The identification of immunodominant linear epitopes of dengue type 2 virus capsid and NS4a proteins using pin-bound peptides

Virus Research 112 (2005) 60–68

The identification of immunodominant linear epitopes of dengue type 2 virus capsid and NS4a proteins using pin-bound peptides Ravulapalli AnandaRao, Sathyamangalam Swaminathan, Navin Khanna ∗ RGP Laboratory, International Centre for Genetic Engineering and Biotechnology, PO Box 10504, Aruna Asaf Ali Marg, New Delhi 110067, India Received 6 December 2004; received in revised form 8 March 2005; accepted 8 March 2005 Available online 26 April 2005

Abstract We have used multi-pin peptide synthesis strategy to identify B-cell epitopes on two small dengue virus proteins, capsid and NS4a. We have identified several linear, immunodominant epitopes on both these proteins. Almost all these epitopes mapped to regions predicted to be hydrophilic based on Kyte and Doolittle profiles. Of the capsid epitopes identified in this study, the most immunogenic ones mapped to the C-terminal ␣4 helix, which lies on the solvent-exposed surface of the capsid dimer. The capsid epitopes were dengue-specific in that they could recognize antibodies in dengue virus-, but not yellow fever virus (YFV)- or Japanese encephalitis virus (JEV)-immune sera. This study has demonstrated the presence of anti-NS4a antibodies in dengue-patient sera definitively, for the first time, using authentic NS4a-derived pin-bound peptides as capture antigens. All the NS4a epitopes mapped to the amino-terminal third of the NS4a molecule. Our study suggests that the immunodominant epitopes of these two dengue proteins might have the potential to be used as a part of a recombinant multi-epitope protein containing carefully chosen E and NS1 epitopes for the detection of dengue infections with a high degree of sensitivity and specificity. © 2005 Elsevier B.V. All rights reserved. Keywords: Dengue virus; Capsid; NS4a; Multi-pin peptide synthesis; Immunodominant epitopes

1. Introduction Dengue infections generally occur in areas where other flaviviral infections (such as Japanese encephalitis and yellow fever) are also common. While preventive vaccines are available for these other flaviviral infections (reviewed in Monath, 1999; Tsai et al., 1999), there is none available for the prevention of dengue (Lai and Monath, 2003; Saluzzo, 2003). Coupled with the lack of a specific antiviral therapy to treat dengue infections, early and accurate diagnosis of dengue infection is of paramount importance in clinical management of dengue patients (WHO, 1997). Increasingly, dengue diagnosis has begun to depend on the detection of dengue-specific serum antibodies (Groen et al., 2000; Cuzzubbo et al., 2001). Commercial ELISA kits that use whole dengue virus preparations, obtained either from infected cell cultures or animal ∗ Corresponding author. Tel.: +91 11 26177357x272; fax: +91 11 26162316. E-mail address: [email protected] (N. Khanna).

0168-1702/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2005.03.022

tissues, as antigens, can lead to misdiagnosis due to the crossreactivity of other non-dengue flavivirus-specific antibodies towards dengue antigens. Replacing the whole viral antigens will eliminate the cross-reactivity and help in the development of improved diagnostic assays in terms of sensitivity and specificity. One approach to address this would be to detect dengue infections through the use of multiple dengue virus-specific peptides to capture dengue-specific antibodies from patient sera. Dengue viruses encode and express three structural (capsid, C; premembrane prM and envelope E) and seven nonstructural (NS) proteins (NS1, 2a, 2b, 3, 4a, 4b, and 5) (Lindenbach and Rice, 2001). Of these ten proteins, antibodies to C, prM, E, NS1 and NS3 have been detected in sera of dengue-infected patients (Churdboonchart et al., 1991; Se-Thoe et al., 1999; Vald´es et al., 2000; Cardosa et al., 2002). A dengue viral protein with an electrophoretic mobility similar to that of NS4a has been observed in western blots using dengue-patient sera, suggesting that dengue infection elicits anti-NS4a antibodies as well (Churdboonchart et al., 1991;

