Respiratory virus mRNA vaccines: mRNA Design, clinical studies, and future challenges

Abstract

Respiratory infectious diseases frequently erupt on a global scale, with RNA viruses, such as SARS‐CoV‐2, RSV, and influenza viruses, posing challenges to vaccine development due to their high mutation rates. Traditional vaccine development cycles are lengthy and struggle to keep pace with rapidly evolving viruses, whereas messenger RNA (mRNA) vaccines have demonstrated significant advantages due to their short development periods, straightforward production, and low costs. After the outbreak of the COVID‐19 pandemic, multiple mRNA vaccines, including Pfizer‐BioNTech and Moderna, rapidly received emergency use authorization, validating their feasibility. The Nobel Prize in Physiology or Medicine in 2023 was awarded to Katalin Karikó and Drew Weissman, underscoring the efficacy of mRNA vaccine technology. In 2024, the U.S. Food and Drug Administration (FDA) approval of Moderna's respiratory syncytial virus (RSV) mRNA vaccine marked the immense potential of mRNA technology in vaccine innovation. This review article summarizes the design, clinical research, and future challenges of mRNA vaccines for respiratory viruses, delving into antigen design, mRNA delivery systems, and advancements in vaccines for multiple respiratory viruses, including innovations in self‐amplifying mRNA and circular mRNA vaccines. Additionally, the development of combination vaccines is underway, aiming to provide protection against multiple viruses through a single administration. Despite the significant progress in mRNA vaccine development, challenges remain regarding raw material costs, stability, and delivery efficiency. In the future, with technological advancements and the accumulation of clinical experience, the design strategies and delivery systems of mRNA vaccines are expected to be continuously optimized, thereby enhancing their safety and efficacy.

 

1. INTRODUCTION

Respiratory infectious diseases have repeatedly surged globally in recent years, posing severe risks to human health, with viral infections accounting for a significant proportion. Among respiratory viruses, most, including coronaviruses, respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, and rhinoviruses, are single‐stranded RNA viruses prone to mutation, with only a few, such as adenoviruses and bocaviruses, classified as DNA viruses. The mutability of RNA viruses continues to complicate the development of neutralizing antibodies and vaccines. For instance, the SARS‐CoV‐2 Omicron variant exhibits extensive mutations, including 36 mutations located on the spike protein, rendering neutralizing antibodies targeting SARS‐CoV‐2 less effective or entirely ineffective against Omicron. ¹ , ² , ³ , ⁴ , ⁵ , ⁶ Consequently, vaccine designs for respiratory viruses require continual updates to address emerging variants. Traditional vaccine development and regulatory timelines are often too protracted to meet the demands posed by rapidly evolving acute viral pathogens. In contrast, messenger RNA (mRNA) vaccines offer significant advantages for emergency vaccine development against respiratory viruses, given their shorter development cycles, simpler production processes, lower costs, and rapid scalability. ⁷

Following the onset of the COVID‐19 pandemic, Pfizer (US) and BioNTech (Germany) jointly developed the BNT162 vaccine, ⁸ while Moderna developed mRNA‐1273 ⁹ , ¹⁰ ; both vaccines received emergency use authorization across multiple nations and were administered globally, demonstrating the feasibility of mRNA vaccines. This pivotal advancement has ushered in a new era of vaccine development. In October 2023, the Nobel Prize in Physiology or Medicine was awarded to Katalin Karikó and Drew Weissman for their foundational discoveries in nucleoside base modification, which enabled the development of effective COVID‐19 mRNA vaccines. This prestigious recognition underscores the validated utility of mRNA vaccine technology in the COVID‐19 pandemic. On May 31, 2024, Moderna announced that its RSV mRNA vaccine, mRESVIA, received U.S. Food and Drug Administration's (FDA) approval, ¹¹ , ¹² marking a milestone that highlights the expansive future potential of mRNA technology in vaccine innovation. Studies have demonstrated that booster doses significantly enhance neutralizing antibody levels and broaden immune responses against variants, thereby playing a critical role in sustaining immunity, especially against rapidly mutating viruses such as SARS‐CoV‐2. ¹³ In the coming years, additional mRNA vaccines targeting respiratory viruses are anticipated to enter the market, offering new preventive and therapeutic solutions for global public health.

This article systematically reviews the current status and advancements of mRNA vaccines used clinically and those undergoing clinical trials. It provides a comprehensive synthesis from multiple perspectives, including antigen design, mRNA delivery, and administration strategies, as well as the clinical progress of various respiratory virus vaccines.

