Identification of a dengue virus type 2 (DEN-2) serotype-specific B-cell epitope and detection of DEN-2-immunized animal serum samples using an epitope-based peptide antigen

Han-Chung Wu1, Mei-Ying Jung1, Chien-Yu Chiu1, Ting-Ting Chao1, Szu-Chia Lai2, Jia-Tsrong Jan2 and Men-Fang Shaio2

1 Graduate Institute of Oral Biology, College of Medicine, National Taiwan University, Taipei, Taiwan 100, Republic of China
2 Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan 100, Republic of China

Correspondence
Han-Chung Wu
hcw0928{at}ha.mc.ntu.edu.tw


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, a serotype-specific monoclonal antibody (mAb), D2 16-1 (Ab4), against dengue virus type 2 (DEN-2) was generated. The specificity of Ab4, which recognized DEN-2 non-structural protein 1, was determined by ELISA, immunofluorescence and immunoblotting analyses. The serotype-specific B-cell epitope of Ab4 was identified further from a random phage-displayed peptide library; selected phage clones reacted specifically with Ab4 and did not react with other mAbs. Immunopositive phage clones displayed a consensus motif, His–Arg/Lys–Leu/Ile, and a synthetic peptide corresponding to the phage-displayed peptide bound specifically to Ab4. The His and Arg residues in this epitope were found to be crucial for peptide binding to Ab4 and binding activity decreased dramatically when these residues were changed to Leu. The epitope-based synthetic peptide not only identified serum samples from DEN-2-immunized mice and rabbits by ELISA but also differentiated clearly between serum samples from DEN-2- and Japanese encephalitis virus-immunized mice. This mAb and its epitope-based peptide antigen will be useful for serologic diagnosis of DEN-2 infection. Furthermore, DEN-2 epitope identification makes it feasible to dissect antibody responses to DEN and to address the role of antibodies in the pathogenesis of primary and secondary DEN-2 infections.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Dengue virus (DEN) is a human pathogen that causes a spectrum of illnesses that range from mild, undifferentiated acute fever to severe syndromes such as dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) (Halstead, 1988; Henchal & Putnak, 1990; Gubler & Clark, 1995). Primary DEN infection can cause painful, debilitating and non-fatal dengue fever (DF). It appears to protect against re-infection by the same serotype. Severe and sometimes fatal DHF and DSS often occur in regions where more than one DEN serotype is circulating (Halstead, 1988; Gubler, 1998). A second, different DEN serotype infection has been associated with increased risk for DHF and may be caused by monocyte/macrophage uptake of virus complexes to non-neutralizing antibodies, subneutralizing cross-reactive antibodies or low-titre neutralizing antibodies (Halstead et al., 1984; Halstead, 1988; Bielefeldt-Ohmann, 1997).

Virus infection triggers production of antibodies directed against viral protein epitopes by activating host humoral immunity. Epitopes can be classified as conformational (discontinuous or linear sequences) or non-conformational (linear sequences) (Sela, 1969; Barlow et al., 1986; Laver et al., 1990). Linear epitopes are short stretches of primary protein structure composed of continuous amino acid residues of the primary sequence. Conformational epitopes consist of several amino acid residues, discrete in primary sequence, that assemble to form antigenic determinants on the tertiary structure of native proteins (Barlow et al., 1986; Laver et al., 1990). Viral protein epitopes are pivotal in the pathogenesis of virus infectious diseases and in the development of effective vaccines and diagnostic reagents.

Advances in peptide technology have led to the development of combinatorial peptide libraries expressed on a solid-phase support or displayed on bacteriophages. Phage-displayed random peptide libraries and the high molecular diversity displayed by these libraries provide opportunities to study various topics: mapping B-cell epitopes (Scott & Smith, 1990; D'Mello et al., 1997; Fu et al., 1997; Wu et al., 2001a, b) and protein–protein contacts (Atwell et al., 1997; Bottger et al., 1997; Nord et al., 1997; Smith et al., 1999), selecting bioactive peptides bound to receptors (Li et al., 1995; Akeson et al., 1996; Wrighton et al., 1996; Koivunen et al., 1999) or proteins (Castano et al., 1995; Pasqualini et al., 1995; DeLeo et al., 1995; Bottger et al., 1996; Kraft et al., 1999), searching for disease-specific antigen mimics (Folgori et al., 1994; Prezzi et al., 1996), determining cell- (Barry et al., 1996; Szardenings et al., 1997; Mazzucchelli et al., 1999) and organ-specific peptides (Pasqualini & Ruoslahti, 1996; Arap et al., 1998, 2002; Rajotte & Ruoslahti, 1999; Essler & Ruoslahti, 2002), producing peptides that mimic the effect of neutralizing antibodies (Heiskanen et al., 1997) and identifying peptides that mimic non-peptide ligands (Cortese et al., 1994).

DEN is a major cause of paediatric morbidity and mortality in tropical regions (Halstead, 1988). A safe vaccine and simple reliable test for serodiagnosis of DEN infection could reduce morbidity and mortality significantly. Serotype-specific B-cell epitopes of DEN are not well documented. Currently, neither a protein nor a peptide antigen has been used to differentiate the four DEN serotypes.