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Se-Thoe et al., 1999). It has been reported that the prevalence of these anti-NS4a antibodies which is ∼5% in primary infections, increases to ∼50% in secondary infections (Se-Thoe et al., 1999). Obviously, peptides corresponding to the antigenic determinants of the proteins that elicit antibodies in dengue-infected patients would be useful as diagnostic reagents. While the epitopes on the major antigens such as E (Aaskov et al., 1989; Innis et al., 1989; Roehrig et al., 1990; Megret et al., 1992; Jianmin et al., 1995) and NS1 (Huang et al., 1999; Garcia et al., 1997; Falconar et al., 1994; Wu et al., 2001) have been mapped extensively, not much information is available in the literature regarding the epitopes of the remaining proteins. In this study, we have focused on two small proteins, the capsid protein (consisting of 100 amino acid (aa) residues) and NS4a (150 aa residues) for several reasons. One, both proteins are reported to elicit antibodies in dengue-infected patients; therefore, peptides carrying immunodominant epitopes of these proteins could be potentially useful as diagnostic agents. Two, not much is known regarding the antigenic structure of these proteins. While there is a report in the literature identifying an epitope on the dengue capsid recognized by a panel of six monoclonal antibodies (Bulich and Aaskov, 1992), there is virtually no information regarding NS4a epitopes. Finally, both these proteins are relatively small in size and amenable to rapid epitope mapping using the multi-pin peptide synthesis approach (Rodda and Tribbick, 1996). In this work, we present data on the linear immunodominant epitopes of dengue type 2 virus (DEN-2) capsid and NS4a proteins. We also show that these epitopes can be used to develop recombinant multi-epitope proteins with enhanced sensitivity of anti-dengue antibody detection in patient serum.

2. Materials and methods 2.1. Computer analysis The predicted protein sequences of DEN-2 capsid (DEN2 NGC, Genbank AF038403) and NS4a (DEN-2 Jamaica, GenBank M20558) were analyzed using MacVector software. Putative hydrophobic and hydrophilic regions along the length of these molecules were predicted using the Kyte and Doolittle method. 2.2. Peptide synthesis A total of 90 peptides were synthesized in this study, of which 37 were derived from the capsid and the rest from NS4a. For convenience, these peptides have been designated serial numbers with the prefix ‘c’ to denote capsid peptides and the prefix ‘n’ to denote NS4a peptides. All peptides were decamers with the exception of the last NS4a peptide, n53, which was an octamer. Solid-phase peptide synthesis, based on Fmoc (9-fluorenylmethoxycarbonyl) chemistry, was performed using the multi-pin peptide synthesis kit purchased

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from Chiron Mimitopes Pty Ltd, Australia. Peptides were synthesized on polyethylene pins arranged in an 8 × 12 matrix (of the same dimensions as well in a 96-well microtiter plate). These pin-bound peptides were used in ELISAs (below). 2.3. Murine hyperimmune sera and dengue-patient sera Hyperimmune murine sera specific for DEN-2 and yellow fever virus (YFV) were obtained from Dr. A.M. Jana, Defense Research Development Establishment, Gwalior, India; Japanese encephalitis virus (JEV)-specific murine hyperimmune serum was from Dr. S. Vrati, National Institute of Immunology, New Delhi, India. Hyperimmune sera were generated by repeated boosting to obtain high antibody titers. The dengue-patient sera were obtained from Dr. R. Agarwal, of Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India and Prof. S. Fernando, University of Sri Jayewardenepura, Sri Lanka. All sera were from DEN-2 infected patients. 2.4. Enzyme-linked immuno sorbent assay (ELISA) for epitope identification ELISA was carried out using the pin-bound capsid and NS4a peptides. All incubations were done using the 96-well microtiter format. Briefly, the assay was as follows. Pins were immersed into 200 ␮l blocking buffer (5% skimmed milk powder (SMP) in 1 × phosphate buffered saline (PBS), pH 7.2) at 37 ◦ C for 4 h. The pins were washed (1 × PBS/0.1% Tween 20) and then immersed in 200 ␮l of 1:100 diluted (diluent: 1 × PBS/5% SMP/1% sodium deoxy cholate (SDC)) murine hyperimmune serum (dengue type 2-, YFV- or JEVspecific) or patient serum. After incubation at 37 ◦ C for an appropriate time (15 min for human sera; 1 h for murine sera), the pins were washed (1 × PBS/0.1% Tween-20/1% SDC) four times (5 min/wash) and incubated with 200 ␮l of 1:7500 diluted secondary antibody-horseradish peroxidase (HRPO) conjugate purchased from Calbiochem. Depending on the assay, three different secondary antibody-HRPO conjugates were used. Anti-mouse IgG-HRPO was used (incubation time: 1 h) when the peptides were scanned with murine hyperimmune serum; either anti-human IgM-HRPO or antihuman IgG-HRPO was used (incubation time: 15 min) in assays designed to scan the peptides with dengue-patient sera. Each of these three HRPO conjugated secondary antibodies was pre-tested to ensure that they do not bind to the pinbound peptides in the absence of the primary antibody. Pins were washed four times as before and incubated with 200 ␮l of 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate solution at 37 ◦ C (15 min for assays with dengue-patient sera and 45 min for those with murine hyperimmune sera). Reactions were stopped by removal of the pins followed by the addition of 100 ␮l 1 M H2 SO4 . Optical densities (ODs) were measured at 450 nm in a tecan microtiter plate reader. For the purpose of assigning linear epitopes, the mean OD obtained

