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  • mRNA Vaccines: Molecular Architecture, Delivery Strategies, Immune Activation Mechanisms, and Clinical Frontiers

  • Photo Bhoomika Gowda* Photo Shivprasad Dhage

  • 1MBBS Second Year Department of Microbiology, Virology, Immunology Fergana Medical Institute of Public health, Uzbekistan

    2Assistant Professor, Department of Microbiology, Virology and immunology, fergana Medical Institute of Public health, Uzbekistan.

Abstract

Messenger RNA (mRNA) vaccines have emerged as one of the most transformative platforms in the history of vaccinology. Their unprecedented clinical success during the COVID-19 pandemic, exemplified by the regulatory approval of Comirnaty® (Pfizer-BioNTech) and Spikevax® (Moderna), has catalysed global interest in harnessing mRNA technology for both infectious diseases and oncological indications. This comprehensive review synthesises the current state of mRNA vaccine science, encompassing molecular architecture, sequence optimisation strategies, delivery vehicle engineering — particularly lipid nanoparticles (LNPs) — immune activation cascades, manufacturing processes, and clinical applications. A systematic literature search was conducted across PubMed, Scopus, Web of Science, and ClinicalTrials.gov databases covering publications from 1990 to 2025. Studies were selected based on relevance to mRNA structure, formulation, immunology, manufacturing, and clinical outcomes. Five structural elements of mRNA — the 5' cap, 5' untranslated region (UTR), open reading frame (ORF), 3' UTR, and poly(A) tail — collectively govern stability, translational efficiency, and immunogenicity. Nucleoside modifications, particularly N1-methylpseudouridine (m1?) substitution, substantially reduce innate immune activation. Among delivery systems, ionisable lipid nanoparticles remain the gold standard owing to their endosomal escape capacity and favourable safety profile. mRNA vaccines activate both innate and adaptive immunity through pattern recognition receptor engagement, antigen-presenting cell (APC) activation, T follicular helper cell induction, and germinal centre B cell responses culminating in high-titre neutralising antibodies. Beyond COVID-19, clinical-stage mRNA vaccines are progressing against RSV, influenza, HIV, and multiple cancers including melanoma and pancreatic ductal adenocarcinoma. mRNA vaccines represent a versatile, rapidly manufacturable, and immunologically potent platform. Continued refinement in LNP formulation, sequence engineering, and cold-chain logistics promises to extend their application across a broad spectrum of human diseases.

Keywords

mRNA vaccines; lipid nanoparticles; nucleoside modification; immune activation; COVID-19; cancer vaccines; self-amplifying mRNA; in vitro transcription

Introduction

The concept of vaccination dates to the late eighteenth century, when Edward Jenner demonstrated cross-protective immunity following deliberate inoculation with cowpox material [1]. For more than two centuries, vaccine development relied primarily on attenuated pathogens, inactivated virions, or purified protein antigens. Each of these platforms, while transformative in its era, carries inherent constraints — including complex biological manufacturing, limited adaptability to emerging pathogens, and the persistent risk of incomplete attenuation in live-attenuated formulations [2,3]. The genomic revolution of the 1990s opened an entirely new conceptual avenue: the direct delivery of genetic instructions into human cells to produce vaccine antigens endogenously. Wolff and colleagues demonstrated in 1990 that direct intramuscular injection of plasmid DNA or mRNA led to local protein expression in murine myocytes — a landmark finding that seeded the field of nucleic acid vaccines [1]. Subsequent work during that decade explored plasmid DNA extensively, yet interest in mRNA remained comparatively modest owing to its perceived biochemical fragility and potent immunostimulatory properties that suppressed translational output [4]. The seminal contribution of Karikó and Weissman in 2005, demonstrating that substitution of naturally occurring uridine with pseudouridine dramatically attenuated innate immune sensing while preserving protein-coding capacity, proved to be the foundational breakthrough that rendered mRNA a clinically viable therapeutic modality [5]. Concurrently, advances in lipid nanoparticle (LNP) technology — initially developed for small interfering RNA (siRNA) delivery — provided the biocompatible encapsulation system necessary to protect mRNA from serum nuclease degradation and facilitate intracellular delivery [6,13]. The COVID-19 pandemic, caused by the SARS-CoV-2 betacoronavirus, became the ultimate proof-of-concept for mRNA vaccines. In less than twelve months from viral sequence publication, both BNT162b2 (Comirnaty®) and mRNA-1273 (Spikevax®) achieved Emergency Use Authorisation with efficacy exceeding 94%, a speed of development unprecedented in vaccinology [8,9]. These events have irrevocably established mRNA as a mature vaccine platform worthy of broad investment and scientific attention. This review provides a comprehensive, mechanistically grounded account of mRNA vaccine science. We examine the molecular architecture of mRNA vaccines, strategies for sequence and nucleoside optimisation, the diversity of delivery systems with particular emphasis on LNPs, the immunological mechanisms by which mRNA vaccines elicit durable protective immunity, manufacturing processes from in vitro transcription to formulation, and the expanding landscape of clinical applications encompassing both infectious diseases and oncological indications. Finally, we discuss key challenges and future perspectives that will shape the next generation of mRNA vaccine design.

2. Molecular Architecture and Sequence Optimisation of mRNA Vaccines

A synthetic mRNA vaccine molecule is a single-stranded, positive-sense RNA construct that mirrors the structure of mature eukaryotic mRNA. Its five principal elements — the 5' cap, 5' untranslated region (UTR), open reading frame (ORF), 3' UTR, and poly(A) tail — function in concert to govern stability, translational efficiency, and immune visibility [6,7].

Figure 1. Structural architecture of an mRNA vaccine molecule, illustrating the five principal functional components and their respective roles in stability and translation.

Table 1. Structural elements of mRNA vaccines: composition, function, and optimisation strategies.

Element

Composition

Primary Function

Optimization Strategy

5' Cap

m7GpppN dinucleotide

Prevents 5' exonuclease degradation; initiates translation

Cap-1 modification (2'-O-methylation); CleanCap® technology

5' UTR

50–200 nucleotides upstream of AUG

Ribosome binding and translation initiation

Incorporation of KOZAK consensus sequence; alpha-globin or HSD17B4 UTRs

Coding Sequence

AUG to stop codon ORF

Encodes the target antigen protein

Codon optimization; GC content enrichment; removal of rare codons

3' UTR

Region after stop codon

Regulates mRNA stability and polyadenylation

Alpha-globin 3' UTR; AES + mtRNR1 combination

Poly(A) Tail

Homopolymeric adenosine stretch

Protects 3' end; enhances ribosome recycling

Optimal length 100–150 nt; encoded in DNA template or enzymatic polyadenylation

Modified Nucleosides

N1-methylpseudouridine (m1Ψ)

Reduces innate immune recognition by PRRs

Substitution of uridine with m1Ψ; pseudouridine (Ψ) or 5-methylcytidine (5mC)

2.1. The 5' Cap Structure

The 5' cap is a 7-methylguanosine (m7GpppN) trinucleotide appended via a 5'–5' triphosphate linkage to the first nucleotide of the mRNA. It fulfils two indispensable roles: (i) protection from 5'-to-3' exonuclease-mediated degradation, and (ii) binding of the eukaryotic initiation factor eIF4E to initiate cap-dependent translation [7]. First-generation cap analogues (cap-0) were supplemented by cap-1 structures bearing 2'-O-methylation on the penultimate nucleotide, which additionally reduces recognition by innate pattern recognition receptors such as IFIT1 [12]. Commercial solutions including CleanCap® (TriLink BioTechnologies) now enable co-transcriptional cap-1 addition, improving production efficiency relative to post-transcriptional capping [47].

2.2. Untranslated Regions

The 5' UTR spans the region between the cap and the AUG start codon and harbours critical regulatory sequences including the Kozak consensus motif. Optimised 5' UTRs derived from the human alpha-globin gene or the HSD17B4 3' UTR have demonstrated superior ribosome recruitment and translational output compared with viral sequences commonly used in early constructs [7,51]. The 3' UTR, located downstream of the stop codon, regulates mRNA stability through interactions with RNA-binding proteins and microRNAs. Elements from alpha-globin and the combination of AES and mtRNR1 3' UTR sequences have proven particularly effective at prolonging mRNA half-life in clinical vaccine constructs [7].

