Virus Research 179 (2014) 133–139

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Fine mapping of a linear epitope on EDIII of Japanese encephalitis virus using a novel neutralizing monoclonal antibody Wen-Lei Deng, Chi-Yu Guan, Ke Liu, Xiao-Min Zhang, Xiu-Li Feng, Bin Zhou ∗ , Xiao-Dong Su, Pu-Yan Chen Key Laboratory of Animal Diseases Diagnosis and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China

a r t i c l e

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Article history: Received 1 May 2013 Received in revised form 24 October 2013 Accepted 24 October 2013 Available online 31 October 2013 Keywords: Japanese encephalitis virus EDIII protein Monoclonal antibody Epitope Spatial conformation

a b s t r a c t The domain III (EDIII) of the envelope protein of Japanese encephalitis virus (JEV) is proposed to play an essential role in JEV replication and infection; it is involved in binding to host receptors and contains specific epitopes that elicit neutralizing antibodies. However, most previous studies have not provided detailed molecular information about the functional epitopes on JEV EDIII protein. In this study, we described a monoclonal antibody (mAb 2B4) we produced and characterized by IFA, PRNT, ELISA and Western blot analyses. The results showed that mAb 2B4 was specific to JEV EDIII protein and possessed high neutralization activity against JEV in vitro. Furthermore, we found that the motif, 394 HHWH397 , was the minimal unit of the linear epitope recognized by mAb 2B4 through screening a phage-displayed random 12-mer peptide library. Using sequence alignment analysis it was found that this motif was highly conserved among JEV strains and was present in West Nile Virus (WNV). Indeed, ELISA data showed that this epitope could be recognized by both JEV-positive swine serum and WNV-positive swine serum. Notably, this linear epitope was highly hydrophilic and was located within the terminal end of a ␤pleated sheet of EDIII. An analysis of the spatial conformation supported the possibility of inducing specific antibodies to this epitope. Taken together, we identified 394 HHWH397 as an EDIII-specific linear epitope recognized by mAb 2B4, which would be beneficial for studying the pathogenic mechanism of JEV; and mAb 2B4 was also a potential diagnostic and therapeutic reagent. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Japanese encephalitis virus (JEV), a member of the genus Flavivirus within the family Flaviviridae, can cause serious epidemics in tropical and subtropical areas with a high mortality rate of approximately 25% in humans and lead to a serious public health problem in southern and eastern Asia (Van den Hurk et al., 2009; Wang et al., 2007). Even though two kinds of vaccines, the attenuated vaccine (SA-14-14-2) and the inactivated vaccines (mouse brain-derived and Vero cell culture-derived), are widely used to vaccinate human to prevent JEV infection, JE is widespread in the south, southeast, and the east regions of Asia, with epidemics breaking out every few years (Pang et al., 2013; Verma, 2012). Therefore, it is important to develop new therapies against JEV. The JEV genome is a single-stranded, positive-sense RNA of approximately 11 kb, containing a single open reading frame (ORF) of 10,296 nucleotides and an encoding fragment of a polypeptide of 3432 amino acid residues. This polypeptide comprises seven

∗ Corresponding author. Tel.: +86 25 84396028; fax: +86 25 84396028. E-mail address: [email protected] (B. Zhou). 0168-1702/$ – see front matter © 2013 Elsevier B.V. All rights reserved.

nonstructural proteins (NS1, NS2A, NS2B, NS3, NS44A, NS4B, and NS5), and three structural proteins, namely, core protein (C), premembrane protein (prM/M), and envelope protein (E) (Sumiyoshi et al., 1987; Hashimoto et al., 1988; Aihara et al., 1991). The E protein is the major glycoprotein on the surface of JEV. It is responsible for viral binding to cellular receptors, membrane fusion and inducing protective neutralizing antibodies in hosts (Lin and Wu, 2003). The E protein consists of three domains: domain I (central domain), domain II (dimerization domain) and domain III (immunoglobulinlike domain). Domain II is reported to serve as a hinge during the pH-dependent conformational change in the endosome (Butrapet et al., 2011), while domain III forms a ␤-barrel composed of seven anti-parallel ␤-strands, resembling the immunoglobulin constant domain (Wu et al., 2003). The E-proteins in flaviviruses are very similar in composition and hence generate cross-reactive immune responses. Considerable efforts have been made toward identification of JEV specific epitopes utilizing approaches based on synthetic peptides and monoclonal antibodies (Kimura-Kuroda and Yasui, 1983; Konishi et al., 1992; See et al., 2002). A series of monoclonal antibodies (mAbs) against JEV, first reported in 1982, were used to analyze particular epitopes of JEV, determine the differences among isolated strains, and investigate


