Abstract
The success of mRNA-based COVID-19 vaccines highlights the promise of nucleic acid platforms. Circular RNA (circRNA), with its inherent structural stability and durable protein expression, may overcome key limitations of linear mRNA. This review explores the rationale for circRNA vaccine development and its unique potential against rapidly mutating viruses (e.g., influenza, coronaviruses) to elicit broad neutralising antibodies. We evaluate preclinical evidence of circRNA vaccines providing more durable and cross-protective immunity and discuss challenges for clinical translation.
Introduction
The rapid development, manufacturing agility, and remarkable efficacy of nucleoside-modified, lipid nanoparticle (LNP)-encapsulated mRNA vaccines against COVID-19 have unequivocally established nucleic acid platforms as a central pillar of 21st-century vaccinology (Polack et al., 2020; Baden et al., 2021). This triumph demonstrated the feasibility of direct in vivoproduction of antigens, eliciting robust humoral and cellular immune responses. However, the journey of linear mRNA as a drug substance is fraught with intrinsic molecular challenges. Its linearity makes it susceptible to rapid degradation by ubiquitous exoribonucleases, limiting its intracellular half-life and, consequently, the duration of antigen expression. Furthermore, the 5’ cap and 3’ poly(A) tail, while essential for translation and stability, are also potent ligands for innate immune sensors like RIG-I and MDA-5. Although nucleoside modifications (e.g., N1-methylpseudouridine) and purification techniques can mitigate this, residual immunogenicity can lead to inflammatory side effects and may also inhibit translation, creating a delicate balance between efficacy and reactogenicity (Karikó et al., 2005; Nance & Meier, 2021). For vaccines targeting persistent infections or requiring long-term protective immunity, the transient nature of protein expression (typically days to a week) may be suboptimal.
Enter circular RNA. Once considered splicing artifacts, circRNAs are now recognized as a vast, conserved class of endogenous RNA molecules formed by a non-canonical “back-splicing” event that ligates a downstream 3’ splice site to an upstream 5’ splice site, creating a covalently closed loop devoid of free ends (Memczak et al., 2013; Jeck et al., 2013). This circular conformation confers profound biochemical stability, rendering them resistant to exonuclease-mediated decay and granting them substantially longer intracellular half-lives than their linear counterparts. Notably, some endogenous circRNAs have been shown to be translatable via internal ribosome entry site (IRES)-mediated mechanisms, producing functional peptides (Legnini et al., 2017; Pamudurti et al., 2017). These innate properties—extreme stability, low immunogenicity, and durable translation potential—make engineered synthetic circRNAs an exceptionally attractive platform for prophylactic and therapeutic applications.
The conceptual leap to harness circRNA as a vaccine platform was pioneered by key proof-of-concept studies demonstrating that exogenous, protein-encoding circRNAs could be engineered and efficiently translated in mammalian cells (Wesselhoeft et al., 2018). Subsequent work has rapidly advanced this platform into the vaccinology arena, with early preclinical studies against SARS-CoV-2 showing highly promising results (Qu et al., 2022; Chen et al., 2023). This review aims to synthesize the current state of this rapidly evolving field. We will first delineate the technical and mechanistic advantages of circRNA vaccines. We will then focus on their most transformative potential: combating antigenically variable viruses that have eluded conventional vaccine approaches. By examining the application of circRNA technology to influenza, coronaviruses, and HIV, we will explore its promise for achieving broad and durable protection. A critical analysis of the expanding preclinical evidence will be provided, followed by a clear-eyed discussion of the formidable but surmountable challenges on the path from bench to bedside. The development of circRNA vaccines represents not merely an incremental improvement, but a potential paradigm shift towards a new generation of more stable, potent, and broadly protective nucleic acid medicines.
Technical Advantages of the circRNA Platform
The superior performance of circRNA vaccines stems from fundamental biophysical and biochemical properties imparted by their circular architecture, which directly addresses key weaknesses of linear mRNA.
1. Covalently Closed Structure and Exonuclease Resistance: The defining feature of circRNA is its continuous, covalently closed loop. This structure eliminates the free 5’ and 3’ ends that are the primary substrates for processive cellular exoribonucleases like XRN1 and the exosome complex. Consequently, engineered circRNAs exhibit extraordinary stability in cells. Studies comparing in vitro-transcribed (IVT) circRNAs to their linear counterparts show half-lives extended by several-fold, often exceeding 48 hours in cell culture compared to less than 10 hours for unmodified linear mRNA (Wesselhoeft et al., 2018). In vivo, this translates to significantly prolonged antigen expression following delivery, a critical factor for driving robust germinal center reactions, affinity maturation, and the development of long-lived plasma cells and memory B cells. Sustained antigen presentation is a key determinant of immune response quality and durability, an area where circRNA holds a distinct theoretical advantage.
