Distinct cytotoxic cell subsets underlie protective and non-protective immunity to African swine fever virus.

Limited understanding of African swine fever (ASF) immunity remains a major barrier to the rational development of safe and effective vaccines. While antibody-mediated protection is still poorly defined, growing evidence highlights a central role for cellular immunity, particularly cytotoxic cells, as components associated with ASF virus (ASFV) infection control. However, the contribution of individual cytotoxic subsets across different virological and immunological contexts is not well characterized. Here, we investigated cytotoxic responses during BA71ΔCD2 live attenuated vaccine-induced protection and during late-stage lethal ASFV infection. Protective immunity was characterized by antigen-specific cytotoxic T-cell responses, whereas acute ASF was associated with broad cytotoxic activation. Early increases in perforin-positive CD4-CD8αβ+ T cells coincided with the onset of protection, and elevated recall responses correlated with survival following lethal challenge, supporting their association with protective immunity. Additional correlates of protection included CD4+CD8αβ+ cytotoxic T cells, IFNγ-producing cells, and specific antibodies, illustrating the multifactorial nature of protective immunity. In contrast, pigs with acute ASF showed broad cytotoxic expansion, perforin-positive natural killer and γδ T cells showing the strongest association with viremia. Together, these findings advance our understanding of cytotoxic responses to ASFV and identify perforin-positive T cells, alongside complementary immune components, as potential correlates of protection for future vaccine development.