Sample collection
In Viet Nam, the first case of dengue hemorrhagic fever (DHF) was identified in 1963 in the Mekong Delta region of southern Viet Nam [15]. Between 1963 and 1995, approximately 1,518,808 DHF cases with 14,133 deaths had been reported in this country [16, 17]. Major epidemics of dengue had occurred in 1969, 1983, 1987, 1998 and 2009 in which the 1987 dengue outbreak with 354,517 cases and 1566 deaths represented the single largest reported dengue outbreak in the world [18]. In 2015, 54 out of 63 provinces reported a combined 97,476 case of dengue including 61 deaths [19]. While all four DENV serotypes co-circulate in Viet Nam, climate differences in Viet Nam result in differences in dengue epidemiology across the country. In Ha Noi, the capital city located in northern Viet Nam, dengue transmission intensity is quite low, usually peaking between October and November during the hot, wet season, with the majority cases being reported in adults.
In this study, 100 paired blood samples were collected from healthy volunteer adults who resided in Ha Noi, Viet Nam. The first sampling was performed before the dengue season in August 2015. This is the pre-exposure group or pre-dengue season group. The second sampling was done at the end of the dengue season in November 2015. This is the post-exposure group or post-dengue season group. During our sampling period, the four DENV serotypes were co-circulating in the area however, DENV-1 and DENV-2 were the dominant serotypes. Sera were separated as quickly as possible, transferred to sterile 2 ml cryo-tubes and stored in −70°C until used. In this study, (1) none of the participants reported fever or symptoms/signs suggesting dengue fever at the time of recruitment, (2) none had a fever within three months of recruitment nor (3) during the approximately three-month study period.
An additional 20 serum samples obtained from acute secondary adult DENV infection patients were also used in this study (the patient group) for further analysis and comparison. The serum samples were obtained from patients within 3 days of the onset of the disease. The patient samples were negative for anti-DENV IgM antibodies but positive for anti-DENV IgG antibodies. Only samples confirmed by virus isolation and real-time polymerase chain reaction (PCR) was used for the study. Thus, the day of the onset of fever was narrowed to less than 4 days to satisfy this criteria. Due to the absence of anti-DENV IgG antibodies in the acute phase, the acute primary DENV infection cases were not used as the patient group for the purpose of comparing the neutralizing activities between patient group and non-patient group (healthy volunteers).
Virus and cell lines
DENV-1 (99St12A strain), DENV-2 (00St22A strain), DENV-3 (SLMC50 strain), and DENV-4 (SLMC318 strain) were used for DENV IgM capture ELISA. These viruses were propagated onto C6/36 mosquito cell lines. Infected culture fluid (ICF) was collected on day 7 post-infection for the preparation of a tetravalent DENV antigen (25 ELISA units/serotype).
DENV-1, 01-44-1HuNIID strain (GenBank accession no. AB111007), DENV-2, 00St22A strain, DENV-3 5528 strain (GenBank accession no. KP893718) and DENV-4 SLMC318 strain were used for PRNT in this study as these strains form clear plaques. In PRNT experiments, virus stock was prepared by using baby hamster kidney cell lines (BHK- Japan Health Science Research Resource Bank, Japan) [6]. Infected culture fluid (ICF) collected on day 5 post-infection was used to inoculate BHK and FcγR-expressing BHK cell lines for virus titration and neutralization tests. BHK cells were cultured in Eagle’s Minimum Essential Medium (EMEM) (Sigma, USA), supplemented with heat-inactivated 10% fetal bovine serum (FBS, Sigma) at 37°C in 5% CO2. FcγR-expressing BHK cells were cultured in EMEM (Sigma), supplemented with heat inactivated 10% fetal bovine serum (FBS, Sigma) and 0.5 mg/ml Neomycin (G418, PAA Laboratories GmbH, Austria) at 37°C in 5% CO2.
Detection of DENV IgM
Following Bundo and Igarashi’s (1985) protocol [20], an in-house DENV IgM capture ELISA (in-house IgM ELISA) was performed to confirm the presence of anti-DENV IgM antibodies in serum samples. First, the polystyrene 96-well microplates (Maxisorp Nalge Nunc International, Roskilde, Denmark) were coated with 100μl (5.5μg/100μl) of 100-time diluted goat anti-human IgG antibodies specific for IgM (μ-chain specific) (Cappel ICN Pharmaceuticals, Aurora, OH) with ELISA coating buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6 containing 0.02% sodium azide) in all wells, except for the blanks at 4°C overnight. Then, to avoid non-specific binding, the wells, except for the blanks were blocked with 100 μl of undiluted Blockace Solution (UK-B 80, Yukijirushi, Sapporo, Japan), and incubated for 1 h at room temperature (RT). The microplates were washed three times with PBS containing 0.05% Tween 20 (PBS-T) (Gibco, NY, USA). Positive and negative controls and patient sera were diluted 1:100 in PBS-T and 100 μl of diluted specimen was added into duplicate wells. The plates were incubated at 37°C for 1 h and nonspecific reactants were removed by washing three times with PBS-T. A tetravalent DENV antigen (25 ELISA unit/serotype) was used as an assay antigen in this experiment. A total of 100 μl of DENV antigen was then added to each well and incubated at 37°C for 1 h. Unbound DENV antigen was then removed by washing the wells three times with PBS-T. Next, a total of 100 μl of horseradish peroxidase (HRPO)-conjugated anti-flavivirus (anti-DENV, anti JEV) mouse monoclonal antibody (12D11/7E8) [21,22,23] at a dilution of 1:2500 (diluent was PBS-T mixed with 10% Blockace) was added to the wells and incubated at 37°C for 1 h. If DENV antigen were retained in the wells by the anti-DENV antibodies in the serum samples, the HRPO conjugated anti-flavivirus mouse monoclonal antibody would bind to the DENV antigen in the wells. Excess conjugate was removed by washing three times with PBS-T. A total of 100 μl of substrate solution, 5 mg o-phenylenediamin dihydrochloride (OPD) (Sigma Chemical, St. Louis, MO) and 0.03% hydrogen peroxide in 10 ml of 0.05 M citrate-phosphate buffer, pH 5.0 was added to each well (substrate buffer was also added in blank wells) and incubated in the dark at RT for 1 h. The color was allowed to develop during this incubation time. To terminate the reaction, 100 μl of stop solution (1 N sulfuric acid) was added to each well (including the blanks) and the optical density at 492 nm (OD492) was measured using a Multiscan ELISA plate reader (Thermolabsystem, Tokyo, Japan). The positive control or the samples with OD492 two times or higher than the negative control were considered positive (OD492 of positive control or samples / OD492 of negative control ≥ 2).
Detection of anti-DENV IgG antibodies
In the IgG assay, an in-house flavivirus IgG indirect ELISA protocol, modified by Inoue et al. (2010) [24] was used to measure DENV IgG titer. In the modified protocol, instead of DENV antigen, a total of 100 μl of purified Japanese encephalitis virus (JEV) antigen (JEV ML-17 strain; 250 ng per well in ELISA coating buffer) was used to coat the 96-well microplates (Maxisorp Nunc, Denmark) with the exception of the blanks at 4°C overnight. To avoid non-specific binding, the wells (except for the blanks) were blocked with 100 μl of undiluted Blockace and incubated for 1 h at RT. The microplates were then washed three times with PBS-T. To each well, 100 μl of serum samples at a 1:1000 dilution in PBS-T + 10% Blockace was added and incubated for 1 h at 37°C. Standard serum containing antibodies against the test antigen and the serum from the non-disease population were added on each plate for positive and negative controls, respectively. The microplates were wash three times with PBS-T. Next, 100 μl of goat anti-human IgG antibodies conjugated to horseradish peroxidase (HRPO) (American Qualex, San Clemente, CA), at a 1:25,000 dilution in PBS-T + 10% Blockace were added to each well and the plates were incubated for 1 h at 37°C. The microplates were again washed three times with PBS-T and 100 μl of OPD substrate solution was added to each well (substrate buffer was also added in the blank wells). The microplates were incubated in the dark at RT for 30 mins and 100 μl of stop solution was finally added to each well (including the blanks) to terminate the reaction. A standard curve was generated by using the OD492 values of 2-fold diluted dengue positive control serum from a 1:1000 dilution to 1:214, in PBS-T + 10% Blockace. The IgG titers of the patient sera were determined by the positive standard curve. A sample titer equal to, or greater than, 1:3000 (cut-off value of positive IgG was at 1:1000 + 3 standard deviations) was considered IgG-positive for dengue virus [24]. To confirm that the IgG antibody detected was not confounded by the cross-reactivity to JEV, all the results obtained by the in-house flavivirus IgG indirect ELISA described above were investigated again by a commercial anti-DENV IgG kit (Dengue IgG indirect Elisa, Pan Bio, Inverness, Australia) according to the manufacturer’s instructions.
