An Epitope-Substituted DNA Vaccine Improves Safety and Immunogenicity against Dengue Virus Type 2

Dengue virus (DENV), a global disease, is divided into four serotypes (DENV1-4). Cross-reactive and non-neutralizing antibodies against envelope (E) protein of DENV bind to the Fcγ receptors (FcγR) of cells, and thereby exacerbate viral infection by heterologous serotypes via antibody-dependent enhancement (ADE). Identification and modification of enhancing epitopes may mitigate enhancement of DENV infection. In this study, we characterized the cross-reactive DB21-6 and DB39-2 monoclonal antibodies (mAbs) against domain I-II of DENV; these antibodies poorly neutralized and potently enhanced DENV infection both in vitro and in vivo. In addition, two enhancing mAbs, DB21-6 and DB39-2, were observed to compete with sera antibodies from patients infected with dengue. The epitopes of these enhancing mAbs were identified using phage display, structural prediction, and mapping of virus-like particle (VLP) mutants. N8, R9, V12, and E13 are the reactive residues of DB21-6, while N8, R9, and E13 are the reactive residues of DB39-2. N8 substitution tends to maintain VLP secretion, and decreases the binding activity of DB21-6 and DB39-2. The immunized sera from N8 substitution (N8R) DNA vaccine exerted greater neutralizing and protective activity than wild-type (WT)-immunized sera, both in vitro and in vivo. Furthermore, treatment with N8R-immunized sera reduced the enhancement of mortality in AG129 mice. These results support identification and substitution of enhancing epitope as a novel strategy for developing safe dengue vaccines.

Introduction secondary infection [26]. Therefore, identification of B-cell epitopes of DENV E protein, which induce cross-reactive and non-neutralizing antibodies, may provide valuable information for vaccine development.
Although various strategies have been employed in an attempt to develop dengue vaccine (including the use of attenuated or inactive virus, and the development of subunit vaccines), a safe and effective vaccine against DENV is not yet available [28]. Thus, there is a need to identify and substitute the epitopes recognized by poorly neutralizing and highly enhancing antibodies to improve the dengue vaccine. In this study, we found that the cross-reactive mAbs DB21-6 and DB39-2 exhibit poor neutralizing activity and high capacity for enhancing DENV infection. We used competitive enzyme-linked immunosorbent assay (ELISA) to determine the relationship between mAbs and sera antibodies from dengue patients. We proceeded to use phage display, bioinformatic analysis, and VLP mutants to identify the epitopes recognized by DB21-6 and DB39-2. To further improve the DNA vaccines against DENV2, we substituted the N8 residue of wild-type (WT) DENV2 E protein with arginine (N8R) in a plasmid for immunization. N8R-immunized sera produced higher neutralizing and protective activity than WT-immunized sera. Moreover, treatment of AG129 mice with N8R-immunized sera reduced mortality, as compared with mice treated with WT-immunized sera. Taken together, we have identified a novel cross-reactive and infection-enhancing epitope in E protein. Our results demonstrate that substitution of this enhancing epitope is a promising strategy for development of a safe dengue vaccine.

Ethics statement
Mouse experiments were carried out in accordance with strict guidelines from the Care and Use Manual of the National Laboratory Animal Center, Taiwan. The protocol was approved by the Committee on the Ethics of Animal Experiments of Academia Sinica (Permit Number: 11-04-166). The human serum samples were collected during an outbreak between 2002 and 2003 in Taiwan. The study protocol was approved by the National Taiwan University Institutional Review Board (NTUH-REC No. 200903086R). The written informed consent was obtained, and all human serum samples were coded for anonymity.

In vitro measurement of ADE with mAbs
Serial dilutions of mAbs were incubated with DENV1 Hawaii (MOI = 1), DENV2 16681 (MOI = 1), DENV3 H87 (MOI = 5), and DENV4 H241 (MOI = 1) for 1 hour at 4°C. The mixtures were then used to infect K562 cells for 2 hours at 37°C. After washing, the cells were incubated with 2% FBS in RPMI medium (Gibco) at 37°C for 3 days. The infected cells were collected and fixed with 3.7% formaldehyde for 10 minutes at 4°C. For staining, the cells were permeabilized with 2% FBS in PBS containing 0.1% saponin (Sigma), followed by staining with 4 μg/ml 4G2 for 0.5 hours at 4°C. The cells were washed and incubated with R-phycoerythrin (RPE)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) for 1 hour at 4°C. The cells were washed, and the percentages of infected cells were determined by flow cytometry. For infection of THP-1 cells, DENV2 16681 (MOI = 1 or 10) was incubated with diluted mAbs for 1 hour at 4°C, and then incubated with cells for 2 hours at 37°C. After 3 days, the cells were fixed, permeabilized, and stained with hDB32-6 [21]. After washing, the cells were incubated with an RPE-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories), and were subsequently analyzed by flow cytometry.

