Development and optimization of a Zika virus antibody-dependent cell-mediated cytotoxicity (ADCC) assay

https://doi.org/10.1016/j.jim.2020.112900Get rights and content

Abstract

Zika virus (ZIKV) has become a global public health issue due to its teratogenicity and ability to cause Guillain-Barré syndrome in adults. Although anti-ZIKV envelope protein neutralizing antibodies correlate with protection, the non-neutralizing function of ZIKV antibodies including antibody-dependent cell-mediated cytotoxicity (ADCC) is incompletely understood. To study the role of ADCC antibodies during ZIKV infections, we generated a stably transfected, dual-reporter target cell line with inducible expression of a chimeric ZIKV prM-E protein on the cell surface as the target cell for the assay. By using this assay, nine of ten serum samples from ZIKV-infected patients had >20% ADCC killing of target cells, whereas none of the 12 healthy control sera had >10% ADCC killing. We also observed a time-dependent ADCC response in 2 patients with Zika. This demonstrates that this assay can detect ZIKV ADCC with high sensitivity and specificity, which could be useful for measurement of ADCC responses to ZIKV infection or vaccination.

Introduction

Zika was first discovered in 1947 in Uganda. From the 1960s to 1980s human infections were found across Africa and Asia. In 2007 Zika virus (ZIKV) spread from Africa and Asia caused the first large outbreak in humans on the Pacific island of Yap. In 2013 the virus crossed the Pacific Ocean to the Americas, leading to the 2015–16 Zika epidemic (Kindhauser, 2016; Musso et al., 2019). As of July 2019, evidence of ZIKV transmission has been reported in 87 countries and territories throughout the world. The Zika outbreak in the Americas peaked during the first half of 2016. In 2018, a total of 31,587 suspected, probable and confirmed cases of Zika disease were reported in the Region of the Americas(World Health Organization, 2019. July). Infection with ZIKV is usually subclinical or results in a mild illness, but it has also been associated with severe neurological manifestations including Guillain–Barré syndrome in adults(Oehler et al., 2014; Parra et al., 2016). If infected during pregnancy, ZIKV can cross the placenta, infect the fetus and cause severe brain malformations and other birth defects depending on the gestational age at the time of infection(de Oliveira et al., 2017; Hoen et al., 2018). The severe birth defects associated with ZIKV infection during pregnancy, prompted the World Health Organization (WHO) to declare Zika a global public health emergency in 2015(Lanciotti et al., 2016; Pan American Health Organization, 2016). Although several Zika vaccine candidates are under evaluation(Gaudinski et al., 2018; Horstick and Runge-Ranzinger, 2018), there is no prevention or treatment currently available.

ZIKV infection induces protective immunity that correlates with antibodies directed against epitopes on the viral envelope protein dimer(Dai et al., 2016; Stettler et al., 2016; Delgado et al., 2018; Lucas et al., 2018). Virus-specific antibodies can appear in serum as early as 2–3 days after onset of fever(Rogers et al., 2017). The antibody response includes anti-ZIKV neutralizing antibodies that neutralize infectious virions prior to host cell entry. However, neutralizing antibodies do not affect virus that has already infected cells while Fc-mediated immune effector functions(Bailey et al., 2019) can be effective against virus already inside host cells.

Non-neutralizing antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies, may play an important role in viral clearing infection by killing virus-infected host cells(Vanderven et al., 2017; Ye et al., 2017). In ADCC, virus-specific antibodies bind to the surface of the infected cell and mediate cellular lysis by activation of effector cells, such as natural killer (NK) cells and monocytes. ADCC is known to correlate with control of or protection against Epstein-Barr virus, HIV, and hepatitis C virus(Frenzel et al., 2014; Oliviero et al., 2017; Chen et al., 2018; Forthal and Finzi, 2018; Holder et al., 2018; Sicca et al., 2018; Yu et al., 2018). Furthermore, ADCC responses also correlate with the efficacy of experimental vaccines against herpes simplex virus, influenza virus, and HIV(Jegaskanda et al., 2017; Vanderven et al., 2017; Ye et al., 2017). The role of ADCC in patients with Zika is not known. Development of a functional assay to detect ZIKV ADCC could clarify the role these antibodies play in control and prevention of ZIKV infection. In this study, we describe development and optimization of an ADCC assay for ZIKV. To do this, we developed a stably transfected target cell line expressing ZIKV precursor membrane protein (prM) and envelope protein (E) on the cell surface. prM and E proteins are ZIKV structural proteins and comprised the virus envelope on the surface. Also, both proteins are major targets of ZIKV vaccine design(Alam et al., 2017; Abbink et al., 2018; Richner and Diamond, 2018). We developed, optimized, and characterized a ZIKV ADCC assay based on NK-mediated target cell killing.

Section snippets

EGFP-ires-luciferase and ZIKV prM-E gene

Enhanced green fluorescent protein (EGFP)-ires-Luciferase reporter gene derived from plasmid pHAGE PGK-GFP-Luciferase-w was cut with NotI and ClaI and then blunted by Klenow Fragment. The blunt fragment was introduced into the EcoRV site of plasmid pcDNA4/TO (Invitrogen, Carlsbad CA) to generate plasmid pcDNA4/TO GFP-ires-Luc. The coding sequence for ZIKV prM and E protein of PRVABC59 (KU501215.1)(Lanciotti et al., 2016) was synthesized and codon-optimized for mammalian cells (GenScript,

ZIKV prM-E expressed by a codon-optimized synthetic construct does not persist on the target cell surface

To generate a target cell line for a ZIKV ADCC assay, we first synthesized a codon-optimized prM-E gene and transfected it into reporter-only cells to determine the feasibility of this approach to express ZIKV prM-E protein on the cell surface. Single clones were selected using the same strategy as mentioned above. We then tested for surface prM-E protein using ZIKV E antibodies by immunofluorescence on transfected reporter-only cells. We found that most prM-E protein was released spontaneously

Acknowledgements

This study was funded by contracts with the National Institute of Allergy and Infectious Diseases at the National Institutes of Health to the Emory Vaccine and Treatment Evaluation Units [VTEU]: HHSN272201300018I (Rouphael). Additional support was provided by the Georgia Research Alliance (GRA), the Emory University School of Medicine, and Children’'s Healthcare of Atlanta (CHOA). We acknowledge in particular the Children's Healthcare of Atlanta and Emory University Pediatric Biostatistics Core

Author contributions statement

X.C., L.J.A. and E.J.A. proposed and designed the experiments. X.C. performed Western Blot, flow cytometry analysis and ADCC experiments. L.D. conducted Fluorescence Microscopy analysis. C.M. did statistical analysis. X.C., L.J.A., C.A.R. and E.J.A. analyzed the data and discussed the interpretation of results. X.C. and E.J.A. wrote the main manuscript text. All authors contributed to review and revisions of the manuscripts.

Declaration of Competing Interest

E.J.A has received personal fees from AbbVie and Pfizer for consulting, and his institution receives funds to conduct clinical research unrelated to this manuscript from MedImmune, Regeneron, PaxVax, Pfizer, GSK, Merck, Novavax, Sanofi-Pasteur, and Micron. L.J.A. has have done paid consultancies on RSV vaccines for Moderna Therapeutics, Inc., Bavarian Nordic, Novavax, Daiichi-Sankyo, ClearPath Vaccines Company, and Pfizer; his laboratory is receiving funding through Emory University from Pfizer

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