Infection assay
Serum samples that were seropositive with both in-house flavivirus IgG indirect ELISA and Dengue IgG indirect Elisa (Pan Bio, Inverness, Australia) were checked for the presence of neutralizing antibodies to specific DENV serotypes using plaque reduction neutralization test (PRNT). The samples were serially diluted 2-fold from 1:10 to 1:2560 in EMEM/2% FBS. The serum samples were heat-inactivated at 56°C for 30 min before use. As the amount of serum sample was limited, two replicates were tested for each of the serum samples to four DENV serotypes. A total of 25 μl of virus mixture containing 35-50 plaque-forming units (35-50 PFU/25μl or 1400-2000 PFU/ml) and 25 μl of the diluted serum samples were mixed to allow virus-antibody neutralization reaction. After 1 h incubation at 37°C, 50 μl of the mixture were inoculated onto BHK cell (FcγR-negative BHK) and FcγR-expressing BHK cell monolayers in 24-well plates and incubated for 60 min at 37°C in 5% CO2 then overlaid with 1 ml of EMEM (Nissui Pharmaceutical, Japan) containing 2% FBS and 1% methylcellulose (Wako Pure Chemical Industries, Japan). The plates were incubated at 37°C in 5% CO2 for 5-7 days until plaque formation was confirmed by the naked eye. The cells were then fixed with 4% paraformaldehyde phosphate buffer solution (Wako, Osaka, Japan) for 30 min at RT and then stained with 1.25% crystal violet (Wako, Osaka, Japan). The number of plaque was counted again by naked eye. PRNT50 end points were calculated using the reciprocal of the final serum dilution showing a 50% or greater reduction in plaque counts in wells compared to the number of plaque from the negative control wells with no antibodies. Positive PRNT samples were defined as having a neutralizing titer of 10 or above to any of the viruses tested (PRNT50 ≥ 10 or Log2 (PRNT50) ≥3.32) [25]. PRNT assay using JEV was also performed in order to confirm the seropositive samples were truly DENV infection. Neutralization antibodies (N.A) against JEV from the sero-positive samples were much lower compared to N.A against DENV.
In the infection-enhancement assay, serum samples were diluted with EMEM/2% FBS. A total of 25 μl of DENV containing 35-50 plaque-forming units (35-50 PFU/25μl or 1400-2000 PFU/ml) were mixed with 25 μl of 1:20 diluted serum samples (a 1:20 dilution was used instead of a 1:10 dilution due to insufficient amount serum samples from the second sampling). The presence of ADE activity was determined by using plaque assay on FcγR-expressing BHK, which was similar to the method for the neutralizing assay. Positive infection-enhancement (measured as fold-enhancement) was defined as fold-enhancement values greater than the cut-off value plus 2 times standard deviation (cut-off value = mean plaque count at a 1:20 serum dilution on FcγR-expressing BHK)/(mean plaque count in the absence of human samples on FcγR-expressing BHK cells).
Data analysis
In this study, all data from PRNT50 result was transformed to base-2 logarithm for analysis. In descriptive analyses, numbers and percentages were used for categorical variables. Mean and standard deviation (SD); median and interquartile range (IQR) were used for continuous variables. Odds ratio (OR) and the 95% confidence interval (95% C.I) were estimated to indicate the probability of DENV infection for each group. For comparison in specific groups, chi-squared test, Fisher’s exact test, Wilcoxon rank-sum (Mann-Whitney) test and t-test was used appropriately. Statistical tests were performed with Stata 14.1 (StataCorp LP, College Station, Texas 77,845 USA) and GraphPad Prism version 7.0a (GraphPad software, La Jolla California USA) with 5% level of significance and two-tailed p values.