Magnitude and Functionality of the NS1-Specific Antibody Response Elicited by a Live-Attenuated Tetravalent Dengue Vaccine Candidate

Abstract

Background: Dengue virus (DENV) can cause life-threatening disease characterized by endothelial dysfunction and vascular leakage. DENV nonstructural protein 1 (NS1) induces human endothelial hyperpermeability and vascular leak in mice, and NS1 vaccination confers antibody-mediated protective immunity. We evaluated the magnitude, cross-reactivity, and functionality of NS1-specific IgG antibody responses in sera from a phase 2 clinical trial of Takeda's live-attenuated tetravalent dengue vaccine candidate (TAK-003).

Methods: We developed an enzyme-linked immunosorbent assay to measure anti-DENV NS1 IgG in sera from DENV-naive or preimmune subjects pre- and postvaccination with TAK-003 and evaluated the functionality of this response using in vitro models of endothelial permeability.

Results: TAK-003 significantly increased DENV-2 NS1-specific IgG in naive individuals, which cross-reacted with DENV-1, -3, and -4 NS1 to varying extents. NS1-induced endothelial hyperpermeability was unaffected by prevaccination serum from naive subjects but was variably inhibited by serum from preimmune subjects. After TAK-003 vaccination, all samples from naive and preimmune vaccinees completely abrogated DENV-2 NS1-induced hyperpermeability and cross-inhibited hyperpermeability induced by DENV-1, -3, and -4 NS1. Inhibition of NS1-induced hyperpermeability correlated with NS1-specific IgG concentrations. Postvaccination sera also prevented NS1-induced degradation of endothelial glycocalyx components.

Conclusion: We provide evidence for functional NS1-specific IgG responses elicited by a candidate dengue vaccine.

Clinical trials registration: NCT01511250.

Keywords: Dengue virus; IgG response; nonstructural protein 1; vaccine.

Figure 1.

Serum NS1 IgG concentrations in recipients of TAK-003. Subject-matched serum samples from placebo or TAK-003 recipients that were either dengue virus (DENV)-naive or DENV-preimmune, obtained at different times pre- or postvaccination, were tested using the DENV-2 NS1 IgG indirect enzyme-linked immunosorbent assay. The OD₄₀₅ values were plotted against log₁₀ dilution factor. Dilution factors were interpolated for each sample at OD₄₀₅ of 0.5, and concentration was calculated relative to the assigned EC₅₀ of the reference standard/DENV-positive serum. Statistical analyses were performed using 1-way ANOVA, with Tukey multiple comparison. ****, P < .0001; *, P = .025; ns, not significant. Abbreviations: LLOQ, lower limit of quantitation.

Figure 2.

Cross-reactivity of the TDV-2 NS1 IgG response. Pre- and postvaccination (day 0 and day 120, respectively) serum samples from dengue virus (DENV)-naive or DENV-preimmune TAK-003 recipients were tested in the NS1 IgG indirect enzyme-linked immunosorbent assay using NS1 antigen from DENV-1, -2, -3, or -4. The concentration of NS1 IgG calculated relative to the DENV-positive control serum is plotted against times postvaccination for each serotype. Statistics were performed using 1-way ANOVA, with Sidak multiple comparison. ****, P < .0001; ns, not significant. Abbreviations: LLOQ: lower limit of quantitation

Figure 3.

Post-TAK-003 vaccination sera protect against dengue virus (DENV)-2 NS1-induced hyperpermeability, and protection correlates with IgG concentration. The effect of pre- or postvaccination sera on DENV-2 NS1-induced endothelial hyperpermeability was evaluated by TEER. Representative graphs are shown for (A) DENV-naive (subject ID: 1023014) and (B) DENV-preimmune (subject ID: 1052010) samples. HPMEC were grown on Transwell semipermeable membranes (0.4 µm pore size), and serum samples (30 µL) were added to the apical chamber in the presence or absence of 5 µg/mL DENV-2 NS1 (DENV-2 NS1, blue squares; day 0 serum alone, green diamonds; day 120 serum alone, red triangles; day 0 serum + DENV-2 NS1, inverted pink triangles; day 120 serum + DENV-2 NS1, light blue ×). Endothelial permeability was measured at indicated time points over 48 hours. Dotted line indicates change of medium. Relative TEER values from 1 independent experiment performed in duplicate are plotted. Error bars indicate SEM. C, Correlation analysis of 6 DENV-naive and 6 preimmune samples from both day 0 and day 120 (24 total samples). Area under the curve (AUC) values were calculated for each curve in each TEER experiment. The absolute reduction in relative TEER for each serum sample (A, B, and Supplementary Figures 3 and 4) was calculated by subtracting the AUC of NS1 + serum from the AUC of NS1 alone. Reduction was plotted against the log₁₀ of IgG concentrations for each sample, and a correlation analysis was performed. The dotted line represents the reduction of TEER by DENV-positive (+) control serum. Naive subjects, day 0 (filled) and day 120 (open) diamonds; preimmune subjects, day 0 (filled) and day 120 (open) squares; positive control, open black circle. Abbreviations: Ab, antibody; DENV-nv-TAK-003, DENV-naive TAK-003 recipients; DENV-pre-TAK-003, DENV-preimmune TAK-003 recipients; HPMEC, human pulmonary microvascular endothelial cells; TEER, transendothelial electrical resistance.

