Development of a Novel Assay to Assess the Avidity of Dengue Virus-Specific Antibodies Elicited in Response to a Tetravalent Dengue Vaccine

Isamu Tsuji, David Dominguez, Michael A Egan, Hansi J Dean, Isamu Tsuji, David Dominguez, Michael A Egan, Hansi J Dean

Abstract

Antibody affinity maturation is a critical step in development of functional antiviral immunity; however, accurate measurement of affinity maturation of polyclonal serum antibody responses to particulate antigens such as virions is challenging. We describe a novel avidity assay employing biolayer interferometry and dengue virus-like particles. After validation using anti-dengue monoclonal antibodies, the assay was used to assess avidity of antibody responses to a tetravalent dengue vaccine candidate (TAK-003) in children, adolescents, and adults during two phase 2 clinical trials conducted in dengue-endemic regions. Vaccination increased avidity index and avidity remained high through 1 year postvaccination. Neutralizing antibody titers and avidity index did not correlate overall; however, a correlation was observed between neutralizing antibody titer and avidity index in those subjects with the highest degree of antibody affinity maturation. Therefore, vaccination with TAK-003 stimulates polyclonal affinity maturation and functional antibody responses, including neutralizing antibodies.

Clinical trials registration: NCT01511250 and NCT02302066.

Keywords: BLI; TAK-003; VLP; antibody; avidity; biolayer interferometry; dengue; vaccine; virus-like particle.

© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.

