Functional analysis and epitope recognition of African swine fever virus pA151R antigen-specific T cells

• ASFV non-structural protein pA151R-specific T cells were characterized in immune-protected pigs using advanced immunological techniques suitable for swine. • T-cell epitopes of pA151R recognized by swine T cells were mapped. Both pA151R-specific and epitope peptide-specific CD4 + and CD8 + T cells expressed IFN-γ and TNF-α, and are capable of killing epitope peptide-pulsed autologous swine target cells. • pA151R-specific T cells potentially contribute to protective immunity and pA151R can be used as a component of protective antigen cocktail for vaccine development. African swine fever (ASF) is a highly lethal hemorrhagic disease of swine caused by African swine fever virus (ASFV). Development of safe and effective ASFV subunit vaccine relies on the identification of protective antigens. In this study, we systematically evaluated the antigenicity of ASFV non-structural protein pA151R recognized by T cells from immune-protected pigs. Recombinant pA151R (rpA151R) was expressed in E. coli and used to generate anti-rpA151R polyclonal antibodies (pAb). This pAb bound both eukaryotically-expressed and native viral pA151R, confirming that rpA151R retains its native antigenicity. Using ASFV attenuated vaccine-immunized pigs, we further analysed the kinetics and functions of pA151R-specific T cells as well as their epitope recognition. The results showed that pA151R-specific T cell responses peaked at 14 days post-immunization in pigs, and secreted IFN-γ, TNF-α, IL-2, and perforin simultaneously, with multifunctional characteristics. T-cell epitope mapping identified seven peptides recognized by these pA151R-specific T cells. Among them, three peptides (P2, P4, and P5) were exclusively recognized by CD4 + T cells, four peptides (P6, P10, P12, and P13) were specific for CD8 + T cells whereas P1, P7, and P9 were recognized by both CD4 + and CD8 + T cells. These peptide-specific CD4 + or CD8 + T cells showed cytotoxicity, killing peptide-pulsed autologous target cells in a dose-dependent manner. These findings demonstrated that pA151R-specific swine T cells are able to contribute to protective immunity against ASFV and pA151R is a potential protective antigen for vaccine development. This study established a benchmark for screening and defining more ASFV protective antigens.