Cap-free, self-amplifying RNA vaccine translates and amplifies in vivo, demonstrating protective efficacy against H5N8 in mice
Background
Self-amplifying RNA (saRNA) vaccines have the potential to induce immune responses with lower doses compared to conventional non-replicating mRNA vaccines because the RNA replicates within cells. However, the large size of the RNA molecule and the manufacturing process, which involves adding a 5' cap necessary for protein translation and RNA stability, need to be considered. The researchers developed a cap-independent saRNA platform, called CLsamRNA, based on a Coxsackievirus B5 replicon, utilizing an internal ribosome entry site (IRES). This design is a preclinical vaccine candidate engineered to initiate translation without a 5' cap and amplify RNA within cells.
Key Findings
The researchers systematically optimized the genetic elements to enhance antigen expression from the CLsamRNA and compared its expression profile with existing VEEV-based saRNA and nucleoside-modified mRNA when delivered in lipid nanoparticles. CLsamRNA encoding the hemagglutinin antigen of H5 subtype highly pathogenic avian influenza induced neutralizing antibodies and antigen-specific cellular immune responses in mice. Cross-reactivity against H5N1 clade 2.3.4.4b was also observed. Notably, a dose of 0.01 μg administered to BALB/c mice provided complete protection against a lethal H5N8 challenge. These results are preclinical findings obtained in animal models and do not reflect efficacy in humans.
Significance and Implications
This study demonstrates that an RNA vaccine platform combining cap-independent translation and self-amplification can generate protective immunity against H5 viruses at very low doses. Distinct early inflammatory responses and lymph node immune gene signatures compared to existing platforms were also observed, warranting further investigation. However, safety, dosage, duration of immunity, and efficacy in humans need to be verified through clinical trials. At this stage, the confirmed conclusion is that it provides a basis for further development as an HPAI H5 vaccine candidate, rather than simplifying manufacturing or commercialization.
Self-amplifying mRNA (samRNA) vaccines can induce potent immune responses at lower doses than conventional non-replicating mRNA vaccines; however, large RNA size and manufacturing considerations associated with 5' capping remain important challenges. Here, we developed a capless samRNA (CLsamRNA) vaccine platform derived from a Coxsackievirus B5 replicon that uses IRES-mediated cap-independent translation. Systematic optimization of key genetic elements enhanced antigen expression from the CLsamRNA backbone. Using reporter RNAs, LNP-formulated CLsamRNA showed rapid early expression and RNA amplification, with expression kinetics distinct from VEEV-based saRNA and nucleoside-modified mRNA comparators. When encoding the hemagglutinin antigen of highly pathogenic avian influenza clade 2.3.4.4 H5 viruses, CLsamRNA induced potent immune responses after LNP formulation. In mouse models, CLsamRNA induced potent neutralizing antibody responses, cross-reactive activity against clade 2.3.4.4b H5N1 virus, Th1-skewed humoral immunity, and strong antigen-specific cellular immune responses. CLsamRNA also elicited platform-specific early inflammatory and lymph-node immune gene signatures associated with antigen presentation, costimulation, and cellular immune priming. Notably, a minimal 0.01 μg dose of CLsamRNA conferred complete protection against lethal H5N8 influenza virus challenge in BALB/c mice. These findings support CLsamRNA as a distinct cap-independent RNA vaccine platform with dose-sparing protective efficacy against HPAI H5 viruses.
Given the ongoing emergence of variants, research on vaccine platforms that can address multiple H5 strains is crucial for highly pathogenic avian influenza. This study provides preclinical evidence that cap-free, self-amplifying RNA can prevent H5N8 infection in mice at low doses and exhibits cross-reactivity against H5N1. Future studies should focus on safety and immunogenicity in humans, reproducibility of large-scale manufacturing, and direct comparison with existing mRNA vaccines. Therefore, this result should be viewed as an achievement that expands the scope of validation for next-generation RNA vaccine candidates, rather than the immediate availability of a usable vaccine.