A Non-Hemadsorbing Live-Attenuated Virus Vaccine Candidate Protects Pigs against the Contemporary Pandemic Genotype II African Swine Fever Virus

African swine fever (ASF) is a highly contagious and severe hemorrhagic transboundary swine viral disease with up to a 100% mortality rate, which leads to a tremendous socio-economic loss worldwide. The lack of safe and efficacious ASF vaccines is the greatest challenge in the prevention and control of ASF. In this study, we generated a safe and effective live-attenuated virus (LAV) vaccine candidate VNUA-ASFV-LAVL3 by serially passaging a virulent genotype II strain (VNUA-ASFV-L2) in an immortalized porcine alveolar macrophage cell line (3D4/21, 50 passages). VNUA-ASFV-LAVL3 lost its hemadsorption ability but maintained comparable growth kinetics in 3D4/21 cells to that of the parental strain. Notably, it exhibited significant attenuation of virulence in pigs across different doses (10³, 10⁴, and 10⁵ TCID₅₀). All vaccinated pigs remained healthy with no clinical signs of African swine fever virus (ASFV) infection throughout the 28-day observation period of immunization. VNUA-ASFV-LAVL3 was efficiently cleared from the blood at 14-17 days post-infection, even at the highest dose (10⁵ TCID₅₀). Importantly, the attenuation observed in vivo did not compromise the ability of VNUA-ASFV-LAVL3 to induce protective immunity. Vaccination with VNUA-ASFV-LAVL3 elicited robust humoral and cellular immune responses in pigs, achieving 100% protection against a lethal wild-type ASFV (genotype II) challenge at all tested doses (10³, 10⁴, and 10⁵ TCID₅₀). Furthermore, a single vaccination (10⁴ TCID₅₀) provided protection for up to 2 months. These findings suggest that VNUA-ASFV-LAVL3 can be utilized as a promising safe and efficacious LAV candidate against the contemporary pandemic genotype II ASFV.