Figures

Figure 1.
Figure 1.
Reactivity of anti-dengue monoclonal antibodies to biotinylated dengue VLPs. A, SDS-PAGE of dengue VLPs. VLP were reduced and 20 μg of VLP was applied to 10%–20% tricine SDS-PAGE and stained by Simple Stain Blue (ThermoFisher Scientific). B, Reactivity of anti-dengue monoclonal antibodies panel. Response of 10 μg/mL of anti-dengue mAbs at 600 seconds association was measured by Octet Red or Octet HTX systems using 5 μg/mL biotinylated VLP in SA or SAX biosensor. Data of 4G2, WNV-E60, 2D22, and DV4-75 were measured 3 times and the averages are shown. 1M7, DENV-E106, and 5J7 were measured once. C, Dose dependence of anti-dengue monoclonal antibodies. 4G2, WNV-E60, and DV4-75 antibody clones were diluted from 0.01 to 10 μg/mL to measure response at 600 seconds of association. All data were measured 3 time and averages ± SD are shown. The mAb clone, specificity, and epitope of dengue E-protein were: 4G2, CR, fusion loop [25]; WNV-E60, CR, fusion loop [26]; 1M7, CR, fusion loop [27]; DENV1-E106, DENV-1 specific, QE [28]; 2D22, DENV-2 specific, QE [29]; 5J7, DENV-3 specific, QE [30]; and DV4-75, DENV-4 specific, QE [31]. Abbreviations: CR, cross-reactive; DENV, dengue virus; IgG, immunoglobulin G; mAb, monoclonal antibody; QE, quaternary E-protein; SA, streptavidin; SAX, high-precision streptavidin; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; VLP, virus-like particle.
Figure 2.
Figure 2.
Optimization of anti-dengue polyclonal antibody purification from human serum. A, Optimization of Protein G Sepharose elution conditions: 0.2 mL of dengue standard serum was mixed with 3 mL of Protein A MAPSII binding buffer and 0.6 mL of 50% Protein A Sepharose or 3 mL of DPBS and 0.6 mL of 50% Protein G Sepharose for 90 minutes, washed with Protein A MAPSII binding buffer or DPBS, and eluted in 0.1 M glycine buffer pH 3.0–2.5. Eluate were neutralized to pH 7.0–7.5 immediately with 1 M Tris-HCl pH 8.0 and exchanged buffer to DPBS using Amicon Ultra-4. Purity was measured by NuPAGE (ThermoFisher Scientific) and purified antibody avidity was measured using biotinylated DENV-1 VLP and Octet RED system (FortéBio). B, Optimization of serum volume: 0.03–0.2 mL of clinical trial DEN-203, volunteer No. 1044010, day 90 serum was mixed with 3 mL of DPBS and 0.6 mL of 50% Protein G Sepharose for 90 minutes and washed with DPBS and eluted in 0.1 M glycine buffer pH 2.7. Eluate was neutralized to pH 7.0–7.5 immediately with 1 M Tris-HCl pH 8.0 and exchanged buffer to DPBS using Amicon Ultra-4. Purity was measured by NuPAGE and purified antibody avidity was measured using biotinylated DENV-1 VLP and Octet RED system. Abbreviations: DENV, dengue virus; DPBS, Dulbecco’s phosphate-buffered saline; VLP, virus-like particle.
Figure 3.
Figure 3.
Optimization of avidity assay. A, Optimization of association and VLP concentration. Antibody avidity index was measured using Octet HTX (FortéBio) and DENV-1, 2, 3, and 4 VLP. Anti-dengue polyclonal antibody (125 μg/mL) purified from clinical trial DEN-203 volunteer serum (DENV-1, volunteer No. 1071001 day 90; DENV-2, volunteer No. 1071009 day 90; DENV-3, volunteer No. 1083005 day 0; and DENV-4, volunteer No. 1093001 day 90) was used to measure association and dissociation rates. Association times were 600, 900, and 1800 seconds at biotinylated DENV VLP 5 μg/mL, and biotinylated VLP concentrations were 1, 3, and 5 μg/mL at 1800 seconds association time. Data were measured 8 times and bar graph shows averages ± SD. B, Optimization of dissociation rate analysis. Biosensorgram of anti-dengue polyclonal antibody purified from DEN-203 volunteer serum (volunteer No. 1081001, day 0; 250 μg/mL) was generated by Otet RED systems and 5 μg/mL DENV-3 VLP. Dissociation calculation was analyzed from 30 to 600 seconds (red) and from 30 to 1200 seconds (blue). Dissociation rate was measured by 9 different biosensorgrams and average ± SD is shown. C, Difference in biotinylated DENV-1 VLP. Five lots of DENV-1 VLP (lot No. 15062616, 18011816, 18042710, 19040109, and 20012911) were biotinylated with 50 excess moles of EZ-Link Sulfo-NHS-Biotin. Antibody avidity index was measured using the Octet HTX system with 7 different anti-dengue polyclonal antibodies purified from DEN-203 volunteer serum (volunteer No. 1044010 day 90, 1053009 day 90, 1053011 day 120, 1071001 day 90, 1071009 day 90, 1083005 day 0, and 1093001 day 90) at 5 μg/mL of biotinylated DENV-1 VLPs. Graph shows average ± 95% confidence interval. D, Linearity of avidity assay. Antibody avidity index was measured using Octet HTX (FortéBio) and biotinylated DENV-1 VLP. Anti-dengue polyclonal antibody from DEN-203 volunteer No. 1053005, day 90, serum was used in the assay. Antibody concentrations were 10 to 70 μg/mL and measured 15 times. Data are average ± SD. Equations were DENV-1 response, y = 0.0103x + 0.087; log10[koff], y = 0.0033x − 3.927; and log10[avidity index], y = 0.0076x + 3.138). Abbreviations: Conc., concentration; DENV, dengue virus; koff, antibody dissociation rate constant; VLP, virus-like particle.
Figure 3.
Figure 3.
