Advancing mRNA vaccines for infectious diseases: key components, innovations, and clinical progress Open Access

Vaccination remains one of humanity’s most powerful tools in the fight against infectious diseases. According to the World Health Organization, vaccines prevent an estimated million of deaths annually [1]. However, traditional vaccine development encounters several challenges, including lengthy production timelines and the rapid mutation of viruses, particularly RNA viruses, which can render vaccines ineffective against emerging strains [2]. Additionally, safety concerns such as the risk of ‘virulence reversion’, where live attenuated vaccines might mutate back to a pathogenic form, further complicate conventional vaccine development (Table 1) [3].

In recent decades, mRNA technology has emerged as a ground-breaking platform for vaccine and therapeutic development. Since the discovery of mRNA in 1961, its potential has been progressively explored, with early demonstrations of in vitro transcription (IVT) and successful validation in mice occurring in 1990s [4,5]. However, the clinical application of mRNA faced substantial hurdles, including poor cellular uptake due to its negatively charged phosphate backbone, susceptibility to enzymatic degradation, and rapid systemic clearance [6,7]. These challenges underscored the crucial need for an efficient and safe delivery system to fully unlock mRNA’s therapeutic potential.

The development of lipid-based delivery systems has been a major milestone in overcoming these obstacles. Liposomes were first explored for nucleic acid delivery in the 1970s, but their short half-life, high sensitivity, and a complex preparation process limited their practical use [8-10]. A significant breakthrough came in the 2010s with the development of four-component lipid nanoparticles (LNPs), which revolutionized nucleic acid delivery [11]. By 2018, the FDA had approved the first LNP-based siRNA therapeutic, paving the way for further advancements. The emergence of the SARS-CoV-2 pandemic in 2020 accelerated research into LNP-mRNA vaccines [12]. Compared with traditional vaccines, mRNA vaccines offer substantial advantages, such as bypassing the need for cell culture and enabling rapid large-scale production, dramatically reducing development timelines.

Despite these advantages, several challenges remain. Issues such as ultra-low temperature storage requirements, potential allergic reactions to LNP components, and the rapid waning of neutralizing antibody titers are the highlight areas that need further improvement [13-16]. Additionally, expanding mRNA vaccine distribution to extrahepatic tissues is another key frontier.

This review aims to provide a comprehensive analysis of the key components of LNP-mRNA vaccines, including antigen-encoding RNA design, LNP composition, and strategies for optimization. Furthermore, it will explore the current landscape of clinical trials for LNP-mRNA vaccines targeting infectious diseases, lessons from failed trails, and advances in extrahepatic tissues delivery, contributing to the ongoing advancements in this rapidly evolving field.

LNP-mRNA vaccines possess the remarkable ability to activate both the innate and the adaptive immune systems. Upon administration, exogenous mRNA that successfully enters cells is rapidly recognized by various pattern recognition receptors (PRRs), triggering a cascade of innate immune responses. Among these PRRs, Toll-like receptors 7 and 8 (TLR7/8) play a crucial role in detecting single-stranded RNA [17]. Additionally, double-stranded mRNA fragments can be sensed by TLR3 in the endosome, as well as cytoplasmic receptors such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Activation of these diverse sensory pathways converges on shared downstream signaling nodes, culminating in the coordinated biosynthesis of type I interferons (primarily IFN-α/β) and pro-inflammatory cytokines. This inflammatory milieu functions as an ‘adjuvant-like’ environment, priming the immune system for subsequent adaptive responses.

Simultaneously, mRNA vaccines are internalized by local antigen-presenting cells (APCs), where they are translated into antigenic proteins. These proteins are then degraded into smaller peptides by the proteasome and presented on major histocompatibility complex class I (MHC-I) molecules to activate CD8⁺ cytotoxic T lymphocytes. In parallel, the antigenic peptides that enter the endosomal/lysosomal pathway are loaded onto MHC-II molecules for presentation to CD4⁺ helper T cells [18,19]. The expression of cytokines, such as type I interferons and co-stimulatory molecules, is crucial for APC-mediated T cell activation. Moreover, secreted antigenic proteins can bind to B cell receptors, triggering humoral immunity. CD4⁺ follicular helper T (Tfh) cells further assist B cells in producing high-affinity antibodies [20]. Research in animal models has demonstrated that LNPs induce high levels of IL-6 and other cytokines, which promote Tfh cells differentiation and germinal B cells formation, thereby accelerating the affinity maturation of antibodies. Moreover, type I interferon signaling enhances CD8⁺ T cells activation and proliferation, further strengthening immune protection [17,21].