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the process of viral replication (Chen et al., 1999; Kimura-Kuroda and Yasui, 1986). The mAbs specific to E protein have high neutralizing activity and therefore provide strong protection from JEV infection by inhibiting the adherence of the virus to the cellular membrane or affecting their fusion (Hwang and Foote, 2005). With the progress in antibody humanization technology and their clinical applications, neutralizing mAbs have been significantly enhanced both in safety and efficiency (Lin and Wu, 2003). Little is known about the spatial configuration of the epitopes on domain III of JEV E protein, where the epitopes are defined as a few contact residues that are energetically important for binding affinity in the antibody–antigen complex (Jin et al., 1992; Prasad et al., 1993). In this study, we produced a neutralizing mouse monoclonal antibody against EDIII of JEV E protein, mapped the epitope, and deduced its spatial conformation. 2. Materials and methods 2.1. Cells and viruses BHK-21 cells were maintained in Dulbecco’s modified essential medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Invitrogen), 0.2% NaHCO3 , 100 ␮g/mL streptomycin and 100 IU/mL penicillin (GIBCO, Invitrogen) at 37 ◦ C with 5% CO2 . The JEV NJ-2008 strain (GenBank accession number GQ918133) was maintained in our laboratory, and virus propagation was performed as described previously (Zhang et al., 2011). Briefly, diluted JVE NJ-2008 was added into BHK-21 cells for 1 h, washed twice and then cultured in the same conditions except that the DMEM contained 2% FBS. When the cytopathic effect (CPE) reached 70–80%, the viruses were collected and stored at −70 ◦ C (Deng et al., 2011). 2.2. MAb preparation We followed the standard procedure of producing monoclonal antibodies (Hua and Bu, 2011). Briefly, 8-week old female BALB/c mice (Yang Zhou University, Yangzhou, China) were subcutaneously immunized three times with 100 ␮g purified JEV emulsified in Freund’s complete adjuvant (day 1) or Freund’s incomplete adjuvant (Sigma) (days 14 and 28). A final booster injection was administered only with EDIII 3 days before cell fusion. Spleen cells from the mice and mouse myeloma SP2/0 cells were fused and maintained according to the standard procedures (Galfrè and Milstein, 1981). Hybridomas were screened for secretion of anti-JEV specific mAbs using an indirect ELISA with purified JEV as the antigen. The hybridoma cells were successively subcloned 3 times by limiting dilution to ensure monoclonality and stability. MAb isotype was determined using the Mouse Monoclonal Antibody Isotyping Kit (Pierce) according to the manufacturer’s introductions. MAb was purified from mouse ascites using protein A affinity columns (GE), and the concentration of the purified mAb was determined by spectrophotometer (Thermo).