2. ANTIGEN DESIGN

The selection of target antigens, optimization of sequence design, and the delivery system are critical determinants of the core competitiveness of mRNA vaccines. ⁸ , ¹⁴ As a key component recognized by the immune system to induce a specific immune response, antigen is pivotal in the development of respiratory virus vaccine. Choosing the appropriate viral protein as the antigen to elicit a robust immune response is a crucial first step. Sequence optimization can further enhance the mRNA's expression levels, prolong its retention time, and reduce immunogenicity. Meanwhile, the delivery system acts as an efficient vehicle to protect and transport the active components to the target sites for timely release; few companies possess this technology, which is patent‐protected and represents a “chokepoint” in mRNA vaccine technology.

2.1. Selection of key antigens

The first step in vaccine design is the selection of core viral antigens. For instance, in developing the RSV mRNA vaccine, Moderna compared several antigenic forms, including the wild‐type full‐length RSV F protein derived from the RSV A2 strain (mF), the truncated secreted trimeric form of RSV F (sF), the pre‐fusion stabilized F protein (DS‐Cav1), the monomeric pre‐fusion single‐chain RSV F form (RSV F5), and other reported full‐length RSV F proteins (RSV F7) and pre‐fusion stabilized RSV F (RSV F8). Immunogenicity testing revealed that the pre‐fusion configuration (DS‐Cav1) was most effective in inducing a potent neutralizing antibody response and generating a strong neutralizing immune response. ¹⁵

In influenza vaccines, the hemagglutinin (HA) and neuraminidase (NA) proteins are commonly targeted due to their critical roles in viral entry and replication. ¹⁶ The high mutation rate of the influenza virus presents major challenges for vaccine development. Key issues include antigenic drift, where frequent mutations in the HA and NA proteins diminish vaccine efficacy ¹⁷ ; the need for broad protection, which requires multivalent vaccines targeting conserved viral regions to ensure widespread immunity; and the demand for rapid adaptation met by mRNA platforms that enable swift updates to match circulating strains, thus effectively addressing the virus's rapid mutations. ¹⁸

Future research should focus on identifying conserved antigens across multiple respiratory viruses to develop broad‐spectrum vaccines that offer protection against a wider range of pathogens.

2.2. Optimization of mRNA sequence

To accurately mimic the biological function of natural mRNA transcripts in protein translation, an mRNA sequence comprises five critical structural elements: the 5′ cap, 5′ untranslated region (UTR), coding sequence (CDS), 3’ UTR, and poly(A) tail, all of which contribute to antigen expression and structural stability. mRNA sequences undergo multiple strategies to enhance antigen immunogenicity, including codon optimization, UTR refinement, AI‐assisted structural design, and the incorporation of immunoregulatory elements.

Nucleoside base modification, for instance, replacing uridine (U) with pseudouridine (Ψ) or other modifications, enhances mRNA stability and translation efficiency. ¹⁹ Codon optimization is a key step aimed at improving the stability and translational efficiency of the mRNA sequence to maximize vaccine efficacy. Codon adaptation is also adjusted to align with the host cell's codon usage, achieving a codon adaptation index (CAI) that promotes optimal expression in host cells. In the approved RSV mRNA vaccine, mRNA‐1345, the coding sequence includes four mutations (as in DS‐Cav1): S155C, S290C, S190F, and V207L, with S155C and S290C mutations forming disulfide bonds, whereas S190F and V207L mutations function as cavity‐filling alterations. Furthermore, the pre‐F protein encoded by mRNA‐1345 contains two additional mutations (A149C and Y458C) in the F1 domain, and the 104–144 amino acid residues are replaced by a GS linker, with the cytoplasmic tail deleted.

Distinct 5’ UTR sequences may be required for different mRNA sequences and target cells. ²⁰ Notably, the 5’ UTR of mRNA‐1345 is shorter than that of the COVID‐19 mRNA vaccine mRNA‐1273, lacking a GC‐rich segment, whereas the 3’ UTR sequences of both vaccines are identical, differing by only two nucleotides. The KOZAK sequence and Poly(A) tail sequence remain consistent, and both terminate with a tandem arrangement of three distinct stop codons. AI‐assisted design enables more efficient mRNA sequence optimization by predicting and refining RNA and protein structures, thereby accelerating drug and vaccine development. ⁷

