Dataset Information

Mammalian Cell-Line-Expressed CD2v Protein of African Swine Fever Virus Provides Partial Protection against the HLJ/18 Strain in the Early Infection Stage.

ABSTRACT: A cell line expressing the CD2v protein of ASFV was generated. The efficient expression of CD2v protein was determined by immunofluorescence and Western blotting. The CD2v protein was Ni-affinity purified from the supernatant of cell cultures. The CD2v-expressing cells showed properties of hemadsorption, and the secreted CD2v protein exhibited hemagglutinating activity. The antigenicity and immunoprotection ability of CD2v were evaluated by immunizing pigs alone, combined with a cell-line-expressed p30 protein or triple combined with p30 and K205R protein. Immunized pigs were challenged with the highly virulent ASFV strain HLJ/18. Virus challenge results showed that CD2v immunization alone could provide partial protection at the early infection stage. Protein p30 did not show synergistic protection effects in immunization combined with CD2v. Interestingly, immunization with the triple combination of CD2V, p30 and K205R reversed the protection effect. The viremia onset time was delayed, and one pig out of three recovered after the challenge. The pig recovered from ASFV clinical symptoms, the rectal temperature returned to normal levels and the viremia was cleared. The mechanism of this protection effect warrants further investigation.

SUBMITTER: Hua RH

PROVIDER: S-EPMC10383863 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Mammalian Cell-Line-Expressed CD2v Protein of African Swine Fever Virus Provides Partial Protection against the HLJ/18 Strain in the Early Infection Stage.

Hua Rong-Hong RH Liu Jing J Zhang Shu-Jian SJ Liu Ren-Qiang RQ Zhang Xian-Feng XF He Xi-Jun XJ Zhao Dong-Ming DM Bu Zhi-Gao ZG

Viruses 20230628 7

A cell line expressing the CD2v protein of ASFV was generated. The efficient expression of CD2v protein was determined by immunofluorescence and Western blotting. The CD2v protein was Ni-affinity purified from the supernatant of cell cultures. The CD2v-expressing cells showed properties of hemadsorption, and the secreted CD2v protein exhibited hemagglutinating activity. The antigenicity and immunoprotection ability of CD2v were evaluated by immunizing pigs alone, combined with a cell-line-expres ...[more]

PMID: 37515155

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Project description:African swine fever (ASF) is a highly contagious hemorrhagic disease of pigs, posing a significant threat to the world pig industry. Several researchers are investigating the possibilities for developing a safe and efficient vaccine against ASF. In this regard, significant progress has been made and some gene-deleted ASFVs are reported as potential live attenuated vaccines. A seven-gene-deleted live attenuated vaccine candidate HLJ/18-7GD (among which CD2v is included) has been developed in our laboratory and reported to be safe and protective, and it is expected to be commercialized in the near future. There is an urgent need for developing a diagnostic method that can clearly discriminate between wild-type-ASFV-infected and vaccinated animals (DIVA). In the present study, a dual indirect ELISA based on p54 and CD2v proteins was successfully established to specifically distinguish serum antibodies from pigs infected with wild-type ASFV or possessing vaccine immunization. To evaluate the performance of the assay, a total of 433 serum samples from four groups of pigs experimentally infected with the wild-type HLJ/18 ASFV, immunized with the HLJ/18-7GD vaccine candidate, infected with the new lower virulent variant, and specific-pathogen-free pigs were used. Our results showed that the positive rate of immunized serum was 96.54% (p54) and 2.83% (CD2v), and the positive rate of the infection by wild-type virus was 100% (p54) and 97.8% (CD2v). Similarly, the positive rate to infection by the new low-virulent ASFV variant in China was 100% (p54) and 0% (CD2v), indicating the technique was also able to distinguish antibodies from wild-type and the new low-virulent ASFV variant in China. Moreover, no cross-reaction was observed in immune sera from other swine pathogens, such as CSFV, PEDV, PRRSV, HP-PRRSV, PCV2, and PrV. Overall, the developed dual indirect ELISA exhibited high diagnostic sensitivity, specificity, and repeatability and will provide a new approach to differentiate serum antibodies between wild virulent and CD2v-unexpressed ASFV infection, which will play a great role in serological diagnosis and epidemiological monitoring of ASF in the future.

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.

Dataset Information

Mammalian Cell-Line-Expressed CD2v Protein of African Swine Fever Virus Provides Partial Protection against the HLJ/18 Strain in the Early Infection Stage.

Publications

Mammalian Cell-Line-Expressed CD2v Protein of African Swine Fever Virus Provides Partial Protection against the HLJ/18 Strain in the Early Infection Stage.

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