Systematic antigenic characterisation of Oropouche virus glycoproteins using a pseudotyped virus platform: implications for vaccine antigen selection

Background Oropouche virus (OROV) is an emerging orthobunyavirus responsible for widespread febrile illness across the Americas, with recent epidemiological modelling estimating over 9.4 million infections between 1960 and 2025. The virus underwent unprecedented geographic expansion during 2023–2024, leading to imported cases in non-endemic regions worldwide. Currently, no licenced vaccine is available, and most vaccine development efforts rely on the historical prototype strain BeAn19991. However, the antigenic properties of the OROV glycoprotein complex remain incompletely defined, hindering rational vaccine design. Methods We generated OROV pseudovirions expressing the Gn and Gc glycoprotein complex (GPC) using a vesicular stomatitis virus (VSV)-based system. A robust in vitro neutralisation assay was established to quantify neutralising antibody titres against these pseudovirus. Representative strains from historical lineages and contemporary circulating variants (2022–2024) were systematically characterised through phylogenetic analysis and cross-neutralisation assays using female guinea pig sera. Findings Phylogenetic analysis of the OROV GPC identified four distinct genetic clades: clade I (Brazilian strains isolated between 1960 and 2006), clade II (Ecuador 2016 and Peru 2024 isolates), clade IIIa (Brazilian strains from 2009 to 2018), and clade IIIb (epidemic strains circulating during 2022–2024). While all representative strains elicited comparable homologous neutralisation titres, significant antigenic divergence was observed among heterologous strains. Sera induced by the prototype strain BeAn19991(belonging to Clade Ⅰ) exhibited markedly reduced neutralising activity (>4-fold reduction) against heterologous OROV strains and provided limited protection against clade IIIb circulating variants. In contrast, sera induced by AJT39490, which belongs to Clade IIIa, demonstrated broad-spectrum cross-neutralising activity against all tested OROV strains, highlighting its cross-protective potential as a candidate antigen with broad cross-neutralising capability. Interpretation This study provides a systematic characterisation of OROV glycoprotein antigenicity using a pseudotyped virus platform. Our findings demonstrate substantial antigenic diversity among OROV strains, revealing that the historical prototype strain BeAn19991 may not provide sufficient protection against contemporary circulating variants. Notably, the broad cross-neutralisation capacity exhibited by AJT39490-induced sera highlights its potential as a candidate antigen for inducing protective immunity against diverse OROV lineages, addressing a critical gap in current OROV vaccine development efforts.