Production and application of mouse monoclonal antibodies targeting linear epitopes in pB602L of African swine fever virus

Abstract

African swine fever (ASF) is an acute hemorrhagic disease of domestic pigs. The causative agent of ASF, ASF virus (ASFV), is a double-stranded DNA virus, the sole member in the family Asfarviridae. The non-structural protein pB602L of ASFV is a molecular chaperone of the major capsid protein p72 and plays a key role in icosahedral capsid assembly. This protein is antigenic and is a target for developing diagnostic tools for ASF. To generate monoclonal antibodies (mAbs) against pB602L, a prokaryotically expressed recombinant pB602L protein was produced, purified, and used as an antigen to immunize mice. A total of eight mouse mAbs were obtained, and their binding epitopes were screened by Western blot using an overlapping set of polypeptides from pB602L. Three linear epitopes were identified and designated epitope 1 (366ANRERYNY373), epitope 2 (415GPDAPGLSI423), and epitope 3 (498EMLNVPDD505). Based on the epitope recognized, the eight mAbs were placed into three groups: group 1 (B2A1, B2F1, and B2D10), group 2 (B2H10, B2B2, B2D8, and B2A3), and group 3 (B2E12). The mAbs B2A1, B2H10, and B2E12, each representing one of the groups, were used to detect pB602L in ASFV-infected porcine alveolar macrophages (PAMs) and pig tissues, using an indirect fluorescence assay (IFA) and immunohistochemical staining, respectively. The results showed that pB602L was detectable with all three mAbs in immunohistochemical staining, but only B2H10 was suitable for detecting the proteins in ASFV-infected PAMs by IFA. In summary, we developed eight anti-pB602L mouse mAbs recognizing three linear epitopes in the protein, which can be used as reagents for basic and applied research on ASFV.

Detection of African swine fever virus antibodies in serum using a pB602L protein-based indirect ELISA

“…Previous studies have shown that pB602L is strongly antigenic and can be used to develop diagnostic tools for ASFV. Previously, a pB602L-based ELISA assay was employed to detect serum antibodies against ASFV, and the test results were mostly consistent with those obtained using the gold standard, western blot assays (16). B Gutiérrez-Castañeda et al showed that pB602L is recognized by hyperimmune antisera from domestic pigs and bushpigs at late time points after infection, suggesting it may be useful diagnostically to distinguish animals persistently infected with virus (17).…”

“…One of the proven markers to characterize this virus is the central variable region (CVR) within the B602L gene (17,27). pB602L is an important nonstructural protein and a molecular chaperone of the major structural protein p72, which is required for the formation of the icosahedral capsid structure of ASFV (16). The B602L gene contains a central variable region, so the amino acid sequence alignment analysis of the B602L protein of different genotypes of ASFV strains was carried out.…”

“…This suggests that an ELISA against these proteins may be of use in distinguishing animals persistently infected with virus from animals immunized with vaccines incorporating virus structural proteins. Previously, a pB602L-based ELISA assay was used to detect ASFV serum antibodies, which also distinguished pigs persistently infected with native ASFV strains from pigs immunized with subunit vaccines of structural proteins (16). Compared with eukaryotic expression systems, prokaryotic expression systems have lower production costs, simple operation, stable protein preparation, and easy purification and are suitable for large-scale production (31,32).…”

“…ASFV infects mononuclear macrophages mainly through megalocytosis and clathrin-mediated endocytosis (1). pB602L is a nonstructural protein that functions as a molecular chaperone of the major structural protein p72, forming aberrant "zipper-like" structures instead of icosahedral virus particles in the absence of pB602L (16). B602L contains a central variable region (CVR) that allows frequently subgenotyping of ASFV isolates based on this region (17).…”

Lactobacillus plantarum Surface-Displayed ASFV (p14.5) Can Stimulate Immune Responses in Mice

“…Its mortality rate is as high as 100% [ 1 ]. The causative agent of ASF, ASF virus (ASFV), is a double-stranded DNA virus, the sole member of the Asfarviridae family [ 2 ].…”