2.3. Self‐amplifying mRNA and circular mRNA vaccines

Self‐amplifying RNA (saRNA) differs from conventional mRNA in its capacity for self‐replication using its own RNA sequence as a template. Typically, mRNA encodes the target protein, which is translated by ribosomes in the cell. In contrast, saRNA not only expresses the target protein but also includes sequences that code for RNA‐dependent RNA polymerase (RdRP). ²¹ , ²² This polymerase enables replication of the saRNA, thereby reducing the required dose and lowering production costs. Therapeutically, this reduces the injection dose and frequency needed in mRNA treatments, extends therapeutic efficacy, and minimizes potential side effects associated with the mRNA and delivery vector. In November 2023, Japan's Ministry of Health, Labour and Welfare approved ARCT‐154, a saRNA COVID‐19 vaccine developed jointly by Arcturus Therapeutics and CSL. This marked the first nonemergency clinical application of saRNA technology. Compared to the 100‐μg dose of Moderna's mRNA vaccine and the 30‐μg dose of BioNTech/Pfizer's mRNA vaccine, ARCT‐154 requires only a 5‐μg dose. Clinical trials have shown that ARCT‐154, as a booster in individuals who received three doses of an mRNA COVID‐19 vaccine, elicited an immune response against SARS‐CoV‐2 Wuhan‐Hu‐1 comparable to that of BNT162b2. Furthermore, in neutralization assays against the Omicron BA.4/5 variant, ARCT‐154 outperformed BNT162b2. Both vaccines demonstrated strong tolerability and safety profiles in the study population. ²³

Circular mRNA (circRNA) vaccines have recently emerged as an innovative approach in vaccine development. In July 2018, Daniel Anderson and colleagues at MIT demonstrated that engineered circRNA could stably and efficiently express proteins in eukaryotic cells, establishing a new application for exogenous circRNA in protein expression. This finding highlighted circRNA as an effective alternative to linear mRNA. Compared to conventional linear mRNA vaccines, circRNA's unique circular structure eliminates the need for a costly 5′ cap and extensive 3′ tail, conferring potential advantages such as increased stability and enhanced immunogenicity. ²⁴ CircRNA's stability under ambient conditions suggests that it could be used in vaccines that can be stored without a cold chain. Currently, most circRNA vaccines remain in experimental and early‐stage clinical trials, as researchers continue to assess their safety and immunogenicity.

2.4. Combination vaccines

Combination vaccines integrate two or more antigen components into a single vaccine, enabling immunoprotection against multiple respiratory viruses through a single administration. This approach streamlines vaccination schedules, improves vaccination efficiency, and reduces the incidence of respiratory diseases. Such vaccines commonly include antigens targeting prevalent respiratory viruses such as influenza, RSV, rhinovirus, and parainfluenza virus.

In June 2024, Moderna's investigational combination vaccine, mRNA‐1083, targeting both COVID‐19 and influenza, reached its primary endpoints in a Phase III trial. In adults aged 50–64 and those 65 and older, mRNA‐1083 induced a more robust immune response against both influenza and COVID‐19 compared to existing single‐antigen vaccines. The vaccine is expected to undergo regulatory review by the end of 2024, with plans to accelerate its market availability under priority review status. ²⁵ , ²⁶

Meanwhile, researchers at the Perelman School of Medicine, University of Pennsylvania, developed an experimental multivalent mRNA vaccine targeting all 20 known subtypes of influenza viruses, covering both influenza A and B strains. This multivalent mRNA vaccine demonstrated broad protective efficacy in mice by inducing antibodies against various influenza subtypes. It leverages the same mRNA platform as Pfizer's and Moderna's SARS‐CoV‐2 vaccines. ¹⁶ Testing in animal models showed that the vaccine alleviated symptoms of influenza and protected animals from death, even when exposed to influenza strains differing from those used in vaccine formulation.

3. DELIVERY SYSTEMS AND ADMINISTRATION ROUTES FOR MRNA VACCINES

3.1. Delivery systems

The considerable molecular weight of mRNA (10⁴–10⁶ Da) and its inherent negative charge impede its ability to traverse the anionic lipid bilayer of cellular membranes. Furthermore, mRNA is prone to degradation and recognition by the immune system in vivo, underscoring the necessity for a robust delivery system that not only protects mRNA but also facilitates its entry into target cells. Commonly employed delivery systems include lipid nanoparticles (LNPs), cationic liposomes, polymers, and extracellular vesicles. Lipid‐based mRNA carriers typically adhere to specific criteria: stable nanostructure effectively protect the mRNA payload, reduced recognition by the mononuclear phagocyte system (MPS) and clearance by renal filtration, and all lipid components should be biodegradable. Targeted biodistribution of lipid nanoparticle–mRNA formulations can be improved by further modifying and optimizing the nanoparticle, or by adjusting the proportions of lipid components. ²⁷

Currently, clinically approved LNP formulations are composed of four types of lipids: ionizable cationic lipids, cholesterol, PEGylated lipids, and phospholipids. Through the application of microfluidic mixing techniques, mRNA is encapsulated within LNPs formed from these components in precise ratios. The selection of both components and their respective proportions is critical for the successful delivery of mRNA via LNPs. Researchers have established extensive libraries of ionizable lipids specifically for mRNA delivery, with prominent examples including DLin‐MC3‐DMA, SM‐102, and ALC‐0315. These lipids are recognized as FDA‐approved cationic lipids for RNA delivery, with SM‐102 and ALC‐0315 serving as key constituents of the FDA‐approved mRNA vaccines mRNA‐1273 and BNT162b, respectively, for COVID‐19 prevention. DLin‐MC3‐DMA, which is FDA‐approved for small interfering RNA (siRNA) delivery in Onpattro, has also been widely used in mRNA applications. ²⁸ Each component plays a vital role in enhancing the overall immunogenicity of RNA‐LNP formulations, which is essential for their therapeutic efficacy. However, the intricate mechanisms and interactions at play—particularly the influence of specific lipid structures, such as the number and length of lipid “tails” and the characteristics of the headgroups—on the immunogenicity of LNP formulations remain inadequately understood. ²⁹

