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. In particular, cytotoxic cells have emerged as key components to control ASF virus (ASFV) infection. However, the contribution of individual cytotoxic subsets across different virological and immunological contexts is not well characterised. Here, we investigated cytotoxic responses during BA71{Delta}CD2 live attenuated vaccine (LAV)-induced protection and during late-stage lethal ASFV infection, and demonstrated the involvement of different cytotoxic subsets in each scenario. Early increases in perforin-producing CD8{beta}+ T cells in blood after immunisation coincided with the onset of protection. At later time points, elevated levels of these cells after in vitro ASFV-specific stimulation correlated with survival to lethal challenge, supporting their central role in protective immunity. Additional correlates of protection during recall responses included CD4+CD8{beta}+ cytotoxic T cells, IFN{gamma}-producing cells, and ASFV-specific antibodies, illustrating the multifactorial nature of immunity to ASF. In contrast, pigs with acute ASF exhibited a distinct cytotoxic profile characterised by broad increases across multiple perforin-producing subsets. Although all of them showed reduced susceptibility to ASFV-induced lymphopenia, only perforin-producing NK and{gamma}{delta} T cells correlated with viremia, suggesting their active involvement during late disease. Together, these findings advance our understanding of cytotoxic responses to ASFV and identify cytotoxic T cells, alongside other immune components, as potential correlates of protection that may guide future vaccine development.