Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody.
Envelope glycoprotein E of flaviviruses is exposed at the surface of the virion, and is responsible for eliciting a neutralizing antibody (Ab) response, as well as protective immunity in the host. In this report, we describe a method for the fine mapping of a linear sequence of the E protein of dengue virus type-2 (DEN-2), recognized by a type-specific and neutralizing monoclonal Ab (mAb), 3H5. First, an Escherichia coli expression vector containing a heat-inducible lambda pL promoter was used to synthesize several truncated, and near-full length E polypeptides. Reactivities of these polypeptides with polyclonal mouse hyperimmune sera, as well as the 3H5 mAb revealed the location of the 3H5-binding site to be within a region of 166 amino acids (aa) between aa 255 and 422. For fine mapping, a series of targeted deletions were made inframe within this region using the polymerase chain reaction (PCR). The hydrophilicity pattern of this region was used as a guide to systematically delete the regions encoding the various groups of surface aa residues within the context of a near-full-length E polypeptide by using PCR. The 3H5-binding site was thus precisely mapped to a region encoding 12 aa (between aa 386 and 397). A synthetic peptide containing this sequence was able to bind to the 3H5 mAb specifically, as shown by enzyme-linked immunosorbent assay. In addition, we show that rabbit Abs raised against the synthetic peptide of 12 aa were able to bind to the authentic E protein, and to neutralize DEN-2 virus in a plaque reduction assay.
Trirawatanapong
T
T
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66103.
Chandran
B
B
Putnak
R
R
Padmanabhan
R
R
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000956:Antigens, Viral / Q000737:chemistry*; D001483:Base Sequence; D001666:Binding Sites, Antibody; D003716:Dengue Virus / Q000276:immunology*; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry; D014759:Viral Envelope Proteins / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; C051821:E-glycoprotein, Dengue virus type 2; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins