Demystifying mRNA vaccines: an emerging platform at the forefront of cryptic diseases

Figures & data

Figure 1. Progression of mRNA technology. The timeline illustrates the advancement of mRNA therapeutics, highlighting key milestones related to both general advancement of mRNA technology and the evolution of mRNA as a vaccine platform.

Figure 2. Activation of the immune system by mRNA vaccines. (1) The innate immune response is triggered via pathogen-associated molecular pattern (PAMP) recognition on non-self RNA. This recognition is mediated by pattern recognition receptors (PRRs) such as melanoma differentiation-associated 5 (MDA-5), nucleotide oligomerisation domain 2 (NOD2), and retinoic acid-inducible gene I (RIG-I) or toll-like receptors (TLRs), which ultimately results in type I interferon (IFN-I) release, allowing upregulation of proinflammatory genes, activation of the Th1 pathway and APC activation. (2) The internalised mRNA can be translated to generate the antigen, which is presented on the major histocompatibility complexes (MHC) on the APCs, initiating the adaptive response. (3) The antigen degraded by proteasomes is presented as peptides on the MHC I complex, leading to interactions with CD8+ cytotoxic T lymphocytes and triggering their maturation for a cellular response (indicated by blue arrows). (4) Additionally, antigens secreted exogenously can be internalised by other APCs, degraded into peptides within lysosomal endosomes, and presented on MHC II complexes on the cell surface (indicated by green arrows). This stimulates CD4+ T-helper cell interactions, which prime B lymphocytes on maturation for an antibody-mediated humoral response and generate memory cells specific to the target pathogen.

Figure 3. Synthesis of IVT-mRNA. (1) IVT-mRNA synthesis begins with a DNA template, usually pDNA, engineered to contain the gene of interest coding for the desired antigen identified by sequencing the genome of the target pathogen. (2) The pDNA template must include a bacteriophage promoter (T7), the ORF, a poly-deoxyribose T sequence (to code for the poly-A tail), and a restriction site which allows for restriction enzyme-mediated linearisation. (3) The linearised template undergoes in vitro transcription using a bacteriophage-derived T7 RNA polymerase. Following transcription, a mixture containing the desired mRNA, phage RNA polymerase, and nucleoside triphosphates is obtained. The 5’-cap can be introduced either enzymatically or be yielded in the transcription stage by including N⁷-methyl-guanosine analogue residues in excess of guanosine triphosphate (GTP) residues present. The 3’-tail can also be enzymatically added in this step, if not already incorporated. (4) The mixture is then purified using DNase I to degrade any contaminants and the template, followed by purification of the desired mRNA transcripts from a mix of abortive transcripts, longer transcripts with a 3’-overhang, oligodeoxynucleotides, and free nucleotides. While purification can be achieved by a series of precipitation and extraction steps, a chromatographic process is best suited for separating the transcripts of varied sizes. Techniques like high-performance liquid chromatography (HPLC) further refine the quality of the product, diminishing possibilities of unnecessarily activating innate immune sensors via contaminants. (5) Rapid mixing of the obtained mRNA with lipid via the utilisation of microfluidics allows for the self-assembly of mRNAs within lipid nanoparticles. (6) The obtained solution containing nanoparticles has to undergo further dialysis or filtration to remove any unencapsulated mRNA or non-aqueous solvent. Post filtration, the purified solution containing mRNA within lipid nanoparticles is ready for administration within the host.

Figure 4. Strategy to optimise mRNA vaccines. mRNA can be modulated according to cell specificity in a number of ways: (1) By replacing nucleoside base(s) with chemically modified nucleoside(s), the translation efficiency and immunogenicity of mRNA can be altered. (2) Codon optimisation employed with GC-rich transcripts reduces immunogenicity to further increase translation efficiency and the safety profile of the mRNA. (3) By introducing stable 3’ UTR sequences, translation efficiency and stability can be modulated. (4) Manipulating the length of the 3’ poly(A) tail can improve mRNA stability. (5) Within the 5’ UTR, incorporation of GCC-(A/G)-CCAUGG sequence or avoidance of sequences similar to that of the ORF can enhance translation efficiency. (6) The 5’ cap promotes translation and further stabilises the mRNA. (7) The mRNA can be encapsulated within a variety of nanoparticle carriers for delivery into target tissues. This figure was created with BioRender.com.