Recently, we identified the serotype-specific B-cell epitope of DEN-1 (Wu et al., 2001a). In this study, we generated a serotype-specific mAb against DEN-2 and used a phage-displayed peptide library to identify the serotype-specific B-cell epitope for DEN-2. This serotype-specific mAb and epitope-based peptide can be used to develop a convenient, efficient serologic test that identifies DEN serotypes.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
DEN-2 strain PL046, a local Taiwanese strain isolated from patients with DF in Hsiao-Liu-Chiu (Islet of Pingtung, Taiwan, Republic of China) was provided by the National Institute of Preventive Medicine, Taiwan, Republic of China. Four prototype DEN strains [DEN-1 (Hawaii), DEN-2 (New Guinea C), DEN-3 (H87) and DEN-4 (H241)] were provided by D. J. Gubler (Centers for Disease Control and Prevention, Fort Collins, USA). These viruses were passaged in Aedes albopictus C6/36 cells and DEN titres were measured by plaque assay in BHK-21 cells. C6/36 cells were grown in RPMI 1640 medium containing 10 % heat-inactivated FBS. BHK-21 cells were grown in RPMI 1640 medium containing 5 % heat-inactivated FBS.

Generation of serotype-specific mAbs against DEN-2.
Hybridomas secreting anti-DEN-2 antibodies were generated according to standard procedures (Kohler & Milstein, 1975). Briefly, the spleen of an immunized mouse was removed. Splenocytes were fused with NSI/1-Ab4-1 (NS-1) myeloma cells and washed twice with DMEM. Fused cells were then mixed in a 15 ml conical tube and 1 ml 50 % (v/v) PEG (Gibco-BRL) was added over 1 min with gentle stirring. The mixture was diluted by the slow (1 min) addition of 1 ml DMEM, twice, followed by the slow addition (2 min) of 8 ml serum-free DMEM. The mixture was then centrifuged at 400 g for 5 min. The fused cell pellet was re-suspended in DMEM supplemented with 15 % FBS, HAT medium and hybridoma cloning factor (ICN). Next, 150 µl per well of the resuspension mixture was distributed in 96-well tissue culture plates. Hybridoma colonies were screened by ELISA for mAbs that bound DEN-infected C6/36 cells. Selected clones were subcloned by limiting dilution. Final hybridoma clones were isotyped using an isotyping kit from Roche Diagnostics. Ascites fluids were produced in pristane-primed BALB/c mice. Hybridoma cell lines were grown in RPMI 1640 medium with 10 % heat-inactivated FBS. mAbs were affinity-purified using a protein G–Sepharose 4B gel and ELISA and Western blot assays were used to measure the activity and specificity of the antibodies isolated.

Preparation of DEN-2 antigen.
C6/36 cells were infected with DEN-2, pelleted and washed three times with PBS. Cells were then lysed in lysis buffer (25 mM Tris/HCl, pH 7·4, 150 mM NaCl and 1 % Nonidet-P40) in the presence of protease inhibitors (1 mM EDTA, 0·1 mM PMSF, 10 µM leupeptin, 10 µM chymostatin and 1 µM pepstatin). Cell debris was removed by centrifugation at 3000 g for 10 min at 4 °C and protein was quantified using the protein dye-binding method described by Bradford (1976).

Western blot analysis.
Cell lysates or proteins were mixed with an equal volume of sample buffer (50 mM Tris/HCl, pH 6·8, 100 mM DTT, 2 % SDS, 0·1 % bromophenol blue and 10 % glycerol), separated by SDS-PAGE and then transferred to a nitrocellulose membrane (Hybond-C Super, Amersham). Non-specific antibody-binding sites were blocked with 5 % skimmed milk in PBS and membranes were incubated with primary antibody. Blots were then treated with HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) and developed with chemiluminescence reagents (ECL, Amersham).

Indirect immunofluorescence assay.
BHK-21 cells were mock-infected or infected with DEN-1, -2, -3 or -4. Cells were then fixed with -20 °C methanol/acetone (1 : 1) for 10 min and washed three times with PBS. Cells were incubated with a 100-fold dilution of mAb or normal ascites fluid. After 60 min of incubation, cells were washed three times with PBST [PBS plus 0·5 % (w/v) Tween-20] for 5 min each time. Cells were then treated with FITC-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories) for 30 min, washed four times with PBST for 5 min each time and mounted for fluorescent microscopic observation.

Phage-display biopanning procedures.
An ELISA plate was coated with 100 µg mAb Ab4 ml-1 in 0·1 M NaHCO3 (pH 8·6) buffer: samples of 100 µl of diluted mAb were then added to each well and incubated at room temperature for 2 h with gentle agitation. The plate was washed, incubated with blocking buffer (1 % BSA in PBS) at 4 °C overnight and then washed rapidly five times with PBST. The phage-displayed peptide library was diluted to 4x1010 phage, added onto the coated plate and rocked gently for 50 min at room temperature. The plate was then washed 10 times with PBST. Bound phage was eluted with 100 µl 0·2 M glycine/HCl (pH 2·2) and 1 mg BSA ml-1 and the eluate was neutralized with 15 µl 1 M Tris/HCl (pH 9·1). Eluted phage was amplified in ER2738 culture at 37 °C with vigorous shaking for 4·5 h. Amplified phage was centrifuged for 20 min at 10 000 g at 4 °C and supernatant was removed to a fresh tube and re-spun. The upper 80 % of the supernatant was removed to a fresh tube and 1/6 vol. PEG/NaCl [20 % (w/v) PEG-8000 and 2·5 M NaCl] was added to precipitate the phage at 4 °C overnight. Phage was isolated by centrifugation for 20 min at 10 000 g at 4 °C. The phage pellet was suspended in 1 ml PBS and centrifuged for 5 min at 4 °C to pellet residual cells. The supernatant was transferred to a fresh microcentrifuge tube and re-precipitated with 1/6 vol. PEG/NaCl on ice for 45 min. Phage was isolated by centrifugation at 4 °C for 10 min and re-suspended in 200 µl PBS containing 0·02 % NaN3. Isolated phage was centrifuged for 1 min to pellet any remaining insoluble matter. The supernatant was transferred to a fresh tube and amplified phage was titrated onto LB medium plates containing IPTG and X-Gal. The protocol for second-round biopanning was identical to the first, with the addition of 2x1011 p.f.u. from first-round biopanning phage to each well. Third-round biopanning was, once again, identical to the first, with the addition of 2x1011 p.f.u. of second-round biopanning phage to each well. Unamplified third-round phage eluate was titrated on LB medium plates containing IPTG and X-Gal. The remaining eluate was stored at 4 °C.

