The described project was supported by funds from the Uniformed Services University of the Health Sciences to JJM. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense, the Uniformed Services University of the Health Sciences or any other agency of the U.S. Government.
WGV performed all the experiments and analyzed the data; JJM designed and supervised the study; WGW and JJM wrote the paper.

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.
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	<li>George, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prior+exposure+to+zika+virus+significantly+enhances+peak+dengue-2+viremia+in+rhesus+macaques.">Prior exposure to zika virus significantly enhances peak dengue-2 viremia in rhesus macaques.</a> Sci Rep. 7 (1), 10498 (2017).</li><li>Stettler, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity,+cross-reactivity,+and+function+of+antibodies+elicited+by+Zika+virus+infection.">Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection.</a> Science. 353 (6301), 823-826 (2016).</li><li>Mattia, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue+reporter+virus+particles+for+measuring+neutralizing+antibodies+against+each+of+the+four+dengue+serotypes.">Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes.</a> PLoS One. 6 (11), e27252 (2011).</li><li>Chiofalo, M. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amine+reactive+dyes:+an+effective+tool+to+discriminate+live+and+dead+cells+in+polychromatic+flow+cytometry.">Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry.</a> J Immunol Methods. 313 (1-2), 199-208 (2006).</li><li>Priyamvada, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+Cell+responses+during+secondary+dengue+virus+infection+are+dominated+by+highly+cross-reactive,+memory-derived+plasmablasts.">B Cell responses during secondary dengue virus infection are dominated by highly cross-reactive, memory-derived plasmablasts.</a> J Virol. 90 (12), 5574-5585 (2016).</li><li>Priyamvada, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+antibody+responses+after+dengue+virus+infection+are+highly+cross-reactive+to+Zika+virus.">Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus.</a> Proc Natl Acad Sci U S A. 113 (28), 7852-7857 (2016).</li></ol>

The authors have nothing to disclose.

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 other13,14. 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 flaviviruses1,2. The potential to cross ...

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 vivo1,2. 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 Fc&#947;III-receptor-expressing leukemia cell line that bind to the Fc region of antibodies and become infected in the presence of sub-neutralizing concentrations of antibody4,5.
ADE assays have been extensively used in studies investigating the risk factors for severe dengue and to delineate the mechanisms of in vitro ADE6,7,8.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 cells6,7,8,9,10,11, both of which are time consuming and labor intensive.

<table border="0" cellpadding="0" cellspacing="0" width="794">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">DENV 1-4 RVP</td>
			<td style="width:139px;">Integral Molecular</td>
			<td style="width:119px;">RVP-501</td>
			<td style="width:323px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">K562 cells</td>
			<td style="width:139px;">ATCC</td>
			<td style="width:119px;">CCL-243</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">RPMI</td>
			<td style="width:139px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">FBS</td>
			<td style="width:139px;">GE lifesceinces</td>
			<td style="width:119px;">SH 30910.03</td>
			<td>10% FBS in RPMI-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Penicillin-Streptomycin</td>
			<td style="width:139px;">MP biomedicals</td>
			<td style="width:119px;">1670049</td>
			<td>100 IU Pen/mL, 100 ug Strep/mL in RPMI-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HEPES</td>
			<td style="width:139px;">Cellegro</td>
			<td style="width:119px;">25-060-Cl</td>
			<td>0.025M in RPMI-10</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">MEM Non-essential Amino Acid Solution (100&#215;)</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">M7145</td>
			<td>1x in RPMI-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sodium Pyruvate</td>
			<td style="width:139px;">Cellegro</td>
			<td style="width:119px;">25-000-Cl</td>
			<td>1mM in RPMI-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">L-glutamine</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">MT25005CI&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sterile V-bottom plates</td>
			<td style="width:139px;">Thomas Scientific</td>
			<td style="width:119px;">333-8001-01V</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">BD Falcon Polypropylene 5 ml FACS tubes</td>
			<td style="width:139px;">VWR</td>
			<td style="width:119px;">60819-728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Non-sterile U-bottom plates</td>
			<td style="width:139px;">Falcon</td>
			<td style="width:119px;">Ref 353910</td>
			<td>A high throughput alternative to FACS tubes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">5mL, sterile, serological pipette</td>
			<td style="width:139px;">Denville</td>
			<td style="width:119px;">P7127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">200uL sterile pipette tips</td>
			<td style="width:139px;">Denville</td>
			<td style="width:119px;">P3020 CPS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">20uL sterile pipette tips</td>
			<td style="width:139px;">Denville</td>
			<td style="width:119px;">P1121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">50-200uL multichannel pipette</td>
			<td style="width:139px;">Denville</td>
			<td style="width:119px;">P3975-8-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">5-50uL multichannel pipette</td>
			<td style="width:139px;">Denville</td>
			<td style="width:119px;">P3975-9-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">20% formadehyde</td>
			<td style="width:139px;">Tousimis</td>
			<td style="width:119px;">&#160;#1008B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Water Bath</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:119px;">TSCOL35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">thermomixer</td>
			<td style="width:139px;">Eppendorf</td>
			<td style="width:119px;">2231000387</td>
			<td>An alternative to a waterbath for heat inactivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">CO2 Incubator</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:119px;">13-998-213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ethanol</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:119px;">E7023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Liquid Bleach</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">NC9724348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Flowjo 9.8</td>
			<td style="width:139px;">TreeStar, Inc.</td>
			<td style="width:119px;"></td>
			<td>Flow cytometry analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACSDiva 6.1.2</td>
			<td style="width:139px;">Becton Dikinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">BD LSR II flow cytometer</td>
			<td style="width:139px;">Becton Dikinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Liquid Bleach</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">NC9724348</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple flow cytometry based assay to determine in vitro antibody dependent enhancement of dengue virus using zika virus convalescent serum

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.