Recent advancements have seen researchers optimize the composition, structure, and manufacturing processes of LNPs to augment the delivery efficiency, safety, and stability of mRNA vaccines. ³⁰ In 2024, a collaborative study by researchers from the School of Pharmacy at Zhejiang University, including Dr. Shuai Liu, Professor Zhen Gu, and Professor Ping Yuan, reported the development of a streamlined three‐component LNP system (comprising nAcx‐Cm lipids, permanent cationic lipids, and PEGylated lipids). This innovative approach enabled simultaneous mRNA accumulation and translation in the lungs and liver, achieving targeted organ delivery while minimizing adverse effects. ³¹

The utilization of extracellular vesicles as delivery systems for mRNA vaccines represents an emerging and promising area of research. This strategy harnesses plant‐derived extracellular vesicles as natural carriers for the safe and effective delivery of mRNA vaccines to target cells. Preliminary studies suggest that oral delivery methods can yield favorable outcomes; however, significant improvements in production scalability, cost‐effectiveness, and delivery efficiency are imperative for future advancements. ³²

3.2. Administration routes

Typically, mRNA vaccines for COVID‐19 are administered via intramuscular injection, specifically into the deltoid muscle of the upper arm. This administration route ensures the rapid entry of the vaccine components into the systemic circulation. Although less common, some mRNA vaccines are delivered through subcutaneous injection, which involves administering the vaccine into the subcutaneous adipose tissue beneath the skin.

Recent studies are investigating alternative delivery methods, such as nasal spray or inhalation, which aim to mimic the natural infection pathways of respiratory viruses and may enhance the immunogenicity of respiratory virus vaccines. Research conducted by scientists at Beth Israel Deaconess Medical Center (BIDMC) involved administering a single dose of the Ad26 vaccine through three different routes: intramuscular injection, intranasal delivery (via nasal spray), and tracheal delivery (using nebulizers or inhalers) in three groups of rhesus macaques. ³³ , ³⁴ The findings revealed that direct administration of the COVID‐19 vaccine to the trachea induced a more robust mucosal immune response compared to both intramuscular and intranasal delivery methods. These results support the feasibility of developing vaccines specifically targeting respiratory virus infections.

Furthermore, Professor Giovanni Camussi from the University of Turin has validated the feasibility of using orange‐derived extracellular vesicles as a delivery system for oral capsule formulations of mRNA vaccines. The S1‐mRNA encapsulated within orange‐derived extracellular vesicles was freeze‐dried and subsequently encapsulated in capsules, demonstrating stability at room temperature for up to 1 year without compromising the integrity of the extracellular vesicles or the mRNA. This oral capsule formulation has also been shown to effectively induce immune responses in rats, efficiently triggering cellular immunity (Figure 1).

FIGURE 1.

Respiratory virus messenger RNA (mRNA) vaccines: mRNA design, delivery, administration, and challenges.

4. CLINICAL RESEARCH PROGRESS OF MRNA VACCINES FOR RESPIRATORY VIRUSES

mRNA vaccines represent a novel and efficient strategy for combating infections caused by rapidly mutating respiratory viruses, offering significant public health advantages for disease prevention and control. The rapid development of mRNA vaccines is facilitated by their reliance on established technological platforms, which allow for swift modifications of antigen sequences. This results in shorter development timelines, simplified production processes, lower costs, and suitability for large‐scale manufacturing. mRNA vaccines have played a pivotal role in the defense against viruses such as SARS‐CoV‐2, RSV, and influenza.

Since the outbreak of the COVID‐19 pandemic, global research institutions and pharmaceutical companies have invested substantial resources into the development of mRNA vaccines targeting SARS‐CoV‐2, leading to the approval of multiple vaccines. The mRESVIA (mRNA‐1345) developed by Moderna is the first non‐COVID mRNA vaccine to receive regulatory approval globally, marking it as the third RSV vaccine approved, following Pfizer's Abrysvo and GlaxoSmithKline's Arexvy. Concurrently, nearly a dozen other mRNA vaccines designed for the treatment and prevention of RSV are currently undergoing clinical trials.

