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

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.

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

“…Reporter virus particles (RVP) were purchased from Integral Molecular (Philadelphia, PA) ZIKV reporter virus particles (Z-RVPs) contain CprM/E from ZIKV strain SPH2015 packaged within the particle a DENV-based replicon with the Renilla luciferase gene [ 32 ]. The Z-RVP reagent is replication deficient but is capable of infecting permissive cells to deliver the replicon which drives the expression of Renilla luciferase.…”

“…Z-RVP reagent assessment. The structure, antigenicity, stability and reproducibility of the Z-RVP reagent has been described [32]. The Z-RVP reagent was assessed for relative infectivity and cytotoxicity.…”

“…Reporter virus particles (RVPs) have been used as a substitute for the live virus reagent in neutralization assays to measure antibodies against several flaviviruses including DENV, YFV, JEV, and West Nile Virus (WNV) [22][23][24][25][26][27][28], and most recently with ZIKV [16,28,29]. RVPs are replication-deficient and deliver their reporter gene to permissive cells [26][27][28][30][31][32].…”

“…Flavivirus RVPs containing the virus surface antigens (prM/M and E) have been shown to be antigenically indistinguishable from infectious virus using virus-specific antibodies [26][27][28][30][31][32]. DENV neutralizing monoclonal antibody (mAb) 4G2 neutralized all four serotypes of DENV-RVPs while non-neutralizing mAb 15F3 did not neutralize any [24].…”

Distinct cellular immune signatures in acute Zika virus infection are associated with high or low persisting neutralizing antibody titers

“…Anti-Zika virus IgM and IgG were measured by antibody-capture ELISA using recombinant ZIKV antigen kindly provided by the US Centers for Disease Control and Prevention (CDC) and as previously described ( 75, 76 ). ZIKV neutralizing titers were measured using a ZIKV reporter viral particle neutralization titration assay (Integral Molecular, Philadelphia, PA) ( 77 ), and index donations were tested for pre-existing DENV IgG with the Detect IgG ELISA (InBios; Seattle, WA).…”