Identification of B-cell epitope of dengue virus type 1 and its application in diagnosis of patients

Abstract

Using a serotype-specific monoclonal antibody (MAb) of dengue virus type 1 (DEN-1), 15F3-1, we identified the B-cell epitope of DEN-1 from a random peptide library displayed on phage. Fourteen immunopositive phage clones that bound specifically to MAb 15F3-1 were selected. These phage-borne peptides had a consensus motif of HxYaWb (a = S/T, b = K/H/R) that mimicked the sequence HKYSWK, which corresponded to amino acid residues 111 to 116 of the nonstructural protein 1 (NS1) of DEN-1. Among the four synthetic peptides corresponding to amino acid residues 110 to 117 of the NS1 of DEN-1, -2, -3, and -4, only one peptide, EHKYSWKS (P14M) of DEN-1, was found to bind to 15F3-1 specifically. Furthermore, P14M was shown to inhibit the binding of phage particles to 15F3-1 in a competitive inhibition assay. Histidine(111) (His(111)) was crucial to the binding of P14M to 15F3-1, since its binding activity dramatically reduced when it changed to leucine(111) (Leu(111)). This epitope-based peptide demonstrated its clinical diagnostic potential when it reacted with a high degree of specificity with serum samples obtained from both DEN-1-infected rabbits and patients. Based on these observations, our DEN-1 epitope-based serologic test could be useful in laboratory viral diagnosis and in understanding the pathogenesis of DEN-1.

FIG. 1

Selection for 15F3-1-binding phage clones. After the third round of biopanning, 20 individual phage clones were isolated; only 14 of them reacted strongly with 15F3-1.

FIG. 2

Characterization of the B-cell epitope of MAb 15F3-1. (A) Binding assay of synthetic peptide with MAb 15F3-1. Only the P14M peptide antigen specifically reacted with 15F3-1. (B) Competitive inhibition of phage clone binding to 15F3-1 by synthetic peptide. Binding of 15F3-1-selected phage clone (HB47-1) to 15F3-1 was inhibited only by peptide P14M.

FIG. 3

The specificity of binding of synthetic peptides from DEN-1, -2, -3, and -4 with antibody 15F3-1 was determined by ELISA. DEN-1 peptides showed two- to sixfold-higher reactivity than DEN-2, -3, and -4 peptides, control peptides (P4M and P7M), or BSA.

FIG. 4

ELISA reactivities of DEN-1 synthetic peptide P14M with serum samples from 21 DEN-1-infected patients versus 21 healthy donors. The cutoff value (dashed lines) was calculated as 0.405. Solid line, mean OD₄₉₀.