A phage-displayed cyclic peptide that interacts tightly with the immunodominant region of hepatitis B surface antigen

Journal of Clinical Virology 34 (2005) 35–41

A phage-displayed cyclic peptide that interacts tightly with the immunodominant region of hepatitis B surface antigen Wen Siang Tan a, b, ∗ , Geok Hun Tan a , Khatijah Yusoff a, b , Heng Fong Seow b, c a

c

Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia b Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Department of Clinical Laboratory Science, Faculty of Medicine and Health Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Received 27 August 2004; received in revised form 10 January 2005; accepted 24 January 2005

Abstract The surface antigen (HBsAg) of hepatitis B virus (HBV) is highly conformational and generally evokes protective humoral immune response in human. A disulfide constrained random heptapeptide library displayed on the coat protein III of filamentous bacteriophage M13 was employed to select specific ligands that interact with HBsAg subtype ad. Fusion phages carrying the amino acid sequence ETGAKPH and other related sequences were isolated. The binding site of peptide ETGAKPH was located on the immunodominant region of HBsAg. An equilibrium binding assay in solution showed that the phage binds tightly to HBsAg with a relative dissociation constant (KDrel ) of 2.9 ± 0.9 nM. The phage bearing this peptide has the potential to be used as a diagnostic reagent and two assays for detecting HBsAg in blood samples are described. © 2005 Elsevier B.V. All rights reserved. Keywords: Filamentous bacteriophage; Biopanning; HBsAg; Dissociation constant; Immunodominant region

1. Introduction Hepatitis B virus (HBV) poses a major public health problem worldwide. The virus is estimated to infect more than one-third of the world’s population and there are about 350 million carriers of HBV worldwide (Jung and Pape, 2002). High prevalence areas have been identified in South-East Asia, China and Africa (reviewed by Lee, 1997). Prolonged carriage of HBV may progress to chronic liver diseases such as cirrhosis and hepatocellular carcinoma. Despite the presence of effective vaccines, about 1–2 million people die of HBV infection each year (Jung and Pape, 2002). In Malaysia, about 5% of healthy blood donors are chronic carriers of HBV (Merican et al., 2000). Abbreviations: HBsAg, hepatitis B surface antigen; HBcAg, hepatitis rel B core antigen; KD , relative dissociation constant; ABTS, 2,2 -azino-di-3ethyl-benzthiazoline-sulfonate; PNPP, p-nitrophenyl phosphate ∗ Corresponding author. Tel.: +603 89466715; fax: +603 89430913. E-mail address: [email protected] (W.S. Tan). 1386-6532/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2005.01.007

Three morphological distinct forms of particle are found in sera of infected individuals (Bayer et al., 1968). They exist as spherical and filamentous particles of 20 nm diameter as well as 42 nm double-shelled Dane particles (Dane et al., 1970). The latter is the infectious form and it consists of an outer envelope derived from the host cell membrane. Embedded in the envelope are three distinct but related forms of the surface antigen (HBsAg): L-HBsAg (large) , M-HBsAg (middle) and S-HBsAg (small). Internal to the envelope is the viral nucleocapsid which is made of many copies of the core antigen (HBcAg). Within the nucleocapsid is the polymerase protein (P) which is covalently attached to a partially doublestranded circular DNA of about 3.2 kb (Ganem, 1991). The three forms of surface antigens, L-HBsAg, M-HBsAg and S-HBsAg are encoded by one single open reading frame of the viral genome by using three different in-frame start codons and a common stop codon. Hence, the proteins differ at their N-termini, but have a large sequence in common within their C-terminal ends. The longest of the three, LHBsAg, has the PreS1 region of 108 or 119 amino acid

