Modified mRNA-LNP Vaccines Confer Protection against Experimental DENV-2 Infection in Mice

Figure 1. Design and Characterization of DENV-2 mRNA LNP Vaccines (A) Schematic illustration of DENV-2 mRNA comprising a 5′ cap, a 5′ UTR, a signal peptide, an antigen (either prME, E80, or NS1), a 3′ UTR, and a 3′ poly(A) tail. (B) Schematic representation of the LNP-encapsulated mRNA. (C) Cryo-electron microscopy image of a preparation of mRNA LNP. (D) Protein expression levels in mRNA-transfected HEK293T cells. Protein expression was analyzed by flow cytometry with an antibody to DENV envelope (4G2) or an antibody to the DENV NS1 protein using staining of mock-transfected HEK293T cells as negative control.

Figure 2. Immunization with Modified DENV-2 mRNA-LNPs Elicits Potent Antigen-Specific T Cell and Neutralizing Antibody Responses (A) Female BALB/c mice were immunized by i.m. injection with three doses of 20 μg nucleoside-modified prME-mRNA LNP, E80-mRNA LNP, NS1-mRNA LNP, or empty LNP at weeks 0, 2, and 4. Sera were collected at week 6. (B–D) Endpoint dilution titers were measured by ELISA plates coated with DENV-2 16681 virion (B), DENV-2 E80 protein (C), or NS1 protein (D). Sera neutralization against DENV-2 16681 was determined by PRNT50 assay (E). Each point represents an individual mouse, while horizontal lines indicate the mean. (F) At week 6, splenocytes were harvested and used for measuring antigen-specific IFNγ-producing T cells using the ELISPOT assay. Four peptide pools (P1, P2, P3, and P4) spanning the entire DENV-2 E80 protein and a single immunodominant epitope peptide from DENV2 NS1 (P265–273) were used for stimulation, while PBS was used as negative control. Four mice were used in each group. Statistical differences were determined using Student’s t test (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Figure 3. Evaluation of Immunogenicity of Various Doses of E80-mRNA or NS1-mRNA Vaccine Groups of female BALB/c mice were vaccinated via i.m. inoculation with 20 μg, 10 μg, or 5 μg E80-mRNA and NS1-mRNA, or empty LNP as negative control at weeks 0, 2, and 4, and sera were collected at week 6 for analysis. (A–C) Endpoint dilution titers were measured by ELISA plates coated with DENV-2 16681 virion (A), E80 protein (B), or NS1 protein (C). (D) Sera neutralization against DENV2 16681 was determined by PRNT50 assay. Each point represents an individual mouse, while horizontal lines indicate the mean. (E) Splenocytes were used to quantify antigen-specific IFNγ-producing T cells using ELISPOT assay with P2, P4, P265–273, or PBS control as stimuli. Three mice were used in each group. Statistical differences were determined using Student’s t test (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Figure 4. mRNA LNP Vaccines Confer Protection against Viral Challenge in BALB/c Mice (A) Female BALB/c mice (n = 10) were vaccinated via i.m. inoculation with 10 μg E80-mRNA, NS1-mRNA, or empty LNP at weeks 0, 2, and 4, and sera were collected at week 6 for analysis. At week 7, vaccinated BALB/c mice (n = 5) were administered 1 mg anti-INFα/βR blocking antibody and challenged with 5 × 106 PFUs of mouse-adapted DENV-2-GZ-LP 1 day later. (B–D) At days 2, 3, and 4 after viral challenge, binding antibody endpoint dilution titers were measured by ELISA with plates coated with DENV-2 16681 virion (B), E80 protein (C), or NS1 protein (D). (E) Sera neutralization against DENV-2 16681 was determined by PRNT50 assay. (F) Splenocytes were used to measure antigen-specific IFN-γ-producing T cells using the ELISPOT assay (n = 5). E protein peptide pools (P2 and P4), NS1 peptide (P265–273), or PBS was used for stimulation. (G and H) Viral loads in serum (G) or spleen (H) were measured by PRNT50 assay (G) or by qRT-PCR (H). Each point represents an individual mouse, while horizontal lines indicate the mean. Statistical differences were determined using Student’s t test (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <…

Figure 5. Characterization of ADE Activities and IgG Subclass Using Immune Sera Obtained from Mice Immunized with mRNA Vaccines (A) Assessing antibody-dependent enhancement of DENV-2 infection of immune sera in K562 cells. Serial dilutions of heat-inactivated mouse sera were pre-incubated with DENV-2, followed by the addition of K562 cells. Cells were harvested 48 h after infection with immune-complexes and stained with a primary antibody against E protein (4G2 antibody) and then with a secondary anti-mouse antibody labeled with Alexa Fluor 488. The percentages of infection were determined by fluorescence-activated cell sorting (FACS). (B) Sera from E80-mRNA vaccine (left) and E80-mRNA+NS1-mRNA vaccine (right) were analyzed for IgG1, IgG2a, IgG2b, and IgG3 ratio by isotype ELISA using coating antigen DENV-2 16681 virion.