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Comparative Study
. 2006 Dec;80(24):12149-59.
doi: 10.1128/JVI.01732-06. Epub 2006 Oct 11.

Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein

Theodore Oliphant  1 , Grant E Nybakken, Michael Engle, Qing Xu, Christopher A Nelson, Soila Sukupolvi-Petty, Anantha Marri, Bat-El Lachmi, Udy Olshevsky, Daved H Fremont, Theodore C Pierson, Michael S Diamond
Affiliations
Comparative Study

Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein

Theodore Oliphant et al. J Virol. 2006 Dec.

Abstract

Previous studies have demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope (E) strongly protect against infection in animals. Herein, we observed significantly less efficient neutralization by 89 MAbs that recognized domain I (DI) or II (DII) of WNV E protein. Moreover, in cells expressing Fc gamma receptors, many of the DI- and DII-specific MAbs enhanced infection over a broad range of concentrations. Using yeast surface display of E protein variants, we identified 25 E protein residues to be critical for recognition by DI- or DII-specific neutralizing MAbs. These residues cluster into six novel and one previously characterized epitope located on the lateral ridge of DI, the linker region between DI and DIII, the hinge interface between DI and DII, and the lateral ridge, central interface, dimer interface, and fusion loop of DII. Approximately 45% of DI-DII-specific MAbs showed reduced binding with mutations in the highly conserved fusion loop in DII: 85% of these (34 of 40) cross-reacted with the distantly related dengue virus (DENV). In contrast, MAbs that bound the other neutralizing epitopes in DI and DII showed no apparent cross-reactivity with DENV E protein. Surprisingly, several of the neutralizing epitopes were located in solvent-inaccessible positions in the context of the available pseudoatomic model of WNV. Nonetheless, DI and DII MAbs protect against WNV infection in mice, albeit with lower efficiency than DIII-specific neutralizing MAbs.

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Figures

FIG. 1.
FIG. 1.
In vitro neutralization and enhancement activity of DI- or DII-specific MAbs. (A) Neutralization of WNV using a PRNT assay on BHK cells. (B) Neutralization of RVPs on Vero cells. (C) Neutralization of RVPs on Raji DC-SIGNR cells. The data are expressed as percentages of the no-MAb control. The data shown are the means from at least three independent experiments. Error bars indicate the standard errors of the means, and statistical significance was determined using an unpaired, two-tailed t test compared to the no-MAb control (*, P ≤ 0.05; **, P ≤ 0.01). (D) Enhancement of RVP infection on K562 cells. The data shown are the enhancement over baseline infection without MAb. All MAbs were studied in a single experiment to allow for comparisons of the power of enhancement. Values of less than 100 indicate neutralization, whereas values greater than 100 indicate enhancement.
FIG. 2.
FIG. 2.
Flow cytometry patterns of loss-of-function DI- or DII-specific MAb variants selected by yeast surface display. Representative histograms are shown for MAbs E53, E100, E113, and E121. Red arrows indicate mutations that result in loss of MAb binding. The data shown are representative of three independent experiments. FL4-H, log fluorescence intensity on the FL4 (660=nm) channel.
FIG. 3.
FIG. 3.
Epitope mapping of DI- and DII-specific neutralizing MAbs. Binding of (A) E18, (B) E53, (C) 7H7, (D) E113, (E) E121, (F) E48, (G) E100, and (H) E101 to mutants expressed on the yeast surface. The binding of each MAb to the mutants was measured by flow cytometry, and total fluorescence was normalized to yeast expressing wild-type DI-DII. The data shown are the means from three independent experiments. Error bars indicate the standard errors of the means. The colors red, yellow, blue, and green indicate domains I, II, and III and the fusion loop, respectively. Mutations that resulted in ≥50% reduction of MAb binding were mapped (shown in magenta and boxed) onto the WNV E protein crystal structure (Protein Data Bank accession code 2HG0). For MAbs E53 and 7H7, residues that compose the primary binding site within DII are boxed and secondary sites in DI are circled. Epitopes are labeled using the same nomenclature defined in Table 1.
FIG. 4.
FIG. 4.
Epitope expression on the WNV virion. Yeast display epitope residues (magenta) for (A) E16, (B) E18, (C) E53, (D) 7H7, (E) E113, and (F) E121 were mapped onto the pseudoatomic model of the mature WNV virion. For E16, the blue indicates additional contact residues as determined by X-ray crystallography. Virions are depicted as 2.0-Å-radius Cα atoms and are colored according to their symmetry axes, twofold (cyan), threefold (green), and fivefold (yellow). Epitopes are boxed on one E protein in each symmetry axis. Secondary binding sites in DI for E53 and 7H7 are circled in each symmetry axis.
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