An mRNA vaccine functions by employing a synthetic messenger RNA (mRNA) molecule to elicit an immune response. This vaccine introduces antigen-encoding mRNA into host cells, which then utilize this genetic blueprint to synthesize specific foreign proteins typically associated with a pathogen (e.g., a virus) or a cancerous cell. The resulting protein molecules subsequently trigger an adaptive immune response, enabling the body to recognize and eliminate the respective pathogen or malignant cells. For cellular uptake, the mRNA is delivered within a co-formulation, encapsulated by lipid nanoparticles that safeguard the RNA strands and facilitate their entry into cells.
An mRNA vaccine is a type of vaccine that uses a copy of a molecule called messenger RNA (mRNA) to produce an immune response. The vaccine delivers molecules of antigen-encoding mRNA into cells, which use the designed mRNA as a blueprint to build foreign protein that would normally be produced by a pathogen (such as a virus) or by a cancer cell. These protein molecules stimulate an adaptive immune response that teaches the body to identify and destroy the corresponding pathogen or cancer cells. The mRNA is delivered by a co-formulation of the RNA encapsulated in lipid nanoparticles that protect the RNA strands and help their absorption into the cells.
The reactogenicity, defined as a vaccine's propensity to induce adverse reactions, for mRNA vaccines is comparable to that observed with conventional non-RNA vaccines. Individuals predisposed to autoimmune responses may experience adverse reactions to messenger RNA vaccines. Key advantages of mRNA vaccines over traditional counterparts include simplified design, accelerated and more economical production, the stimulation of both cellular and humoral immunity, and the absence of interaction with genomic DNA. Although certain messenger RNA vaccines, such as the Pfizer–BioNTech COVID-19 vaccine, necessitate ultracold storage prior to distribution, other mRNA vaccines, like the Moderna vaccine, do not impose such stringent requirements.
Within the field of RNA therapeutics, messenger RNA vaccines garnered significant attention, particularly as COVID-19 vaccines. In December 2020, both Pfizer–BioNTech and Moderna secured authorization for their respective mRNA-based COVID-19 vaccines. Specifically, on December 2, the UK Medicines and Healthcare products Regulatory Agency (MHRA) became the inaugural regulatory body to approve an mRNA vaccine, granting authorization for the Pfizer–BioNTech vaccine's widespread deployment. Subsequently, on December 11, the US Food and Drug Administration (FDA) issued an emergency use authorization for the Pfizer–BioNTech vaccine, followed a week later by a similar authorization for the Moderna vaccine. In 2023, Katalin Karikó and Drew Weissman were jointly awarded the Nobel Prize in Physiology or Medicine for their pivotal discoveries regarding modified nucleosides, which were instrumental in developing effective mRNA vaccines against COVID-19.
Historical Context
Foundational Research
The initial successful transfection of engineered mRNA, encapsulated within a liposomal nanoparticle, into a cellular environment was documented in 1989. A year subsequent to this, 'naked' (unprotected) laboratory-synthesized mRNA was directly injected into murine muscle tissue. These pioneering investigations provided the first empirical evidence that in vitro transcribed mRNA, incorporating a selected gene, could effectively deliver genetic information to facilitate the production of a desired protein within living cellular tissue, thereby laying the groundwork for the conceptualization of messenger RNA vaccines.
In 1993, liposome-encapsulated mRNA, engineered to encode a viral antigen, demonstrated the capacity to stimulate T cells in murine models. The subsequent year saw the development of self-amplifying mRNA, achieved by incorporating both a viral antigen and a replicase-encoding gene. This methodology was successfully employed in mice to induce both humoral and cellular immune responses against a viral pathogen. By the following year, mRNA encoding a tumor antigen was shown to elicit a comparable immune response against cancerous cells in mice.
