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Development and characterization of monoclonal antibodies against the extracellular domain of African swine fever virus structural protein, CD2v.

ABSTRACT: African swine fever virus (ASFV), a DNA double-stranded virus with high infectivity and mortality, causing a devastating blow to the pig industry and the world economy. The CD2v protein is an essential immunoprotective protein of ASFV. In this study, we expressed the extracellular region of the CD2v protein in the 293F expression system to achieve proper glycosylation. Monoclonal antibodies (mAbs) were prepared by immunizing mice with the recombinant CD2v protein. Eventually, four mAbs that target the extracellular region of the ASFV CD2v protein were obtained. All four mAbs responded well to the ASFV HLJ/18 strain and recognized the same linear epitope, ¹⁵⁴SILE¹⁵⁷. The specific shortest amino acid sequence of this epitope has been accurately identified for the first time. Meaningfully, the ¹⁵⁴SILE¹⁵⁷ epitope was highly conformed in the ASFV Chinese epidemic strain and Georgia2008/1 strains according to the analysis of the conservation and have a fair protective effect. These findings contribute to further understanding of the protein function of CD2v and provide potential support for the development of diagnostic tools and vaccines for ASFV.

SUBMITTER: Liu S

PROVIDER: S-EPMC9716131 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Development and characterization of monoclonal antibodies against the extracellular domain of African swine fever virus structural protein, CD2v.

Liu Siyuan S Ding Peiyang P Du Yongkun Y Ren Dongna D Chen Yilan Y Li Minghui M Sun Xueke X Wang Siqiao S Chang Zejie Z Li Ruiqi R Zhang Gaiping G

Frontiers in microbiology 20221118

African swine fever virus (ASFV), a DNA double-stranded virus with high infectivity and mortality, causing a devastating blow to the pig industry and the world economy. The CD2v protein is an essential immunoprotective protein of ASFV. In this study, we expressed the extracellular region of the CD2v protein in the 293F expression system to achieve proper glycosylation. Monoclonal antibodies (mAbs) were prepared by immunizing mice with the recombinant CD2v protein. Eventually, four mAbs that targ ...[more]

PMID: 36466651

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Project description:The CD2-like (CD2V) protein is a crucial antigen of African swine fever virus (ASFV). CD2V interacts with the cellular AP-1 protein, participates in intracellular transport of virus, and induces neutralizing antibodies to partly protect swine from virus attack. In this study, a specific CD2V dimeric protein was designed to enhance antigenicity and immunogenicity, expressed in a Bac-to-Bac baculovirus expression vector system and purified by Ni-affinity chromatography. After animal immunization, five monoclonal antibodies (mAbs) (7E12, 22B3, 18A3, 13G11, and 43C2) against CD2V were developed. The variable regions of heavy chains and light chains of the mAbs were sequenced to prove that the five mAbs differed from one another. The mAbs of CD2V could combine with ASFV by immunoperoxidase monolayer assay (IPMA). B cell epitopes of CD2V were screened using the five mAbs by indirect enzyme-linked immunosorbent assay (ELISA) and Dot-ELISA. Therefore, three B cell epitopes (147FVKYT151, 157EYNWN161, and 195SSNY198) were identified. This is the first time that mAbs of the ASFV CD2V protein have been developed and the sequencing of heavy chains and light chains of mAbs has been completed. Linear B cell epitopes, which were core targets of immunoprotection of the CD2V protein, were identified by mAbs for the first time. This study provides efficient epitopes for the development of ASFV subunit vaccines. IMPORTANCE The ASFV CD2V protein is a crucial antigen on the outer envelopes of virus particles. A modified ASFV CD2V dimeric protein was expressed in the Bac-to-Bac baculovirus expression vector system. Five monoclonal antibodies (mAbs) against CD2V were developed, sequenced, and applied to identify CD2V protein B cell epitopes. Three B cell epitopes, 147FVKYT151, 157EYNWN161, and 195SSNY198, were identified. This is the first time CD2V mAbs have been developed, the sequencing of heavy chains and light chains of CD2V mAbs have been completed, and CD2V B cell epitopes have been identified by using scanning peptide method and bioinformatics methods.

Project description:African swine fever virus (ASFV) is a high-consequence pathogen posing a substantial threat to global food security. This large DNA virus encodes more than 150 open reading frames, many of which are uncharacterized. The EP402R gene encodes CD2v, a glycoprotein expressed on the surface of infected cells and the only viral protein known to be present in the virus external envelope. This protein mediates binding of erythrocytes to both cells and virions. This interaction is known to prolong virus persistence in blood thus facilitating viral transmission. The sequence of the extracellular domain of CD2v shows similarity with that of mammalian CD2 proteins and is therefore likely to feature two immunoglobulin (Ig)-like domains. A combination of protein structure modeling and extensive mutagenesis was used to identify residues mediating binding of transiently expressed CD2v to erythrocytes. The N-terminal Ig-like domain AGFCC'C″ β sheet was identified as the putative CD2v erythrocyte-binding area. This region differed from the putative CD58 ligand binding site of host CD2, suggesting that CD2v may bind to a ligand(s) other than CD58. An attenuated genotype I ASFV was constructed by replacing the wild-type EP402R gene for a mutant form expressing CD2v bearing a single amino acid substitution, which abrogated the binding to erythrocytes. Pigs immunized with the recombinant virus developed early antibody and cellular responses, low levels of viremia, mild clinical signs post-immunization, and high levels of protection against challenge. These findings improve our understanding of virus-host interactions and provide a promising approach to modified live vaccine development.ImportanceA better understanding of the interactions between viruses and their hosts is a crucial step in the development of strategies for controlling viral diseases, such as vaccines and antivirals. African swine fever, a pig disease with fatality rates approaching 100%, causes very substantial economic losses in affected countries, and new control measures are clearly needed. In this study, we characterized the interaction between the ASFV CD2v protein and host erythrocytes. The interaction plays a key role in viral persistence in blood since it can allow the virus to "hide" from the host immune system. We identified the amino acids in the viral protein that mediate the interaction with erythrocytes and used this information to construct a mutant virus that is no longer able to bind these cells. This virus induces strong immune responses that provide high levels of protection against infection with the deadly parental virus.

Dataset Information

Development and characterization of monoclonal antibodies against the extracellular domain of African swine fever virus structural protein, CD2v.

Publications

Development and characterization of monoclonal antibodies against the extracellular domain of African swine fever virus structural protein, CD2v.

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