The first phase of the project focused on the evaluation of three naturally attenuated ASFV vaccine prototypes. The candidate Lv17/WB/Rie1 (genotype II) provided 100% protection in domestic pigs without clinical signs and 92% protection in orally vaccinated wild boar, representing the first evidence of a promising oral vaccine for this species. The second prototype, NH/P68 (genotype I), was adapted to the MA104 cell line and demonstrated 100% protection in domestic pigs with reduced side effects, while studies in wild boar continued. The third prototype, ASF/ARRIAH/CV-1, was characterised but had not yet undergone in vivo evaluation during the initial period. Several DIVA antigens were identified, deletion mutants were generated from Lv17/WB/Rie1 for in vivo testing, and preliminary work began on identifying suitable cell lines for vaccine production.
During the second reporting period, full-genome analyses guided the generation of 11 deletion mutants of Lv17/WB/Rie1 and one mutant of NH/P68 using CRISPR-CAS9. WSL and MA104 cell lines were advanced for production, and the Lv17/WB/Rie1-ΔCD mutant was shown to be safe in wild boar, with both low-dose/high-dose and high-dose single-administration schemes providing high protection. A preliminary production process was established, and both freeze-dried and liquid formulations were found feasible. A triplex RT-PCR assay was developed, preliminary serological DIVA tools were produced, and epidemiological modelling progressed with updated European ASF scenarios, eco-epidemiological analyses and the definition of region-specific baiting strategies for wild boar.
In the final period, a total of 17 deletion mutants were generated, including ΔCD, ΔCD-I8 and ΔCD-D3. In domestic pigs, the ΔCD prototype provided 100% protection, showed an improved safety profile, and did not transmit to contact animals; the derived mutants also maintained efficacy with enhanced safety. In wild boar, double-dose oral vaccination with ΔCD-based candidates conferred full protection under experimental conditions. For vaccine production, MA104 and IPAM cell lines were further optimised, a GMP-compliant Master Cell Seed was established and vaccine batches were confirmed to be genetically stable and suitable for lyophilisation. The triplex real-time PCR DIVA assay was adapted to a commercial format, recombinant proteins were produced for serological DIVA testing, and both molecular and serological DIVA tools were validated using experimental and field samples, including through an international proficiency test.
Substantial progress was also made in epidemiological modelling, risk mapping, wild boar movement and behaviour analyses, and ASF modelling in Europe and Asia. Field baiting studies assessed bait uptake, selectivity and deployment conditions to support future oral vaccination. Dissemination and exploitation activities included extensive stakeholder engagement, scientific communication efforts and the preparation of five patent applications related to vaccine candidates and DIVA diagnostics. Together, these results provide a strong basis for the future exploitation of the vaccine prototypes, diagnostic tools and modelling platforms developed during the project.