2. Reduced Innate Immunogenicity: The innate immune system is exquisitely tuned to recognize viral nucleic acids through pattern recognition receptors (PRRs). Linear mRNA, especially if unmodified, is a potent agonist for endosomal Toll-like receptors (TLRs 3, 7, 8) and cytosolic sensors like RIG-I and MDA-5, which bind to its 5’ triphosphate or double-stranded regions. While nucleoside modification dampens this response, it can be incomplete. The circular conformation of circRNA physically lacks the termini recognized by RIG-I. Furthermore, the absence of a 5’ cap structure (replaced by an IRES or similar element for translation initiation) removes another potential PAMP (pathogen-associated molecular pattern). As a result, properly purified synthetic circRNAs exhibit markedly lower induction of type I interferon (IFN-α/β) and pro-inflammatory cytokines in antigen-presenting cells like dendritic cells (Qu et al., 2022). This “stealthier” profile is doubly beneficial: it minimizes vaccine-associated reactogenicity (e.g., fever, fatigue) and removes IFN-mediated inhibition of cap-dependent translation. This allows the host cell’s translational machinery to focus almost exclusively on producing the encoded antigen, potentially leading to higher protein yields per RNA molecule.
3. Prolonged and High-Level Protein Expression: The combination of nuclease resistance and low immunogenicity culminates in sustained, high-level antigen production. The translation of circRNA is typically driven by an engineered IRES element, such as those from encephalomyocarditis virus (EMCV) or cricket paralysis virus (CrPV). These elements direct cap-independent ribosome recruitment and initiation. Advanced engineering, including the optimization of IRES sequences, the inclusion of splicing elements and inverted repeats to enhance circularization efficiency, and the strategic design of the open reading frame, has led to circRNAs that can produce protein at levels matching or exceeding those of state-of-the-art nucleoside-modified linear mRNAs over extended periods (Chen et al., 2023). This prolonged antigen bioavailability is particularly advantageous for generating high-quality, cross-reactive B cell responses against conserved but subdominant epitopes, which require extended germinal center activity for effective targeting.
Antiviral Applications: Targeting Viral Evolution and Immune Evasion
The unique pharmacokinetic profile of circRNA—durable antigen expression with minimal inflammation—makes it an ideal platform for vaccinating against viruses that utilize rapid mutation or immune evasion as their primary survival strategy. Here, we explore its application against three major viral challenges.
1. Influenza: Toward a Universal Vaccine. Seasonal influenza vaccines require annual reformulation due to antigenic drift in the hemagglutinin (HA) head domain. A universal influenza vaccine aims to provide durable protection against diverse strains, ideally groups 1 and 2 influenza A viruses. A leading strategy focuses the immune response on the conserved HA stalk region. However, the immunodominant head often distracts the response. The circRNA platform is uniquely suited for this challenge. By encoding stabilized, headless HA stalk antigens or computationally designed “mosaic” HA sequences, a circRNA vaccine can drive the persistent production of these less-immunogenic but conserved epitopes. This prolonged exposure, without the inflammatory context that might bias responses toward immunodominant variable epitopes, could more effectively prime and expand rare B cell clones targeting the stalk, driving their affinity maturation over time. Preclinical studies are testing circRNAs encoding HA constructs with stabilized stems, ferritin nanoparticle-displayed stalks, or multi-hemagglutinin constructs, with the goal of eliciting broadly neutralizing antibodies (bnAbs) that neutralize across subtypes (e.g., H1, H3, H5).
2. β-Coronaviruses: Preparing for SARS-CoV-3 and Beyond. The emergence of SARS-CoV-2 variants of concern (Alpha, Delta, Omicron) demonstrated the vulnerability of first-generation vaccines to antigenic shift. The goal for pan-sarbecovirus or universal coronavirus vaccines is to protect against current and future zoonotic threats. circRNA vaccines are being deployed in two key strategies. First, they can encode prefusion-stabilized spike proteins from multiple sarbecoviruses (e.g., SARS-CoV-1, SARS-CoV-2, pangolin coronaviruses, bat coronaviruses) either as a mixture or as a single immunogen designed in silicoto present conserved epitopes, such as those in the receptor-binding domain (RBD). Second, they can express “mosaic” or “consensus” spike sequences that represent an ancestral or averaged version of the sarbecovirus clade. The durable in vivoexpression from a circRNA vaccine could be critical for maturing B cell responses against these shared, often cryptic, epitopes. Early proof-of-concept studies have shown that circRNA-LNP vaccines expressing the SARS-CoV-2 spike or RBD induce potent neutralizing antibodies in mice and non-human primates, with some demonstrating superior breadth against variants like Omicron compared to linear mRNA vaccines (Qu et al., 2022; Chen et al., 2023).