Subsequent Development
The inaugural human clinical trial, which utilized ex vivo dendritic cells transfected with mRNA encoding tumor antigens (a therapeutic cancer mRNA vaccine), commenced in 2001. Four years later, a significant breakthrough was reported: the successful application of modified nucleosides as a strategy to facilitate mRNA transport into cells without triggering the host's innate immune defenses. By 2008, clinical trial outcomes for an mRNA vaccine directly administered into the body to target cancer cells were published.
BioNTech, established in 2008, and Moderna, founded in 2010, were both created with the objective of advancing mRNA biotechnologies. Concurrently, the US research agency DARPA initiated the ADEPT biotechnology research program, aimed at developing nascent technologies for the US military. Recognizing the potential of nucleic acid technology for pandemic defense, DARPA commenced investments in this domain. DARPA grants were perceived as a strong endorsement, subsequently stimulating further investment in mRNA technology from other governmental bodies and private investors. During this period, DARPA notably provided a $25 million grant to Moderna.
The initial human clinical trials utilizing an mRNA vaccine to combat an infectious agent, specifically rabies, commenced in 2013. Subsequently, over the ensuing years, clinical investigations into mRNA vaccines targeting various other viruses were initiated. These mRNA vaccines, intended for human application, underwent study for infectious agents including influenza, Zika virus, cytomegalovirus, and Chikungunya virus.
Acceleration
The onset of the COVID-19 pandemic, coupled with the sequencing of its etiological agent, SARS-CoV-2, in early 2020, precipitated the swift advancement of the inaugural approved mRNA vaccines. By December of that year, BioNTech and Moderna secured authorization for their respective mRNA-based COVID-19 vaccine candidates. Notably, in December 2020, the UK Medicines and Healthcare products Regulatory Agency (MHRA) made history as the first global regulatory body to sanction an mRNA vaccine, issuing emergency authorization for the widespread deployment of Pfizer–BioNTech's BNT162b2 COVID-19 vaccine. Concurrently, the US Food and Drug Administration also granted emergency use authorization for both the Pfizer–BioNTech and Moderna COVID-19 vaccines during December 2020.
Combination vaccines
A bivalent messenger RNA vaccine designed to confer protection against both influenza and COVID-19 was introduced in 2026.
Mechanism
The fundamental objective of vaccination is to elicit a robust adaptive immune response, leading to the generation of antibodies specifically directed against a particular pathogen. These distinct molecular markers present on the pathogen, which serve as targets for antibodies, are termed antigens.
Conventional vaccines induce an antibody response through the administration of various components into the body, including isolated antigens, attenuated (weakened) viruses, inactivated (non-viable) viruses, or recombinant viral vectors engineered to express specific antigens (harmless carrier viruses containing an antigen transgene). Crucially, these antigenic components and viral preparations are cultivated and processed ex vivo.
Conversely, mRNA vaccines operate by introducing a transient, synthetically engineered fragment of a viral RNA sequence into the vaccinated individual. These mRNA fragments are subsequently internalized by dendritic cells via phagocytosis. Utilizing their intrinsic cellular machinery, specifically ribosomes, dendritic cells translate the mRNA to synthesize the corresponding viral antigens. The body naturally degrades these mRNA fragments within a few days post-administration. While other non-immune cells possess the potential to internalize vaccine mRNA, synthesize antigens, and present them on their surfaces, dendritic cells exhibit a significantly higher propensity for absorbing the mRNA globules. Importantly, the translation of these mRNA fragments occurs within the cytoplasm and does not interfere with the host's genomic DNA, which is compartmentalized within the cell nucleus.
