Potent Dengue virus neutralization by a therapeutic antibody with low monovalent affinity requires bivalent engagement

We recently described our most potently neutralizing monoclonal antibody, E106, which protected against lethal Dengue virus type 1 (DENV-1) infection in mice. To further understand its functional properties, we determined the crystal structure of E106 Fab in complex with domain III (DIII) of DENV-1 envelope (E) protein to 2.45 Å resolution. Analysis of the complex revealed a small antibody-antigen interface with the epitope on DIII composed of nine residues along the lateral ridge and A-strand regions. Despite strong virus neutralizing activity of E106 IgG at picomolar concentrations, E106 Fab exhibited a ∼20,000-fold decrease in virus neutralization and bound isolated DIII, E, or viral particles with only a micromolar monovalent affinity. In comparison, E106 IgG bound DENV-1 virions with nanomolar avidity. The E106 epitope appears readily accessible on virions, as neutralization was largely temperature-independent. Collectively, our data suggest that E106 neutralizes DENV-1 infection through bivalent engagement of adjacent DIII subunits on a single virion. The isolation of anti-flavivirus antibodies that require bivalent binding to inhibit infection efficiently may be a rare event due to the unique icosahedral arrangement of envelope proteins on the virion surface

Data collection and refinement statistics^a.

a<p>Numbers in parentheses refer to the highest resolution shell.</p>b<p>R_merge = Σ|I−<i>|/Σ<i>, where I is the intensity of each individual reflection.</i></i></p><i><i>c<p><i>R</i> = ∑(F_P−F_calc)/∑F_P.</p>d<p>Molprobity score defined as 0.42574 * log(1+clashscore)+0.32996 * log(1+max(0,pctRotOut-1))+0.24979 * log(1+max(0,100−pctRamaFavored-2))+0.5.</p></i></i

Modeling of E106 binding to DENV.

<p>(<b>A</b>) The E106 epitope is highlighted in magenta on a DENV-1 model of the DENV-2 cryo-electron microscopic reconstruction (PDB code 1P58). All atoms of the model are displayed and colored by domains (DI, red; DII, gold; DIII, blue). Magnified view of the boxed region in (A) with E106 Fab docked onto the epitopes at the primary candidates for bivalent antibody binding namely (<b>B</b>) adjacent 2-fold epitopes (49 Å CH1 separation) which is permuted as an outer 5-fold ring and (<b>C</b>) adjacent inner and outer 5-fold epitopes (24 Å CH1 separation). E106 Fab heavy and light chain is represented by green and cyan spheres, respectively. The C-terminal CH1 residue is represented by ‘C’. (<b>D</b>) Schematic of the possible arrangements of E106 MAb bivalent binding to the lateral ridge plus A-strand epitope (magenta) on a single virion. The distances labeled in magenta and green are E106 epitope (T329) and C-terminal CH1 residue (R214) distances respectively. One Fab pair (green, heavy chain; cyan, light chain) is shown across the 5- and 2-fold and another involves adjacent 2-fold symmetry axes (the 2-fold permutes as an outer 5-fold ring, dashed gray line). 5-, 3- and 2-fold axes of symmetry are connected by gray dashed lines for clarity and represented by pentagons, triangles, and ovals, respectively. Epitopes that are occluded as a result of bivalent binding are shown in gray.</p

Fab versus MAb neutralization and binding.

<p>(<b>A</b>) DENV-1 neutralization by E106 MAb (filled square, red) and Fab (empty square, salmon) (upper panel) and corresponding fold reduction (lower panel). The control E103 MAb (filled circle, blue) and corresponding Fab (empty circle, purple) is included for comparison. (<b>B</b>) Qualitative ELISA binding of MAbs and Fabs to DENV-1. E106 MAb binds virions to a similar extent as E103 MAb but E106 Fab binding is significantly less than E103 Fab (P<0.0001). The negative control is E16. The results are from two ELISA experiments performed in duplicate and without background subtraction. The limit of detection was determined by performing the assay in the absence of virus.</p

DENV-1 DIII-E106 Fab structural interface.

<p>The structural interface of DENV-1 DIII-E106 Fab (described by the surface area of antibody, Ab<i>^a</i> and antigen, Ag<i>^b</i> that is buried <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Krissinel1" target="_blank">[24]</a> as well as shape complementarity, Sc<i>^c</i> in the complex <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Lawrence1" target="_blank">[23]</a>) is compared to previously determined anti-DENV and anti-WNV Fab complexes. PDB codes E16 – WNV E DIII, 1ZTX <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Nybakken1" target="_blank">[20]</a>; 1A1D-2 – DENV2 E DIII, 2R29 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Lok1" target="_blank">[21]</a>; E53 – WNV E, 3I50 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Cherrier1" target="_blank">[43]</a>; E111 – DENV1 E DIII, 4FFY <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Austin1" target="_blank">[33]</a>; 2H12 – DENV1 DIII, 4AL8 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Midgley1" target="_blank">[17]</a>; 2H12 – DENV3 DIII, 4ALA <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Midgley1" target="_blank">[17]</a>; 2H12 – DENV4 DIII, 4AM0 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Midgley1" target="_blank">[17]</a>; 4E11 – DENV1 DIII, 3UZQ <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Cockburn1" target="_blank">[10]</a>; 4E11 – DENV2 DIII, 3UZV <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Cockburn1" target="_blank">[10]</a>; 4E11 – DENV3 DIII, 3UZE <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Cockburn1" target="_blank">[10]</a>; 4E11 – DENV4 DIII, 3UYP <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Cockburn1" target="_blank">[10]</a>.</p

Fab and MAb binding to DENV-1 RVPs.