3. HIV and Other Challenging Pathogens. HIV presents perhaps the ultimate challenge: a globally diverse virus with an envelope trimer (Env) decorated with a dense glycan shield and highly variable loops, and capable of establishing latent reservoirs. Eliciting bnAbs against HIV is a monumental task, as the rare bnAb precursor B cells require extended periods of affinity maturation through sequential exposure to a series of evolving Env immunogens. The circRNA platform could be transformative for such complex prime-boost regimens, often called “germline-targeting” and “sequential immunization” strategies. A circRNA vaccine could deliver a stabilized, germline-targeting Env immunogen for a prolonged period, providing a sustained stimulus to engage and initiate the expansion of rare naive bnAb-precursor B cells. Subsequent boosts with circRNAs encoding a series of carefully designed Env “evolution” immunogens could then guide the B cell lineage maturation over time, all within the context of a single immunization or a reduced series. This approach could also be applied to other pathogens with hypervariable surface proteins, such as hepatitis C virus (HCV) and respiratory syncytial virus (RSV), where focusing the response on conserved neutralization sites is paramount.
Preclinical Proof-of-Concept: Evidence from Animal Models
The promising theoretical advantages of circRNA vaccines are now being substantiated by a growing body of preclinical data. Key studies in small and large animal models have focused on immunogenicity, protective efficacy, and durability, primarily against SARS-CoV-2.
Immunogenicity and Neutralizing Antibody Responses: Initial studies established that LNP-formulated, IVT circRNAs encoding reporter or antigenic proteins are efficiently translated in vivofollowing intramuscular injection in mice. Qu et al. (2022) demonstrated that a circRNA vaccine encoding the SARS-CoV-2 Spike protein, delivered via an LNP optimized for circRNA, induced potent antigen-specific antibody and T-cell responses in mice. Notably, the induced neutralizing antibody titers against the ancestral strain were comparable to or higher than those induced by a modified linear mRNA-LNP vaccine. More importantly, the circRNA vaccine induced antibodies with greater neutralizing breadth against variants of concern, including Beta and Delta, suggesting qualitative differences in the antibody response. A follow-up study by Chen et al. (2023) provided further compelling evidence. They showed that a circRNA vaccine encoding an Omicron-specific RBD elicited significantly higher and more durable neutralizing antibody titers against Omicron BA.1, BA.2, and BA.5 subvariants in mice compared to a linear mRNA counterpart. The circRNA vaccine also induced robust and durable germinal center B cell responses in draining lymph nodes, a cellular correlate of long-lived immunity.
Protective Efficacy and Durability: The ultimate test of any vaccine platform is protection from challenge. In the hamster model of SARS-CoV-2 pathogenesis, immunization with a circRNA-LNP vaccine encoding the full-length Spike protein provided complete protection from weight loss and significantly reduced viral loads in the lungs and nasal turbinates following challenge with the ancestral virus (Qu et al., 2022). Perhaps the most persuasive data to date comes from non-human primate (NHP) studies, the gold standard for vaccine translation. Rhesus macaques immunized with a circRNA vaccine developed high-titer neutralizing antibodies and antigen-specific T-cell responses. Upon challenge with a high dose of SARS-CoV-2, vaccinated animals showed rapid control of viral replication in both the upper and lower respiratory tract, with viral loads reduced by orders of magnitude compared to controls. Crucially, emerging data suggests the antibody responses induced by circRNA vaccines may be more durable. Studies tracking antibody kinetics over several months in mice and NHPs indicate a slower decay in neutralizing titers following circRNA vaccination compared to linear mRNA, consistent with the platform’s design for sustained antigen production.
Cross-Protection and Universal Vaccine Potential: Preliminary data against influenza is also emerging. In murine models, circRNA vaccines encoding conserved HA stalk antigens or computationally optimized broadly reactive HA antigens have been shown to elicit antibodies that cross-react with heterosubtypic HAs (e.g., from both group 1 and 2 influenza A viruses) and provide partial protection against lethal challenge with mismatched viral strains. These studies, while early-stage, provide proof-of-principle that the durable antigen expression from circRNA can be leveraged to focus the immune response on subdominant, conserved epitopes, moving closer to the goal of a universal influenza vaccine.
