Shin, Yong Won
(R&D Center, GC Pharma)
,
Chang, Ki-Hwan
(R&D Center, GC Pharma)
,
Hong, Gwang-Won
(R&D Center, GC Pharma)
,
Yeo, Sang-Gu
(Division of VPD Control and NIP, Korea Centers for Disease Control and Prevention, Ministry of Health and Welfare, Osong Health Technology Administration Complex)
,
Jee, Youngmee
(Division of VPD Control and NIP, Korea Centers for Disease Control and Prevention, Ministry of Health and Welfare, Osong Health Technology Administration Complex)
,
Kim, Jong-Hyun
(Division of VPD Control and NIP, Korea Centers for Disease Control and Prevention, Ministry of Health and Welfare, Osong Health Technology Administration Complex)
,
Oh, Myoung-don
(Department of Internal Medicine, College of Medicine, Seoul National University)
,
Cho, Dong-Hyung
(School of Life Science, Kyungpook National University)
,
Kim, Se-Ho
(Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University)
Although smallpox was eradicated in 1980, it is still considered a potential agent of biowarfare and bioterrorism. Smallpox has the potential for high mortality rates along with a major public health impact, eventually causing public panic and social disruption. Passive administration of neutralizin...
Although smallpox was eradicated in 1980, it is still considered a potential agent of biowarfare and bioterrorism. Smallpox has the potential for high mortality rates along with a major public health impact, eventually causing public panic and social disruption. Passive administration of neutralizing monoclonal antibodies (mAbs) is an effective intervention for various adverse reactions caused by vaccination and the unpredictable nature of emerging and bioterrorist-related infections. Currently, vaccinia immune globulin (VIG) is manufactured from vaccinia vaccine-boosted plasma; however, this production method is not ideal because of its limited availability, low specific activity, and risk of contamination with blood-borne infectious agents. To overcome the limitations of VIG production from human plasma, we isolated two human single-chain variable fragments (scFvs), (SC34 and SC212), bound to vaccinia virus (VACV), from a scFv phage library constructed from the B cells of VACV vaccine-boosted volunteers. The scFvs were converted to human IgG1 (VC34 and VC212). These two anti-VACV mAbs were produced in Chinese Hamster Ovary (CHO) DG44 cells. The binding affinities of VC34 and VC212 were estimated by competition ELISA to $IC_{50}$ values of $2{\mu}g/ml$ (13.33 nM) and $22{\mu}g/ml$ (146.67 nM), respectively. Only the VC212 mAb was proven to neutralize the VACV, as evidenced by the plaque reduction neutralization test (PRNT) result with a $PRNT_{50}$ of ~0.16 mg/ml (${\sim}1.07{\mu}M$). This VC212 could serve as a valuable starting material for further development of VACV-neutralizing human immunoglobulin for a prophylactic measure against post-vaccination complications and for post-exposure treatment against smallpox.
Although smallpox was eradicated in 1980, it is still considered a potential agent of biowarfare and bioterrorism. Smallpox has the potential for high mortality rates along with a major public health impact, eventually causing public panic and social disruption. Passive administration of neutralizing monoclonal antibodies (mAbs) is an effective intervention for various adverse reactions caused by vaccination and the unpredictable nature of emerging and bioterrorist-related infections. Currently, vaccinia immune globulin (VIG) is manufactured from vaccinia vaccine-boosted plasma; however, this production method is not ideal because of its limited availability, low specific activity, and risk of contamination with blood-borne infectious agents. To overcome the limitations of VIG production from human plasma, we isolated two human single-chain variable fragments (scFvs), (SC34 and SC212), bound to vaccinia virus (VACV), from a scFv phage library constructed from the B cells of VACV vaccine-boosted volunteers. The scFvs were converted to human IgG1 (VC34 and VC212). These two anti-VACV mAbs were produced in Chinese Hamster Ovary (CHO) DG44 cells. The binding affinities of VC34 and VC212 were estimated by competition ELISA to $IC_{50}$ values of $2{\mu}g/ml$ (13.33 nM) and $22{\mu}g/ml$ (146.67 nM), respectively. Only the VC212 mAb was proven to neutralize the VACV, as evidenced by the plaque reduction neutralization test (PRNT) result with a $PRNT_{50}$ of ~0.16 mg/ml (${\sim}1.07{\mu}M$). This VC212 could serve as a valuable starting material for further development of VACV-neutralizing human immunoglobulin for a prophylactic measure against post-vaccination complications and for post-exposure treatment against smallpox.
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제안 방법
Primers were designed to fit into the N- and Cterminal regions of each variable heavy chain (VH), variable kappa chain (Vκ), and variable lambda chain (Vλ) region (Table 1).
In this study, a phage display library of human single-chain variable fragment (scFv) antibodies was constructed from the B cells of healthy volunteers boosted by the VACV vaccine. Human scFvs against VACV were then isolated, and their neutralizing efficacy was demonstrated following conversion to human immunoglobulin G1 (IgG1).
The sequencing of colonies displaying binding to VACV in ELISA was performed by Genotech (Korea) using primer P035 (Table 1). Two scFv clones, SC34 and SC212, were screened based on their binding to VACV and by sequence analysis for the next step.
This study solved the disadvantages of human plasma-derived VIG production by isolating human mAbs capable of neutralizing VACV from a phage-display scFv library prepared from B cells of VACV-vaccinated volunteers. As a result, a human anti-VACV mAb VC212 having a PRNT50 of ~0.
이론/모형
VACV neutralization by anti-VACV mAb was determined by a plaque-reduction neutralization test (PRNT) [14]. For PRNT, 1 × 106 Vero E6 cells (Korea Centers for Disease Control and Prevention (KCDC), Korea) were seeded into 6-well plates and incubated overnight.
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