Yamanaka Atsushi, Suzuki Ryosuke, Konishi Eiji
BIKEN Endowed Department of Dengue Vaccine Development, Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand; BIKEN Endowed Department of Dengue Vaccine Development, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
Vaccine. 2014 Jul 23;32(34):4289-95. doi: 10.1016/j.vaccine.2014.06.017. Epub 2014 Jun 17.
Dengue fever and dengue hemorrhagic fever are endemic throughout tropical and subtropical countries. Four serotypes of dengue viruses (DENV-1 to DENV-4), each with several genotypes including various subclades, are co-distributed in most endemic areas. Infection-neutralizing and -enhancing antibodies are believed to play protective and pathogenic roles, respectively. Measurement of these functional antibodies against a variety of viral strains is thus important for evaluating coverage and safety of dengue vaccine candidates. Although transportation of live virus materials beyond national borders is increasingly limited, this difficulty may be overcome using biotechnology that enables generation of an antibody-assay antigen equivalent to authentic virus based on viral sequence information. A rapid system to produce flavivirus single-round infectious particles (SRIPs) was recently developed using a Japanese encephalitis virus (JEV) subgenomic replicon plasmid. This system allows production of chimeric SRIPs that have surface proteins of other flaviviruses. In the present study, SRIPs of DENV-1 (D1-SRIPs) were evaluated as an antigen for functional antibody assays. Inclusion of the whole mature capsid gene of JEV into the replicon plasmid provided higher D1-SRIP yields than did its exclusion in cases where a DENV-1 surface-protein-expressing plasmid was used for co-transfection of 293T cells with the replicon plasmid. In an assay to measure the balance between neutralizing and enhancing activities, dose (antibody dilution)-dependent activity curves in dengue-immune human sera or mouse monoclonal antibodies obtained using D1-SRIP antigen were equivalent to those obtained using DENV-1 antigen. Similar results were obtained using additional DENV-2 and DENV-3 systems. In a conventional Vero-cell neutralization test, a significant correlation was shown between antibody titers obtained using D1-SRIP and DENV-1 antigens. These results demonstrate the utility of D1-SRIPs as an alternative antigen to authentic DENV-1 in functional antibody assays. SRIP antigens may contribute to dengue vaccine candidate evaluation, understanding of dengue pathogenesis, and development of serodiagnostic systems.
登革热和登革出血热在热带和亚热带国家呈地方性流行。登革病毒有4种血清型（DENV-1至DENV-4），每种血清型都有几种基因型，包括各种亚分支，在大多数流行地区共同分布。感染中和抗体和增强抗体分别被认为起着保护和致病作用。因此，针对多种病毒株检测这些功能性抗体对于评估登革热候选疫苗的覆盖范围和安全性很重要。尽管将活病毒材料运输到国外的限制越来越多，但利用生物技术，根据病毒序列信息生成与真实病毒等效的抗体检测抗原，或许可以克服这一困难。最近利用日本脑炎病毒（JEV）亚基因组复制子质粒开发了一种快速生产黄病毒单轮感染性颗粒（SRIP）的系统。该系统可以生产具有其他黄病毒表面蛋白的嵌合SRIP。在本研究中，评估了DENV-1的SRIP（D1-SRIP）作为功能性抗体检测的抗原。当使用表达DENV-1表面蛋白的质粒与复制子质粒共转染293T细胞时，将JEV的整个成熟衣壳基因纳入复制子质粒比不纳入时能产生更高产量的D1-SRIP。在一项检测中和与增强活性平衡的试验中，使用D1-SRIP抗原在登革热免疫人血清或小鼠单克隆抗体中获得的剂量（抗体稀释）依赖性活性曲线与使用DENV-1抗原获得的曲线相当。使用额外的DENV-2和DENV-3系统也得到了类似结果。在传统的Vero细胞中和试验中，使用D1-SRIP和DENV-1抗原获得的抗体滴度之间显示出显著相关性。这些结果证明了D1-SRIP作为功能性抗体检测中替代真实DENV-1的抗原的实用性。SRIP抗原可能有助于登革热候选疫苗的评估、登革热发病机制的理解以及血清诊断系统的开发。
