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A high throughput reporter virus particle microneutralization assay for quantitation of Zika virus neutralizing antibodies in multiple species.

ABSTRACT: Zika virus is a Flavivirus, transmitted via Aedes mosquitos, that causes a range of symptoms including Zika congenital syndrome. Zika has posed a challenging situation for health, public and economic sectors of affected countries. To quantitate Zika virus neutralizing antibody titers in serum samples, we developed a high throughput plate based Zika virus reporter virus particle (RVP) assay that uses an infective, non-replicating particle encoding Zika virus surface proteins and capsid (CprME) and a reporter gene (Renilla luciferase). This is the first characterization of a Zika virus RVP assay in 384-well format using a Dengue replicon Renilla reporter construct. Serially diluted test sera were incubated with RVPs, followed by incubation with Vero cells. RVPs that have not been neutralized by antibodies in the test sera entered the cells and expressed Renilla luciferase. Quantitative measurements of neutralizing activity were determined using a plate-based assay and commercially available substrate. The principle of limiting the infection to a single round increases the precision of the assay measurements. RVP log10EC50 titers correlated closely with titers determined using a plaque reduction neutralization test (PRNT) (R2>95%). The plate-based Zika virus RVP assay also demonstrated high levels of precision, reproducibility and throughput. The assay employs identical reagents for human, rhesus macaque and mouse serum matrices. Spiking studies indicated that the assay performs equally well in different species, producing comparable titers irrespective of the serum species. The assay is conducted in 384-well plates and can be automated to simultaneously achieve high throughput and high reproducibility.

SUBMITTER: Bohning K

PROVIDER: S-EPMC8064526 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Recent worldwide outbreaks of Zika virus (ZIKV) infection and the lack of an approved vaccine raise serious concerns regarding preparedness to combat this emerging virus. We used a virus-like particle (VLP)-based approach to develop a vaccine and a microneutralization assay for ZIKV. A synthetic capsid-premembrane-envelope (C-prM-E) gene construct of ZIKV was used to generate reporter virus particles (RVPs) that package a green fluorescent protein (GFP) reporter-expressing West Nile virus (WNV) replicon. The assay was adapted to a 96-well format, similar to the plaque reduction neutralization test (PRNT), and showed high reproducibility with specific detection of ZIKV neutralizing antibodies. Furthermore, C-prM-E and prM-E VLPs were tested as vaccine candidates in mice and compared to DNA vaccination. While the ZIKV prM-E construct alone was sufficient for generating VLPs, efficient VLP production from the C-prM-E construct could be achieved in the presence of the WNV NS2B-3 protease, which cleaves C from prM, allowing virus release. Immunization studies in mice showed that VLPs generated higher neutralizing antibody titers than those with the DNA vaccines, with C-prM-E VLPs giving slightly higher titers than those with prM-E VLPs. The superiority of C-prM-E VLPs suggests that inclusion of capsid may have benefits for ZIKV and other flaviviral VLP vaccines. To facilitate the VLP platform, we generated a stable cell line expressing high levels of ZIKV prM-E proteins that constitutively produce VLPs as well as a cell line expressing ZIKV C-prM-E proteins for RVP production. While several vaccine platforms have been proposed for ZIKV, this study describes a safe, effective, and economical VLP-based vaccine against ZIKV.IMPORTANCE To address the growing Zika virus epidemic, we undertook this study with two objectives: first, to develop a safe, effective, and economical vaccine for ZIKV, and second, to develop a rapid and versatile assay to detect the anti-ZIKV immune response. We generated a cell line stably expressing ZIKV prM-E that produces large amounts of VLPs in the supernatant and a ZIKV C-prM-E cell line that produces reporter virus particles upon transfection with a GFP replicon plasmid. The prM-E VLPs induced a strong neutralizing antibody response in mice that was better when the capsid was included. VLP-based vaccines showed significantly better neutralizing antibody responses than those with their DNA counterparts. The RVP-based microneutralization assay worked similarly to the PRNT assay, with a rapid GFP readout in a 96-well format. Our VLP-based platform provides a source for a ZIKV vaccine and diagnosis that can rapidly be adapted to current outbreaks.

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A high throughput reporter virus particle microneutralization assay for quantitation of Zika virus neutralizing antibodies in multiple species.

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