and uninfected suckling mice were collected for IFA. BHK-21 cells were inoculated with serially diluted infected CSF (10−1 , 10−2 , 10−3 , 10−4 ) and uninfected CSF (non-diluted). Rabbit serum against JEV (1:50) was used as a positive control and uninfected cells were used as a negative control. 2.4. Expression of recombinant proteins In order to estimate the binding specificity of mAb 2B4 against JEV, we expressed three recombinant proteins: rNS1 (Flamand et al., 1992), rEDIII (Ge et al., 2008) and rMEP (Wei et al., 2010). They were used as coated antigens in ELISA, respectively. Three recombinant plasmids, containing rNS1 gene, rEDIII gene, and rMEP gene, were maintained in our laboratory. The multiple-epitope fragment (MEP) from the E protein of JEV included eight epitopes (amino acid residues 75–92, 149–163, 258–285, 356–362, 373–399, 397–403, 60–68, 436–445); rEDIII included a 27aa (aa373-399) from E protein of JEV; rNS1 included a full 352-amino-acid NS1 protein of JEV. The expression of the three recombinant proteins was performed as described previously (Ge et al., 2008; Wei et al., 2010; Zhou et al., 2011). The purification steps were performed according to the manufacturer’s instructions (Novagen, Ni-NTA His·Bind Resin). Only freshly purified proteins were used for ELISA and Western blot. 2.5. Indirect ELISA In the indirect ELISA, the above three recombinant proteins were coated into plates and the cell lysate of BHK-21 was used as the negative control. After incubation at 4 ◦ C overnight, the plates were washed extensively with PBS and blocked with 200 ␮L PBS with 1% BSA for 2 h at 37 ◦ C. The plates were washed again and incubated with 5 ␮g/mL mAb 2B4 diluted in PBST (100 ␮L) for 1 h at 37 ◦ C. After another wash cycle, HRP-conjugated goat anti-mouse IgG (1:5000) (Santa Cruz) was added to each well and incubated at 37 ◦ C for 45 min. Plates were washed three times and then incubated with TMB substrate (Promega). The reaction was stopped by the addition of 2 N H2 SO4 to the medium, and the optical density (OD) at 450 nm wavelength was measured using a microplate reader (ELX800, ClonTeck). Data were presented as means ± standard deviation (S.D.) from triplicate experiments. A P (virus strain)/N (negative control) value >2.1 was considered positive. 2.6. Western blot analysis rNS1, rEDIII, rMEP and the cell lysate of BHK-21 were separated using 12% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in PBST and then reacted with 5 ␮g/mL mAb 2B4 at room temperature (RT) for 2 h. Subsequently, bound antibody was detected using HRPconjugated goat anti-mouse IgG, and visualized using enhanced chemiluminescence (ECL; GE Healthcare Life Sciences).

2.3. Indirect immunofluorescence assay (IFA) 2.7. Plaque reduction neutralization test (PRNT) The specificity of purified mAb against JEV in infected cells was determined by IFA. Infected BHK-21 cells were fixed with 4% PFA in PBS at room temperature for 10 min. Next, serial dilutions of mAb were added as primary antibody and rabbit serum against JEV (1:50) was used as a positive control. After incubation for 1 h at 37 ◦ C, cells were washed three times with PBS. Cells were then incubated with a 500-fold dilution of FITC-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Santa Cruz) for 30 min at 37 ◦ C. After three washes with PBS, positive cells were detected using a fluorescent microscope. Cerebrospinal fluids (CSF) from JEV-infected

A standard plaque reduction neutralization test (PRNT) (Lin et al., 1996) was performed using JEV NJ-2008 strain. Briefly, mixtures (200 ␮L/well) of 100 plaque forming units (PFU) of JEV and serial dilutions of purified mAb 2B4 were prepared in 0.5 mL of DMEM medium, and mAb WH303, a monoclonal antibody against CSFV-E2, was performed as a negative control. Neutralization was allowed to take place for 2 h at 37 ◦ C. The neutralization reaction was performed in 24-well plates as described previously (Cabrala et al., 2012). Finally, plaques were stained with TrueBlue substrate