Identification of immunopositive phage clones by ELISA.
An ELISA plate was coated with 100 µl antibody (100 µg ml-1) in 0·1 M NaHCO3 (pH 8·6) at room temperature for 2 h and blocked with blocking buffer at 4 °C overnight. Serially diluted phage was added to the antibody-coated plate and incubated at room temperature for 1 h. The plate was then washed six times with PBST and diluted HRP-conjugated anti-M13 antibody (Pharmacia) diluted 1 : 5000 in blocking buffer was added. The plate was incubated at room temperature for 1 h with agitation and washed six times with PBST. HRP/substrate solution was added to each well and incubated at room temperature. The reaction was stopped with 3 N HCl and the plate was read using a microplate reader set at a wavelength of 490 nm.

DNA sequencing and computer analysis.
DNA sequences of purified phage were determined according to the dideoxynucleotide chain termination method using an automated DNA sequencer (ABI PRISM 377, Perkin-Elmer). The primer used for phage DNA sequencing was 5'-CCCTCATAGTTAGCGTAA-3'; this primer locates to the antisense strand of gene III of the M13 phage and is 96 nt away from the inserted DNA. Inserted oligonucleotide sequences of phage DNA were selected and translated to peptide sequences. Peptide sequences were aligned using the DNASIS software designed for the Apple Macintosh (Hitachi Software Engineering).

Antibody-binding assay.
ELISA plates were coated with 50 µl per well of individual peptide antigens at a concentration of 10 µg ml-1 and blocked with 1 % BSA. For the binding assay, 2-fold serial peptide antigens (concentrations ranging from 20 to 0·31 µg ml-1) were coated to the plates and 2 µg antibody ml-1 was added to each well and incubated at room temperature for 1 h. Plates were incubated with HRP-conjugated anti-mouse IgG and colour was subsequently developed with o-phenylenediamine dihydrochloride (Sigma) and hydrogen peroxide in the dark. The reaction was stopped with 3 N HCl and the absorbance was measured at 490 nm.

Animal immunization.
Female, 4- to 6-week-old, BALB/c mice were immunized by intraperitoneal (i.p.) injection of DEN and Japanese encephalitis virus (JEV) with adjuvant on days 0, 14 and 28; mice were bled on day 38. Outbreed laboratory rabbits were infected by intravenous (i.v.) injection with DEN-2. Serum samples used in IgM assays were collected 7 days after an initial i.v. infection. Hyperimmune sera were obtained following four inoculations with virus. Immunized serum samples of mice and rabbits were collected and antibody levels were measured by ELISA.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of serotype-specific mAbs against DEN-2
Immunoblotting and ELISA assays determined the reactivity of mAb D2 16-1 (Ab4) with DEN-2 and other DEN serotypes. Ab4 reacted only to NS1 of DEN-2 and did not cross-react with DEN-1, -3 or -4 (Fig. 1). Immunoblotting analysis showed that the NS1 protein of DEN-2 was dimeric on a native gel (Fig. 1A, NS12) and monomeric on a denaturing gel (Fig. 1B, NS1). ELISA confirmed the specificity of Ab4. C6/36 cells were infected with DEN-1, -2, -3 and -4. Cells were then fixed for ELISA analysis using Ab4, normal mouse IgG (NM-IgG) and normal mouse serum (NMS). Ab4 detected DEN-2-infected cells but not DEN-1-, -3- or -4-infected cells (Fig. 1C). To confirm the DEN-2 serotype-specificity of Ab4, we also performed an indirect immunofluorescence assay. BHK-21 cells were infected with DEN-1, -2, -3 or -4. Only DEN-2-infected BHK-21 cells were detected by Ab4; DEN-1-, -3- and -4-infected cells were not reactive (data not shown). We concluded that Ab4 is a DEN-2 serotype-specific mAb that does not cross-react with other DEN serotypes.



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Fig. 1. Identification the serotype-specificity of DEN-2 mAb (Ab4) by ELISA and immunoblotting assay. Four serotypes of DEN antigens from DEN-infected C6/36 cell lysates were size-fractionated on polyacrylamide gels with (denature gel, B) or without (native gel, A) {beta}-mercaptoethanol. The blot was incubated with Ab4 mAb. Only DEN-2 (D2) could be recognized by Ab4 both on native (A) and denatured (B) gel. NS12: dimeric form of NS1 protein. In ELISA (C), only DEN-2-infected C6/36 cells could be detected by Ab4 but not DEN-1 (D1), -3 (D3) and -4 (D4)-infected cells. Normal mouse IgG (NM-IgG) and normal mouse serum (NMS) had no such reactivity with four serotype of DENs. Error bars indicate SD.