In conclusion, mRNA vaccines effectively drive APCs maturation and lymphocytes activation. The innate immune system establishes an inflammatory environment rich in cytokines and interferons, providing a foundation for robust B and T cell responses. The coordinated activation of innate and adaptive immunity ultimately confers strong and durable protective immunity (Figure 1).

LNP-mRNA vaccines can be categorized based on RNA type. Currently, three major forms of RNA can be produced via IVT: conventional linear mRNA, self-amplifying RNA (saRNA), and circular RNA (circRNA) (Figure 2) [22].

Conventional mRNA, the most widely used type, consists of a 5´ cap, a 5´ untranslated region (UTR), an open reading frame (ORF) encoding the antigen, a 3´ UTR, and a poly (A) tail. While IVT technology enables rapid intracellular protein expression, conventional mRNA is highly susceptible to enzymatic degradation and requires high doses to achieve protective efficacy.

saRNA, derived from positive-sense RNA viruses, represents the next generation of RNA technology. By encoding a replicase while replacing viral structural genes with antigen sequences, saRNA can self-replicate within cells, significantly reducing the required vaccine dose and administration frequency [23]. This mitigates challenges such as the ‘PEG dilemma’ observed in repeated vaccinations [24]. Researchers have indicated that saRNA vaccines elicit robust immune responses at much lower doses than conventional mRNA vaccines. For instance, McKay et al. developed an saRNA-based SARS-CoV-2 vaccine that induced strong antibody titers, a Th1-biased immune response, and superior neutralization against pseudoviruses and wildtype strains, exceeding the antibody potency found in convalescent COVID-19 patients [25].

Additionally, saRNA generates immunostimulatory signals via dsRNA intermediates formed during replication, further enhancing the innate immune response [26-28]. Nevertheless, saRNA molecules are relatively large, typically exceeding 9 kb as they encompass both replicase and antigen sequences. This characteristic imposes higher performance requirements on delivery vectors. Their production process is more complex than conventional mRNA. Moreover, while saRNA-induced innate immunity can enhance immunogenicity, excessive activation may suppress antigen expression, requiring careful optimization [27].

circRNA, characterized by a closed-loop structure lacking a 5´ cap and 3´ poly (A) tail, exhibits enhanced nuclease resistance and prolonged in vivo stability [29]. Notably, circRNA vaccines remain at 4℃ for at least four weeks, whereas conventional linear mRNA-LNP vaccines degrade under similar conditions within three weeks [30]. Due to their closed structure, circRNAs evade innate immune recognition more effectively, reducing excessive inflammatory responses and potentially improving safety [31]. Besides, studies have demonstrated that engineered and optimized circRNA exhibits remarkable superiority in protein production. In vitro, the level of active protein generated by circRNA is substantially higher than that produced by an equivalent amount of linear mRNA. This advantage is observed not only in vitro but also in vivo, where circRNA shows both enhanced translational efficiency and prolonged protein expression [32].

Advancements in delivery systems have further expanded the potential of circRNA vaccines. For instance, a mannose-modified LNP vector has been developed to enhance lymph node targeting, leading to prolonged antigen availability and stronger Tfh and germinal center B cell responses. Mouse studies revealed that lyophilized mLNP-circRNA vaccines retained stability at 4℃ for up to 24 weeks without loss of immunogenicity [33]. These findings suggest that by enhancing delivery methods, such as targeted delivery and improved stability during lyophilization, circRNA vaccines can offer greater convenience for storage and transportation while preserving high efficacy. The concept of circRNA vaccines has been validated in multiple disease models. For example, Wei et al. developed a circRNA vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. In studies with macaque monkey experiments, this vaccine successfully elicited high-titer neutralizing antibodies and a significant T cell response, and it offered effective protection against both Delta- and Omicron-mutant strains [30]. Despite these promising advances, circRNA vaccines remain in the early stages of research, with key challenges such as large-scale production, in vivo metabolism, and long-term safety requiring further investigation [34].

