Multiplexed FluoroSpot for the Analysis of Dengue and Zika Virus Specific and Cross-Reactive Memory B-cells

Abstract

Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne pathogens that have a significant impact on human health. Immune sera, monoclonal antibodies and memory B-cells (MBCs) isolated from patients infected with one DENV type can be cross-reactive with the other three DENV serotypes and even more distantly related flaviviruses such as ZIKV. Conventional ELISpots effectively measure antibody secreting B-cells (ASCs) but since they are limited to the assessment of a single antigen at a time, it is challenging to distinguish serotype-specific and cross-reactive MBCs in the same well. We developed a novel multifunction FluoroSpot assay using fluorescently labeled DENV and ZIKV (FLVs) that measures the cross-reactivity of antibodies secreted by single B-cells. Conjugation efficiency and recognition of FLVs by virus-specific antibodies were confirmed by flow cytometry. Using a panel of DENV immune, ZIKV immune and naïve PBMC, FLVs were able to simultaneously detect DENV serotype-specific, ZIKV-specific, DENV serotype-cross-reactive and DENV/ZIKV cross-reactive antibodies secreted by individual MBCs. Our findings indicate that the FLVs are sensitive and specific tools to detect specific and cross-reactive MBCs. These reagents will allow the assessment of the breadth as well as the durability of DENV/ZIKV B-cell responses following vaccination or natural infection. This novel approach using FLVs in a FluoroSpot assay can be applied to other diseases such as Influenza where prior immunity with homo or hetero subtype-specific MBCs may influence subsequent infections.

Introduction

Dengue viruses (DENV) and Zika virus (ZIKV) are members of the genus Flavivirus and belong to the family Flaviviridae, which also includes other medically important viruses such as yellow fever virus, West Nile virus, Japanese encephalitis virus and tick-borne encephalitis virus (1). DENV and ZIKV are transmitted to humans by the bite of mosquitos, predominantly Aedes aegypti (2, 3). Due in part to the global spread of the vector, the four types of DENV circulate in much of the world and cause episodic outbreaks. The recent outbreak of ZIKV in Brazil in 2015 was associated with neurological symptoms, microcephaly in infants born to pregnant women and novel patterns of transmission via sexual intercourse (4).

B-cells, are massively expanded during primary and secondary DENV infections and several studies have evaluated the specificity of the antibodies secreted by plasmablasts (5–9). Long-lived plasma cells and memory B-cells reside in the bone marrow and in the peripheral blood, respectively, and contribute to Abs detected in the sera years after infection (10, 11). One challenge to studying these cell populations is the lack of commercially available reagents to identify antigen-specific B-cells. We recently used amine labeling of DENV to identify DENV-specific B-cells in the circulation (12, 13). We identified multiple subsets of antigen-specific B-cells in the circulation during acute primary and secondary DENV infection, including naïve B-cells, MBCs and plasmablasts with a much lower frequency of DENV-specific MBCs six months later (13). Exposure to ZIKV or administration of a ZIKV vaccine will likely occur in children and adults on a background of pre-existing immunity to DENV (14, 15). Therefore, understanding the magnitude and quality of cross-reactive responses at the B-cell and antibody (Ab) levels between ZIKV and DENV is important (16).

The envelope (E), pre-membrane (prM) and the non-structural protein 1 (NS1) proteins are the main targets for the humoral immune response after DENV and ZIKV infection (17). However, there are several challenges in measuring antibody responses (14, 18). The four DENV serotypes differ from each other by 30–35%, and DENV differs from ZIKV by 41–46%, in amino acid sequence of the E protein (19). Cross-reactive antibodies specific for E with poor, moderate or potent neutralizing activity and against prM and NS1 have been characterized in individuals following natural infection (20–24). A number of human mAbs from DENV immune donors only bind quarternary epitopes presented on mature viruses and these Abs cannot be detected using recombinant (rE) protein (25). Multiple studies have not been able to reliably distinguish DENV from ZIKV infection serologically, given the high degree of antigenic similarity between these two viruses (26, 27) but newer assays using recombinant virus particles effectively measure neutralizing antibody responses to ZIKV (28). Cross-reactive neutralizing antibodies between DENV and ZIKV are not sustained long term in DENV and ZIKV immune individuals (29, 30). Several in vitro studies have recently reported antibody-dependent enhancement (ADE) activity of antibodies using sera from DENV or ZIKV immune donors (23, 31), while two macaque studies showed no significant impact of DENV antibodies on viral load or severity of subsequent ZIKV induced disease (32, 33).

The B-cell ELISpot assay to enumerate the number of antibody secreting cells (ASCs) was first described in 1983 (34). The ELISpot assay has been modified to detect antigen specific plasmablasts and memory B-cells expanded in vitro into ASCs (6, 35–37). While the B-cell ELISpot assay is a powerful and highly sensitive method to analyze antibodies from ASCs, it is limited to detection of a single antigen in a well. To visualize cross-reactive ASCs, a Quad Color FluoroSpot assay was recently developed in which standardized concentrations of DENV antigens were added to antibodies secreted by MBCs captured on plates by Ig-specific antibodies (38). Spots representing individual B-cells were identified using DENV serotype-specific fluorescently labeled Abs.