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(KPL, Gaithersburg, MD, USA), visualized under microscope and counted. All the experiments were run in triplicates. 2.8. Neutralization assay in mice 25 ␮g mAb 2B4 (according to the pre-experiment result of LD50) (Saxena et al., 2003) was added to 100 LD50 JEV NJ-2008 strain and incubated at 4 ◦ C overnight. Then the mixtures were intracerebrally injected into 1-day-old suckling mice (N = 10) (Murali-Krishna et al., 1996). mAb M0 (a gift from Prof. Shengbo Cao, Hazhong Agricutural University, Wuhan, China) and 1H7A8 (IgG1, ␬) (GenScript, Nanjing, China) were used as positive and negative controls, respectively. The injected mice were monitored daily for 28 days for clinical signs of infection, including ruffled hair, a hunched back, paralysis, and death. When signs of encephalitic paralysis developed, mice were euthanized as the experimental end point. 2.9. Determination of epitopes by phage-displayed random peptide library Epitope prediction was performed as previously described (Larralde et al., 2007; Maxwell et al., 2005; Lin et al., 2006). Briefly, purified 2B4 antibodies were immobilized on 96-well plates, phages from the Ph.D-12 Phage Display Peptide Library (New England BioLabs Inc., USA) were added and incubated with immobilized antibodies at 37 ◦ C for 1 h. The plates were washed three times with TBST, and the bound phages were amplified by direct infection of E. coli ER2738. The amplified phages were purified by precipitation with 20% PEG 8000/2.5 M NaCl for the next cycle screening. Three rounds of selection were routinely performed. The immunopositive phage clones were screened by the assay mentioned above and further characterized by DNA sequencing. The MegAlign program of DNAStar and BLAST algorithm for maximum homology were used to determine the consensus sequence. 2.10. Homology analysis and cross-reaction examination To investigate the homology of the epitope among flaviviruses, sequence alignment of the epitope from the homologous region of aa373–399 of EDIII protein of JEV and other flaviviruses was completed by using the DNASTAR Lasergene program (Windows version; DNASTAR Inc., Madison, WI). 2.11. Detection of the reactivity of the epitope with positive swine serum To verify whether the displayed epitope could be detected by JEV-positive serum or WNV-positive serum, a peptide with the consensus sequence (393 NHHWHK398 ) was synthesized and conjugated to the C-terminus of albumin from bovine serum (BSA). BSA-conjugated peptides were diluted serially, coated onto ELISA reader plates, and reacted with JEV-positive swine serum (1:100), WNV-positive swine serum (1:100) and JEV-negative swine serum, respectively. Bound swine serum was detected using HRP-conjugated goat anti-swine IgG (Santa cruze). The reactions were stopped by addition of 2 M H2 SO4 and the absorbance at 450 nm was measured by a ELISA plate reader.


EDIII protein was simulated to determine its peptide structure. Then, the theoretical spatial location of this linear epitope (394 HHWH397 ) within the peptide (aa373–399) was determined by the above software program. 3. Results 3.1. Production and characterization of mAb After cell fusion and screening, several hybridomas that produced specific monoclonal antibodies were obtained. Among the monoclonal antibodies we chose 2B4 for further studies because it had the strongest reactivity with JEV determined by indirect ELISA (data not shown). 2B4 was composed of IgG1 heavy chain paired with a ␬-type light chain, as determined using the Mouse MonoAb-ID Kit (HRP). To test the specificity of 2B4, we performed immunofluorescence staining assay. As shown in Fig. 1A, specific staining was readily observed in JEV infected cells, but not in uninfected cells and cells infected by other three viruses. The relatively strong fluorescence stains were detected by 2B4 in the infected CSF samples diluted at 10−1 –10−3 , while no distinct reaction was detected in the control CSF samples (Fig. 1B). It was suggested that 2B4 could detect clinical JEV infection in CSF. 3.2. Reactivity of 2B4 with viral proteins The reactivity of mAb 2B4 with expressed recombinant viral proteins was evaluated by ELISA, and the binding specificity of mAb to rEDIII and rMEP was determined by Western blot analysis. The ELISA results showed that mAb 2B4 strongly interacted with rEDIII and rMEP (Fig. 2A). In addition, proteins rEDIII and rMEP were readily detected with 2B4 antibody by Western blot; in contrast, no signal was detected from rNS1 protein and lysates of BHK-21 cells (Fig. 2B). These results showed that 2B4 specifically reacted with rEDIII and rMEP, suggesting that these two proteins shared the same epitope. 3.3. Neutralization activity of mAb 2B4 To test whether mAb 2B4 neutralized JEV, we investigated the ability of mAb 2B4 to neutralize JEV NJ-2008 strain using PRNT. As shown in Fig. 3, the data showed that mAb 2B4 with the concentration 0.5 ␮g/mL resulted in the 50% reduction on viral plaque formation, suggesting that a PRNT50 titer of mAb 2B4 was 0.5 ␮g/mL. Whereas, the control mAb WH303 did not neutralize JEV even though the concentration of 1.0 ␮g/mL was performed. To further investigate whether mAb 2B4 was capable of neutralizing JEV in vitro, we injected 2B4-treated JEV into mice, and the mice survival rate was detected. As shown in Fig. 4A, 90% of the mice challenged by 2B4-treated virus survived infection, whereas only 50% of the mice challenged by 1H7A8-treated and untreated virus survived infection. Compared to mAb M0 (as a positive control), the results demonstrated that 2B4 led to a higher survival rate (90%) and possessed higher neutralizing activity in vitro (Fig. 4B). The mAb 2B4 not only provided partial protection from the challenge with a median lethal dose of JEV, but also could postpone the death of infected mice. 3.4. Epitope prediction

