Extended Data Fig. 3: mRNA-1273 poly(A) tail extension in vivo and in macrophages.

Related to Fig. 2. a, Abundance of mRNA-1273 in tissues surrounding injection sites or in the dLNs, 2 h, 8 h, or 24 h following immunization, expressed as ΔCt (cycle threshold) mRNA-1273 Ct related to ActB Ct values. N = 5 independent biological replicates (mice) for each time point. Values from the same mouse at given time point indicated with matching shapes. Mean values ± s.e.m. indicated in red. b, eDRS-based mRNA-1273 poly(A) lengths distribution in muscle-resident cells collected from tissues surrounding injection sites 24 h after immunization with mRNA-1273 vaccine. c, Relative abundances of CD45⁺ (top left), CD45⁺ CD11C⁺ (top right), CD45⁺ F4/80&CD64⁺ MHCII^−/low CD11c⁺ (macrophages, bottom left), and CD45⁺ CD11c⁺ MHCII⁺ F4/80&CD64⁻ (DCs, bottom right) cell populations in muscles (injection sites) 6 h, 12 h, 24 h and 48 h following immunization with mRNA-1273. Mean values ± s.e.m. indicated in blue. N = 6 independent biological experiments (mice) for each time point. For gating strategy see Supplementary Fig. 5. d, cDNA-seq measured mRNA-1273 poly(A) lengths distribution in muscle-resident DCs (CD45⁺, I-A/I-E (MHCII)^high, CD11c⁺, F4/80⁻ and CD64⁻) and macrophages (CD45⁺, CD11c⁺, F4/80⁺, CD64⁺), sorted from vaccine injection sites 12 h and 24 h after administration. e, Levels of mRNA-1273 in muscle-resident macrophages and DCs, sorted out from injection sites 6 h, 12 h, 24 h and 48 h after mRNA-1273 administration. Values are shown as fold change of vaccine molecules per cell, relative to 6 h time point (top) or ΔCt (cycle threshold) mRNA-1273 Ct related to ActB Ct values (bottom). Mean values ± s.e.m. indicated in red. N = 3 independent biological experiments (mice) for each time point. f,g, Western blot analysis of spike protein expression showing efficient translation of mRNA-1273 in mBMDMs (f) or hMDMs (g) up to 96 h (f) or 72 h (g) after mRNA LNPs delivery. Western blot on spike protein, CD80 as the activation marker (g), α-tubulin and GAPDH levels and Ponceau S staining (bottom) served as loading controls. Representative pictures from 2 independent biological experiments. h, Representative raw DRS currents of mRNA-1273 poly(A) tails detected in samples 24 h after administration to mBMDMs. Unprocessed poly(A) tail with intact pentamer at the poly(A) 3’end (first panel), re-adenylated poly(A) tail with intact pentamer inside poly(A) (second panel), and re-adenylated poly(A) tails without signs of pentamer signature (third, fourth and fifth panel). Expected position of the mΨCmΨAG pentamer signature is marked with grey shading. Out of 100 randomly selected raw nanopore currents, only 2 contained pentamer remnants inside poly(A) stretch. i, eDRS-based BNT162b2 poly(A) lengths distribution in mBMDMs 12 h and 24 h after vaccine administration (related to Fig. 2d, 2 time points shown for clarity). Density plot, with values scaled in range 0–1, is shown. j, Spike protein production in mBMDMs up to 72 h after mRNA-1273 (left) or BNT162b2 (right) delivery. Western blot on spike protein, GAPDH, β-actin, and Ponceau staining (bottom) served as loading controls. Bottom, quantification of spike protein levels (normalized to β-actin levels, 4 independent biological experiments) ; mean values ± s.e.m. indicated in colour. k, Amount of spike protein produced 24 h and 48 h after a dose-dependent administration (0.2-10 µg of mRNA LNPs per well in 6-well plate) of mRNA-1273 (left) or BNT162b2 (right), assessed by ELISA from culture medium. N = 2 independent biological experiments. For b and d, reads are divided into containing (blue) or lacking (green) mΨCmΨAG sequence, with median values and fractions of elongated or shortened tails shown. Bottom panels: relative abundance of each group, with group sizes indicated. P values calculated with two-sided Wilcoxon test (on reads lacking mΨCmΨAG) with Benjamini–Hochberg correction. Data represent 2 independent biological experiments (b). For d, sequencing of material pooled from 6 mice was done.