Following the synthesis of viral antigens by the host cell, the standard mechanisms of the adaptive immune system are engaged. Antigens undergo proteolytic degradation by proteasomes. Subsequently, Major Histocompatibility Complex (MHC) class I and class II molecules bind to these antigenic fragments and translocate them to the cellular membrane, thereby initiating the "activation" of the dendritic cell. Upon activation, dendritic cells egress to regional lymph nodes, where they present the processed antigen to both T lymphocytes and B lymphocytes. This presentation event stimulates the proliferation and differentiation of these lymphocytes, leading to the generation of antibodies precisely specific to the antigen, culminating in the establishment of immunological protection.
mRNA
The core constituent of an mRNA vaccine is its meticulously designed mRNA construct. This in vitro transcribed mRNA originates from an engineered plasmid DNA, which incorporates an RNA polymerase promoter and a sequence corresponding to the desired mRNA construct. Through the enzymatic action of T7 phage RNA polymerase on the plasmid DNA, the mRNA can be synthesized in a laboratory setting. The overall efficacy of the vaccine is intrinsically linked to the stability and structural integrity of this engineered mRNA.
The in vitro transcribed mRNA shares fundamental structural elements with endogenous mRNA found in eukaryotic cells. These include a 5' cap, a 5'-untranslated region (UTR) and a 3'-UTR, an open reading frame (ORF) responsible for encoding the target antigen, and a 3'-poly(A) tail. Strategic modifications to these various components of the synthetic mRNA can significantly augment its stability and translational efficiency, thereby enhancing the overall efficacy of the vaccine.
Synthetic 5'-cap analogues can enhance mRNA stability and augment protein translation. Furthermore, modifications to regulatory elements within the 5'-untranslated region (UTR) and 3'-UTR, along with optimization of the poly(A) tail length, contribute to mRNA stabilization and elevated protein synthesis. Nucleotide modifications in mRNA can simultaneously mitigate innate immune activation and extend the mRNA's half-life within the host cell. Protein translation is influenced by the nucleic acid sequence and codon usage. Enhancing the guanine-cytosine (GC) content of the sequence improves mRNA stability and half-life, thereby boosting protein production. Substituting rare codons with synonymous codons commonly utilized by the host cell further augments protein synthesis.
Delivery
Effective vaccine function necessitates the entry of adequate mRNA into the host cell cytoplasm to stimulate the production of target antigens. However, the cellular uptake of mRNA molecules presents several significant challenges. mRNA molecules are inherently too large for passive diffusion across the cell membrane and possess a negative charge, leading to electrostatic repulsion with the similarly charged membrane. Furthermore, mRNA is susceptible to rapid degradation by ribonucleases (RNAases) present in biological fluids such as skin and blood.
To surmount these delivery obstacles, diverse methodologies have been developed. Vaccine delivery approaches are broadly categorized based on whether mRNA transfer into cells occurs within (in vivo) or outside (ex vivo) the living organism.
Ex vivo
Dendritic cells present antigens on their surface, facilitating interactions with T cells and thereby initiating an immune response. In an ex vivo approach, dendritic cells can be harvested from patients, programmed with the target mRNA, and subsequently re-administered to elicit an immune response.
The most straightforward mechanism for ex vivo dendritic cell uptake of mRNA molecules is endocytosis; however, this pathway is relatively inefficient in laboratory settings but can be substantially enhanced via electroporation.
In vivo
Following the discovery that direct administration of in vitro transcribed mRNA results in antigen expression within the body, in vivo strategies have been extensively explored. These methods present several advantages compared to ex vivo techniques, notably by eliminating the expense associated with harvesting and adapting patient-derived dendritic cells and by more closely mimicking a natural infection process.
Various injection routes, including intradermal, intravenous, or intramuscular administration, yield distinct levels of mRNA uptake, thus establishing the selection of the administration route as a critical determinant for effective in vivo delivery. Comparative research has indicated that direct injection into lymph nodes elicits the most robust T-cell response.
Naked mRNA injection
Naked mRNA injection refers to the administration of vaccine mRNA solely within a buffer solution. This method of mRNA delivery has been recognized since the 1990s. Initial global clinical investigations employed intradermal injections of naked mRNA for vaccination purposes. Diverse administration techniques for naked mRNA have been utilized, including subcutaneous, intravenous, and intratumoral injections. While naked mRNA delivery can induce an immune response, its efficacy is comparatively limited, and the mRNA frequently undergoes rapid degradation post-injection.
