A reliable, rapid, and high-throughput assay for DENV neutralization antibodies is critical for laboratory and clinical studies of DENV infection and vaccine. Considering the limitations of plaque based assay, some novel methods for neutralizing assays have been described [12–18]. Che and coworkers recently developed a novel ELISPOT based neutralization test, demonstrating a well correlation with the conventional PRNT assay . Pseudo infectious DENV reporter virus particles (RVP) carrying green fluorescent protein (GFP) reporter were also used to measure neutralization antibodies with rapidity, stability and reproducibility [15, 16, 20]. Infection with RVP could be monitored by the GFP signals using flow cytometry. However, GFP is not suitable for real-time quantification, and production of RVP requires special cell lines and replicon based plasmids. Live reporter virus carrying luciferase reporter replicates almost the same as wild type virus, representing a more advanced tool. Many reporter viruses, including SARS-related corona virus, human hepatitis C virus, parainfluenza virus, HIV, adenovirus, have been described and well applied for antiviral screening, live imaging, or function studies [21–25]. Live reporter DENV engineering a reporter gene at the capsid gene has been developed . Recently, we described the stable luciferase DENV reporter virus Luc-DENV and used it for high-throughput screening for antiviral drugs . In this study, we demonstrated the utility of Luc-DENV for measuring neutralization and enhancing antibodies. Using three identified neutralizing mAbs, Luc-based assay showed well correlation with the PRNT-based assay. 4G2 and 2B8 are both IgG1 isotype mAbs, and 2A10G6 belongs to IgG2a isotype. 2B8 recognizes the domain III of DENV E protein and inhibit viral binding, while 2A10G6 and 4G2 inhibit fusion. All three mAbs were active in inhibiting plaque forming and Luc expression in Luc-DNEV infected Vero cells. The value of PRNT50 and LRNT50 are well correlated (R2 > 0.95). The Luc-based assay was readily applied in evaluation of clinical samples from vaccinated animals and infected patients.
ADE infection of DENV has been well demonstrated in vitro and in vivo, and represents one of the major impediments against vaccine development. Previously, different methods based on infection rate [27, 28], progeny viral yield , and number of infectious centers [30, 31] have been reported to measure the ADE activity in FcR expressing cells including K562, U937 or THP-1 cells. The FACS analysis has been commonly used to quantify the infection rate in C6/36 cells, Raji B, and human peripheral blood mononuclear cells [32, 33]. Progeny viral yield can be detected either by conventional plaque assay or NS1-based ELISA , ELISPOT , and real-time RT-PCR . Recently, Moi et al. successfully established stable BHK-21 cell lines that express FcRIIA, which facilitate both neutralization and ADE assay.
The plaque based assay determined the infectious particles released from virus-infected cells, whereas the RLU based assay described in this study offered a simple method which detected viral protein expression in cells. Linear correlation was established between the two assays for both neutralization and ADE assays (Figure 1D and Figure 2B). The newly developed assay method is comparable to the traditional plaque assay, with some unique advantages. First, this Luc-based assay is more substantial and time saving. The conventional plaque test used 12-well plates and 5–7 days observation for the plaque forming, the new test is compared performing the same protocol involved 24-well plates and cost no more than 2 days. Second, this new assay method has a more wide-range scope of application with high repetitiveness and reliability. Luc-DENV replicates well in multiple cells including BHK-21, K562, Vero and THP-1 and A549 cells, and luciferase activity can also be detected stably in various cells. Neutralization and ADE assays can be performed in the same cells . Third, this new assay method is easy to adapt for a high-throughput manner , which is of critical importance for large-scale clinical samples assays during clinical trials of dengue vaccine.