Enhancing the stability, reducing immunogenicity, and improving protein expression of mRNA can be achieved through various optimization strategies. Key approaches include nucleoside modification, codon optimization, and structural refinement [35-37].

A major challenge of exogenous mRNA is its innate immunogenicity, which can trigger excessive inflammatory responses and interfere with antigen expression, potentially reducing vaccine efficacy and causing adverse effects [38]. Researchers have indicated that inappropriate or excessive activation of type I interferons, such as IFN-α, may disrupt antigen expression, reduce vaccine effectiveness, and potentially lead to severe adverse effects [39]. To mitigate this, various chemical modifications have been introduced to reduce immunogenicity while maintaining translation efficiency [40]. For instance, natural adenosine can be replaced with N¹-methyladenosine (m¹A) or N6-methyladenosine (m⁶A), and cytosine can be substituted with 5-methylcytosine (5-mC) [41,42]. Additionally, uridine can be replaced with 5-methoxyuridine (5moU), pseudouridine (Ψ), or N¹-methylpseudouridine (m¹Ψ) [43]. Notably, both 5-mC and Ψ have been shown to reduce mRNA immunogenicity and enhance protein translation in both in vivo and in vitro studies [44].

Codon usage can be tailored to host cell preferences to enhance translation efficiency and protein yield. Strategies include substituting rare codons with more prevalent ones (to prevent ribosomal stalling) and increasing GC content to improve stability. For instance, codon-optimized E7 genes have shown enhanced immunotherapeutic efficacy in HPV-16 vaccines [45]. COVID-19 . After purifiedmRNA vaccines developed by Moderna and Pfizer/BioNTech, codon optimization improved antigen expression and vaccine effectiveness [46]. Additionally, optimizing codon sequences can reduce unwanted activation by minimizing RIG-I-mediated innate immune activation [47].

The non-coding regions of mRNA, including the 5´ cap, 5´ UTR, 3´ UTR, and 3´ Poly (A) tail, play essential roles in regulating mRNA stability, translation, and cellular function.

5´ cap particularly the m7G cap structure (cap-0, cap-1, and cap-2) protects mRNA from exonuclease degradation and enhances translation efficiency while reducing immunogenicity [48].

UTRs influence translation efficiency, stability and localization. Using highly expressed natural UTRs, such as α- and β-globin, can prevent degradation and improve mRNA longevity [49].

Poly (A) tail, its length typically around 100 nt, is crucial for mRNA stability and expression efficiency. However, studies suggest that a 75 nt poly (A) tail may achieve optimal expression in human cells, indicating a context-dependent balance [50,51]. Novel designs, such as segmented poly (A) tail, further extend stability and translation efficiency [52].

Machine learning and deep learning algorithms are revolutionizing mRNA vaccine design by enabling precise sequence optimization.

Advanced models such as convolutional neural networks, residual neural networks, and graph neural networks can analyze large-scale sequence data to extract intricate features, including codon usage preferences and secondary structures. Leveraging these deep learning architectures, researchers can subsequently optimize mRNA stability and translation efficiency by fine-tuning its design based on the complex patterns identified [53]. For example, during COVID-19 vaccine development, researchers introduced the LinearDesign algorithm, which optimized mRNA stability and codon usage within 11 minutes, leading to a 128-fold increase in antibody titers in mice [54]. Additionally, machine learning models have been employed to fine-tune UTR design and stabilize ORF structures (CDSFold model) to enhance vaccine efficacy [55]. Epitope prediction is a cornerstone of mRNA vaccine design. As the specific regions on antigen proteins where antibodies bind, epitopes play a critical role in vaccine efficacy, making their precise identification essential. Commonly used prediction tools, such as DeepAb, BepiPred, and DiscoTope, employ deep learning algorithms to analyze complex datasets [56]. By integrating computational techniques with bioinformatics, these tools efficiently screen for candidate sequences with robust immunogenic potential, thereby significantly enhancing both the precision and efficiency of mRNA vaccine development.