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using normal/pre-immune sera plus twice the standard deviation (S.D.) of the mean was used as the cut-off value. The pin-bound peptides were re-used after stripping off of the bound antibody and conjugate by sonication in hot detergent (1% SDS at 60 ◦ C) under reducing conditions (0.1% ␤mercaptoethanol), and verifying antibody conjugate removal, as per the manufacturer’s instructions. Control ELISAs were run in parallel, in which DEN-2 virus was used as the capture antigen (instead of the pin-bound peptides) to detect anti-dengue antibodies in the test sera. Each ELISA was performed twice. 2.5. In-house ELISA for the detection of serum anti-dengue antibodies The assay was essentially similar to that described above, except that serum antibodies were captured in microtiter wells coated with different recombinant protein antigens or DEN-2 virus lysates, instead of the pin-bound peptides. The recombinant protein antigens used were r-DME-G (AnandaRao et al., 2005), its second-generation version, r-DME-G2 (this study) or DEN-2 E protein (Bisht et al., 2002).

3. Results 3.1. Epitope selection The protein sequences of capsid (100 aa) and NS4a (∼150 aa) of DEN-2 were predicted from nucleotide sequences retrieved from the GenBank database (capsid was from DEN-2 NGC; NS4a was from DEN-2 Jamaica). Kyte and Doolittle hydrophilicity plots of these proteins generated using MacVector software are shown in Fig. 1. From the hy-

drophilicity plot of the capsid protein, two major regions of hydrophilicity were discernible, one at the amino (N)terminal end spanning aa residues 1–25, and the other at the carboxy (C)-terminal end (aa residues 70–100). The central domain, sandwiched between these two hydrophilic regions, was largely hydrophobic and therefore not likely to be potentially antigenic. In contrast, the NS4a Kyte and Doolittle plot predicted the N-terminal one-third of the molecule to be hydrophilic and the C-terminal two-thirds of the molecule to be largely hydrophobic. Since regions of hydrophilicity are likely to be surface-exposed and therefore potentially antigenic, we focused on these regions of the two molecules for pepscan analysis. Accordingly, we synthesized overlapping decapeptides offset by one residue corresponding to the hydrophilic regions of capsid and NS4a proteins. In addition, we also designed sequential, non-overlapping decapeptides spanning the entire hydrophobic regions in the two proteins. All these peptides were synthesized on pin surfaces using Fmoc chemistry as described in Section 2. 3.2. Identification of capsid epitopes For mapping immunodominant epitopes on the DEN-2 capsid, a library of 37 peptides (designated as c1–c37) was generated. Of these, peptides c1–c13 and c19–c37 represented overlapping peptides corresponding to the amino- and carboxy-terminal regions of the capsid protein. Five peptides, c14–c18, were non-overlapping peptides spanning the central hydrophobic region of the capsid molecule (see Fig. 2). These peptides (pin-bound) were scanned in ELISAs, against murine hyperimmune sera to identify potential B-cell epitopes. We used three different hyperimmune sera (obtained from mice immunized with DEN-2, JEV or YFV) as the source of primary antibodies and anti-mouse IgG-HRPO as

Fig. 1. Kyte and Doolittle hydropathy profiles of DEN-2 virus capsid (A) and NS4a (B) proteins generated using MacVector software. The horizontal axis indicates amino acid residue number and the vertical axis indicates the hydropathy score. Positive scores indicate hydrophilicity and negative scores indicate hydrophobicity.