Table 1. A systematic comparison of COVID-19 vaccines

	BNT162b2 (Pfizer/BioNTech)	mRNA-1273 (Moderna)	ChAdOx1-S/ AZD1222 (Oxford/Astrazeneca)	Ad26.COV2.S (Johnson & Johnson/Janssen)	CoronaVac (Sinovac)	BBIBP-CorV (Sinopharm)
Vaccine platform	Nucleoside-modified mRNA that encodes the prefusion-stabilised viral spike (S) glycoprotein of SARS-CoV-2	Nucleoside-modified mRNA that encodes the prefusion-stabilised viral spike (S) glycoprotein of SARS-CoV-2	Recombinant, replication-deficient chimpanzee adenovirus vector that encodes the viral spike (S) glycoprotein of SARS-CoV-2	Replication-incompetent, adenovirus type 26 (AD26)-vectored monovalent vaccine that encodes the viral spike (S) glycoprotein of SARS-CoV-2	Vero cell-based, aluminium hydroxide-adjuvanted, and β-propiolactone-inactivated vaccine based on the CZ02 strain	Vero cell-based, aluminium hydroxide-adjuvanted, and β-propiolactone-inactivated vaccine based on the 19nCOV-CDC-TAN-HB02 strain (HB02 strain)
Delivery system	mRNA encapsulated by lipid nanoparticle	mRNA encapsulated by lipid nanoparticle	Adenovirus-based delivery system; adenoviruses are non-enveloped icosahedral particles (virions)	Double-stranded DNA encapsulated by an icosahedral protein structure	··	··
Dosage	30 μg of nucleoside-modified mRNA; 2 doses	100 μg of nucleoside-modified mRNA; 2 doses	0.5 ml of 5 × 10^10 recombinant particles; 2 doses	0.5 ml of 5 × 10^10 AD26.COV2.S viral particles; 1 dose	0.5 ml of 3 μg inactivated vaccine; 2 doses	0.5 ml of 4 μg inactivated vaccine; 2 doses
Clinical trial population	Argentina, Brazil, Germany, South Africa, Turkey, and USA; 43,448 participants	USA; 30,000 participants	Brazil, South Africa, and UK; 23,745 participants	Argentina, Brazil, Chile, Colombia, Mexico, Peru, South Africa, and USA; 43,783 participants	Brazil, Chile, China, Indonesia, and Turkey: 25,000 participants	Bahrain, Egypt, Jordan, UAE; 41,301 participants
T-cell activation	Robust expression of T-helper 1 (Th1) and T follicular helper (Tfh) type CD4+ responses as well as a robust IFNγ+ IL-2+ CD8+ T-cell responses in participants aged ≥16 years	Robust CD4+ T-cell responses and low CD8+ T-cell responses in adults aged 18–55 years and in older adults ≥65 years	CD4+ and CD8+ T-cell responses in adults aged 18–55 years	CD4+ and CD8+ T-cell responses by day 15 and up to day 29 in the majority of adults aged 18–55 years and in older adults aged ≥65 years	CD4+ and CD8+ T-cell responses in adults aged 18–59 years and in older adults ≥60 years	CD4+ and CD8+ T-cell responses in adults aged 18–59 years and in older adults ≥60 years
Recommended for ages	≥12 years	≥18 years	≥18 years	≥18 years	≥18 years	≥18 years