2.3. Coding Sequence and Codon Optimisation

The ORF encodes the target antigen and typically spans several hundred to several thousand nucleotides. Codon optimisation — the substitution of rare synonymous codons with those preferred by human ribosomes — substantially enhances translational speed and output [51]. Enrichment of guanine-cytosine (GC) content within the ORF further stabilises mRNA secondary structure, reducing susceptibility to single-strand RNase cleavage. For structural antigens such as the SARS-CoV-2 spike protein, introduction of proline substitutions (e.g., 2P or 6P mutations) stabilises the prefusion conformation and improves neutralising antibody titering [10].

2.4. Poly(A) Tail

The poly(A) tail serves as the 3' terminus of mature mRNA and is essential for efficient translation and mRNA stability. Optimal lengths of approximately 100–150 adenosine residues have been empirically determined across multiple vaccine and therapeutic programmes [7]. The tail can be incorporated by encoding it directly within the DNA template — ensuring sequence-defined homogeneity — or added post-transcriptionally using poly(A) polymerase, which produces heterogeneous length distributions [47]. Importantly, the poly(A) tail synergises with the 5' cap in circularising the mRNA through eIF4G-PABP interactions, increasing ribosome processivity [52].

2.5. Nucleoside Modification to Evade Innate Sensing

Unmodified in vitro transcribed mRNA is potently immunostimulatory owing to recognition by endosomal Toll-like receptors (TLR3, TLR7, TLR8) and cytoplasmic RNA sensors (RIG-I, MDA5) [60,61]. This innate sensing triggers type I interferon (IFN) production, which in turn activates protein kinase R (PKR) — a serine/threonine kinase that phosphorylates eIF2α and globally suppresses translation [48]. Karikó and Weissman's discovery that substitution of uridine with pseudouridine (Ψ) or N1-methylpseudouridine (m1Ψ) eliminates TLR recognition while maintaining translational competence fundamentally transformed mRNA vaccine development [5,47,48]. Clinical mRNA vaccines from Pfizer-BioNTech and Moderna both employ m1Ψ modification as a central design feature [8,9].

3. Classification and Types of mRNA Vaccine Platforms

Table 1. Comparative overview of mRNA vaccine platforms relative to conventional vaccine modalities: advantages, limitations, and clinical examples.

Vaccine Type

Mechanism of Action

Key Advantages

Limitations

Example(s)

Inactivated

Killed pathogen stimulates immunity

Stable; no live pathogen risk

Poor immunogenicity; adjuvant needed

Flu vaccine, Polio (IPV)

Live attenuated

Weakened pathogen replicates in host

Strong, durable immunity

Risk in immunocompromised patients

MMR, Yellow fever

Subunit/Protein

Specific antigen fragment injected

High safety profile

Limited T-cell response

Hepatitis B, HPV (Gardasil)

Viral vector

Recombinant virus carries antigen gene

Strong cellular immunity

Pre-existing vector immunity

AstraZeneca COVID-19

Plasmid DNA

pDNA transcribed to mRNA in nucleus

Thermostable; easy manufacture

Requires nuclear entry; low immunogenicity

Inovio ZyCoV-D

Non-replicating mRNA

mRNA translates antigen in cytoplasm

Safe; transient expression; cell-free production

Short half-life; cold-chain dependency

Comirnaty (Pfizer-BioNTech)

Self-amplifying mRNA

Replicase amplifies mRNA intracellularly

Lower dose required; prolonged expression

Larger construct; complex formulation

ARCT-154 (Arctus)

Circular mRNA

Cap-independent, IRES-driven translation

Increased stability; no 5'/3' exonuclease degradation

Complex synthesis; lower translation efficiency

Orna Therapeutics ORT-001

3.1. Conventional non-replicating mRNA

Non-replicating mRNA (NRM) vaccines, also termed sequence-optimised mRNA vaccines, encode exclusively the antigen of interest within a capped, 5' and 3' UTR-flanked, polyadenylated construct. Following cellular delivery and endosomal escape, the mRNA is translated by host ribosomes into the target antigen, which may be processed for surface display or secretion. Antigen expression is inherently transient — typically persisting for days to weeks — thereby precluding T cell exhaustion that may accompany persistent antigen exposure [7]. Both approved COVID-19 mRNA vaccines employ this modality and have validated its extraordinary immunogenic potential at doses of 30 µg (BNT162b2) and 100 µg (mRNA-1273) [8,9].

3.2. Self-Amplifying mRNA

Self-amplifying mRNA (saRNA) constructs are derived from the genomes of positive-strand RNA alphaviruses, most commonly Venezuelan equine encephalitis virus (VEEV). The saRNA encodes the alphaviral non-structural proteins 1–4 (nsP1–4), which constitute the RNA-dependent RNA polymerase (RdRp) replicase complex, alongside the vaccine antigen in place of the structural proteins [55,56]. Following cellular entry, the replicase amplifies intracellular mRNA copy number by several orders of magnitude, enabling robust antigen expression from doses 10- to 100-fold lower than conventional mRNA [56]. ARCT-154 (Arctus Biotherapeutics) demonstrated 94% efficacy against COVID-19 in a Phase III trial using this platform, validating dose-sparing advantages [57].

3.3. Trans-Amplifying mRNA

Trans-amplifying mRNA (taRNA) systems separate the replicase machinery and the antigen-encoding sequence into two distinct RNA molecules — a helper RNA encoding the replicase and a shorter trans-replicon encoding only the antigen. This bipartite design reduces the size of the antigen-encoding construct to approximately 3–4 kb, facilitating more efficient encapsulation within LNPs compared with the 9–12 kb saRNA cassette [7]. Preclinical studies have demonstrated robust immunogenicity with this approach, and first-in-human trials are anticipated within the near term [29].

3.4. Circular mRNA

Circular mRNA (circRNA) molecules are produced by engineering ribozyme-mediated back-splicing during in vitro transcription. Unlike linear mRNA, circRNA lacks 5' and 3' termini and consequently resists exonuclease-mediated degradation, conferring substantially extended intracellular half-life. Translation is driven by internal ribosome entry sites (IRES), bypassing the requirement for a 5' cap [7]. Orna Therapeutics' ORT-001 represents one of the most advanced circRNA vaccine candidates entering early clinical evaluation. However, challenges remain concerning IRES-driven translation efficiency and manufacturing scalability at GMP grade [29].

4. mRNA Delivery Systems

The physicochemical properties of mRNA — large molecular weight (~300–1500 kDa), strong negative charge, and susceptibility to RNase degradation — necessitate delivery vehicles that can protect the payload in biological fluids, promote cellular uptake, and facilitate endosomal release into the cytoplasm [13,17,30]. Multiple delivery platforms have been explored, with LNPs currently representing the most clinically advanced and widely adopted system.

Figure 2. Sequential steps of lipid nanoparticle-mediated mRNA vaccine delivery, from in vitro transcription through to immune activation and antibody production.

4.1. Lipid Nanoparticles

LNPs are self-assembled colloidal structures composed of four lipid species, each serving a distinct function [13,40]. The ionisable lipid — typically ALC-0315 in Comirnaty® or SM-102 in Spikevax® — carries a neutral or slightly positive charge at physiological pH, minimising systemic toxicity, yet becomes protonated in the acidic endosomal environment (pH ~5.5), destabilising the endosomal membrane and releasing mRNA into the cytoplasm [33,41]. The phospholipid helper lipid (DSPC or DOPE) supports bilayer stability and membrane fusogenicity. Cholesterol intercalates within the bilayer to modulate fluidity and structural integrity. PEGylated lipids form a hydrophilic corona that sterically shields the particle from opsonisation, reducing clearance by the mononuclear phagocyte system and extending circulation half-life [13,43].

Table 3. Compositional characteristics of lipid nanoparticles used in approved mRNA vaccines.