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Fig. 2. Schematic representation of capsid peptides synthesized for epitope mapping. The predicted amino acid sequence of DEN-2 virus capsid is shown on the top. A total of 37 decapeptides corresponding to the capsid were synthesized using multi-pin peptide synthesis kit (chiron). Overlapping peptides were synthesized corresponding to the putative hydrophilic regions and contiguous (non-overlapping) peptides for the putative hydrophobic regions. The numbered lines below the protein sequences denote the peptides synthesized. These lines have been positioned exactly below the corresponding regions of the proteins from where their sequences were derived. (In the text, the capsid peptides have been given the prefix ‘c’).

the secondary antibody. In this experiment, several reactive peptides along the entire length of the capsid molecule were picked up. These results are presented in the main panel of Fig. 3. The inset shows a control experiment, run in parallel, wherein DEN-2 virus was used as the capture antigen, to detect anti-dengue antibodies in DEN-2 murine hyperimmune serum. There were three clusters of overlapping peptides of which one cluster (c1–c3) mapped to the N-terminal region and two clusters (c25–c28 and c33–c36) mapped to the carboxy-terminal region of the capsid protein. The sequences of these peptides and their relative epitope activities are summarized in Table 1. The first cluster, defined by the core sequence ‘NQRKKARN’ mapped to the N-terminus

Fig. 3. IgG-specific epitope activities of synthetic capsid peptides of DEN-2 virus determined using murine hyperimmune sera. A total of 37 pin-bound peptides (shown in Fig. 2) were scanned using DEN-2 (grey bars), YFV (black bars) and JEV (open bars) hyperimmune mice sera by ELISA, as described in materials and methods. (The inset shows a control experiment in which DEN-2 virus was used as the capture antigen to detect IgG antibodies in DEN-2 hyperimmune murine serum (grey bar) and normal murine serum (open bar)). The data represent the average of two separate experiments.

(aa residues 3–10). Interestingly, this epitope overlaps (by 2 aa residues) with the N-terminal capsid epitope reported by Bulich and Aaskov (1992). It is pertinent that the latter epitope was mapped with monoclonal antibodies, while in this Table 1 Immunoreactive dengue type 2 virus capsid peptides Peptide #

Peptide sequence (aa residue #s)

ELISA (OD450 )a Murineb Humanc

c1 c2 c3 c13 c14d c15d c16d c17d c19 c22 c23 c24 c25 c26 c27 c28 c33 c34 c35 c36 c37

MNNQRKKARN (1–10) NNQRKKARNT (2–11) NQRKKARNTP (3–12) FNMLKRERNR (13–22) VSTVQQLTKR (23–32) FSLGMLQGRG (33–42) PLKLFMALVA (43–52) FLRFLTIPPT (53–62) KKSKAINVLR (73–82) KAINVLRGFR (76–85) AINVLRGFRK (77–86) INVLRGFRKE (78–87) NVLRGFRKEI (79–88) VLRGFRKEIG (80–89) LRGFRKEIGR (81–90) RGFRKEIGRM (82–91) EIGRMLNILN (87–96) IGRMLNILNR (88–97) GRMLNILNRR (89–98) RMLNILNRRR (90–99) MLNILNRRRR (91–100)

– 0.38 0.49 – 0.43 0.10 – 0.27 0.11 – – – 0.26 1.50 0.12 0.61 0.68 1.10 1.17 0.20 –

0.29 0.10 – 0.13 – – 0.11 0.13 – 0.14 0.14 0.13 0.38 0.14 0.12 – – – – 0.19 0.28

(–) indicates low (< 0.1) or no reactivity. a ELISA ODs represent corrected values obtained after subtracting cut-off OD (0.495 for murine and 0.664 for human sera). b Hyperimmune mouse serum. c Dengue-patient serum (IgG- and IgM-positive using Dengue Duo Test). d Non-overlapping peptides.