36

W.S. Tan et al. / Journal of Clinical Virology 34 (2005) 35–41

residues (depending on serotype), followed by the PreS2 region of 55 residues and the S region which comprises 226 amino acid residues (Heermann et al., 1984). The second largest protein, the M-HBsAg, contains the PreS2 and S regions. The smallest of these polypeptides, S-HBsAg, contains 226 residues of the S region. HBsAg can be expressed in a variety of heterologous systems, including yeasts (Valenzuela et al., 1982; Miyanohara et al., 1983; Hitzeman et al., 1983; Murray et al., 1984), animal cells (Dubois et al., 1980; Hirschman et al., 1980; Moriarty et al., 1981; Christman et al., 1982; Gough and Murray, 1982; Siddiqui, 1983; Shih et al., 1984) and plants (Mason et al., 1992). In these systems, the recombinant HBsAg assembles into 20 nm particles which are morphologically and antigenically similar to the spherical particles derived from human serum. Serologically, the HBsAg contains dominant neutralizing epitopes, denoted as the ‘a’ determinant epitopes. They are located within a double-looped structure, the first loop lies between amino acid residues 107–137, and the other between residues 138–149 (Zuckerman and Zuckerman, 2003). These sets of epitopes which are known as the immunodominant region are highly conformational (AshtonRickardt and Murray, 1989) and its three-dimensional structure at atomic resolution has yet to be determined by Xray crystallography or nuclear magnetic resonance. In this paper, we describe the isolation of ligands that interact with this region from a disulfide-constrained heptapeptide library and the application of the fusion phage harbouring the ETGAKPH sequence for detecting HBsAg in serum samples.

2. Materials and methods 2.1. Isolation of peptides that interact with HBsAg by biopanning HBsAg purified from human plasma [subtype ad (Biodesign); 3 ␮g/ml in TBS (50 mM Tris–HCl, pH 7.5, 150 mM NaCl); 100 ␮l] was coated onto a microtiter plate well overnight at 4 ◦ C and then blocked with blocking buffer (0.1 M NaHCO3 , pH 8.6, 5 mg/ml BSA, 0.02% NaN3 ; 200 ␮l) for 2 h at room temperature (∼27 ◦ C). The well was then added with a disulfide-constrained heptapeptide phage display library (New England Biolabs, USA) that had been diluted to 1 × 1011 plaque forming unit (pfu) in TBS (50 mM Tris–HCl, pH 7.5, 150 mM NaCl; 110 ␮l). After 1 h of incubation at room temperature, the mixture was discarded and the well washed with TBST [TBS, pH 7.5 containing 0.05% (v/v) Tween 20]. Bound phage was eluted with glycine-HCl (0.1 M, pH 2.2; 120 ␮l) and neutralized with Tris–HCl (1 M, pH 9.0; 15 ␮l). The eluted phage was then amplified by inq fecting E. coli strain ER2738 [F pro A+ B+ lac1  (lacz) m15 R zzf::Tn10 (Tet )/fhu Az gln v (lac-proAB) thi-1  (hsdsmcrB) 5] and the biopanning process was repeated for another

two rounds. Streptavidin and BSA were used as positive and negative controls, respectively. 2.2. Phage titration A single colony of E. coli ER2738 was inoculated into 5 ml of Luria Bertani (LB) broth and incubated with shaking until OD600 about 0.5. Top agarose (1% Bacto-tryptone, 0.5% yeast extract, 0.5% NaCl, 0.1% MgCl2 ·6H2 O, 0.7% agarose) was aliquoted (3 ml) into tubes and equilibrated at 45 ◦ C in a water bath. Phage (10 ␮l) and mid-log phase E. coli cells were added to the equilibrated top agarose, mixed and poured onto a LB agar plate containing IPTG (0.2 mg/ml; 4 ␮l) and X-gal (20 mg/ml; 40 ␮l). The plates were allowed to cool, and incubated overnight at 37 ◦ C. Plaques formed were counted and the amount of phage was determined as plaque forming unit. All assays were performed in triplicates. 2.3. Determination of nucleotide sequence Single plaques from each round of biopanning experiment were picked from LB agar plates used in the phage titration assay. The phages were grown in LB broth containing E. coli host cells. Single-stranded DNA of the phage was extracted and the nucleotide sequence of the inserts in the gpIII gene was determined by sequencing using the EQ DTCS kit (Beckman Coulter, USA) and analyzed with the CEQ 8000 DNA sequencer (Beckman Coulter, USA). 2.4. Large-scale preparation and purification of phages Phages were propagated in E. coli host cells grown in LB broth (1 L). The phage particles were precipitated by PEG and purified through cesium chloride density gradient centrifugation as described by Smith and Scott (1993). 2.5. Antibody-phage competition assay Microtiter plate wells were coated with HBsAg (subtype ad; 3 ␮g/ml; 100 ␮l) as described above. Anti-S-HBsAg monoclonal antibody (Chemicon, 1:1000 dilution; 50 ␮l) was added with a series of different concentrations of phage carrying the peptide ETGAKPH (109 –1011 pfu; 50 ␮l). The mixtures (100 ␮l) were added into the HBsAg coated wells, incubated for 1 h at room temperature (∼27 ◦ C) and washed six times with TBST. Anti-HBcAg monoclonal antibody (mAb C1-5, Pushko et al., 1994) was used as the negative control. Bound phages were eluted and titered as described above. The assay was carried out in triplicate determinations. 2.6. Interaction of phage ETGAKPH with the immunodominant region of HBsAg Recombinant bacteriophage T7-HBsAg111–156 (Tan et al., 2003; 1011 pfu, 100 ␮l) carrying the immunodominant region (residues 111–156 of subtype adyw; Pasek et al., 1979;

