Neutralizing Human Fab Fragments against Measles Virus Recovered by Phage Display

Abstract

Five human recombinant Fab fragments (Fabs) specific for measles virus (MV) proteins were isolated from three antibody phage display libraries generated from RNAs derived from bone marrow or splenic lymphocytes from three MV-immune individuals. All Fabs reacted in an enzyme-linked immunosorbent assay with MV antigens. In radioimmunoprecipitation assays two of the Fabs, MV12 and MT14, precipitated an Ϸ80-kDa protein band corresponding to the hemagglutinin (H) protein from MV-infected Vero cell cultures, while two other Fabs, MT64 and GL29, precipitated an Ϸ60-kDa protein corresponding the nucleocapsid (N) protein. In competition studies with MV fusion, H-and N protein-specific monoclonal antibodies (MAbs), the H-specific Fabs predominantly blocked the binding of H-specific MAbs, while the N-specific Fabs blocked MAbs to N. In addition, N-specific Fabs bound to denatured MV N protein in Western blotting. The specificity of the fifth Fab, MV4, could not be determined. By plaque reduction assays, three of the five Fabs, MV4, MV12, and MT14, exhibited neutralizing activity (80% cutoff) against MV (LEC-KI strain) at concentrations ranging between Ϸ2 and 7 g ml ؊1 . Neutralization capacity against MV strains Edmonston and Schwarz was also detected, albeit at somewhat higher Fab concentrations. In conclusion, three neutralizing Fabs were isolated, two of them reactive against the H glycoprotein of MV and another reactive against an undefined epitope. This is the first study in which MV-neutralizing human recombinant Fab antibodies have been isolated from phage display libraries.

A Recombinant Human Monoclonal Antibody to Human Metapneumovirus Fusion Protein That Neutralizes Virus In Vitro and Is Effective Therapeutically In Vivo

“…Several of these Fabs exhibited neutralizing activity in vitro, but the majority of F-binding Fabs that were isolated did not neutralize virus, consistent with previous reports using phage display technology as a means of sampling specific antiviral human antibody repertoires (2,14,18,86). However, of the 14 F-specific Fabs we identified by ELISA screening, 12 bound hMPV-infected cells, and 4 of these exhibited in vitro neutralizing activity.…”

“…The preparation of combinatorial phage display libraries from variable heavy-and light-chain antibody genes provides an efficient method for the isolation of human antibody Fabs. The construction of antibody libraries on the surface of M13 phage and their application for the generation of human MAbs against numerous viruses have been described in several reports (2,3,9,14,15,18,60,86). Many of these studies reported the isolation of antibodies that neutralize virus in vitro and in vivo.…”

Distinct Kinetics for Binding of the CD46 and SLAM Receptors to Overlapping Sites in the Measles Virus Hemagglutinin Protein

“…Antigenic site IV must be more distant because the antibody I-44 had lower inhibitory activity. These data show that the receptor binding surface in MVH is a major target for antibody responses neutralizing virus infectivity (21,22). The similarities in blocking receptor binding to MVH among the different monoclonal antibody tested suggest that the shift in receptor usage by MV may not necessarily be driven by specific immune responses.…”

“…Mouse monoclonal antibodies binding to MVH (I-12, I-29, I-41, I-44, 16-CD11, and 16-DE6) were prepared from hybridomas (21), and anti-MVH-specific MV4, MV12, and MT14 were human recombinant neutralizing Fabs derived by the phage display method (22).…”

Isolation of Human Monoclonal Antibodies That Neutralize Human Rotavirus

“…Recently, several recombinant human Fab fragments exhibiting neutralizing activity toward viruses such as human immunodeficiency virus types 1 and 2, Ebola virus, measles virus, Puumala virus, and respiratory syncytial virus were prepared by means of a phage display system (1,3,6,7,28,37,50). Since the amount of surface proteins with neutralization epitopes on viruses is small and the immunogenicity of inner proteins is quite high, it is generally much more difficult to obtain MAbs with neutralizing capacity than nonneutralizing MAbs.…”