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. 2003 Jan 28;116(2):139–150. doi: 10.1016/0378-1119(92)90509-N

Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody

Thaweesak Trirawatanapong a, Bala Chandran b, Robert Putnak c, R Padmanabhan a,∗
PMCID: PMC7125935  PMID: 1634111

Abstract

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 λ 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.

Keywords: Recombinant DNA, λ pl promoter, expression in Escherichia coli, epitope mapping, PCR, deletion mutagenesis, mAb, synthetic peptide antigen

Abbreviations: A, absorbance; aa, amino acid(s); Ap, ampicillin; bp, base pair(s); C, capsid protein; cDNA, DNA complementary to RNA; DEN-2, dengue virus type 2; DMEM, Dulbecco's modified Eagle's medium; DOC, deoxycholate; E, envelope protein; ELISA, enzyme-linked immunosorbent assay; HI, hemagglutination-inhibition; HMAF, hyperimmune mouse ascitic fluid containing polyclonal antibodies against a mixture of DEN-2 antigens; HPLC, high-performance liquid chromatography; HSV-2, herpes simplex virus type 2; JE, Japanese encephalitis virus; kb, kilobase(s) or 1000 bp; KLH, keyhole limpet hemocyanin; LB, Luria-Bertani (medium); M, membrane protein; mAb, monoclonal antibody; MBS, m-maleimidobenzoylsulfosuccinimide ester; m.o.i., multiplicity of infection; NGS-C, New Guinea C strain; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PBS, phosphate-buffered saline (10 mM Na phosphate,pH 7.2/150 mM NaCl); PolIk, Klenow (large) fragment of E. coli DNA polymerase I; PCR, polymerase chain reaction; pfu, plaque forming units; PMSF, phenylmethylsulfonyl fluoride; prM, precursor to membrane protein; PRNT, plaque reduction neutralization test; RBS, ribosome binding site; SDS, sodium dodecyl sulfate; Taq, Thermus aquaticus YTI; TBE, tick-borne encephalitis; WN, West Nile virus; YF, yellow fever virus

Received by J.J. Schlesinger

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