Simple SummaryAfrican swine fever (ASF) causes acute hemorrhagic disease in domestic pigs. This disease causes huge economic losses worldwide, and there are no safe and effective vaccines. The ASF virus (ASFV) has at least 68 structural proteins. One of these is CD2v, an outer envelope protein usually used as a target for ASF vaccine development. However, the effective immunological regions of this protein have not been fully characterized. Herein, we used monoclonal antibodies to identify a novel B cell epitope located in the intracellular region of CD2v. Importantly, the epitope can elicit both humoral and cellular immune responses in a mouse model. This study provides new insights into ASF epitope vaccine development.AbstractThe 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 is264EPSPREP270, 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.