A single immunization with core–shell structured lipopolyplex mRNA vaccine against rabies induces potent humoral immunity in mice and dogs

Figures & data

Figure 1. Construction and characterization of the LPP-mRNA-G vaccine in vitro and in vivo. (A) Schematic of nucleoside-modified RABV mRNA. Each structural element is optimized and modified to modulate mRNA stability, translation ability, and immunostimulatory characteristics. (B) Schematic of the LPP-mRNA-G vaccine construct. (C) Representative transmission electron microscopy image of LPPs in solution following mRNA encapsulation. Scale bar, 50 nm. (D) LPP particle size by dynamic light scattering. (E) Zeta potential of LPPs at pH 4.0 and 7.4. For c and e, one representative result from three independent experiments is shown.

Figure 2. Transfection efficiency of LPP-mRNA-G in cells and biodistribution in the mouse body. (A) HEK-293 T cells were transfected with LPP-mRNA-G. G protein levels in cell lysates at 24 h were measured by western blotting. (B) Transfection efficiency of LPP-mRNA-G. The mRNA-G was transfected into HEK-293 T cells using LPP or the commercial transfection reagent, jetPRIME. The expression of G protein was observed under fluorescence microscopy after incubation for 3, 12, 72, or 96 h. Scale bar, 200 µm. (C) Duration and distribution of LPP-mRNA-G in vivo. Groups of ICR mice (n = 3) were vaccinated with one injection of 15 µg LPP-mRNA-G. Three mice per group were euthanized at 8, 24, 48, and 72 h after inoculation. Draining lymph node, muscle, brain, heart, liver, kidney, spleen, and lung samples were harvested from each mouse for quantitation of mRNA-G levels using b-DNA assays. The pie chart represented the of G-mRNA accumulative distribution of the sum at each timepoint (8, 24, 48, and 72 h) post immunization (top right corner panel). Data are presented as the mean. (D) Duration and distribution of G protein production from LPP-mRNA-G in vivo. Groups of ICR mice (n = 3) were vaccinated with one injection of 10 µg LPP-mRNA-G. Three mice per group were euthanized at 8, 24, 48, and 72 h after inoculation. Draining lymph node, muscle, brain, heart, liver, kidney, spleen, and lung samples of each mouse were harvested for quantitation of G protein expression levels by ELISA. The pie chart represented the of G protein accumulative distribution of the sum at each timepoint (8, 24, 48, and 72 h) post immunization (top right corner panel). Data are presented as the mean. (E) Expression levels of G protein were further detected by immunofluorescence assays using monoclonal antibodies against G protein in lymph nodes and muscle tissues. mRNA concentrations were determined by in situ hybridization using probes specific for the mRNA component of LPP-mRNA. Scale bars, 200 or 50 µm.

Figure 3. A single dose of LPP-mRNA-G vaccination induces high levels of antibody production in mice. (A) Vaccine immunization strategy. Groups of mice were immunized by intramuscular injection of 5, 1, or 0.2 µg of LPP-mRNA-G. A licensed commercial inactivated rabies vaccine (ITV) was included as a positive control and the same volume of PBS was injected as a placebo. Serum was collected for IgG and VNA testing for 8 consecutive weeks. The total IgG and VNA produced in PBS-inoculated mice were undetectable. (B) Detection of RABV IgG antibody by ELISA. One-way ANOVA was used to evaluate intergroup differences. ns, no significant difference; *, P < 0.05; **, P < 0.01. Error bars indicate means with SEM (n = 10). (C) VNA titers were measured using fluorescent-antibody virus neutralization (FAVN) assays. One-way ANOVA was used to evaluate intergroup differences. ns, no significant difference; *, P < 0.05; **, P < 0.01. Error bars indicate means with SEM (n = 10).