Measurement of in vivo ADE with mAbs in AG129 mice
Type I and II interferon receptor-deficient mice (AG129; 5-to 6-weeks-old) were purchased from B&K Universal. The AG129 mice were given intraperitoneal (i.p.) injections of 5 μg mAbs in 200 μl PBS on days -1 and 1. The mouse IgG1 isotype antibody was used as a negative control. On day 0 of infection, mice were intravenously (i.v.) inoculated with 1 × 10 5 pfu of the mouse-adapted DENV2 S221 (obtained from Sujan Shresta) [30], in 100 μl PBS. The survival rates of AG129 mice were recorded for 30 days.

Measurement of viremia by quantitative RT-PCR
AG129 mice were infected with 1 × 10 5 pfu DENV2 S221 by i.v. inoculation on day 0, and treated with 5 μg mAbs via i.p. injection on days -1 and 1. Viral RNA was extracted from pooled and infected mice sera using the QIAamp viral RNA minikit (Qiagen). Quantitative RT-PCR was performed based on previously published procedures [31], using the LightCycler 480 system (Roche). The standard curve was generated with DENV2 S221 (at concentrations from 10 1 to 10 7 pfu/ml). Viremia measurements were expressed as pfu equivalents/ml, which was calculated based on the threshold cycle value (Ct) according to the standard curve for DENV2 S221.

Competitive ELISA of mAbs and patient serum samples
A total of 21 DENV2-infected patient serum samples were collected from 11 DF patients and 10 DHF patients during an outbreak between 2002 and 2003 in Taiwan. Diagnosis of DENV infection was based on IgM antibody-capture ELISA (MAC-ELISA), reverse-transcriptase PCR (RT-PCR), or virus isolation in cell cultures, as previously described [15]. These serum samples were collected between days 4 and 22 from the onset of symptoms; such sera contained antidengue antibodies. All of these patients were determined to have classical DF or DHF based on the criteria published by the World Health Organization (WHO) in 2009 [32]. The characteristics of patient serum samples enrolled in this study are also provided (S1 Table). Competitive ELISA was performed as previously described [33]. Briefly, the plates were coated with polyclonal rabbit anti-DENV hyper-immune sera at 4°C overnight. After blocking, the diluted DENV2 viral supernatants (1 × 10 6 pfu) were added for 2 hours at room temperature (RT). The diluted mAbs and patient sera (1:100 dilution) were incubated for 2 hours at RT. After washing, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories) was added for 1 hour at RT. The peroxidase substrate o-phenylenediamine dihydrochloride (OPD, Sigma-Aldrich) was then added, and the reaction was stopped with 3N HCl. The optical density (OD) was measured at 490 nm. Normal human serum (NHS) was used as a control. The percentage of competition was calculated as follows: competition (%) = [1−(OD of patient serum-mAb mixture/OD of NHS-mAb mixture)] × 100.

Phage display biopanning
Phage display biopanning was performed as previously described [21]. Briefly, the plate was coated with 100 μg/ml mAbs at 4°C for 6 hours. After washing and blocking, 4 × 10 10 pfu of phage-displayed peptide library (New England BioLabs, Inc.) were incubated for 50 mins at RT. After washing, bound phage was eluted with 100 μl 0.2 M glycine/HCl (pH 2.2) and neutralized with 15 μl 1 M Tris/HCl (pH 9.1). The eluted phage was then amplified in ER2738 for subsequent rounds of selection. The phage was titrated onto LB plates containing IPTG and X-Gal. The second and third rounds of selection were identical to the first round except for the addition of 2 × 10 11 pfu of amplified phage.

Identification of immunopositive phage clones by ELISA
The plate was coated with 50 μg/ml mAbs. After washing and blocking, the amplified phages were added, and incubated for 1 hour at RT. After washing, diluted HRP-conjugated anti-M13 antibody (GE Healthcare) was added at RT for 1 hour. The plates were developed, and subsequently terminated by 3N HCl. The OD was measured at 490 nm.
Identification of epitopes using flow cytometry-based binding assay to cells expressing WT and mutant DENV2 prM/E proteins The pCBD2-2J-2-9-1 plasmid expressing prM-E proteins of DENV2 has been previously characterized and described [14][15][16]. Site-directed mutagenesis was performed to replace each of the selected amino acid residues, as described in the previous study [21]. After mutagenesis, the plasmids were sequenced to ensure the absence of any further mutations at non-target sites. BHK-21 cells were transfected with constructs expressing the wild-type (WT) or mutant DENV2 E protein using polyjet in vitro DNA transfection reagent (SignGen Laboratories). After 2 days, the cells were fixed, and permeabilized with 2% FBS in PBS containing 0.1% saponin (Sigma). For staining, cells were incubated with DB21-6, DB39-2, 4G2, and mixed mAbs (DB32-6, 3H5, and DB25-2) at a concentration of 1, 1, 1, and 1 μg/ml, respectively, at 4°C for 0.5 hours. After washing, the cells were incubated with RPE-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories), and analyzed by flow cytometry. The relative index of a mAb to a mutant E protein was measured using the formula: [intensity of the mutant E/ intensity of WT E (recognized by a mAb)]/[intensity of mutant E/intensity of WT E (recognized by mixed mAbs)].

