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. 2020 Nov 18;14(11):e0008730.
doi: 10.1371/journal.pntd.0008730. eCollection 2020 Nov.

Antigenicity, stability, and reproducibility of Zika reporter virus particles for long-term applications

Affiliations

Antigenicity, stability, and reproducibility of Zika reporter virus particles for long-term applications

J Charles Whitbeck et al. PLoS Negl Trop Dis. .

Abstract

The development of vaccines against flaviviruses, including Zika virus (ZIKV) and dengue virus (DENV), continues to be a major challenge, hindered by the lack of efficient and reliable methods for screening neutralizing activity of sera or antibodies. To address this need, we previously developed a plasmid-based, replication-incompetent DENV reporter virus particle (RVP) production system as an efficient and safe alternative to the Plaque Reduction Neutralization Test (PRNT). As part of the response to the 2015-2016 ZIKV outbreak, we developed pseudo-infectious ZIKV RVPs by modifying our DENV RVP system. The use of ZIKV RVPs as critical reagents in human clinical trials requires their further validation using stability and reproducibility metrics for large-scale applications. In the current study, we validated ZIKV RVPs using infectivity, neutralization, and enhancement assays with monoclonal antibodies (MAbs) and human ZIKV-positive patient serum. ZIKV RVPs are antigenically equivalent to live virus based on binding ELISA and neutralization results and are nonreplicating based on the results of live virus replication assays. We demonstrate reproducible neutralization titer data (NT50 values) across different RVP production lots, volumes, time frames, and laboratories. We also show RVP stability across experimentally relevant time intervals and temperatures. Our results demonstrate that ZIKV RVPs provide a safe, high-throughput, and reproducible reagent for large-scale, long-term studies of neutralizing antibodies and sera, which can facilitate large-scale screening and epidemiological studies to help expedite ZIKV vaccine development.

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Conflict of interest statement

I have read the journal's policy and the authors of this manuscript have the following competing interests. Integral Molecular offers ZIKV RVPs as a commercial reagent. J. C. Whitbeck, A. Thomas, K. Kadash-Edmondson, A. Grinyo-Escuer, Lewis J. Stafford, E. Davidson, B. J. Doranz are all current or former members of Integral Molecular. B. J. Doranz is a shareholder of Integral Molecular.