 
Screening of phage-displayed peptide library with DEN-2 serotype-specific antibody
Ab4, a serotype-specific mAb of DEN-2, is an IgG1 isotype (data not shown). Its B-cell epitope was identified by the phage-display method. To select immunopositive phage clones by DEN-2-specific mAbs, Ab4 ascites fluid was purified by a protein G affinity column. Purified antibodies were immobilized on ELISA plates and bound phage clones were selected after biopanning three times. Further screening of immunopositive phage clones was performed by single phage clone isolation and amplification for Ab4 screening by ELISA. Of the 20 phage clones selected, 17 (Ab4-C1, C2, C3, C4, C5, C6, C7, C8, C11, C12, C13, C14, C15, C17, C18, C19 and C20) had significant enhancement of antibody Ab4 reactivity (Fig. 2); they did not bind to either NMS or NM-IgG (Fig. 2).



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Fig. 2. Identification of Ab4-selected phage clones by ELISA. A phage-displayed random peptide library was screened by Ab4. After three rounds of screening, 17 phage clones from 20 selected phage clones showed significant reactivity to antibody Ab4 but not to normal mouse serum (NMS) or normal mouse IgG.

 
To prove that the selected phage clone bound Ab4 specifically, antibodies were incubated with a 10-fold serial dilution of the selected phage clone (Ab4-C4) and the control phage clone (HB47-1, selected by an anti-DEN-1 mAb). Only the selected phage clone (Ab4-C4) bound the antibody specifically and dose dependently. The control phage clone (HB47-1) did not react with the antibody (Fig. 3A). To confirm further that the selected phage clone Ab4-C4 bound Ab4 specifically, eight (Ab9-1, 12-1, 32-7, 42-1, 43-1, 45-3, 48-6 and 52-2) mAbs against DEN-2 and NM-IgG were incubated with Ab4-C4 and an ELISA was performed. The immunopositive phage clone Ab4-C4 showed high Ab4 antibody specificity and did not react with these eight mAbs or with NM-IgG (Fig. 3B). The control phage clone (HB47-1) did not bind Ab4 (Fig. 3B).



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Fig. 3. (A) Specific reactivity of selected phage clone with antibody Ab4. The Ab4-selected phage clone (Ab4-C4) could react the antibody specifically but control phage clone (DEN-1) could not. (B) Identification of the specificity of Ab4-selected phage clones with mAbs. Nine mAbs against DEN-2 and purified normal mouse IgG were immobilized on a 96-well microplate and incubated with 1x109 p.f.u. of Ab4-selected phage (Ab4-C4) clones and control phage clones (DEN-1). The Ab4-selected phage clones bound only to Ab4 and did not react with the other eight antibodies or with normal mouse IgG (NM-IgG).

 
Characterization of the B-cell epitope
Twelve immunopositive phage clones (Ab4-C2, C3, C4, C8, C11, C12, C13, C14, C15, C17, C19 and C20) that were highly reactive with Ab4 were amplified and phage DNAs were isolated for DNA sequencing. The sequencing primer, 5'-CCCTCATAGTTAGCGTAA-3', is located in the antisense strand of gene III of the M13 phage and has 96 nt separated from the inserted DNA. Inserted nucleotides of the selected phage clones were sequenced. All contained 36 inserted nucleotides (translated to 12 aa residues) (Table 1). Inserted oligonucleotide sequences of phage DNAs were selected and translated to peptide sequences. Peptide sequences were aligned using DNASIS to analyse the epitopes and the binding motif of Ab4. Interestingly, three residues, His (H)–Arg (R)/Lys (K)–Leu (L)/Ile (I), were highly conserved in these immunopositive phage clones; the other nine residues were random (Table 1). We concluded that the binding motif for Ab4 is His–Arg/Lys–Leu/Ile and that all phage clones selected contained this motif (Table 1).


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Table 1. Alignment of phage-displayed peptide sequences selected by DEN-2 serotype-specific mAb Ab4

Phage-displayed consensus amino acids are shown in bold.

 
Binding assay of a mimic synthetic peptide
To verify that peptide sequences identified by phage display were recognized by antibody Ab4, synthetic peptide-binding assays were carried out. Phage-displayed peptides were synthesized in multiple antigen peptide (MAP) form because ELISA sensitivity for an eight-chain MAP is greater than for a single-chain peptide (Tam & Zavala, 1989; Wu et al., 2001a). As shown in Fig. 4, synthetic peptide P7M (SHRLHNTMPSES), corresponding to immunopositive phage clone Ab4-C4 (Table 1), which had been selected with Ab4 from the phage-displayed peptide library, bound antibody in a concentration-dependent manner. Unrelated control MAP peptides, KGTFDPLQEPRT (P4M) and EHKYSWKS (P14M), were not reactive.



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Fig. 4. ELISA reactivity of synthetic peptide corresponding with selected phage clone Ab4-C4. The phage-displayed peptide SHRLHNTMPSES (P7M) was synthesized and immobilized on a 96-well plate as an antigen to detect Ab4. The synthetic MAP peptide (P7M), corresponding with the selected phage clone, reacted with Ab4 specifically. Two control MAP peptides P4M and P14M had no reactivity with Ab4.

 
Using the phage-display method, we determined that the binding motif for Ab4 was His–Arg–Leu/Ile. His and Arg were found in most immunopositive phage clones (Table 1). We proposed that these two amino acid residues play a crucial role in binding to the antibody. To prove this hypothesis, we synthesized two peptides: P7M-m1 (SLRLHNTMPSES) and P7M-m2 (SHLLHNTMPSES). There is only a difference of one amino acid residue between these peptides and P7M (SHRLHNTMPSES). ELISA indicated that binding activity was decreased dramatically when positively charged His or Arg in P7M was changed to aliphatic side-chain Leu in P7M-m1 or P7M-m2, respectively (Fig. 5A). Control peptide P4M (KGTFDPLQEPRT) was not reactive with Ab4 (Fig. 5A).



