Structure and function analysis of therapeutic monoclonal antibodies against dengue virus type 2 - PubMed (original) (raw)

. 2010 Sep;84(18):9227-39.

doi: 10.1128/JVI.01087-10. Epub 2010 Jun 30.

S Kyle Austin, Michael Engle, James D Brien, Kimberly A Dowd, Katherine L Williams, Syd Johnson, Rebeca Rico-Hesse, Eva Harris, Theodore C Pierson, Daved H Fremont, Michael S Diamond

Affiliations

Structure and function analysis of therapeutic monoclonal antibodies against dengue virus type 2

Soila Sukupolvi-Petty et al. J Virol. 2010 Sep.

Abstract

Dengue virus (DENV) is the most prevalent insect-transmitted viral disease in humans globally, and currently no specific therapy or vaccine is available. Protection against DENV and other related flaviviruses is associated with the development of antibodies against the viral envelope (E) protein. Although prior studies have characterized the neutralizing activity of monoclonal antibodies (MAbs) against DENV type 2 (DENV-2), none have compared simultaneously the inhibitory activity against a genetically diverse range of strains in vitro, the protective capacity in animals, and the localization of epitopes. Here, with the goal of identifying MAbs that can serve as postexposure therapy, we investigated in detail the functional activity of a large panel of new anti-DENV-2 mouse MAbs. Binding sites were mapped by yeast surface display and neutralization escape, cell culture inhibition assays were performed with homologous and heterologous strains, and prophylactic and therapeutic activity was evaluated with two mouse models. Protective MAbs localized to epitopes on the lateral ridge of domain I (DI), the dimer interface, lateral ridge, and fusion loop of DII, and the lateral ridge, C-C' loop, and A strand of DIII. Several MAbs inefficiently inhibited at least one DENV-2 strain of a distinct genotype, suggesting that recognition of neutralizing epitopes varies with strain diversity. Moreover, antibody potency generally correlated with a narrowed genotype and serotype specificity. Five MAbs functioned efficiently as postexposure therapy when administered as a single dose, even 3 days after intracranial infection of BALB/c mice. Overall, these studies define the structural and functional complexity of antibodies against DENV-2 with protective potential.

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Figures

FIG. 1.

FIG. 1.

MAb neutralization of different DENV-2 genotypes. Increasing concentrations of purified DV2-87 (A), DV2-96 (B), and DV2-106 (C) were mixed with 102 PFU of DENV-2 strains corresponding to all four genotypes, and neutralization was assessed by a standard PRNT assay in BHK21 cells. Graphs were generated after regression analysis using statistical software. The data are representative of at least three independent experiments.

FIG. 2.

FIG. 2.

Epitope mapping of anti-DENV-2 MAbs on the E protein. (A) Localization of neutralizing epitopes on DENV-2 DI-DII as determined by yeast surface display. Structure of DENV-2 DI-DII (strain 16681), with amino acid residues that significantly affect binding of indicated neutralizing MAbs marked. (B to D) Localization of neutralizing epitopes on DENV-2 DIII as determined by yeast surface display. Structure of DENV-2 DIII (strain 16681), with amino acid residues that affect binding of representative neutralizing MAbs marked in orange. (B) DV2-76 (A strand); (C) DV2-106 (lateral ridge); (D) DV2-104 (C-C′ loop).

FIG. 3.

FIG. 3.

Characterization of neutralization escape mutants. (A) A neutralization assay was performed with selected escape variants (G177D or K307R) after plaque purification. These escape variants were obtained after three to six passages of DENV-2 (strain 16681) under selection with either DV2-48 or DV2-67 on BHK21 cells. Note that the G177D and K307R variants are resistant to DV2-48 and DV2-67, respectively. Data are representative of two independent experiments performed in duplicate. (B to D) Confirmation of resistant phenotype with DENV-2 RVPs. Mutated RVPs (G177D, K291R, K307R, or M196V) were used to confirm that single amino acid substitutions in the E protein reduce or eliminate neutralization by the selecting MAb (DV2-48 [B]; DV2-67 [C]; and DV2-96 [DR] control DIII MAb). In each series, the reduced neutralizing capacity of the indicated MAb is compared to the neutralizing capacity of wild-type DENV-2 RVP generated in parallel. The data are representative of two or three separate experiments performed in triplicate. Error bars indicate standard error within a single experiment. Lines represent curve fits generated by nonlinear regression analysis. For panel C, diluted hybridoma supernatant was used instead of purified antibody.

FIG. 4.

FIG. 4.

Structural analysis of genotypic variation. (A) Diagram of DI-DII (top, top view; bottom, bottom view) showing sequence variation of different DENV-2 genotypes. Amino acid sequences that differ among DENV-2 genotypes are indicated, with solvent-accessible or -inaccessible residues depicted in green and cyan, respectively. (B) Ribbon diagram of DENV-2 DIII with β-strands and loops labeled accordingly from published X-ray crystallographic structure. Amino acid sequences that vary among the different DENV-2 genotypes are indicated, with solvent-inaccessible and -accessible residues depicted in blue and green, respectively. Magenta coloring indicates residues that are not predicted to be solvent accessible in the mature virion based on cryo-electron microscopic reconstruction (26) but may be accessible during other stages of the virus life cycle.

FIG. 5.

FIG. 5.

Strongly neutralizing DENV-2 MAbs inhibit at a postattachment stage. To determine whether the MAbs neutralize DENV-2 infection after cellular attachment, BHK21 cells were prechilled and 102 PFU of DENV-2 (strain 16681) was added to each well for 1 h at 4°C. (C) After extensive washing at 4°C, saturating concentrations of the MAbs (50 μg/ml) were added for 1 h at 4°C, and then the neutralization assay was completed. In comparison, a standard preincubation neutralization test with all steps performed at 37°C (A) or 4°C (B) is shown for reference. (A and B) In this case, virus and MAb are incubated together for 1 h at 37°C or 4°C, prior to addition to cells. Data shown are the average of three independent experiments, with error bars representing standard deviation. Asterisks indicate statistically significant differences. Note that DV2-29 loses some of its neutralization potency when preincubated at 4°C compared to 37°C.

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