Detection of secreted VLPs by capture ELISA
BHK-21 cells were transfected with vectors expressing the WT or mutant E protein of DENV2, as described above. At 48 hours post-transfection, culture supernatants were collected. The plates were coated with polyclonal rabbit anti-DENV hyper-immune sera at 4°C overnight. After blocking, two-fold dilutions of supernatants containing WT or mutant VLPs were added for 2 hour at RT. The wells were then incubated with diluted DB32-6 and 4G2 at RT for 2 hour. After washing, a 1:2000 dilution of HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories) was added for 1 hour at RT. Finally, the plates were developed, and the reaction was subsequently terminated with 3N HCl. The OD was measured at 490 nm.

Preparation of plasmids for immunization
Plasmids expressing WT E protein of DENV2 or a mutant E protein in which the N8 residue was substituted with R (N8R) were used for immunization. For coating, 25 mg of 1.0 μm gold powder was resuspended with 50 mM spermidine (Sigma-Aldrich, St. Louis, MO). Then, 50 μg of plasmid DNA was added, followed by the addition of 1M CaCl 2 (Sigma-Aldrich, St. Louis, MO); the solution was mixed and precipitated for 10 mins at RT. After collection by centrifugation, the gold-DNA complex was washed with absolute ethanol and resuspended in 0.1 mg/ml of polyvinylpyrrolidone (PVP) (360 kDa; Sigma Chemicals, Inc.) solution. The slurry was injected into a TefzelR tube (McMaster-Carr, Chicago, IL), and then coated. After the ethanol had dried off, the tube was cut into 0.5-inch bullets and stored at -20°C. The gold in each bullet contained 1 μg of DNA. Before use, the bullets were loaded into the Helios gene gun device (Bio-Rad, Hercules, CA) for delivery of plasmids.

Immunization of mice
The abdominal epidermis of 6 week-old female BALB/c mice was injected with a gene gun using a helium pressure setting of 400 lb/inch 2 . Each mouse was immunized by administering 4 bullets containing 1 μg plasmid DNA. Mice were immunized at 0, 3, and 6 weeks. Serum samples were collected before immunization and 3 weeks after the third immunization (pre-, 1 st , 2 nd , 3 rd immunized sera). The serum samples were pooled from five to six mice for each immunized group and evaluated by ELISA, neutralization assay, and in vivo ADE assay.
Evaluation of immunized sera against DENV2 by ELISA C6/36 cells infected with DENV2 16681 were used as antigens. C6/36 cells were seeded into each well (2 × 10 4 cells/well) of 96-well ELISA plates. After one day, 2 × 10 3 pfu of DENV2 16681 (MOI = 0.1) was added to infect the cells at 37°C for 2 hours. The wells were washed with PBS, and then cultured in 2% FBS culture medium at 28°C for 5 days. Next, the infected cells were fixed with 1:1 methanol/acetone at 4°C for 10 mins. The plates were blocked with 5% skimmed milk at 4°C for 24 hours. Diluted immunized sera were then added for incubation at RT for 2 hours. The plates were then washed three times with phosphate-buffered saline containing 0.1% (w/v) Tween 20 (PBST 0.1 ), and subsequently incubated with HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories). Finally, the plates were developed, and the reaction terminated with 3N HCl. The OD was measured at 490 nm.
In vitro and in vivo neutralization assays with immunized sera DENV2 16681 (MOI = 1) was incubated with 3 rd immunized sera for 1 hour at 4°C. Next, the mixtures were used to infect BHK-21 cells for 2 hours at 37°C. After 3 days, the cells were fixed, permeabilized, and stained with 4 μg/ml 4G2. After washing, the cells were incubated with RPE-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories), and analyzed by flow cytometry. Inhibition percentage (%) = [1−(the percentage of infected cells incubated with immunized sera/without immunized sera)] × 100.
The ICR mice were purchased from the Laboratory Animal Center, National Taiwan University College of Medicine. Serially-diluted immunized sera were incubated with 1 × 10 4 pfu (25-fold lethal dose, 25-fold LD 50 ) of DENV2 16681 for 0.5 hours at 4°C. Two-day-old suckling mice were inoculated with 20 μl of the mixtures through intracranial (i.c.) injection. After challenge, the survival rates were recorded for 28 days.