LNPs are the most widely used delivery systems for mRNA vaccines, demonstrating efficient mRNA protection and intracellular delivery during the COVID-19 pandemic (Figure 3). Typically composed of ionizable lipids, cholesterol, helper lipids, and PEGylated lipids, LNPs enable rapid, scalable production with high biosafety. However, challenges such as hepatic accumulation, potential immunogenicity, and targeted delivery limitations remain. Addressing these issues is crucial for expanding LNP applications in RNA vaccines.

Ionizable lipids (ILs), constituting 30–50% of LNP composition, play a pivotal role in RNA encapsulation and endosomal escape. They typically contain a hydrophilic ionizable cationic headgroup, a biodegradable linker, and one or more hydrophobic tails [57]. Their pKa (typically 6–7) is optimized to facilitate RNA binding in acidic environments while remaining neutral at physiological pH, minimizing toxicity [58]. When LNPs are internalized by cells via endocytosis, the acidic environment of the endosomes encourages fusion with the endosomal membrane, ensuring efficient release of the mRNA cargos into the cytoplasm.

DODAP and DODMA are among the first ILs used for RNA delivery [59]. The first FDA-approved siRNA-LNP therapeutic, patisiran (Onpattro^®), utilized Dlin-MC3-DMA ILs [60]. This breakthrough led to further IL innovations, including ALC-0315 (Pfizer/BioNTech BNT162b2) and SM-102 (Moderna mRNA-1273) [61]. ILs such as Dlin-MC3-DMA and L-608 (Merck 32) have been evaluated in respiratory syncytial virus (RSV) vaccine trials [62]. And ATX-100 was used in Arcturus' COVID-19 saRNA vaccine ARCT-154 [63]. In addition, some ILs remain undisclosed, such as those in the ARCoV vaccine co-developed by Suzhou Abogen, Walvax and the Academy of Military Medical Sciences of China (Figure 4) [64].

Recent IL advancements include polycyclic adamantane-based ILs (e.g. 11-AM) and cyclic imidazole-modified ILs (e.g. 93-O17S), which show potential for T cell targeting [65]. Furthermore, A18-Iso5-2DC18, with a cyclic amine headgroup, exhibits strong STING activation, promoting dendritic cell maturation [66]. Notably, an LNP incorporating 4N4T (four tertiary amine nitrogen atoms and four hydrophobic tails) lipids elicited stronger Th1-based immune responses and higher RBD-specific IgG and neutralizing antibody titers than SM-102-based vaccines [67].

Cholesterol, comprising 20–50% of LNPs, enhances membrane stability, delivery efficiency, and circulatory half-life [68]. Natural cholesterol is commonly used in current mRNA vaccines, but alternatives like β-sitosterol, a natural analogue, may further boost transfection efficiency by influencing cellular uptake [69]. However, the long-term safety of β-sitosterol and the costs associated with large-scale production still require further evaluation.

Helper lipids, the most commonly used are phospholipids, typically 10–20% of LNPs, forming a bilayer membrane that stabilizes RNA encapsulation and promotes efficient delivery. Saturated phosphatidylcholine lipids, such as 1,2-distearoyl-sn-glycero-3-phosphocholine, are widely used in RNA vaccines. In addition to their critical role in membrane formation, altering the polarity of phospholipid has been shown to influence the organ-targeting characteristics of LNPs [70].

PEGylated lipids make up a small fraction of the LNPs, typically around 1.5%. Despite their low abundance, increasing the content of PEGylated lipids improves the size distribution and overall stability of LNPs. This enhancement provides a protective shield against protein adsorption in the bloodstream, which in turn prevents recognition and clearance by the reticuloendothelial system and the mononuclear phagocyte system, thereby extending the circulation half-life of LNPs [68,71]. Research indicates that the length of the carbon chain in these PEGylated lipids affects their desorption rate. Presently, commercially available RNA vaccines use PEGylated lipids with 14-carbon chains, such as the ALC-0159 and DMG-PEG₂₀₀₀.

