Dengue Virus Infection-Enhancing Activity in Serum Samples with Neutralizing Activity as Determined by Using FcγR-Expressing Cells

Background Progress in dengue vaccine development has been hampered by limited understanding of protective immunity against dengue virus infection. Conventional neutralizing antibody titration assays that use FcγR-negative cells do not consider possible infection-enhancement activity. We reasoned that as FcγR-expressing cells are the major target cells of dengue virus, neutralizing antibody titration assays using FcγR-expressing cells that determine the sum of neutralizing and infection-enhancing activity, may better reflect the biological properties of antibodies in vivo. Methods and Findings We evaluated serum samples from 80 residents of a dengue endemic country, Malaysia, for neutralizing activity, and infection-enhancing activity at 1∶10 serum dilution by using FcγR-negative BHK cells and FcγR-expressing BHK cells. The serum samples consisted of a panel of patients with acute DENV infection (31%, 25/80) and a panel of donors without acute DENV infection (69%, 55/80). A high proportion of the tested serum samples (75%, 60/80) demonstrated DENV neutralizing activity (PRNT50≥10) and infection-enhancing activity. Eleven of 18 serum samples from patients with acute secondary DENV infection demonstrated neutralizing activity to the infecting serotype determined by using FcγR-negative BHK cells (PRNT50≥10), but not when determined by using FcγR-expressing cells. Conclusion Human serum samples with low neutralizing activity determined by using FcγR-negative cells showed DENV infection-enhancing activity using FcγR-expressing cells, whereas those with high neutralizing activity determined by using FcγR-negative cells demonstrate low or no infection-enhancing activity using FcγR-expressing cells. The results suggest an inverse relationship between neutralizing antibody titer and infection-enhancing activity, and that neutralizing activity determined by using FcγR-expressing cells, and not the activity determined by using FcγR-negative cells, may better reflect protection to DENV infection in vivo.


Introduction
Dengue fever (DF) and dengue hemorrhagic fever (DHF) is caused by infection with dengue virus (DENV), a flavivirus, which consists of four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). DENV affects up to 100 million people annually living in the tropics and sub-tropical areas. Clinical manifestations of DENV infection ranges from asymptomatic and relatively mild dengue fever (DF), to severe, life-threatening illness, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [1,2]. In endemic regions, the risk for developing severe infection was speculated to be higher as compared to non-endemic regions due to the higher possibility of secondary exposure to heterologous DENV serotypes [3,4]. The number of dengue patients has increased in Malaysia over the past 10 years with 7,103 cases and 45 deaths in 2000, to 41,486 cases and 88 deaths in 2009, to, 46,171 cases and 134 deaths in 2010 [5,6]. All four DENV serotypes co-circulate in Malaysia [7,8]. High prevalence of severe dengue virus infections and dengue-related deaths in recent years is speculated to be associated to rapid urbanization and global travel, leading to the spread of dengue virus, and thus to higher prevalence of infected individuals [9][10][11].
Primary infection with one DENV serotype does not confer protection to infection with a heterologous serotype [12,13]. Epidemiological studies have demonstrated that DHF occurs at a higher rate in secondary infection than in primary infection [14][15][16][17]. DENV sub-neutralizing, infection-enhancing antibodies induced during primary infection is speculated to play a central role in the pathogenesis of DHF [18][19][20][21]. During secondary infection, sub-neutralizing antibodies form infectious immunecomplexes with DENV, resulting in higher levels of viral progeny in FccR-expressing cells, a phenomenon known as antibody-dependent enhancement (ADE) [22,23]. It has been speculated that ADE may play a role not only in causing DHF but in worsening a spectrum of DENV illness [24].
We previously demonstrated that higher neutralizing antibody titers were detected using FccR-negative BHK cells as compared to FccR-expressing BHK cells [25]. In the present study, we examined DENV infection-enhancing activity in serum samples with varying levels of DENV neutralizing activity using FccRexpressing BHK cells.