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Fig. 5. (A) Identification of amino acid residue for binding to Ab4. The reactivity of P7M with Ab4 was decreased markedly when His2 (H) or Arg3 (R) in the P7M was changed to Leu2 or Leu3 in the peptides of P7M-m1 and P7M-m2. The control peptide P4M had no reactivity with Ab4. Error bars indicate SD. (B) Peptide-competitive inhibition assay by immunoblotting analysis. The reactivity of Ab4 with NS1 was inhibited by phage-displayed peptide P7M; control peptide P7M-m1 had no effect.

 
To confirm further that the phage-displayed peptide was the epitope of Ab4, a peptide-competitive inhibition assay was performed to determine whether the synthetic peptide P7M competed with NS1 proteins for reactivity with Ab4. The reaction activity of Ab4 with NS1 proteins was inhibited markedly by P7M. The mutated peptide P7M-m1 had no effect on Ab4 reaction with NS1 proteins (Fig. 5B). Our observations suggest strongly that P7M is the B-cell epitope of Ab4 and that His and Arg are important residues for binding to the mAb.

Detection of DEN-2-infected animal sera using epitope-based synthetic peptides
We evaluated whether the epitope-based peptide antigen P7M could be used as a diagnostic tool to detect immunized animal serum samples. Two different species of animals were immunized with DEN-2. Serum samples were collected to test detection efficacy. To test whether antibodies produced from DEN-2-immunized rabbit sera would react with synthetic peptide P7M, we collected hyperimmune serum samples after four inoculations with DEN-2 for ELISA. All serum samples from DEN-2 hyperimmune rabbits had higher ELISA antibody reactivity with P7M peptide than with pre-immune serum samples (Fig. 6B). Serum samples obtained from five JEV hyperimmune and eight normal BALB/c mice had no significant ELISA reactivity with P7M; however, serum samples from all eight DEN-2 hyperimmune BALB/c mice had significant ELISA reactivity with peptide antigen (Fig. 6A). This suggests that P7M can differentiate between JEV and DEN-2 in immunized mouse serum samples. Using different animals and different immunization techniques (i.p. inoculation in mice and i.v. inoculation in rabbits), we determined that P7M could identify DEN-2-immunized animal serum samples. To test further the specificity of P7M, we used this peptide antigen to detect DEN-1-, -2- and -3-immunized mouse serum samples. P7M detected all eight DEN-2 hyperimmune BALB/c mice by ELISA (Fig. 6C). In contrast, all serum samples obtained from DEN-1 and -3 hyperimmune mice were seronegative. These results indicated that vertebrates like rabbits and mice immunized with DEN-2 can produce antibodies recognized by this epitope-based synthetic peptide antigen.



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Fig. 6. ELISA reactivities of P7M with immunized animal serum samples (100-fold dilution). (A) Eight DEN-2-immunized mouse serum samples (DEN-2) could be identified by the peptide P7M but five JEV-immunized mice and eight normal mouse serum samples (NMS) revealed no such reactivity. (B) ELISA reactivities of P7M with pre-immunized and DEN-2-immunized rabbit sera. Five DEN-2-immunized rabbit serum samples could be identified by P7M. (C) ELISA reactivities of P7M with DEN-1, -2- and -3-immunized mouse serum samples. Eight DEN-2-immunized mouse serum samples could be identified by P7M but six DEN-1- and -3-immunized mouse serum samples and eight NMS revealed no such reactivity.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The identification of viral B-cell epitopes is important for peptide selection for subunit vaccines, development of virus-specific serological diagnostic reagents and understanding virus–antibody interactions at a molecular level. This is the first study to characterize a serotype-specific B-cell epitope of a DEN-2 mAb using the phage-display method. We found that the selected phage-displayed 12-mer peptide sequence had a consensus motif, His–Arg/Lys–Leu/Ile. We also detected antibodies from serum samples of DEN-2-immunized animals using the epitope-based peptide antigen. It is feasible that our serotype-specific mAb, phage-displayed epitope and epitope-based peptide antigen can be used to develop diagnostic laboratory tests for serologic DEN identification.

B-cell epitopes of DEN-2 have been documented using overlapping synthetic peptides (PEPSCAN) to analyse antisera (Aaskov et al., 1989; Innis et al., 1989; Roehrig et al., 1990). However, this approach requires many overlapping synthetic peptides, has difficulty identifying conformational epitopes and cannot determine the amino acid-binding motif. A variety of DEN-2 antigenic domains have been studied by antigen fragments using recombinant or enzyme-cleavage proteins (Mason et al., 1990; Megret et al., 1992; Trirawatanapong et al., 1992; Roehrig et al., 1998). Even though these methods have identified antigenic domains consisting of 50–200 aa residues, they have not been able to specify an exact epitope consisting of only three to eight amino acids. At present, no serotype-specific B-cell epitope has been identified by any of these methods.

Phage display is a powerful tool for analysing linear and conformational epitopes or mimotopes, which are generally difficult to characterize. Using this method, we have identified the serotype-specific B-cell epitope of DEN-1 (Wu et al., 2001a) and neutralizing B-cell epitopes on Clostridium botulinum neurotoxin (Wu et al., 2001b). In the present study, we isolated the serotype-specific B-cell epitope of DEN-2 and confirmed further that the His–Arg–Leu motif was crucial for peptide antigen–antibody binding. In the B-cell DEN-1 epitope study, phage-displayed peptides had a consensus motif of HxYaWb (a=S/T and b=K/H/R). This motif mimics the HKYSWK sequence, which corresponds to aa 111–116 of NS1 of DEN-1 (Wu et al., 2001a). These findings indicate that the B-cell epitope of this DEN-1 antibody is linear. Typically, B-cell epitopes identified by this method have an easily recognizable consensus sequence, often corresponding to the peptide sequence found in the natural antigen. However, sometimes the phage-displayed consensus sequence cannot be found in the sequence of natural antigens. For example, our present study and earlier published data (Wu et al., 2001b) as well as other reports (Felici et al., 1993; Folgori et al., 1994) have demonstrated that phage-displayed epitopes interact with antigen-binding sites of antibodies. However, consensus sequences may not show any similarity with the sequence of the natural antigen (Felici et al., 1993; Folgori et al., 1994). In these cases, epitopes can mimic natural epitopes (mimotopes) or conformational epitopes.

