Fig 1.
Characterization of dengue targeting monoclonal antibodies from antibody-secreting B cells of the rhesus macaque.
(A) The longitudinal geometric mean neutralization titers of the rhesus sera for DENV1, DENV2, DENV3, and DENV4, respectively. The LiCor50 Titers indicate the dilution rate of the sera maintains 50% of the neutralizing activity. (B) The pie chart shows the distribution of antibodies that bound DEN-80E strongly, weakly, or not at all, as determined by ELISA screening. The antibodies were considered to be strong binders when OD450 was above or equal to 0.5 (3%), weak binders when OD450 was between 0.1 and 0.5 (14%), and non-binders when OD450 was below 0.1 (83%). (C) Percentage of binders specific for single and multiple dengue serotypes among antibodies binding DEN-80E. (D-I) Reactivity for DEN-80E binding in ELISA screening for antibodies binding DEN-80E of DENV1 (D), DENV2 (E), DENV3 (F), DENV4 (G), 2 or 3 dengue serotypes (H), all four dengue serotypes (I). The X-axis indicates individual antibodies, the black dotted lines indicate the cut-off OD450 of 0.1, and NC means negative control.
Table 1.
Binding activity of DENV-specific mAbs.
Fig 2.
Neutralization activity and kinetic analysis of the potent neutralizing antibodies.
(A) Neutralization activity of the indicated antibodies to DENV. The virus was mixed with 5-fold serial dilutions of antibodies. Then neutralization activity was evaluated by micro-neutralization assay in duplicates. Data are represented as mean ± SEM. The curves are fitted by the Response—Variable slope (four parameters) model. (B) Binding kinetics of selected antibodies on immobilized DEN-80E. The graphs show kinetic binding of six potent neutralizing antibodies at the concentration of 25 nM to immobilized antigens DEN1-80E, DEN2-80E, DEN3-80E, and DEN4-80E as indicated.
Table 2.
Neutralization activity for the selected DENV mAbs.
Table 3.
Kinetic binding constants of the DENV neutralizing mAbs.
Table 4.
Epitope mapping of the selected neutralizing antibodies.
Fig 3.
Critical residues for binding of dengue serotype-specific macaque antibodies.
(A) Critical resides (green spheres) are mapped on the mature E protein dimer structure (PDB, 1UZG). Domain I is red, domain II yellow, domain III blue, and fusion loop cyan. The asymmetric monomer rotated 180 degrees is shown in gray. (B) The key residues of the d511/d628 epitope (shown on PDB, 1OK8). D329 (BC loop) and K361 (DE loop) are on the lateral ridge of DENV2 domain III, which is a reported epitope region of mouse anti-dengue neutralizing antibodies. Bottom panel: side view of the mature virus capsid indicates that the residues D329 and K361 display the side chains on the external surface of domain III (PDB, 3J27). (C) The key residues of the d182 epitope (PDB, 3G7T). T329 is in the BC loop of DENV1 domain III. V300 is in the DI-III hinge. (D) The key residues of the d559 epitope (PDB, 1OK8, modified). Y81 and K83 are in the bc loop of DENV4 domain II. Atoms colored red for O, blue for N. Molecular graphic images were produced using UCSF Chimera (http://www.rbvi.ucsf.edu/chimera). (E-H) Neutralization percentage of the indicated wild type (WT) DENV reporter virus particles, and its critical amino acid residue substitution mutants. Antibodies d182 (E), d511 (F), d628 (G), and d559 (H) were tested at the concentration of 3 μg/ml. The assays were performed in triplicates and data were analyzed using GraphPad Prism.
Fig 4.
Critical residues for binding of cross-reactive antibody d462.
The structural presentation of the critical residues of the epitope on DENV4 domain I. (A) Critical resides (green spheres) are mapped on both monomers of the mature E protein dimer structure (PDB, 1UZG). Domain I is red, domain II yellow, domain III blue and fusion loop cyan. The asymmetric monomer rotated 180 degrees is gray. (B) Sequence alignment of the epitope region on the E protein. Residues V160, V173 and D177 on DENV4 are not identical among different serotypes. However, they have similar chemical characteristics for DENV3 in position 173 (Valine compare to Alanine) and 177 (Aspartic acid compare to Glutamic acid). The NCBI accession numbers of the sequences are ACJ04226 (DENV1), AGS49173 (DENV2), AJA37731 (DENV3) and ACW82884 (DENV4) respectively. (C) Antibody d462 epitope on an E protein raft (PDB, 4CBF). The black line shows the boundary of a single asymmetric unit. The residues that interact with d462 are shown as green spheres. Enlarged view (right) shows the epitope region on F0, G0 β-strands, and the G0H0 loop on E protein domain I. The epitope of d462 (V160, V173, D177) is overlapped with that of the reported chimpanzee antibody 5H2 (E172, K174, D177, E180, R293). (D) Structural comparison of the epitope region on DENV4 (PDB, 3UC0) and DENV3 (PDB, 1UZG), indicating the structural similarity of the key residues. DENV4 is shown in red ribbon and DENV3 is shown in gray ribbon. The residues on DENV4 are shown as green sticks, on DENV3 as gray sticks. Atoms are colored red for O, blue for N. The analysis was performed using “Structural Comparison: Match -> Align” of UCSF chimera. Molecular graphic images were produced using the UCSF Chimera (http://www.rbvi.ucsf.edu/chimera). (E) Percentage neutralization of the DENV4 reporter virus particles, and its critical amino acid residue substitution mutants. Antibody d462 was tested at the concentration of 3 μg/ml. The assays were performed in triplicates and data were analyzed using GraphPad Prism.