However, the widespread use of PEG in consumer products has led to pre-existing anti-PEG antibodies (APAs) in the population, which may accelerate LNP clearance (accelerated blood clearance, ABC), potentially reducing vaccine efficacy [72]. Repeated administration might further compromise vaccine effectiveness. Notably, between December 14 and 23, 2020, clinical data reported in the Vaccine Adverse Event Reporting System identified 21 cases of anaphylaxis in 1.89 million Pfizer/BioNTech vaccines. These reactions are most likely associated with sensitivity to PEG [73].

To address the challenges associated with PEG dilemma, various strategies have been explored, including enhancing mRNA expression efficiency to reduce dose requirements, replacing mRNA with saRNA that requires lower doses, and substituting PEGylated lipids with alternative materials. For example, Dong et al. demonstrated that replacing PEGylated lipids with pSar lipids improved mRNA delivery while maintaining safety [61]. Optimizing PEG terminal groups has also emerged as a promising strategy. Methoxy-PEGylation (MeO-PEG) is widely applied in numerous marketed nanomedicines, including MeO-PEG-DMG and ALC-0159 [74,75]. Zhan et al. discovered that hydroxyl-PEGylation (OH-PEG) at the PEG terminus effectively prevents binding by pre-existing APAs in humans [76]. Another innovative approach is substituting PEG with brush-shaped polymer lipids (BPLs), which exhibit lower APA affinity, prolonged circulation, and enhanced mRNA delivery. Xiao et al. developed high-density BPLs, demonstrating superior performance over conventional DMG-PEG2000 LNPs, particularly in repeated-dose studies [77]. These advancements provide new strategies to mitigate PEG-related limitations, paving the way for safer and more effective LNP-mRNA vaccines.

The recent achievements of mRNA vaccines, as seen in bnt162b2 (Pfizer-Biontech) and mRNA-1273 (Moderna), have sparked interest in their application to various infectious diseases like influenza, RSV, Zika virus, HIV, and more (Table 2). With their swift design, scalable production, and targeted immunogenicity, mRNA vaccines hold promise in addressing persistent challenges in vaccine development, presenting effective solutions for both emerging and re-emerging infectious diseases.

mRNA-based COVID-19 vaccines represent the most extensively explored mRNA vaccine platform to date. Comirnaty^® (BNT162b2) was the first mRNA vaccine authorized for emergency use by the FDA on December 11, 2020, and by the EMA on December 21, 2020 [78]. Shortly thereafter, Moderna’s mRNA-1273 vaccine also received approval. Both vaccines encode the full-length spike protein of SARS-CoV-2 and demonstrated 95.0% and 94.1% efficacy, respectively, in Phase III clinical trials [79]. To combat viral mutations, new vaccine formulations continue to be developed. For example, CSPC Pharmaceutical Group Ltd. developed SYS6006, an mRNA-based vaccine encoding the spike protein sequence with key mutations from Delta, Omicron BA.4, BA.5, and BF.7 strains, which received emergency use authorization (EUA) in China [80-82]. Moderna’s mRNA-1283, which encodes only the RBD and N-terminal domain of the spike protein, exhibited enhanced immune responses against the Omicron variant BA.4/BA.5 and the original virus strains compared to mRNA -1273.222 in Phase III clinical trials [83]. Additionally, mRNA-1283 exhibits improved stability at standard refrigeration temperatures (2–8℃), simplifying storage and distribution [84].

Combination vaccines represent a promising next-generation strategy. Moderna’s mRNA-1083, which integrates the influenza vaccine mRNA-1010 with the COVID-19 vaccine mRNA-1283, is currently in Phase III trials after demonstrating comparable or superior efficacy to licensed quadrivalent influenza vaccines and mRNA-1273 in earlier trials [85,86]. Beyond conventional mRNA vaccines, saRNA vaccines are under investigation. Arcturus Therapeutics' ARCT-154, an saRNA-based COVID-19 vaccine, demonstrated broader efficacy, longer-lasting immune protection, and reduced antigen dose requirements compared with BNT162b2, marking the first published clinical evidence of saRNA vaccine efficacy [87,88].

Clinical trials have also assessed the safety and immunogenicity of COVID-19 mRNA vaccines in special populations. Studies indicate that these vaccines elicited strong immune responses with acceptable tolerability in both elderly and pediatric populations [89-91]. Moreover, a six-month follow-up study of individuals aged 12 and older who received two doses of BNT162b2 reported an efficacy rate exceeding 86% and a favorable long-term safety profile [92]. These findings provide crucial support for broad vaccine deployment in diverse age groups.