Further analysis of the Ab4 epitope indicated that most immunopositive phage clones contained His and Arg (Table 1). These amino acids may play an important role in antibody binding. To test this hypothesis, we synthesized two peptides corresponding to P7M in which His and Arg were substituted with Leu. Specificity and reactivity of these peptides for Ab4 were determined by ELISA: P7M peptide containing His and Arg had greater reactivity and this reactivity was dramatically decreased when positively charged His and Arg were transformed to the aliphatic side-chain Leu in P7M-m1 and P7M-m2, respectively (Fig. 5). Mutational analysis of the epitope showed that a single amino acid change within this peptide affected antibody-binding activity dramatically. His and Arg are important for Ab4 binding, confirming further that charged residues are important in antigenic epitope–antibody interactions (Strauss et al., 1991; Wu et al., 2001a).

Serologic diagnosis of DEN infection uses haemagglutination inhibition (HI) or IgM-capture ELISA. HI assays have various practical limitations: they do not provide an early diagnosis and require paired sera, acetone extraction of serum, serial dilution of sera and are not specific for different flavivirus infections. IgM- and IgG-capture ELISAs require preparation of DEN antigen and antibody as a preliminary step and their use is confined largely to specialized virology centres. Two new DEN diagnostic tests, MRL Diagnostics Dengue Fever Virus IgM Capture ELISA and PanBio Rapid Immunochromatographic Test, have been used to detect DEN antibodies. However, they cannot distinguish between the four DEN serotypes and have 45–50 % cross-reactivity with JEV-infected patients (Vaughn et al., 1998, 1999; Lam & Devine, 1998). Therefore, it is necessary to develop specific diagnostic reagents for DEN serotyping. DHF and DSS have been associated strongly with sequential infections by different DEN serotypes. It has been hypothesized that non-neutralizing cross-reactive antibodies acquired during the first DEN infection enhance the second infection by a different DEN serotype (Halstead, 1988). The molecular mechanism underlying this explanation has not been determined because serotype-specific peptide antigens that distinguish the four serotypes DEN infection have not been isolated and identified.

The similarity of amino acid sequences for the four DEN serotypes ranges from 63·2 to 78·7 % (Westaway & Blok, 1997). High similarity of amino acid sequences within the four DEN serotypes makes them difficult to distinguish using antigens obtained from overlapping synthetic peptides (PEPSCAN) or from recombinant or enzyme-cleavage antigen fragments. Generation of serotype-specific mAbs against DEN and investigation of B-cell epitopes makes it possible to identify serotype-specific epitopes and to develop epitope-based peptides for the diagnosis of DEN infection (Wu et al., 2001a). In this study, we identified the serotype-specific epitope of DEN-2 and used an epitope-based peptide as an antigen to detect DEN-2 antibodies in serum samples from immunized mice and rabbits. Applications of this epitope-based peptide to human serum samples are in progress. Specifically, we are evaluating whether it can distinguish the four DEN serotypes and determine whether a DEN infection is primary or secondary. Using a synthetic peptide for DEN diagnosis is relatively simple and should become routine. Because a second infection with a different DEN serotype increases the risk for DHF and DSS, serotype identification is important. At present, neither a protein nor a peptide antigen is being used to differentiate the four serotypes of DEN or JEV infection. We believe that a serotype-specific mAb and the epitope-based peptide of DEN-2 will be valuable for developing a serotype-specific diagnostic reagent.


   ACKNOWLEDGEMENTS
 
This work was supported by grant NSC 90-2320-B-002-207 from the National Science Council, Republic of China to H.-C. W. and grant NHRI-CN-CL8902P from the National Health Research Institute, Department of Health, Taipei, Republic of China to H.-C. W.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Aaskov, J. G., Geysen, H. M. & Mason, T. J. (1989). Serologically defined linear epitopes in the envelope protein of dengue 2 (Jamaica strain 1409). Arch Virol 105, 209–221.[Medline]

Akeson, A. L., Woods, C. W., Hsieh, L. C. & 8 other authors (1996). AF12198, a novel low molecular weight antagonist, selectively binds the human type I interleukin (IL)-1 receptor and blocks in vivo responses to IL-1. J Biol Chem 271, 30517–30523.[Abstract/Free Full Text]

Arap, W., Pasqualini, R. & Ruoslahti, E. (1998). Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279, 377–380.[Abstract/Free Full Text]

Arap, W., Haedicke, W., Bernasconi, M. & 7 other authors (2002). Targeting the prostate for destruction through a vascular address. Proc Natl Acad Sci U S A 99, 1527–1531.[Abstract/Free Full Text]

Atwell, S., Ultsch, M., De Vos, A. M. & Wells, J. A. (1997). Structural plasticity in a remodeled protein–protein interface. Science 278, 1125–1128.[Abstract/Free Full Text]

Barlow, D. J., Edwards, M. S. & Thornton, J. M. (1986). Continuous and discontinuous protein antigenic determinants. Nature 322, 747–748.[Medline]

Barry, M. A., Dower, W. J. & Johnston, S. A. (1996). Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries. Nat Med 2, 299–305.[Medline]

