Garg Himanshu, Sedano Melina, Plata Gabrielle, Punke Erin B, Joshi Anjali
Center of Emphasis in Infectious Diseases, Department of Biomedical Sciences, Texas Tech University Health Sciences Center, El Paso, Texas, USA
Center of Emphasis in Infectious Diseases, Department of Biomedical Sciences, Texas Tech University Health Sciences Center, El Paso, Texas, USA.
J Virol. 2017 Sep 27;91(20). doi: 10.1128/JVI.00834-17. Print 2017 Oct 15.
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. 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.
近期寨卡病毒（ZIKV）感染在全球范围内爆发，且缺乏获批疫苗，这引发了人们对对抗这种新兴病毒的准备工作的严重担忧。我们采用基于病毒样颗粒（VLP）的方法来研发一种寨卡病毒疫苗及微中和试验。寨卡病毒的合成衣壳 - 前膜 - 包膜（C - prM - E）基因构建体被用于生成报告病毒颗粒（RVP），其包装了表达绿色荧光蛋白（GFP）报告基因的西尼罗河病毒（WNV）复制子。该试验采用了96孔板形式，类似于蚀斑减少中和试验（PRNT），并显示出对寨卡病毒中和抗体的特异性检测具有高重现性。此外，C - prM - E和prM - E病毒样颗粒作为候选疫苗在小鼠中进行了测试，并与DNA疫苗进行了比较。虽然单独的寨卡病毒prM - E构建体足以生成病毒样颗粒，但在西尼罗河病毒NS2B - 3蛋白酶存在的情况下，可从C - prM - E构建体高效生产病毒样颗粒，该蛋白酶可将C从prM上切割下来，从而使病毒得以释放。小鼠免疫研究表明，病毒样颗粒产生的中和抗体滴度高于DNA疫苗，C - prM - E病毒样颗粒产生的滴度略高于prM - E病毒样颗粒。C - prM - E病毒样颗粒的优越性表明，包含衣壳可能对寨卡病毒和其他黄病毒属病毒样颗粒疫苗有益。为促进病毒样颗粒平台的发展，我们生成了一个稳定表达高水平寨卡病毒prM - E蛋白的细胞系，其可组成性地产生病毒样颗粒，以及一个表达寨卡病毒C - prM - E蛋白用于生产报告病毒颗粒的细胞系。虽然已经提出了几种针对寨卡病毒的疫苗平台，但本研究描述了一种安全、有效且经济的基于病毒样颗粒的寨卡病毒疫苗。为应对日益严重的寨卡病毒疫情，我们开展了本研究，有两个目标：第一，研发一种安全、有效且经济的寨卡病毒疫苗；第二，研发一种快速且通用的试验来检测抗寨卡病毒免疫反应。我们生成了一个稳定表达寨卡病毒prM - E的细胞系，其在上清液中产生大量病毒样颗粒，以及一个寨卡病毒C - prM - E细胞系，在用GFP复制子质粒转染后产生报告病毒颗粒。prM - E病毒样颗粒在小鼠中诱导了强烈的中和抗体反应，当包含衣壳时效果更好。基于病毒样颗粒的疫苗显示出比其DNA对应物显著更好的中和抗体反应。基于报告病毒颗粒的微中和试验与蚀斑减少中和试验的工作方式类似，在96孔板形式中可快速读取GFP信号。我们基于病毒样颗粒 的平台为寨卡病毒疫苗和诊断提供了一个来源，可迅速适应当前的疫情爆发。
