Institute of Preventive Medicine, National Defense Medical Center, 237010 No. 172, Dapu Rd., Sanxia Dist, Taipei, 11490, Taiwan.
Department and Graduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei, 11490, Taiwan.
BMC Infect Dis. 2024 Sep 30;24(1):1076. doi: 10.1186/s12879-024-09973-y.
Viral neutralization (NT) assays can be used to determine the immune status of patients or assess the potency of candidate vaccines or therapeutic monoclonal antibodies (mAbs). Focus reduction neutralization test (FRNT) is a conventional neutralization test (cVNT) with superior specificity for measurement of neutralizing antibodies against a specific virus. Unfortunately, the application of FRNT to the chikungunya virus (CHIKV) involves a highly pathogenic bio-agent requiring biosafety level 3 (BSL3) facilities, which inevitably imposes high costs and limits accessibility. In this study, we evaluated a safe surrogate virus neutralization test (sVNT) that uses novel CHIKV replicon particles (VRPs) expressing eGFP and luciferase (Luc) to enable the rapid detection and quantification of neutralizing activity in clinical human serum samples.
This unmatched case-control validation study used serum samples from laboratory-confirmed cases of CHIKV (n = 19), dengue virus (DENV; n = 9), Japanese encephalitis virus (JEV; n = 5), and normal individuals (n = 20). We evaluated the effectiveness of sVNT, based on mosquito cell-derived CHIK VRPs (mos-CHIK VRPs), in detecting (eGFP) and quantifying (Luc) neutralizing activity, considering specificity, sensitivity, and reproducibility. We conducted correlation analysis between the proposed rapid method (20 h) versus FRNT assay (72 h). We also investigated the correlation between sVNT and FRNT in NT titrations in terms of Pearson's correlation coefficient (r) and sigmoidal curve fitting.
In NT screening assays, sVNT-eGFP screening achieved sensitivity and specificity of 100%. In quantitative neutralization assays, we observed a Pearson's correlation coefficient of 0.83 for NT50 values between sVNT-Luc and FRNT.
Facile VRP-based sVNT within 24 h proved highly reliable in the identification and quantification of neutralizing activity against CHIKV in clinical serum samples.
病毒中和（NT）测定可用于确定患者的免疫状态，或评估候选疫苗或治疗性单克隆抗体（mAb）的效力。焦点减少中和试验（FRNT）是一种常规中和试验（cVNT），对测量针对特定病毒的中和抗体具有更高的特异性。不幸的是，将 FRNT 应用于基孔肯雅病毒（CHIKV）涉及到一种具有高致病性的生物制剂，需要生物安全 3 级（BSL3）设施，这不可避免地带来了高昂的成本和限制了可及性。在本研究中，我们评估了一种安全的替代病毒中和试验（sVNT），该试验使用新型 CHIKV 复制子颗粒（VRP）表达 eGFP 和荧光素酶（Luc），以快速检测和定量临床人血清样本中的中和活性。
这项非匹配病例对照验证研究使用了实验室确诊的 CHIKV 病例（n=19）、登革热病毒（DENV；n=9）、日本脑炎病毒（JEV；n=5）和正常个体（n=20）的血清样本。我们根据蚊子细胞来源的 CHIK VRP（mos-CHIK VRP）评估了 sVNT 的有效性，以检测（eGFP）和定量（Luc）中和活性，考虑了特异性、敏感性和重现性。我们进行了快速方法（20 小时）与 FRNT 测定（72 小时）之间的相关性分析。我们还研究了 sVNT 和 FRNT 在 NT 滴定中的相关性，采用 Pearson 相关系数（r）和 S 型曲线拟合。
在 NT 筛选试验中，sVNT-eGFP 筛选的敏感性和特异性均为 100%。在定量中和试验中，我们观察到 sVNT-Luc 和 FRNT 的 NT50 值之间存在 0.83 的 Pearson 相关系数。
在 24 小时内，基于 VRP 的简便 sVNT 在鉴定和定量临床血清样本中的 CHIKV 中和活性方面具有高度可靠性。