Figures

Fig 1
Fig 1. ZIKV RVPs are infectious and antigenically equivalent to live Zika virus.
(A) Schematic of the ZIKV RVP, composed of capsid, prM/M, and E proteins from a defined ZIKV strain (SPH2015), and an RNA reporter genome replicon. (B) Luciferase ZIKV RVPs were tested for infectivity with cell lines that are commonly used for flavivirus infectivity studies, including BHK-DC-SIGN, Vero, and Raji-DC-SIGN-R cells. At 72 h after infection, cells were lysed and analyzed for luciferase activity (RLU). Values for BHK-DC-SIGN cells (filled symbols) are plotted on the left y-axis. Values for Raji-DC-SIGN-R and Vero cells (open symbols) are plotted on the right y-axis. All values represent n = 3 replicate wells, and error bars represent SD. Regression analysis indicates a linear relationship for the three datasets with RVP volume (R2 > 0.99). (C) Serial dilutions of the indicated MAbs were incubated with ZIKV RVPs for 1 h, followed by infection of BHK-DC-SIGN cells. After 72 h, cells were lysed and analyzed for RLU. All neutralization results are shown as normalized % infection (% of luciferase signal in the absence of MAb). For 1N5, 4E8, and 1C19, n = 2 and error bars indicate range. For all other data sets n = 7 or 8 and error bars represent SD. Gray symbols represent non-neutralizing MAbs, black symbols represent neutralizing MAbs. (D) Epitopes of 11 anti-ZIKV MAbs used in this paper, mapped onto the ZIKV structure. MAbs that neutralize ZIKV are indicated in bold and underlined.
Fig 2
Fig 2. ZIKV RVPs are antigenically equivalent to live virus.
(A) ELISA was performed using ZIKV RVPs with various conformational MAbs. Detection MAbs were two anti-DENV MAbs with ZIKV cross-neutralizing activity (C8, C10), five neutralizing anti-ZIKV MAbs (ZIKV-117, ZIKV195, ZIKV116, A9E, LM-081), and two negative control MAbs (CHIKV-specific CKV063 and DENV1-specific 1F4). The capture MAb was a mouse anti-fusion loop MAb (4G2). Mean signal to background ratio (S:B) of three concentrations of detection MAb (1, 2, and 4 μg/mL) is shown with the SD (error bars). Dashed horizontal line represents S:B = 5. (B) Flow cytometry analysis of LM-081 binding to viral envelope proteins expressed in human cells shows that LM-081 binds specifically to ZIKV and not to other flaviviruses, including DENV serotypes 1–4. (C) Shotgun mutagenesis epitope mapping of LM-081 revealed two critical residues in the fusion loop on one E monomer (N103, G106), and one critical residue on Domain III of an adjacent monomer (I317). (D) Shotgun mutagenesis epitope mapping of anti-DENV EDE MAbs C8 and C10 reveal critical residues (green spheres), shown on the ZIKV E ectodomain (PDB # 5IRE; [11]).
Fig 3
Fig 3. ZIKV RVPs are infectious and stable under commonly used conditions.
(A) Cryopreserved ZIKV RVP aliquots were thawed, mixed, incubated at 4, 25, or 37°C, and used to infect BHK-DC-SIGN cells. Infectivity was compared to that of freshly thawed ZIKV RVPs. Each data point is the mean of 3 replicate wells. (B) Infectivity of ZIKV RVPs sampled immediately after production or after multiple freeze/thaw cycles. Each data point represents the mean of at least 4 replicate wells. RVP infectivity did not diminish upon freeze/thaw when tested from 0 to 5 cycles. (C) Five different lots of ZIKV RVPs (produced at various times over 23 months) were tested for infectivity using BHK-DC-SIGN target cells. RVPs were added to 96-well black plates and subjected to serial, 2-fold dilutions before adding target cells. Each data point is the mean of 8 replicate wells. All error bars represent SD.
Fig 4
Fig 4. ZIKV RVPs can be used to derive reproducible antibody neutralization titers.
(A) MAb C8 neutralization of RVPs was performed using various volumes (5–40 μL/well) of ZIKV RVPs from a single lot (#258A). RVPs were combined with serial 3-fold dilutions of C8 and incubated for 1 h at 37°C prior to infecting BHK-DC-SIGN cells. (B) NT50 values from A are plotted for each of the eight replicates run for each RVP volume. The mean NT50 value for each volume is shown by a horizontal line, and the mean NT50 value (μg/mL) and SD for each volume is as shown. (C) C8 neutralization of RVPs was performed on 5 different days, using one lot of ZIKV RVPs at 20 μL/well. (D) ZIKV neutralization by C8 was performed with 5 different lots of ZIKV RVPs, produced at various times over ~2 years. Each lot was used at 20 μL/well. Data points for curves in A-D are means of 8 replicates, error bars indicate SD.
Fig 5
Fig 5. ZIKV RVPs are reproducibly neutralized in different laboratories by human serum from a ZIKV positive patient and by MAbs.
(A) ZIKV RVP neutralization experiments using serum from a ZIKV infected individual (closed symbols) were conducted in three different laboratories: Integral Molecular (INTG), Vitalant Research Institute (VRI) and Integrated Biotherapeutics (IBT). Serum from an uninfected individual was used as a negative control (open symbols). Neutralization experiments were performed in different laboratories using MAbs (B) C10 and (C) LM-081. Data points for RVP neutralization assays represent the mean and standard deviation for 5–8 replicate data points. For PRNT assays, data points represent the mean and range of 2 replicates.
Fig 6
Fig 6. Antibody-dependent enhancement (ADE) of ZIKV infection.
ZIKV RVPs were mixed and incubated for 1 h at 37°C with serial dilutions of ZIKV-reactive neutralizing MAbs LM081, C8, 1C19 (filled symbols) or non-neutralizing MAbs 1N5, 4E8, 4G2 (unfilled symbols). K562 cells were then added to each well and incubated for 72 h at 37°C. Culture medium was removed, cells were lysed, and plates were analyzed for luciferase activity. Each data point is the average of 2 wells and is plotted as a percentage of the no antibody control. Error bars indicate range.
Fig 7
Fig 7. Control of ZIKV RVP maturation state.
(A) Western blot showing maturation state of ZIKV RVPs produced in the absence or presence of transfected human furin. (B) Infectivity of ZIKV RVPs produced under normal conditions or in the presence of transfected furin. Each data point is the mean of 2 wells, error bars represent the range. (C) Neutralization of infectivity by ZIKV RVPs produced under normal conditions in the absence or presence of transfected furin. ZIKV RVPs were incubated with serial dilutions of MAbs C8, C10, LM-081, 4G2, and CKV063. Each data point represents the average of 8 wells; error bars indicate SD.

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