The segmented RNA genome of influenza viruses allows for the emergence of multiple subtypes and frequent antigenic drift, posing challenges for vaccine development. Currently, at least 18 distinct influenza A virus subtypes are circulating globally [93]. Multivalent vaccines encoding antigens for multiple subtypes offer a promising solution, which is difficult with conventional vaccines technology but feasible with RNA-based vaccine platforms.

Moderna’s mRNA-1010, a quadrivalent seasonal influenza vaccine encoding the full-length hemagglutinins (HA) of four influenza strains (A/H1N1, A/H3N2, B/Victoria, and B/Yamagata). In Phase I/II clinical trials (NCT04956575), single doses of mRNA-1010 induced hemagglutination inhibition (HAI) titers higher than those from standard inactivated vaccines against influenza A strains, while producing comparable HAI titers for influenza B strains [94,95]. However, the results of Phase III clinical trial were less favorable. mRNA-1010 was associated with higher adverse event rates compared with traditional vaccines, similar immunogenicity against influenza A, and inferior immunogenicity against influenza B [96]. These findings highlight the need for further optimization of mRNA vaccine platforms or antigen design to enhance efficacy and safety. Beyond conventional mRNA vaccines, Pfizer has developed saRNA-based influenza vaccine candidates. A Phase I clinical trial has been completed, though results have not yet been disclosed (NCT05227001, data from https://clinicaltrials.gov).

RSV is a single-stranded, negative-sense RNA virus and the leading cause of bronchitis and pneumonia in infants, young children, the elderly, and immunocompromised individuals. On May 31, 2024, the FDA approved Moderna’s mRNA-1345, encoding a stabilized prefusion F glycoprotein, marking the first RSV mRNA vaccine for older adults (≥60 years) [97], Sanofi’ SP0256, an RSV mRNA vaccine for older adults, showed promising Phase I/II clinical trials results and is currently in a Phase IIb trial in combination with human metapneumovirus (hMPV) (NCT06134648, NCT06237296). Sanofi also plans to develop a trivalent mRNA vaccine targeting RSV, hMPV, and parainfluenza virus [98].

While RSV mRNA vaccines have demonstrated preventive efficacy, safety concerns remain. The most common adverse reactions (ARs) include injection site pain, fatigue and myalgia (systemic), which are generally mild to moderate and transient [99]. However, in July 2024, the FDA placed an emergency hold on the Phase I clinical trial of mRNA-1365-P101 due to a potential safety signal [100]. Specifically, an imbalance in severe lower respiratory tract infections caused by RSV was observed, with a higher incidence in the vaccine group than in controls. The underlying cause remains unclear, highlighting the need for continued safety evaluations as mRNA vaccines become more widely used. To date, no broad-spectrum universal vaccine has been successfully developed for all age groups.

Developing an HIV vaccine has posed a longstanding challenge. Despite decades of research, numerous HIV vaccine candidates have failed in Phase III trials, and no vaccine has been approved for human use [101]. Moderna is currently conducting Phase I clinical studies to evaluate the safety and immunogenicity of the eOD-GT8 60mer mRNA vaccine (mRNA-1644) and the Core-g28v2 60mer mRNA vaccine (mRNA-1644v2-Core) in HIV-1 uninfected adults (NCT05001373). This trial aims to assess the ability of mRNA-based vaccines to elicit specific B-cell responses and drive their early maturation toward generating broadly neutralizing antibodies (bnAbs) [102]. Additionally, Moderna has launched another Phase I trial for the mRNA-1574 vaccine candidate, which is designed to encode native-like HIV trimers. The main endpoints of this trial are to evaluate the safety profile and immunogenicity of this experimental vaccine, with a specific focus on immune responses targeting HIV trimers (NCT05217641) [103]. As of now, results from the above trials have not been disclosed.

