A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Summary

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

Abstract

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

Introduction

Antibody dependent enhancement (ADE) of infection is a process whereby partially cross-reactive antibody responses induced by a serotype of virus enhances uptake of another serotype of virus, leading to increased viral replication and viremia. ADE has been extensively documented in Dengue virus (DENV) infections where four major serotypes are prevalent. In a subset of patients, ADE is associated with Dengue hemorrhagic fever (DHF). We have recently shown that Zika virus (ZIKV) infection induced significantly high levels of DENV cross-reactive antibody responses that caused ADE of DENV in vitro and likely contributed to the enhancement of DENV viremia in vivo^1,2. Antibody dependent enhancement assays are a valuable tool to assess the capacity of antibodies to enhance secondary infection with related viruses and provide valuable insights into the pathogenesis of flavivirus infections and inform the development of vaccines.

The assay described here uses DENV RVPs along with K562 cells that are normally impermissible to infection. RVPs are structurally intact replication incompetent DENV viral particles that encode a sub-genomic green fluorescent protein (GFP) replicon that is expressed after a single round of replication³. As such, cells that become infected with RVPs fluoresce green and can be readily detected using flow cytometry or microscopy. The RVPs used in this assay were obtained from commercial sources. They can, however, be generated against other viruses and used in the assay described in this manuscript. Meanwhile, K562 cells are an FcγIII-receptor-expressing leukemia cell line that bind to the Fc region of antibodies and become infected in the presence of sub-neutralizing concentrations of antibody^4,5.

ADE assays have been extensively used in studies investigating the risk factors for severe dengue and to delineate the mechanisms of in vitro ADE^6,7,8.The ADE assay described here can be quickly and easily used to determine the capacity of serum to enhance in vitro infection using RVPs and flow cytometry, as compared to other assays used currently, which require either the determination of plaque forming units (pfu) in Vero cells or antibody staining of infected cells^{6,7,8,9,10,11}, both of which are time consuming and labor intensive.

Protocol

The serum samples used to demonstrate the protocol described here were obtained from rhesus macaques that were housed and cared for in accordance with local, state and federal policies in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facility. All animal experiments were reviewed and approved by Institutional Animal Care and Use Committee and samples were acquired through a tissue sharing protocol.

1. Day 1

NOTE: Perform all the steps described below in a sterile laminar flow biosafety cabinet used for tissue culture in a BSL-2 laboratory.

1. Thaw the serum samples at room temperature and transfer 100 µL of each serum sample to a sterile tube. Heat inactivate for 30 min at 56°C either in a water bath or a temperature-adjustable thermomixer. Make 10-fold serial dilutions of the serum sample ranging from 1:1 to 1:1,000 using cold RPMI-10 (RPMI with 10% fetal bovine serum).
2. Transfer 10 µL of serially diluted serum sample to each well of a sterile 96-well V-bottom plate. Include two sets of control wells, RPMI-10 with RVPs only and without serum sample, and RPMI-10 only without RVPs and without serum sample. Set up each serum sample and RPMI-10 controls in triplicates.
3. Remove the Dengue-1, 2, 3 and 4 RVPs from the -80 °C freezer and thaw them in a 37 °C water bath. Transfer the thawed RVPs immediately to ice. Obtain approximately 170 µL RVPs for each serum sample (10 µL of RVPs x 3 wells for each serum dilution (1:1, 1:10, 1:100, 1:1,000) and 10 µL of RVPs x 3 wells for each of the RPMI-10 with RVP only control wells).
4. Pipette 10 µL of RVPs into each well of the 96-well V-bottom plate containing the serum sample and to the RPMI-10 with RVPs (no serum) control wells. Do not add RVPs to the RPMI-10 only (no serum sample and no RVP) wells. Mix thoroughly by pipetting up and down 5–10 times. Add 10 µL cold RPMI-10 in lieu of RVPs to each cold RPMI-10 only (no serum sample and no RVP) control wells.
5. Transfer the 96-well V-bottom plate to an incubator and incubate the plate for 1 hour at 37 °C in the presence of 5% CO₂. While the 96-well V-bottom plate is incubating, clean the surface of the biosafety cabinet with 70% ethanol and run the UV light for 15 min.
6. Remove a fully confluent T75 flask of K562 cells from the incubator, mix the cells well using a sterile 5 mL pipet, and transfer 5 mL of cells to a sterile 15 mL conical tube.
   NOTE: The K562 cells are maintained in RPMI-10 and subcultured at 1 x 10⁶/mL.
7. Count the number of cells by removing 10 µL cells from the sterile 15 mL conical tube and mix with 10 µL tryphan blue. Count the cells using a hemocytometer to determine the total number of cells in the 15 mL conical tube.
8. Centrifuge the 15 mL conical with cells at ~1,200 x g for 10 minutes, decant the supernatant, and resuspend the cells in warm RPMI-10 at a concentration of 80,000 cells/30 µL of media (2.66 x 10⁶ cells /mL).
9. Remove the 96-well V-bottom plate from the incubator (step 1.6), transfer 30 µL of K562 cells to each well of the 96-well V-bottom plate, and mix thoroughly by pipetting up and down 5–10 times. Transfer the 96-well V-bottom plate to an incubator and incubate the plate for 1 hr at 37 °C in the presence of 5% CO₂.
10. After incubating for 1 h, remove the 96-well V-bottom plate from the incubator and centrifuge at ~1,200 x g for 5 minutes. After centrifugation, decant the media from the wells by turning the plate upside down into a container containing 10% bleach.
11. Wash the cells in each well by resuspending them in 125 µL of warm RPMI-10, mix thoroughly with a pipette, and centrifuge the plate at ~1,200 x g for 5 min. Decant the media by turning the plate upside down into a container containing 10% bleach. Repeat this wash step two times.
12. After washing, add 100 µL of warm RPMI-10 to each well, mix up and down with the pipet, and incubate the plate for 48 h at 37 °C in the presence of 5% CO₂.