Fig 5.
Critical residues for binding of the broadly neutralizing antibody d448.
The structural presentation of the critical residues of the epitope on DENV4 domain II. (A) Critical resides (green spheres) are mapped on both monomers of the mature E protein dimer structure (PDB, 1UZG). Domain I is red, domain II yellow, domain III blue and fusion loop cyan. The asymmetric monomer rotated 180 degrees is gray. (B) Sequence alignment of the epitope region on the E protein. Epitope residues D215, P219, Q256, and G266 are conserved among the different serotypes; only one residue M237 in DENV4 is not conserved. (Leucine in the other three serotypes) The NCBI accession numbers of the sequences are ACJ04226 (DENV1), AGS49173 (DENV2), AJA37731 (DENV3) and ACW82884 (DENV4) respectively. (C) Side view of the E:M:M:E heterotetramer. The transmembrane domain of M protein is in magenta, the space-filling model of the ectodomain of M protein is in light blue. Four critical residues (P219, M237, Q256, and G266) of the epitope are located in the hydrophobic core of the E-M interface. One residue (D215), out of the hydrophobic core, contacts the perimembrane helix of the M protein (PDB, 1UZG). (D) Enlarged view shows the contact of D215 with R38 on M protein. The analysis was performed using “Structural Analysis: Distances” of UCSF chimera. (E) Enlargement of the hydrophobic core. The stick model, shown in light blue, presents the first 20 amino acids of M protein (M1-20). The space-filling model of E shows the three hydrophobic pockets on E protein, where the M1-20 binds. The red dotted lines show the boundaries of the pockets. The critical residues of the epitope are indicated. (F) In pocket 2, H209 and T212 on E form a hydrophobic core with His7 on M, and in pocket 3, T19 on M is encompassed by the recess of T206 and H261 on E. The amino acid G266 forms a barrier between pocket 2 and pocket 3, contacting the conserved residues of H7 and H19 on M protein. (G) The amino acids P219, Q256, M237 are on the edge of pocket 1, consistent with L216, L218, and M260 on E protein, these six residues encompass the valine in the position 2 (V2) on M in the pocket 1 center. Atoms are shown in red for O, blue for N, and magenta for S. Molecular graphic images were produced using the UCSF Chimera (http://www.rbvi.ucsf.edu/chimera). (H) Neutralization percentage of the DENV2 reporter virus particles, and its critical amino acid residue substitution mutants. (I) Neutralization percentage of the DENV4 reporter virus particles, and its critical amino acid residue substitution mutants. Antibody d448 was tested at the concentration of 3 μg/ml. The assays were performed in triplicates and data were analyzed using GraphPad Prism.
Fig 6.
The cross-reactivity of antibody d448 to flaviviruses.
(A) Binding of d448 to the E proteins of flaviviruses was analyzed by ELISA. E proteins (100 ng/well) were coated on 96-well plate. Serial dilution of the d448 antibody was added in the coated plate for the detection of the binding, and measurement of the EC50. (B) The neutralization activity of d448 was determined by assaying Vero cell infectivity in the presence of serial dilutions of purified antibody. DENV2 (16681 strain) was used as a positive control. Zika virus (SMGC-1 strain), yellow Fever virus (17D strain), and West Nile virus (Kunjin, MRM61C strain) were used in the study.
Fig 7.
Germline usage and the mutation analysis of the antibodies.
General repertoire properties of the rhesus macaque antibodies, including germline gene usage, germline gene divergence and the length of heavy chain complementarity determining region 3 (CDR3). The analysis was performed using IgBLAST with IMGT V domain delineation (https://www.ncbi.nlm.nih.gov/igblast/). The entire panel contains all isolated mAbs and neutralizing mAbs. (A) Heavy chain V-gene germline usage of total mAbs. (B) Kappa chain V-gene germline usage of total mAbs. (C) Lambda chain V-gene germline usage of total mAbs. (D) The natively paired V-gene germline usage of total mAbs. All the heavy chains are associated with their natively paired light chain (kappa or lambda). (E) The germline mutation rate of the native antibodies. Each dot represents one unique sequence. The mean value and the standard deviation are indicated with the black bar, no significant difference regarding the germline gene divergence of the total mAbs and the neutralizing mAbs. The labels are VH-Total: the heavy chain of the total mAbs; VH-Neu: the heavy chain of the neutralizing mAbs; VL-Total: the light chain of the total mAbs; VL-Neu: the light chain of the neutralizing mAbs. (F) The heavy chain CDR3 length of the cloned mAbs (blue), and the neutralizing mAbs (red). (G) The heavy chain V germline distribution of the cloned antibodies (blue), and the neutralizing antibodies (red). The percentage of antibodies with the VH4 germline is shown.