Identification of Two Novel Linear B Cell Epitopes on the CD2v Protein of African Swine Fever Virus Using Monoclonal Antibodies

Abstract

African swine fever virus (ASFV) is a highly infectious viral pathogen that endangers the global pig industry, and no effective vaccine is available thus far. The CD2v protein is a glycoprotein on the outer envelope of ASFV, which mediates the transmission of the virus in the blood and recognition of the virus serotype, playing an important role in ASFV vaccine development and disease prevention. Here, we generated two specific monoclonal antibodies (mAbs), 6C11 and 8F12 (subtype IgG1/kappa-type), against the ASFV CD2v extracellular domain (CD2v-ex, GenBank: MK128995.1, 1–588 bp) and characterized their specificity. Peptide scanning technology was used to identify the epitopes recognized by mAbs 6C11 and 8F12. As a result, two novel B cell epitopes, 38DINGVSWN45 and 134GTNTNIY140, were defined. Amino acid sequence alignment showed that the defined epitopes were conserved in all referenced ASFV strains from various regions of China including the highly pathogenic, epidemic strain, Georgia2007/1 (NC_044959.2), with the same noted substitutions compared to the four foreign ASFV wild-type strains. This study provides important reference values for the design and development of an ASFV vaccine and useful biological materials for the functional study of the CD2v protein by deletion analysis.

Development of novel monoclonal antibodies for blocking NF-κB activation induced by CD2v protein in African swine fever virus

“…Although multiple linear B-cell epitopes of CD2v have recently been identified (34)(35)(36)(37)(38)(39)(40), their functions remain unknown (Table 1). To analyze the antigenic epitopes recognized by our anti-CD2v mAbs, we designed and constructed five CD2v extracellular domain truncation mutants (D1-D5) (Figures 6A-C).…”

“…Through peptide-based ELISA, we identified two novel epitopes. The first epitope, recognized by mAb 3J25, was identified as 25 TIILDSNITNDNN 37 . It overlapped with the linear epitope of amino acids 28-51 in the CD2v extracellular domain, which was identified by Zhang's group (35).…”

“…In recent years, the development of monoclonal antibodies (mAbs) targeting linear epitopes of CD2v has made progress, though their bioactivity has not been evaluated extensively (Table 1) (34)(35)(36)(37)(38). Hence, we opted for the Expi293F eukaryotic expression system to express the recombinant N-CD2v-His protein (extracellular region of CD2v), as it offers superior post-translational modifications, particularly glycosylation, ensuring that the antigens expressed closely resemble the in vivo structure of CD2v (Figure 1A).…”

Novel Epitope Mapping of African Swine Fever Virus pI215L Protein Using Monoclonal Antibodies

“…ASFV is a large, enveloped virus with icosahedral morphology and belongs to the Asfarviridae family, isolates of which have linear dsDNA genomes of 170–194 kbp, encoding more than 150 polypeptides [ 5 ]. Monoclonal antibodies against various ASFV proteins, including the major capsid protein p72, the viral internal envelope protein p30 and p17, the outer envelope protein CD2v, the type II transmembrane protein p54, the nonstructural protein pK205R, and the uncharacterized protein pCP312R and pC129R, have been generated for ASFV infection research and development of ASFV diagnosis [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. pI215L, a nonstructural protein of ASFV, is found within the virion and expressed at a high level in infected porcine alveolar macrophages (PAMs) from 4 h post-infection (hpi) [ 14 , 15 ], indicating a possible role of pI215L protein in the early steps of infection.…”

Special Issue “African Swine Fever and Other Swine Viral Diseases in Africa”

“…Four of the seven studies described in this Special Issue are concerned with vaccine development. One of these is a general paper describing two novel epitopes on the CD2v protein of ASFV, a glycoprotein on the outer envelope of the virus that is of considerable interest in vaccine development [ 1 ]. The remaining three studies emanate from east Africa and are concerned with developing a vaccine to protect against infection with genotype IX ASFV, which was first described from a domestic pig isolate in Uganda, is dominant in East Africa and is a virus of concern due to increased propensity to spread in domestic pigs.…”