<p>(<b>A</b>) E106 MAb binding to DENV-1 RVPs by bio-layer interferometry. E106 MAb binds with high apparent affinity to DENV-1 RVPs (K_Dapp of 13±2 nM) with a slow dissociation (t_1/2>400 sec). Raw data is in red and fits are in gray. (<b>B</b>) Direct binding of E106 Fab to DENV-1 RVPs. Results are representative of several independent experiments that showed low affinity binding (K_D of >1 µM) and fast off-rate kinetics (t_1/2<2 sec). Raw data is in salmon and fits are in gray. BLI determined binding of the control E103 (<b>C</b>) MAb and (<b>D</b>) Fab to DENV-1 RVPs. E103 MAb and Fab binding to DENV-1 RVPs are comparable, with a calculated apparent K_Dapp of 0.8±0.1 nM and K_D of 7±1 nM, respectively, as expected for a predominantly monovalent interaction. Raw data is in blue (E103 MAb) and purple (E103 Fab) and fits are in gray. Results are representative of at least two independent experiments.</p

Crystal structure of E106 Fab in complex with DENV-1 E DIII.

<p>(<b>A</b>) The E106 Fab epitope on DENV-1 E DIII is comprised of residues in the A-strand (K307 and K310), the end of the B-strand (K325 and Y326), and the connecting BC (E327, T329, and D330) and DE (K361 and E362) loops. The immunoglobulin-like DIII is shown in blue (epitope regions in magenta), with the E106 heavy chain in green and light chain in cyan. (<b>B</b>) Heavy chain residue W98 binds in a deep pocket contributed by the aliphatic groups of side chain DENV-1 E DIII residues K307, K325, and E327 and main chain of Y326, in stereo (top panel). The electron density map is contoured at 1.1σ. Ball and stick representation of the molecular interactions involving T329 (the residue that escapes neutralization <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Shrestha1" target="_blank">[13]</a> in stereo (bottom panel). (<b>C</b>) Surface representation of DENV-1 E DIII (top) and E106 Fab (bottom) highlighting residues making direct contacts in the complex (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072.s001" target="_blank">Table S1</a>). DENV-1 E DIII residues previously identified by yeast surface display are displayed in magenta <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Shrestha1" target="_blank">[13]</a>. (<b>D</b>) The structural epitope on DENV-1 DIII is shown in ribbon representation. (<b>E</b>) Sequence of DIII of DENV-1 aligned with that of DENV-2, -3, -4 and WNV highlighting the E106 structural epitope, which is conserved in DENV-1 genotypes but not DENV serotypes or WNV. Identical residues are represented by a dot, deleted residues by a hash. For comparison, The DIII structural epitopes of WNV E16, DENV-2 1A1D-2, DENV cross-reactive 4E11 and 2H12 MAbs and DENV-1-E111 contact residues are labeled with green, purple, light purple, orange and cyan asterisks respectively <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Cockburn1" target="_blank">[10]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Midgley1" target="_blank">[17]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Nybakken1" target="_blank">[20]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Lok1" target="_blank">[21]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072#ppat.1004072-Austin1" target="_blank">[33]</a>.</p

E106 Fab and MAb binds with low affinity to DENV-1-DIII.

<p>Analysis of DENV-1 DIII binding to (<b>A</b>) E106 Fab and (<b>B</b>) E106 MAb as measured by SPR. The kinetically fit sensorgrams (fits in gray, raw data in black) for which a 5.8±3.0 µM affinity (Fab) and 4.2±1.2 µM (MAb) was calculated on the left panel is similar to the equilibrium data fit 4.8±2.1 µM (Fab) and 3.2±0.8 µM (MAb) which is shown on the right panel. (<b>C</b>) ITC confirms a micromolar affinity of E106 Fab for DENV-1 DIII. Results are representative of at least two independent experiments.</p

Functional characteristics of neutralization by E106 MAb.

<p>(<b>A–B</b>) Time and temperature dependence of neutralization. DENV-1 RVPs were pre-incubated with E106 MAb for one (<b>A</b>) or five hours (<b>B</b>) at three different temperatures (4°C, 37°C and 40°C) before infecting Raji-DCSIGNR cells. Infection was carried out at 37°C and assessed 48 h later by flow cytometry. Error bars represent the standard error of the mean of duplicate infections. Results are representative of two independent experiments. (<b>C</b>) Pre- or post-attachment neutralization test. BHK21-15 cells were pre-chilled to 4°C, and 10² PFU of DENV-1 (strain 16007) was added to each well for one hour at 4°C. After extensive washing at 4°C, increasing concentrations of E106 MAb were added for one hour at 4°C, and the PRNT determined (triangles, Post). A standard pre-incubation PRNT with all steps performed at 4°C is shown for reference (squares, Pre). Data shown are representative from three experiments performed in duplicate. (<b>D</b>) E106 MAb neutralization is insensitive to the maturation state of the virus. E106 MAb neutralization of immature, standard, and mature 16007 RVP preparations, independently validated with the fusion-loop specific E60 MAb, are shown. A representative neutralization assay of three experiments is depicted. Error bars represent the standard error of the mean of duplicate infections. The inset depicts the EC50 values of neutralization of the distinct RVP preparations (immature, standard and mature) by E106 MAb and the control E60 MAb.</p