Challenges and Future Directions
Despite its considerable promise, the circRNA vaccine platform is nascent and faces significant scientific, manufacturing, and regulatory hurdles that must be cleared for clinical translation.
1. Scalable GMP Manufacturing: The production of clinical-grade circRNA is more complex than that of linear mRNA. The standard method involves in vitrotranscription of a linear precursor containing permuted intron-exon sequences or hepatitis delta virus (HDV) ribozyme motifs that facilitate self-splicing and circularization. This reaction must be followed by extensive purification to remove linear RNA contaminants, immunostimulatory byproducts, and enzymes. Chromatographic purification (e.g., using HPLC or FPLC) is effective but can be low-yield and challenging to scale. Developing robust, high-yield, and cost-effective enzymatic or enzymatic/chemical hybrid synthesis and purification processes that meet stringent GMP standards for identity, purity, potency, and sterility is a paramount priority for the field.
2. Delivery System Optimization: While LNPs have been spectacularly successful for mRNA delivery, they may require optimization for circRNA. The size, structure, and potentially different protein-binding properties of circRNA could influence LNP formulation parameters (lipid ratios, ionizable lipid structure), encapsulation efficiency, and biodistribution. Research is needed to design LNPs specifically tailored for the efficient delivery of circRNA to key antigen-presenting cells in the lymph nodes and muscle, and to minimize off-target delivery. Alternative delivery modalities, such as polymer-based nanoparticles or novel cationic formulations, are also being explored.
3. Comprehensive Safety and Toxicology Profiling: The long-term in vivopersistence of circRNA is a double-edged sword. While beneficial for durable immunity, it raises theoretical safety questions that must be rigorously addressed. These include: the potential for rare integration events (though RNA does not integrate by known mechanisms), the immunotoxicity of any protein product expressed for extended periods, and the possibility of autoimmune reactions against sustained self-antigen production (relevant for cancer vaccines or therapeutic applications). Comprehensive preclinical toxicology studies in two relevant animal species, assessing local and systemic reactogenicity, pharmacokinetics/pharmacodynamics of antigen expression, and potential organ pathology, are essential. The impact of pre-existing immunity to the viral IRES elements used (e.g., EMCV IRES) also requires investigation.
4. The Regulatory Path: Regulatory agencies (FDA, EMA, etc.) have established pathways for mRNA vaccines, but circRNA presents novel characteristics. Developers will need to engage early with regulators to define critical quality attributes (CQAs), determine appropriate analytical methods for characterizing circularity and purity, and design clinical trials that adequately assess the unique pharmacokinetic profile (duration of antigen expression) and long-term safety. A key regulatory discussion will center on the definition of the product’s persistence and its implications for dosing intervals and risk-benefit assessment.
5. Direct Comparison and Clinical Validation: Ultimately, the value of circRNA must be proven in head-to-head comparisons with the best-in-class linear mRNA vaccines in clinical trials. Superiority or non-inferiority endpoints should include not only peak immunogenicity but, crucially, the durability and breadth of the immune response, as well as reactogenicity profiles. The first-in-human clinical trials for circRNA vaccines are anticipated in the coming 1-2 years, and their results will be pivotal in determining the platform’s future.
Conclusion
The circRNA vaccine platform emerges from the shadow of its linear mRNA predecessor not as a mere alternative, but as a potentially superior technological evolution for specific, critical applications. Its core advantages—exceptional molecular stability, diminished innate immunogenicity, and capacity for prolonged protein expression—are precisely tailored to address the limitations of first-generation nucleic acid vaccines. This makes it a particularly compelling candidate for confronting the most persistent challenges in infectious disease: viruses that mutate rapidly or that require sophisticated immune focusing to elicit broad protection.
The preclinical data, while early, is highly encouraging, demonstrating robust immunogenicity, cross-protective efficacy, and promising durability in relevant animal models. The path to the clinic, however, is steep, requiring innovations in manufacturing, delivery, and a thorough understanding of its long-term safety profile. If these challenges can be met, circRNA technology could enable a new class of vaccines: single-shot vaccines that provide years of protection against seasonal influenza, universal coronavirus vaccines that protect against future pandemics at their source, and precision-engineered immunogens capable of guiding the immune system to produce rare, broadly neutralizing antibodies against foes like HIV. The circularization of RNA, a natural biological quirk, may thus prove to be the key that unlocks a new frontier in durable, broadly protective immunization.
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