Uncovering early-stage vaccine candidates against Fasciola hepatica through integrative comparative proteomic analysis and proof-of-concept CRISPR-Cas9 gene editing

Fasciolosis, mainly caused by the liver fluke Fasciola hepatica , is a globally distributed parasitic disease that affects both livestock and humans, generating significant economic losses and public health concerns. Current control strategies rely mainly on anthelmintic treatments, but the increasing emergence of drug-resistant parasite populations highlights the urgent need for alternative approaches. Although numerous vaccine candidates have been investigated over the past decades, the development of an effective commercial vaccine has been hindered by inconsistent protection across host species and experimental conditions, one of the main limitations being the difficulty in selecting and validating suitable antigens. We implemented a bioinformatic prioritization pipeline by integrating stage-specific proteomic datasets (including ex vivo, in vivo, and secretomes) of early F. hepatica infection. Candidates were selected based on their consistent detection across all datasets and analysed their structural stability, predicted solubility, and antigenic potential. Spatial localization of the prioritized proteins was evaluated using confocal immunofluorescence in newly excysted juveniles (FhNEJ) and migratory stages. Furthermore, a CRISPR-Cas9-based gene editing approach was established in FhNEJ to assess the feasibility of functional disruption at this stage. Comparative analysis identified a core proteome of six proteins consistently detected across all early infection datasets. From this set, cathepsin B3 (FhCB3), legumain 1 (FhLeg1), and serpin 1 (FhSrp1) were prioritized as high-potential vaccine antigens. Immunofluorescence confirmed that these proteins are localized on the parasite surface, ensuring their exposure to the host immune system. CRISPR-Cas9 induced targeted mutations with editing efficiencies of 3.2% for FhCB3 and 2.1% for FhLeg1, demonstrating the feasibility of CRISPR-based gene editing in FhNEJ. This study establishes an integrated framework for the rational prioritization of vaccine candidates and provides a technical milestone with the first application of CRISPR-Cas9 in FhNEJ. These findings support the use of host-exposed antigens as promising targets for the development of sustainable control strategies against fasciolosis.