Review of in vitro methods for foot-and-mouth disease vaccine immunogenicity assessment

Vaccination is widely used to control foot-and-mouth disease (FMD), necessitating robust approaches for evaluating vaccine potency to ensure a consistent supply of effective and safe vaccines. Traditional (in vivo) potency testing methods rely on live animal models, raising ethical concerns, logistical constraints, procedural variability and significant financial costs. In vitro approaches have therefore been increasingly explored to complement these methods and address these challenges. Recent advancements in in vitro methodologies have focused on assessing vaccine-induced immune responses and virus-neutralizing activity using a variety of innovative cell-based assays, recombinant protein-based immunoassays, and molecular techniques, all of which have shown promise as surrogate tools that support vaccine potency evaluation. However, several challenges persist in optimizing these in vitro methods. Key obstacles include the standardization of assay protocols, ensuring reproducibility across different laboratories, and addressing the complexities of immune responses that occur in vivo, which are difficult to replicate in in vitro environments. Furthermore, there remains a need for robust validation of in vitro assays using a broad range of field isolates to ensure their relevance to diverse viral strains. This review examines the current landscape of in vitro approaches supporting FMD vaccine-induced immune responses, detailing both the advancements made in assay development and the challenges that remain to be overcome. Furthermore, there is discussion of the role of these methods in complementing conventional animal-based testing, particularly as non-invasive endpoints aligned with the principles of the 3Rs (replacement, reduction, and refinement), with potential to improve efficiency, ethical standards, and global FMD control strategies.