mRNA vaccines are also being explored for various other viral infections. Moderna developed mRNA-1325 (NCT03014089) and mRNA-1893 (NCT04064905), both encoding the Zika premembrane (prM) and envelope (E) proteins. Phase I clinical trials demonstrated that mRNA-1893 elicited stronger serum-neutralizing antibody responses and better tolerability than mRNA-1325 after two doses [104,105]. For cytomegalovirus (CMV), Moderna’s Mrna-1647, comprising six mRNA sequences encoding the CMV pentamer complex and glycoprotein B (gB) antigens, demonstrated potent and durable humoral responses in Phase I/II trials, with superior neutralizing activity and antibody-dependent cellular cytotoxicity compared to the traditional gB/MF59 vaccine [106,107]. Currently, this vaccine is undergoing Phase III trials. In malaria research, BioNTech’s BNT165b1, an mRNA vaccine targeting Plasmodium falciparum circumsporozoite protein (PfCSP), entered Phase I clinical trials in 2022 (NCT05581641), representing a step toward effective malaria vaccines [108].

Despite significant progress, several mRNA vaccine projects have faced challenges, underscoring the need for continuous optimization in vaccine design. For example, the early Zika virus mRNA vaccine (mRNA-1325) revealed that single amino acid differences disrupted virus-like particle formation, reducing immunogenicity and limiting efficacy [105]. Similarly, CureVac’s COVID-19 vaccine (CVnCoV) exhibited only 47% efficacy in Phase III trials, largely due to its lack of nucleoside modifications, which heightened innate immune activation and diminished potency [109]. In contrast, vaccines from Pfizer-BioNTech and Moderna, incorporating modified nucleosides, demonstrated significantly higher efficacy. Additionally, CureVac’s rabies vaccine (CV7201), which used protamine as a delivery system, showed route-dependent efficacy and caused adverse reactions in 78% of Phase I participants [110]. Development shifted to CV7202, which employed LNPs for improved delivery efficiency and reduced reactogenicity [111]. As viral variants continue to emerge, single-epitope vaccines may lack broad-spectrum protection. Future designs should consider multivalent strategies and optimize mRNA sequences, delivery systems, and adaptability to evolving pathogens to enhance vaccine efficacy and safety.

mRNA-LNP vaccines, such as mRNA-1273 and BNT162b2, require frozen storage at −20℃ and −60℃ to −80℃, respectively, posing logistical challenges in resource-limited regions [112]. Lyophilization (freeze-drying) offers a promising solution by enhancing stability and shelf-life, potentially enabling ambient temperature storage [113]. However, the process must preserve key characteristics, such as particle size, polydispersity index, encapsulation efficiency, mRNA integrity, and immunogenicity [114]. Recent studies have demonstrated the feasibility of lyophilizing mRNA-LNP formulations. For example, Muramatsu et al. showed that mRNA-LNPs formulated in 5 mM Tris buffer (pH 8) with 10% sucrose and 10% maltose maintained their stability at room temperature for 12 weeks and at 4℃ for 24 weeks [115]. Similarly, Ai et al. described a lyophilized Omicron mRNA vaccine that remained stable at 25℃ for over six months, retaining robust humoral and cellular immune responses [116]. Moderna’s CMV vaccine (mRNA-1647) is undergoing Phase III clinical trials in healthy participants aged 16 to 40 years (NCT05085366) [117]. This lyophilized formulation has demonstrated stability at 5℃ for up to 18 months. Beyond lyophilization, alternative drying techniques, such as spray drying, spray freeze drying, thin-film freeze-drying, and supercritical spray drying, are being explored to optimize stability and vaccine performance [118]. Further research is needed to refine these approaches for large-scale implementation.

LNP-mRNA systems exhibit an inherent liver affinity, mainly due to ApoE-mediated uptake by hepatocytes, especially Kupffer cells and hepatic sinusoidal endothelial cells, making targeted delivery to extrahepatic tissues a significant challenge [119,120].

