of BMP-2 and VEGF-A mRNAs exhibited a offers an alternative therapeutic option for PKU patients who
synergistic effect, effectively promoting both osteogenic and eliminates the need for a lifelong Phe-restricted diet. In line with
angiogenic processes.225 This synergistic action resulted in super- this possibility, ModernaTx, Inc. (Cambridge, MA, USA) has included
ior healing outcomes when compared to treatments involving PAH PKU mRNA-3283 in its product development pipeline
BMP-2 or VEGF-A alone. The findings strongly indicate that (www.modernatx.com/research/product-pipeline). MMA is an
employing a combination of multiple growth factor-encoding organic acidaemia that poses a high risk of morbidity as well as
mRNAs, along with cell therapy and a biomaterial scaffold, holds death and currently has no approved treatments addressing its
great promise as a viable strategy to attain favorable outcomes for underlying cause.236 This autosomal recessive disorder hinders the
bone regeneration. metabolism of propionate derived from certain proteins and
Together, mRNA vaccines show promising potential in the fats.237 As a result, there is a notable accumulation of methylma-
promotion of tissue generation. Apart from the abovementioned lonic acid in body fluids and tissues. The primary cause of this
damage, mRNA vaccines may be able to promote the generation disease is commonly attributed to a deficiency in the mitochon-
of other tissues. drial enzyme known as methylmalonyl-coenzyme A (CoA) mutase
(MUT). Repeated intravenous injection of LNP-encapsulated MUT
mRNA into hypomorphic Mut−/−, TgINS-CBA-G715V mice resulted in a
MRNA VACCINES IN RARE DISEASES decrease in plasma MMA concentrations as well as an enhanced
Rare diseases are defined as medical conditions that impact a survival rate.238,239 Significantly, comprehensive safety studies
small proportion of the population, characterized by their low revealed no discernible alterations in liver function tests, inflam-
prevalence and often limited understanding due to their rarity. matory cytokine generation, or the production of anti-MMA
Patients may struggle to find appropriate medical care and antibodies. A phase I/II clinical trial is presently underway to assess