Suggested route and site of administration	Intramuscular, deltoid muscle	Intramuscular, deltoid muscle	Intramuscular, deltoid muscle	Intramuscular, deltoid muscle	Intramuscular, deltoid muscle	Intramuscular, deltoid muscle
Efficacy *	95%	94.1%	63.1%	66.1%	50.7%	78.1%
Effectiveness against new variants of concern **	93.7% against Alpha 75% against Beta 88% against Delta	100% against Alpha 96.4% against Beta	74.5% against Alpha 67% against Delta	··	··	··
Cost per dose ***	Approximately USD 20	Approximately USD 20	Below USD 6(as low as USD 2.15)	USD 10	··	USD 19–36
Preparation	Diluted in 1.8 ml of 0.9% sodium chloride solution before use	Ready to use	Ready to use	Ready to use	Ready to use	Ready to use
Safety concerns	Known history of anaphylaxis and immediate allergic reaction (e.g. anaphylaxis, urticaria, angioedema, respiratory distress) to the first dose	Known history of anaphylaxis	Known history of anaphylaxis	Known history of anaphylaxis and capillary leak syndrome	Known history of anaphylaxis	Known history of anaphylaxis
Storage temperature	Undiluted: −90 to −60°C for 6 months from the time of formulation Undiluted, thawed: 2–8°C for 120 hours before dilution (up to 30°C for 2 hours) Diluted: 2–30°C for 6 hours	Frozen, unopened: −25 to −15°C until expiration date Thawed, unopened: 2–8°C for 30 days (8–25°C for 12 hours) Thawed, punctured: 2–25°C for 6 hours after withdrawing first dose	Frozen, unopened: 2–8°C for 6 months Opened: 2–8°C for 48 hours (up to 30°C for 6 hours)	Frozen, unopened: −20°C for 24 months Thawed, unopened: 2–8°C for 3 months Thawed, opened: 2–8°C for 6 hours	Unopened: 2–8°C for 12 months or until expiration date	Unfrozen, pre-filled syringe: 2–8°C for 24 months
Light sensitivity	Avoid direct exposure to light	Avoid direct exposure to light	Avoid direct exposure to light	Avoid direct exposure to light	Avoid direct exposure to light	Avoid direct exposure to light
Side effects	Very common (≥1/10): Injection site swelling, pain, headache, chills, arthralgia, myalgia, and pyrexia (higher frequency after 2^nd dose Common (≥1/100 to ˂1/10): Injection site redness and nausea Uncommon (≥1/1000 to ˂1/100): Injection site itching, lymphadenopathy, insomnia, pain in extremities, and malaise Rare (≥1/10,000 to ˂ 1/1000): Bell<apos;>s palsy (acute peripheral facial paralysis), myocarditis, and pericarditis	Very common (≥1/10): Injection site pain, headache, nausea, vomiting, fatigue, chills, fever, myalgia, arthralgia, stiffness, and lymphadenopathy Common (≥1/100 to ˂1/10): Injection site redness or swelling, rash, vomiting, and diarrhoea Uncommon (≥1/1000 to ˂1/100): Injection site itching Rare (≥1/10,000 to ˂ 1/1000): Facial swelling, Bell<apos;>s