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...

Watch this Scientific Journal Video about A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum at JoVE.com

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

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.

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

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

The overall goal of this procedure is to determine the capacity of sera collected from Zika virus infected rhesus macaques to mediate antibody dependent enhancement of dengue virus infection in vitro. This method can help answer key questions related to antibody dependent enhancement of infection of dengue caused by Zika virus induced cross-reactive antibodies. The main advantage of this technique is that it is high troper, simple, and easy to perform.Demonstrating this procedure will be William Valiant, an emerging infectious disease graduate student, in my laboratory. Begin by thawing the serum samples at room temperature. When thawed, transfer 100 microliters of each serum sample to a sterile tube and heat and activate for 30 minutes at 56 degrees Celsius.Next, make 10-fold serial dilutions of the serum sample ranging from one-to-one, to one-to-one thousand in cold RPMI 10. Then transfer 10 microliters of each diluted serum sample to the wells of a sterile 96 well V-bottom plate. Next, thaw dengue one, two, three, and four proporter viral particles in a 37 degrees Celsius water bath, ensuring a volume of approximately 170 microliters of each RVP per serum sample.Once thawed, immediately transfer the tubes of RVPs to ice. Then pipet 10 microliters of RVPs into the wells containing serum sample dilutions into the no serum control wells. Mix thoroughly by pipetting up and down five to ten times.Transfer the 96 well V-bottom plate to an incubator and incubate for an hour at 37 degrees Celsius, in the presence of 5%carbon dioxide. While the 96 well V-bottom plate is incubating, clean the surface of the bio-safety cabinet with 70%ethanol, and run the UV light for 15 minutes. Then remove a fully confluent T-75 flask of K562 cells from the incubator.Mix the cells well using a sterile five millimeter pipet and transfer five milliliters to a sterile 15 milliliter conical tube. Next, remove 10 microliters of cells from the tube, mix with 10 microliters of trypan blue and load into a hemocytometer. Count the cells and calculate the total number of cells.After centrifuging the cells at 12 hundred times G for 10 minutes, decant the supernatant and re-suspend the cells in warm RPMI 10 at a concentration of 80 thousand cells per 30 microliters of media. After removing the 96 well V-bottom plate from the incubator, transfer 30 microliters of K562 cells to each wall of the 96 well V-bottom plate. And mix thoroughly by pipetting up and down five to ten times.Then return the plate to the incubator for one hour. Following the incubation, centrifuge the plate at 12 hundred times G for five minutes. After centrifugation, decant the media from the wells by turning the plate upside down into a container containing 10%bleach.Then wash the cells in each wall by re-suspending them in 125 microliters of warm RPMI 10 and centrifuging as before. After decanting the last wash, add 100 microliters of warm RPMI 10 to each well. Mix up and down with the pipet and incubate the plate for 48 hours at 37 degrees Celsius in the presence of 5%carbon dioxide.Two days later, remove the plate from the incubator and transfer to a bio-safety cabinet. Using a multichannel pipet, mix the contents of each well and transfer to 96 well U-bottom plate. Rinse each well of the 96 well V-bottom plate with 100 microliters of 1%paraformaldehyde and PBS and transfer the remaining cells and PBS to the respective wells in the 96 well U-bottom plate.Mix thoroughly using a multichannel pipet. Cover the plate with aluminum foil and let it sit in the incubator for 30 minutes to fix the cells. Prepare the flow cytometer by running unstained K562 cells to calibrate the side and forward scatter settings using the ethyl-one channel for GFP florescence.Then acquire 30 thousand to 50 thousand cells from each sample. Using flow cytometry analysis software, set the first gate based on the forward scatter area versus forward scatter height to include single cells and exclude autofluorescent dublets from analysis. Calculate the average percentage of GFP positive cells for each condition and then calculate fold enhancement of infection for each serum dilution.Graph fold enhancement on the y-axis versus dilution on the x-axis and perform statistical analysis using ANOVA followed by Tukey's post-hoc test for multiple comparisons. Data from a representative animal using dengue virus one reporter virus particles are shown here. The percentage of GFP positive cells for the no serum control sample was 0.253%The undiluted serum sample was 2.58%GFP positive.The proportion of GFP positive cells reached a high of 12.8%at the one to ten serum dilution following to 0.735%at a one to one hundred dilution and 0.022%at a one to one thousand dilution. Antibody dependent enhancement of dengue infection was examined for the four dengue virus sera types. The data shows that the serum collected 16 weeks post-infection significantly enhanced infection of K562 cells by all dengue virus sera types at a dilution of one to ten.Once mastered, this technique can be done in 48 hours if it is performed properly. While attempting this procedure, it is important to remember to acquire the data within one to two hours after the final fixation step of 30 minutes is completed. Don't forget that working with infected serum can be extremely hazardous and precautions such as PPE, and BSL2 Level practices should always be taken while preforming this procedure.

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Immunology and Infection

8.6K visualizações.  Uniformed Services University. The overall goal of this procedure is to determine the capacity of sera collected from Zika virus infected rhesus macaques to mediate antibody dependent enhancement of dengue virus infection in vitro. This method can help answer key questions related to antibody dependent enhancement of infection of dengue caused by Zika virus induced cross-reactive antibodies. The main advantage of this technique is that it is high troper, simple, and easy to perform.Demonstrating this procedure will ...