Polymer and peptide vectors
Cationic polymers can be combined with mRNA to form protective complexes known as polyplexes. These polyplexes shield the recombinant mRNA from ribonucleases and facilitate its cellular entry. Protamine, a natural cationic peptide, has been employed for mRNA encapsulation in vaccine applications.
Lipid nanoparticle vector
The U.S. Food and Drug Administration (FDA) first approved lipid nanoparticles as a drug delivery system in 2018, specifically for the siRNA drug Onpattro. The encapsulation of mRNA molecules within lipid nanoparticles represented a pivotal advancement for developing effective mRNA vaccines, resolving several critical technical challenges associated with delivering mRNA into host cells. Prior research on lipid-mediated delivery of siRNA to cells established a foundational basis for analogous investigations into mRNA delivery using lipids. Nevertheless, the development of novel lipids was necessitated to encapsulate mRNA strands, given their considerably greater length compared to siRNA strands.
The lipid primarily offers a protective barrier against degradation, thereby enhancing translational efficiency. Furthermore, modifying the lipid's external surface enables precise targeting of specific cell types via ligand-receptor interactions. Nevertheless, numerous investigations have underscored the challenges associated with researching this delivery mechanism, revealing discrepancies in cellular uptake between in vivo and in vitro applications of nanoparticles. Nanoparticles can be introduced into the body and disseminated through various pathways, including intravenous injection or the lymphatic system.
A significant challenge associated with lipid nanoparticles stems from the reliance on microfluidics for several key technological advancements enabling their practical application. Scaling up microfluidic reaction chambers presents difficulties because microfluidics inherently leverages the unique behaviors of liquids at the microscale. Overcoming this limitation necessitates operating a substantial number of microfluidic reaction chambers concurrently, a novel endeavor demanding specialized, custom-engineered apparatus. This particular aspect constituted the primary manufacturing bottleneck for COVID-19 mRNA vaccines. Pfizer addressed the scaling issue by implementing a parallelized strategy. Following confirmation that impingement jet mixers were not amenable to direct scaling, Pfizer fabricated approximately 100 small mixers, each comparable in size to a U.S. half-dollar coin. These were then interconnected with pumps and filters via an intricate network of piping, and a computerized system was established to precisely regulate flow and pressure within the mixers.
A further challenge concerning the widespread application of this delivery method pertains to the availability of the specialized lipids essential for lipid nanoparticle formation, particularly ionizable cationic lipids. Prior to 2020, these lipids were produced in limited quantities, typically measured in grams or kilograms, primarily for medical research and a select number of orphan drugs. With the established safety and efficacy of mRNA vaccines in 2020, the limited number of manufacturers capable of producing these critical lipids faced the formidable task of rapidly escalating production to meet demands for several tons.
Viral Vectors
Beyond non-viral delivery approaches, RNA viruses have been genetically modified to elicit comparable immunological responses. Common RNA viruses employed as vectors encompass retroviruses, lentiviruses, alphaviruses, and rhabdoviruses, each exhibiting distinct structural and functional characteristics. These viral vectors have been investigated in clinical studies across various diseases within animal models, including mice, chickens, and primates.
Advantages
Traditional Vaccines
mRNA vaccines present distinct benefits when compared to conventional vaccines. As mRNA vaccines are not derived from active or even inactivated pathogens, they inherently lack infectivity. Conversely, conventional vaccines necessitate pathogen production, a process that, at high volumes, could elevate the risk of localized viral outbreaks at manufacturing sites. A further biological advantage of mRNA vaccines is their capacity to stimulate both cellular and humoral immunity, owing to the intracellular production of antigens.
mRNA vaccines possess a manufacturing advantage due to their rapid design capabilities. For instance, Moderna developed its mRNA-1273 vaccine for COVID-19 within a mere two days. Furthermore, their production can be expedited, made more cost-effective, and standardized (leading to reduced error rates), thereby enhancing responsiveness during significant outbreaks.
