Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells
Neutralizing antibodies (nAbs) can be used before or after infection to prevent or treat viral diseases. However, there are few efficacious nAbs against classical swine fever virus (CSFV) that have been produced, especially the porcine-originated nAbs. In this study, we generated three porcine monoc...
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MDPI AG
2023-03-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/15/4/863 |
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| author | Lihua Wang Rachel Madera Yuzhen Li Douglas P. Gladue Manuel V. Borca Michael T. McIntosh Jishu Shi |
| author_facet | Lihua Wang Rachel Madera Yuzhen Li Douglas P. Gladue Manuel V. Borca Michael T. McIntosh Jishu Shi |
| author_sort | Lihua Wang |
| collection | DOAJ |
| description | Neutralizing antibodies (nAbs) can be used before or after infection to prevent or treat viral diseases. However, there are few efficacious nAbs against classical swine fever virus (CSFV) that have been produced, especially the porcine-originated nAbs. In this study, we generated three porcine monoclonal antibodies (mAbs) with in vitro neutralizing activity against CSFV, aiming to facilitate the development of passive antibody vaccines or antiviral drugs against CSFV that offer the advantages of stability and low immunogenicity. Pigs were immunized with the C-strain E2 (CE2) subunit vaccine, KNB-E2. At 42 days post vaccination (DPV), CE2-specific single B cells were isolated via fluorescent-activated cell sorting (FACS) baited by Alexa Fluor™ 647-labeled CE2 (positive), goat anti-porcine IgG (H + L)-FITC antibody (positive), PE mouse anti-pig CD3ε (negative) and PE mouse anti-pig CD8a (negative). The full coding region of IgG heavy (H) chains and light (L) chains was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Overall, we obtained 3 IgG H chains, 9 kappa L chains and 36 lambda L chains, which include three paired chains (two H + κ and one H + λ). CE2-specific mAbs were successfully expressed in 293T cells with the three paired chains. The mAbs exhibit potent neutralizing activity against CSFVs. They can protect ST cells from infections in vitro with potent IC<sub>50</sub> values from 14.43 µg/mL to 25.98 µg/mL for the CSFV C-strain, and 27.66 µg/mL to 42.61 µg/mL for the CSFV Alfort strain. This study is the first report to describe the amplification of whole-porcine IgG genes from single B cells of KNB-E2-vaccinated pig. The method is versatile, sensitive, and reliable. The generated natural porcine nAbs can be used to develop long-acting and low-immunogenicity passive antibody vaccine or anti-CSFV agents for CSF control and prevention. |
| first_indexed | 2024-03-11T04:27:06Z |
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| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-11T04:27:06Z |
| publishDate | 2023-03-01 |
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| spelling | doaj.art-5de860d963b241e49b470de77fc12fd62023-11-17T21:44:52ZengMDPI AGViruses1999-49152023-03-0115486310.3390/v15040863Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B CellsLihua Wang0Rachel Madera1Yuzhen Li2Douglas P. Gladue3Manuel V. Borca4Michael T. McIntosh5Jishu Shi6Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USADepartment of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USADepartment of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USADepartment of Agriculture, Agricultural Research Service, Plum Island Animal Disease Center, Greenport, NY 11944, USADepartment of Agriculture, Agricultural Research Service, Plum Island Animal Disease Center, Greenport, NY 11944, USADepartment of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32611, USADepartment of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USANeutralizing antibodies (nAbs) can be used before or after infection to prevent or treat viral diseases. However, there are few efficacious nAbs against classical swine fever virus (CSFV) that have been produced, especially the porcine-originated nAbs. In this study, we generated three porcine monoclonal antibodies (mAbs) with in vitro neutralizing activity against CSFV, aiming to facilitate the development of passive antibody vaccines or antiviral drugs against CSFV that offer the advantages of stability and low immunogenicity. Pigs were immunized with the C-strain E2 (CE2) subunit vaccine, KNB-E2. At 42 days post vaccination (DPV), CE2-specific single B cells were isolated via fluorescent-activated cell sorting (FACS) baited by Alexa Fluor™ 647-labeled CE2 (positive), goat anti-porcine IgG (H + L)-FITC antibody (positive), PE mouse anti-pig CD3ε (negative) and PE mouse anti-pig CD8a (negative). The full coding region of IgG heavy (H) chains and light (L) chains was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Overall, we obtained 3 IgG H chains, 9 kappa L chains and 36 lambda L chains, which include three paired chains (two H + κ and one H + λ). CE2-specific mAbs were successfully expressed in 293T cells with the three paired chains. The mAbs exhibit potent neutralizing activity against CSFVs. They can protect ST cells from infections in vitro with potent IC<sub>50</sub> values from 14.43 µg/mL to 25.98 µg/mL for the CSFV C-strain, and 27.66 µg/mL to 42.61 µg/mL for the CSFV Alfort strain. This study is the first report to describe the amplification of whole-porcine IgG genes from single B cells of KNB-E2-vaccinated pig. The method is versatile, sensitive, and reliable. The generated natural porcine nAbs can be used to develop long-acting and low-immunogenicity passive antibody vaccine or anti-CSFV agents for CSF control and prevention.https://www.mdpi.com/1999-4915/15/4/863classical swine fever (CSF)porcinemonoclonal antibodyneutralizingC-strainE2 |
| spellingShingle | Lihua Wang Rachel Madera Yuzhen Li Douglas P. Gladue Manuel V. Borca Michael T. McIntosh Jishu Shi Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells Viruses classical swine fever (CSF) porcine monoclonal antibody neutralizing C-strain E2 |
| title | Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells |
| title_full | Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells |
| title_fullStr | Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells |
| title_full_unstemmed | Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells |
| title_short | Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells |
| title_sort | development of porcine monoclonal antibodies with in vitro neutralizing activity against classical swine fever virus from c strain e2 specific single b cells |
| topic | classical swine fever (CSF) porcine monoclonal antibody neutralizing C-strain E2 |
| url | https://www.mdpi.com/1999-4915/15/4/863 |
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