Generation and characterization of monoclonal antibodies against dengue virus type 1 for epitope mapping and serological detection by epitope-based peptide antigens

Abstract

Dengue virus (DEN), the pathogen behind dengue hemorrhagic fever, remains a public health problem in Asia and South America. In this study, monoclonal antibodies (MAbs) against DEN serotype 1 (DEN-1) were generated by fusing NSI/1-Ag4-1 mouse myeloma cells with lymphocytes from BALB/c mice immunized with DEN-1. Twelve MAbs were found to react specifically to the DENs by enzyme-linked immunosorbent assay, immunofluorescence analysis, and immunoblotting analysis. Five MAbs, namely, DA4-7, DA6-7, DA9-5, DA10-2, and DA11-13, were found to react with envelope proteins of DEN-1. Two serotype-specific MAbs of DEN-1, DA6-7 and DA11-13, were further shown to neutralize DEN-1 infection by a plaque reduction neutralization test. The neutralizing epitopes of these MAbs were further identified from a random peptide library displayed on phage. Immunopositive phage clones reacted specifically with these MAbs and did not react with normal mouse serum. Epitope-based peptide antigens were proved able to detect antibodies in serum samples collected from DEN-1-infected patients but not in those taken from DEN-2-infected patients or healthy controls. We believe that these MAbs and neutralizing epitopes will provide information that will lead to the development of DEN-1 serotype-specific diagnostic reagents and vaccines.

FIG. 1.

Identification of MAbs against E and NS1 proteins of DENs by immunoblot analysis. Four serotypes of DEN antigens (D1 to D4) from DEN-infected C6/36 cell lysates were size fractionated in polyacrylamide gels. The blots were incubated with MAbs. (A) MAbs DA4-7, DA6-7, DA9-5, DA10-2, and DA11-13, recognizing E proteins (55 kDa) of DENs, were identified by immunoblot analysis using a nonreducing gel. (B) MAbs DA6-6, DA8-4, DA11-1, DA11-9, DA15-2, DA31-6, and DA32-9 against the dimeric form of NS1 proteins (75 kDa) were identified by immunoblot analysis using a nonreducing gel.

FIG. 2.

In vitro neutralization of DEN strains by neutralizing MAbs DA6-7 and DA11-13. The ascitic fluids containing DA6-7 and DA11-13 were purified using a protein G Sepharose column. (A) The neutralizing activities of the purified MAbs were tested by PRNT against DEN-1 strain 766733. (B) Inhibition of DEN infection by neutralizing MAbs, using an immunofluorescence assay. DEN-1 strain 766733 was incubated with 100 μg/ml of neutralizing or control MAb for 1 hour and then used to infect BHK-21 cells. The DEN-infected cells were detected with the 4G2 MAb.

FIG. 3.

Identification of neutralizing epitopes of DA6-7 and DA11-13 by phage display. (A) A phage-displayed random peptide library was screened by DA6-7. After three rounds of biopanning, 15 of 30 selected phage clones showed significant reactivity to antibody DA6-7 but not to control NMS. (B) A phage-displayed random peptide library was screened by DA11-13. After three rounds of biopanning, 32 of 36 selected phage clones showed significant reactivity to antibody DA11-13 but not to NMS.

FIG. 4.

Specific reactivities of selected phage clones to neutralizing MAbs. An ELISA plate was coated with DA6-7 (A), DA11-13 (B), or NM-IgG (C). The antibodies were then incubated with 10-fold serially diluted phage clones (10⁹, 10⁸, 10⁷, 10⁶, and 0 PFU). The DA6-7-selected phage clone (DA6-7-C4) bound to DA6-7 specifically but did not react with DA11-13 and NM-IgG. DA11-13-selected phage clones (DA11-13-C1, -C3, -C12, and -C36) bound to DA11-13 specifically but did not react with DA6-7 and NM-IgG.

FIG. 5.

Phage competitive inhibition assay by immunoblot analysis. (A) The reactivity of DA6-7 with E protein was inhibited by phage clone DA6-7-C4. The reactivity of DA11-13 with E protein was inhibited by phage clones DA11-13-C2 (B) and DA11-13-C4 (C).

FIG. 6.

(A and B) Capture ELISA for serum samples from patients with DEN-1 infection. Serum samples (200-fold dilution) from DEN-infected patients were analyzed. Representative data are shown to illustrate the MAb responses. All of the serum samples from DEN-1- and DEN-2-infected patients could be detected by DA6-7-captured DEN-1 (A) and DA11-13-captured DEN-1 (B). (C and D) ELISA reactivities of phage clones with serum samples (200-fold dilution) from DEN-infected patients. (C) Six of eight serum samples from DEN-1-infected patients could be identified by DA6-7-C4, but all serum samples from DEN-2-infected patients and NHS from healthy control subjects did not reveal such reactivity. (D) Five of eight serum samples from DEN-1-infected patients could be identified by DA11-13-C1, but eight serum samples from DEN-2-infected patients and NHS revealed no such reactivity. Cutoff values are represented by solid lines.