Here we present a novel approach to detecting cross-reactive ASCs, wherein we directly labeled viruses representing all four serotypes of DENV as well as ZIKV. We hypothesized that fluorescently labeled DENV and ZIKV would be useful to measure DENV-specific, ZIKV-specific, DENV serotype cross-reactive and DENV-ZIKV cross-reactive memory B-cell responses. We used well-characterized PBMC from DENV immune, ZIKV immune and naïve donors to simultaneously detect ASCs producing antibodies that recognize one, two, three or four serotypes of DENV and/or ZIKV.

Materials and Methods

Samples

A panel of 19 DENV-immune, ZIKV immune and naïve subjects was studied (Table 1). All DENV and ZIKV peripheral blood mononuclear cells (PBMC) were collected after written informed consent. The studies were approved by the institutional review boards at the University of Rhode Island, National Institute of Allergy and Infectious Diseases (NIAID) and the Human Subjects Research Review Board for the Commanding General of the US Army Medical Research and Material Command. Blood samples of travelers were collected at the National Institutes of Health Clinical Center from patients displaying symptoms of a suspected ZIKV infection following return to the United States from areas where ZIKV was known to be circulating. PBMC were purified and cryopreserved until used.

Table 1.

Description of donors used in the study

Donor No.	Immune status	Country of origin	Exposure
1	DENV-Immune	USA	Vaccine
2	DENV-Immune	USA	Vaccine
3	DENV-Immune*	USA	Natural infection
4	DENV-Immune*	USA	Vaccine
5	DENV-Immune*	USA	Vaccine
6	DENV-Immune*	USA	Vaccine
7	DENV-Immune*	USA	Vaccine
8	DENV-Immune*	Philippines	Natural infection
9	ZIKV-immune#	USA	Natural infection
10	ZIKV-immune#	USA	Natural infection
11	ZIKV-immune#	USA	Natural infection
12	ZIKV-immune#	USA	Natural infection
13	DENV-naïve**	USA	-
14	DENV-naive**	USA	-
15	DENV-naive**	USA	-
16	DENV-naive**	USA	-
17	DENV-naive**	USA	-
18	DENV-naive**	USA	-
19	DENV-naive**	USA	-

^*DENV IgG ELISA: DENV 1–4 VLPs ELISA.

^#Infection diagnosed by symptoms, confirmed by PCR and RVP ZIKV neutralization assay (28).

^**Based on history provided by the donor.

Preparation of purified, concentrated flaviviruses

DENV −1 strain WP-74, DENV-2 strain S16803, DENV-3 strain CH53489 and DENV-4 strain TVP-360 were propagated in Vero cells, concentrated by tangential flow ultrafiltration, and purified by sucrose gradient ultracentrifugation as described previously (39). The concentrations of purified DENV were determined by A260/280 measurement on a NanoDrop. Protein concentrations of DENV-1, DENV-2, DENV-3 and DENV-4 were 0.44 mg/ml, 0.49 mg/ml, 0.89 mg/ml and 0.84 mg/ml respectively. The ZIKV Paraiba_01 strain was propagated in C6/36 cells. The infected cell culture supernatant was harvested on day 5 post infection. The supernatant was layered on top of a 30% sucrose solution containing 10 mM Tris, 100 mM NaCl, and 1 mM EDTA. The virus was pelleted by ultracentrifugation in a swinging-bucket rotor at 26,000 rpm for 4 hr at 4°C to remove low-molecular-weight contaminants such as soluble proteins. The virus pellet was resuspended in phosphate-buffered saline. The purity of the viral preparations was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein concentration of ZIKV was 0.8 mg/ml.

Labeling of DENV preparations

We labeled purified DENV and ZIKV using small scale 3×100–200 μg Lightening-Link Rapid antibody protein labeling kits DL405, DL488, DL594 or DL650 (Innova Biosciences, Cambridge, UK). Briefly, 10 μl of lightening link rapid modifier was added to 100 μl of virus preparation with gentle pipetting and the mixture was added to the pellet of lyophilized dye. After gentle aspiration, the reaction was allowed to proceed 15–30 minutes at room temperature protected from light. Lightening link rapid quencher (10 μl) was added to terminate the reaction. After 15 minutes, RPMI 1640 (no phenol red) supplemented with 5% fetal bovine serum (FBS) was added in a volume to normalize initial protein concentrations of the viral preparations. Aliquots of 50 μl labeled virus were stored at −80°C until further use.

