Technical Review: Pulmonary delivery of small circular RNA vaccines for influenza prevention

Authors: Yu Zhang, You Xu, Wei Tang, Shurong Zhou, Xiang Liu, Zitong Wang, Guizhi Zhu

Affiliations: Chinese Academy of Sciences, Hangzhou, Zhejiang, China; Virginia Commonwealth University, Richmond, Virginia, USA; National University of Singapore, Singapore; University of Michigan, Ann Arbor, Michigan, USA

Background Introduction

This research falls within the interdisciplinary field of RNA-based vaccine development and nanomedicine, combining elements of molecular biology, immunology, and pharmaceutical engineering. The study addresses critical limitations of current influenza vaccines, which typically provide only 40-60% protective effectiveness and require annual reformulation due to viral antigenic drift. Traditional mRNA vaccines, while promising, face challenges including limited biostability, inflammatory responses, and activation of protein kinase R (PKR) that hampers antigen translation.

The researchers therefore focus on circular RNA (circRNA) as an alternative to linear mRNA. Unlike conventional mRNA, circRNA lacks terminal structures, making it highly resistant to exonuclease degradation and providing enhanced stability and sustained protein expression. The study specifically targets the matrix protein 2 ectodomain (M2e), a highly conserved but poorly immunogenic influenza A antigen, as a candidate for universal influenza vaccination.

Schematic illustration of pulmonary delivery of a small circRNA flu vaccine. Small circRNA is comprised of RNA coding sequence (CDS) for antigens, and a short IRES and a Kozak consensus sequence that together initiate antigen translation. Small circRNA was synthesized by ligating RNA precursor oligonucleotide(s) using RNA ligase and DNA splint(s), followed by circRNA purification. The small sizes and terminus-less small circRNA confer high thermostability and biostability. Small circRNA was loaded into LNPs, and the resulting circRNA vaccines (i.e., circRNA LNPs) were administered into mice by i.t. administration for pulmonary delivery to APCs. As a result, circRNA vaccines potentiated and prolonged antigen-specific adaptive immunomodulation. In young adult mice and aged mice, circRNA-M2e vaccines elicited M2e-specific CD4+ T cell responses, which conferred protection of immunized mice from lethal challenge of influenza.

Materials and Methodology

The researchers first designed and synthesized a 156-nucleotide small circRNA-M2e. Stability was assessed through serum degradation assays comparing circRNA-M2e with benchmark m1ψ-modified mRNA. Three lipid nanoparticle (LNP) formulations were then screened for delivery efficiency of this circRNA. For the optimal formulation, translation efficiency and immunogenicity were then evaluated in HeLa and DC2.4 cells using ELISA, Western blotting, and immunofluorescence staining. Finally, an in vivo study was conducted on a murine model to evaluate biodistribution, immunogenicity, protective efficacy, and age-related immune response.

Results

Stability and Translation Efficiency

CircRNA-M2e demonstrated significantly enhanced stability compared to conventional mRNA, with superior resistance to serum-mediated degradation after 30 minutes at 37°C. In cellular assays, circRNA-M2e exhibited higher translational efficiency than m1ψ-mRNA-M2e while producing comparable levels of essential proinflammatory cytokines (IL-6, IL-12, IFN-β) necessary for antigen presentation. This enhanced stability provides crucial advantages for vaccine storage, transport, and sustained antigen expression.

LNP Optimization

SM-102 LNPs emerged as the superior delivery vehicle, showing the strongest lung bioluminescence signal among tested formulations. The optimized LNPs demonstrated spherical morphology with 106.7 ± 4.3 nm diameter and 91% encapsulation efficiency. Importantly, LNP delivery significantly enhanced circRNA retention in lung tissue for at least 72 hours while reducing systemic dissemination to liver and kidney, indicating targeted pulmonary accumulation essential for local immune activation.

Cellular Targeting and Safety

Flow cytometric analysis revealed that LNPs efficiently delivered circRNA to crucial lung-resident antigen-presenting cells, including B cells, alveolar macrophages, and dendritic cells. Safety assessments showed transient inflammatory cell infiltration that resolved within 24 hours, normal lung histology, and absence of significant respiratory inflammatory responses, though temporary systemic cytokine elevation occurred. These findings indicate a favorable safety profile with targeted immune activation.

Immunological Efficacy

The circRNA-M2e vaccine elicited robust and multifaceted immune responses superior to conventional approaches. In young adult mice, the vaccine induced strong M2e-specific CD4+ T cell responses with enhanced Th2 and Th17 differentiation, as evidenced by increased IL-4 and IL-17A secretion. Humoral immunity analysis revealed significantly elevated IgG1 and IgG2a antibody levels, indicating balanced Th1/Th2 responses crucial for comprehensive viral protection. Remarkably, the vaccine maintained efficacy in immunosenescent 18-month-old mice, demonstrating 203% higher M2e-specific CD4+ T cell responses compared to conventional mRNA vaccines.

Protective Efficacy

The most significant finding was complete protection (6/6 survival) against lethal PR8 influenza challenge compared to 4/6 survival with conventional mRNA vaccines. Protected mice showed rapid weight recovery, minimal clinical symptoms, and maintained normal body temperature, demonstrating superior prophylactic efficacy against a 4×LD50 viral dose. This enhanced protection validates the therapeutic potential of the circRNA platform for universal influenza prevention.

Conclusion

Zhang and colleagues demonstrated that pulmonary-delivered small circRNA vaccines represent a promising advancement in influenza prevention technology. The research establishes circRNA's superior stability, sustained antigen expression, and enhanced immunogenicity compared to conventional mRNA. Additionally, it showcased effective protection against lethal influenza challenge in both young and aged animal models. The targeted M2e antigen approach offers potential for universal, strain-independent influenza vaccination.

This paper utilized the PreciGenome NanoGenerator Flex-S for LNP synthesis. Consistent pressure based microfluidics and small batch volumes are both ideal for LNP formulation screening, where reliable results are key for focused optimization.

For further details, check out the link to the article here:

https://www.sciencedirect.com/science/article/abs/pii/S0168365925008107?via%3Dihub