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study the N-terminal epitope has been mapped using polyclonal serum. The second and third cluster of peptides identified regions with the core sequences ‘RGFRKEI’ (aa residues 82–88) and ‘RMLNILN’ (aa residues 90–96) towards the Cterminus of the capsid molecule. In all instances, flanking residues did seem to have a role in the overall antigenicity as evidenced by the ELISA ODs of individual peptides in each cluster. Two peptides (c14 and c17) corresponding to the putative hydrophic region of the capsid were identified with reactivities comparable to that of the N-terminal epitope. Of the entire panel of 37 peptides assayed, peptides c26 and c35 represented the most immunogenic regions of the capsid molecule. Interestingly, none of these peptides reacted with JEV hyperimmune murine serum. This was essentially true for YFV hyperimmune serum as well with the single exception of peptide c37 corresponding to the carboxy-terminal 10 aa residues of the capsid protein. This peptide showed a slight reactivity to YFV hyperimmune serum (Fig. 3). We then tested these peptides against a pool of human dengue-patient sera. To obtain this pool, we first screened sera of suspected dengue patients, using a commercially available dengue diagnostic kit, the Dengue Duo Test kit (PanBio Pty Ltd., Australia). This kit uses a mixture of purified recombinant E proteins of all four DEN serotypes to simultaneously detect the presence of both IgM and IgG classes of anti-dengue antibodies, in a single sample (Cuzzubbo et al., 2001). Sera (n = 15) that were positive for anti-dengue antibodies (both IgM and IgG classes) were pooled together for screening the capsid peptides. Serum antibodies that were captured by the pin-bound capsid peptides were detected using anti-human IgG-HRPO. For comparison, these peptides were screened in parallel using a control serum pool, generated using sera that were seronegative for both IgM and IgG classes of anti-dengue antibodies. The data are shown in Fig. 4A. Data from parallel ELISAs done using DEN-2 virus coated microtiter wells are depicted in Fig. 4B. The human serum pool also reacted with the same three peptide

Fig. 4. IgG-specific epitope activities of synthetic capsid peptides of DEN-2 virus determined using human sera. (A) The experiment is the same as in Fig. 3, except that the capsid peptides were scanned using normal human (black bars) and dengue-patient sera (open bars). The horizontal axis denotes the peptide number and the vertical axis denotes absorbance measured at 450 nm. The cut-off absorbance used to identify reactive peptides is shown by the dotted line. (B) Control experiment done in parallel using DEN-2 virus as the capture antigen, in place of the pin-bound peptides. Antibodies captured from normal human (black bars) and dengue-patient (open bars) sera were revealed using either anti-human IgM (M) or anti-human IgG (G) conjugate. The data represent the average of two separate experiments.

clusters recognized by the murine anti-dengue hyperimmune serum, but with some subtle differences (see Table 1). For example, in the first cluster the dengue-infected human serum pool showed reactivity towards peptides c1 and c2 and did not react with peptide c3. Similarly, in the second cluster (c25–c28), the human serum reacted with three (c25–c27) out of four peptides. In addition, the pooled human serum also recognized peptides c22–c24, which were not picked up by the murine serum. Of the four peptides in the third cluster (c33–c36), the human serum reacted with just a single peptide, c36. Interestingly, the dengue-infected human serum pool recognized peptide c37, which failed to be identified using the murine serum. ELISAs were also carried out using the pin-bound capsid peptides to identify IgM-specific anti-dengue antibodies in the dengue-patient serum pool. This experiment was similar to the one described above, except that the secondary antibody used was anti-human IgM-HRPO conjugate. In contrast to the experiment above, only 7 peptides (c16, c17, c18, c28, c30, c32, and c33) displayed detectable reactivity towards IgM class of anti-dengue antibodies, of the 37 tested. Of these, peptides c17 and c33, displayed relatively higher ELISA reactivities, with OD450 values of 0.17 and 0.19, respectively. The remaining five peptides showed relatively weaker reactivities (<0.1 OD450 ). 3.3. Identification of NS4a epitopes We synthesized a panel of 53 peptides, designated n1–n53, for NS4a epitope analysis (Fig. 5). Forty-three overlapping peptides (offset by one residue) spanned the putative hydrophilic amino-terminal region of the NS4a molecule (aa residues 1–50). The rest of the molecule, predicted to be largely hydrophobic, was analyzed using a set of 10 contiguous peptides (peptides n44–n53). None of the 53 NS4a peptides produced a detectable signal in ELISA using DEN2-specific hyperimmune murine serum as the source of primary antibodies, indicating that NS4a-specific antibodies are either absent or present at very low undetectable titers in the murine serum (data not shown). In contrast to this however, we could discern several NS4a peptides that reacted with dengue-infected patient serum (IgG+ /IgM+ based on PanBio Dengue Duo test). In this experiment, antibodies captured by the pin-bound NS4a peptides were detected using anti-human IgG-HRPO conjugate. The results are shown in Fig. 6. The data revealed three adjacent clusters of overlapping peptides spanning the region of NS4a between aa residues 13 and 50. The sequences of these NS4a peptides and their relative reactivities are summarized in Table 2. The first cluster of peptides (n13–n17) mapped to the region of NS4a spanning aa residues 13–26. All these peptides shared the common core sequence “MTQKAR’. The second cluster of peptides (n25–27 and n29) defined by the core sequence ‘AVLHTA’ mapped to aa 25–38 of NS4a. A third cluster consisting of peptides n37, n39 and n41 could be discerned mapping to a region of NS4a corresponding to aa residues 37–50. All these three peptides