Figure 4. LPP-mRNA-G facilitates the generation of Tfh cells, GC B cells, PCs, and RABV-specific ASCs in mice. (A) Representative flow cytometric plots of Tfh cells. C57BL/6 mice were immunized with 5 µg of LPP-mRNA-G, 0.1 dose of ITV vaccine, or DMEM. At 7 and 14 dpi, inguinal LN cells were stained with markers of Tfh cells (CD4⁺ CXCR5⁺ PD1⁺). (B) Statistical analyses of CD4⁺ CXCR5⁺ PD1⁺ Tfh cell plots. One-way ANOVA was used to evaluate intergroup differences. *, P < 0.05; ***, P < 0.001. Error bars indicate means with SD (n = 5). (C) Representative flow cytometric plots of GC B cells. At 7 and 14 dpi, inguinal LN cells were stained with markers of GC B cells (B220⁺ GL7⁺ CD95⁺). (D) tatistical analyses of B220⁺ GL7⁺ CD95⁺ GC B cell plots. One-way ANOVA was used to evaluate intergroup differences. *, P < 0.05; ***, P < 0.01. Error bars indicate means with SD (n = 5). (E) Representative flow cytometric plots of PCs. At 7 and 14 dpi, BM cells were stained with markers of PCs (B220^low CD138⁺). (F) Statistical analyses of B220^low CD138⁺ PC plots. One-way ANOVA was used to evaluate intergroup differences. *, P < 0.05; ***, P < 0.0001. Error bars indicate means with SD (n = 5). (G) Representative images of ELISpot assays. At 7 and 14 dpi, inguinal LN cells were prepared and RABV-specific ASCs were counted by ELISpot assay. (H) Statistical analyses of the number of RABV-specific ASCs. One-way ANOVA was used to evaluate intergroup differences. *, P < 0.05; ***, P < 0.0001. Error bars indicate means with SD (n = 5).

Figure 5. LPP-mRNA-G induces strong secondary antibody response in mice. (A) Representative flow cytometric plots of MBCs. C57BL/6 mice were immunized with 5 µg of LPP-mRNA-G, 0.1 dose of ITV vaccine, or DMEM. At 100 dpi, inguinal LN cells were stained with markers of MBCs (B220 ⁺CD38 ⁺CD138⁻). (B) Statistical analyses of B220 ⁺CD38 ⁺CD138⁻ MBC plots. One-way ANOVA was used to evaluate intergroup differences **, P < 0.01. Error bars indicate means with SD (n = 5). (C) Representative flow cytometric plots of PCs. Mice (n = 5) were boosted 5 µg of LPP-mRNA-G, 0.1 dose of ITV vaccine, or DMEM at 100 dpi after primary immunization. At 14 days after the second dose, single BM cells were stained with markers of PCs (B220^low CD138⁺). (D) Statistical analyses of B220^low CD138⁺ PC plots. One-way ANOVA was used to evaluate intergroup differences. **, P < 0.01. Error bars indicate SD of the mean (n = 5). (E) Representative images of ELISpot assays. At 14 days after the second dose, inguinal LN cells were prepared and RABV-specific ASCs were counted by ELISpot assay. (F) Statistical analyses of the number of RABV-specific ASCs. One-way ANOVA was used to evaluate intergroup differences. ****, P < 0.0001. Error bars indicate means with SD (n = 5). (G) Detection of RABV IgG antibody by ELISA before and after the second dose (14 dpi). One-way ANOVA was used to evaluate intergroup differences. ns, no significant difference; ***, P < 0.001. Error bars indicate means with SEM (n = 5). (H) VNA titers were measured by fluorescent-antibody virus neutralization (FAVN) assays before and after the second dose (14 dpi). One-way ANOVA was used to evaluate intergroup differences. ns, no significant difference; ***, P < 0.001. Error bars indicate means with SEM (n = 5).

Figure 6. A single immunization of LPP-mRNA-G provides full protection against lethal RABV challenge in mice. (A) Scheme of LPP-mRNA-G immunization and RABV challenge. Each mouse received intramuscular injections of 5, 1, or 0.2 µg LPP-mRNA-G followed by intracranial injection of 50 LD₅₀ of the RABV strain, CVS-24. Mice receiving a 0.1 dose of ITV vaccine, were used as positive controls. (B) Survival rates. A log-rank test was used to evaluate intergroup differences in survival rates. *, P < 0.05; **, P < 0.01. (C) Changes in body weight. Error bars indicate means with SD (n = 10).

Figure 7. A single dose of LPP-mRNA-G elicits potent antibody production in dogs. (A) Scheme of LPP-mRNA-G immunization and evaluation. Beagles received three different doses (10, 2, or 0.4 µg) of LPP-mRNA-G by intramuscular injection. After inoculation, injection sites were continuously observed for signs of adverse reactions. Body temperature and body weight were measured daily. Animals in the simulation group received injections of PBS. Beagles receiving one dose ITV vaccine, were used as positive controls. Sera were collected at the indicated time points. (B) Detection of RABV IgG antibody by ELISA. Error bars indicate SEM of the means (n = 5). (C) VNA titers were measured using fluorescent-antibody virus neutralization (FAVN) assays. Error bars indicate means with SEM (n = 5). (D) The proportion of dogs with seroconversion induced by vaccination. VNA values greater than 0.5 IU/ml were considered positive.

Data availability

All data associated with this study are present in the paper or the Supplementary Materials. Request for resources, data and reagents should be directed to the lead contact, Ling Zhao ([email protected]). All unique reagents described in this study are available upon request to the lead author with a completed Materials Transfer Agreement.