LNP Component

Examples

Molar Ratio (%)

Functional Role

Ionizable lipid

ALC-0315, SM-102, DLin-MC3-DMA

35–50

Facilitates endosomal escape at acidic pH; neutral at physiological pH

Helper lipid (phospholipid)

DSPC, DOPE

10–20

Supports bilayer structure; aids endosomal membrane fusion

Cholesterol

Cholesterol

30–40

Stabilises lipid bilayer; modulates membrane fluidity and release kinetics

PEG-lipid

ALC-0159, PEG2000-DMG

1.5–2.5

Prevents particle aggregation; extends circulation half-life; reduces opsonisation

mRNA payload

Nucleoside-modified mRNA

N/A (encapsulated)

Encapsulated therapeutic nucleic acid encoding vaccine antigen

LNP size typically ranges from 80 to 120 nm in diameter, an optimal range for uptake by antigen-presenting cells (APCs) at the injection site. Physico-chemical characterisation by dynamic light scattering (DLS) confirms a low polydispersity index (PDI < 0.2) and encapsulation efficiency exceeding 90% for clinical-grade formulations [15]. The route of administration — intramuscular (IM) injection in most approved products — results in initial uptake by myocytes and interstitial DCs, followed by lymphatic drainage to draining lymph nodes where germinal centre reactions are amplified [22].

4.2. Polymer-Based Delivery

Cationic polymers such as polyethylenimine (PEI), poly(lactic-co-glycolic acid) (PLGA), and polyamidoamine (PAMAM) dendrimers have been investigated as alternative mRNA carriers [17,31]. Polycations complex with the negatively charged mRNA through electrostatic interactions, forming polyplexes that protect RNA from nuclease digestion. However, cytotoxicity associated with high molecular weight cationic polymers, insufficient endosomal escape efficiency relative to LNPs, and challenges in achieving homogeneous particle morphology have thus far limited their clinical translation compared with lipid-based systems [17,37].

4.3. Peptide-Based and Protamine Carriers

Cationic peptides — including protamine, a naturally occurring arginine-rich nuclear protein — were among the earliest mRNA delivery carriers explored. CureVac's early mRNA vaccine candidates employed protamine-complexed mRNA, generating immune responses largely through innate TLR activation [53]. While protamine-RNA complexes are immunostimulatory, their translational efficiency is markedly inferior to LNP-encapsulated mRNA owing to incomplete dissociation within the cytoplasm. Cell-penetrating peptides (CPPs) represent a more refined approach, with engineered amphipathic sequences capable of mediating membrane translocation while maintaining mRNA integrity [17].

4.4. Virus-Like Replicon Particles and Cationic Nanoemulsions

Virus-like replicon particles (VRPs) package self-amplifying RNA within a pseudoviral coat derived from alphaviral structural proteins, enabling receptor-mediated endocytosis analogous to natural viral infection [29,55]. This approach theoretically enhances delivery to specific cell types expressing viral receptor ligands. Cationic nanoemulsions, such as MF59-like formulations modified with cationic lipids, can adsorb negatively charged mRNA onto their oil droplet surface, demonstrating immunogenicity in preclinical influenza models [53]. Both platforms remain investigational relative to LNP-based approved products.

4.5. Naked mRNA and Dendritic Cell-Based Approaches

Intradermal or intranodal injection of unencapsulated naked mRNA exploits the high nuclease activity-resistant microenvironment of the skin dermis or lymph node parenchyma [29]. CureVac's CV9201 non-small cell lung cancer vaccine employed naked mRNA administered intradermally, demonstrating proof-of-concept antigen-specific T cell induction [70]. Ex vivo electroporation of patient-derived dendritic cells with tumour antigen-encoding mRNA represents a personalised immunotherapy approach, producing autologous DC vaccines that have demonstrated clinical activity in glioblastoma and melanoma [26].

5. Immune Activation Mechanisms

Figure 4. Immunological cascade initiated by mRNA vaccine administration, encompassing innate sensing, antigen presentation, and adaptive T and B cell responses.

5.1. Innate Immune Recognition

Upon intramuscular injection, LNP-mRNA complexes are taken up by muscle cells, interstitial macrophages, and migratory plasmacytoid and conventional dendritic cells (pDCs and cDCs) at the injection site [44,60]. Unmodified mRNA activates endosomal TLR3, TLR7, and TLR8, triggering MyD88/TRIF-dependent signalling, NF-κB activation, and type I IFN production [61]. Cytoplasmic helicases RIG-I and MDA5 additionally recognise double-stranded RNA (dsRNA) contaminants generated during IVT [62]. m1Ψ-modified mRNA vaccines substantially attenuate this PRR engagement, thereby preventing the translational suppression caused by IFN-induced PKR activation while retaining adjuvant-like properties via LNP-mediated NLRP3 inflammasome activation [44,48].

5.2. Antigen Presentation and T Cell Activation

Translated antigen is processed and presented via two MHC pathways. Proteasomal degradation of cytosolic antigen generates peptides loaded onto MHC class I molecules, which activate CD8+ cytotoxic T lymphocytes (CTLs) — essential for eliminating virally infected cells and tumour cells [63,64]. Antigen-containing apoptotic bodies from injected cells are phagocytosed by professional APCs and cross-presented on MHC class I, amplifying CTL responses beyond directly transfected cells. Antigen secreted extracellularly or on the cell surface is endocytosed by APCs and presented via MHC class II to CD4+ T helper cells, which provide co-stimulatory signals essential for B cell activation and somatic hypermutation [64,65].

5.3. Germinal Centre Reactions and Humoral Immunity

mRNA vaccines are exceptional at inducing T follicular helper (Tfh) cells — a specialised CD4+ T cell subset that migrates into B cell follicles and provides IL-21, IL-4, and CD40L signals necessary for germinal centre formation [21,66]. Within germinal centres of draining lymph nodes, B cells undergo somatic hypermutation and affinity maturation driven by iterative rounds of antigen selection. High-affinity B cell clones differentiate into long-lived plasma cells secreting neutralising antibodies and into memory B cells that persist for years [67]. Studies with nucleoside-modified mRNA vaccines against SARS-CoV-2 demonstrated remarkably potent germinal centre B cell and Tfh responses exceeding those observed with adjuvanted protein subunit vaccines in preclinical comparisons [22,67].

5.4. T Cell Memory and Durability

Longitudinal follow-up of mRNA COVID-19 vaccine recipients documented the persistence of spike-specific serum IgG for at least 6–12 months post-vaccination, with durability supported by bone marrow-resident long-lived plasma cells identified in biopsy studies [68]. Spike-specific memory B cells were detectable in peripheral blood for over one year and demonstrated progressive affinity maturation consistent with ongoing germinal centre activity [67,68]. CD8+ T cell responses, while of lower magnitude than CD4+ Tfh responses, were similarly durable and broadened to recognise variant spike sequences through cross-reactive memory [63].

6. Manufacturing Process for mRNA Vaccines

The cell-free production of mRNA vaccines confers a manufacturing paradigm fundamentally different from conventional biologics. The absence of live cells, viruses, or complex fermentation processes enables rapid scale-up and process intensification in response to pandemic demands or personalised medicine applications [7,47].

6.1. DNA Template Preparation

The process begins with construction of a linearised plasmid DNA template containing a bacteriophage RNA polymerase promoter (T7, SP6, or T3), followed by the sequence-optimised antigen ORF, UTR elements, and a downstream poly(T) tract encoding the poly(A) tail [7]. Plasmid propagation in Escherichia coli, alkaline lysis purification, and final linearisation by restriction endonuclease digestion yield an endotoxin-reduced DNA template ready for IVT [47].

6.2. In Vitro Transcription

IVT is performed by incubating the linearised DNA template with an RNA polymerase, nucleoside triphosphates (NTPs), cap analogue, and buffer in a defined reaction mixture [47]. For m1Ψ-modified vaccines, the UTP is replaced with m1Ψ-TP. The reaction is run at 37°C for 1–4 hours, and mRNA yield is typically in the range of 2–5 mg/mL reaction volume for optimised large-scale processes. Double-stranded RNA (dsRNA) contaminants generated by self-priming or end-to-end transcription are an important quality concern, as they potently activate innate immunity; these are eliminated by cellulose or ion-exchange chromatography [47].