Although no mRNA vaccines for influenza have yet been approved, preliminary results from early‐phase clinical trials suggest that influenza virus mRNA vaccines show promise in terms of safety and efficacy. The flexibility of mRNA technology enables rapid adjustments to vaccine sequences, allowing for adaptation to circulating strains of influenza virus, thereby enhancing the response to viral mutations. Researchers are actively developing multivalent mRNA vaccines that can simultaneously protect against multiple subtypes of influenza virus, offering broader immunity. However, further research and large‐scale clinical trials are essential to validate their efficacy and safety.

4.1. Clinical progress of SARS‐CoV‐2 mRNA vaccines

SARS‐CoV‐2 is a single‐stranded positive‐sense RNA enveloped virus whose genome encodes several proteins, including the envelope protein (E), membrane protein (M), nucleocapsid protein (N), and spike protein (S). During the infection of human cells, the spike protein trimers of the virus bind to the angiotensin‐converting enzyme 2 (ACE2) receptors on the surface of host cells. This interaction is subsequently cleaved by the transmembrane serine protease 2 (TMPRSS2), inducing conformational changes that expose the fusion peptide of the spike protein and facilitate viral membrane fusion. ³⁵ , ³⁶ After the outbreak of the COVID‐19 pandemic, the virus and its various variants (including Alpha, Beta, Delta, Gamma, Omicron BA.5, BA.7, KP.1.1, KP.2, KP.3.1.1 etc. ¹⁸ , ³⁷ , ³⁸ ) spread widely across the globe, significantly disrupting daily life and posing immense challenges to public health systems. This situation prompted collaboration among multiple countries and companies to accelerate vaccine and therapeutic development, leading to the rapid emergence of COVID‐19 mRNA vaccines that received Emergency Use Authorization or approval, providing essential tools for epidemic control. These vaccines have demonstrated high efficacy and safety in preventing SARS‐CoV‐2 infection, alleviating symptoms, and reducing the risk of severe disease. ³⁹

Dr. Peter Marks, Director of the FDA's Center for Biologics Evaluation and Research, has emphasized the importance of administering updated vaccines to prevent current circulating variants, particularly as public immunity may be waning. In June 2024, the FDA requested vaccine manufacturers to produce vaccines targeting the JN.1 strain and, where feasible, the KP.2 strain. On August 22, 2024, updated versions of the COVID‐19 vaccines developed by Pfizer (PFE.US) and Moderna (MRNA.US) received approval, specifically targeting the KP.2 strain. Both companies indicated that the updated vaccines are expected to elicit a stronger immune response against JN.1 and other circulating subtypes compared to last year's vaccines that targeted the Omicron XBB.1.5 variant. In contrast, NVAX.US's NVX‐CoV2373 vaccine, which employs protein‐based technology, has demonstrated a slower update pace compared to the mRNA vaccines developed by Pfizer and Moderna. The company has applied for authorization of a new JN.1 vaccine but has not yet received FDA approval (Table 1).

TABLE 1.

The approved messenger RNA (mRNA) vaccines against COVID‐19.

No.	Drug name	Target	Original research institution	Phase	Approved date
1	Bivalent COVID‐19 mRNA vaccine (CSPC Pharmaceutical)	SARS‐CoV‐2 S protein containing BA.5 key mutations	CSPC Pharmaceutical Group Limited	Approved	2023‐12‐01
2	SARS‐CoV‐2 mRNA vaccine (variants) (Walvax Biotechnology)	SARS‐CoV‐2 S protein	Yunnan Walvax Biotechnology Co., Ltd. | Shanghai Blue Magpie Biomedical Co., Ltd. | Fudan University	Approved	2023‐12‐01
3	Zapomeran	SARS‐CoV‐2 S protein	Arcturus Therapeutics Holdings, Inc.	Approved	2023‐09‐05
4	Bemremeran	SARS‐CoV‐2 S protein	Daiichi Sankyo Co., Ltd.	Approved	2023‐08‐02
5	HIPRA SARS‐CoV‐2 (Laboratorios HIPRA)	SARS‐CoV‐2 S protein	Laboratorios HIPRA SA	Approved	2023‐03‐30
6	COVID‐19 mRNA vaccine (SYS6006)	SARS‐CoV‐2 S protein	CSPC Pharmaceutical Group Limited	Approved	2023‐03‐22
7	COVID‐19‐mRNA vaccine (Stemirna Therapeutics)	SARS‐CoV‐2 S protein	SMICell (Shanghai) Biotechnology Co., Ltd.	Approved	2022‐12‐08
8	Elasomeran/Davesomeran	SARS‐CoV‐2 S protein	Moderna, Inc.	Approved	2022‐11‐03
9	ARCoV(Walvax Biotechnology)	SARS‐CoV‐2 S protein	Yunnan Walvax Biotechnology Co., Ltd. | Academy of Military Sciences of the Chinese People's Liberation Army | Suzhou Abogen Biosciences Co., Ltd.	Approved	2022‐09‐30
10	COVID‐19 vaccine(HDT Bio)	SARS‐CoV‐2 S protein	HDT Bio Corp.	Approved	2022‐06‐29
11	Elasomeran/Imelasomeran	SARS‐CoV‐2 S protein	Moderna, Inc.	Approved	2021‐05‐21
12	Elasomeran(mRNA‐1273)	SARS‐CoV‐2 S protein	Moderna, Inc.	Approved	2020‐12‐23
13	BNT162b2	SARS‐CoV‐2 S protein	BioNTech SE	Approved	2020‐12‐02

