A multiple-target mRNA-LNP vaccine induces protective immunity against experimental multi-serotype DENV in mice

Abstract

Dengue virus (DENV) is a mosquito-borne virus with a rapid spread to humans, causing mild to potentially fatal illness in hundreds of millions of people each year. Due to the large number of serotypes of the virus, there remains an unmet need to develop protective vaccines for a broad spectrum of the virus. Here, we constructed a modified mRNA vaccine containing envelope domain III (E-DIII) and non-structural protein 1 (NS1) coated with lipid nanoparticles. This multi-target vaccine induced a robust antiviral immune response and increased neutralizing antibody titers that blocked all four types of DENV infection in vitro without significant antibody-dependent enhancement (ADE). In addition, there was more bias for Th1 than Th2 in the exact E-DIII and NS1-specific T cell responses after a single injection. Importantly, intramuscular immunization limited DENV transmission in vivo and eliminated vascular leakage. Our findings highlight that chimeric allogeneic structural and non-structural proteins can be effective targets for DENV vaccine and that they can prevent the further development of congenital DENV syndrome.

Keywords: Dengue virus (DENV); Envelope domain III (E-DIII); Immune response; Multi-serotype; Non-structural protein 1 (NS1); mRNA vaccine.

Fig. 1

Design and evaluation of physicochemical properties of dengue virus (DENV) mRNA vaccine candidates. A Schematic diagram of the construction of two DENV mRNA vaccine candidates. Through codon optimization, the heterochimeric E-DIII and NS1 constructs were fused to an N-terminal tPA signal sequence (tPA), a C-terminal vesicular stomatitis virus G protein transmembrane and cytoplasmic domain (VSV-G TM ​+ ​CD), and cloned into a DNA plasmid. The combination of serotype 1 (DENV-1) and serotype 2 (DENV-2) is named DENV-a, and DENV-b is formed by the chimera of serotype 3 (DENV-3) and serotype 4 (DENV-4). B Schematic diagram showing the proposed mechanism for mRNA vaccine candidate translation in cytoplasm. The dotted arrows indicate the order in which the proteins are translated for processing. C Immunofluorescence co-localization of DENV-a and DENV-b expressing viral proteins. The E-DIII (Green) was labeled using the mAb 4E11, the NS1 (Red) was detected with 1A3. The nuclei were stained with DAPI (blue). Scale bar, 25 ​μm. D Western blotting was used to detect HEK-293T cells transfected with DENV-a and DENV-b lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) for 24 ​h. The first column represents the molecular mass (M) of the protein. E Transmission electron microscopy images showing representative DENV-a and DENV-b nanoparticles, respectively (scale bar, 100 ​nm).

Fig. 2

Screening of different doses of DENV mRNA candidate vaccine. A Grouping of vaccine candidates at different doses (2, 5, and 20 ​μg). Female C57BL/6 mice (n ​= ​5) aged from 6 to 8 weeks were immunized every 2 weeks, and serum was collected for analysis at 6 weeks after enhanced immunization. Endpoint dilution titers of each group were measured by ELISA plates coated with DENV-1 E-DIII and DENV-2 NS1 protein (B), DENV-4 E-DIII and DENV-3 NS1 protein (C). The empty-LNP group was given a placebo. D–F Neutralizing antibody titers against DENV-1(D), DENV-2 (E), DENV-3 (F), and DENV-4 (G) were analyzed using the FRNT₅₀ assay. The dashed line indicates the detection limit. Data are presented as means ​± ​SEM. Significant differences were determined by one-way ANOVA (∗P ​< 0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001 and ∗∗∗∗P ​< ​0.0001; ns indicates not significant).

