The African swine fever virus (ASFV) causes high mortality in domestic pigs. ASFV encodes an important protein target for subunit vaccine development, CD2v, but its most effective immunological regions are not known. Herein, we generated a monoclonal antibody (mAb) named IF3 by immunizing mice against the intracellular region of the CD2v protein (CD2v-IR). 1F3 specifically recognized CD2v, which is expressed transiently in transfected Sf9 cells and also in inactivated ASFV-infected porcine alveolar macrophage (PAM) cells. The epitope recognized by 1F3 is ²⁶⁴EPSPREP²⁷⁰, which is highly conserved in ASFV genotypes. Immunization of mice with this epitope elicited an increased IgG response, including IgG1 and IgG2a subtypes, and also increased CD8⁺ T cells and cytokine expression. Overall, these results indicate that this epitope induces both humoral and cellular immune responses that may be used for ASFV-related subunit vaccine design and development.

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) of African swine fever virus (ASFV) is an essential enzyme required for efficient virus replication. Previous crystallography data have indicated that dUTPase (E165R) may serve as a therapeutic target for inhibiting ASFV replication; however, the specificity of the targeting site(s) in ASFV dUTPase remains unclear. In this study, 19 mouse monoclonal antibodies (mAbs) were produced, in which four mAbs showed inhibitory reactivity against E165R recombinant protein. Epitope mapping studies indicated that E165R has three major antigenic regions: 100–120 aa, 120–140 aa, and 140–165 aa. Three mAbs inhibited the dUTPase activity of E165R by binding to the highly conserved 149–RGEGRFGSTG–158 amino acid sequence. Interestingly, 8F6 mAb specifically recognized ASFV dUTPase but not Sus scrofa dUTPase, which may be due to structural differences in the amino acids of F151, R153, and F154 in the motif V region. In summary, we developed anti-E165R-specific mAbs, and identified an important antibody-binding antigenic epitope in the motif V of ASFV dUTPase. Our study provides a comprehensive analysis of mAbs that target the antigenic epitope of ASFV dUTPase, which may contribute to the development of novel antibody-based ASFV therapeutics.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is an acute, febrile, highly contagious, and lethal disease that poses a severe threat to the global pig farming industry. Currently, no globally recognized, safe, and effective commercial ASF vaccine has been developed, making vaccination a crucial strategy for outbreak control. The ASFV structural proteins p72, p30, and p54 are key targets for vaccine development. In this study, we developed a novel baculovirus vector-based system for surface display of a recombinant protein comprising epitopes from p72, p30, and p54. Upon infection, the recombinant protein was expressed and anchored on the plasma membrane of Sf-9 cells. Purified virus analysis revealed that the Bac-recombinant protein enhanced gene delivery and transgene expression in mammalian cells compared to the Bac-Wild Type (Bac-WT). In a murine model, the Bac-recombinant protein induced significantly higher IFN-γ and IL-4 levels than Bac-p30 and the negative control. However, further evaluation in swine models is required to confirm its protective potential against ASFV. Furthermore, it also elicited a robust antibody response, generating high-titer Bac-recombinant protein-specific antibodies. Therefore, these findings suggest that the ASFV Bac-recombinant protein is a promising candidate for a vector-based vaccine.