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Fig. 5. Schematic representation of NS4a peptides synthesized for epitope mapping. The predicted amino acid sequence of DEN-2 virus NS4a protein is shown on the top. A total of 53 peptides corresponding to NS4a were synthesized using multi-pin peptide synthesis kit (chiron). Overlapping peptides were synthesized corresponding to the putative hydrophilic regions and contiguous (non-overlapping) peptides for the putative hydrophobic regions. The numbered lines below the protein sequences denote the peptides synthesized. These lines have been positioned exactly below the corresponding regions of the proteins from where their sequences were derived. (In the text, the NS4a peptides have been given the prefix ‘n’).

shared the core sequence ‘YNHALS’. In addition, we also found a single amino-terminal peptide (n2), corresponding to aa residues 2–11, also displaying significant reactivity towards dengue-patient serum. Of the NS4a peptides tested, n14 and n25 were the most immunogenic peptides. Next, we sought to determine if our NS4a peptides could react with dengue-specific IgM antibodies. To this end, we repeated the above scanning experiment, using anti-human IgM-HRPO to detect IgM class of NS4a-reactive antibodies in dengue-

patient sera. The results showed that none of the 53 NS4a peptides displayed any measurable reactivity in this ELISA (data not shown), suggesting that NS4a does not evoke a detectable IgM response during dengue infection. 3.4. Capsid and NS4a epitopes enhance the sensitivity of a first generation diagnostic antigen We recently designed a novel recombinant protein by assembling together IgG-specific, linear immunodominant epitopes of E and NS1 (AnandaRao et al., 2005). We showed that this protein, r-DME-G (recombinant dengue multi-epitope protein, IgG-specific), could detect anti-dengue antibodies in Table 2 Immunoreactive dengue type 2 virus NS4a peptides

Fig. 6. IgG-specific epitope activities of synthetic NS4a peptides of DEN-2 virus determined using dengue-patient serum. Fifty-three pin-bound peptides (shown in Fig. 5) were scanned with normal human (black bars) and dengue-patient sera (open bars) in an ELISA. The horizontal axis denotes the peptide number and the vertical axis denotes absorbance measured at 450 nm. The dotted line indicates the cut-off absorbance, used to identify reactive NS4a peptides. (The inset shows a control experiment in which DEN-2 virus was used as the capture antigen to detect IgG antibodies in normal human (black bar) and dengue-patient (open bar) serum). The data represent the average of two separate experiments.

Peptide #

Peptide sequence (aa residue #s)

ELISA (OD450 )a

n2 n13 n14 n15 n16 n17 n25 n26 n27 n29 n37 n39 n41

LTLNLITEMG (2–11) LPTFMTQKAR (13–22) PTFMTQKARD (14–23) TFMTQKARDA (15–24) FMTQKARDAL (16–25) MTQKARDALD (17–26) LDNLAVLHTA (25–34) DNLAVLHTAE (26–35) NLAVLHTAEA (27–36) AVLHTAEAGG (29–38) GGRAYNHALS (37–46) RAYNHALSEL (39–48) YNHALSELPE (41–50)

0.56 0.45 0.89 0.26 0.51 0.27 0.75 0.31 0.44 0.29 0.23 0.21 0.37

a ELISA ODs represent corrected values obtained after subtracting cut-off OD (0.35).

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Fig. 7. In-house ELISA for the detection of antibodies in dengue-patient serum. Anti-dengue antibodies in normal human (black bars) and denguepatient (open bars) sera were detected using different proteins (1: no capture antigen; 2: r-DME-G2; 3: r-DME-G; and 4: r-envelope) as capture antigens. Captured antibodies were visualized using anti-human IgG-HRPO conjugate. Data shown represent the average of triplicate assays.

a panel of dengue-patient sera, pre-confirmed to be seropositive for anti-dengue antibodies using a commercially available dengue diagnostic kit. To address the question if the C and NS4a epitopes identified above would be potentially useful in developing diagnostic intermediates, we developed a second-generation r-DME-G protein (r-DME-G2). To this end, the gene encoding the r-DME-G protein was re-designed to include sequences encoding the capsid and NS4a epitopes identified in our pepscan analysis. The re-designed gene was expressed in Escherichia coli, and the expressed protein purified by affinity chromatography (data not shown). The capacity of the r-DME-G2 protein to detect anti-dengue antibodies in pooled dengue-patient sera was then compared with that of the first generation r-DME-G protein, in an in-house ELISA. In this assay, microtiter wells were coated with the different antigens and then incubated with an appropriately diluted aliquot of pooled dengue-patient serum. Captured antibodies were visualized using anti-human IgG-HRPO. Controls were run in parallel, in which recombinant E protein was used as the capture antigen. The results are depicted in Fig. 7. The data clearly show that, the r-DME-G2 protein (containing epitopes from E, NS1, C and NS4a) is far more superior to the r-DME-G protein (containing only E and NS1 epitopes).

