The impact of nucleoside base modification in mRNA vaccine is influenced by the chemistry of its lipid nanoparticle delivery system

Abstract

The use of modified nucleosides is an important approach to mitigate the intrinsic immunostimulatory activity of exogenous mRNA and to increase its translation for mRNA therapeutic applications. However, for vaccine applications, the intrinsic immunostimulatory nature of unmodified mRNA could help induce productive immunity. Additionally, the ionizable lipid nanoparticles (LNPs) used to deliver mRNA vaccines can possess immunostimulatory properties that may influence the impact of nucleoside modification. Here we show that uridine replacement with N1-methylpseudouridine in an mRNA vaccine encoding influenza hemagglutinin had a significant impact on the induction of innate chemokines/cytokines and a positive impact on the induction of functional antibody titers in mice and macaques when MC3 or KC2 LNPs were used as delivery systems, while it impacted only minimally the titers obtained with L319 LNPs, indicating that the impact of nucleoside modification on mRNA vaccine efficacy varies with LNP composition. In line with previous observations, we noticed an inverse correlation between the induction of high innate IFN-α titers in the macaques and antigen-specific immune responses. Furthermore, and consistent with the species specificity of pathogen recognition receptors, we found that the effect of uridine replacement did not strictly translate from mice to non-human primates.

Keywords: MT: Oligonucleotides: Therapies and Applications; N1-methylpseudouridine; delivery system; immune response; innate immunity; ionizable lipids; lipid nanoparticle; mRNA modification; mRNA vaccine.

Graphical abstract

Figure 1

Kinetics of Cy5-Fluc mRNA fluorescence and expression after i.m. delivery via different LNPs (A) Representative IVIS images of cyanine-5 fluorescence signal in mice injected with 5 μg of naked cyanine-5-labeled, 5- methoxyuridine-modified CleanCap FLuc mRNA or the same mRNA encapsulated within MC3, KC2, or L319 LNPs. (B) Quantification of the fluorescence signal by total radiance efficiency (AU = (ph/s)/(μW/cm²)) in the injected site at 0, 2, 4, 6, 24, 48 and 72 h post injection. (n = 4 to 5 mice per group). Data are presented as mean values +/− SEM. (C) Representative bioluminescence images of luciferase expression in the Cy5-Fluc mRNA-injected mice. (D) Quantification of the bioluminescence signal (AU = photons/s) in the injected site at 6, 24, 48 and 72 h post injection. (n = 4 to 5 mice per group). Data are presented as mean values +/− SEM.

Figure 2

Comparison of MC3, KC2, and L319 LNPs in mice and NHPs with unmodified and 1MpU-modified mRNA (A) Individual and mean HI titers measured in sera collected from Balb/c mice (n = 8/group) after a single immunization (D20) and two immunizations (D42) with 5 μg of a monovalent A/California/07/2009 (H1N1) split influenza vaccine (Vaxigrip; MIV) or with 5 μg of unmodified mRNA (UNR) or 1MpU-modified mRNA (MNR) encoding full-length hemagglutinin (HA) of closely related influenza virus strain A/Netherlands/602/2009 (H1N1). The mRNA was encapsulated into MC3 (green), KC2 (purple), or L319 (orange) LNPs. Immunization was performed by 2 i.m. injections into the quadriceps given 3 weeks apart (D0, D21) under a final volume of 50 μL of PBS. For each lipid, UNR versus MNR comparison was performed by ANOVA after log transformation of the HI titers. When difference was significant, p-value was reported directly on the graph (B) 500 μL fractions of the mRNA vaccines used for the immunization of mice (i.e., 50 μg of UNR or MNR) were used to immunize cynomolgus macaques. The macaques (n = 4/group) were immunized by 2 i.m. injections into the deltoid given 4 weeks apart (D0, D28). A control group received 15 μg of the monovalent A/California/07/2009 Vaxigrip (MIV) following the same schedule. Individual and mean HI titers measured after a single immunization (D28) and after two immunizations (D55 and D85) are shown. A given symbol was attributed to each individual macaque from the different groups and kept the same throughout the graphs. (C) 8 weeks following the second immunization (D85), PBMCs were collected from the macaques for the determination of antibody-secreting cells (ASCs). The percentage of antigen-specific ASCs to the total IgG + ASCs is shown for each individual macaque represented by its symbol. The bar represents the mean for the group.

Figure 3

T cell responses in NHPs immunized with MC3, KC2, and L319 LNPs loaded with unmodified and 1MpU-modified mRNA 4 weeks following the second immunization (D55), PBMCs were collected from the macaques and analyzed for (A) IFN-γ and (B) IL-13 secreting cells by FluoroSpot assay. Cell counts from each individual macaque are shown after subtracting counts obtained with PBMCs collected from the same animal pre-immunization. Each macaque can be identified on the graph with its symbol attributed in Figure 2. The bar represents the mean for the group.

Figure 4

Innate responses in NHPs immunized with MC3, KC2, and L319 LNPs loaded with unmodified and 1MpU-modified mRNA 6 and 24 h following the first immunization, serum samples from macaques of Figure 2 were tested for innate chemokines/cytokines by MSD assay. Measured levels of (A) eotaxin, (B) MCP-1, (C) I-TAC, (D) IL-6, (E) IL-1RA, and (F) IFN-α2a are represented in pg/mL after subtracting background levels measured pre-immunization for each individual macaque identified by its symbol attributed in Figure 2. The bar represents the mean for the group. Levels of IL-1β, IL-8, and IL-17A did not increase over background and are not represented.

Figure 5

Blood biomarkers in NHPs immunized with MC3, KC2, and L319 LNPs loaded with unmodified and 1MpU-modified mRNA 7 days before immunization (D-7) and 2 days following each immunization (D2, D30), plasma samples from the macaques were analyzed for standard blood parameters: alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, lipase, and C-reactive protein (CRP). Protein concentrations are shown for each individual macaque identified by its symbol attributed in Figure 2. Values are expressed in IU/L for ALP, ALT, AST, and lipase, in μmol/L for creatinine, and in mg/L for CRP. The bars represent the mean from each group. Lower and upper dashed lines in each graph represent the fifth and 95^th percentile, respectively, of reference pre-dose values commonly observed in cynomolgus macaques based on historical data. The fifth to 95^th percentile range covers 90% of these pre-dose data.