Validation of reagents using antibody coated beads and U937 DC-SIGN cells

Labeling efficiency as well as retention of binding capacity of FL-DENV and ZIKV was verified on all preparations. Goat anti mouse IgG (Fc) beads (100 ul aliquots of 3.0 micron beads; Spherotech, Inc MPFc-30–5, Lake Forest, IL) were incubated with the optimal concentrations of mouse anti-dengue 2H2 Ab (Thomas Scientific, Swedesboro, NJ) or 3H5 Ab (Millipore Sigma, Burlington, MA), anti-flavi 4G2 Abs (Novus Biologicals, Littleton, CO) or mouse anti human CD28 Abs (BD Biosciences, San Jose, CA) and then washed and resuspended in 1 ml of PBS containing 10% FBS. Test aliquots of FL-DENV or ZIKV were incubated with 15 μl aliquots of functionalized beads for 30 minutes on a rotating platform at room temperature, washed two times and read on the flow cytometer. A pattern of reactivity was revealed to show both specificity of binding as well as relative fluorescence intensity of the labeled virion preparations. For binding assays, FL DENV-1, 2, 3 or 4 were added to 1×10⁵ U937 DC-SIGN cells. Following incubation at 37°C for approximately 45 minutes, cells were washed and fixed in 100ul of 1:4 BD Cytofix Fixation Buffer (diluted in PBS) by BD Biosciences. Binding of FL-DENV was assessed on a BD LSR flow cytometer.

Stimulation of PBMC

Cryopreserved PBMC were thawed and washed twice with RPMI 1640 media with 10% FBS (Sigma-Aldrich, St. Louis, MO) and 100 U/mL penicillin/streptomycin (HyClone TM, Australia). Cells were counted and diluted to 2×10⁶/mL in RPMI / 10% FBS. PBMC were stimulated with 2.5 μg/mL R848 (InvivoGen, San Diego, CA) and 1000 U/mL recombinant human (rh) IL-2 (Peprotech, Rocky Hill, NJ) for 7 days at 37°C and 5% CO₂. Supernatants from MBC cultures were collected and assessed for antibody secretion by ELISA. Cells were collected and tested in ELISpot and FluoroSpot assays.

DENV and ZIKV Elisa

PBMC culture supernatants were tested for DENV and ZIKV IgG antibodies in ELISA assays. Maxisorp ELISA plates (Thermo Fisher Scientific Inc, USA) were coated with 10 ng/well DENV or ZIKV VLPs (The Native Antigen Company, Oxford, UK) and incubated overnight at 4 °C. The plates were blocked with 1% bovine serum albumin (BSA) for 90 minutes, and 50 μl supernatant was added per well for 1 hour at 37 °C. Plates were washed with phosphate-buffered saline (PBS), containing 0.05% Tween-20. Goat anti-human IgG-Fc fragment coupled to horseradish peroxidase (HRP; A80–104P; Bethyl Laboratories Inc., Montgomery, TX, USA) was added as the secondary antibody at a 1:5000 dilution. Then, 100 μl TMB (3, 3, 5, 5′- tetramethylbenzidine) peroxidase substrate (Thermo Scientific, Rockford, IL, UK) was added. After approximately 15 minutes, the enzyme reaction was stopped by addition of 50 μl sulfuric acid, and the plates were read at 450 nm. The supernatants from each condition were tested in duplicate. The average of duplicates was calculated after background values were subtracted.

Modified ELISpot

ELISpot assays were performed as previously described (6). Briefly, wells of Millipore ELISpot plates, MAIPSWU10 (Millipore, Billerica, MA) were treated with 20 μl per well 35% ethanol, then coated with 100 μl DENV or DENV VLP (7.5 μg/mL). To detect total IgG, the wells were coated with 80 μl anti-human Ig capture antibody (CTL, Shaker Heights, OH). The plates were stored at 4°C overnight. PBMC cultures were collected and counted, and 2×10⁵ or 5×10⁴ cells in 100 μl was added to each well in duplicate to assess DENV-specific or total IgG ASCs, respectively. The plates were incubated overnight at 37°C and 5% CO₂. After two washes with PBS and 0.05% Tween PBS, 80 μl biotinylated mAb directed against human IgG (CTL) was added to each well. The plates were incubated for 2 h at room temperature, washed three times with 0.05% Tween PBS, and developed with alkaline phosphatase-conjugated streptavidin for 1 hour. The plates were washed two times with PBS-Tween followed by two times with deionized water, and then 80 μl of the substrate solution was added for 15 minutes before rinsing three times with tap water. The background reactivity (spot counts in negative control wells) observed within the ELISpot assay was 1–2 spots per well.

FluoroSpot Assay

For FluoroSpot assays, IPFL plates (PUV96; CTL) were treated with 20 μl per well 70% ethanol for 1 minute. Ig capture antibody (Human IgG Single color FluoroSpot assay, CTL) were added to wells and plates were incubated overnight at 4°C. Following washing and blocking of the plates, MBC cultures (2×10⁵) were added to duplicate wells for each condition then incubated overnight at 37°C and 5% CO₂. The plates were washed and blocked with 1% BSA for 1 hour at 37°C. Optimized concentrations of FL DENV and ZIKV diluted in 100 μl of PBS was then added. The plates were incubated for 1 hour at room temperature. Plates were washed three times with PBS followed by two times with deionized water and rinsed with tap water. The plates were allowed to dry completely. To determine total IgG-secreting cells, 5×10⁴ MBC cultures were added to wells coated with human Ig and visualized using a polyclonal PE labeled IgG detection Ab (CTL).