Serum samples
Eighty serum samples obtained from 80 residents in Perak, Malaysia were used in the study. Perak is located in north-western region of Peninsular Malaysia, and is endemic for dengue, and other flavivirus infections [26][27][28]. Incidence of DENV infection in Perak was 2,288 in 2010, 2,734 in 2009, and, 4,119 in 2008 [6]. The serum samples were collected in 2008 and were provided by National Public Health Laboratory, Malaysia. Characteristics of the patient population sampled are summarized in Table 1.

Ethics statement
The study protocol was approved by the ethics committee of the National Institute Infectious Diseases, Japan (Reference no. 210). Patients were de-identified and study data was analyzed anonymously.

Dengue diagnostics
Patient serum samples used in the present study were examined for the presence of dengue viral RNA by reverse-transcriptase polymerase chain reaction (RT-PCR), and NS1 antigen by enzymelinked immunosorbant assay (ELISA). Viral RNA was extracted using High Pure RNA extraction kit (Roche Diagnostics, Germany) and DENV serotypes were determined by serotype-specific reverse transcriptase polymerase chain reaction (RT-PCR) [29]. For serological tests, serum samples were heat-inactivated at 56uC for 30 minutes before use. Detection of the NS1 antigen was performed using Platelia Dengue NS1 Antigen (Bio-Rad Laboratories, France) according to manufacturers' instructions. After reaction was terminated, optical density readings (OD) were obtained with a spectrophotometer at wavelengths of 450 nm/620 nm and the index of each sample was calculated with the following formula: OD of samples/OD of calibrators. The index value of each sample was interpreted according to manufacturer's instructions; index values of ,0.9, 0.9-1.1, and, .1.1 were considered negative, equivocal, and positive, respectively [30].

Plaque reduction neutralization test (PRNT)
Heat-inactivated human serum samples were serially diluted 2folds from 1:5 to 1:1250 with EMEM/2% FCS. As the amount of serum samples was limited, two replicates were tested for each of the serum samples to four dengue serotype. Virus-antibody mixture was prepared by mixing 25 ml of DENV-1, DENV-2, DENV-3, or, DENV-4 at titers of 2000 PFU/ml with 25 ml of serum samples serially diluted 2 folds from 1:5 to 1:1250. For

Author Summary
Dengue has become a major international public health concern in recent decades. There are four dengue virus serotypes. Recovery from infection with one serotype confers life-long protection to the homologous serotype but only partial protection to subsequent infection with other serotypes. Secondary infection with a serotype different from that in primary infection increases the risk of development of severe complications. Antibodies may play two competing roles during infection: virus neutralization that leads to protection and recovery, or infectionenhancement that may cause severe complications. Progress in vaccine development has been hampered by limited understanding on protective immunity against dengue virus infection. We report the neutralization activity and infection-enhancement activity in individuals with dengue in Malaysia. We show that infectionenhancement activity is present when neutralizing activity is absent or low, and cross-reactive neutralizing activity may be hampered by infection-enhancing activity. Conventional assays for titration of neutralizing antibody do not consider infection-enhancement activity. We used an alternative assay that determines the sum of neutralizing and infection-enhancement activity in sera from dengue patients. In addition to providing insights into antibody responses during infection, the alternative assay provides a new platform for the study of immune responses to vaccine.
infection with DENV alone, the mixture was prepared by mixing 25 ml of DENV-1, DENV-2, DENV-3, or, DENV-4 strains at titers of 2000 PFU/ml with 25 ml of EMEM supplemented with 10% FCS. After incubation at 37uC for 60 minutes, 50 ml of virusantibody mixture was inoculated on FccR-negative BHK and FccR-expressing BHK monolayers in 12-well plates. The plates were then incubated for 60 minutes at 37uC in 5% CO 2 . After virus absorption, the cells were washed once with 1 ml of EMEM (Sigma) and overlaid with 1 ml EMEM (Nissui Pharmaceutical, Japan) containing 2% FCS (Sigma) and 1% methylcellulose (Wako Pure Chemical Industries, Japan). The plates were incubated at 37uC in 5% CO 2 for 5-7 days, when plaque formation could be confirmed by naked eye. Cells were then fixed with 10% formalin (Wako Pure Chemical Industries) and stained with methylene blue (Wako Pure Chemical Industries). Number of plaques was counted with naked eye. Plaque-reduction neutralizing test (PRNT 50 ) end points are expressed as the last serum dilution showing a 50% or greater reduction in plaque counts as compared to the number of plaques determined from wells of cells infected in the absence of antibodies [31].

