title: Negative effects of a disulfide bond mismatch in anti-rabies G protein single-chain antibody variable fragment FV57.
abstract: Rabies virus (RV) causes a fatal infectious disease requiring efficient post-exposure prophylaxis (PEP), which includes a rabies vaccine and rabies immunoglobulin (RIG). The single-chain antibody variable fragment (scFv), a small engineered antibody fragment derived from an antibody variable heavy chain and light chain, has the potential to replace the current application of RIG. In previous studies, we constructed and evaluated an anti-rabies virus G protein scFv (FV57) based on the monoclonal antibody CR57. Of the five cysteines in FV57, four are linked in intra-chain disulfide bonds (Cys-VH28/Cys-VH98 and Cys-VL16/Cys-VL84), and one is free (Cys-VL85). However, the thiol in Cys-VL85 neighboring Cys-VL84 in the CDR3 of the light chain is likely to mismatch with the thiol in Cys-VL16 during the renaturing process. In order to study effects of the mismatched disulfide bond, Cys-VL85 and Cys-VL84 of FV57 were mutated to serine to construct mutants FV57(VL85S) and FV57(VL84S). Furthermore, the disulfide bonds in the light chain of FV57, FV57(VL85S) and FV57(VL84S) were deleted by mutating Cys-VL16 to serine. All mutants were prepared and evaluated along with the original FV57. The results indicated that the mismatched disulfide bond of FV57 linking the light chain FR1 and CDR3 would confer deleterious negative effects on its activity against RV, likely due to spatial hindrance in the light chain CDR3. Moreover, avoidance of the disulfide bond mismatch provided an additional 30% protective efficacy against RV infection in the mouse RV challenge model. Thus, modifications of FV57 to eliminate the disulfide bond mismatch may provide a candidate therapeutic agent for effective PEP against rabies.
authors.affiliation: National Engineering Laboratory for HIV Vaccine, Jilin University, Changchun, 130012, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Duan
authors.affiliation: National Engineering Laboratory for HIV Vaccine, Jilin University, Changchun, 130012, China; Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, 130012, China.
authors.forename: Tiejun
authors.initials: T
authors.lastname: Gu
authors.affiliation: National Engineering Laboratory for HIV Vaccine, Jilin University, Changchun, 130012, China; Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, 130012, China.
authors.forename: Xizhen
authors.initials: X
authors.lastname: Zhang
authors.affiliation: National Engineering Laboratory for HIV Vaccine, Jilin University, Changchun, 130012, China; Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, 130012, China.
authors.forename: Chunlai
authors.initials: C
authors.lastname: Jiang
authors.affiliation: National Engineering Laboratory for HIV Vaccine, Jilin University, Changchun, 130012, China.
authors.forename: Ruosen
authors.initials: R
authors.lastname: Yuan
authors.affiliation: National Engineering Laboratory for HIV Vaccine, Jilin University, Changchun, 130012, China.
authors.forename: Zhuang
authors.initials: Z
authors.lastname: Li
authors.affiliation: National Engineering Laboratory for HIV Vaccine, Jilin University, Changchun, 130012, China.
authors.forename: Dandan
authors.initials: D
authors.lastname: Wang
authors.affiliation: National Engineering Laboratory for HIV Vaccine, Jilin University, Changchun, 130012, China.
authors.forename: Xiaoxu
authors.initials: X
authors.lastname: Chen
authors.affiliation: School of Biosciences and Biotechnology, University of Camerino, Camerino, 62032, Italy.
authors.forename: Chunlai
authors.initials: C
authors.lastname: Wu
authors.affiliation: National Engineering Laboratory for HIV Vaccine, Jilin University, Changchun, 130012, China; Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, 130012, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: National Engineering Laboratory for HIV Vaccine, Jilin University, Changchun, 130012, China; Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, 130012, China. Electronic address: ygwu@jlu.edu.cn.
authors.forename: Yongge
authors.initials: Y
authors.lastname: Wu
authors.affiliation: National Engineering Laboratory for HIV Vaccine, Jilin University, Changchun, 130012, China; Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, 130012, China. Electronic address: weikong@jlu.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Kong
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D057134:Antibodies, Neutralizing / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D000914:Antibodies, Viral / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology*; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics; D005260:Female; D006023:Glycoproteins / Q000276:immunology*; D051379:Mice; D009154:Mutation; D056990:Post-Exposure Prophylaxis; D011818:Rabies / Q000276:immunology / Q000517:prevention & control*; D011819:Rabies Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D011820:Rabies virus / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D014759:Viral Envelope Proteins / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000956:Antigens, Viral; D006023:Glycoproteins; D011819:Rabies Vaccines; D057127:Single-Chain Antibodies; D014759:Viral Envelope Proteins; C064951:glycoprotein G, Rabies virus; D003545:Cysteine
keywords: Disulfide bond mismatch; Glycoprotein; Rabies virus; scFv