Stabilized dengue virus 2 envelope subunit vaccine redirects the neutralizing antibody response to all E-domains

Abstract

The four dengue virus (DENV) serotypes cause several hundred million infections annually. Several live-attenuated tetravalent dengue vaccines (LAVs) are at different stages of clinical testing and regulatory approval. A major hurdle faced by the two leading LAVs is uneven replication of vaccine serotypes stimulating a dominant response to one serotype at the expense of the other three, leading to the potential for vaccine antibody (Ab)-enhanced, more severe infections by wild-type (WT) DENV serotypes that fail to replicate in the vaccine. Protein subunit vaccines are a promising alternative since antigen dosing can be precisely controlled. However, DENV envelope (E) protein subunit vaccines have not performed well to date, possibly due to differences between the monomeric structure of soluble E and the E homodimer of the viral surface. Previously, we have combined structure-guided computational and experimental approaches to design and produce DENV2 E antigens that are stable homodimers at 37℃ and stimulate higher levels of neutralizing Abs (NAbs) than the WT E antigen in mice. The goal of this study was to evaluate if DENV2 E homodimers stimulate NAbs that target different epitopes on E protein compared to the WT E monomer. Using DENV4/2 chimeric viruses and Ab depletion methods, we mapped the WT E-elicited NAbs to simple epitopes on domain III of E. In contrast, the stable E homodimer stimulated a more complex response toward all three surface-exposed domains of the E protein. Our findings highlight the impact of DENV2 E oligomeric state on the quality and specificity of DENV NAbs and the promise of DENV E homodimers as subunit vaccines.IMPORTANCEThe ideal dengue virus (DENV) vaccine should elicit a balanced and highly protective immune response against all four DENV serotypes. Current tetravalent live-attenuated DENV vaccines have faced challenges due to uneven replication of vaccine virus strains stimulating a strong immune response to one serotype and weak responses to the other three. Protein subunit vaccines provide novel opportunities to stimulate a balanced response because dosing can be precisely controlled and independent of vaccine virus replication. Here, we compare immune responses elicited by a new DENV serotype 2 protein vaccine designed to match the structure of proteins on the viral surface. We find that proteins designed to match the viral surface stimulate better immune responses targeting multiple sites on the viral surface compared to previous protein vaccines. Our results justify further testing and development of these second-generation DENV protein subunit vaccines.

Keywords: dengue; envelope protein; epitope mapping; flavivirus; neutralizing antibodies; protein subunit vaccine.

Fig 1

Immunogenicity of recombinant DENV2 E protein vaccines: two-dose study. (A) DENV2 soluble E structure (PDB 1OAN) of the vaccine antigens: WT and SD*FL with stabilizing amino acid mutations shown. (B) Six-week-old female Balb/c mice were immunized with 5 µg of WT or SD*FL rE ± Alum (125 µg) at week 0 (W0), and boosted with the same antigen ± Alum at week 3 (W3). IgG-binding titer at W12 against (C) DENV2, (D) DENV2 rE monomer (M2P4), and (E) DENV2 rE-stabilized dimer with mutated FL (SD*FL) was measured by serially diluting the mouse sera, and the resulting area under the curve (AUC) was reported. Due to limited sample availability, the number of mice in the SD*FL group in experiments (D) and (E) was reduced to n = 4. (F) To determine the ratio of dimer:monomer-binding Ab, the AUC value from dimer and monomer binding was used. The horizontal line represents the mean titer of all mice within the group. Statistical analysis was done using unpaired t-test (two-tailed, P < 0.05). (G) DENV2 50% neutralizing Ab titers at week 12. The limit of detection for this assay was 1:10. The horizontal line represents the geometric mean titer of all mice within the group. Statistical analysis was done on log-transformed values, followed by unpaired t-test (two-tailed, P < 0.05). The color and symbols match individual mice tested in different assays.

Fig 2

Immunogenicity of recombinant DENV2 E protein vaccines: three-dose study. (A) Six-week-old female Balb/c mice were immunized with 5 µg of WT or SD*FL rE following two different vaccination schedules: two-dose schedule—W0 and W3; three-dose schedule—W0, W3, and W8. Serum samples from each animal were collected pre- (W3, W8) and post-boost (W5, W10, W12). IgG binding against (B) DENV2, (C) DENV2 rE monomer (M2P4), and (D) DENV2 rE dimer with mutated fusion loop (SD*FL) was measured on W12 sera, and the resulting area under the curve (AUC) was determined. (E) We took the AUC value of the monomer and dimer binding to quantify the dimer:monomer Ab ratio. The horizontal line represents the mean titer of all mice within the group. Statistical analysis was done using unpaired t-test (two-tailed, P < 0.05). (F) DENV2 50% neutralizing Ab titers at week 12. The limit of detection for this assay was 1:10. The horizontal line represents the geometric mean titer of all mice within the group. Statistical analysis was done on log-transformed values, followed by unpaired t-test (two-tailed, P < 0.05). The color and symbols match individual mice tested in different assays.

Fig 3

Mapping DENV2 E-domains targeted by neutralizing Ab using DENV4/2 chimeric viruses. (A) DENV1–4 50% neutralizing Ab titers at week 12. (B) DENV4/2 chimera schematic with DENV2 domain substitution highlighted in yellow (PBD 4CBF). The neutralizing Ab titer elicited by WT and SD*FL rE vaccines is reported as the Neut₅₀ value against the parental viruses (A) DENV2 and DENV4 and against the various domains of DENV2 on a DENV4 backbone, (C) DENV4/2 EDI, (D) DENV4/2 EDII, and (E) DENV4/2 EDIII. The limit of detection for these assays is 1:60. The horizontal line represents the geometric mean titer of all mice within the group. Statistical analysis was done on log-transformed values, followed by unpaired t-test (two-tailed, P < 0.05). The same mouse in different experiments is represented with the same color and symbols.

Fig 4

Characterization of EDIII-binding antibody. (A) A schema for DENV2 EDIII depletion. First, we conjugated Magne HaloTag beads with either EDIII or HaloTag. Next, we treated the mouse sera with either EDIII- or HaloTag (control)-coated beads. Magnetic pulldown will separate any antibodies that bind to the coated beads, leaving behind unbound antibodies (non-simple EDIII or non-HaloTag antibodies) in the sera. Downstream-depleted samples analysis includes (B) DENV2 EDIII IgG binding, (C) DENV2 stabilized dimer with intact FL (SD) IgG binding, and (D) DENV2 neutralization, with limit of detection of DF120. (E) Percentage of NAb that targets other areas outside of simple EDIII region. Each data point is the average of each sample’s duplicates. (B, C, E) The horizontal line represents the mean of all mice within the group. Statistical differences between Halo- vs EDIII-depleted samples (B, C) were determined by paired t-test (one-tailed, P < 0.05). Unpaired t-test (one-tailed, P < 0.05) was used to determine the statistical differences for (E). (D) The horizontal line represents the geometric mean titer of all mice within the group. Statistical analysis was done on log-transformed values, followed by paired t-test (one-tailed, P < 0.05). The color and symbols match individual mice tested in different assays. AUC, area under the curve.