Studies of in vivo ADE with immunized sera
AG129 mice were given i.p. injections of dilutions of immunized sera on days -1 and 1, and were i.v. inoculated with 1 × 10 5 pfu of DENV2 S221 on day 0. The survival rates were recorded for 30 days.

Statistical analysis
Survival rate was expressed using Kaplan-Meier survival curves, and statistical analyses were performed using GraphPad Prism 5. For competition assays of mAbs and patient sera, Student's t tests were used to identify significant differences and calculate P values ( Ã P<0.05, ÃÃÃ P<0.001, NS not significant). For evaluation of immunized sera against DENV2 by ELISA, two-way ANOVA with Bonferroni post-hoc test was used to determine the significant differences and calculate P values ( ÃÃ P<0.01, NS not significant). GraphPad Prism 5 was used to analyze 50% inhibition titers against DENV2, based on inhibition percentages from pooled immunized sera.
To estimate the in vitro neutralizing activity, we infected BHK-21 cells with a mixture of individual mAbs and DENV1-4. Previous studies have reported that 4G2 is an anti-flavivirus antibody with neutralizing and enhancing activity at certain concentrations [16]. We observed that 4G2 exerts higher neutralization activity than DB21-6 and DB39-2 against DENV2 (S2 Enhancing activities of DB21-6 and DB39-2 To investigate in vitro enhancement of DENV infection through ADE [34,35], we performed in vitro ADE assays, and detected the increases in the percentage of dengue-infected cells by flow cytometry [36]. The FcγRIIA-bearing K562 cells, which do not express type 1 interferon (IFN) [37], were used to measure the enhancement of infected cells through extrinsic ADE. The serially-diluted mAbs were incubated with DENV1-4, and then used to infect K562 cells. The infection percentage was measured by flow cytometry, revealing infection enhancement over a broad range of mAb concentrations ( Fig 1A). As compared to the other mAbs, 4G2 caused enhancement of DENV1-4 infection in K562 cells at lower antibody concentrations. DB21-6 and DB39-2 enhanced DENV1-4 infection in K562 cells at high antibody concentrations ( Fig 1A). To further confirm enhancement of infection, we proceeded to examine the enhancement of DENV2 16681 infection by DB21-6 and DB39-2 in FcγRI-and FcγRIIA-bearing THP-1 cells. Infection in THP-1 cells was enhanced to a greater extent by DB21-6 and DB39-2 than by 4G2 (Fig 1B). DENV2 S221 was previously used to study enhancement of mortality via ADE in AG129 mice [30]. To evaluate the in vitro enhancement of DENV2 S221 infection by mAbs, we performed ADE assays using K562 cells and THP-1 cells. As for DENV1-4 infection, high concentrations of DB21-6 and DB39-2 enhanced DENV2 S221 infection in K562 cells (S3A Fig). In addition, DB21-6 and DB39-2 enhanced DENV2 S221 infection in THP-1 cells at high concentrations of antibody (S3B Fig). These results suggest that DB21-6 and DB39-2 can enhance DENV2 S221 infection in vitro. Next, we confirmed the in vivo enhancing activities in AG129 mice. The AG129 mice treated with 5 μg DB21-6 and infected with DENV2 S221 exhibited increased mortality as compared to control infected mice (Fig 2A). In addition, AG129 mice treated with 5 μg of DB39-2 also exhibited elevated mortality (Fig 2B). In order to determine viremia in DENV2 S221-infected AG129 mice following treatment with DB21-6 or DB39-2, the viral RNA levels were measured by quantitative RT-PCR. The results indicate that viral loads were significantly increased after DB21-6 or DB39-2 treatment of infected AG129 mice, as compared to isotype control Ab treatment (Fig 2C). These results indicate that DB21-6 and DB39-2 have non-neutralizing activities, and enhance mortality in AG129 mice.