Data Acquisition

The plates were scanned using an S5 (for dual color) or S6 (for quad color) CTL ImmunoSpot analyzer. Spots representing antibodies secreted by antigen-specific MBCs were detected using different filters (440/40 for DL405 DENV-2, 520/40 for DL488 DENV-4 or ZIKV, 630/60 for DL594 DENV-3 and 690/50 for DL650 DENV-1). The settings for the sensitivity of spot counting were established and adjusted manually for each plate using antigen stimulated and negative controls and to exclude artefacts or background spots. The number of specific or cross-reactive spots were evaluated by the reader and verified manually. The numbers of antigen-specific ASCs were adjusted to ASC/1×10⁶ input cells for statistical analysis. FCS data files of wells were collected on a BD LSR II with BD FACSDiva 8.0.1 software to display and analyze specific and cross-reactive spots. Data analysis and transformation was performed using FlowJo v10 (Treestar, Ashland, OR, USA).

Statistical Analysis

Statistical analysis was performed using GraphPad Prism v 6.00 (Graph Pad Software, La Jolla, California, USA). FluoroSpot results were analyzed with Mann-Whitney test for paired comparisons. ELISA binding results were analyzed using one-way ANOVA and T-test. A p-value of ≤0.05 was considered significant

Results

Generation of FL-DENV

The sucrose gradient purified DENV for each of the four serotypes were titrated in a virus capture ELISA and virus particles bound to capture antibody were detected using the flavivirus cross-reactive antibody 4G2 (Figure 1A). The end-point titers for DENV-1, −2, −3, and −4 were 3,200, 12,800, 12,800, and 51,200, respectively. We conjugated DENV-1, 2, 3 and 4 to amine reactive, water soluble DyLight (DL) dyes with different excitation wavelengths-DL650, DL405, DL594 and DL488, respectively. The specific dyes with absorption spectra ranging from 400–670 nm were selected to match the principal output wavelengths for flow cytometry and FluoroSpot instrumentation. FLVs were initially evaluated for binding to beads coated with flavivirus-specific Abs 2H2, 4G2 and 3H5 or control anti-CD28 Abs. In all cases, there was a clear separation in binding of FLVs between control and flavivirus-cross-reactive 2H2 and 4G2 Ab coated beads, although the intensity of fluorescence varied based on the dye used (Figure 1B). As expected, there was no shift in fluorescence when labeled DENV-1, 3 or 4 preparations were added to DENV-2-specific antibody (3H5) coated beads. To determine whether the labeling process inhibited the ability of DENV to bind susceptible cells, we evaluated the binding of FLVs to U937 DC-SIGN cells. After incubation of DL650 DENV-1, DL405 DENV-2, DL594 DENV-3 or DL488 DENV-4 with U937 DC-SIGN cells at 4°C for 45 minutes, we found significant binding of all FLVs to U937 DC-SIGN cells (Figure 1C). Finally, to determine whether there was competition for binding of FLVs to antibody coated beads, DL594 DENV-3 and DL488 DENV-4 were added alone or together to 4G2 coated beads and fluorescence evaluated. We found a shift in fluorescence in the PE and FITC channels, respectively, when 4G2 beads bound with DENV-3 or −4 were added alone (Figure 1D). When DENV-3 and −4 were added together, staining was detected along a diagonal and not skewed towards a particular fluorescence channel, indicating that both FLVs were able to bind 4G2 coated beads equally. Taken together, our data indicate productive labeling of FLVs with no apparent effect on the structure or binding capabilities of the viruses.

FIGURE 1.

Validation of Labeled DENV preps. (1A) Different concentrations of DENV-1, DENV-2, DENV-3 or DENV-4 were used in a virus-capture ELISA assay. (1B) Binding of DL650 DENV-1, DL405 DENV-2, DL594 DENV-3 and DL488 DENV-4 to anti-CD28 (control), 2H2 (DENV cross-reactive), 4G2 (flavivirus cross-reactive) and 3H5 (DENV-2 specific) coated beads. Every FL-DENV preparation was evaluated for binding to flavivirus and control antibody coated beads. (1C) U937 DC-SIGN cells were incubated with 2–5 μl DL650 DENV-1, DL405 DENV-2, DL594 DENV-3 or DL488 DENV-4 for 45 minutes and binding was evaluated. (1D) DL594 DENV-3 or DL488 DENV-4 were added alone or together to assess binding to CD28 (control) or 4G2 coated beads.

