Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells.
Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells.
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DOI：
10.3390/v15040863
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发表时间：
2023-03-28
期刊：
影响因子：
--
通讯作者：
Shi J
中科院分区：
文献类型：
--
作者：
Wang L;Madera R;Li Y;Gladue DP;Borca MV;McIntosh MT;Shi J
中和抗体（nAb）可以在感染前或感染后使用，以预防或治疗病毒性疾病。然而，目前针对猪瘟病毒（classical swine fever virus，CSFV）的nAbs，特别是猪源性nAbs的研究还很少。本研究制备了3株具有体外中和活性的抗猪瘟病毒单克隆抗体（mAb），旨在为研制稳定、低免疫原性的猪瘟病毒被动抗体疫苗或抗病毒药物奠定基础。用C株E2（CE 2）亚单位疫苗KNB-E2免疫猪。在疫苗接种后42天（DPV），通过荧光激活细胞分选（FACS）分离CE 2特异性单个B细胞，所述荧光激活细胞分选（FACS）由Alexa Fluor™ 647标记的CE 2（阳性）、山羊抗猪IgG（H + L）-FITC抗体（阳性）、PE小鼠抗猪CD 3 ε（阴性）和PE小鼠抗猪CD 8 α（阴性）诱饵。通过逆转录-聚合酶链反应（RT-PCR）扩增IgG重链（H）和轻链（L）的完整编码区。总的来说，我们获得了3条IgG H链、9条κ L链和36条λ L链，其中包括三条配对链（两条H + κ和一条H + λ）。在293 T细胞中成功表达了具有三条配对链的CE 2特异性mAb。mAb表现出针对CSFV的有效中和活性。它们可以保护ST细胞免受体外感染，CSFV C株的有效IC 50值为14.43 µg/mL至25.98 µg/mL，CSFV Alfort株为27.66 µg/mL至42.61 µg/mL。本研究首次报道了从KNB-E2免疫猪的单个B细胞中扩增全猪IgG基因。该方法具有通用性、灵敏度和可靠性。所产生的天然猪nAb可用于开发长效和低免疫原性的被动抗体疫苗或用于CSF控制和预防的抗CSFV剂。
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 IC50 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.
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DOI：
10.1097/aci.0000000000000787
发表时间：
2021-12-01
影响因子：
2.8
作者：
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