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
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ABSTRACT |
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INTRODUCTION |
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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 proteinprotein 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.
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METHODS |
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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 GSepharose 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.
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RESULTS |
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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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DISCUSSION |
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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 50200 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 HisArgLeu motif was crucial for peptide antigenantibody 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 111116 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 epitopeantibody 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 4550 % 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.
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ACKNOWLEDGEMENTS |
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Received 13 March 2003;
accepted 11 June 2003.