W.S. Tan et al. / Journal of Clinical Virology 34 (2005) 35–41

GenBank accession number: J02202), and T7 wild-type (1011 pfu; 100 ␮l) were coated on microtiter plate wells for overnight at 4 ◦ C. Phage bearing the peptide sequence ETGAKPH (101 –1010 pfu, 100 ␮l each) was added and incubated for 1 h at room temperature. The wells were washed six times with TBST, anti-M13 antibody conjugated to horse radish peroxidase (HRP; Amersham Pharmacia Biotech; 1:3000 dilution; 100 ␮l) was added and incubated at room temperature for 1 h. HRP substrate solution [ABTS (2,2 azino-di-3-ethyl-benzthiazoline-sulfonate) in sodium citrate; 200 ␮l] was added and absorbance at 405 nm was measured with a microtiter reader (model 550, Bio-Rad, USA). 2.7. Equilibrium binding assay in solution Affinity assay in solution was carried out as described by Dyson et al. (1995) and Ramanujam et al. (2002, 2004). Molecular mass value of 24 kDa was used for molar concenrel value was determined by tration calculation of S-HBsAg. KD fitting the binding data points with the equation for two bindrel + [S]} + {[S]B rel ing sites: Y = {[S]Bmax1 /(KD1 max2 /KD2 + [S]} TM (Grafit version 3.0, Erithacus Software Ltd., Surrey, UK). The fraction bound, Y, was calculated from the equation Y = [pfu0 −pfux ]/pfu0 , pfux is the free phage at different concentrations of HBsAg ([S]). pfu0 is the free phage when rel and K rel represent the relative dissociation con[S] = 0. KD1 D2 stants for the high- and low-affinity sites, respectively. Bmax1 and Bmax2 refer to maximal binding for the high- and lowaffinity sites, respectively. 2.8. Detection of HBsAg by immobilizing fusion phage on microtiter plate well Polystyrene wells were coated with different concentrations of phage carrying the peptide sequence ETGAKPH (101 –1010 pfu; 100 ␮l) overnight at 4 ◦ C and blocked with milk diluent (10%; KPL, Maryland) for 2 h at room temperature. Samples: purified HBsAg subtype ad (3 ␮g/ml; 100 ␮l), HBsAg-positive human serum (1:5000 dilution; 100 ␮l), HBsAg-negative human serum (1:5000 dilution; 100 ␮l) or 10% milk diluent (100 ␮l) were added to the wells and incubated for 1 h at room temperature. The wells were washed six times with TBST. Mouse anti-HBsAg monoclonal antibody conjugated to alkaline phosphatase (1:1000 dilution, Chemicon; 100 ␮l) was added, incubated at room temperature for 1 h and washed six times with TBST. P-nitrophenyl phosphate substrate (PNPP; 1 mg/ml) was added and A405 was measured.

37

were washed six times with TBST and blocked with milk diluent (10%). Phage bearing the peptide sequence ETGAKPH (101 –1010 pfu; 100 ␮l) was added into each well and incubated at room temperature for 1 h. The wells were washed six times with TBST and bound phage was determined as described above (Section 2.6).