2.12. Spatial conformation of 394 HHWH397 on EDIII The spatial conformation was simulated with a computer software program (PEP-FOLD, an online resource for de novo peptide structure prediction (Maupetit et al., 2009) for the theoretical location of this critical epitope on EDIII. Briefly, the spatial conformation of the peptide (373 EMEPPFGDSYIVVGRGDKQIN HHWH KA399 ) on

A phage displayed peptide library was performed to map the epitope determinants of mAb 2B4. As shown in Fig. 5A, the results indicated that mAb 2B4 specifically reacted with the ten selected phage clones that were selected following three rounds of enrichment of the peptide library with 2B4. Next, these positive phage clones were subjected to DNA sequence analysis. The consensus


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Fig. 1. The mAb 2B4 recognized JEV-infected BHK-21 cells. (A) The cells were infected with JEV NJ-2008 strain. At 72 hpi, the cells were fixed and primed with mAb 2B4 (1) and rabbit polyclonal antibody (pAb) (2), respectively; uninfected BHK-21cells, CSFV-infected PK-15 cells, PRRSV-infected Marc-145cells, and PCV2-infected PK-15 cells were also primed with mAb 2B4 as the negative controls (3–6). (B) The cells were inoculated with serially diluted JEV-infected cerebrospinal fluids (CSF) (10−1 , 10−2 , 10−3 , 10−4 ) and control CSF (non-diluted). At 72 hpi, the cells were fixed and primed with mAb 2B4 (1–4); JEV-positive rabbit pAb (5) was used as a positive control, uninfected cells were also primed with mAb 2B4 as a negative control (6). The green fluorescence demonstrated reactivity of mAb 2B4 with JEV-infected cells. (For interpretation of the references to color in this text, the reader is referred to the web version of the article.)

sequences were aligned using the DNASTAR Lasergene program (DNASTAR). As shown in Fig. 5B, the 394 HHWH397 motif was shared by almost all the selected peptides, which might be concluded as the target epitope. This sequence was also proved to match the 394 HHMH397 sequence in EDIII of JEV, indicating that peptide library screen successfully identified the epitope on EDIII recognized by 2B4.

3.5. Homology and cross-reactivity analysis Once the linear epitope was mapped, we compared residues of the EDIII sequences within 10 different JEV isolates. It was found that this linear epitope was highly conserved among these JEV strains (data not shown). Furthermore, we also performed sequence alignment between this epitope and the homologous sequences of other flaviviruses, the results displayed that the 394 HHMH397 motif was shared by both JEV and WNV strains (Fig. 6). 3.6. Identification of the displayed epitope using synthetic peptide The ELISA result indicated that 2B4 showed positive reactivity with a synthetic peptide (Fig. 7A). Furthermore, the reactivity of swine serum with various concentrations of synthetic peptide was determined by ELISA. The synthetic peptide (minimum 0.5 ␮g/mL) could be detected by both JEV-positive swine serum and WNVpositive swine serum (Fig. 7B), suggesting that 394 HHWH397 motif

Fig. 2. Identification of mAb 2B4 binding to JEV viral proteins by ELISA and Western blot analyses. (A) The ELISA data showed that mAb 2B4 had strong interactions with rEDIII and rMEP. Error bars represent the standard deviations. (B) mAb 2B4 reacted with both rEDIII and rMEP proteins and the results showed a single band, respectively.