FL-DENV to detect DENV specific MBCs

A schematic comparing the conventional ELISpot and FluoroSpot assays using specific antibodies or FLVs is shown in Figure 2. Since circulating memory B-cells do not actively secrete Abs, PBMC from DENV immune and naïve subjects were stimulated with the TLR7/8 agonist, r848, and recombinant (r)IL2 in vitro for seven days to convert MBCs into ASCs (40). Stimulated cells were plated onto IPLF plates coated with human immunoglobulin (Ig) capture antibodies and incubated at 37°C overnight. To determine whether FLVs could be used to identify Abs secreted by DENV-specific MBC cultures, we added optimal concentrations of DL594 DENV-3 (red) or DL488 DENV-4 (green), or unlabeled DENV into separate wells. The assay was optimized using ethanol pre-treatment, blocking and washing steps, and varying concentrations of individual FLVs to give a distinct signal when ASCs from immune donors were used and minimal staining when ASCs from naïve donors were used (Figure S1). Representative images of negative control wells containing MBC cultures from a naïve subject with labeled DENV, an immune subject with unlabeled DENV, and unstimulated PBMC from an immune donor with labeled DENV are shown. Red or green spots were detected in wells containing MBC cultures from DENV immune donors with DL594 DENV-3 or DL488 DENV-4, respectively. No spots were detected when the wavelength-mismatched filter was used, indicating lack of signal spillover between detection channels (Figure S1).

FIGURE 2:

Comparison of the ELISpot and FluoroSpot assays: (2A) In the conventional ELISpot the membrane is coated with DENV antigens. Antibodies secreted by MBC cultures bind the antigen. An anti-human biotinylated detection Ab is added followed by streptavidin conjugated with an enzyme, and finally an enzyme substrate is added which produces colored spots. (2B) In the antibody labeled FluoroSpot assay, the membrane is coated with capture Ig antibody. Abs secreted by MBC cultures bind the capture antibody. Purified DENV is added and followed by DENV-specific fluorescently labeled Ab. (2C) In the virus labeled FluoroSpot, the membrane is coated with capture Ab. Abs secreted by MBC cultures bind the capture antibody. After washing and blocking steps, FL-DENV are added.

We evaluated the specificity of the assay by analyzing PBMC from six DENV immune and seven DENV naïve subjects (Figure 3). Representative images of wells containing MBC cultures from a naïve and immune donor with FL DENV-1, 2, 3, 4 and total IgG are shown in Figure 3A. The frequency of DENV-1, 2, 3 or 4 binding ASCs was higher in DENV immune donors, compared to naïve donors, which was statistically significant (Figure 3B, D, F, H). We found 3–12 spots per well when using MBC cultures from naïve donors in the FluoroSpot. Cell culture supernatants collected 7 days post stimulation from the same immune PBMC also had detectable DENV-specific Abs (Figure 3C, E, G, I). We found no Abs that recognized DENV-1, 2, 3 or 4 in supernatants from unstimulated PBMC or MBC cultures from naïve donors.

FIGURE 3.

Analysis of DENV-specific B-cells using labeled DENV-3 or DENV-4. (3A) PBMC from DENV-immune and naïve subjects were stimulated in vitro for seven days with r848 + rIL-2 and seeded onto FluoroSpot plates coated with Ig capture antibody. Images of total IgG secreting, DENV-1 specific, DENV-2 specific, DENV-3 specific, DENV-4 specific MBCs and control wells are shown. Frequencies of (3B) DENV-1 specific, (3D) DENV-2 specific (3F) DENV-3 specific and (3H) DENV-4 specific ASCs per 10⁶ input cells in MBC cultures from six DENV immune and seven DENV naïve donors. We found a statistically significant difference (**P value < 0.01) in the frequencies of DENV-specific ASCs between DENV naive (n=3–6) and immune donors (n=5–6). (3C) DENV-1, (3E) DENV-2, (3G) DENV-3 and (3I) DENV-4 IgG detected by ELISA using supernatants from MBC cultures from the same immune and naïve donors. Values are the mean of duplicate wells for each condition and the average O.D. values in MBC cultures (n=5–6 donors). (****P value < 0.0001) were obtained using a one-way ANOVA test.

Detection of DENV-specific and cross-reactive B-cells in a multiplexed FluoroSpot assay

Optimized concentrations of DL594 DENV-3 and DL488 DENV-4 were added together in the same well in order to distinguish DENV-specific and cross-reactive ASCs. Images were initially analyzed using a single filter and then a computerized overlay image was generated to identify overlapping spots. In the overlay image, the red spots represent ASCs that bound DL594 DENV-3, green spots represent ASCs that bound DL488 DENV-4, and yellow spots represent cross-reactive ASCs that bound DENV-3 and 4, all of which were seen in the ASC cultures from the immune but not naïve donor (Figure 4A). We evaluated ASCs using MBC cultures from an immune donor who received a monovalent DENV-4 vaccine (Figure 4B). Since the donor was exposed only to DENV-4, we expected to see DENV-4 specific spots (green) and DENV-3/4 cross-reactive spots (yellow) and no DENV-3 specific spots (red). In the overlay image, both green and yellow spots were detected with minimal red spots. Furthermore, we found a wide range in the size and color intensity of the green and yellow spots (Figure 4B, expanded image) which we speculate indicate differences in relative avidities for the different viruses. We compared the results from the FluoroSpot to a conventional ELISpot assay and found similar frequencies of DENV-3 and DENV-4 specific ASCs (Figure 4C). However, since the conventional ELISpot assay cannot differentiate between specific and cross-reactive Abs secreted by individual ASCs, we could not compare the frequency of cross-reactive cells between the two assay formats. Supernatants collected from immune PBMC cultures also contained Abs that bound DENV-3 and DENV-4 (Figure 4D).