4. Discussion Several dengue diagnostic kits are commercially available (Groen et al., 2000; Cuzzubbo et al., 2001). Many of these use whole dengue virus preparations as antigens to detect antidengue antibodies. The use of the whole dengue virus antigen is expensive and prone to serological cross-reactivity due to similarity with other flaviviruses such as JEV and YFV. To eliminate these shortcomings, the whole virus antigen must be replaced with a more appropriate antigen in dengue diagnostics. In this context, knowledge of immunogenic epitopes encoded by pathogens is a pre-requisite for developing diagnostic peptides for the detection of infections with a high degree of sensitivity and specificity. While the immunogenic

epitopes on the E and NS1 proteins have been well documented (Aaskov et al., 1989; Innis et al., 1989; Roehrig et al., 1990; Megret et al., 1992; Jianmin et al., 1995; Huang et al., 1999; Garcia et al., 1997; Falconar et al., 1994; Wu et al., 2001), very little information is available regarding the antigenicity of the remaining dengue viral proteins. The capsid and NS4a are two small viral proteins involved in genome packaging (Lindenbach and Rice, 2001) and in anti-host viral defense (Mu˜noz-Jord´an et al., 2003). Importantly, from the viewpoint of diagnostics, these two proteins are also implicated in the induction of B-cell antibody responses in dengue virus-infected individuals (Vald´es et al., 2000; Churdboonchart et al., 1991; Se-Thoe et al., 1999). The purpose of this study was to locate linear B-cell epitopes on dengue virus capsid and NS4a proteins, which might be potentially useful in diagnosis. We therefore performed pepscan analyses using pooled dengue-patient serum, rather than single sera. As DEN-2 is currently the most prevalent of the four serotypes in India, we performed our pepscan analysis with DEN-2 virus-infected patient sera. On the capsid, we identified several IgG-specific antigenic regions, defined by reactive overlapping peptide clusters, using dengue-specific murine hyperimmune serum as well as dengue-patient sera. Both sera identified three major peptide clusters, one in the amino-terminal, and two in the carboxyterminal regions. In addition, both sera picked up epitopes, defined by single, non-overlapping peptides, in the central capsid region (Table 1). For example, the most immunogenic peptides identified using the murine hyperimmune serum were peptides c26, c35, and c3, belonging to clusters 2, 3 and 1, respectively. Using the dengue-patient serum pool, essentially similar data were obtained. However, rather than peptide c26, it was peptide c25, that was picked up most efficiently by the human anti-dengue antibodies. Based on our data, the core sequences of peptide clusters recognized by dengue-patient sera are NNQRKKARN (defined by overlapping peptides c1 and c2), RGFR (defined by overlapping peptides c22–c27) and MLNILNRRR (defined by overlapping peptides c36 and c37). Interestingly, the carboxy-terminally located immunodominant peptides are located on an ␣ helix, exposed on the surface of the capsid dimer (Ma et al., 2004). In general, the observed ELISA reactivities were higher using the murine hyperimmune serum, as expected. Aside from these IgG-specific epitopes, our studies also identified two peptides, c17 and c33, which appeared to recognize antidengue human antibodies of the IgM class. While peptide c17 interacted with IgG antibodies as well, peptide c33 did not. Peptide 33 presumably represents a unique IgM-specific epitope. The results also showed that none of the 37 capsid peptides reacted with either YFV or JEV hyperimmune mice sera. A comparison of the core sequences of each of these immunodominant regions (clustal analysis), showed that there was a high degree of similarity amongst the four dengue serotypes. However, peptide sequences in corresponding regions of YFV and JEV capsids diverged considerably from