Competition assay of mAbs and infected patient sera
We proceeded to perform competition assay to determine whether sera antibodies from dengue patients compete with mAbs for binding to DENV2. The characteristics of patient serum samples enrolled in this study are provided (S1 Table). The sera antibodies from infected patients were observed to compete with DB21-6 and DB39-2. The competition percentages of DB21-6 and DB39-2 were significantly higher in serum samples from DHF patients than those from DF patients (Fig 2D), while the competition percentage of neutralizing DB32-6 [21] was similar for sera from either DF or DHF patients (Fig 2D). We also performed the same experiment with more concentrated serum (1:50 dilution) or diluted serum (1:200 dilution), and obtained similar results (S4 Fig). These results suggest that serum samples from DHF patients contain higher levels of antibodies, which compete for binding with DB21-6 and DB39-2 mAbs.
Identification of enhancing epitopes of DB21-6 and DB39-2 In order to identify the enhancing epitopes of DB21-6 and DB39-2, we used a phage-displayed peptide library to screen the reactive phage clones. After three biopanning rounds, the phage titers were increased to 12,871-fold (DB21-6) and 5,000-fold (DB39-2), respectively, compared to that of the first round ( Fig 3A). The individual phage clones from the third round of biopanning were randomly selected. As shown by ELISA, most selected phage clones exhibited significant reactivity to the mAbs, but not to normal mouse IgG (NMIgG). Of the 30 selected phage clones, 29 clones reacted with DB21-6 ( Fig 3B). The immunopositive phage clones were amplified, and their phage DNA was isolated for DNA sequencing. Eleven phage clones with individual peptide sequences were identified (Table 1). Similarly, of the 47 selected phage clones, 46 reacted with DB39-2 ( Fig 3B). Thirteen of the 46 immunopositive phage clones that reacted with DB39-2 possess individual peptide sequences ( Table 1). Alignment of peptide sequences revealed the binding motif of DB21-6 and DB39-2 to be N-R-x-x-V-E (Table 1). In addition, modeling of the peptide sequences with the pepitope server (http://pepitope.tau.ac.il/) predicted that the epitope residues on the E protein are N8, R9, V12, and E13 (Table 1).
To further verify the epitope of DB21-6 and DB39-2, we performed site-directed mutagenesis of the phage-displayed epitope using pCBD2-2J-2-9-1 as template. After confirmation of variants by sequencing, we transfected cells with the mutant plasmids, and detected binding activity by flow cytometry. The binding percentages for each transfectant were normalized to those of anti-EDIII mAbs (DB32-6, 3H5, and DB25-2) [21], and relative indices were calculated ( Fig 4A). 4G2, which binds to residues at the fusion loop of EDII [16], was used as a control to verify the structural change of E proteins caused by mutations (Fig 4A). Based on the relative indices, we found that mutations at N8, R9, V12, and E13 prevented binding by DB21-6. The same method was used to identify the epitope residues of DB39-2 as N8, R9, and E13. Structural modeling was applied to show that the recognition residues are located in domain I of E protein (Fig 4B). The distance between these residues from the same monomers was analyzed using a structure modeling program, and was found to be less than 30°A (Fig 4C); interestingly, this distance can be spanned by a single IgG molecule [16]. This suggests that the N8, R9, V12, and E13 residues constitute the epitope of DB21-6. In addition, the N8, R9, and E13 residues constitute the epitope of DB39-2. Alignments revealed that the binding motif of DB21-6 and DB39-2 corresponds to the N8, R9, V12, and E13 residues, which are conserved in DENV1-4 (S2 and S3 Tables). Finally, we used VLP-capture ELISA to demonstrate that the mutations at R9, V12, and E13 affect DENV2 VLP secretion (Fig 4D). The effects of these mutations on the ability to secrete VLPs might be due to a change in the structure of E protein. However, the N8R substitution did not affect DENV2 VLP secretion (Fig 4D). N8 substitution tends to maintain VLP secretion and reduces the binding activity of DB21-6 and DB39-2.

Examination of humoral immune responses in mice
The BALB/c mice were immunized with vector, WT, or N8R plasmids at 0, 3, and 6 weeks. After three rounds of immunization, the serum samples were collected and pooled within each immunized group. Next, the immunized sera were examined by ELISA. A remarkable increase of antibody titer against DENV2 was observed after immunization (S5A Fig). The 3 rd WT-and N8R-immunized sera against DENV2 exhibited significantly higher absorbance values than those of vector-immunized sera (Fig 5A). Analysis of immunized sera with anti-IgG1 and IgG2a antibodies revealed that the IgG1/IgG2a ratios increased between the second and third immunization (S5B and S5C Fig). In addition, the immunized mice maintained their anti-DENV2 responses after 15 weeks (S5D Fig). Table 1. Alignment of phage-displayed peptide sequences selected by DB21-6 and DB39-2.

