Potent Neutralizing Human Monoclonal Antibodies Preferentially Target Mature Dengue Virus Particles: Implication for Novel Strategy for Dengue Vaccine

Abstract

The four serotypes of dengue virus (DENV) cause the most important mosquito-borne viral disease in humans. The envelope (E) protein is the major target of neutralizing antibodies and contains 3 domains (domain I [DI], DII, and DIII). Recent studies reported that human monoclonal antibodies (MAbs) recognizing DIII, the D1/DII hinge, the E-dimer epitope, or a quaternary epitope involving DI/DII/DIII are more potently neutralizing than those recognizing the fusion loop (FL) of DII. Due to inefficient cleavage of the premembrane protein, DENV suspensions consist of a mixture of mature, immature, and partially immature particles. We investigated the neutralization and binding of 22 human MAbs to DENV serotype 1 (DENV1) virions with differential maturation status. Compared with FL MAbs, DIII, DI/DII hinge, and E-dimer epitope MAbs showed higher maximum binding and avidity to mature particles relative to immature particles; this feature may contribute to the strong neutralizing potency of such MAbs. FL-specific MAbs required 57 to 87% occupancy on mature particles to achieve half-maximal neutralization (NT₅₀), whereas the potently neutralizing MAbs achieved NT₅₀ states at 20 to 38% occupancy. Analysis of the MAb repertoire and polyclonal sera from patients with primary DENV1 infection supports the immunodominance of cross-reactive anti-E antibodies over type-specific antibodies. After depletion with viral particles from a heterologous DENV serotype, the type-specific neutralizing antibodies remained and showed binding features shared by potent neutralizing MAbs. Taken together, these findings suggest that the use of homogeneous mature DENV particles as an immunogen may induce more potent neutralizing antibodies against DENV than the use of immature or mixed particles.IMPORTANCE With an estimated 390 million infections per year, the four serotypes of dengue virus (DENV) cause the most important mosquito-borne viral disease in humans. The dengue vaccine Dengvaxia was licensed; however, its low efficacy among dengue-naive individuals and increased risk of causing severe dengue in children highlight the need for a better understanding of the role of human antibodies in immunity against DENV. DENV suspensions contain mature, immature, and partially immature particles. We investigated the binding of 22 human monoclonal antibodies (MAbs) to the DENV envelope protein on particles with different maturation states. Potently neutralizing MAbs had higher relative maximum binding and avidity to mature particles than weakly neutralizing MAbs. This was supported by analysis of MAb repertoires and polyclonal sera from patients with primary DENV infection. Together, these findings suggest that mature particles may be the optimal form of presentation of the envelope protein to induce more potent neutralizing antibodies against DENV.

Keywords: dengue virus; envelope; mature particles; monoclonal antibody; neutralization.

FIG 1

Binding of different categories of human anti-E MAbs to mature, immature, or mixed particles. (A) Outline of preparation of mature, mixed, and immature DENV1 (D1) virions from 293T-furin cells, 293T cells, and 293T cells in the presence of ammonium chloride, respectively. (B) WB analysis of mature, mixed, or immature DENV1 virions purified by sucrose gradient ultracentrifugation and probed with DENV-immune human serum (anti-prM/E) and rabbit serum against M-peptide (anti-M). The values on the left are molecular masses (in kilodaltons). (C) The relative prM contents of the different particles were determined by a virion ELISA, which used a murine MAb (FL0251) for capture and human MAbs (anti-E MAb DVG17.12, anti-prM MAb DVB 59.3) for detection. (D to M) Binding curves of different categories of human MAbs to mature, immature, or mixed particles based on virion-capture ELISAs, including FL-specific MAbs (D, E), DIII MAbs (F, G), EDE1 MAbs (H, I), EDE2 MAbs (J, K), and DI/IIh MAb (L, M). The relative OD (rOD) compared to the OD of DENV-immune human serum is presented. Data are the means and standard deviations for duplicates from one representative experiment of two.