FIGURE 4.

Evaluation of DENV serotype specificity. (4A) Analysis of DENV-specific ASCs using labeled DENV-3 and DENV-4. Level one analysis was performed to identify ASCs that bound DENV-3 or DENV-4. In level 2 analysis, images were superimposed and used to generate a computerized overlay image based on exact location parameters of each spot center, identifying ASCs that bound DENV-3 (red spots), DENV-4 (green spots), or both DENV-3 and −4 (yellow spots). (4B) Detailed analysis of DENV-3/DENV-4 ASCs using PBMC from a well-characterized immune donor who received a monovalent DENV-4 vaccine. (4C) Cells from the same donor as used in 4B was tested in a conventional ELISpot assay using DENV-3 or DENV-4 to coat the wells. (4D) Supernatants from the MBC cultures were tested by IgG ELISA and Abs that bound DENV-3 and −4 VLPs were identified. Values are the mean of duplicate wells for each condition from three experiments performed.

Analysis of the level of cross-reactivity using the FluoroSpot assay

To demonstrate that the FluoroSpot assay could detect cross-reactive ASCs, we used well-defined PBMC from subjects who had natural DENV infections or received candidate DENV vaccines (Table 1). ASCs that bound a single (red or green spots) or two DENV serotypes (yellow spots) were detected in PBMC from all subjects (Figure 5A), however the frequencies of specific and cross-reactive B-cells varied amongst the donors (Figure 5B). The ratios of DENV-specific and cross-reactive ASCs to total IgG secreting cells in immune subjects ranged from 0.2–5% (Table 2). FCS files were generated using the ImmunoSpot software, which enabled bivariate analysis of spots using FlowJo software and showed clear separation of red, green and yellow spots (Figure 5C). In-depth analysis in FlowJo, identified cells that recognized DENV-3 and −4 equally, cells that recognize DENV-3 better than DENV-4 and cells that recognized DENV-4 better than DENV-3 (Figure 5D). The FluoroSpot results analyzed using FlowJo reflected the patterns we detected in the FluoroSpot overlay images, which identified red, green and yellow spots of different intensities and sizes, including red spots with varying sizes of yellow centers and green spots with varying sizes of yellow centers. Taken together, the dual-color FluoroSpot retained the sensitivity of the single-color assay and in addition was able to identify ASCs that bound two different DENV serotypes.

FIGURE 5.

Analysis of the specificity and cross-reactivity of DENV-immune ASCs. (5A) Overlay images of wells of MBC cultures from five DENV immune donors. (5B) Frequencies and spot counts (per 10⁶ input cells) of DENV-3 specific, DENV-4 specific and DENV-3/4 cross-reactive ASCs. (5C) FCS files generated using the ImmunoSpot software were subjected to bivariate analysis using FlowJo analysis software. Y axis is red fluorescence (DENV-3) and X axis is green fluorescence (DENV-4), spots in upper right quadrant (circular gate) are DENV-3/4 cross-reactive spots (5D) Flow plots with axes shown in linear scale to highlight the varying relative avidities of the cross-reactive spots. Values are the mean of duplicate wells for each condition. Data shown are representative data from one of two experiments performed.

Table 2:

Summary of DENV-specific and cross-reactive MBCs

Donor No.	Total IgG / 106 input cells	Number (Frequency) relative to total IgG
		DENV-3 spots (%)	DENV-4 spots (%)	DENV-3/4 spots (%)
2	14060	355.0 (2.52)	725.0 (5.15)	310.0 (2.20)
3	16620	575.0 (3.45)	215.0 (1.29)	295.0 (1.77)
4	16380	217.5 (1.32)	525.0 (3.20)	47.5 (0.28)
6	18660	860.0 (4.60)	192.5 (1.03)	87.5 (0.46)
7	18540	702.5 (3.78)	310.0 (1.67)	240.0 (1.29)

Multiplexed FluoroSpot assay using four different FL-DENV

We next used four different FL-DENV together to assess the cross-reactivity of B-cells in MBC cultures. Shown in Figure 6 are images of 1 well which contained MBC cultures from a DENV immune donor who reported receiving a tetravalent DENV vaccine. Using a single filter (Figure 6A), dual filters (Figure 6B) and four filters (Figure 6C), we found serotype-specific MBCs (gold, blue, red or green spots representing DENV-1, 2,3 or 4 specific MBCs), MBCs that recognized two DENV serotypes (purple, pink), three serotypes as well as all four serotypes (white spots). The size and intensity of the DENV-specific and cross-reactive spots varied in MBC cultures from this donor.

FIGURE 6.

FluoroSpot for analysis of DENV-1–4 specific and cross-reactive spots. (6A) Representative images of a single well (one of two wells tested) containing MBC cultures from a DENV immune subject labeled with FL-DENV 1–4. Single filters were used to identify DENV-1, 2, 3 and 4 specific spots. (6B) Images of wells using dual color filters. (6C) An enlarged image of the well shown in 6A displays the computerized overlay of all four colors, enabling identification of specific and cross-reactive ASCs. Representative DENV-specific and cross-reactive ASCs are indicated with different symbols - green spots (DENV-4), blue spots (DENV-2), gold spots (DENV-1), purple spots (DENV-2/DENV-3) and white spots (DENV-1–4).