DB21-6 phage clones
Peptide sequences a PC21-15 a The phage-displayed consensus amino acids are indicated by boldface type. b The amino acid sequences 8 to 13 in E protein of DENV1-4 were retrieved from GenBank (accession number AIU47321, AAB58782, AAA99437, and AAX48017).   The immunized sera were evaluated for their neutralizing activity against DENV2. Both WTand N8R-immunized sera exhibited high neutralizing activities, while vector-immunized sera did not (Fig 5B). Interestingly, DENV2 infection was more effectively neutralized by N8Rimmunized sera than by WT-immunized sera (Fig 5C). To further evaluate whether immunized sera could broadly neutralize the diverse DENV2 strains, BHK-21 cells were infected with mixtures of immunized sera and four different DENV2 strains: 16681, NGC, PL046, and Malaysia 07587. Remarkably, the WT-and N8R-immunized sera exhibited high neutralizing activities against various types of DENV2 strain (S6 Fig). Next, we examined the protective effect of immunized sera against DENV2 16681 in vivo. The survival rates of mice treated with WT-immunized sera at dilutions of 1:100 and 1:200 were significantly higher than that of mice treated with vector-immunized sera at a dilution of 1:100 (Fig 5D), while the survival rates of mice treated with N8R-immunized sera at dilutions of 1:100, 1:200, and 1:400 were significantly higher than that of mice treated with vector-immunized sera at a dilution of 1:100 (Fig 5E). In addition, treatment with WT-immunized sera afforded 50% protection at a dilution of 1:200, while N8R-immunized sera afforded 50% protection at a dilution of 1:400 (Fig 5D and 5E). Hence, N8R-immunized sera possessed higher neutralizing and protective activity than WT-immunized sera both in vitro and in vivo.

Reduction of the in vivo enhancing activity of the immunized sera
In order to study the in vivo enhancement of mortality, we passively transferred different dilutions of WT-, N8R-, or vector-immunized sera into AG129 mice. Following infection with DENV2 S221, the survival rate of mice treated with WT-or N8R-immunized sera (1:25 dilution) was higher than that of mice treated with vector-immunized sera (Fig 6A). However, mice treated with WT-immunized sera at a dilution of 1:100 showed higher mortality than mice treated with vector-immunized sera (Fig 6B). Notably, the survival rate of mice treated with N8R-immunized sera at a dilution of 1:100 was higher than that of mice treated with vector-immunized sera (Fig 6B). In addition, no enhancement of mortality was observed in mice treated with N8R-immunized sera (Fig 6B). Finally, treatment with WT-or N8R-immunized sera at a dilution of 1:400 did not have a neutralizing or enhancing effect on the survival rates of mice (Fig 6C). These results indicate that the N8R substitution of E protein can reduce in vivo enhancement of mortality.
To further characterize these enhancing antibodies are produced in immunized sera, we performed competitive ELISA to inhibit the binding of HRP-conjugated DB21-6 or DB39-2 mAbs by immunized sera (Fig 6D). The competition percentages of HRP-conjugated DB21-6 and DB39-2 were significantly higher in WT-immunized sera than those in N8R-immunized sera (Fig 6E). These results suggest that N8R substitution would redirect immunodominance by reducing the generation of enhancing antibodies.