Antibody-dependent enhancement (ADE) assay
For enhancement assay against 4 DENV serotypes, 25 ml serum samples diluted at 1:5 with EMEM/2% FCS were used. Virusantibody mixture was prepared by mixing 25 ml of DENV-1, DENV-2, DENV-3, or, DENV-4 strains at titers of 1000-2000 PFU/ml with 25 ml of 1:5 diluted serum samples and incubated at 37uC for 60 minutes. For negative controls, virus mixture was prepared by mixing 25 ml of DENV-1, DENV-2, DENV-3, or, DENV-4 strains at titers of 1000-2000 PFU/ml with 25 ml of 2% FCS/EMEM and incubated at 37uC for 60 minutes. Fifty microliters of the virus-antibody mixture was then applied to FccR-expressing BHK monolayers in 12-well plates. The plates were then incubated for 60 minutes at 37uC in 5% CO 2 . After virus absorption, the cells were washed with 1 ml of EMEM and overlaid with 1 ml EMEM containing 2% FCS and 1% methylcellulose. The plates were incubated at 37uC in 5% CO 2 for 5 days. Cells were then fixed with 10% formalin and stained with methylene blue. Plaques were counted with naked eye. Fold enhancement values were determined using the following ratio: (mean plaque count at 1:10 serum dilution)/ (mean plaque count in the absence of human serum samples, negative control). The sum of the mean of the negative control plus two times of the standard deviation (SD) value obtained from 4 wells of negative control was used as cut-off to differentiate enhancing and non-enhancing activity [32,33]. Enhancing activity was defined as positive when values are greater than the mean plaque count in the absence of human serum samples plus a greater than 2 times SD.