3. Results 3.1. Peptides that interact with HBsAg The peptide sequences selected from three rounds of biopanning against HBsAg are shown in Table 1. In the first round, 20% of the phages carried the peptide sequence ETGAKPH. This dominant sequence increased to 50% and 75% in the second and third rounds, respectively. This suggests that there was an enrichment of peptide sequences that interact with HBsAg. About 17% of the phages in the third round harbored peptide sequences ETGEKPQ and QTGEKPQ, which are highly related to this dominant sequence. The fourth and last residues of these peptides are replaced by Glu (E) and Gln (Q), respectively. 3.2. Inhibition of phage binding to HBsAg by antibody The dominant phage carrying the peptide sequence ETGAKPH was selected for further analysis. A monoclonal antibody that binds specifically to S-HBsAg was used in an antibody-phage competition assay. Fig. 1 shows that the fusion phage competes with the monoclonal antibody for a binding site on HBsAg. A monoclonal antibody against the HBcAg, which does not interact with the HBsAg, did not inhibit the binding. This result implies that the peptide could bind to S-HBsAg.

Table 1 Heptapeptides obtained from three rounds of biopanning against HBsAg Rounds of biopanning Heptapeptide sequence Frequency of sequence (%) First round

ETGAKPH NPHPQQP NPPPPQP HPPHPQP QHGAMGL

20 20 20 20 20

Second round

2.9. Detection of HBsAg by immobilizing the antigen on microtiter well

ETGAKPH QTNHMGL QTKHQGR QPNHMGL QSKPQGL

50 20 10 10 10

Third round

Samples [HBsAg subtype ad (3 ␮g/ml), HBsAg-positive human serum (1:5000 dilution), HBsAg-negative human serum (1:5000 dilution) or milk diluent (10%)] were coated to the microtiter plate wells for overnight at 4 ◦ C. The wells

ETGAKPH QTGEKPQ ETGEKPQ QAFFPNA

75 8.3 8.3 8.3

After three rounds of selection and amplification, 15, 30 and 36 individual clones from the first, second and third rounds, respectively, were sequenced.

38

W.S. Tan et al. / Journal of Clinical Virology 34 (2005) 35–41

Fig. 1. Inhibition of antibodies binding HBsAg with phage bearing the peptide ETGAKPH. Monoclonal anti-HBsAg antibody ( ) or monoclonal anti-HBcAg antibody ( ) was incubated with different concentrations of the phage. The mixtures were then added into wells that had been coated with HBsAg. The phage which interacted with the immobilized HBsAg was titered. Phage bearing the peptide ETGAKPH in the absence of antibody (
) was used as a control. Assays were performed in triplicates and the error bars represent the standard deviation from the arithmetic mean.

3.3. Peptide ETGAKPH interacts with the immunodominant region In order to locate the binding site of peptide ETGAKPH on S-HBsAg, the fusion phage was used to interact with the immunodominant region (amino acid residues 111–156) of HBsAg displayed on phage T7 (Tan et al., 2003). Fig. 2 shows that the peptide ETGAKPH interacts with the immunodominant region but not with the wild-type phage T7. 3.4. Equilibrium binding assay in solution rel beThe method employed here for measuring the KD tween the phage carrying the peptide ETGAKPH and HBsAg (0–125 nM) in solution was adapted from Dyson et al. (1995). In this assay, various concentrations of HBsAg were incubated with a constant concentration of phage (1010 pfu)

Fig. 3. Interaction of HBsAg with ETGAKPH phage. The binding data were fitted with the equation for two binding sites using the GraFitTM software.