Optimization of avidity assay. A, Optimization of association and VLP concentration. Antibody avidity index was measured using Octet HTX (FortéBio) and DENV-1, 2, 3, and 4 VLP. Anti-dengue polyclonal antibody (125 μg/mL) purified from clinical trial DEN-203 volunteer serum (DENV-1, volunteer No. 1071001 day 90; DENV-2, volunteer No. 1071009 day 90; DENV-3, volunteer No. 1083005 day 0; and DENV-4, volunteer No. 1093001 day 90) was used to measure association and dissociation rates. Association times were 600, 900, and 1800 seconds at biotinylated DENV VLP 5 μg/mL, and biotinylated VLP concentrations were 1, 3, and 5 μg/mL at 1800 seconds association time. Data were measured 8 times and bar graph shows averages ± SD. B, Optimization of dissociation rate analysis. Biosensorgram of anti-dengue polyclonal antibody purified from DEN-203 volunteer serum (volunteer No. 1081001, day 0; 250 μg/mL) was generated by Otet RED systems and 5 μg/mL DENV-3 VLP. Dissociation calculation was analyzed from 30 to 600 seconds (red) and from 30 to 1200 seconds (blue). Dissociation rate was measured by 9 different biosensorgrams and average ± SD is shown. C, Difference in biotinylated DENV-1 VLP. Five lots of DENV-1 VLP (lot No. 15062616, 18011816, 18042710, 19040109, and 20012911) were biotinylated with 50 excess moles of EZ-Link Sulfo-NHS-Biotin. Antibody avidity index was measured using the Octet HTX system with 7 different anti-dengue polyclonal antibodies purified from DEN-203 volunteer serum (volunteer No. 1044010 day 90, 1053009 day 90, 1053011 day 120, 1071001 day 90, 1071009 day 90, 1083005 day 0, and 1093001 day 90) at 5 μg/mL of biotinylated DENV-1 VLPs. Graph shows average ± 95% confidence interval. D, Linearity of avidity assay. Antibody avidity index was measured using Octet HTX (FortéBio) and biotinylated DENV-1 VLP. Anti-dengue polyclonal antibody from DEN-203 volunteer No. 1053005, day 90, serum was used in the assay. Antibody concentrations were 10 to 70 μg/mL and measured 15 times. Data are average ± SD. Equations were DENV-1 response, y = 0.0103x + 0.087; log10[koff], y = 0.0033x − 3.927; and log10[avidity index], y = 0.0076x + 3.138). Abbreviations: Conc., concentration; DENV, dengue virus; koff, antibody dissociation rate constant; VLP, virus-like particle.
Figure 4.
Figure 4.
Biosensorgram of dengue vaccine recipients and avidity assay parameters. A and B, Biosensorgrams at 0, 28, 90, 120, 180, and 360 days of clinical trial DEN-203 (A) volunteer No. 1022005 and (B) volunteer No. 1044010. Antibody avidity index was measured using the Octet RED system (FortéBio) and DENV-2 virus-like particles. Anti-dengue polyclonal antibody was used at 250 μg/mL to measure response and dissociation rates. Black line, dissociation calculation curve by Langmuir 1:1 binding model. C, Antibody response, koff, and avidity index of dengue vaccine recipient’s serum (volunteer No. 1022005 and No. 1044010). Assays were done in duplicate and averages are shown. Abbreviations: DENV, dengue virus; koff, antibody dissociation rate constant.
Figure 5.
Figure 5.
Avidity index data generated from TAK-003 recipients: (A) clinical trial DEN-203 baseline seronegative n = 37; (B) DEN-203 baseline seropositive n = 19; and (C) clinical trial DEN-204 baseline seronegative n = 21 and baseline seropositive n = 15. Data were obtained using the Octet HTX system (FortéBio) and DENV-1, DENV-2, DENV-3, and DENV-4 virus-like particles. A and B, Serum samples in trial DEN-203 were collected on study days 0 (baseline/prevaccination; first TAK-003 dose administered), 28, 90 (second TAK-003 dose administered), 120, 180, and 360. C, Serum samples in trial DEN-204 were collected on study day 0 (baseline/prevaccination; first TAK-003 dose administered) and day 180 (3 months after administration of second dose). Avidity index = response/koff. ****P < .0001, ***P < .0002, **P < .0021, *P < .0332 vs day 0 (Wilcoxon signed-rank test). Negative avidity index data were extrapolated to 1 for drawing purpose. Bars show minimum and maximum, boxes 25 and 75 percentile, and lines median. Abbreviations: DENV, dengue virus; koff, antibody dissociation rate constant; ns, not significant.
Figure 6.
Figure 6.
Correlation between avidity assay parameters. Avidity index and MNT titers: (A) DENV-1, (B) DENV-2, (C) DENV-3, and (D) DENV-4. Data from clinical trial DEN-203 (24 base line seronegative and 19 seropositive volunteers) were used apart from baseline seronegative day 0 data. The number of data points is 226–228: open symbols, baseline seropositive and closed symbols, baseline seronegative. Correlation between response, koff, and MNT titer are shown in Supplementary Figure 5 and correlation analysis data in Supplementary Table 6. Abbreviations: DENV, dengue virus; koff, antibody dissociation rate constant; MNT, microneutralization test.
Figure 7.
Figure 7.
Correlation between DENV-2 avidity index and MNT antibody titer for subjects divided by low, medium and high koff ranges. A, Correlation analysis using all data sets, n = 230. B–D, Correlation analysis data divided into 3 ranges by log10 koff values: (B) log10[koff] −4.7 to −4.6, n = 77; (C) log10[koff] −4.6 to −4.0, n = 77; (D) log10[koff] −4.0 to −2.8, n = 76. Data were from 24 baseline seronegative and 19 seropositive volunteers and data under response LoD (0.015 nm; Supplementary Table 5) were eliminated from the analysis. Correlation of DENV-1, DENV-3, and DENV-4 are shown in Supplementary Figure 9. Correlation analysis date parameters are shown in Supplementary Table 7. Abbreviations: DENV, dengue virus; koff, antibody dissociation rate constant; LoD, limit of detection; MNT, microneutralization test.

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