Analysis of DENV/ZIKV cross-reactive MBCs

To detect DENV/ZIKA cross-reactive MBCs we analyzed PBMC from DENV and ZIKV immune subjects using a pan-DENV/ZIKV dual color FluoroSpot. We labeled a pan-DENV preparation using equal amounts (measured by protein concentration) of the four DENV serotypes with DL594 (red) and a preparation of purified ZIKV with DL488 (green). PBMC from ZIKV immune donors were obtained approximately two months post natural ZIKV infection and PBMC from DENV immune donors were obtained months to years after infection or vaccination (Table 1). Using DL594 pan-DENV and DL488 ZIKV, we found many more green spots compared to red or yellow spots in the wells, from 4 of 4 ZIKV immune donors indicating low cross-reactivity between DENV and ZIKV ASCs in MBC cultures (Figure 7A). While, the frequency of cross-reactive MBCs was low, they were higher in PBMC from donors 9 and 10 compared to PBMC from donors 11 and 12 (Figure 7B, Table 3). Interestingly, donors 9 and 10 reported receiving a yellow fever vaccination. We next evaluated MBC cultures from DENV immune donors. MBC cultures from one of four DENV immune donors secreted Abs that bound DENV and ZIKV strongly which resulted in many more yellow spots in the well (Figure 7C left-most image). This donor received a monovalent DENV-4 vaccine and had traveled to an area where yellow fever was endemic (41). Supernatants from MBC cultures contained Abs that bound DENV and ZIKV VLPs (Figure 7E and F). PBMC from DENV immune donors had a higher frequency of DENV-specific ASCs compared to DENV/ZIKV cross-reactive ASCs (Figure 7D; Table 2).

FIGURE 7.

ZIKA and Pan-DENV dual color FluoroSpot analysis. (7A) Cells from four ZIKV immune and (7C) four DENV immune subjects were tested in ZIKV/Pan-DENV dual color FluoroSpot assays. Overlaid images are shown, displaying green spots (ZIKV-specific ASCs), red spots (DENV-specific ASCs) and yellow spots (ZIKV/DENV cross-reactive ASCs). Frequencies of DENV-specific, ZIKV-specific and DENV/ZIKV cross-reactive ASCs per 10⁶ cells in MBC cultures from (7B) four DENV immune and (7D) four ZIKV immune donors were tabulated. Values are the mean of duplicate wells for each type of ASC. (7E and F) Supernatants from the MBC cultures were tested by IgG ELISA and Abs that bound DENV and ZIKV VLPs were identified. (*P value < 0.05, **P value < 0.01, ****P value < 0.0001) were obtained using T-test.

Table 3:

Summary of ZIKV-specific and ZIKV/DENV cross-reactive MBCs

Donor No.	Total IgG /106 input cells	Number (Frequency) relative to total IgG
		ZIKV spots (%)	DENV spots (%)	ZIKV/DENV spots (%)
9	15280	850 (5.56)	275 (1.79)	140 (0.91)
10	8760	950 (10.84)	350 (3.99)	290 (3.31)
11	10900	690 (6.33)	170 (1.55)	105 (0.96)
12	11600	600 (5.17)	75 (0.64)	50 (0.43)

Discussion

We established a novel FluoroSpot assay using DENV and ZIKV labeled with different DyLight dyes which allowed us to distinguish DENV-specific, DENV serotype-cross-reactive, ZIKV-specific and DENV/ZIKV cross-reactive ASCs at the single cell level. Using PBMC from DENV immune, ZIKV immune and naïve donors, our data indicate that FLVs can be used to study the specificity of Abs secreted by memory B-cells generated after natural DENV or ZIKV infection or post vaccination. In each experiment performed, we used FLVs to evaluate PBMC from flavivirus-naïve donors as specificity controls. Although no DENV-binding Abs were detected in these individuals by ELISA, a very low number of spots were seen in the FluoroSpot assay. Given that members of flavivirus family circulate globally, it is difficult to exclude the possibility that these background spots represent true flavivirus cross-reactive immunity to other flaviviruses. Our findings clearly demonstrate that the FluoroSpot is better adapted than the conventional ELISpot to distinguish cross-reactive B-cell responses.

In this study, we were able to efficiently label DENV-1–4 and ZIKV with DyLight dyes using commercially available protein labeling kits, similar to our previous study using Alexa dyes (13). We chose to label DENV and ZIKV with lightning link DyLight dyes since they were easy to use and did not involve spin or separation steps. The excitation and emission spectra of the DyLight dyes used were also compatible with common laser and filter sets. We have not extensively compared Alexa Fluor labeled DENV and Dylight labeled DENV in multiparametric flow cytometry assays used to identify MBCs in the circulation. We have not performed structural or detailed infectivity studies on Dylight labeled DENV and ZIKV. It is possible that some aberrant epitopes are presented because of the modifications made to the viruses during the labeling process. Furthermore, we cannot rule out that the labeling process blocked some epitopes on the surface of the virion. All labeled FLVs however, bound beads that were coated with multiple flavivirus-specific (2H2, 4G2 and 3H5) but not control (CD28) antibodies and the susceptible cell line U937 DC-SIGN. Furthermore, antibodies in supernatants from stimulated dengue immune PBMC bound unlabeled and FL-DENV equally in ELISA assays (data not shown).