Zhang et al.
24
the safety, pharmacokinetics, and pharmacodynamics of adminis- diseases long-term, despite still in an attempt stage. More studies
tering LNP-encapsulated human MUT mRNA (mRNA-3705) to are warranted to validate their efficacy against rare diseases.
individuals diagnosed with isolated methylmalonic acidemia
(NCT04899310 and NCT05295433). PA is a pediatric disorder
caused by a mitochondrial deficiency in propionyl-CoA carboxylase CONCLUSIONS AND PERSPECTIVES
(PCC), which is an enzyme consisting of a heterododecamer mRNA vaccines have become a hotspot in disease prevention and
encoded by the PCCA and PCCB genes that plays a vital role in treatment, becoming predominant in preclinical and clinical trials,
catalyzing the carboxylation of propionyl-CoA to methylmalonyl- especially in infectious diseases and cancers.141 Nevertheless,
CoA within the body.240 This deficiency hampers the metabolism except for the anti-COVID-19 mRNA vaccine, few have been
of propionate, resulting in the accumulation of toxic metabolites approved for disease treatment thus far. Several challenges are
within the body, such as 2-methylcitrate, 3-hydroxypropionate, and not completely addressed that may limit the application of mRNA
propionyl carnitine. Intravenous injection of LNP-encapsulated vaccines. Striking a balance between achieving optimal antigen
PCCA and PCCB mRNAs led to the generation of therapeutic levels production and ensuring adequate adjuvant effects poses a
of PCCA and PCCB in the livers of a hypomorphic disease model significant challenge. The adjuvant effect of mRNA vaccines
(Pcca−/−[p. A138T]) in mice.241 During a 6-month duration, the promotes innate and adaptive immunity, but excessive innate
repeated administration of PCCA and PCCB mRNAs encapsulated immunity inhibits mRNA translation.8,246 5′ capping, nucleoside
in LNPs was well tolerated. This treatment approach resulted in a modification, poly(A) tail modification, and HPLC purification are
reduction of toxic metabolite levels in the plasma, although strategies already used to decrease innate immunity.17,247,248 The
complete normalization was not achieved. Liver transaminase interaction of the delivery carrier mRNA and innate immune
levels remained within the normal range, and no adverse reactions system requires further investigation to achieve an effective
were observed. These findings support the ongoing Phase I/II study balance. Another challenge is the large-scale manufacturing of
of mRNA-3927 (LNP-encapsulated PCCA and PCCB mRNAs) to mRNA. As a consequence of the lack of a continuous manufactur-
evaluate the safety and pharmacodynamic activity of the therapy ing process, synthesis, purification, and formulation must be
in PA patients aged 1 year or older (NCT05130437 and performed in different facilities in three states in the USA, largely
NCT04159103). GSD1a is a genetic metabolic disorder resulting limiting the rapid manufacture of mRNA vaccines. For instance,
from an autosomal recessive mutation in the gene responsible for the manufacture of millions of doses of BNT162b2 takes 60 days,
coding the catalytic subunit of glucose-6-phosphatase (G6Pase).242 far from satisfying the vaccination needs of 6 billion people
This enzyme hydrolyses glucose-6-phosphate, producing free worldwide (derived from https://www.nytimes.com/interactive/
glucose. As the main hub for gluconeogenesis, the liver serves as 2021/health/pfizer-coronavirus-vaccine.html). A continuous man-
the primary organ affected by disruptions in this process. GSD1a is ufacturing process may enhance the efficiency of mRNA vaccine
characterized by symptoms such as hypoglycemia, hypertriglycer- production by combining three facilities into a fluidic system.
idaemia, anemia, renal disease, and an increased lifelong risk of Continuous manufacturing may ensure the recycling and reuse of
HCC. A recent study demonstrated that repeated intravenous raw compounds (e.g., enzymes or NTPs), and avoiding transport
injection of LNP-encapsulated hG6PC-a mRNA in a liver-specific may significantly reduce time and costs. Proper temperature
G6pc knockout mouse (L. G6pc−/−) resulted in a significant control is crucial for maintaining the efficacy of vaccines. Most