until equilibrium was reached (15 h; data not shown). The concentration of free phage at equilibrium was determined by incubating an aliquot of the reaction mixture in HBsAgcoated wells. Fig. 3 shows that the concentration of the free phage reduces progressively with increased concentration of HBsAg. The binding data were fitted with the equation for rel values were 2.9 ± 0.9 nM (K rel ) two binding sites and the KD D1 rel and 0.83 ± 0.63 mM (KD2 ). 3.5. Direct phage-ELISA for detecting HBsAg Different concentrations of phage carrying the peptide ETGAKPH were immobilized on a microtiter plate and blocked with milk diluent. Then, samples were added and bound HBsAg was detected by anti-HBsAg antibody conjugated to AP, followed by the addition of PNPP substrate. The results of the assay are depicted in Fig. 4a. The data demonstrate that 1010 pfu of phage is sufficient to give a significant absorbance value of about 1.0 and 0.85 for HBsAg-positive human serum (1:5000 dilution) and purified HBsAg, respectively. HBsAgnegative human serum and milk diluent gave significantly low readings. The sensitivity of the assay was determined by a serial dilutions of purified HBsAg, and it was found that it can detect as low as 1 pg/ml of HBsAg (Fig. 4b). These results show that the phage ETGAKPH has the potential to be used in the development of diagnostic assays for the detection of HBsAg. 3.6. Indirect phage-ELISA for detecting HBsAg

Fig. 2. Interaction of phage ETGAKPH with the immunodominant region of HBsAg displayed on phage T7. Phage T7 displaying the immunodominant region of HBsAg ( ), wild-type T7 ( ) or 10% milk diluent (
) was immobilized on microtiter plate wells. Different concentrations of phage bearing the peptide sequence ETGAKPH were added to the wells. Anti-M13 phage antibody conjugated to HRP was added, followed by ABTS, and absorbance at 405 nm was measured. Assays were performed in triplicates and the error bars represent the standard deviation from the arithmetic mean.

In this assay, samples containing HBsAg were immobilized on microtiter plate wells and the unsaturated area of the wells was blocked by milk diluent. Then, different concentrations of phage ETGAKPH were added to interact with the immobilized HBsAg. Anti-phage antibody conjugated to HRP was added to interact with the bound phage followed by the ABTS substrate. The results of the assay for analysis of purified HBsAg and HBsAg-positive human serum im-

W.S. Tan et al. / Journal of Clinical Virology 34 (2005) 35–41

39

Fig. 4. (a) Direct phage-ELISA for detecting HBsAg. Wells were coated with different concentrations of phage carrying the ETGAKPH peptide. HBsAgpositive serum ( ), HBsAg-negative serum ( ), purified human HBsAg () or 10% milk diluent (
) was added to react with the peptide. Mouse antiHBsAg antibody was added to interact with the HBsAg. Then anti-mouse antibody conjugated to AP was added followed by PNPP substrate. (b) Sensitivity of the direct phage-ELISA. Wells were coated with 1010 pfu of phage ETGAKPH. The assay was carried out as (a) but different concentrations of human HBsAg were used. The straight dot line across the figure is the cutoff value, calculated as the mean value of the negative control plus three standard deviations. Assays were performed in triplicates and the error bars represent the standard deviation from the arithmetic mean.

Fig. 5. (a) Indirect phage-ELISA for detecting HBsAg. HBsAg-positive serum ( ), HBsAg-negative serum ( ), purified HBsAg () or 10% milk diluent (
) was immobilized on wells. Different concentrations of phage carrying the peptide ETGAKPH were added to interact with the immobilized HBsAg. Anti-M13 phage antibody conjugated to HRP followed by ABTS substrate. (b) Sensitivity of indirect phage-ELISA. The assay was carried out as (a) but different concentrations of human HBsAg were coated on wells and detected with 1010 pfu of phage ETGAKPH. The straight dot line across the figure is the cut-off value, calculated as the mean value of the negative control plus three standard deviations. Assays were performed in triplicates and the error bars represent the standard deviation from the arithmetic mean.

mobilized on a solid support are shown in Fig. 5a. Both the samples gave rise to significantly high absorbance readings compared to the negative controls: milk diluent and HBsAgnegative human serum. This assay was able to detect HBsAg down to a concentration of 1 pg/ml Fig. 5b.

cation of these antibodies are difficult and time-consuming, which dramatically increase their costs. Recently, it has been shown that the vast structural diversity of peptides displayed on the surface of bacteriophages can be used to isolate specific ligands for a particular target of interest (reviewed in Smith and Petrenko, 1997). These ligands should be useful as recognition reagents for detection of various biological structures including HBsAg. In the present study, a disulfide-constrained library was employed to select for conformational ligands that interact with HBsAg, because it is a conformational antigen which is highly cross-linked by disulfide bonds in Dane particles and 20 nm envelope particles (Mangold et al., 1997). Moreover, it has been demonstrated that higher affinity ligands can be isolated more easily with a disulfide-constrained library compared to a linear library (O’Neil et al., 1992; Gho et al.,