Discussion
DENV infections stimulate immune responses and elicit a small proportion of protective antibodies. However, a high proportion of non-protective antibodies are also generated, which 50% inhibition against DENV2 infection. (D and E) After the 3 rd immunization, the pooled sera were serially diluted and incubated with 25-fold LD 50 of DENV2 for 0.5 hour at 4°C. Next, the mixtures were injected into ICR suckling mice by the intracerebral route (i.c.). The survival rates were recorded for 28 days. The number of animals tested for each immunized sera ranged from 4 to 16 per group. Kaplan-Meier survival curves and P values are shown (***P<0.001, **P<0.01, compared to vector-immunized sera). Data shown are from one representative experiment of two independent experiments. doi:10.1371/journal.pntd.0003903.g005 Fig 6. In vivo enhancement of mortality in AG129 mice treated with immunized sera. (A) AG129 mice were i.v. infected with 1 × 10 5 pfu DENV2 S221 on day 0, and given i.p. injections of WT-(n = 4), N8R-(n = 4), or vector-(n = 4) immunized sera (1:25 dilution) on days -1 and 1. Kaplan-Meier survival curves (1:25 dilution) are shown, and P values were analyzed using GraphPad Prism 5. The survival rates of mice treated with WT-(P = 0.3834) or N8R-(P = 0.1278) immunized sera did not significantly differ from that of mice treated with vector-immunized sera. (B) AG129 mice were i.v. infected with 1 × 10 5 pfu DENV2 (S221) on day 0, and given i.p. injections of WT-(n = 5), N8R-(n = 5), or vector-(n = 7) immunized sera (1:100 dilution) on days -1 and 1. Kaplan-Meier survival curves (1:100 dilution) are shown, and P values were analyzed using GraphPad Prism 5. The survival rate of mice treated with WT-(***P = 0.0007) immunized sera was significantly lower than that of mice treated with vector-immunized sera. However, the survival rate of mice treated with N8R-(P = 0.3538) immunized sera did not differ from that of mice treated with vector-immunized sera. (C) AG129 mice were i.v. infected with 1 × 10 5 pfu DENV2 (S221) on day 0, and given i.p. injections with WT-(n = 7), N8R-(n = 7), or vector-(n = 6) immunized sera (1:400 dilution) on days -1 and 1. The survival rates were recorded for 30 days. Kaplan-Meier survival curves (1:400 dilution) are shown, and P values were analyzed using GraphPad Prism 5. The might be associated with enhancement of viral infections. Here, we characterized the ability of DB21-6 and DB39-2 to increase the percentage of dengue virus-infected cells. Furthermore, we confirmed that these mAbs enhance mortality in AG129 mice. Through competition assay, we found that sera antibodies from infected patients compete for binding with these mAbs. Using phage-display, structure prediction, and VLP mutants, we mapped the epitopes of enhancing mAbs DB21-6 and DB39-2 on EDI protein. To investigate how to reduce the enhancing effects while maintaining neutralizing activity, we substituted the N8 residue of E protein, and immunized mice with WT or N8R plasmids with a gene gun delivery system. After three immunizations, N8R-immunized sera produced neutralizing activity against DENV2, and reduced enhancement of mortality as compared to WT-immunized sera. Thus, substitution of enhancing epitope residues can increase the immune response against viral infection while reducing the potential for ADE.
The antibodies induced by E protein of DENV play important roles in neutralizing effects and regulation of viral infection [21,[38][39][40]. There are three structural domains (domain I, II, and III) in E protein. In previous reports, some mouse mAbs that bind to domain III of E protein were found to exhibit neutralizing activity and obstruct viral infection [21][22][23]. However, the anti-E or prM antibodies are cross-reactive and weakly neutralizing, which may enhance viral infection through ADE [26,36]. Here, we demonstrated that cross-reactive DB21-6 and DB39-2 against EDI-II have poor neutralizing activities against DENV1-4 (S2 Fig). In addition, we found that DB21-6 and DB39-2 have strong ADE activities in vitro (Fig 1). Previous studies have shown that anti-fusion loop 4G2 enhances viral infections in both in vitro ADE assays and AG129 mice [41]. We also observed that 4G2 has partially neutralizing activity against DENV1-4 (S2 Fig) and enhances in vitro viral infections at low antibody concentrations ( Fig  1A). Notably, we also found that DB21-6 and DB39-2 enhanced DENV1-4 infection in K562 cells at high concentrations ( Fig 1A). Furthermore, infection of DENV2 was enhanced to a greater extent by DB21-6 and DB39-2 than by 4G2 in THP-1 cells (Fig 1B). In addition, DB21-6 and DB39-2 enhanced mortality in AG129 mice (Fig 2A and 2B) and increases the viral loads in infected mice sera (Fig 2C). These results indicate that DB21-6 and DB39-2 have strong enhancing activity both in vitro and in vivo.
ADE is regarded as an important mechanism leading to the development of severe dengue disease, including DHF/DSS [5]. Cross-reactive and non-neutralizing antibodies binding to viruses can enhance infection of FcγR-bearing cells by ADE, resulting in increased viral load and/or production of cytokines [6]. High viral load is correlated with dengue disease severity and DHF [42,43]. Thus, there is a need to be able to confirm the presence of enhancing antibodies in dengue patient sera. Our results indicate that the competition percentages of DB21-6 and DB39-2 were significantly higher in DHF patient sera than those in DF patient sera ( Fig  2D), suggesting that the higher levels of enhancing antibodies, DB21-6 and DB39-2, in serum samples of dengue patients are associated with severe dengue disease. We hypothesize that the DENV infected patients might suffer more severe symptoms, such as DHF, when the expression level of the enhancing antibodies is higher.