enhancement in fasting glycemia and a decrease in GSD1a vaccines can be stored at 2–8 °C for extended periods, and mRNA
biomarkers, such as glycogen, G6P, and triglycerides.243 Both vaccines such as BNT162b2 and mRNA-1273 must be kept at
treated and control animals exhibited similar levels of cytokines, −80 °C and −20 °C, respectively. This poses a significant challenge
including IFN-γ, IL-1β, TNFα, and IL-6, in their serum. The treatment for their distribution. The instability of the LNP-mRNA system is
did not induce anti-G6Pase responses, liver injury, alterations in the reason for the strict temperature requirement for storing
body weight, or any signs of distress. These results support further mRNA vaccines. Despite various lyoprotectants (e.g., lactate,
investigation of LNP-encapsulated mRNA as a potential treatment mannose, and trehalose) have been incorporated into mRNA-
for inherited metabolic disorders. Currently, a clinical study is protamine formulations, enabling successful long-term storage at
underway to assess the safety, tolerability, pharmacokinetics, and room temperature after freeze-drying, as claimed in several
pharmacodynamics of a single intravenous dose of LNP- patents, it is important to note that the efficacy of preserving
encapsulated hG6PC-a mRNA (mRNA-3745) in patients with GSD1a mRNA delivery efficiency in vivo has been limited when 20%
(NCT05095727). OTC is a crucial enzyme in the urea cycle that is (weight by volume) sucrose or trehalose is added to LNPs and
found in the liver and facilitates the conversion of carbamoyl subjected to freeze-drying. The alteration of the nanostructure of
phosphate and ornithine into citrulline and phosphate.244 This the LNP-mRNA system due to freeze-drying and reconstitution is
process plays vital roles in the elimination of ammonia from the believed to potentially impact the LNPs’ interactions with plasma,
body. High levels of ammonia can cause varying degrees of which can lead to a decline in mRNA delivery efficiency in vivo. To
neuropsychiatric symptoms. Despite various available treatments, date, there is no known resolution to the requirement for
such as protein-restricted diets and ammonia scavengers, it is extremely cold storage and transportation conditions for LNP-
important to note that there is currently no definitive treatment for mRNA vaccines, which could impose significant constraints on the
addressing the root cause of OTC deficiency. Prieve et al. widespread use of mRNA vaccines in the future. Safety is another
demonstrated that NP-encapsulated hOTC mRNA (ARCT-810) concern in the use of mRNA vaccines. The extensive deployment
successfully treated a hyperammonemic murine model of OTC of the COVID-19 mRNA vaccine created a chance to thoroughly
deficiency (Otcspf-ash), resulting in the normalization of plasma study the adverse reactions associated with mRNA vaccines.
ammonia and orotic acid levels, an enhanced survival, as well as a According to safety monitoring by the Centers for Disease Control
good safety profile.245 A phase I study has been completed and Prevention (CDC, https://www.cdc.gov/coronavirus/2019-
assessing the safety, tolerability and pharmacokinetics of ARCT-810 ncov/vaccines/safety/adverse-events.html), some people have
in healthy adult subjects, but the result has not been reported reported no side effects after administration of the COVID-19
(NCT04416126). Two phase IB clinical trials (NCT05526066 and mRNA vaccine, while many have experienced mild to moderate
NCT04442347) are currently underway to assess the safety, side effects such as headache, fatigue, and soreness at the
tolerability, and pharmacokinetics of a single dose of ARCT-810 injection site, which are generally temporary and typically resolve
in clinically stable OTC-deficient patients. within a few days. Although several reactions are rare after
Together, there is a lack of therapeutic agents that can cure these vaccination, multiple cases have been reported. Anaphylaxis, a
rare diseases. mRNA vaccines render it possible to control these severe type of allergic reaction, has occurred in about 5 cases per