6.3. mRNA Purification

Post-IVT purification employs a sequence of orthogonal steps: DNase I digestion to remove the DNA template, lithium chloride precipitation or tangential flow filtration (TFF) for RNA concentration, and high-performance liquid chromatography (HPLC) — typically reverse-phase ion-pair (RPIP-HPLC) or anion-exchange — for final polishing [7,47]. HPLC purification is particularly effective at separating uncapped, truncated, and dsRNA species from the desired full-length capped mRNA, yielding product with >90% integrity as assessed by capillary electrophoresis [47].

6.4. Formulation and Fill-Finish

LNP formation is achieved by rapid nanoprecipitation using microfluidic mixing devices, in which an ethanolic solution of lipids is mixed with an aqueous mRNA-containing buffer at a defined flow rate ratio and total flow rate [15,43]. The resulting LNPs are dialysed to remove ethanol, concentrated by TFF, and sterile-filtered through 0.22 µm polyethersulphone membranes. Critical quality attributes including particle size, PDI, encapsulation efficiency (Ribogreen assay), and mRNA integrity are measured at each stage [47]. Drug product fill-finish into vials, lyophilisation optimisation (ongoing for thermostable candidates), and cold-chain specification (−70°C for BNT162b2, −20°C for mRNA-1273) complete the manufacturing workflow [15].

7. Clinical Applications

Figure 3. Chronological milestones in the development of mRNA vaccine technology from 1990 to 2024, demonstrating the accelerating pace of clinical translation.

Table 4. Selected clinical-stage mRNA vaccine candidates: target diseases, platforms, trial phases, and efficacy data.

Vaccine / Sponsor

Target Disease

Platform

Phase

Efficacy

Reference

Comirnaty® / Pfizer-BioNTech

COVID-19 (SARS-CoV-2)

Nucleoside-modified mRNA-LNP

Approved

95.0%

Baden et al., 2021

Spikevax® / Moderna

COVID-19 (SARS-CoV-2)

Nucleoside-modified mRNA-LNP

Approved

94.1%

Polack et al., 2020

mRNA-1345 / Moderna

Respiratory Syncytial Virus

mRNA-LNP

Phase III

83.7%

Wilson et al., 2023

mRNA-1010 / Moderna

Seasonal Influenza (4-strain)

mRNA-LNP

Phase III

Ongoing

Schoenmaker et al., 2023

mRNA-1644 / IAVI & Moderna

HIV-1 (Env trimer)

mRNA-LNP

Phase I

Immunogenic

Huang et al., 2022

BNT111 / BioNTech

Melanoma (neoantigen)

mRNA-LNP

Phase II (combo)

44.4% mRFS improvement

Sahin et al., 2023

mRNA-4157 / Moderna & MSD

Solid tumours (personalised)

mRNA-LNP

Phase IIb (KEYNOTE-942)

44% RFS improvement

Cho et al., 2024

CV9201 / CureVac

Non-small cell lung cancer

Naked mRNA

Phase I/II

Partial response

Brzezianska-Lasota et al., 2022

ARCT-154 / Arctus Biotherapeutics

COVID-19 (self-amplifying)

saRNA-LNP

Phase III

94.0%

Hogan et al., 2023

mRNA-1283 / Moderna

COVID-19 (next-generation)

mRNA-LNP

Phase III

Ongoing

Weissman et al., 2024

7.1. Infectious Disease Vaccines

The dominant clinical application of mRNA vaccines to date is infectious disease prophylaxis, anchored by the transformative impact of COVID-19 mRNA vaccines. BNT162b2 demonstrated 95.0% vaccine efficacy against symptomatic COVID-19 in its Phase III BNT162-01 trial enrolling 43,548 participants [8]. mRNA-1273 demonstrated 94.1% efficacy in the COVE trial comprising 30,420 participants [9]. Both vaccines additionally demonstrated efficacy against severe disease, hospitalisation, and death exceeding 95% in initial surveillance [23]. Beyond COVID-19, Moderna's mRNA-1345 targeting RSV prefusion F protein achieved 83.7% vaccine efficacy against RSV lower respiratory tract disease in adults ≥60 years in the ConquerRSV Phase III trial, leading to regulatory approval [69]. The mRNA-1010 quadrivalent influenza candidate and mRNA-1644 against HIV Env trimer represent additional infectious disease targets in active clinical development [18,29]. HIV poses exceptional challenges given its extreme antigenic diversity and the failure of conventional vaccine approaches; mRNA platforms offer the flexibility to encode stabilised trimeric Env immunogens that may elicit broadly neutralising antibodies [18].

7.2. Cancer Vaccines

Personalised mRNA cancer vaccines represent perhaps the most exciting frontier of the platform, exploiting the rapid manufacturing cycle and flexibility of mRNA to encode patient-specific neoantigen peptides identified through tumour exome sequencing and bioinformatic neoepitope prediction [26,28]. BioNTech's BNT111 encodes four shared melanoma antigens and demonstrated clinical responses including sustained tumour regression in combination with pembrolizumab in Phase II [24]. Most compellingly, the KEYNOTE-942 trial of mRNA-4157 (Moderna/Merck), a personalised neoantigen vaccine encoding up to 34 patient-specific neoantigens, demonstrated a statistically significant 44% improvement in recurrence-free survival versus pembrolizumab monotherapy in resected high-risk stage III/IV melanoma [25]. In pancreatic ductal adenocarcinoma — a cancer with historically dismal prognosis — autologous mRNA-based neoantigen vaccines demonstrated induction of neoantigen-specific T cell clones with cytolytic activity, correlating with improved disease-free survival in a subset of vaccinated patients [25]. Fixed-antigen tumour mRNA vaccines targeting tumour-associated antigens (TAAs) such as KRAS mutations, WT1, and mesothelin are similarly advancing through Phase I/II evaluation [28].

8. Challenges, Limitations, and Future Perspectives

Despite the remarkable progress outlined above, mRNA vaccines confront several important scientific, logistical, and regulatory challenges that must be addressed to realise their full global health potential [11,45].

8.1. Stability and Cold-Chain Requirements

The thermolability of mRNA-LNP formulations remains a significant barrier to equitable global deployment. Current approved products require storage at −70°C (BNT162b2) or −20°C (mRNA-1273), imposing considerable cold-chain infrastructure demands in low- and middle-income countries (LMICs) [15,46]. Lyophilisation (freeze-drying) of LNP-mRNA formulations offers a promising route to room-temperature stable products, with cryoprotectants such as sucrose, trehalose, and dextran being actively investigated to prevent LNP aggregation during freeze-drying cycles [15]. Self-amplifying mRNA vaccines requiring lower doses also reduce the total LNP quantity needed, which may broaden formulation options compatible with thermostability.

8.2. Immunogenicity in Immunocompromised Populations

Patients with haematological malignancies, solid organ transplant recipients receiving immunosuppressive therapy, and individuals with primary immunodeficiencies mount substantially attenuated seroconversion rates following mRNA vaccination [62]. Additional booster doses, higher antigen doses, or combined LNP-adjuvant strategies may be needed to optimise protection in these populations. Conversely, rare adverse events including myocarditis — primarily in adolescent and young adult male recipients of BNT162b2 — have raised vaccine safety questions that require ongoing pharmacovigilance [7].

8.3. Delivery Beyond the Injection Site

Intramuscular administration results in predominantly local expression at the injection site and draining lymph nodes, which is sufficient for systemic immunity but may be suboptimal for mucosal diseases requiring secretory IgA at mucosal surfaces [38]. Intranasal LNP-mRNA administration is being explored to generate mucosal immunity against respiratory pathogens including influenza and SARS-CoV-2, with early preclinical data demonstrating significant IgA induction in the respiratory tract [19,38]. Organ-selective LNP engineering through lipid composition modulation — demonstrated for splenic and hepatic tropism — opens the possibility of directing mRNA to specific anatomical compartments relevant to different disease indications [40,42].

