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. 2004 Dec;78(24):13975-86.
doi: 10.1128/JVI.78.24.13975-13986.2004.

Localization and characterization of flavivirus envelope glycoprotein cross-reactive epitopes

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Localization and characterization of flavivirus envelope glycoprotein cross-reactive epitopes

Wayne D Crill et al. J Virol. 2004 Dec.

Abstract

The flavivirus E glycoprotein, the primary antigen that induces protective immunity, is essential for membrane fusion and mediates binding to cellular receptors. Human flavivirus infections stimulate virus species-specific as well as flavivirus cross-reactive immune responses. Flavivirus cross-reactive antibodies in human sera create a serious problem for serodiagnosis, especially for secondary flavivirus infections, due to the difficulty of differentiating primary from secondary cross-reactive serum antibodies. The presence of subneutralizing levels of flavivirus cross-reactive serum antibodies may result in a dramatic increase in the severity of secondary flavivirus infections via antibody-dependent enhancement. An understanding of flavivirus E-glycoprotein cross-reactive epitopes is therefore critical for improving public health responses to these serious diseases. We identified six E-glycoprotein residues that are incorporated into three distinct flavivirus cross-reactive epitopes. Two of these epitopes which are recognized by distinct monoclonal antibodies contain overlapping continuous residues located within the highly conserved fusion peptide. The third epitope consists of discontinuous residues that are structurally related to the strictly conserved tryptophan at dengue virus serotype 2 E-glycoprotein position 231.

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Figures

FIG. 1.
FIG. 1.
DENV-2 envelope glycoprotein homodimer. The image shows a top view looking down towards the viral surface and depicts the locations of flavivirus cross-reactive epitope residues, which are highlighted by green space-filling representations. The structural and functional domains I, II, and III are shown in red, yellow, and blue, respectively, and disulfide bridges are shown as orange bars.
FIG. 2.
FIG. 2.
Structural locations of cross-reactive epitope residues for flavivirus cross-reactive MAbs in the atomic structure of the DENV-2 E-glycoprotein dimer and bar graphs indicating reductions in MAb reactivities as assayed by IFA and/or Ag-ELISA for mutations at these E-glycoprotein positions. (A) Flavivirus group-reactive A1 epitope residues (MAbs 4G2 and 6B6C-1) from the fusion peptide region of DII. This view is looking downward toward the viral membrane surface at an angle of ∼45° while looking in toward the fusion peptide region about 45° off of parallel to the dimer's longitudinal axis. DI, DII, and DIII are shown in red, yellow, and blue, respectively. DI and DIII from the alternate subunit are depicted as space-filling Van der Waals surfaces to highlight the close fitting of the fusion peptide into this region. Fusion peptide residues 100 to 108 are depicted in green, with the participating amino acids colored by atom and labeled. Glycan moieties attached to Asn153 and Asn67 are depicted with tan space-filling models (labeled CHO153 and CHO67, respectively), and disulfide bridges are shown as orange bars. Lys310 and Arg323 from DIII are depicted as light cyan blue sticks to emphasize that along with the glycan on residue 153, these moieties cover and protect fusion peptide residues Trp101 and Gly102. (B) Flavivirus subgroup-reactive epitope residues (MAb 1B7-5). The angle of this view and the coloring and labeling scheme are the same as those for panel A. The residues identified in this study are depicted as sticks and are labeled as colored individual atoms. (C) Bar graph showing decreases in cross-reactive MAb reactivities in Ag-ELISA for DENV-2 VLPs with substitutions at the listed residues. The fold decreases in MAb reactivities were determined by dividing the wild-type antigen end-point MAb titers by the mutant antigen end-point titers. Anti-A1 reactive MAbs 4G2 and 6B6C-1 are flavivirus group-reactive antibodies, and the anti-A5 MAb 1B7-5 is a flavivirus subgroup-reactive antibody. †, substitutions at G104 and W231 produced plasmids which were unable to secrete measurable VLP antigen into the tissue culture medium. Therefore, fold decreases in MAb reactivities for these two constructs are from IFA data. MAb reactivities with a wild-type unmutated plasmid did not have end points extending nearly as far in the less sensitive immunofluorescence assay as they did in Ag-ELISA (Table 3), and therefore the reductions for substitutions at G104 and W231 were not as large as those for other constructs that were measured by Ag-ELISA, even though the substitutions at these two positions appeared to completely abolish MAb reactivity.

References

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