Zhang et al.
25
million vaccine doses administered. Thrombosis with thrombocy- the still technological obstacles limiting the precise detection and
topenia syndrome is a rare yet significant adverse event quantification of immunogenic tumor neoantigens and an
characterized by the formation of blood clots in major blood insufficient understanding of the accurate biological mechanism
vessels and a decrease in platelet count. It has been reported in of tumor immune evasion. Conventional exome sequencing does
around 4 cases per million doses administered, signifying its not capture noncanonical peptides derived from the genomic
infrequent occurrence but considerable severity. More importantly, “dark matter” that may include most of the new epitopes
the cases of myocarditis and pericarditis are increasing after the expressed by tumors.249,250 Experimental and in silico approaches
administration of mRNA vaccines. During the study period, more for identifying neoantigens are largely biased toward MHC I
than 350 million mRNA vaccines were administered, and the CDC epitopes and insensitive to MHC II and rare allotypes, causing a
scientists observed that the incidence of myocarditis was highest significant underestimation of the frequency of targetable
among males in the following age groups following the second immunogenic neoantigens. Moreover, a therapeutic vaccine
dose of an mRNA vaccine: 12–15 years (70.7 cases per million doses usually works better in the context of adjuvant therapy or in
of Pfizer-BioNTech), 16–17 years (105.9 cases per million doses of cases of minimal residual disease, where the tumor burden is low
Pfizer-BioNTech), and 18–24 years (52.4 cases and 56.3 cases per and the immunosuppressive microenvironment is not firmly
million doses of Pfizer-BioNTech and Moderna, respectively). As of established.251 Instead, the T-cell response triggered by persona-
March 2, 2023, 715 reports have been verified to meet the CDC’s lized vaccination would be largely slowed down by various
working case definition for myocarditis, and the findings are as immunosuppressive cells252–254 (e.g., cancer-associated fibro-
follows: 5–11 years (23 verified reports of myocarditis after blasts, vascular endothelial cells, tumor-associated macrophages,
23,376,785 doses administered), 12–15 years (376 verified reports tumor-associated neutrophils, suppressive myeloid cells, regula-
of myocarditis after 25,913,772 doses administered), and 16–17 tory T cells, and regulatory B cells) and immunosuppressive
years (316 verified reports of myocarditis after 14,180,263 doses regulators (e.g., PD-1, PD-L1, CTLA-4, IDO-1, TGF-β, IL-10, and IL-35)
administered). The mechanisms causing these rare adverse events in the tumor immune microenvironment (TIME) of large load
remain to be addressed. Finally, the durability of mRNA vaccines tumors. In this context, a combined therapy is required for
against COVID-19, such as the Pfizer-BioNTech and Moderna effective control of tumors. Vaccination enables the turn from the
vaccines, may decline over time. The virus is constantly evolving, immunological “cold” tumor into the “hot” phenotype and induces
and new variants may emerge that are not as well recognized by PD-L1 upregulation in the TIME.251 This phenomenon guides the
the immune system as the original virus, leading to decreased combination of PD-1/PD-L1 blockade and personalized vaccina-
effectiveness of the vaccine over time, especially if the variants tion. The clinical trial NCT03897881 evaluating pembrolizumab in
become more prevalent. In addition, the immune response combination with neoantigen vaccination against melanoma is
generated by the vaccine may decrease over time as the immune active but not recruiting patients; for example, the study is
system’s memory of the virus fades. This is a normal process that ongoing, and the participants are under therapy or being
occurs with any vaccine, but the rate of decline may be faster with evaluated but not enrolled. Similarly, cancer vaccines preclinically
mRNA vaccines due to their unique mechanism of action. synergize with the inhibition of other inhibitory molecules (e.g.,
Furthermore, the vaccine may not provide as strong or long- CTLA-4, TIM-3, LAG-3, IDO, or TGF-β) and the stimulation of
lasting protection against certain populations, such as immuno- costimulatory molecules (e.g., GITR, OX40, and CD137).251
compromised individuals or elderly individuals. Several approaches Additionally, a phase I clinical trial for glioblastoma
may improve the overall effectiveness of the vaccine and extend its (NCT02709616) tested a personalized vaccination together with
duration, including administering booster shots of the mRNA temozolomide and radiotherapy. Recently, Huang et al. estab-
vaccine at specific intervals, using different types of vaccines (such lished a pipeline to construct tumor immune subtypes, which act
as a combination of mRNA and traditional vaccines), and optimizing as biomarkers that reflect the immune status in tumors and their
the storage and transportation conditions for mRNA vaccines. TIME (e.g., immune infiltration and function, as well as the
Altogether, the technique for mRNA vaccine preparation and expression of immune checkpoints and immunological cell death
application is not perfect and remains to be further ameliorated. modulators).255,256 The immune subtype might provide precise
In addition to these universal issues underlying mRNA vaccines, guidance for combined cooperation with the mRNA vaccine,
there are specific challenges in different diseases. Given the warranting further clinical investigation.
application of mRNA vaccines in immunological diseases, rare
diseases, and tissue damage are still at an early stage, and there
are insufficient studies assessing their efficacies and challenges in ACKNOWLEDGEMENTS
the context of these diseases. Therefore, infectious diseases and This work was supported by the National Natural Science Foundation of China
cancer, in which mRNA vaccines are more prevalently used, are (31970696 and U20A20378), Zhejiang Provincial Natural Science Foundation for
selected as examples for discussing the obstacles of mRNA Distinguished Young Scholar (LR22H160010), National Key Research and Develop-
ment Program (2019YFC1316000), Zhejiang Provincial Key Research and Develop-
vaccines in specific diseases. There are two main categories of
ment Program (2019C03019), and Zhejiang Provincial College Student Science and
infectious viruses: those that are newly emerging or reemerging Technology Innovation Activity Plan-College Student Innovation and Entrepreneur-
and those that cause chronic infections. The protection efficacy of ship Incubation Program (2023R401203).
mRNA vaccines against the rapidly emerged coronavirus has been
exceptional, and their low production cost and ease of
manufacture suggest that they could be instrumental in control AUTHOR CONTRIBUTIONS
of future pandemics resulted from rapidly emerging viruses. X.H. and T.L. conceived the manuscript and share senior authorship. X.H., G.Z., and
However, these emerging or reemerging viruses tend to mutate T.T. contributed equally to the literature review and writing. G.Z. and Y.C. contributed
rapidly, presenting a challenge in developing mRNA vaccines that to rephrasing and proofreading. X.H. and G.Z. designed the figures. All authors
are broad or seasonal in nature. Additionally, generating effective discussed and edited the manuscript and approved the final version.
neutralizing antibodies against chronic infectious viruses is
typically difficult, as they are adept at evading innate immunity.
Unlike infectious diseases, cancer is caused by genetic and ADDITIONAL INFORMATION
epigenetic factors, and it is characterized by complex and Supplementary information The online version contains supplementary material
heterogeneous antigen expression, thus requiring the use of a available at https://doi.org/10.1038/s41392-023-01579-1.
personalized mRNA vaccine. However, several challenges limit the
Competing interests: The authors declare no competing interests.
clinical application of personalized cancer mRNA vaccines, such as