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Fig. 8. Major IgG-specific DEN-2 epitopes identified in this study. Line maps of DEN-2 capsid (A) and NS4a (B) ‘core’ epitope sequences identified by pepscan analysis. Shown below are the corresponding peptide sequences (identified by clustal analysis) of these two proteins of other flaviviruses (abbreviations: D-1, D-3 and D-4: dengue virus type 1, 3, and 4, respectively; JEV: Japanese encephalitis virus; YFV: yellow fever virus).

those of the dengue capsids (Fig. 8A). This explains the lack of reactivity of the dengue capsid peptides when scanned with either YFV- or JEV-hyperimmune murine sera. We conclude that the capsid epitopes identified in this study are truly dengue virus-specific and therefore have the potential to be useful in the detection of dengue infections. All NS4a peptides with detectable epitope activity were mapped to the amino-terminal putative hydrophilic region of the protein. As with the capsid, we found multiple adjacent clusters of reactive peptides, using dengue-patient serum. A comparison of the core sequences of these NS4a peptide clusters amongst dengue and other flaviviruses such as YFV and JEV (Fig. 8B), showed that the core sequence AVLHTA defined by the second cluster is unique to the dengue viruses alone. On the other hand, the core epitope sequence defined by the remaining two peptide clusters showed varying degrees of similarity with the corresponding NS4a peptides of YFV and JEV. The previous reports in the literature that described the detection of NS4a-specific antibodies in dengue patient were based on the observation of a protein with the predicted electrophoretic mobility of NS4a in western blots (Churdboonchart et al., 1991; Se-Thoe et al., 1999). A more recent report demonstrated that a recombinant NS3/NS4a fusion protein displayed ELISA reactivity using dengue-patient sera (Dos Santos et al., 2004). However, this study did not unambiguously identify NS4a-specific antibodies in the patient sera. Our current study provides definitive evidence for the occurrence of NS4a-specific antibodies in dengue-patient serum using authentic dengue type 2 NS4a-derived peptides as capture antigens. Further, our data show that these NS4aspecific antibodies that we detected are IgG type antibodies. We were unable to detect any NS4a peptides that showed reactivity when tested against murine dengue-specific hyperimmune serum. This perhaps is a reflection of effective clearance of virus-infected cells during the booster immunizations

performed in the course of generating the murine hyperimmune serum. This would effectively preclude any translation and replication of the viral genome, which is a must before non-structural proteins like NS4a are produced and perceived by the immune system. In a recent study, we demonstrated that a recombinant dengue multi-epitope protein, r-DME-G, created by splicing together linear immunodominant epitopes of E and NS1 can serve as a useful dengue diagnostic antigen (AnandaRao et al., 2005). To gauge the diagnostic utility of the C and NS4a epitopes identified in this study, we developed a secondgeneration molecule by incorporating these into the r-DMEG molecule. Preliminary data suggest that the modified antigen, r-DME-G2, displays relatively enhanced sensitivity in detecting anti-dengue antibodies. In dengue endemic areas, secondary infections are most common. Thus, it is very likely that many of the dengue-patient sera used to generate the pooled serum in our studies are from secondary infections. This is consistent with all these sera testing positive using the PanBio kit, which is designed to detect secondary infections (Cuzzubbo et al., 2001). The prevalence of antibodies to various structural and non-structural dengue proteins is generally higher in secondary compared to primary dengue infections (Churdboonchart et al., 1991; Se-Thoe et al., 1999; Vald´es et al., 2000). For example, the seroprevalence of antiNS4a antibodies is ∼50% in secondary infections (Se-Thoe et al., 1999). It is therefore conceivable that the recognition of these antibodies in the pooled dengue-patient serum by the r-DME-G2 protein contributes to its improved sensitivity. In conclusion, our study has identified several immunodominant IgG-specific epitopes on both the capsid and NS4a proteins using a multi-pin peptide scanning approach. The capsid epitopes are specific to dengue virus and do not react with YFV- and JEV-specific antibodies. This work shows for the first time the existence of anti-NS4a antibodies in

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dengue-infected human serum. The immunodominant peptides of capsid and NS4a, in conjunction with the welldocumented dengue-specific epitopes of E and NS1, could be developed into diagnostic reagents for the detection of dengue infections with a high degree of sensitivity and specificity.

Acknowledgements We thank Drs. A.M. Jana and S.Vrati for providing us the hyperimmune mice sera used in this study. We gratefully acknowledge Prof. S. Fernando and Dr. R. Agarwal for the dengue-patient sera. The work was supported by ICGEB core funds. R.A. is supported by a senior research fellowship from the Council of Scientific and Industrial Research, India.

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