2. Day 3

1. Remove the 96-well V-bottom plate containing 100 µL of cells and media/well from the incubator and move it to a biosafety cabinet . Using a multichannel pipette, mix the contents of each well and transfer the cells and media to a 96 well U-bottom plate or to pre-labeled 5 mL polypropylene tubes.
2. Rinse each well in the 96-well V-bottom plate with 100 µL of 1% Paraformaldehyde (PFA) in 1x Phosphate Buffered Saline (PBS) and transfer to the respective wells in the 96 well U-bottom plate or labeled 5 mL polypropylene tubes from step 2.1 to yield a final concentration of 0.5% PFA/well or tube. Mix thoroughly using a multichannel pipette, cover the plate with aluminum foil, and let it sit in the incubator for 30 minutes to fix the cells.
3. Prepare the flow cytometer (see Table of Materials for example) by running unstained K562 cells to calibrate the side and forward scatter along with the fluorescence settings. The only fluorescence channel required is FL1, as GFP is the only fluorescence emitted from the cells. Acquire ~30,000–50,000 cells from each sample.
   NOTE: Any flow cytometer capable of reading a single fluorescence can be used for acquiring the data, as cells infected with RVPs will only emit green fluorescence due to the presence of GFP, and no other fluorescence channel are needed.

3. Data Analysis

1. Analyze data acquired on the flow cytometer using any flow cytometry analysis software (see Table of Materials).
   1. Set the first gate based on FSC-A (forward scatter – area) vs FSC-H (forward scatter – height) to include single cells and exclude auto-fluorescent doublets from analysis¹². Then, gate singlet-gated cells based on SSC-A (side scatter – area) vs FSC-A to exclude any autofluorescent debris.
   2. Analyze SSC-A vs FSC-A gated cells for the expression of GFP based on SSC-A vs GFP-A. Determine the percentage of GFP+ cells for each dilution of the serum and control samples by setting a gate around GFP+ cells.
2. Determine the average percentage of GFP+ cells for each serum sample dilution and control wells by dividing the total percentage of GFP+ cells in the triplicate wells by 3.
3. Calculate fold-enhancement of infection by dividing the average percentage of GFP+ cells in the serum samples for each dilution divided by the average percentage of GFP+ cells in the RPMI-10 with RVPs (no serum sample) control wells.
4. Graph fold-enhancement (y-axis) versus dilution (x-axis), and perform statistical analysis using ANOVA followed by Tukey's post-hoc test for multiple comparisons.

Results

Sera from 4 rhesus macaques were collected 16 weeks after infection with ZIKV and tested for their potential to enhance DENV-1, 2, 3 and 4 infection in K562 cells. The animals had peak viremia of ~1 x 10⁵ copies of ZIKV RNA/mL of plasma by day 3 after infection that declined to levels that were below the limit of detection by 7 days after infection. No viremia was detected in the plasma at 16 weeks post-infection. Then, the RVP assay was performed and the collected data were an...

Discussion

Flaviviruses such as DENV and ZIKV share significant evidence of homology in both their structural and non-structural proteins that generate antibodies that cross-react with each other^13,14. These cross-reactive antibody responses have been shown to enhance of infection both in vivo and in vitro with either a heterologous serotype or other related flaviviruses^1,2. The potential to cross ...

Materials

Name	Company	Catalog Number	Comments
DENV 1-4 RVP	Integral Molecular	RVP-501
K562 cells	ATCC	CCL-243
RPMI	Corning	10-040-CV
FBS	GE lifesceinces	SH 30910.03	10% FBS in RPMI-10
Penicillin-Streptomycin	MP biomedicals	1670049	100 IU Pen/mL, 100 ug Strep/mL in RPMI-10
HEPES	Cellegro	25-060-Cl	0.025M in RPMI-10
MEM Non-essential Amino Acid Solution (100×)	Sigma	M7145	1x in RPMI-10
Sodium Pyruvate	Cellegro	25-000-Cl	1mM in RPMI-10
L-glutamine	Fisher Scientific	MT25005CI
Sterile V-bottom plates	Thomas Scientific	333-8001-01V
BD Falcon Polypropylene 5 ml FACS tubes	VWR	60819-728
Non-sterile U-bottom plates	Falcon	Ref 353910	A high throughput alternative to FACS tubes
5mL, sterile, serological pipette	Denville	P7127
200uL sterile pipette tips	Denville	P3020 CPS
20uL sterile pipette tips	Denville	P1121
50-200uL multichannel pipette	Denville	P3975-8-B
5-50uL multichannel pipette	Denville	P3975-9-B
20% formadehyde	Tousimis	#1008B
Water Bath	ThermoFisher	TSCOL35
thermomixer	Eppendorf	2231000387	An alternative to a waterbath for heat inactivation
CO2 Incubator	ThermoFisher	13-998-213
Ethanol	Sigma Aldrich	E7023
Liquid Bleach	Fisher Scientific	NC9724348
Flowjo 9.8	TreeStar, Inc.		Flow cytometry analysis software
BD FACSDiva 6.1.2	Becton Dikinson
BD LSR II flow cytometer	Becton Dikinson
Liquid Bleach	Fisher Scientific	NC9724348