Luciferase unleashed: Lighting the path to vaccine approval using a novel high-throughput chikungunya virus neutralization assay

Chikungunya virus (CHIKV) neutralization assays, which measure virus-neutralizing activity, play a pivotal role in evaluating vaccine effectiveness and monitoring immunity. As CHIKV continues to spread beyond endemic tropical regions, causing more frequent outbreaks and debilitating disease, it remains a serious global health concern. Vaccination can help alleviate these burdens by inducing neutralizing antibodies, which are also produced following natural infection. Neutralizing antibodies can prevent virus infection and are considered a key correlate of protection against disease caused by CHIKV. Accurate assessment of neutralizing antibodies is therefore essential for both vaccine evaluation and public health surveillance. This work introduces a validated luciferase-based high-throughput CHIKV neutralization assay (CHIKV-luc NT80) for quantifying serum neutralizing antibodies. The assay uses a genetically engineered reporter virus (CHIKV181/25-luc) that expresses luciferase, allowing in vitro viral infectivity to be measured using luminescence rather than manual plaque counting. Compared to the traditional plaque reduction neutralization test (PRNT), this method is faster, easier, more reproducible, and better suited for large-scale clinical studies associated with a high biological sample burden, due to its scalability. The use of a stringent 80% neutralization titer threshold (NT80) also provides a more stringent measure of antibodies compared to conventional NT50 values. The assay was compared to other methods, having high concordance (r ≈ 0.9) and low bias (≤15%), supporting its analytical reliability, qualified for phase 2 clinical trials, and validated for phase 3 clinical sample testing. The assay contributed to the establishment of the first World Health Organization International Standard for anti-CHIKV (code number 1502/19), and its results harmonized well with other virus neutralization methods. Neutralization titer results from preclinical and clinical serum sample testing supported the characterization of a surrogate threshold of protection, and licensure of VIMKUNYA (CHIKV virus-like particle [VLP] vaccine).