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( Jimin Wi ),( Mun Sik Jeong ),( Hyo Jeong Hong ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.7
Hepatitis B virus (HBV) is a major cause of liver cirrhosis and hepatocellular carcinoma. With recent identification of HBV receptor, inhibition of virus entry has become a promising concept in the development of new antiviral drugs. To date, 10 HBV genotypes (A-J) have been defined. We previously generated two murine anti-preS1 monoclonal antibodies (mAbs), KR359 and KR127, that recognize amino acids (aa) 19-26 and 37-45, respectively, in the receptor binding site (aa 13-58, genotype C). Each mAb exhibited virus neutralizing activity in vitro, and a humanized version of KR127 effectively neutralized HBV infection in chimpanzees. In the present study, we constructed a humanized version (HzKR359-1) of KR359 whose antigen binding activity is 4.4-fold higher than that of KR359, as assessed by competitive ELISA, and produced recombinant preS1 antigens (aa 1-60) of different genotypes to investigate the binding capacities of HzKR359-1 and a humanized version (HzKR127-3.2) of KR127 to the 10 HBV genotypes. The results indicate that HzKR359-1 can bind to five genotypes (A, B, C, H, and J), and HzKR127-3.2 can also bind to five genotypes (A, C, D, G, and I). The combination of these two antibodies can bind to eight genotypes (A-D, G-J), and to genotype C additively. Considering that genotypes A-D are common, whereas genotypes E and F are occasionally represented in small patient population, the combination of these two antibodies might block the entry of most virus genotypes and thus broadly neutralize HBV infection.
( Dain Kim ),( Hyeseon Yoon ),( Sangkyu Kim ),( Jimin Wi ),( Heesu Chae ),( Gyunghee Jo ),( Jun-yeol Yoon ),( Heeyoun Kim ),( Chankyu Lee ),( Se-ho Kim ),( Hyo Jeong Hong ) 한국미생물 · 생명공학회 2018 Journal of microbiology and biotechnology Vol.28 No.12
Cross-reactive material 197 (CRM<sub>197</sub>) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. CRM<sub>197</sub> has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect CRM<sub>197</sub> and CRM<sub>197</sub> conjugate vaccines, we generated a human anti-CRM<sub>197</sub> monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-CRM197 polyclonal antibody. The affinity (K<sub>D</sub>) of 3F9 for CRM<sub>197</sub> was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of CRM<sub>197</sub>. The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was <1 ng/ml CRM<sub>197</sub>. In addition, the 3F9 antibody bound to the CRM<sub>197</sub>-polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of CRM<sub>197</sub> and CRM<sub>197</sub> conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against CRM197 and to develop a sandwich ELISA for CRM<sub>197</sub> conjugate vaccines.
( Gyunghee Jo ),( Mun Sik Jeong ),( Jimin Wi ),( Doo Hyun Kim ),( Sangkyu Kim ),( Dain Kim ),( Jun-yeol Yoon ),( Heesu Chae ),( Kyun-hwan Kim ),( Hyo Jeong Hong ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.8
The hepatitis B virus (HBV) envelope contains small (S), middle (M), and large (L) proteins. PreS1 of the L protein contains a receptor-binding motif crucial for HBV infection. This motif is highly conserved among 10 HBV genotypes (A-J), making it a potential target for the prevention of HBV infection. In this study, we successfully generated a neutralizing human monoclonal antibody (mAb), 1A8 (IgG1), that recognizes the receptor-binding motif of preS1 using a phage-displayed human synthetic Fab library. Analysis of the antigen-binding activity of 1A8 for different genotypes indicated that it can specifically bind to the preS1 of major HBV genotypes (A-D). Based on Bio-Layer interferometry, the affinity (K<sub>D</sub>) of 1A8 for the preS1 of genotype C was 3.55 nM. 1A8 immunoprecipitated the hepatitis B virions of genotypes C and D. In an in vitro neutralization assay using HepG2 cells overexpressing the cellular receptor sodium taurocholate cotransporting polypeptide, 1A8 effectively neutralized HBV infection with genotype D. Taken together, the results suggest that 1A8 may neutralize the four HBV genotypes. Considering that genotypes A-D are most prevalent, 1A8 may be a neutralizing human mAb with promising potential in the prevention and treatment of HBV infection.