survival rate of mice treated with WT-(P = 0.8701) or N8R-(P = 0.1587) immunized sera did not significantly differ from that of mice treated with vectorimmunized sera. (D) Schematic describing the competition assay of mAbs and immunized sera. The ELISA plates were coated with polyclonal rabbit anti-DENV hyper-immune sera at 4°C overnight. After blocking, the diluted DENV2 viral supernatants were added for 2 hours at RT. The HRP-conjugated enhancing mAbs (Innova Biosciences HRP Conjugation Kit) and immunized sera (1:40 dilution) were incubated for 2 hours at RT. After washing, the OPD was added, and the reaction was stopped with 3N HCl. The OD was measured at 490 nm. (E) Competition between mAbs and immunized sera for binding to DENV2. The percentage of competition is shown. Normal mouse serum (NMS) was used as a control. The percentage of competition was calculated as follows: competition (%) = [1−(OD of immunized serum-mAb mixture/OD of NMS-mAb mixture)] × 100. The P values (***P<0.001) were analyzed using twoway ANOVA with Bonferroni post-hoc test. Identification of binding domain and epitope residues in the E protein may provide helpful information for investigation of neutralizing and enhancing mechanisms of dengue infection. Phage display is a powerful method for developing epitope-based diagnostics and identifying B-cell epitopes [21,44]. After screening a phage-displayed peptide library, we found that the phage clones selected using DB21-6 and DB39-2 mAbs displayed peptide sequences containing a consensus motif, N-R-x-x-V-E (Table 1). These displayed peptide sequences may be suitable for detecting enhancing antibodies in serum samples from dengue patients, and for providing information on the pathogenesis of dengue. By alignment of displayed peptide sequences and structural modeling, the candidate epitopes were predicted and verified using VLP mutants (Figs 3 and 4). The epitope residues of enhancing mAb DB21-6 are N8, R9, V12, and E13 in domain I of DENV2 E protein (Fig 4A and 4B), and the epitope residues of enhancing mAb DB39-2 are N8, R9, and E13 in domain I of DENV2 E protein (Fig 4A and 4B). We aligned the N8, R9, V12, and E13 residues, and found that these residues were conserved in DENV1-4 (S2 Table). Thus, cross-reactive DB21-6 and DB39-2 can bind to DENV1-4. A previous report indicated that G106 and L107 are the epitope residues of enhancing mAb 4G2 [16]. In our studies, we also confirmed that W101, G106, L107, and F108 in the fusion loop are the epitope residues of 4G2 ( Fig 4A). The epitope residues recognized by 4G2 are different from those recognized by DB21-6 and DB39-2. These findings suggest that DB21-6/DB39-2 and 4G2 enhance DENV infection through different mechanisms. In addition, the enhancing epitopes of DB21-6 (N8, R9, V12, and E13) and DB39-2 (N8, R9, and E13) are novel and have not previously been reported. Therefore, further verification of these enhancing epitopes and the detailed molecular mechanism(s) by which these enhancing antibodies propagate dengue infection are worth investigating through cryo-electron microscopy (cryo-EM).
The E protein is targeted by most reported dengue vaccines, and is thus regarded as an important target [28]. Sanofi Pasteur published data from a phase III study on tetravalent dengue vaccine, which conferred moderate protection (56%) against dengue disease [45]. Furthermore, the vaccine provided low protection (35%) against DENV2, but more than 75% protection against DENV3 and 4, and 50% against DENV1. Improvements in vaccine efficacy and the effect of the substitution of the enhancing epitope on safety are yet to be examined. Previous studies have shown that DNA vaccine candidates against DENV1 or DENV2 with substitutions in the fusion loop (at G106 and L107) and the cross-reactive epitopes of EDIII (at K310, E311, and P364) confer protective immunity [46,47]. In addition, enhancement of mortality by enhancing antibodies against the fusion loop was reduced in mice immunized with such vaccines. In this study, we have identified new enhancing antibodies and a novel enhancing epitope that are different from those previously reported. Mutations at R9, V12, and E13 may change the structure of E protein and affect VLP secretion. However, our VLP-capture ELISA results suggest that the N8R substitution does not affect DENV2 VLP secretion ( Fig  4D), which is crucial for its use in immunization. Moreover, we used an in vitro neutralizing assay and in vivo protection assays to show that both WT-and N8R-immunized sera exerted protective activities against DENV2 (Fig 5). Interestingly, N8R-immunized sera had higher in vitro neutralizing activity and in vivo protective activity than the WT-immunized sera (Fig 5B-5E). These results suggest that immunization with the N8R DNA vaccine may increase neutralizing and protective immunity against DENV2.
An earlier investigation used mouse-adapted DENV2 S221 to study severe dengue disease via ADE in AG129 mice [30]. Here, we passively transferred diluted vector-, WT-, or N8Rimmunized sera, and then challenged AG129 mice with DENV2 S221. WT-and N8R-immunized sera were protective at a 1:25 dilution, as compared to vector-immunized sera. However, the mortality of mice was enhanced by treatment with WT-immunized sera at a 1:100 dilution, as compared to treatment with vector-immunized sera. Importantly, the mortality of mice treated with N8R-immunized sera at a 1:100 dilution was not enhanced (Fig 6B). When the dilution was increased to 1:400, no enhanced mortality was observed (Fig 6C). Our results indicate that substituting the enhancing epitope can reduce the ADE phenomenon and increase protective activity in vivo.
In this study, substitution of enhancing epitope and preservation of neutralizing epitope in immunized mice provide protective immunity. Such an approach would redirect immunodominance ( Fig 6E) and improve immunogenicity by satisfying the required neutralizing occupancy [48]. In summary, we have identified a novel enhancing epitope, enabling us to reduce the potential for ADE through N8R substitution in DENV2 E protein. This may be a viable approach for developing new dengue vaccines that can increase the anti-DENV immune response.