Fig. 4: Highly presented antigens provide protection as mRNA vaccine candidates.

a C57BL/6J mice were vaccinated utilizing full length, mRNA-encoded Listeria antigens formulated in α-GC adjuvanted cationic LNPs. All seven antigen candidates were each tested in one independent experiment (experiments 1 and 2). Additional experiments for LMON_0149 and LLO_E262K are shown in Supplementary Fig. 5. Mice were vaccinated intravenously with cationic LNPs comprising 10 µg of Listeria antigen mRNA. As negative control and inter-experiment reference, 10 µg ovalbumin (OVA) mRNA was injected in both experiments. In experiment 1, a combination vaccine was administered containing 5 µg LLO_E262K and 5 µg LMON_2272 mRNA. In experiment 2, PBS injection was included as additional negative control, while low-dose Listeria monocytogenes EGD infection (1 × 10⁴ CFUs) served as positive control. Two weeks after prime vaccination, an identical booster was administered and 2 weeks later animals were challenged by i.v. injection of 7.5 × 10⁵ bacteria. Mice were euthanized 72 h post-challenge and bacterial loads in spleen and liver were assessed by CFU counting. Created with BioRender.com. b Bar charts depicting CFU counts in spleen (upper) and liver (lower) relative to the OVA negative control. All Listeria vaccines reduced the bacterial burden, while only LMON_0149, EF-Tu and the combination vaccine reached statistical significance in both organs (representative results from single experiments, two-tailed Mann-Whitney test, data are presented as mean values ± SD, n = 5 individual animals except in liver for LMON_2272 (n = 3), FtsI (n = 3), EF-Tu (n = 4), Listeria infection (n = 3), and in spleen for OVA (n = 4) and Listeria infection (n = 4), where plating of the excluded replicates did not yield CFUs). Pearson (c) and Spearman rank (d) correlations were calculated with GraphPad Prism 9.3 between the number of identified bacterial immunopeptides per vaccine candidate and vaccination efficacy expressed as % CFU reduction. For the combination vaccine, peptide numbers for both antigens were summed up. In both liver and spleen, a positive correlation between the number of presented immunopeptides and protective efficacy is indicated by positive r values, although without reaching statistical significance. Asterisks indicate p values with *p value < 0.05 and **p value < 0.01. Source data are provided as a Source Data file.