A capless hairpin-protected mRNA vaccine encoding the full-length Influenza A hemagglutinin protects mice against a lethal Influenza A infection

Fig. 1. Capless mRNA vectors with double hairpins at the 5’ end demonstrated efficient IRES-initiated eGFP expression in MDCK cells. a mRNA vector schematics (see the text for structural details). b eGFP expression in MDCK cells 12 and 36 hours after transfection with equimolar amounts of the indicated mRNA vectors. (n = 4 independent experiments, *P < 0.05 shown for 12 hours only).

Fig. 2. DNA expression plasmids that transcribe mRNAs with self-folding double hairpins at the 5’ end cannot initiate translation in the absence of an IRES. a Vector schematics; relevant parts of the DNA plasmids and their projected mRNA transcripts are shown together. © in mRNA transcripts I, III, V, and VII indicates that the transcript has likely been capped in vivo; ? in Vectors II, IV, VI, and VIII indicates that the capping status of the transcript is unknown because it is not clear if mRNA with double hairpins in the 5’ end can be capped in vivo; ‘start’ - the transcription start site; ‘cleavage’ - the cleavage site within the SV40 polyadenylation signal; A and B - individual hairpin sequences folding into a double hairpin in mRNAs transcribed from DNA vectors II, IV, VI and VIII. b eGFP and mCherry expression in MDCK cells 48 hours after transfection with equimolar amounts of the indicated DNA plasmid. (n = 4 independent experiments).

Fig. 3. Double hairpins at the 5’ end interfere with cap-dependent translation of mCherry. a mRNA vector schematics; © in vectors 1C and 2C indicates that the transcript has been capped using an Anti-Reverse Cap Analog (ARCA); A and B: individual hairpin sequences that comprise the double hairpin in mRNA vectors 2 and 2C. b eGFP and mCherry expression in MDCK cells 12 h after mRNA transfection with equimolar amounts of each vector (n = 4 independent experiments).

Fig. 4. Capless IRES-initiated mRNA vectors with double hairpins at the 5’ end require a poly(A) tail for HA expression in MDCK cells and mouse myoblasts and induce anti-HA antibody production when delivered as a vaccine. a mRNA vector schematics. b HA expression in MDCK cells and mouse myoblasts (MB) 12 h after transfection with equimolar concentrations of the indicated mRNA vectors. ELISA data are normalized to the signal after transfection with vector E(HA) separately for MDCK cells and myocytes. (n = 4 independent experiments done at separate times, *P < 0.05). c HA expression in MDCK cells 12 h after transfection with equimolar concentrations of phosphatase-treated or -untreated mRNA vectors. ELISA data are normalized to the signal after transfection with the untreated mRNA vector E(HA). (n = 4 independent experiments done at separate times, *P < 0.05). d Anti-HA IgG titers three weeks after the last vaccination in individual mice immunized with a HA-mRNA vaccine that contained either the E(HA) or E(HA)-ph mRNA vectors. Control: data for non-immunized mice. For all comparisons, blood was collected 7 days before the first immunization and 21 days after the third immunization.

Fig. 5. Immunization with a vaccine that includes a capless mRNA that has double hairpins at the 5’ end and encodes the EMCV IRES linked to the HA ORF protects mice against a lethal dose of Influenza A. a Timeline of the experiment. b Schematic of the mRNA vectors used in the vaccines. c Anti-HA antibody titer 7 days before the first vaccination and then 21 days after the last vaccination (42 days after the first vaccination). d Survival data for vaccinated groups for the three weeks after challenge with the influenza A virus; no animals died after 3 weeks. e Weight loss in vaccinated mice after Influenza A (A/PR/8/34) virus infection ((50 × LD50) (LD50 = 95 PFU/mouse) for the first three weeks after challenge. f Clinical scores assigned for eGFP-mRNA vaccinated mice for the first ten days after a live Influenza A virus challenge. g Clinical score assigned for HA-mRNA vaccinated mice for the first ten days after a live Influenza A virus challenge. The following clinical scores were assigned: 0 = normal, 1 = slightly ruffled, 2 = ruffled fur, 3 = ruffled fur and inactive, 4 = hunched/moribund, and 5 = dead.