In addition to traditional linear mRNA vaccines, the ARCT‐154 vaccine, codeveloped by CSL and Arcturus Therapeutics, was approved in April 2024 as the first commercially available saRNA vaccine. SaRNA technology allows for high‐level and sustained expression of target proteins at low doses. Furthermore, a team led by Wei Wensheng at Peking University has developed a circular RNA vaccine encoding the receptor‐binding domain (RBD) of the SARS‐CoV‐2 spike protein (Table 1). This vaccine has been shown to induce greater production of neutralizing antibodies and more effective T‐cell responses compared to traditional linear mRNA vaccines, with data published in Cell in 2022. ⁴⁰

4.2. Clinical progress of influenza virus mRNA vaccines

Influenza viruses, belonging to the Orthomyxoviridae family, are enveloped negative‐sense RNA viruses that cause significant seasonal morbidity and mortality worldwide. Clinical manifestations typically arise 1 to 4 days postinfection and persist for approximately 1 week. Although the symptoms are often self‐limiting in healthy individuals, the burden of disease escalates dramatically among vulnerable populations, including those with underlying health conditions. According to World Health Organization (WHO) reports, seasonal influenza leads to approximately 1 billion cases annually, with 3 to 5 million severe cases and an estimated 2 90 000 to 6 50 000 deaths attributed to respiratory diseases. ⁴¹

Influenza viruses are categorized into four primary types: A, B, C, and D, with types A and B being the most relevant for seasonal epidemics. Influenza A viruses are further subdivided into multiple subtypes based on the surface proteins HA and NA, with H1N1 and H3N2 being the currently predominant strains. The ability of influenza viruses to undergo rapid antigenic drift necessitates the frequent production of updated vaccines; thus, adaptive mutations in vaccine strains generated in embryonated chicken eggs can alter protective efficacy. Consequently, vaccine reformulation is required biannually to mitigate viral transmission. Organizations such as the WHO convene regularly to assess circulating strains and provide recommendations for vaccine formulations suitable for global distribution. The mRNA vaccine platform offers distinct advantages, including rapid scalability and the avoidance of the need for embryonated eggs or cell cultures, thereby circumventing the risks of adaptive mutations while effectively addressing the rapid antigenic variability of the virus. ¹⁷

Moderna is developing multiple candidate influenza vaccines currently in clinical evaluation, including mRNA‐1010, which has demonstrated both safety and immunogenicity in Phase III clinical trials. ⁴² Other candidates in the pipeline include next‐generation influenza vaccines, such as mRNA‐1020 and mRNA‐1030, which target more conserved antigens and are currently in Phase II trials. The application of mRNA technology has brought novel opportunities for the development of combination vaccines targeting multiple respiratory pathogens. The mRNA platform allows for enhanced flexibility in the design and production of vaccine components against various pathogens. Among the Phase I trial candidates is mRNA‐1045, a combination vaccine targeting both influenza and RSV. Pfizer and BioNTech have expanded their collaboration to establish a robust pipeline of mRNA vaccines. Preliminary findings from Phase III trials of their candidate PF‐07252220 for influenza and respiratory diseases indicate superior efficacy in individuals aged 18 to 64 compared to existing influenza vaccines. However, this candidate achieved secondary immunogenicity endpoints solely for influenza A strains, with results from trials involving individuals aged 65 and older anticipated later this year. Both companies have indicated the potential for adjustments to the candidate, although such modifications could result in delays in securing any possible approvals. Initial results suggest that certain combination vaccines hold promise in eliciting immune responses; however further large‐scale clinical trials are essential to confirm their long‐term efficacy and safety. Concurrently, ongoing optimization studies regarding vaccine formulation, dosing, and administration protocols are being conducted to enhance the efficacy and acceptability of the vaccines. The interplay between different pathogen components and the balance of immune responses necessitates continued research and innovation for effective optimization (Table 2).

TABLE 2.

Messenger RNA (mRNA) vaccines against flu virus and combined antigens.