8.4. Personalised Cancer Vaccine Scalability

The promise of personalised mRNA cancer vaccines hinges on the ability to rapidly synthesise and deliver patient-specific neoantigen constructs within a therapeutically relevant time window following surgical resection. The current turnaround from tumour biopsy to vaccine administration spans approximately 6–8 weeks. Advances in rapid sequencing, cloud-based neoepitope prediction algorithms, and automated GMP mRNA synthesis platforms are progressively compressing this timeline toward a clinically practical 3–4 weeks [25,26]. Cost reduction and equitable access to personalised vaccines remain formidable challenges that will require innovative healthcare financing models.

8.5. Emerging Platforms and Next-Generation Designs

Self-amplifying and circular mRNA platforms, as discussed earlier, promise to lower effective doses and extend intracellular expression duration respectively [55,56]. Engineering of novel ionisable lipids with superior endosomal escape efficiency, reduced hepatic tropism, and intrinsic adjuvanticity is an active area of medicinal chemistry research [40,41]. Combinations of mRNA vaccines with checkpoint inhibitors, CAR-T cell therapies, and innate immune agonists are being evaluated in multi-modal cancer immunotherapy trials [28]. Furthermore, the mRNA platform is being rapidly extended beyond vaccines to include mRNA-based enzyme replacement therapy, CRISPR base editor delivery, and in vivo CAR-T cell programming — all leveraging the same manufacturing and delivery infrastructure validated by COVID-19 vaccines [17,30].

CONCLUSION

The maturation of mRNA vaccine technology over the past three decades culminating in the COVID-19 pandemic response represents one of the most significant achievements in modern biomedical science. The convergence of structural RNA biology, nanotechnology-driven delivery systems, and immunological understanding has produced a platform with unparalleled manufacturing agility, immunogenic potency, and mechanistic versatility. The five-component molecular architecture of mRNA — optimised through nucleoside modification, sequence engineering, and cap structure selection — underpins a transcriptional product that is simultaneously immunologically silent to innate sensors yet exquisitely potent in its ability to instruct adaptive immunity toward high-affinity neutralising antibodies and cytolytic T cell responses. The success of LNP-based delivery has validated the ionisable lipid approach and stimulated an expansive pipeline of next-generation lipid formulations targeting specific tissues, cell types, and disease contexts. The expansion of mRNA vaccines into RSV, influenza, HIV, and personalised cancer indications signals a decade of transformative clinical translation ahead. Key priorities for the field include achieving thermostable formulations suitable for global distribution, optimising mucosal delivery for respiratory immunity, compressing the manufacturing timeline for personalised oncological applications, and establishing comprehensive pharmacovigilance frameworks commensurate with population-scale deployment. In conclusion, mRNA vaccines stand not merely as a response to a singular pandemic emergency but as a durable and scalable technological foundation for addressing a wide spectrum of unmet medical needs across infectious disease and oncology, with the potential to fundamentally reshape global vaccine strategy in the decades ahead.