4. Discussion HBsAg is one of the immunologic markers of HBV infection, and therefore, many immunological methods, especially ELISA, have been developed to detect the antigen in blood samples. Most of the presently available methods depend on polyclonal or monoclonal antibodies which are isolated from sera of immunized animals or culture media of hybridomas, as recognition reagents. However, the production and purifi-

40

W.S. Tan et al. / Journal of Clinical Virology 34 (2005) 35–41

1997; Ho et al., 2003). Panning of the disulfide-constrained library with purified human HBsAg, yielded predominantly hydrophilic peptide with the sequence ETGAKPH. This peptide contains one hydroxylic side chain (T), one negatively charged (E) and two positively charged (K, H) residues, suggesting that the peptide binds to a hydrophilic region on the surface of HBsAg and the complementary sites may be held together by a hydrogen bond and salt bridges. To date, the three-dimensional structure of HBsAg at atomic resolution is not known. The proposed model of S-HBsAg in the 20 nm spherical HBsAg particles is composed of four transmembrane helices (residues 8–28, 78–100, 160–184 and 189–210), one hydrophilic loop (residues 101–159) exposed on the surface, one loop (residues 28–77) projects inside the particle and one hydrophobic C-terminal end (residues 217–226) believed to be exposed on the surface (Stirk et al., 1992). However, Paulij et al. (1999) identified a monoclonal antibody that interacts with residues 178–186 of S-HBsAg, leading the authors to propose that the region between residues 160–207 may not span the membrane twice but is rather exposed on the surface of the particles. As shown in the antibody-phage competition assay, a monoclonal antibody that binds specifically to the S-HBsAg inhibited the binding of the fusion phage to purified human HBsAg, suggesting that the peptide ETGAKPH interacts with S-HBsAg. Based on the hydrophilic characteristic of peptide ETGAKPH, it is most likely that the peptide interacts with the hydrophilic loop (residues 101–159) located on the surface of the particle. The immunodominant region (residues 111–156) which is highly conformational is located within this hydrophilic loop and the T7 phage displaying this region interacted with the phage carrying the peptide ETGAKPH, confirming that the peptide interacts with the hydrophilic loop exposed on the surface of HBsAg particles. An equilibrium binding assay in solution adapted from Dyson et al. (1995) showed that the binding between the phage ETGAKPH and the HBsAg particles purified from hurel values. The man plasma gave rise to two widely differing KD rel rel first KD value is in nanomolar range (KD1 = 2.9 ± 0.9 nM) indicating that the interaction is as tight as antigen–antibody rel value, which is in sub-millimolar interaction. The second KD rel range (KD2 = 0.83 ± 0.63 mM), could be due to the binding of the phage to a small proportion of denatured HBsAg in the reaction. The immunodominant region is highly antigenic and antibodies against these conformational epitopes are widely used in standard diagnostic assays for detecting HBsAg in blood samples. As phage bearing the peptide ETGAKPH interacts tightly with this region, we therefore explore the feasibility of using the phage as HBsAg detection reagent in ELISA. Both the direct and indirect phage-ELISA were able to detect HBsAg to as low as 1 pg/ml. The high sensitivity of these rel value (2.9 nM) assays is most probably due to the low KD of phage-HBsAg interaction. Furthermore, the presence of three to five copies of gpIII protein on a phage particle could significantly enhance the binding of the phage to HBsAg.