No.	Drug name	NCT number	Indication	Original research institution	Phase
1	PF‐07252220	NCT05540522	Influenza	BioNTech SE | Pfizer Inc.	III
2	mRNA‐1010	NCT05827068	Influenza	Moderna, Inc.	III
3	mRNA‐1083	NCT06097273 NCT06508320	Influenza	Moderna, Inc.	III
4	mRNA‐1073	NCT05375838	Influenza and COVID‐19	Moderna, Inc.	I/II
5	mRNA‐1020 /mRNA1030	NCT05333289	influenza	Moderna, Inc.	I/II
6	mRNA‐1012	NCT05827068	Influenza	Moderna, Inc.	II
7	mRNA‐1045	NCT05585632	Influenza, COVID‐19, SARS, and RSV	Moderna, Inc.	I
8	mRNA‐1230	NCT05585632	Influenza, COVID‐19, SARS, and RSV	Moderna, Inc.	I
9	mRNA‐1365	NCT05743881	Influenza and HMPV	Moderna, Inc.	I
10	GSK‐4382276A	NCT06431607	Influenza	GSK Plc	II

4.3. Clinical progress of RSV mRNA vaccines

RSV belongs to the genus Orthopneumovirus within the family Pneumoviridae and the order Mononegavirale and comprises single‐stranded, negative‐sense RNA viruses. Human RSV is a leading cause of lower respiratory tract infections among infants, young children, and the elderly. Globally, it is characterized by two antigenic subtypes, A and B. RSV infections affect over 33 million individuals annually, with more than 3 million requiring hospitalization and approximately 59 600 deaths occurring among children under 5 years of age. ⁴³

The substantial susceptibility to RSV, coupled with the severity of the associated symptoms, highlights the significant market demand for an effective vaccine. Despite more than six decades of research, the development of an effective RSV vaccine has faced numerous challenges due to the unique structural properties of RSV surface proteins. ⁴⁴ However, the recent introduction of two recombinant subunit vaccines—GSK's Arexvy and Pfizer's Abrysvo—has provided new avenues for RSV prevention, whereas Moderna's mRNA vaccine, mRESVIA, was approved in June of this year for individuals aged 60 and older. The bivalent recombinant subunit vaccine ABRYSVO has a broader age indication than both Moderna's mRESVIA and GSK's Arexvy, encompassing high‐risk adults aged 18 to 59 as well as pregnant individuals.

In addition to the already‐approved mRESVIA, several candidate RSV mRNA vaccines are advancing through clinical trial stages. Companies such as Moderna and Pfizer are actively pursuing RSV mRNA vaccine projects across various phases of clinical evaluation. ⁴⁵ Immunogenicity studies have yielded promising outcomes, with several trials indicating that the vaccines can elicit specific antibody responses and activate immune cell responses against RSV. There is an ongoing research to assess vaccine safety and efficacy across different age groups, including trials in children and older adults, who are particularly vulnerable to RSV infection (Table 3).

TABLE 3.

Messenger RNA (mRNA) vaccines against respiratory syncytial virus (RSV) virus that have entered the clinical trial.

No.	Drug name	NCT number	Immunogen	Original research institution	Phase	Approved date
1	mRNA‐1345	NCT06060457	RSV F protein	Moderna, Inc.	Approved	2024‐05‐31
2	SP‐0256	NCT06251024	–	Sanofi	II	–
3	STR‐V003	NCT06344975	RSV F protein	Starna Therapeutics (Suzhou) Co., Ltd.	I/II	–
4	RSV mRNA LNP CL‐0059 vaccine (Sanofi)	NCT05639894	–	Sanofi	I/II	–
5	RSV mRNA LNP CL‐0137 vaccine (Sanofi)	NCT05639894	–	Sanofi	I/II	–
6	JCXH‐108	NCT06564194	RSV F protein	JiaChen WestSea (Hangzhou) Biotechnology Co., Ltd.	I/II	–
7	mRNA‐1172	–	–	Moderna, Inc.	I	–
8	mRNA MRK‐1777	–	RSV F protein	ModernaTX, Inc.	I	–
9	SYS6016	CTR20243880	RSV F protein	CSPC Jushi Biopharmaceutical Co., Ltd.	I	–
10	IN‐006	NCT06645665 NCT06287450	RSV F protein	Shenzhen Shenxin Biotech Co., Ltd.	I	–

5. LIMITATIONS, REGULATORY CONCERNS, AND CHALLENGES IN THE DEVELOPMENT OF RESPIRATORY VIRUS MRNA VACCINES

Despite significant advancements in mRNA vaccine development for respiratory viruses, these vaccines still encounter several limitations and regulatory challenges. For instance, mRNA is inherently unstable and necessitates cold chain storage, which restricts distribution in resource‐limited settings. Additionally, manufacturing challenges persist, with scalability and the cost of raw materials posing significant hurdles. ⁴⁶ Furthermore, ensuring safety and efficacy across diverse populations demands rigorous clinical trials and robust post‐market surveillance. ⁴⁷ Several significant challenges remain, as detailed below.

