SYN023, a Humanized Monoclonal Antibody Cocktail, Broadly Neutralizes Genetically Diverse Lyssaviruses and Validates Epitope Prediction Accuracy

Background/Objectives: Lyssaviruses pose a persistent global public health threat, underscoring the need for broad-spectrum neutralizing antibodies to improve rabies post-exposure prophylaxis (PEP). The humanized monoclonal antibody cocktail SYN023 is approved for PEP in China and under active global regulatory review. This study aimed to validate pseudovirus-based neutralization assays, verify SYN023 epitope prediction accuracy, and systematically characterize its pan-lyssavirus neutralizing activity. Methods: Assay validation was performed by directly comparing neutralization titers obtained with authentic rabies virus (RABV) and pseudotyped virus. Epitope analysis was performed, followed by in vivo evaluation in a canine PEP challenge model. The pseudovirus-based neutralization assay was used to assess SYN023 broad-spectrum activity across diverse lyssaviruses. Results: Neutralization titers from authentic and pseudotyped viruses showed high concordance (R2 = 0.8341, p < 0.0001), supporting the pseudovirus system as a reliable surrogate assay. Epitope analysis confirmed that SYN023’s epitope is fully conserved in the highly virulent New York City (NYC) RABV strain. In canine PEP challenge studies using the NYC strain, SYN023 conferred strong protection at 0.1–0.5 mg/kg. SYN023 exhibited potent neutralizing activity against phylogenetically diverse non-rabies lyssaviruses, including Mokola virus (MOKV; EC50 = 14.74 µg/mL), whereas three clinically validated reference antibodies failed to neutralize MOKV even at concentrations up to 1000 µg/mL. Conclusions: This study demonstrates that the pseudovirus-based neutralization assay is a reliable surrogate assay. It also confirms the functional accuracy of the epitope mapping strategy and demonstrates the broad neutralization spectrum of SYN023 against a diverse panel of lyssaviruses.