These data suggest that phage ETGAKPH can be used as a sensitive diagnostic reagent for the detection of HBsAg. The preferred assay format is a double phage sandwich ELISA, in which the wells are coated with the phage to capture HBsAg in blood samples, followed by a recombinant phage labeled with a marker. The marker can be a fluorescent protein, a radioactive compound, an enzyme or a dye. In addition, more work has to be carried out to study the stability of the phage and the specificity of the ligand to different HBsAg serotypes. The reproducibility of the assay should also be verified with more HBsAg positive and negative serum samples. Production and purification of bacteriophages are relatively easier and less expensive compared to those for monoclonal and polyclonal antibodies. These have made phageborn peptide/polypeptide an alternative choice and of course a favorite new comer in disease diagnosis. There are many examples demonstrating the potential of phage-born peptides as potential diagnostic reagents. These include a 9-mer peptide fused to the gpVIII protein for the detection of cucumber mosaic virus (Gough et al., 1999), a cyclic heptapeptide on the gpIII protein for the pathotyping of Newcastle disease virus (Ramanujam et al., 2004) and a 12-mer gpIII protein for the identification of the anti-Vi antigen of Salmonella enerica Serovar Typhi (Tang et al., 2003). These together with the results in this study demonstrate that phage displayed peptides show promise as reagents for detection of biological samples.

Acknowledgements This study was supported by the grant no. 09-02-04-0355EA001 from the Ministry of Science, Technology and Innovation of Malaysia (MOSTI). G.H. Tan is supported by the National Science Fellowship of Malaysia.

References Ashton-Rickardt PG, Murray K. Mutations that change the immunological subtype of hepatitis B virus surface antigen and distinguish between antigenic and immunogenic determinant. J Med Virol 1989;29:204–14. Bayer M, Blumberg B, Werner B. Particles associated with Australia antigen in the sera of patients with leukemia, Down’s syndrome and hepatitis. Nature 1968;218:1057–9. Christman JK, Gerber M, Price PM, Flordellis C, Edelman J, Acs G. Amplification of expression of hepatitis B surface antigen in 3T3 cells cotransfected with a dominant-acting gene and cloned viral DNA. Proc Natl Acad Sci USA 1982;79:1815–9. Dane DS, Cameron CH, Briggs M. Virus-like particles in serum of patients with Australia-antigen-associated hepatitis. Lancet 1970;I:695–8. Dubois MF, Pourcel C, Rousset S, Chany C, Tiollais P. Excretion of hepatitis B surface antigen particles from mouse cells transformed with cloned viral DNA. Proc Natl Acad Sci USA 1980;77:4549–53. Dyson MR, Germaschewski V, Murray K. Direct measurement via phage titer of the dissociation constants in solution of fusion phage-substrate complexes. Nucl Acids Res 1995;23:1531–5.

W.S. Tan et al. / Journal of Clinical Virology 34 (2005) 35–41 Ganem D. Assembly of Hepadnaviral virions and subviral particles. Curr Top Microbiol Immunol 1991;168:61–3. Gho YS, Lee JE, Oh KS, Bae DG, Chae CB. Development of antiangiogenin peptide using a phage-displayed peptide library. Cancer Res 1997;57:3733–40. Gough KC, Cockburn W, Whitelam GC. Selection of phage-display peptides that bind to cucumber mosaic virus coat protein. J Virol Methods 1999;79:169–80. Gough NM, Murray K. Expression of the hepatitis B virus surface, core and E antigen genes by stable rat and mouse cell lines. J Mol Biol 1982;162:43–67. Heermann KH, Goldmann U, Schwartz W, Seyffarth T, Baumgarten H, Gerlich WH. Large surface proteins of hepatitis B virus containing the Pre-S sequence. J Virol 1984;52:396–402. Hirschman SZ, Price P, Garfinkel E, Christman J, Acs G. Expression of cloned hepatitis B virus DNA in human cell cultures. Proc Natl Acad Sci USA 1980;77:5507–11. Hitzeman RA, Chen CY, Hagie FE, Patzer EJ, Liu CC, Estell DA, Miller JV, Yaffe A, Kleid DG, Levinson AD, Oppermann H. Expression of hepatitis B virus surface antigen in yeast. Nucl Acids Res 1983;11:2745–63. Ho KL, Yusoff K, Seow HF, Tan WS. Selection of high affinity binding phages on hepatitis B core particles with a disulfide constrained phage display library. J Med Virol 2003;69:27–32. Jung M-C, Pape GR. Immunology of hepatitis B infection. Lancet 2002;2:43–50. Lee WM. Hepatitis B virus infection. N Engl J Med 1997;337:1733–45. Mangold CMT, Unckell F, Werr M, Streeck RE. Analysis of intermolecular disulfide bonds and free sulfhydryl groups in hepatitis B surface antigen particles. Arch Virol 1997;142:2257–67. Mason HS, Lam DM, Arntzen CJ. Expression of hepatitis B surface antigen in transgenic plants. Proc Natl Acad Sci USA 1992;89:11745–9. Merican I, Guan R, Amarapuka D, Alexander MJ, Chutaputti A, Chien R, Hasnian SS, Leung N, Lesmana L, Phiet PH, Sjalfoellah Noer HM, Sollano J, Sun HS, Xu DZ. Chronic hepatitis B virus infection in Asian countries. J Gastroenterol Hepatol 2000;15:1356–61. Miyanohara A, Toh-e A, Nozaki C, Hamada F, Ohtomo N, Matsubara K. Expression of hepatitis B surface antigen gene in yeast. Proc Natl Acad Sci USA 1983;80:1–5. Moriarty AM, Hoyer BH, Shih JW, Gerin JL, Hamer DH. Expression of the hepatitis B virus surface antigen gene in cell culture by using a simian virus 40 vector. Proc Natl Acad Sci USA 1981;78:2606–10. Murray K, Bruce SA, Wingfield P, van Eerd P, de Reus A, Schellekens H. Hepatitis B virus antigen made in microbial cells immunise against viral infection. EMBO J 1984;3:645–50.