FIG 2

Comparison of neutralizing potency, maximum binding, and dissociation constants of different categories of human anti-E MAbs to mature, immature, or mixed particles. (A) DIII, EDE, and DI/IIh MAbs were more potent neutralizers than FL-specific MAbs. (B, C) Comparison of the B_max (B) and K_d (C) for mature or immature DENV1 particles within each category of MAbs, including FL, DIII, and EDE plus DI/IIh MAbs. (D, E) Comparison of the relative B_max (D) and relative K_d (E) for mature versus immature DENV1 particles, B_max (m/imm) and K_d (m/imm), respectively, between FL, DIII, and EDE plus DI/IIh MAbs. B_max and K_d were determined by nonlinear regression analysis (GraphPad Prism software, version 6.0). Data are the means for duplicates from two experiments. P values were based on the two-tailed Mann-Whitney test.

FIG 3

Relationship between neutralizing (NT) potency and binding parameters to mature, mixed, or immature particles. (A) Relationship between neutralizing potency and binding avidity to mixed DENV1 particles. (B, D, F) Relationship between neutralizing potency and binding avidity (B), B_max to mature DENV1 particles (D), and relative B_max (mature/immature [m/imm]) (F). (C, E, G) Relationship between neutralizing potency and binding avidity (C), B_max to immature DENV1 particles (E), and relative B_max (immature/mature [imm/m]) (G). Neutralizing potency and binding avidity were defined by 1/NT₅₀ and 1/K_d, respectively. B_max and K_d were determined by nonlinear regression analysis (GraphPad Prism software, version 6.0). Data are means for duplicates from two experiments. The Spearman correlation test was performed.

FIG 4

Relationship between neutralization and percent occupancy of mature and mixed particles for different categories of anti-E MAbs. (A to E) Representative example of FL (A), DIII (B), DI/IIh (C), EDE1 (D), and EDE1 (E) MAbs and percent occupancy of these MAbs at the NT₅₀ concentration. (F, G) Comparison of percent occupancy of mixed (F) or mature (G) particles at the NT₅₀ concentration between FL MAbs and potent neutralizing MAbs (including DIII, DI/IIh, and EDE MAbs). The curves were determined by nonlinear regression analysis (GraphPad Prism software, version 6.0). Data are the means and/or standard deviations (for A to E) for duplicates from two experiments. P values were based on the two-tailed Mann-Whitney test.

FIG 5

Analysis of repertoire of anti-E MAbs derived from the same individual. (A) The five MAbs derived from a case with primary DENV1 (D1) infection (DVG) include four GR MAbs and one DENV1 TS MAb. (B) The seven MAbs derived from a case with secondary DENV1 infection (donor 12) include five GR MAbs, one CR MAb, and one DENV1-TS MAb. The binding curves, K_d, and B_max for mature (m), mixed (mix), or immature (imm) particles, as well as the NT₅₀ are presented. GR, group reactive; CR, complex reactive; TS, type specific. Data are the means and/or standard deviations for duplicates from one representative experiment of two.

FIG 6

Analysis of polyclonal human sera. (A) Serum from a case with a prior primary DENV1 infection (patient ID26) was mock or DENV3 (D3) depleted and tested with a virion ELISA. (B) Neutralization curve for DENV1 of mock-depleted or DENV3-depleted serum from patient ID26. (C, D) Binding curves of mature or immature DENV1 particles for mock-depleted (C) or DENV3-depleted (D) serum. The endpoint titers for binding to mature or immature particles and the ratio (mature/immature) were determined based on four-parameter nonlinear regression analysis (GraphPad Prism software, version 6.0). (E) Endpoint titers for mature or immature particles and relative endpoint titers (mature/immature) of mock-depleted or DENV3-depleted sera from 12 patients following primary DENV1 infection. Data are the means and/or standard deviations for duplicates from two experiments. The two-tailed Wilcoxon signed-rank test was used.

FIG 7

Determination of the proportion of TS antibodies in polyclonal sera. (A, B) Serum from a case with prior primary DENV1 infection (ID33) was mock or DENV3 (D3) depleted and titrated on mature (A) or immature (B) DENV1 particles. The endpoint titers of mock-depleted or DENV3-depleted sera were determined based on four-parameter nonlinear regression analysis (GraphPad Prism software, version 6.0). The proportion (in percent) of TS antibodies is equal to (endpoint titer of DENV3-depleted serum/endpoint titer of mock-depleted serum) × 100. The percentage of GR plus CR antibodies equals 100 − percentage of TS antibodies. (C, D) Comparison of the percentage of TS antibodies and the percentage of GR plus CR antibodies in sera from 8 patients following primary DENV1 infection based on mature (C) and immature (D) particles. Data are the means for duplicates from two experiments. The two-tailed Mann-Whitney test was used.