Bielefeldt-Ohmann, H. (1997). Pathogenesis of dengue virus diseases: missing pieces in the jigsaw. Trends Microbiol 5, 409–413.[CrossRef][Medline]

Bottger, V., Bottger, A., Howard, S. F., Picksley, S. M., Chene, P., Garcia-Echeverria, C., Hochkeppel, H. K. & Lane, D. P. (1996). Identification of novel mdm2 binding peptides by phage display. Oncogene 13, 2141–2147.[Medline]

Bottger, A., Bottger, V., Sparks, A., Liu, W. L., Howard, S. F. & Lane, D. P. (1997). Design of a synthetic Mdm2-binding mini protein that activates the p53 response in vivo. Curr Biol 7, 860–869.[Medline]

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef][Medline]

Castano, A. R., Tangri, S., Miller, J. E., Holcombe, H. R., Jackson, M. R., Huse, W. D., Kronenberg, M. & Peterson, P. A. (1995). Peptide binding and presentation by mouse CD1. Science 269, 223–226.[Medline]

Cortese, R., Felici, F., Galfre, G., Luzzago, A., Monaci, P. & Nicosia, A. (1994). Epitope discovery using peptide libraries displayed on phage. Trends Biotechnol 12, 262–267.[Medline]

DeLeo, F. R., Yu, L., Burritt, J. B., Loetterle, L. R., Bond, C. W., Jesaitis, A. J. & Quinn, M. T. (1995). Mapping sites of interaction of p47-phox and flavocytochrome b with random-sequence peptide phage display libraries. Proc Natl Acad Sci U S A 92, 7110–7114.[Abstract]

D'Mello, F., Partidos, C. D., Steward, M. W. & Howard, C. R. (1997). Definition of the primary structure of hepatitis B virus (HBV) pre-S hepatocyte binding domain using random peptide libraries. Virology 237, 319–326.[CrossRef][Medline]

Essler, M. & Ruoslahti, E. (2002). Molecular specialization of breast vasculature: a breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature. Proc Natl Acad Sci U S A 99, 2252–2257.[Abstract/Free Full Text]

Felici, F., Luzzago, A., Folgori, A. & Cortese, R. (1993). Mimicking of discontinuous epitopes by phage-displayed peptides. II. Selection of clones recognized by a protective monoclonal antibody against the Bordetella pertussis toxin from phage peptide libraries. Gene 128, 21–27.[CrossRef][Medline]

Folgori, A., Tafi, R., Meola, A., Felici, F., Galfre, G., Cortese, R., Monaci, P. & Nicosia, A. (1994). A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera. EMBO J 13, 2236–2243.[Abstract]

Fu, Y., Shearing, L. N., Haynes, S., Crewther, P., Tilley, L., Anders, R. F. & Foley, M. (1997). Isolation from phage display libraries of single chain variable fragment antibodies that recognize conformational epitopes in the malaria vaccine candidate, apical membrane antigen-1. J Biol Chem 272, 25678–25684.[Abstract/Free Full Text]

Gubler, D. J. (1998). Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 11, 480–496.[Abstract/Free Full Text]

Gubler, D. J. & Clark, G. G. (1995). Dengue/dengue hemorrhagic fever: the emergence of a global health problem. Emerg Infect Dis 1, 55–57.[Medline]

Halstead, S. B. (1988). Pathogenesis of dengue: challenges to molecular biology. Science 239, 476–481.[Medline]

Halstead, S. B., Venkateshan, C. N., Gentry, M. K. & Larsen, L. K. (1984). Heterogeneity of infection enhancement of dengue 2 strains by monoclonal antibodies. J Immunol 132, 1529–1532.[Abstract/Free Full Text]

Heiskanen, T., Lundkvist, A., Vaheri, A. & Lankinen, H. (1997). Phage-displayed peptide targeting on the Puumala hantavirus neutralization site. J Virol 71, 3879–3885.[Abstract]

Henchal, E. A. & Putnak, J. R. (1990). The dengue viruses. Clin Microbiol Rev 3, 376–396.[Medline]

Innis, B. L., Thirawuth, V. & Hemachudha, C. (1989). Identification of continuous epitopes of the envelope glycoprotein of dengue type 2 virus. Am J Trop Med Hyg 40, 676–687.[Medline]

Kohler, G. & Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497.[Medline]

Koivunen, E., Arap, W., Rajotte, D., Lahdenranta, J. & Pasqualini, R. (1999). Identification of receptor ligands with phage display peptide libraries. J Nucl Med 40, 883–888.[Abstract]

Kraft, S., Diefenbach, B., Mehta, R., Jonczyk, A., Luckenbach, G. A. & Goodman, S. L. (1999). Definition of an unexpected ligand recognition motif for {alpha}v{beta}6 integrin. J Biol Chem 274, 1979–1985.[Abstract/Free Full Text]

Lam, S. K. & Devine, P. L. (1998). Evaluation of capture ELISA and rapid immunochromatographic test for the determination of IgM and IgG antibodies produced during dengue infection. Clin Diagn Virol 10, 75–81.[CrossRef][Medline]

Laver, W. G., Air, G. M., Webster, R. G. & Smith-Gill, S. J. (1990). Epitopes on protein antigens: misconceptions and realities. Cell 61, 553–556.[Medline]

Li, B., Tom, J. Y., Oare, D., Yen, R., Fairbrother, W. J., Wells, J. A. & Cunningham, B. C. (1995). Minimization of a polypeptide hormone. Science 270, 1657–1660.[Abstract]

