Impact of B18R-Encoding Messenger Ribonucleic Acid Co-Delivery on Neutralizing Antibody Production in Self-Amplifying Messenger Ribonucleic Acid Vaccines

Abstract

Objectives: The COVID-19 pandemic has brought mRNA vaccines to the forefront due to their widespread use. In this study, we explored the potential advantages of the self-amplifying mRNA (saRNA) vaccine over conventional mRNA vaccines. Methods: Initially, we optimized lipid nanoparticle formulations and employed dT20 affinity chromatography purification to improve the intracellular expression of saRNA. Subsequently, we demonstrated that saRNA exhibited sustained expression for up to one month, both in vitro and in vivo, in contrast to mRNA. Finally, we developed a saRNA-based COVID-19 vaccine and achieved superior immune protection in mice compared to mRNA vaccine by co-delivering the B18R-encoding mRNA. Results: The co-delivery of B18R-mRNA with the saRNA vaccine significantly enhanced neutralizing antibody responses, outperforming those induced by the mRNA vaccine alone. This co-delivery strategy effectively regulated the early innate immune activation triggered by saRNA, facilitating a more robust adaptive immune response. Conclusions: The optimization strategies we used in this study highlight the potential of saRNA vaccines to offer stronger and more durable immune protection. The insights gained from this study not only promote the advancement of saRNA vaccine development but also provide practical guidance for their broader application in the fight against infectious diseases.

Keywords: B18R; chromatography; lipid nanoparticles; self-amplifying mRNA; vaccine.

Figure 1

Optimization of saRNA enhances expression. (a) Transfection efficiency of saRNA encapsulated in LNPs formulated with different ionizable lipids in Huh7 cells. At a transfection dose of 500 ng per well, detection was performed 48 h post-transfection. (b) The physicochemical properties of SM102-LNP encapsulating saRNA. Integrity analysis of IVT-synthesized saRNA before purification (c) and after purification (d). Z1: Intact saRNA; Z2: Impurity from incomplete transcripts. (e) Comparison of positive rate and MFI in Huh7 cells using LNP–saRNA both before and after purification. At a transfection dose of 200 ng per well, detection was performed 48 h and 72 h post-transfection. **** p < 0.0001.

Figure 2

Validation of prolonged expression of saRNA. (a) Fluorescence of Huh7 cells transfected with saRNA at a dose of 200 ng per well was continuously monitored during cell passages. Scale bar: 400 μm. (b) Positive rate and MFI of varying doses of LNP–saRNA in Huh7 cells. Detection was performed 48 h and 72 h post-transfection. (c) Long-term expression comparison profile between 200 ng of LNP–saRNA and equimolar LNP–mRNA over multiple cell passages. Each time point corresponds to one passage, with a total of 10 consecutive passages completed. (d) Positive rate and MFI of varying doses of LNP–saRNA in Pan02 cells. Detection was performed 24 h and 48 h post-transfection. In vivo imaging (e) and quantitative comparison profile (f) of prolonged expression in mice, with 5 μg of LNP–saRNA injected intramuscularly into the left hindlimb muscle and an equimolar amount of LNP–mRNA injected intramuscularly into the right hindlimb. All cellular experiments were conducted using 24-well plates. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Figure 3

Vaccine expression analysis. (a) Schematic diagram of the mRNA and saRNA vaccines. Western blot analysis of different signal peptide variants of the mRNA vaccine (b) and saRNA vaccine (c) expression in Huh7 and Pan02 cells. Cell supernatants were collected 48 h after transfection with 200 ng of the vaccines. (d) Quantitative measurement of RBD concentrations in Huh7 and Pan02 cells transfected with the 200 ng saRNA vaccine or mRNA vaccine using the ELISA method. Cell supernatants were collected 48 h after transfection. (e) Western blot analysis of B18R expression in Huh7 and Pan02 cells. Cell supernatants were collected 48 h after transfection with 500 ng of the LNP–mRNA-encoding B18R. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Figure 4

Mice immunization experiment. (a) Schematic diagram of the immunization schedule and sample collection. IgG antibody titer (b) and NT₅₀ (c) at day 42 post prime immunization. (d) IgG2a/IgG1 ratio at various time points. ** p < 0.01; *** p < 0.001; ns, not significant.