Fig. 3. The PRNT result of mAb 2B4. The data showed that a PRNT50 titer of mAb 2B4 was 0.5 ␮g/mL. On the contrary, mAbWH303, as a negative control, could not neutralize JEV.

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Fig. 4. The survival curve of mice in the analysis of mAb neutralization of JEV. The mice were monitored daily for 30 days. The survival percentages were shown and the numbers of mice (N) in each experimental group were shown in the legend to each panel. Each descent means one occurrence of death in mice. Survival curve comparisons were performed using Graphpad Prism 5.0 software statistical analysis. The results showed that 90% of the mice challenged by 2B4-treated virus survived infection (A) and 80% of the mice challenged by M0-treated virus survived infection (B), whereas only 50% of the mice challenged by non-treatment virus survived infection.

could induce humoral immune response in vivo. However, synthetic peptide was not recognized by JEV-negative swine serum. 3.7. Spatial conformation of 394 HHWH397 The spatial conformation of the polypeptide (aa373–399) in EDIII protein was simulated by a computer software program to determine its theoretical location. As shown in Fig. 8, the polypeptide mainly formed a ␤-pleated sheet, and the 394 HHWH397 motif was located at the terminal of the fold. With relatively more positive charge due to histidine (His), the 394 HHWH397 motif was highly hydrophilic, which enabled this epitope to induce specific antibodies in immune response.

Fig. 5. Identification of the displayed epitopes by the phage displayed peptide library. (A) MAb recognition of clones selected from the phage displayed peptide library. Ten clones selected after three rounds of biopanning from phage display peptide library were tested for binding to mAb by phage ELISA. C1–C10 for binding to mAb 2B4; the anti-CSFV mAb WH303 served as a negative control. (B) Alignment of 12-mer peptide sequences from ELISA-positive clones defined the linear epitopes for the mAb 2B4. The peptides from ten phage clones that reacted with the mAb2B4 were aligned. Conserved amino acid residues were boxed and consensus sequence motifs were provided below the alignments. The matching sequences 394 HHWH397 on JEV-EDIII were provided at the bottom of alignment as comparison.

4. Discussion Monoclonal antibodies against JEV have become a powerful tool to map JEV-specific epitopes and investigate the antigenic structure. The envelope (E) protein of JEV was reported to be a strong immunogen for the production of neutralizing antibodies and CTLs (Kaur et al., 2002; Gangwar et al., 2011). Identification of the epitope on E protein will be of great help to investigate the mechanism of E-mediated protection (Wu et al., 2004). Previous studies have demonstrated that a few mAbs were prepared to identify many significant epitopes on E protein, which were effective in inducing protective immunity against JEV. Neutralizing epitopes on the lateral surface of domain III of E protein (EDIII) have been mapped, including residues aa333 (Cecilia and Gould, 1991), aa373–399 (Seif et al., 1995), aa306, aa331, and aa387 of JEV (Wu et al., 1997). In this study, we prepared mAb 2B4 with high reacting affinity specifically against EDIII domain of JEV E protein. Furthermore, we identified a neutralizing epitope recognized by this mAb. In the previous studies, the immunogenicity of 373 EMEPPFGDSYIVVGRGDKQINHHWHKA399 on EDIII protein was confirmed (Li et al., 2010a,b), which conferred protection from JEV infection in BALB/c mice. In addition, we optimized

Fig. 6. Sequence alignment between the identified linear epitope and the homologous regions of other flaviviruses. The virus strains listed in this figure were selected as representative strains. Explanation of abbreviations: DENV 1–4, dengue virus 1–4; WNV, West Nile virus; TBEV, tick-borne encephalitis virus; YFV, yellow fever virus. The amino acid sequences in red fonts of each virus were expressed and tested for cross-reactivity with the mAb 2B4. (For interpretation of the references to color in this text, the reader is referred to the web version of the article.)


