Structure-guided affinity maturation of a single-chain variable fragment antibody against the Fu-bc epitope of the dengue virus envelope protein

Abstract

Dengue is one of the most dominant arthropod-borne viral diseases, infecting at least 390 million people every year throughout the world. Despite this, there is no effective treatment against dengue, and the only available vaccine has already been withdrawn owing to the significant adverse effects. Therefore, passive immunotherapy using monoclonal antibodies is now being sought as a therapeutic option. To date, many dengue monoclonal antibodies have been identified, most of which are serotype-specific, and only a few of which are cross-reactive. Furthermore, antibodies that cross-react within serotypes are weakly neutralizing and frequently induce antibody-dependent enhancement, which promotes viral entry and replication. Therefore, broadly neutralizing antibodies with no risk of antibody-dependent enhancement are required for the treatment of dengue. Here, we developed a single-chain variable fragment (scFv) antibody from an anti-fusion loop E53 antibody (PDB: 2IGF). We introduced previously predicted favorable complementarity-determining region (CDR) mutations into the gene encoding the scFv antibody for affinity maturation, and the resultant variants were tested in vitro against the highly conserved fusion and bc epitope of the dengue virus envelope protein. We show some of these scFv variants with two to three substitution mutations in three different CDRs possess affinity constants (KD) ranging from 20 to 200 nM. The scFv-mutant15, containing D31L, Y105W, and S227W substitutions, showed the lowest affinity constant, (KD = 24 ± 7 nM), approximately 100-fold lower than its parental construct. We propose that the scFv-derivative antibody may be a good candidate for the development of an effective and safe immunotherapy. Dengue is one of the most dominant arthropod-borne viral diseases, infecting at least 390 million people every year throughout the world. Despite this, there is no effective treatment against dengue, and the only available vaccine has already been withdrawn owing to the significant adverse effects. Therefore, passive immunotherapy using monoclonal antibodies is now being sought as a therapeutic option. To date, many dengue monoclonal antibodies have been identified, most of which are serotype-specific, and only a few of which are cross-reactive. Furthermore, antibodies that cross-react within serotypes are weakly neutralizing and frequently induce antibody-dependent enhancement, which promotes viral entry and replication. Therefore, broadly neutralizing antibodies with no risk of antibody-dependent enhancement are required for the treatment of dengue. Here, we developed a single-chain variable fragment (scFv) antibody from an anti-fusion loop E53 antibody (PDB: 2IGF). We introduced previously predicted favorable complementarity-determining region (CDR) mutations into the gene encoding the scFv antibody for affinity maturation, and the resultant variants were tested in vitro against the highly conserved fusion and bc epitope of the dengue virus envelope protein. We show some of these scFv variants with two to three substitution mutations in three different CDRs possess affinity constants (KD) ranging from 20 to 200 nM. The scFv-mutant15, containing D31L, Y105W, and S227W substitutions, showed the lowest affinity constant, (KD = 24 ± 7 nM), approximately 100-fold lower than its parental construct. We propose that the scFv-derivative antibody may be a good candidate for the development of an effective and safe immunotherapy. Dengue virus is one of the most prevalent mosquito-borne human pathogens, affecting about half of the world's population (1Bhatt S. Gething P.W. Brady O.J. Messina J.P. Farlow A.W. Moyes C.L. Drake J.M. Brownstein J.S. Hoen A.G. 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For cost-effective manufacture and to address ADE's biosafety issues, many expression systems and protein engineering techniques are now being researched (16Nandi S. Kwong A.T. Holtz B.R. Erwin R.L. Marcel S. McDonald K.A. Techno-economic analysis of a transient plant-based platform for monoclonal antibody production.MAbs. 2016; 8: 1456-1466Crossref PubMed Scopus (97) Google Scholar, 17Ramadhany R. Hirai I. Sasaki T. Ono K. Ramasoota P. Ikuta K. Kurosu T. Antibody with an engineered Fc region as a therapeutic agent against dengue virus infection.Antiviral Res. 2015; 124: 61-68Crossref PubMed Scopus (16) Google Scholar). The dengue virus is a member of the Flaviviridae family that forms both mature and immature states during its life cycle. The outer of a mature virus is of of the Zhang M.G. J. E. S. of dengue for flavivirus maturation, and Full Text Full Text PDF PubMed Scopus Google Scholar, X. P. X. J.M. G. 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PubMed Scopus Google Scholar). we have a of scFv by substitution mutations in the complementarity-determining and tested them in vitro against the protein. The favorable mutations from the in vitro were to scFv analysis also to significant about the the of the scFv antibody and the epitope of the scFv and were in and via to a of the as by and we by an in vitro A. antibody against highly conserved region of dengue envelope protein by in of scFv PubMed Scopus Google Scholar, A. and of protein from envelope Microbiol. PubMed Scopus Google Scholar). As by the scFv to the protein with a of which is with by the A. antibody against highly conserved region of dengue envelope protein by in of scFv PubMed Scopus Google Scholar). To the in the by using and the D31L, Y105W, and at in the three CDRs and of scFv were on in and A. antibody against highly conserved region of dengue envelope protein by in of scFv PubMed Scopus Google Scholar). 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