Zhang et al.
26
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Signal Transduction and Targeted Therapy www.nature.

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mRNA vaccines in disease prevention and treatment

Signal Transduction and Targeted Therapy (2023)8:365 ; https://doi.org/10.1038/s41392-023-01579-1

INTRODUCTION possess multiple beneﬁcial features over traditional vaccines.3,8

Received: 10 January 2023 Revised: 1 July 2023 Accepted: 30 July 2023

© The Author(s) 2023

This comprehensive review provides an in-depth exploration of

MRNA VACCINE DEVELOPMENT

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365

NCT03688178 Glioblastoma CMV pp65 LAMP DC Varlilumab Phase II Recruiting

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365

NCT00510133 AML hTERT, LAMP DC No Phase II Completed

Immunological adjuvants are usually used to stimulate and

T-cell activator), and the constitutively active TLR 4 (DC antigen

subjected to vaccination with autologous monocyte-derived DCs

report revealed that the mRNA encoded CD40L, TLR4, CD70,

HLA-II targeting signal, and a TAA (MAGE-A3, MAGE-C2, tyrosi-

nase, or gp100). After vaccination, two patients exhibited a

No disease > two years. In addition, Jansen et al. showed a phase II

patients were free of disease as compared to 35% in the control

compromise the efﬁcacy activated by mRNA vaccines, one study

investigated the effect of combined treatment of TriMixDC-MEL

long-term clinical responses, with an overall survival of 28% (11/

39) and progression-free survival of 18% (7/39) after 5 years of

follow-up. Eighty percent (12/15) of patients with immune

OX40L, IL-23, IL-36γ

monitoring were vaccine responders, and among them,

10 showed T-cell responses against at least two antigens.

GenenTech, and Moderna are leading companies in the

pharmaceutical industry, and as such, they have announced

vaccine encoding TAAs (gp100, Melan-A, Tyrosinase, MAGE-A1,

MAGE-A3, and survivin) in a group of 21 patients with metastatic

CD4+ regulatory T cells or myeloid suppressor cells were

signiﬁcantly reduced in the peripheral blood of vaccinated

reproductive elevation in vaccine-speciﬁc T cells. BNT111 is a

liposomal RNA vaccine encoding four TAAs, MAGE-A3, NY-ESO-1,

PTEN, and tyrosinase, and its safety and effectiveness were

evaluated in 2015 after intravenous administration in a phase I

patients, with 42 suffering from measurable stage III/IV melanoma.

response. The disease was controlled for a long time in most of

during a follow-up period of up to two years. The observed clinical

T-cell immune responses speciﬁcally targeting the vaccine

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365

Signal Transduction and Targeted Therapy (2023)8:365