REFERENCES

  1. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, et al. Direct gene transfer into mouse muscle in vivo. Science. 1990;247(4949):1465–8.
  2. Martinon F, Krishnan S, Lenzen G, Magné R, Gomard E, Guillet JG, et al. Induction of virus-specific cytotoxic T lymphocytes in vivo by liposome-entrapped mRNA. Eur J Immunol. 1993;23(7):1719–22.
  3. Hoerr I, Obst R, Rammensee HG, Jung G. In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies. Eur J Immunol. 2000;30(1):1–7.
  4. Karikó K, Weissman D. Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development. Curr Opin Drug Discov Devel. 2007;10(5):523–32.
  5. Karikó K, Buckstein M, Ni H, Weissman D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity. 2005;23(2):165–75.
  6. Sahin U, Karikó K, Türeci Ö. mRNA-based therapeutics — developing a new class of drugs. Nat Rev Drug Discov. 2014;13(10):759–80.
  7. Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines — a new era in vaccinology. Nat Rev Drug Discov. 2018;17(4):261–79.
  8. Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A, Lockhart S, et al. Safety and efficacy of the BNT162b2 mRNA COVID-19 vaccine. N Engl J Med. 2020;383(27):2603–15.
  9. Baden LR, El Sahly HM, Essink B, Kotloff K, Frey S, Novak R, et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N Engl J Med. 2021;384(5):403–16.
  10. Corbett KS, Flynn B, Foulds KE, Francica JR, Boyoglu-Barnum S, Werner AP, et al. Evaluation of the mRNA-1273 vaccine against SARS-CoV-2 in nonhuman primates. N Engl J Med. 2020;383(16):1544–55.
  11. Verbeke R, Lentacker I, De Smedt SC, Dewitte H. Three decades of messenger RNA vaccine development. Nano Today. 2019; 28:100766.
  12. Alvarez-Benedicto E, Farbiak L, Marquez Ramirez M, Wang X, Johnson LT, Mian O, et al. Optimization of phospholipid chemistry for improved lipid nanoparticle (LNP) delivery of messenger RNA (mRNA). Biomater Sci. 2022;10(2):549–59.
  13. Hou X, Zaks T, Langer R, Dong Y. Lipid nanoparticles for mRNA delivery. Nat Rev Mater. 2021;6(12):1078–94.
  14. Kulkarni JA, Witzigmann D, Thomson SB, Chen S, Leavitt BR, Cullis PR, et al. The current landscape of nucleic acid therapeutics. Nat Nanotechnol. 2021;16(6):630–43.
  15. Schoenmaker L, Witzigmann D, Kulkarni JA, Verbeke R, Kersten G, Jiskoot W, et al. mRNA-lipid nanoparticle COVID-19 vaccines: structure and stability. Int J Pharm. 2021; 601:120586.
  16. Wadhwa A, Aljabbari A, Lokras A, Foged C, Thakur A. Opportunities and challenges in the delivery of mRNA-based vaccines. Pharmaceutics. 2020;12(2):102.
  17. Kowalski PS, Rudra A, Miao L, Anderson DG. Delivering the messenger: advances in technologies for therapeutic mRNA delivery. Mol Ther. 2019;27(4):710–28.
  18. Huang Y. Preclinical and clinical advances of GalNAc-decorated nucleic acid therapeutics. Mol Ther Nucleic Acids. 2022; 27:952–64.
  19. Li M, Zhao M, Fu Y, Li Y, Gong T, Zhang Z, et al. Enhanced intranasal delivery of mRNA vaccine by overcoming the nasal epithelial barrier via inhalable lipid nanoparticles. J Control Release. 2016; 228:9–19.
  20. Maruggi G, Zhang C, Li J, Ulmer JB, Yu D. mRNA as a transformative technology for vaccine development to control infectious diseases. Mol Ther. 2019;27(4):757–72.
  21. Pardi N, Hogan MJ, Naradikian MS, Parkhouse K, Cain DW, Jones L, et al. Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses. J Exp Med. 2018;215(6):1571–88.
  22. Laczkó D, Hogan MJ, Toulmin SA, Hicks P, Lederer K, Gaudette BT, et al. A single immunization with nucleoside-modified mRNA vaccines elicits strong cellular and humoral immune responses against SARS-CoV-2 in mice. Immunity. 2020;53(4):724–37.
  23. Jackson LA, Anderson EJ, Rouphael NG, Roberts PC, Makhene M, Coler RN, et al. An mRNA vaccine against SARS-CoV-2 — preliminary report. N Engl J Med. 2020;383(20):1920–31.
  24. Sahin U, Oehm P, Derhovanessian E, Jabulowsky RA, Vormehr M, Gold M, et al. An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma. Nature. 2020;585(7823):107–12.
  25. Rojas LA, Sethna Z, Soares KC, Ene CI, Gerdtsson E, Liao CY, et al. Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer. Nature. 2023;618(7963):144–50.
  26. Sahin U, Derhovanessian E, Miller M, Kloke BP, Simon P, Löwer M, et al. Personalised RNA mutanome vaccines mobilise poly-specific therapeutic immunity against cancer. Nature. 2017;547(7662):222–6.
  27. Hewitt SL, Bai A, Bailey D, Ichikawa K, Zielinski J, Kenber R, et al. Durable anticancer immunity from intratumoral administration of IL-23, IL-36γ, and OX40L mRNAs. Sci Transl Med. 2019;11(477):eaat9143.
  28. Miao L, Zhang Y, Huang L. mRNA vaccine for cancer immunotherapy. Mol Cancer. 2021;20(1):41.
  29. Zhang C, Maruggi G, Shan H, Li J. Advances in mRNA vaccines for infectious diseases. Front Immunol. 2019; 10:594.
  30. 30. Dong Y, Siegwart DJ, Anderson DG. Strategies, design, and chemistry in siRNA delivery systems. Adv Drug Deliv Rev. 2019; 144:133–47.
  31. Hajj KA, Whitehead KA. Tools for translation: non-viral materials for therapeutic mRNA delivery. Nat Rev Mater. 2017;2(10):17056.
  32. Miao L, Li L, Huang Y, Delcassian D, Chahal J, Han J, et al. Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation. Nat Biotechnol. 2019;37(10):1174–85.
  33. Reichmuth AM, Oberli MA, Jaklenec A, Langer R, Blankschtein D. mRNA vaccine delivery using lipid nanoparticles. Ther Deliv. 2016;7(5):319–34.
  34. Kauffman KJ, Dorkin JR, Yang JH, Heartlein MW, DeRosa F, Mir FF, et al. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with machine learning. Nano Lett. 2015;15(11):7300–6.
  35. Maier MA, Jayaraman M, Matsuda S, Liu J, Barros S, Querbes W, et al. Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics. Mol Ther. 2013;21(8):1570–8.
  36. Oberli MA, Reichmuth AM, Dorkin JR, Mitchell MJ, Fenton OS, Jaklenec A, et al. Lipid nanoparticle assisted mRNA delivery for potent cancer immunotherapy. Nano Lett. 2017;17(3):1326–35.
  37. Li B, Zhang X, Dong Y. Nanoscale platforms for messenger RNA delivery. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2019;11(2):e1530.
  38. Lokugamage MP, Vanover D, Beyersdorf J, Hatit MZC, Rotolo L, Echeverri ES, et al. Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs. Nat Biomed Eng. 2021;5(9):1059–68.
  39. Patel S, Ashwanikumar N, Robinson E, Xia Y, Mihai C, Griffith JP, et al. Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA. Nat Commun. 2020;11(1):983.
  40. Eygeris Y, Gupta M, Kim J, Sahay G. Chemistry of lipid nanoparticles for RNA delivery. Acc Chem Res. 2022;55(1):2–12.
  41. Fenton OS, Kauffman KJ, McClellan RL, Kaczmarek JC, Zeng MD, Andresen JL, et al. Synthesis and biological evaluation of ionizable lipid materials for the in vivo delivery of messenger RNA to B lymphocytes. Adv Mater. 2017;29(33):1606944.
  42. Hassett KJ, Benenato KE, Jacquinet E, Lee A, Woods A, Yuzhakov O, et al. Optimization of lipid nanoparticles for intramuscular administration of mRNA vaccines. Mol Ther Nucleic Acids. 2019; 15:1–11.
  43. Zeng C, Zhang C, Walker PG, Dong Y. Formulation and delivery technologies for mRNA vaccines. Curr Top Microbiol Immunol. 2022; 440:71–110.
  44. Ndeupen S, Qin Z, Jacobsen S, Bouteau A, Estanbouli H, Igyártó BZ. The mRNA-LNP platform's lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory. iScience. 2021;24(12):103479.
  45. Jain S, Venkataraman A, Wechsler ME, Peppas NA. Messenger RNA-based vaccines: past, present, and future directions in the context of the COVID-19 pandemic. Adv Drug Deliv Rev. 2021; 179:114000.
  46. Lutz J, Lazzaro S, Habbeddine M, Schmidt KE, Baumhof P, Mui BL, et al. Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines. NPJ Vaccines. 2017; 2:29.
  47. Karikó K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther. 2008;16(11):1833–40.
  48. Anderson BR, Muramatsu H, Nallagatla SR, Bevilacqua PC, Sansing LH, Weissman D, et al. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res. 2010;38(17):5884–92.
  49. Vaidyanathan S, Azizian KT, Haque AKMA, Henderson JM, Hendel A, Shore S, et al. Uridine depletion and chemical modification increase Cas9 mRNA activity and reduce immunogenicity without HPLC purification. Mol Ther Nucleic Acids. 2018; 12:530–42.
  50. Weissman D, Karikó K. mRNA: fulfilling the promise of gene therapy. Mol Ther. 2015;23(9):1416–7.
  51. Thess A, Grund S, Mui BL, Hope MJ, Baumhof P, Fotin-Mleczek M, et al. Sequence-engineered mRNA without chemical nucleoside modifications enables an effective protein therapy in large animals. Mol Ther. 2015;23(9):1456–64.
  52. Holtkamp S, Kreiter S, Selmi A, Simon P, Koslowski M, Huber C, et al. Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood. 2006;108(13):4009–17.
  53. Brito LA, Chan M, Shaw CA, Hekele A, Carsillo T, Schaefer M, et al. A cationic nanoemulsion for the delivery of next-generation RNA vaccines. Mol Ther. 2014;22(12):2118–29.
  54. Ulmer JB, Mason PW, Geall A, Mandl CW. RNA-based vaccines. Vaccine. 2012;30(30):4414–8.
  55. Geall AJ, Verma A, Otten GR, Shaw CA, Hekele A, Banerjee K, et al. Nonviral delivery of self-amplifying RNA vaccines. Proc Natl Acad Sci U S A. 2012;109(36):14604–9.
  56. Bloom K, van den Berg F, Arbuthnot P. Self-amplifying RNA vaccines for infectious diseases. Gene Ther. 2021;28(3–4):117–29.
  57. Vogel AB, Lambert L, Kinnear E, Busse D, Erbar S, Reuter KC, et al. Self-amplifying RNA vaccines give equivalent protection against influenza to mRNA vaccines but at much lower doses. Mol Ther. 2018;26(2):446–55.
  58. Hogan MJ, Pardi N. mRNA vaccines in the COVID-19 pandemic and beyond. Annu Rev Med. 2022; 73:17–39.
  59. Tenchov R, Bird R, Bhattacharjee AK, Zhou QA. Lipid nanoparticles — from liposomes to mRNA vaccine delivery, a landscape of research diversity and advancement. ACS Nano. 2021;15(11):16982–7015.
  60. Chen N, Xia P, Li S, Zhang T, Wang TT, Zhu J. RNA sensors of the innate immune system and their detection of pathogens. IUBMB Life. 2017;69(5):297–304.
  61. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature. 2001;413(6857):732–8.
  62. Pichlmair A, Schulz O, Tan CP, Näslund TI, Liljeström P, Weber F, et al. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates. Science. 2006;314(5801):997–1001.
  63. Teijaro JR, Farber DL. COVID-19 vaccines: modes of immune activation and future challenges. Nat Rev Immunol. 2021;21(4):195–7.
  64. Kedl RM, Rees WA, Hildeman DA, Schaefer B, Mitchell T, Kappler J, et al. T cells compete for access to antigen-bearing antigen-presenting cells. J Exp Med. 2000;192(8):1105–14.
  65. Cohn L, Chatterjee B, Esselborn F, Smed-Sörensen A, Nakamura N, Chalouni C, et al. Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation. J Exp Med. 2013;210(5):1049–63.
  66. Lindgren G, Ols S, Liang F, Thompson EA, Lin A, Hellgren F, et al. Induction of robust B cell responses after influenza mRNA vaccination is accompanied by circulating hemagglutinin-specific ICOS+ PD-1+ CXCR3+ T follicular helper cells. Front Immunol. 2017; 8:1539.
  67. Lederer K, Castaño D, Atria DG, Oguin TH, Wang S, Manzoni TB, et al. SARS-CoV-2 mRNA vaccines foster potent antigen-specific germinal center responses associated with neutralizing antibody generation. Immunity. 2020;53(6):1281–95.
  68. Widge AT, Rouphael NG, Jackson LA, Anderson EJ, Roberts PC, Makhene M, et al. Durability of responses after SARS-CoV-2 mRNA-1273 vaccination. N Engl J Med. 2021;384(1):80–2.
  69. Oberemok VV, Laikova KV, Yurchenko KA, Marochkin NA, Fomochkina II, Kubyshkin AV. SARS-CoV-2 will constantly sweep its tracks: a vaccine containing CpG motifs in 3'UTR will chase the virus even when mutated. Inflammation. 2020;43(6):2183–91.
  70. Nabel GJ. Designing tomorrow's vaccines. N Engl J Med. 2013;368(6):551–60.
  71. Schlake T, Thess A, Fotin-Mleczek M, Kallen KJ. Developing mRNA-vaccine technologies. RNA Biol. 2012;9(11):1319–30.
  72. Wilson E, Goswami J, Baqui AH, Doreski PA, Perez-Marc G, Zaman K, et al. Efficacy and safety of an mRNA-based RSV preF vaccine in older adults. N Engl J Med. 2023;389(24):2233–44.

