Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

Abstract

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 (CprM-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.KEYWORDS C-prM-E, diagnostics, microneutralization, neutralization, PRNT, reporter, reporter virus particles, vaccine, Zika, prM-E S ince the identification of Zika virus (ZIKV) from a rhesus monkey in Uganda in 1947 (1, 2) until 2010, the virus predominantly circulated between Aedes mosquitoes and nonhuman primates. Periodic episodes were inde...

Virus Like Particles (VLP) as multivalent vaccine candidate against Chikungunya, Japanese Encephalitis, Yellow Fever and Zika Virus

“…While a number of arboviral VLPs have been developed including some for JEV and DENV, most of these studies utilize prME expression and the particles lack Capsid protein 15 . The inclusion of Capsid protein in all of our VLP preps provides an additional antigen and better immunogenicity as evident from our Zika studies 13,14 . Furthermore, previous studies on arboviral VLPs including CHIKV have been conducted in monovalent forms and there is no study on whether a multivalent VLP vaccine against related arboviruses would provide effective immunity 22 .…”

“…Bicistronic vector expressing flaviviral CprME and Zika NS2B3 produces Capsid protein containing VLPs. The presence of Capsid protein in a flaviviral vaccine can provide an additional immunogen and higher neutralizing antibody titers as seen in our Zika VLP vaccine studies 13,14 . In order to produce CprME VLPs, NS2B3 expression can be achieved either in trans as in Fig.…”

“…For ZIKV, YFV and JEV it included the CprME genes and for CHIKV the C-E3-E2-E1 genes. We have previously developed a VLP based platform for Zika Virus using a codon optimized synthetic gene construct 13 and used a similar approach for developing structural protein constructs for the other arboviruses with minor modifications. We developed a consensus sequence for CHIKV, JEV and YFV to include the most representative circulating virus sequences in our vaccine platform.…”

“…In case of flaviviruses, the structural proteins (CprME) from a separate plasmid can complement a subgenomic WNV replicon expressing a GFP reporter gene to generate RVPs. This RVP based system has been shown to work well for different flaviviruses including WNV and Zika 13,21 . We generated RVPs expressing JEV and YFV and ZIKV structural proteins and tested them for infectivity in Vero cells.…”

“…The success of the multivalent Human Papilloma Virus (HPV) vaccine has demonstrated the safety and efficacy of VLPs as vaccines 12 . Previously, we developed a VLP vaccine candidate for Zika virus and found that the immunogenicity of Capsid protein containing VLPs (CprME) was better than prME VLPs 13 . Furthermore, the use of stable cell lines to generate VLPs provides a much needed platform for the advancement and commercialization of this technology 14 .…”