palsy (acute peripheral facial paralysis), myocarditis, and pericarditis	Very common (≥1/10): Injection site pain, tenderness, warmth, itching, bruising, fatigue, chills, headache, nausea, vomiting, myalgia, and arthralgia Common (≥1/100 to ˂1/10): Injection site swelling, redness, and fever (≥38°C) Uncommon (≥1/1000 to ˂1/100): Lymphadenopathy, decreased appetite, dizziness, abdominal pain, hyperhidrosis (abnormal sweating), pruritus, and rash Very rare (˂1/10,000): Neuroinflammatory disorder (transverse myelitis) has been reported but a causal relationship has not been established	Very common (≥1/10): Injection site pain, headache, nausea, fatigue and myalgia Common (≥1/100 to ˂1/10): Injection site swelling, redness, chills, arthralgia, cough, and fever (≥38°C) Uncommon (≥1/1000 to ˂1/100): Rash, muscle weakness, arm or leg pain, feeling weak and generally unwell, sneezing, sore throat, back pain, tremor, and hyperhidrosis (abnormal sweating) Rare (≥1/10,000 to ˂ 1/1000): Allergic reaction and hives	Very common (≥1/10): Injection site pain, headache, and fatigue Common (≥1/100 to ˂1/10): Injection site swelling, itching, redness, induration, chills, myalgia, arthralgia, nasal congestion, cough, runny nose, sore throat, pruritus, loss of appetite, nausea, diarrhoea, and abdominal pain Uncommon (≥1/1000 to ˂1/100):): Injection site warmth, swelling, fever (≥37.3°C), tremor, dizziness, drowsiness, flushing, vomiting, mucocutaneous rash, and hypersensitivity Rare (≥1/10,000 to ˂ 1/1000): Ocular congestion, eyelid swelling, hot flashes, nose bleeds, diminished sense of smell, abdominal distension, constipation, and muscle spasms	Very common (≥1/10): Injection site pain and headache Common (≥1/100 to ˂1/10): Fever, fatigue, myalgia, arthralgia, cough, dyspnoea, nausea, diarrhoea, and pruritus Uncommon (≥1/1000 to ˂1/100): Redness, swelling, induration, itching, dizziness, anorexia, oropharyngeal pain, dysphagia, runny nose, constipation, and hypersensitivity Rare (≥1/10,000 to ˂ 1/1000): Lethargy, drowsiness, insomnia, sneezing, nasopharyngitis, nasal congestion, dry throat, influenza, hypoesthesia, limb pain, palpitations, abdominal pain, rash, abnormal skin mucosa, acne, ophthalmodynia, ear discomfort, and lymphadenopathy Very rare (˂1/10,000): Chills, taste dysfunction, loss of taste, paresthesia, tremor, attention disorder, epistaxis, asthma, throat irritation, tonsillitis, physical discomfort, neck pain, jaw pain, neck lump, mouth ulcers, toothache, oesophagus disorders, gastritis, faecal discoloration, ophthalmodynia, blurred vision, eye irritation, earache, tension, hypertension, hypotension, urinary incontinence, and delayed menstruation

