Validation and evaluation of the rapid fluorescent focus inhibition test (RFFIT) for the quantification of neutralizing antibodies as an indirect measure of rabies vaccine relative potency: An alternative to the NIH test.

The quality control of rabies vaccines is essential for ensuring their safety and effectiveness in preventing the fatal disease that causes approximately 59,000 human deaths annually [1]. Vaccine potency is an attribute that reflects the vaccine's ability to elicit a protective immune response and must meet international standards: >2.5 IU/dose for human vaccines and > 1.0 IU/dose for veterinary vaccines [2,3]. Rabies vaccine potency is traditionally assessed using the National Institutes of Health (NIH) test, an in vivo method mandated by regulation, which has shown limitations such as high variability, ethical concerns, high costs, and time-consuming procedures. The Rapid Fluorescent Focus Inhibition Test (RFFIT), a semi-in vitro method, quantifies rabies virus neutralizing antibodies (RVNA) in serum. This RVNA titer value can be later translated into a potency value when calibrated against a reference vaccine, offering a more efficient alternative. Compared to NIH tests, RFFIT has lower variability, shorter processing time, and poses fewer ethical concerns, all while maintaining accuracy and reliability. This study validates RFFIT for RVNA determination, which will later be used to indirectly measure rabies vaccine potency, following international bioanalytical validation guidelines and evaluating its specificity, accuracy, precision, linearity, linear range, limit of quantification, and stability. Our results demonstrate that the RFFIT meets all acceptance criteria, showing robustness and reproducibility. Furthermore, when testing potency for 14 commercial veterinary vaccine batches, RFFIT showed a 92.85 % concordance with the NIH test, reinforcing its potential as a direct replacement for the NIH test. RFFIT could enhance vaccine quality control, providing a more efficient and standardized approach, implying a significant reduction in the number of animals used for potency testing.