Reference

  1. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, et al. Direct gene transfer into mouse muscle in vivo. Science. 1990;247(4949):1465–8.
  2. Martinon F, Krishnan S, Lenzen G, Magné R, Gomard E, Guillet JG, et al. Induction of virus-specific cytotoxic T lymphocytes in vivo by liposome-entrapped mRNA. Eur J Immunol. 1993;23(7):1719–22.
  3. Hoerr I, Obst R, Rammensee HG, Jung G. In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies. Eur J Immunol. 2000;30(1):1–7.
  4. Karikó K, Weissman D. Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development. Curr Opin Drug Discov Devel. 2007;10(5):523–32.
  5. Karikó K, Buckstein M, Ni H, Weissman D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity. 2005;23(2):165–75.
  6. Sahin U, Karikó K, Türeci Ö. mRNA-based therapeutics — developing a new class of drugs. Nat Rev Drug Discov. 2014;13(10):759–80.
  7. Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines — a new era in vaccinology. Nat Rev Drug Discov. 2018;17(4):261–79.
  8. Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A, Lockhart S, et al. Safety and efficacy of the BNT162b2 mRNA COVID-19 vaccine. N Engl J Med. 2020;383(27):2603–15.
  9. Baden LR, El Sahly HM, Essink B, Kotloff K, Frey S, Novak R, et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N Engl J Med. 2021;384(5):403–16.
  10. Corbett KS, Flynn B, Foulds KE, Francica JR, Boyoglu-Barnum S, Werner AP, et al. Evaluation of the mRNA-1273 vaccine against SARS-CoV-2 in nonhuman primates. N Engl J Med. 2020;383(16):1544–55.
  11. Verbeke R, Lentacker I, De Smedt SC, Dewitte H. Three decades of messenger RNA vaccine development. Nano Today. 2019; 28:100766.
  12. Alvarez-Benedicto E, Farbiak L, Marquez Ramirez M, Wang X, Johnson LT, Mian O, et al. Optimization of phospholipid chemistry for improved lipid nanoparticle (LNP) delivery of messenger RNA (mRNA). Biomater Sci. 2022;10(2):549–59.
  13. Hou X, Zaks T, Langer R, Dong Y. Lipid nanoparticles for mRNA delivery. Nat Rev Mater. 2021;6(12):1078–94.
  14. Kulkarni JA, Witzigmann D, Thomson SB, Chen S, Leavitt BR, Cullis PR, et al. The current landscape of nucleic acid therapeutics. Nat Nanotechnol. 2021;16(6):630–43.
  15. Schoenmaker L, Witzigmann D, Kulkarni JA, Verbeke R, Kersten G, Jiskoot W, et al. mRNA-lipid nanoparticle COVID-19 vaccines: structure and stability. Int J Pharm. 2021; 601:120586.
  16. Wadhwa A, Aljabbari A, Lokras A, Foged C, Thakur A. Opportunities and challenges in the delivery of mRNA-based vaccines. Pharmaceutics. 2020;12(2):102.
  17. Kowalski PS, Rudra A, Miao L, Anderson DG. Delivering the messenger: advances in technologies for therapeutic mRNA delivery. Mol Ther. 2019;27(4):710–28.
  18. Huang Y. Preclinical and clinical advances of GalNAc-decorated nucleic acid therapeutics. Mol Ther Nucleic Acids. 2022; 27:952–64.
  19. Li M, Zhao M, Fu Y, Li Y, Gong T, Zhang Z, et al. Enhanced intranasal delivery of mRNA vaccine by overcoming the nasal epithelial barrier via inhalable lipid nanoparticles. J Control Release. 2016; 228:9–19.
  20. Maruggi G, Zhang C, Li J, Ulmer JB, Yu D. mRNA as a transformative technology for vaccine development to control infectious diseases. Mol Ther. 2019;27(4):757–72.
  21. Pardi N, Hogan MJ, Naradikian MS, Parkhouse K, Cain DW, Jones L, et al. Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses. J Exp Med. 2018;215(6):1571–88.
  22. Laczkó D, Hogan MJ, Toulmin SA, Hicks P, Lederer K, Gaudette BT, et al. A single immunization with nucleoside-modified mRNA vaccines elicits strong cellular and humoral immune responses against SARS-CoV-2 in mice. Immunity. 2020;53(4):724–37.
  23. Jackson LA, Anderson EJ, Rouphael NG, Roberts PC, Makhene M, Coler RN, et al. An mRNA vaccine against SARS-CoV-2 — preliminary report. N Engl J Med. 2020;383(20):1920–31.
  24. Sahin U, Oehm P, Derhovanessian E, Jabulowsky RA, Vormehr M, Gold M, et al. An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma. Nature. 2020;585(7823):107–12.
  25. Rojas LA, Sethna Z, Soares KC, Ene CI, Gerdtsson E, Liao CY, et al. Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer. Nature. 2023;618(7963):144–50.
  26. Sahin U, Derhovanessian E, Miller M, Kloke BP, Simon P, Löwer M, et al. Personalised RNA mutanome vaccines mobilise poly-specific therapeutic immunity against cancer. Nature. 2017;547(7662):222–6.
  27. Hewitt SL, Bai A, Bailey D, Ichikawa K, Zielinski J, Kenber R, et al. Durable anticancer immunity from intratumoral administration of IL-23, IL-36γ, and OX40L mRNAs. Sci Transl Med. 2019;11(477):eaat9143.
  28. Miao L, Zhang Y, Huang L. mRNA vaccine for cancer immunotherapy. Mol Cancer. 2021;20(1):41.
  29. Zhang C, Maruggi G, Shan H, Li J. Advances in mRNA vaccines for infectious diseases. Front Immunol. 2019; 10:594.
  30. 30. Dong Y, Siegwart DJ, Anderson DG. Strategies, design, and chemistry in siRNA delivery systems. Adv Drug Deliv Rev. 2019; 144:133–47.
  31. Hajj KA, Whitehead KA. Tools for translation: non-viral materials for therapeutic mRNA delivery. Nat Rev Mater. 2017;2(10):17056.
  32. Miao L, Li L, Huang Y, Delcassian D, Chahal J, Han J, et al. Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation. Nat Biotechnol. 2019;37(10):1174–85.
  33. Reichmuth AM, Oberli MA, Jaklenec A, Langer R, Blankschtein D. mRNA vaccine delivery using lipid nanoparticles. Ther Deliv. 2016;7(5):319–34.
  34. Kauffman KJ, Dorkin JR, Yang JH, Heartlein MW, DeRosa F, Mir FF, et al. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with machine learning. Nano Lett. 2015;15(11):7300–6.
  35. Maier MA, Jayaraman M, Matsuda S, Liu J, Barros S, Querbes W, et al. Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics. Mol Ther. 2013;21(8):1570–8.
  36. Oberli MA, Reichmuth AM, Dorkin JR, Mitchell MJ, Fenton OS, Jaklenec A, et al. Lipid nanoparticle assisted mRNA delivery for potent cancer immunotherapy. Nano Lett. 2017;17(3):1326–35.
  37. Li B, Zhang X, Dong Y. Nanoscale platforms for messenger RNA delivery. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2019;11(2):e1530.
  38. Lokugamage MP, Vanover D, Beyersdorf J, Hatit MZC, Rotolo L, Echeverri ES, et al. Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs. Nat Biomed Eng. 2021;5(9):1059–68.
  39. Patel S, Ashwanikumar N, Robinson E, Xia Y, Mihai C, Griffith JP, et al. Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA. Nat Commun. 2020;11(1):983.