Initially, the mass production of the Pfizer–BioNTech vaccine necessitated 110 days, a duration later optimized by Pfizer to 60 days, which remained considerably quicker than the manufacturing timelines for conventional influenza and polio vaccines. Within this broader timeframe, the active production phase spans approximately 22 days: two weeks are dedicated to the molecular cloning and purification of DNA plasmids, four days for DNA-to-RNA transcription and mRNA purification, and another four days for encapsulating mRNA within lipid nanoparticles, followed by the fill and finish stages. The predominant portion of the time allotted for each production cycle is dedicated to stringent quality control measures at every step.
DNA Vaccines
mRNA vaccines offer distinct advantages over DNA vaccines, in addition to the theoretical benefits shared with DNA-based approaches compared to conventional vaccines. A key benefit is that mRNA translation occurs within the cytosol, eliminating the requirement for nuclear entry and thereby mitigating the risk of genomic integration into the host. The incorporation of modified nucleosides, such as pseudouridines or 2'-O-methylated nucleosides, into mRNA constructs can suppress immune stimulation, preventing premature degradation and fostering a more sustained effect through improved translational efficiency. Furthermore, the open reading frame (ORF) and untranslated regions (UTR) of mRNA can undergo sequence engineering to achieve specific objectives, such as augmenting guanine-cytosine content or selecting particular UTRs known to enhance translation. The inclusion of an additional ORF encoding a replication mechanism can further amplify antigen translation and, consequently, the immune response, thereby reducing the initial material quantity required.
Limitations
Storage Challenges
The inherent fragility of mRNA necessitates ultra-low temperature storage for certain vaccines to prevent degradation, which would compromise their immunogenicity. For instance, the Pfizer–BioNTech BNT162b2 mRNA vaccine requires storage within a range of −80 to −60 °C (−112 to −76 °F). In contrast, Moderna's mRNA-1273 vaccine can be maintained at −25 to −15 °C (−13 to 5 °F), akin to standard home freezer conditions, and exhibits stability at 2 to 8 °C (36 to 46 °F) for up to 30 days. In November 2020, Nature reported, "While it's possible that differences in LNP formulations or mRNA secondary structures could account for the thermostability differences [between Moderna and BioNtech], many experts suspect both vaccine products will ultimately prove to have similar storage requirements and shelf lives under various temperature conditions." Ongoing research is exploring various platforms to enable mRNA vaccine storage at elevated temperatures.
Regulatory and Development Context
Prior to 2020, no mRNA-based therapeutic platform, whether a drug or vaccine, had received regulatory approval for human application, raising concerns regarding potential unknown effects. The exigencies of the 2020 COVID-19 pandemic accelerated the demand for rapid production capabilities of mRNA vaccines, rendering them highly appealing to national health organizations. This rapid development prompted discussions concerning the appropriate initial authorization pathways for mRNA vaccines, such as emergency use authorization or expanded access authorization, following the completion of the eight-week post-final human trial period.
Adverse Effects
The reactogenicity profile of mRNA vaccines generally aligns with that observed in conventional, non-RNA vaccines. Nevertheless, individuals predisposed to autoimmune responses may experience adverse reactions to mRNA vaccines. The vaccine's mRNA strands can potentially induce an unintended immune response, leading the body to perceive itself as infected, which can manifest as symptomatic illness in the recipient. To mitigate this, mRNA sequences within vaccine formulations are engineered to closely resemble those naturally produced by host cells.
Clinical trials of novel COVID-19 mRNA vaccines documented robust yet transient reactogenic effects. The majority of individuals are not anticipated to experience severe adverse effects, which encompass symptoms like fever and fatigue. Severe adverse effects are specifically defined as those that impede the performance of daily activities.
Clinical Efficacy
The COVID-19 mRNA vaccines developed by Moderna and Pfizer–BioNTech demonstrated short-term efficacy rates exceeding 90 percent against the ancestral SARS-CoV-2 virus. Before the advent of mRNA technology, drug trials targeting pathogens other than COVID-19 frequently proved ineffective, often necessitating abandonment during early trial phases. The precise mechanisms underlying the notable efficacy of these novel mRNA vaccines remain to be fully elucidated.
