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 v....

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. ....

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

List of materials used in this article

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