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.

Recombinant Protein Micelles to Block Transduction by SARS-CoV-2 Pseudovirus

“…To assess the ability of functional oleosin-based micelles to block viral entry, we used SARS-CoV-2 pseudovirus particles (PVs) as a model to test the inactivation effectiveness of different micellar compositions. PVs are essentially liposomes with the SARS-CoV-2 Spike S1 protein at the membrane, making them antigenically alike to the wild-type virus . PVs have a second-generation lentiviral core containing a green fluorescence protein (GFP) gene, which is expressed upon transduction .…”

“…Evidence of higher-diameter peaks in both Figure 2B, corresponding to Oleo-LCBx, and Figure S2, corresponding to all the blends, could also mean that some Spike S1 protein at the membrane, making them antigenically alike to the wild-type virus. 36 PVs have a second-generation lentiviral core containing a green fluorescence protein (GFP) gene, which is expressed upon transduction. 37 PVs have been proven to serve as a viable, non-replicating model for testing transductions by dengue, zika, and SARS-CoV-2.…”

A reporter virus particle seroneutralization assay for tick-borne encephalitis virus overcomes ELISA limitations

“…Despite the availability of effective vaccines, TBEV continues to pose a considerable threat to public health due to its expanding geographical distribution and the increasing number of reported cases (1) RVP-based SNTs for different orthoflaviviruses have been established using different cell lines, including BHK-J cells (24), BHK-21 cells (26), Vero cells (25), and HUH-7.5 cells (27). In our experiments, we found that HUH7 cells were well suited for the effective execution of TBEV, YFV, DENV, ZIKV, WNV, and JEV RVP SNTs.…”

Genotype-specific features reduce the susceptibility of South American yellow fever virus strains to vaccine-induced antibodies

“…Neutralizing activity in sera from the U.S. donors was measured at days 0,10,14,16,28,90,180,270 and 360 post vaccination (Figure 1A-B) with a widely employed West Nile virus-based reporter viral particle system (RVP) (Pierson et al, 2006). RVPs faithfully recapitulate the behavior of authentic flaviviruses in studies of virus structure, function, and antibody recognition and neutralization (Bohning et al, 2021;Bradt et al, 2019;Dowd et al, 2011;Goo et al, 2016Goo et al, , 2017Mattia et al, 2011) (Figure S1). Serum neutralizing titers against RVPs bearing the structural proteins C-prM-E from the YF-17D vaccine strain (RVP17D) and the virulent African strains Asibi (RVPAsibi) and China (ex-Angola) (RVPChina) (Figure 1A) rose sharply, peaked at days 14-16, and remained stable thereafter, as reported previously (Lindsey et al, 2018;Wec et al, 2020;Wieten et al, 2016).…”