First, advancing the production of mRNA vaccines faces several significant challenges. The high costs of raw materials, such as nucleotides, lipids, and enzymes, are driven by increasing demand, thereby escalating overall expenses. The manufacturing process is highly complex and requires specialized equipment and facilities, which in turn limits production capacity. Generally, the necessity for ultra‐cold storage conditions further complicates distribution, particularly in low‐resource settings.

Second, the stability and delivery efficiency of mRNA vaccines represent critical bottlenecks that require urgent attention. The supply chain for mRNA vaccines is constrained by stringent cold chain requirements, necessitating low‐temperature transport and storage and resulting in relatively short shelf lives. For instance, early mRNA vaccines, such as mRNA‐1273, can be stored at 2–8℃ for 1 month and at room temperature for only 12 h. In contrast, BNT162b2 requires storage at −60℃. Given the limited availability of global cold chain logistics, these rigorous transportation and storage conditions may lead to delays or losses in vaccine distribution. Current research efforts are focused on leveraging advanced delivery methods and AI‐based mRNA sequence design to mitigate these logistical constraints and extend the usability of mRNA vaccines. ⁴⁸ , ⁴⁹

Above all, safety and efficacy are paramount indicators of success in mRNA vaccine development. To ensure the safety of mRNA‐based therapeutics and vaccines, a multidisciplinary approach is essential, encompassing advanced in vitro toxicity screening methods and omics data collection for early risk identification. Continuous monitoring of the rapidly evolving fields of LNP and mRNA engineering is also necessary. Specifically, this involves selecting antigen sequences that effectively activate the immune system while minimizing the risk of undesired immune responses. Optimizing mRNA sequences to reduce potential adverse effects, employing chemical modifications—such as the use of pseudouridine to evade immune recognition and mitigate inflammatory responses—and refining delivery systems are critical steps in this process. For instance, a research team led by Satoshi Uchida at Tokyo Medical and Dental University (TMDU) has utilized a needle‐free injector (PYRO) that employs instantaneous liquid pressure to facilitate the internalization of naked mRNA molecules into cellular structures. In murine models, the jet injection of naked mRNA vaccines in the skin elicited robust humoral immune responses while establishing germinal centers in lymph nodes and inducing CD4+ and CD8+ T‐cell responses, thereby providing effective immunoprotection. Importantly, this approach limits the distribution of mRNA to the injection site, reducing the risk of systemic inflammatory reactions. ¹⁴

Furthermore, rigorous quality control throughout the production process is essential to ensure that impurities in the vaccine remain within acceptable limits, which is vital for regulatory approval and the assurance of vaccine safety and efficacy.

Despite these challenges, the future of mRNA vaccines is promising. With ongoing technological advancements, innovative research methodologies, and the accumulation of clinical experience, the design strategies and delivery systems for mRNA vaccines are expected to continuously improve, thereby further enhancing their safety and efficacy in clinical use. ⁵⁰

6. CONCLUSION

mRNA vaccines have transformed the landscape of vaccinology with their capacity for rapid development, scalability, and adaptability. In the context of respiratory diseases, mRNA vaccines have showcased several key strengths. Their platform provides unparalleled flexibility, enabling rapid modifications to target new variants or emerging viruses, thereby demonstrating exceptional design agility. Additionally, mRNA vaccines have demonstrated high immunogenicity and effectiveness in providing robust protection against multiple respiratory pathogens, reaffirming their real‐world efficacy.

Looking ahead, mRNA technology holds significant promise for addressing future respiratory virus outbreaks. Its versatility supports the development of multivalent vaccines capable of targeting a broad spectrum of respiratory viruses, including human metapneumovirus (hMPV), thereby enhancing global preparedness against emerging infectious threats. As research progresses, mRNA vaccines are poised to remain at the forefront of innovative vaccine strategies, offering a powerful tool to combat both current and future respiratory disease challenges.

AUTHOR CONTRIBUTIONS

Linlin Zheng: Funding acquisition; investigation; writing – original draft. Han Feng: Data curation; funding acquisition; investigation; project administration; writing – review and editing.

FUNDING INFORMATION

This work was supported by grants from the Ministry of Science and Technology of the People's Republic of China (2021YFA1300301 and 2018YFA0507101), the National Natural Science Foundation of China (31730054 and 31770900), and the Beijing Natural Science Foundation (5212016).

CONFLICT OF INTEREST STATEMENT

Han Feng is an editorial board member of Animal Models and Experimental Medicine (AMEM) and a corresponding author of this article. To minimize bias, he was excluded from all editorial decision making related to the acceptance of this article for publication.

ETHICS STATEMENT

None.

Zheng L, Feng H. Respiratory virus mRNA vaccines: mRNA Design, clinical studies, and future challenges. Anim Models Exp Med. 2025;8:1731‐1740. doi: 10.1002/ame2.70018