Figure 4.

A–C, Post-TAK-003 vaccination sera cross-protect against dengue virus (DENV)-1, -3, and -4 NS1-induced hyperpermeability, and protection correlates with IgG concentration. The effect of pre- or postvaccination sera on DENV-1, -3, or -4 NS1-induced endothelial hyperpermeability was evaluated by TEER. Correlation analysis of 3 DENV-naive and 3 preimmune samples pre- (day 0) and post- (day 120) vaccination was performed as follows. Area under the curve (AUC) values were calculated for each curve in each TEER experiment. The absolute reduction in relative TEER for each serum sample (A, B, and Supplementary Figures 6, 7, and 8) was calculated by subtracting the AUC of NS1 + serum from the AUC of NS1 alone. Reduction was plotted against the log₁₀ of IgG concentrations for each sample, and a correlation analysis was performed. The dotted line represents the reduction of TEER by DENV-positive (+) control serum. Naive subjects, day 0 (filled) and day 120 (open) diamonds; preimmune subjects, day 0 (filled) and day 120 (open) squares; positive control, open black circle. Abbreviations: Ab, antibody; DENV-nv-TAK-003, DENV-naive TAK-003 recipients; DENV-pre-TAK-003, DENV-pre-immune TAK-003 recipients; TEER, transendothelial electrical resistance.

Figure 5.

Correlation of cross-reactive magnitude and functionality: TAK-003-vaccinated versus dengue virus (DENV)-infected sera. Postvaccination (day 120) serum from 4 DENV-naive recipients of TAK-003 were pooled (Nv-TAK-003 pool). DENV (+) serum or Nv-TAK-003 pool serum (neat and 2-fold serially diluted sera) were evaluated by (A) DENV-1, (B) -2, (C) -3, and (D) -4 NS1 TEER and enzyme-linked immunosorbent assays, and reduction in relative TEER was correlated with NS1 IgG concentration against each DENV serotype. A dose-dependent correlation was observed between serum-driven protection in TEER and corresponding NS1 IgG concentration across all 4 serotypes, in both DENV-naive TAK-003 recipients and DENV-infected serum pools. The dotted line represents the reduction of TEER by neat DENV-positive (+) control serum. r values are indicated for each serum type, across each serotype. Abbreviations: Ab, antibody; TEER, transendothelial electrical resistance.

Figure 6.

Sera from TAK-003-vaccinated patients prevents dengue virus (DENV)-2 NS1-induced sialic acid and heparan sulfate degradation on human pulmonary microvascular endothelial cells (HPMEC). The effect of DENV-naive (1023014) or preimmune (1052010) sera on DENV-2 NS1-induced disruption of sialic acid (Sia) and heparan sulfate (HS). A, The integrity of the endothelial glycocalyx-like layer on HPMEC was assessed by the surface expression of sialic acid (red, top row) and heparan sulfate surface expression (green, bottom row) at 6 hours posttreatment with serum (as indicated) + DENV-2 NS1 (5 µg/mL) at 37°C, as visualized via confocal microscopy. Nuclei were stained with Hoechst (blue). Images are representative of 1 independent experiment performed in duplicate (×20; scale bar, 50 μm). B and C, Quantification of mean fluorescence intensity (MFI) of (B) sialic acid and (C) heparan sulfate expression from (A) and Supplementary Figure 5 from 1 independent experiment. Values normalized to MFI from the NS1 + positive control serum group (represented by dotted line at 100%) and expressed as percentage of control. Error bars indicate SEM.