Several strategies are being explored to enhance selective organ targeting (SORT) [121]. Lipid modifications, LNPs synthesized with 5A2-SC8 lipids, combined with the addition of the cationic lipid DOTAP, can reduce ApoE binding and enhance targeting to other tissues, such as the lungs and spleen [122]. Adjusting the surface charge of LNPs is another effective approach to reduce liver uptake [123]. Moreover, surface modification of LNPs, such as coupling targeted molecules (like antibodies, peptides, or polysaccharides) to the LNP surface, represents another promising method for improving extrahepatic tissues delivery efficiency [124]. The choice of administration route also plays a crucial role in delivery efficiency, with nasal and intradermal administration enabling direct access to certain tissues. Furthermore, studies indicate that LNP size influences biodistribution, with nanoparticles in the 20–200 nm range demonstrating enhanced uptake by dendritic cells (DCs), thereby improving immune responses [120].

While extrahepatic mRNA delivery remains an area of active research, substantial progress has been made in targeting the lungs, tumors, and central nervous system through lipid chemistry innovations and ligand-based modifications. Future advancements must focus on refining targeting accuracy, ensuring safety, and optimizing large-scale production to expand the clinical applicability of mRNA vaccines.

LNP-mRNA vaccines represent a distinct advancement over traditional vaccines, with the successful application of BNT162b2 and mRNA-1273 during COVID-19 pandemic, highlighting the unprecedented adaptability and efficacy of mRNA vaccine technology in addressing infectious diseases. A comprehensive understanding of mRNA vaccines composition and optimization strategies is essential for further advancing this transformative technology. Optimizing RNA stability and translation efficiency has been a key focus, involving modifications such as alternative RNA types, nucleotide chemical alterations, codon optimization, and refined non-coding regions. Simultaneously, advancements in LNP formulations have enhanced cellular uptake, reduced off-target effects, and improved biodistribution. However, several challenges remain that must be addressed to realize the full potential of mRNA vaccines.

The intrinsic instability of mRNA necessitates a strict cold chain, limiting accessibility in resource-limited regions where ultra-low temperature storage is often impractical. The development of lyophilized mRNA-LNPs, which enable storage at 4℃ for up to six months, represents a major breakthrough. However, the impact of lyophilization buffer composition, processing conditions, and storage parameters on LNP integrity requires further investigation. Another major challenge is LNP liver accumulation, largely due to ApoE-mediated uptake. To overcome this, extensive research has focused on organ-specific LNP technologies, including high-throughput lipid screening, formulation optimization, and antibody-modified delivery systems. A particularly promising approach is SORT technology, which incorporates charged lipids (SORT lipids) into conventional LNP formulations, enabling targeted mRNA delivery to extrahepatic tissues.

Beyond technological challenges, ensuring equitable access to mRNA vaccines remains a critical global priority. The WHO’s fair allocation mechanism for COVID-19 vaccines through COVAX highlights the need to reduce distribution disparities and ensure mRNA vaccines reach low- and middle-income countries (LMICs). To achieve this, optimizing cold chain logistics, improving vaccine thermostability, and reducing production costs are essential. Strengthening local mRNA manufacturing capabilities in LMICs could also enhance regional vaccine security and pandemic preparedness, as evidenced by WHO-led initiatives promoting technology transfer hubs for mRNA vaccine production.

Furthermore, concerns regarding immunogenicity and long-term safety demand continued investigation. While recent clinical trials have shown promising immune responses and safety profiles, variations in vaccine efficacy across different populations emphasize the need for further validation. Additionally, the precise in vivo immune mechanisms of mRNA vaccines remain incompletely understood, necessitating deeper exploration to optimize immune response durability and broad applicability across diverse LNP formulations.

Addressing these scientific, logistical, and ethical challenges is crucial for expanding mRNA vaccine accessibility worldwide. Continued technological innovation, coupled with global cooperation in vaccine allocation and infrastructure development, will be instrumental in realizing the full potential of mRNA vaccines in combating infectious diseases on a global scale.

The authors declare that there are no competing interests associated with the manuscript.

Sha Li, Lu Zheng, Jingyi Zhong: Concepualization; Literature search; Data curation; Writing-original draft.

Sha Li: Conceptualization; Visualization; Writing-original draft; Writing-review & editing. Xihui Gao (Corresponding author): Supervision; Writing-review & editing.

This work was financially supported by the Shanghai Municipal Science and Technology Major Project [ZD2021CY001].

IVT

in vitro transcription

LNPs

lipid nanoparticles

SORT

selective organ targeting

TLRs

Toll-like receptors

sa-RNA

self-amplifying RNA