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Fig. 7. Reactivity of synthetic peptides containing epitope 394 HHWH397 with 2B4, JEV-positive swine serum, and WNV-positive swine serum was tested by ELISA, respectively. (A) Synthetic peptides were evaluated by ELISA for reactivity with 2B4. The ELISA data showed that mAb 2B4 had strong interaction with synthetic peptides. (B) Synthetic peptides were evaluated by ELISA for reactivity with swine serum. The results showed that the synthetic peptides containing the linear epitope, reacted with both JEV-positive swine serum and WNV-positive swine serum. Error bars represent the standard deviations.

a proposed multi-epitope peptide (MEP) by using an epitopebased vaccine strategy combining six B-cell epitopes (amino acid residues 75–92, 149–163, 258–285, 356–362, 373–399 and 397–403) and two T-cell epitopes (amino acid residues 60–68 and 436–445) from the E protein of JEV (Wei et al., 2010). In this study, we identified the critical linear epitope on aa373–399 through

screening a phage-displayed random 12-mer peptide library with mAb 2B4. Moreover, the further results of MegAlign and BLAST analyses demonstrated that 394 HHWH397 was a linear neutralizing epitope recognized by mAb 2B4. We found that the synthetic peptide (aa393–398) could effectively react with both 2B4 and JEV-positive swine serum. Strikingly, this synthetic peptide could also react with WNV-positive swine serum because the motif of 394 HHWH397 was exactly shared by both JEV and WNV, suggesting that this epitope might have similar function in these two viruses. A previous study had reported that the KKPGGPG epitope was a JEV serocomplex-specific linear B-cell epitope, which also reacted with both JEV-positive serum and WNV-positive serum (Sun et al., 2011a,b). To our knowledge, JEV and WNV are two members of the Japanese encephalitis virus serocomplex, which cause crossreactivity among these flaviviruses. Interestingly, some epitopes recognized by mAbs are shared by both JEV and WNV, mainly because these virus antigens contain the same highly conserved immunodominant E glycoprotein. However, whether mAb 2B4 could recognize a WNV antigen still needs further investigation. Gangwar et al. (2011) had reported that mAb NHA-II appeared to be cross-reactive as it docked in the groove region between domains I and III of both the JEV and WNV E proteins. Further research should be carried out to determine whether 2B4 had neutralizing activity toward WNV as it did to JEV. Furthermore, most previous studies have not provided detailed molecular information about the spatial configuration of the functional epitopes on JEV EDIII protein. In this study, we analyzed the spatial conformation of EDIII (aa373–399) and revealed that the 394 HHWH398 sequence was located at the terminal of a ␤-sheet fold. This motif, with three histidine residues (His, an aromatic ring of positive charge, hydrophilic and often presents in active sites of enzymes), presented highly hydrophilic activity to induce immune response. This characteristic was further supported in the identification of 394 HHWH397 by JEV-positive swine serum. Therefore, our results also provided an important foundation for further studies of

Fig. 8. Localization of 394 HHWH397 by structural modeling. (A) All-atom structure conformation of aa373–399: 373 EMEPPFGDSYIVVGRGDKQIN HHWH KA399 . (B) Secondary structure of aa373–399. (C) Delineation of this epitope on aa373–399. 394 HHWH397 was marked black in the image. The polypeptides (aa373–399) were mainly constituted by ␤-pleated sheet, and the linear epitope (aa394–397) located on the terminal of the fold.

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Fine mapping of a linear epitope on EDIII of Japanese encephalitis virus using a novel neutralizing monoclonal antibody.

The domain III (EDIII) of the envelope protein of Japanese encephalitis virus (JEV) is proposed to play an essential role in JEV replication and infec...
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