Lipid Nanoparticle Development for A Fluvid mRNA Vaccine Targeting Seasonal Influenza and SARS-CoV-2

Abstract

mRNA vaccines represent a promising alternative to conventional vaccines, as demonstrated by the rapid deployment of mRNA vaccines during the recent COVID-19 pandemic. In this work, we have adapted and fine-tuned various reported mRNA lipid nanoparticle (LNP) synthesis and preparation procedures, evaluated a range of ionizable cationic lipids, and identified top-performing LNP formulations. The impact of uridine modification on mRNA's ability to trigger immune responses has also been explored. Our findings indicate that both unmodified mRNA and N1-methyl pseudouridine-modified mRNA successfully induced an antigen-specific antibody response in mice, while the methoxy uridine-modified mRNA did not. Based on these studies, we constructed a bivalent Fluvid mRNA vaccine, consisting of LNPs encapsulating uridine-unmodified mRNA encoding either a transmembrane domain-deleted hemagglutinin or the full-length native spike protein. This vaccine stimulated robust T cell and B cell immune responses and conferred 100% protective efficacy against challenge with either influenza or SARS-CoV-2 viruses in the mouse model, without compromising efficacy compared to administering each monovalent vaccine individually. Our data suggest that the multivalent mRNA vaccine can offer protection against different viruses by generating humoral and cellular responses against multiple antigens at the same time.

Fig. 1. Buffer optimization.

A Freeze-Thaw (F/T) effect on particle size, polydispersity index (PDI) and encapsulation of GFP mRNA LNPs prepared in different buffers (PNS buffer, phosphate-based buffer and Tris-based buffer). B 10% sucrose stabilizes mRNA LNP stored at 4 °C for 2 or 7 days, or upon 1 or 2 Freeze-Thaw (F/T) cycles. mRNA LNP was prepared in Tris/acetate pH 7.4 containing 10% sucrose.

Fig. 2. Characterization and comparison of various spike mRNA LNPs made of six different cationic lipids.

A Schematic of proposed mRNA Lipid nanoparticle structure. All LNPs were produced as described in “Methods”. B Weight change after immunizations. Mice (n = 5 per group) received two intramuscular injections at d0 (prime) and d14 (boost). C Anti-spike IgG at d14, d28, and d42 post prime immunizations. D Spike- specific IgG midpoint titers determined by Sigmoidal fit model from plasma titrations on Array. Statistical analysis was performed between DOTMA mRNA LNP group and other groups on each time point. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Only significant p values are shown.

Fig. 3. Neutralizing antibodies elicited by the top three LNP cationic lipids.

A SARS-CoV-2 Virus microneutralization assay on plasma samples collected at different time points from mice immunized with unmodified spike mRNA LNPs containing top ionizable lipids (ALC0315, SM102 or DODMA). Mice (n =5 per group) received two intramuscular injections at d0 (prime) and d14 (boost). Dashed line indicates the cutoff titer of 40. B Controls. Positive, hyperimmune plasma, n = 5. Naïve, normal mouse plasma, n = 3. Statistical analysis was performed between formulation groups vs Naïve. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Only significant p values are shown. nd, not determined.

Fig. 4. Three DODMA DOPE formulations with various compositions.

A Particle size, polydispersity index (PDI), zeta potential (ZP) and encapsulation efficiency. All LNPs were produced as described in “Methods”. B Body weight change following intramuscular immunizations. C Antibody profiling of d14, d28, and d42 plasma samples for anti-spike IgG reactivity. Statistical analysis was performed between 10% DSPC mRNA LNP group and other groups for each corresponding time point. *, p < 0.05; **, p < 0.01. Only significant p values are shown. N = 5 per group.

Fig. 5. Uridine unmodified vs modified mRNA LNPs.

A Structures of uridine, methoxy uridine (5MoU) and N1-methyl pseudouridine (m1Ψ). B Time course of IgG antibodies induced by mRNA LNPs encapsulated with unmodified or chemically modified spike mRNA. Statistical analysis was performed between 5MoU mRNA LNP group and other groups on the same time points. *, p < 0.05; **, p < 0.01. Only significant p values are shown. N = 5 per group.

Fig. 6. Multiplex cytokine profiling of the blood at 3 h post prime (d0) and 3 h post boost (d14) (n = 5).

Statistical analysis of various groups vs Buffer is shown on selected cytokines, using Kruskal–Wallis test with Dunn’s multiple groups correction. The full statistical analysis for all 13 cytokines is shown in Table 4. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Fig. 7. Antibody responses induced by mRNA LNP formulations.

A Total IgG responses against HA (H1N1) and spike (Wuhan strain) were detected from blood samples collected at d14 and d28 on protein microarray. B Spike (variant)- specific IgG antibody response. Statistical analysis of various groups vs Buffer was performed using Kruskal–Wallis test with Dunn’s multiple groups correction. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. N = 5 per group.

Fig. 8. Weight change following viral challenges.

A Intranasal challenge with influenza H1N1 virus (A/California/07/2009 (H1N1) x A/Puerto Rico/8/1934) at 10³ TCID₅₀/mL dose, on day 35 post prime immunization. B Intranasal challenge with 10⁴ TCID₅₀/mL SARS-CoV-2, mouse-adapted, MA10 Variant, on d42 post prime immunization. N = 5 per group.

Fig. 9. Multiplex cytokine profiling in overnight stimulated splenocyte cultures at 7 days post boost immunization.

Mouse splenocytes were stimulated with 10μg/mL (A) HA(H1N1) or (B) spike (Wuhan strain) for 18 h. Comparison between immunized groups vs PBS control group was performed using a Kruskal–Wallis test with Dunn’s multiple groups correction. The statistical analysis for all 12 cytokines is shown in Table 5. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. All other comparisons were non-significant. N = 5 per group.