41

O’Neil KT, Hoess RH, Jackson SA, Ramachandran NS, Mousa SA, DeGrado WF. Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library. Proteins 1992;14:509–15. Pasek M, Goto M, Gilbert W, Zink B, Schaller H, Mackay P, Leadbetter G, Murray K. Hepatitis B virus genes and their expression in Escherichia coli. Nature 1979;282:575–9. Paulij WP, de Wit PLM, S¨unnen CMG, van Roosmalen MH, Petersen-van Ettekoven A, Cooreman MP, Heijtink RA. Localization of a unique hepatitis B virus epitope sheds new light on the structure of hepatitis B virus surface antigen. J Gen Virol 1999;80:2121–6. Pushko P, Sallberg M, Borisova G, Ruden U, Bichko V, Wahren B, Pumpens P, Magnius L. Identification of hepatitis B virus core protein regions exposed or internalized at the surface of HBcAg particles by scanning with monoclonal antibodies. Virology 1994;202:912– 20. Ramanujam P, Tan WS, Nathan S, Yusoff K. Selection of peptide inhibitors that inhibit the replication of Newcastle disease virus. Arch Virol 2002;147:981–93. Ramanujam P, Tan WS, Nathan S, Yusoff K. Pathotyping of Newcastle disease virus with a filamentous bacteriophage. BioTechniques 2004;36:296–300. Shih MF, Arsenakis M, Tiollais P, Roizman B. Expression of hepatitis B virus S gene by herpes simplex virus type 1 vectors carrying alpha- and beta-regulated gene chimeras. Proc Natl Acad Sci USA 1984;81:5867–70. Siddiqui A. Expression of hepatitis B virus surface antigen gene in cultured cells by using recombinant plasmid vectors. Mol Cell Biol 1983;3:143–6. Smith GP, Petrenko VA. Phage display. Chem Rev 1997;97:391–410. Smith GP, Scott JK. Libraries of peptides and proteins displayed on filamentous phage. Meth Enzymol 1993;217:228–57. Stirk HJ, Thornton JM, Howard CR. A topological model for hepatitis B surface antigen. Intervirology 1992;33:148–58. Tan GH, Yusoff K, Seow HF, Tan WS. Display of the immunodominant region of hepatitis B surface antigens on bacteriophage T7. J Clin Virol 2003;28:S80. Tang SS, Tan WS, Devi S, Wang L, Pang T, Thong KL. Mimotopes of Vi antigen of Salmonella enterica serovar typhi identified from phage display peptide library. CDLI 2003;10:1078–84. Valenzuela P, Medina A, Rutter WJ, Ammerer G, Hall BD. Synthesis and assembly of hepatitis B virus surface antigen particles in yeast. Nature 1982;298:347–50. Zuckerman JN, Zuckerman AJ. Mutations of the surface protein of hepatitis B virus. Antiviral Res 2003;60:75–8.