Neutralizing and infection-enhancing activities of serum samples collected in Perak, Malaysia
Eighty serum samples were tested for the presence of neutralizing antibody to each of the four DENV serotypes by using BHK cells (Table 1). Neutralizing antibody (PRNT 50 $10) was detected in 69% (55/80) to DENV-1, 60% (48/80) to DENV-2, 56% (45/80) to DENV-3 and, 20% (16/80) to DENV-4. Of the 80 samples, 12 samples and 13 samples were obtained from patients with acute DENV-1 and DENV-3 infections, respectively (Table 1). Neutralizing activity to DENV-1 was detected in 58% (7/12) of the DENV-1 infected patients and that to DENV-3 was detected in 62% (8/13) of the DENV-3 infected patients. In contrast, enhancing-activity to DENV-1 was detected in 33% (4/12) of the DENV-1 infected patients and that to DENV-3 was detected in 61% (8/13) of the DENV-3 patients ( Table 2). Five DENV-1 infected patients and two DENV-3 infected patients demonstrated neither neutralizing nor infection-enhancing activity to any of the four DENV serotypes (Table S4). Four (58%) of the 7 serum samples from acute secondary DENV-1 infected patients demonstrated infection-enhancing activities to DENV-1 serotype and similarly, 8 (73%) of 11 serum samples from acute secondary DENV-3 infected patients enhanced DENV-3 infection. The results indicate that serum samples from dengue patients and non-dengue patients in dengue endemic region, Malaysia, possess neutralizing and infection-enhancing activities (Table S1, Table S2). The history of past dengue infections of each of the patients was however, not known. Because Malaysia is endemic for dengue infection, and some of the serum samples exhibited high levels of neutralizing antibody titers to DENV, it is highly likely that the patients with high neutralizing antibody activity has been previously exposed to DENV infection. Difference in neutralizing antibody titers to 4 DENV serotypes determined by using FccR-negative BHK cells and FccR-expressing BHK cells The 18 samples derived from acute secondary dengue patients which demonstrated both neutralizing and infection-enhancing activities were examined for neutralizing antibody titers to each of the 4 DENV serotypes. The serum samples were from 7 acute secondary DENV-1 patients and 11 acute secondary DENV-3 patients (Table 1). Using FccR-negative BHK cells, 7 serum samples possessed neutralizing antibody titers only to DENV-2 and 11 samples showed neutralizing antibody to more than one serotype ( Table 3, Table S3). All the serum samples except one (17 of Table 4) exhibited heterotypic neutralizing activity using BHK cells but monotypic neutralizing activity using FccR-expressing cells, as those observed in patients with acute secondary dengue infection (Table 3).
The results indicate that neutralizing antibody titers determined by using FccR-expressing BHK cells were lower than those determined by using FccR-negative BHK cells, and that for some Table 3. Level of neutralizing activity in serum samples obtained from patients with secondary acute dengue infection and from selected non acute dengue patients against each of the 4 DENV serotypes.
The results indicate that sera from patients with DENV infection possess the ability to enhance the infection by the infecting serotype. The ability of serum samples with DENV neutralizing activity to enhance each of the 4 DENV serotypes at 1:10 serum dilution was analyzed (Figure 1 (Figure 1). Serum samples with high levels of neutralizing activity to DENV-2 (40,42,44,49, 54 and 58) exhibited peak fold enhancement ranging from 5.8-7.6 at higher serum dilutions of 1:100-1:1000 ( Figure 2).
Neutralizing titers were higher in samples from non acute dengue patients with neutralizing activity to multitypic DENV than in those from patients with acute primary and secondary DENV infection. In contrast, enhancing activity was significantly lower in non acute dengue patients with neutralizing activity to multitypic DENV than in those from patients with acute secondary DENV infection (Table S5). The results suggest that serum samples with high neutralizing activity possess no or only low levels of infection-enhancing activity to respective serotypes at low serum dilutions. In contrast, serum samples without neutralizing activity possess high levels of enhancing activity.