Mason, P. W., Zugel, M. U., Semproni, A. R., Fournier, M. J. & Mason, T. L. (1990). The antigenic structure of dengue type 1 virus envelope and NS1 proteins expressed in Escherichia coli. J Gen Virol 71, 2107–2114.[Abstract]

Mazzucchelli, L., Burritt, J. B., Jesaitis, A. J., Nusrat, A., Liang, T. W., Gewirtz, A. T., Schnell, F. J. & Parkos, C. A. (1999). Cell-specific peptide binding by human neutrophils. Blood 93, 1738–1748.[Abstract/Free Full Text]

Megret, F., Hugnot, J. P., Falconar, A. & 8 other authors (1992). Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein. Virology 187, 480–491.[Medline]

Nord, K., Gunneriusson, E., Ringdahl, J., Stahl, S., Uhlen, M. & Nygren, P. A. (1997). Binding proteins selected from combinatorial libraries of an {alpha}-helical bacterial receptor domain. Nat Biotechnol 15, 772–777.[Medline]

Pasqualini, R. & Ruoslahti, E. (1996). Organ targeting in vivo using phage display peptide libraries. Nature 380, 364–366.[CrossRef][Medline]

Pasqualini, R., Koivunen, E. & Ruoslahti, E. (1995). A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins. J Cell Biol 130, 1189–1196.[Abstract]

Prezzi, C., Nuzzo, M., Meola, A., Delmastro, P., Galfre, G., Cortese, R., Nicosia, A. & Monaci, P. (1996). Selection of antigenic and immunogenic mimics of hepatitis C virus using sera from patients. J Immunol 156, 4504–4513.[Abstract]

Rajotte, D. & Ruoslahti, E. (1999). Membrane dipeptidase is the receptor for a lung-targeting peptide identified by in vivo phage display. J Biol Chem 274, 11593–11598.[Abstract/Free Full Text]

Roehrig, J. T., Johnson, A. J., Hunt, A. R., Bolin, R. A. & Chu, M. C. (1990). Antibodies to dengue 2 virus E-glycoprotein synthetic peptides identify antigenic conformation. Virology 177, 668–675.[Medline]

Roehrig, J. T., Bolin, R. A. & Kelly, R. G. (1998). Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica. Virology 246, 317–328.[CrossRef][Medline]

Scott, J. K. & Smith, G. P. (1990). Searching for peptide ligands with an epitope library. Science 249, 386–390.[Medline]

Sela, M. (1969). Antigenicity: some molecular aspects. Science 166, 1365–1374.[Medline]

Smith, W. C., McDowell, J. H., Dugger, D. R., Miller, R., Arendt, A., Popp, M. P. & Hargrave, P. A. (1999). Identification of regions of arrestin that bind to rhodopsin. Biochemistry 38, 2752–2761.[CrossRef][Medline]

Strauss, E. G., Stec, D. S., Schmaljohn, A. L. & Strauss, J. H. (1991). Identification of antigenically important domains in the glycoproteins of Sindbis virus by analysis of antibody escape variants. J Virol 65, 4654–4664.[Medline]

Szardenings, M., Tornroth, S., Mutulis, F., Muceniece, R., Keinanen, K., Kuusinen, A. & Wikberg, J. E. (1997). Phage display selection on whole cells yields a peptide specific for melanocortin receptor 1. J Biol Chem 272, 27943–27948.[Abstract/Free Full Text]

Tam, J. P. & Zavala, F. (1989). Multiple antigen peptide: a novel approach to increase detection sensitivity of synthetic peptides in solid-phase immunoassays. J Immunol Methods 124, 53–61.[CrossRef][Medline]

Trirawatanapong, T., Chandran, B., Putnak, R. & Padmanabhan, R. (1992). Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody. Gene 116, 139–150.[CrossRef][Medline]

Vaughn, D. W., Nisalak, A., Kalayanarooj, S., Solomon, T., Dung, N. M., Cuzzubbo, A. & Devine, P. L. (1998). Evaluation of a rapid immunochromatographic test for diagnosis of dengue virus infection. J Clin Microbiol 36, 234–238.[Abstract/Free Full Text]

Vaughn, D. W., Nisalak, A., Solomon, T., Kalayanarooj, S., Nguyen, M. D., Kneen, R., Cuzzubbo, A. & Devine, P. L. (1999). Rapid serologic diagnosis of dengue virus infection using a commercial capture ELISA that distinguishes primary and secondary infections. Am J Trop Med Hyg 60, 693–698.[Abstract/Free Full Text]

Westaway, E. G. & Blok, J. (1997). Taxonomy and evolutionary relationships of flaviviruses. In Dengue and Dengue Hemorrhagic Fever, pp. 147–173. Edited by D. J. Gubler & G. Kuno. New York: CAB International.

Wrighton, N. C., Farrell, F. X., Chang, R. & 7 other authors (1996). Small peptides as potent mimetics of the protein hormone erythropoietin. Science 273, 458–463.[Abstract]

Wu, H. C., Huang, Y. L., Chao, T. T., Jan, J. T., Huang, J. L., Chiang, H. Y., King, C. C. & Shaio, M. F. (2001a). Identification of B-cell epitope of dengue virus type 1 and its application in diagnosis of patients. J Clin Microbiol 39, 977–982.[Abstract/Free Full Text]

Wu, H. C., Yeh, C. T., Huang, Y. L., Tarn, L. J. & Lung, C. C. (2001b). Characterization of neutralizing antibodies and identification of neutralizing epitope mimics on the Clostridium botulinum neurotoxin type A. Appl Environ Microbiol 67, 3201–3207.[Abstract/Free Full Text]

Received 13 March 2003; accepted 11 June 2003.