* Efficacy defines the measurement of the vaccine<apos;>s ability in preventing disease in a controlled and double-blinded clinical trial

** Effectiveness of a vaccine is measured in the real world and counted 14 or more days after the second dose

*** All prices are subject to trademark agreements

·· Information not available on peer-reviewed sources

Table 2. mRNA-based vaccines and therapeutics in clinical trials

	Antigen encoded by mRNA	Route of administration	Target condition	Phase	Status	Clinical trial number
Infectious disease vaccines
mRNA-1273	Full-length, prefusion-stabilised spike (S) protein of SARS-CoV-2	Intramuscular	COVID-19	Phase 4	Recruiting	NCT04780659
BNT162 (Comirnaty)	Full-length, membrane-anchored spike (S) protein of SARS-CoV-2	Intramuscular	COVID-19	Phase 4	Recruiting	NCT04760132
CVnCoV	Full-length, prefusion-stabilised spike (S) protein of SARS-CoV-2	Intramuscular	COVID-19	Phase 3	Active, not recruiting	NCT04674189
ARCT-021	Spike (S) protein of SARS-CoV-2	Intramuscular	COVID-19	Phase 2	Active, not recruiting	NCT04668339
ARCoV	RBD domain of SARS-CoV-2 spike (S) protein	Intramuscular	COVID-19	Phase 2	Completed	ChiCTR2100041855
ChulaCov19	Undisclosed	Intramuscular	COVID-19	Phase 1	Not yet recruiting	NCT04566276
PTX-COVID19-B	Full-length, membrane-anchored spike (S) protein of SARS-CoV-2	Intramuscular	COVID-19	Phase 1	Active, not recruiting	NCT04765436
CoV2 SAM (LNP)	Spike (S) protein of SARS-CoV-2	Intramuscular	COVID-19	Phase 1	Active, not recruiting	NCT04758962
mRNA-1273.351	Full-length, prefusion-stabilised spike (S) protein of SARS-CoV-2 B.1.351 variant	Intramuscular	COVID-19	Phase 1	Active, not recruiting	NCT04785144
MRT5500	Full-length, prefusion-stabilised spike (S) protein of SARS-CoV-2	Intramuscular	COVID-19	Phase 1/2	Recruiting	NCT04798027
CV7202	Rabies virus glycoprotein, RABV-G	Intramuscular	Rabies	Phase 1	Active, not recruiting	NCT03713086
mRNA-1647	Subunits of the CMV pentamer complex and the glycoprotein B (gB) protein	Intramuscular	Cytomegalovirus infection	Phase 1	Completed	NCT03382405
mRNA-1944	A fully human IgG antibody originally isolated from B cells of a patient with a prior history of potent immunity against Chikungunya infection	Intravenous	Chikungunya	Phase 1	Completed	NCT03829384
Cancer vaccines
CV8102	A noncoding single-stranded RNA complexed with a cationic peptide that functions as a strong immunomodulator based on TLR7/8 and RIG-1 activation	Intratumoural	Cutaneous melanoma, adenoidcystic carcinoma, squamous cell cancer of skin, head, and neck	Phase 1	Recruiting	NCT03291002
BI1361849 (CV9202)	Six non-small cell lung cancer (NSCLC)-associated antigens (NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and MUC-1)	Intradermal	Non-small cell lung cancer	Phase 1/2	Active, not recruiting	NCT03164772
BNT111 (FixVac)	Liposomal RNA (RNA-LPX) vaccine, which targets four non-mutated, tumour-associated antigens that are prevalent in melanoma	Intravenous	Advanced melanoma (adjuvant and metastatic)	Phase 1	Active, not recruiting	NCT02410733
MVT-5873 (BNT321)	A fully human IgG1 monoclonal antibody targeting sialyl Lewis A (sLea), an epitope on CA19-9 that is expressed in pancreatic and other gastrointestinal cancers that plays a role in tumor adhesion and metastasis formation, and is a marker of an aggressive cancer phenotype	Intravenous	Pancreatic cancer	Phase 1	Recruiting	NCT02672917
BNT122 (Autogene cevumeran)	Targets locally advanced or metastatic solid tumors	Intravenous	Metastatic melanoma and other solid tumors	Phase 2	Recruiting	NCT03289962
mRNA-4157	Personalised cancer vaccine (PCV) with a combination of validated defined neoantigens, predicted neoepitopes, and mutations in driver genes	Intramuscular	Solid tumours	Phase 1	Recruiting	NCT03313778
mRNA-5671/V941	Four common mutations of an oncogene known as KRAS	Intramuscular	Non-small cell lung cancer, pancreatic neoplasms, and colorectal neoplasms	Phase 1	Recruiting	NCT03948763
Curative and protein supplementation therapeutics
AZD8601	Vascular endothelial growth factor-A (VEGF-A)	Epicardial	Myocardial ischemia	Phase 2	Active, not recruiting	NCT03370887
MRT5005	Cystic fibrosis transmembrane conductance regulator (CFTR)	Inhalation	Cystic fibrosis	Phase 1/2	Recruiting	NCT03375047
LUNAR-OTC (ARCT-810)	Ornithine transcarbamylase (OTC)	Intravenous	Ornithine transcarbamylase deficiency	Phase 1b	Recruiting	NCT04442347
mRNA-3927	Propionyl-CoA carboxylase	Intravenous	Propionic acidemia	Phase 1/2	Recruiting	NCT04159103
NTLA-2001	Cas9	Intravenous	Transthyretin amyloidosis	Phase 1	Recruiting	NCT04601051