  40. Eygeris Y, Gupta M, Kim J, Sahay G. Chemistry of lipid nanoparticles for RNA delivery. Acc Chem Res. 2022;55(1):2–12.
  41. Fenton OS, Kauffman KJ, McClellan RL, Kaczmarek JC, Zeng MD, Andresen JL, et al. Synthesis and biological evaluation of ionizable lipid materials for the in vivo delivery of messenger RNA to B lymphocytes. Adv Mater. 2017;29(33):1606944.
  42. Hassett KJ, Benenato KE, Jacquinet E, Lee A, Woods A, Yuzhakov O, et al. Optimization of lipid nanoparticles for intramuscular administration of mRNA vaccines. Mol Ther Nucleic Acids. 2019; 15:1–11.
  43. Zeng C, Zhang C, Walker PG, Dong Y. Formulation and delivery technologies for mRNA vaccines. Curr Top Microbiol Immunol. 2022; 440:71–110.
  44. Ndeupen S, Qin Z, Jacobsen S, Bouteau A, Estanbouli H, Igyártó BZ. The mRNA-LNP platform's lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory. iScience. 2021;24(12):103479.
  45. Jain S, Venkataraman A, Wechsler ME, Peppas NA. Messenger RNA-based vaccines: past, present, and future directions in the context of the COVID-19 pandemic. Adv Drug Deliv Rev. 2021; 179:114000.
  46. Lutz J, Lazzaro S, Habbeddine M, Schmidt KE, Baumhof P, Mui BL, et al. Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines. NPJ Vaccines. 2017; 2:29.
  47. Karikó K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther. 2008;16(11):1833–40.
  48. Anderson BR, Muramatsu H, Nallagatla SR, Bevilacqua PC, Sansing LH, Weissman D, et al. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res. 2010;38(17):5884–92.
  49. Vaidyanathan S, Azizian KT, Haque AKMA, Henderson JM, Hendel A, Shore S, et al. Uridine depletion and chemical modification increase Cas9 mRNA activity and reduce immunogenicity without HPLC purification. Mol Ther Nucleic Acids. 2018; 12:530–42.
  50. Weissman D, Karikó K. mRNA: fulfilling the promise of gene therapy. Mol Ther. 2015;23(9):1416–7.
  51. Thess A, Grund S, Mui BL, Hope MJ, Baumhof P, Fotin-Mleczek M, et al. Sequence-engineered mRNA without chemical nucleoside modifications enables an effective protein therapy in large animals. Mol Ther. 2015;23(9):1456–64.
  52. Holtkamp S, Kreiter S, Selmi A, Simon P, Koslowski M, Huber C, et al. Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood. 2006;108(13):4009–17.
  53. Brito LA, Chan M, Shaw CA, Hekele A, Carsillo T, Schaefer M, et al. A cationic nanoemulsion for the delivery of next-generation RNA vaccines. Mol Ther. 2014;22(12):2118–29.
  54. Ulmer JB, Mason PW, Geall A, Mandl CW. RNA-based vaccines. Vaccine. 2012;30(30):4414–8.
  55. Geall AJ, Verma A, Otten GR, Shaw CA, Hekele A, Banerjee K, et al. Nonviral delivery of self-amplifying RNA vaccines. Proc Natl Acad Sci U S A. 2012;109(36):14604–9.
  56. Bloom K, van den Berg F, Arbuthnot P. Self-amplifying RNA vaccines for infectious diseases. Gene Ther. 2021;28(3–4):117–29.
  57. Vogel AB, Lambert L, Kinnear E, Busse D, Erbar S, Reuter KC, et al. Self-amplifying RNA vaccines give equivalent protection against influenza to mRNA vaccines but at much lower doses. Mol Ther. 2018;26(2):446–55.
  58. Hogan MJ, Pardi N. mRNA vaccines in the COVID-19 pandemic and beyond. Annu Rev Med. 2022; 73:17–39.
  59. Tenchov R, Bird R, Bhattacharjee AK, Zhou QA. Lipid nanoparticles — from liposomes to mRNA vaccine delivery, a landscape of research diversity and advancement. ACS Nano. 2021;15(11):16982–7015.
  60. Chen N, Xia P, Li S, Zhang T, Wang TT, Zhu J. RNA sensors of the innate immune system and their detection of pathogens. IUBMB Life. 2017;69(5):297–304.
  61. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature. 2001;413(6857):732–8.
  62. Pichlmair A, Schulz O, Tan CP, Näslund TI, Liljeström P, Weber F, et al. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates. Science. 2006;314(5801):997–1001.
  63. Teijaro JR, Farber DL. COVID-19 vaccines: modes of immune activation and future challenges. Nat Rev Immunol. 2021;21(4):195–7.
  64. Kedl RM, Rees WA, Hildeman DA, Schaefer B, Mitchell T, Kappler J, et al. T cells compete for access to antigen-bearing antigen-presenting cells. J Exp Med. 2000;192(8):1105–14.
  65. Cohn L, Chatterjee B, Esselborn F, Smed-Sörensen A, Nakamura N, Chalouni C, et al. Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation. J Exp Med. 2013;210(5):1049–63.
  66. Lindgren G, Ols S, Liang F, Thompson EA, Lin A, Hellgren F, et al. Induction of robust B cell responses after influenza mRNA vaccination is accompanied by circulating hemagglutinin-specific ICOS+ PD-1+ CXCR3+ T follicular helper cells. Front Immunol. 2017; 8:1539.
  67. Lederer K, Castaño D, Atria DG, Oguin TH, Wang S, Manzoni TB, et al. SARS-CoV-2 mRNA vaccines foster potent antigen-specific germinal center responses associated with neutralizing antibody generation. Immunity. 2020;53(6):1281–95.
  68. Widge AT, Rouphael NG, Jackson LA, Anderson EJ, Roberts PC, Makhene M, et al. Durability of responses after SARS-CoV-2 mRNA-1273 vaccination. N Engl J Med. 2021;384(1):80–2.
  69. Oberemok VV, Laikova KV, Yurchenko KA, Marochkin NA, Fomochkina II, Kubyshkin AV. SARS-CoV-2 will constantly sweep its tracks: a vaccine containing CpG motifs in 3'UTR will chase the virus even when mutated. Inflammation. 2020;43(6):2183–91.
  70. Nabel GJ. Designing tomorrow's vaccines. N Engl J Med. 2013;368(6):551–60.
  71. Schlake T, Thess A, Fotin-Mleczek M, Kallen KJ. Developing mRNA-vaccine technologies. RNA Biol. 2012;9(11):1319–30.
  72. Wilson E, Goswami J, Baqui AH, Doreski PA, Perez-Marc G, Zaman K, et al. Efficacy and safety of an mRNA-based RSV preF vaccine in older adults. N Engl J Med. 2023;389(24):2233–44.

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Bhoomika Gowda
Corresponding author

1MBBS Second Year Department of Microbiology, Virology, Immunology Fergana Medical Institute of Public health, Uzbekistan

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Shivprasad Dhage
Co-author

2Assistant Professor, Department of Microbiology, Virology and immunology, fergana Medical Institute of Public health, Uzbekistan.

Bhoomika Gowda*, Shivprasad Dhage, mRNA Vaccines: Molecular Architecture, Delivery Strategies, Immune Activation Mechanisms, and Clinical Frontiers, Int. J. of Pharm. Sci., 2026, Vol 4, Issue 5, 2731-2748. https://doi.org/10.5281/zenodo.20134544

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