Discussion
The relationship between neutralizing activity determined using FccR-negative cells and infection-enhancing activity determined using FccR-expressing cells was examined in the present study.
We determined that a high proportion of the serum samples from residents of a DENV endemic country, Malaysia, possessed DENV neutralizing and infection-enhancing activity (75%, 60/ 80; Table 2). The neutralizing antibody titers were higher when determined by using FccR-negative cells than when determined by using FccR-expressing cells (Table 3). Mammalian cells such as Vero cells and BHK cells are commonly used in plaque reduction neutralizing tests [39]. However, in the absence of FccR, these cells exclusively detect neutralizing antibody titers. Other investigators have suggested that the use of FccR-expressing cells,  including monocyte-lineage THP-1 cells and K562 cells, dendritic cells, FccR-expressing CV-1 and BHK cells, and, DC-SIGN expressing RAJI cells and U937 cells may better reflect the in vivo neutralizing titers [40][41][42][43][44][45][46]. Surrogate plaque titration assays are, however, required to determine virus titers in non-adherent monocyte-lineage cells. Infection-enhancement was also detected using an FccR-expressing cell line, THP-1 cells, for a subset of serum samples (#39, 40, 28 and 77) which exhibited high infection-enhancement activity to DENV-4 using FccR-expressing BHK cells (data not shown). Thus, the results of ADE assays were consistent between THP-1 cells and FccR-expressing BHK. In addition, serum samples (#46, 40,42,43,44,45,52 and 54) that demonstrated neutralizing antibody titers to DENV-2 (1:10 to .1:1280) along with neutralizing activities to other serotypes in FccR-negative BHK cells exhibited neutralizing antibody only to DENV-2 in FccR-expressing BHK cells. Cross-reactive neutralizing activity determined using FccR-negative cells was not detected using FccR-positive cells. This could be due to hampered neutralizing activity to heterologous serotypes by infectionenhancing activity [25,42,47]. Importantly, our results suggest that the FccR-expressing BHK cells provides more informative data on serotype-specific neutralizing activity, and may better reflect protection in vivo.
Presence of infection-enhancing activity in antibodies with neutralizing activity to the infecting serotype has been reported previously [37,38,47]. Interestingly, despite the result that 11 serum samples from patients with DENV infection exhibited neutralizing activity to the infecting serotype at 1:10-1:80 as determined by using FccR-negative BHK cells, the neutralizing titer to the respective infecting serotypes as determined by using FccR-expressing BHK cells was ,1:10 in all of the 11 serum samples tested. It has been reported that the main target cells of DENV infection in vivo are FccR-expressing cells, such as monocytes and macrophages [34][35][36]. The results suggest that DENV-antibody complexes which are incapable of infecting FccR-negative cells, may retain the ability to infect FccR-expressing cells due to the presence of FccR. However, further studies are required to identify the relationship between infection-enhancing activity and neutralizing activity during infection in vivo.
All four DENV serotypes are found co-circulating in Malaysia. Although data was not available for the pre-dominant dengue virus serotype in Perak prior to 2008, DENV-2 was the dominant circulating serotype in peninsular Malaysia between 1998-2000 and 2006-2007 (Chua et al., unpublished data). High levels of neutralizing activity against DENV-2 were detected in some of the serum samples (Table 3), indicate that these individuals may have been previously exposed to DENV-2. Previous studies have suggested that ADE activity influences disease severity in patients with secondary DENV infection [32,48]. As low levels of serum dilutions may better reflect in vivo conditions, serum samples at dilutions of 1:10 were used in the present study. The results showed infection-enhancing activity (fold enhancement) of 0.7-6.7 to the infecting serotype at serum dilutions of 1:10. The assay using FccR-expressing BHK cells detected infection-enhancing activity to infecting serotypes at low serum dilutions in serum samples with neutralizing activity, suggesting the presence of ADE activity in vivo. Interestingly, there was an inverse relationship between infection-enhancement activity to a DENV serotype and high neutralizing activity as determined by using FccR-negative BHK cells to the respective serotypes ( Figure 2, Table 3, Table 4).
Previous studies showed that higher dilutions of patient serum samples, in quantities that are not sufficient to support neutralization, enhance DENV infection [32,47]. Patient serum samples #49, 58, 42, 45, and 54, require higher concentrations for neutralization in the presence of FccR than in the absence of FccR. In addition, serum samples #57, 23, 28, 30, 74, 77, and 78 exhibited similar neutralizing antibody titers in both FccRexpressing BHK cells and FccR-negative BHK cells [25]. Some antibodies may require lower threshold occupancy for neutralization, and, thus, virus neutralization may occur at similar concentrations both in FccR-negative and FccR-expressing cells. Alternatively, binding of some antibodies may lead to DENV conformational changes [49], and therefore, result in virus neutralization both in the presence and absence of FccR, at similar antibody concentrations. The FccR-expressing BHK cellbased assay system is unique as antibody neutralizing activity could be analyzed and compared simultaneously using one cell line (BHK cell line) either in the absence or presence of FccR by a conventional plaque assay.
It is known that primary infection with one DENV serotype induces long-term protection to infection with the same serotype [50]. By using serum samples from a DENV-endemic area, we demonstrated that infection-enhancing activity which was determined only by using FccR-expressing cells hampers neutralizing activity that was determined using FccR-negative BHK cells. Moreover, infection-enhancing activity was also detected in serum samples with low or negative neutralizing activity that were determined using FccR-negative BHK cells. Although in vitro systems may not faithfully reflect all aspects of DENV infection in vivo, the results suggest that as compared to the neutralizing activity determined by FccR-negative cell culture system, the sum of neutralizing and enhancing activity determined by the FccRexpressing cells may better reflect protection to DENV infection in vivo. Further studies are, however, needed to define the relationship between protection and neutralizing titers determined by using FccR-positive cells.

Supporting Information
Table S1 Levels of neutralizing and infection-enhancing activity in serum samples obtained from 42 non-acute dengue patients against each of the four dengue virus serotypes.