We chose to initially focus on labeling DENV-3 and −4 to develop the dual-color FluoroSpot for multiple reasons. Amino acid sequences in the E-protein indicate significant homology between DENV-1 and −3 and DENV-2 and −4 (42); thus, one might expect to find more cross-reactive MBCs using these serotypes together, as the E protein is a dominant target of antibody recognition. Fluorescence was detected on a diagonal when FL DENV-3 and −4 were added together, indicating equivalent binding by both DENV-3 and −4 to 4G2 coated beads. The amount of prM and E proteins present in each of the four DENV preparations varied but DENV-3 and −4 had similar amounts of prM and E proteins by Western Blot analysis compared to DENV-1 and −2 (data not shown). Since FLVs were diluted in a volume calculated to normalize initial protein concentrations of the viral preparations, it is possible that in the multiplexed FluoroSpot assay where four FL-DENV were used together we underestimated the extent of cross-reactivity between serotypes that differed in prM and E protein content. While most of the spots should represent prM or E-specific MBCs, a small number could be NS1-specific as our purified viruses contain trace amounts of secreted NS1.

A quad color FlouroSpot assay was recently developed using four fluorescently labeled DENV serotype-specific mAbs to detect DENV serotype-specific and cross-reactive B-cells (38). Our approach involves direct labeling of the viruses themselves and has several advantages. Our FluoroSpot protocol is simple to use. PBMC stimulated 6–7 days prior were added to Ig coated wells and ASCs detected using FLVs in our assay while purified DENV was added to ASCs followed by detection with antibodies that recognized each of the four serotypes in the recently published study. Using FLVs is not dependent on having a specific mAb for detection, so our assay system is more flexible in terms of which viruses/antigens can be used and multiplexed together. For example, we compared responses to DENV and ZIKV using all four DENV serotypes labeled with the same dye. Alternatively, it would be feasible to compare responses to different strains within a serotype, such as representative of different genotype or wild-type versus vaccine strains. Using an antigen-specific mAb in the detection step may compete with antibodies from ASCs that potentially recognize the same epitope, which could affect sensitivity of the signal. Labeling of rNS1 or rE proteins in the future, would be easier compared to validating five different NS1 or E-specific antibodies for detection of NS1 and rE-specific B cells.

We detected a wide range of spot size and color intensity. We speculate that the size and intensity in each channel relate to the rate of antibody secretion and antibody avidity for each virus. The large, bright yellow spots would be expected to reflect DENV-3/4 or DENV/ZIKV cross-reactive ASCs with high avidity, while small yellow spots represent low avidity ASCs. Analysis of the data using FCS files and FlowJo software supported the presence of Abs with varying avidities, although information regarding which spot on the FluoroSpot well corresponds to which dot on the flow plot is not preserved when exporting the FCS files.

Pre-existing immunity against DENV has been proposed to influence the response to subsequent ZIKV infection, potentially leading to protection or to enhanced infection and possibly disease (14). It is likely that a ZIKV vaccine will be available in the next few years, and therefore it is crucial that we understand whether prior experience with DENV is expected to enhance ZIKV vaccine-induced responses, or potentially hinder it, and whether pre-existing ZIKV immunity influences subsequent DENV infection. Our data using PBMC from DENV and ZIKV immune donors indicate minimal cross-reactivity between DENV and ZIKV MBCs using PBMC from seven of eight donors tested. The timing when samples are obtained post vaccination or natural infection is likely to influence the extent of MBC cross-reactivity between these two more distantly related flaviviruses.

The data from this study showed that FLVs are sensitive and specific tools for the analysis of flavivirus-specific and cross-reactive ASCs. With further development of the FluoroSpot and the incorporation of new filters and fluorophores, it is likely that inclusion of an increased number of antigens or analysis of other functions of B-cells, such as cytokine production, and determination of the involvement of the Ig subclasses in DENV-specific and cross-reactive responses is achievable. The FluoroSpot assay should enable assessment of the breadth as well as the durability of DENV/ZIKV B-cell responses generated in the context of vaccination or natural infection. Moreover, this method may be very useful in vaccine efficacy trials where the number of cells available are limited and to investigate the contribution of cross-reactive immunity to vaccine-induced protection. In addition, the assay can be adapted to assess ASCs specific for other diseases, such as Influenza, in which prior immunity may influence subsequent responses to infection with different virus strains.

Abbreviations:

ASCs,

antibody secreting B-cells

DENV,

Dengue virus

FLVs,

fluorescently labeled viruse

FL-DENV,

fluorescently labeled dengue virus

MBCs,

memory B-cells

PBMC,

peripheral blood mononuclear cells

VLPs,

Virus-like particles

ZIKV,

Zika virus