Fig. 3

Continuous humoral immune evaluation of DENV vaccines candidates. DENV-a, DENV-b, and DENV-ab vaccines candidates (5 ​μg) were intramuscularly injected into female mice aged from 6 to 8 weeks (n ​= ​5). Serum was collected at the specified time to determine the antibody neutralization titers and endpoint titers. (A–D) Neutralization titers of serum of immunized mice against DENV-1 (A), DENV-2 (B), DENV-3 (C), and DENV-4 (D) were analyzed by PRNT₅₀ assays at 6 weeks after prime vaccination. (E–G) End-point titers of E-DIII and NS1 specific IgG subclasses in mice of DENV-a (H), DENV-b (I) and DENV-ab (J) after booster immunization with 5 ​μg mRNA vaccine (week 6). W on the X-axis on represents weeks. (H–J) The Th1/Th2 dominance determined as the end-point titers of IgG2a and IgG1. All of the significant differences were relative to the DENV-ab group. Data are presented as means ​± ​SEM. Significant differences are shown as ∗P ​< ​0.05, ∗∗P ​< ​0.01, and ∗∗∗∗P ​< ​0.0001; ns indicates not significant.

Fig. 4

Cytokine profile of responses induced by vaccine candidates. Spleen cells were re-stimulated with chimeric E-DIII ​+ ​NS1 protein, and cytokine secretion was analyzed by fluorescence-activated cell sorting and ELISPOT assays at 30 weeks after enhanced immunization (n ​= ​5). All tests were independent and included positive and negative controls. (A, B) FCM analysis of the intracellular cytokine IFN-γ induced by CD4⁺T cells and CD8⁺T cells after stimulation with mRNA-LNP vaccine candidates. C The IFN-γ secretion of 2 ​× ​10⁵ spleen cells was determined by ELISPOT assays upon re-stimulation with 10 ​μg/mL of E-DIII ​+ ​NS1 chimeric protein. D Representative FCM analysis plots of intracellular cytokines and gates from single mice in each group. Each mouse had a total of 2 ​× ​10⁶ ​cells. Cells were pre-gated on live/dead-CD45⁻CD3⁻CD4/CD8. Data are presented as means ​± ​SEM. Statistical analysis was performed using one-way ANOVA. (∗P ​< ​0.05, ∗∗P ​< ​0.01, and ∗∗∗∗P ​< ​0.0001; ns. indicates not significant).

Fig. 5

Evaluation of challenge protection in vaccine-immunized mice. A Flow chart of the animal experiment. Three weeks after immunization enhancement, C57BL/6 mice (n ​= ​5) were subcutaneously inoculated with a total of 4 ​× ​10⁵ ​PFU of DENVs (the PFU ratio of the four viruses was 1:1:1:1:1). One day before immunization, C57BL/6 mice were intraperitoneally inoculated with 1 ​mg of anti-IFNαR1 monoclonal antibody. Lungs were collected at 4 days post-infection (dpi). NT₅₀ titers against DENV (B) and endpoint titers of IgG (C) were measured by FRNT₅₀ and ELISA assays. Body weight changes (D) and viral titers in blood (E) were recorded on 2, 3, and 4 dpi. F Quantification of Evans blue dye extracted from the lungs of DENV-infected mice treated with vaccine or empty LNP. Lung tissue was collected and soaked in 1 ​mL formamide to determine the absorbance value at OD₆₁₀. G Representative images of Evans blue-stained lung tissue from mice. PBS is shown as a negative control. Data are presented as means ​± ​SEM. Statistical analysis was performed using one-way ANOVA (∗P ​< ​0.05, ∗∗P ​< ​0.01, and ∗∗∗P ​< ​0.001; ∗∗∗∗P ​< ​0.0001; ns indicates not significant).

Fig. 6

Antibody-dependent analysis of DENV 1–4 infection in K562 ​cells with monotype vaccine candidate and mixed vaccine candidate, respectively. C57BL/6 mice were immunized with 5 ​μg monotype vaccine candidate (DENV-a and DENV-b) or mixed vaccine candidate (DENV-ab) mRNA LNP and mixed with DENV-1, DENV-2, DENV-3, and DENV-4 in serially diluted serum at week 6 and incubated with Fc-γ receptor expressing K562 ​cells. The cell infection was quantitated by flow cytometry (FCM). Representative curves for each serotype are shown for groups A, B, C, and D. All significant differences were relative to the DENV-ab group. Red represents comparisons between DENV-ab and DENV-a, and green represents differences with DENV-b. Data are presented as means ​± ​SEM. Significant differences were determined by one-way ANOVA (∗P ​< ​0.05, ∗∗P ​< ​0.01, and ∗∗∗P ​< ​0.001; ∗∗∗∗P ​< ​0.0001).