Physician-scientist Margaret Liu posited that the efficacy of the novel COVID-19 mRNA vaccines might stem from the "sheer volume of resources" dedicated to their development. Alternatively, she suggested that the vaccines could be "triggering a nonspecific inflammatory response to the mRNA that could be heightening its specific immune response, given that the modified nucleoside technique reduced inflammation but hasn't eliminated it completely." Liu further hypothesized that "this may also explain the intense reactions such as aches and fevers reported in some recipients of the mRNA SARS-CoV-2 vaccines." Although these reactions were described as severe, they were transient; another perspective attributes them to a response to the lipid drug delivery molecules. In June 2021, the U.S. FDA issued a warning concerning a potential elevated risk of myocarditis and pericarditis in certain individuals following vaccination.
Vaccine Hesitancy
Misinformation suggests that messenger RNA (mRNA) vaccines could modify nuclear DNA. However, mRNA within the cytosol undergoes rapid degradation, preventing its entry into the cell nucleus. While retroviruses, which are also single-stranded RNA like many SARS-CoV-2 vaccines, can enter the cell nucleus and utilize reverse transcriptase to synthesize DNA from RNA, they possess specific nuclear import mechanisms that exogenous mRNA, such as that in vaccines, lacks. Furthermore, even if exogenous mRNA could enter the nucleus, DNA synthesis from RNA requires reverse transcriptase and appropriate primers, both of which are associated with retroviruses but absent for vaccine-derived mRNA.
Amplification
Messenger RNA (mRNA) vaccines are categorized into two types: non-amplifying (conventional) mRNA and self-amplifying mRNA. The Pfizer–BioNTech and Moderna vaccines utilize non-amplifying mRNA. Both mRNA modalities are currently under investigation for their potential as vaccine platforms against various pathogens and cancer.
Non-amplifying
Early messenger RNA (mRNA) vaccines employed a non-amplifying mRNA construct. This type of mRNA features a single open reading frame, which encodes the specific antigen of interest. Consequently, the total quantity of mRNA accessible within the cell directly corresponds to the amount administered via the vaccine. The dosage efficacy is thus constrained by the deliverable mRNA quantity. To mitigate toxicity, non-amplifying vaccines incorporate N1-Methylpseudouridine as a substitute for uridine.
Self-amplifying
Self-amplifying messenger RNA (saRNA) vaccines are designed to replicate their mRNA within the cell following transfection. SaRNA constructs contain two open reading frames: the first, similar to conventional mRNA, encodes the antigen of interest, while the second codes for an RNA-dependent RNA polymerase and its associated helper proteins, which facilitate the replication of the mRNA construct within the cell. This replication capability enables the use of smaller vaccine doses. Due to its larger molecular size, the underlying mechanisms and subsequent evaluation of self-amplifying mRNA may differ from those of non-amplifying mRNA.
Research into self-amplifying RNA (saRNA) vaccines includes the development of a malaria vaccine. The first authorized saRNA COVID-19 vaccine was Gemcovac, approved in India in June 2022. Subsequently, ARCT-154, developed by Arcturus Therapeutics, received authorization. A variant of ARCT-154, produced by Meiji Seika Pharma, was authorized in Japan in November 2023.
In 2021, GSK initiated a Phase 1 clinical trial for a self-amplifying RNA (saRNA) COVID-19 vaccine. Concurrently, Gritstone bio commenced a Phase 1 trial for an saRNA COVID-19 vaccine intended as a booster, with interim findings released in 2023. This vaccine is engineered to target both the SARS-CoV-2 spike protein and other viral proteins less susceptible to genetic variation, aiming to enhance protection against SARS-CoV-2 variants. A critical requirement for saRNA vaccine reproduction is the presence of uridine.
- DNA vaccine
- RNA therapeutics
- References
References
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