Construction of Human Fab (Y1/K) Library and Identification of Human Monoclonal Fab Possessing Neutralizing Potency against Japanese Encephalitis Virus

Abstract

A combinatorial human Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from Japanese encephalitis virus hyper‐immune volunteers on pComb3H phagemid vector. The size of the constructed Fab library was 3.3 × 108 Escherichia coli transformants. The library was panned 3 times on the purified Japanese encephalitis virus (JEV) virion, and phage clones displaying JEV antigen‐specific Fab were enriched. The enriched phage pool was then screened for clones producing Fab molecule with JEV neutralizing activity by the focus reduction‐neutralizing test. Among 188 randomly selected clones, 9 Fab preparations revealed neutralizing activities against JEV strain Nakayama. An E. coli transformed with TJE12B02 clone, which produced human monoclonal Fab with the highest neutralizing activity was cultured in a large scale, and the Fab molecule was purified using affinity chromatography. The purified FabTJE12B02 showed the 50% focus reduction endpoint at the concentration of 50.2 μg/ml (ca. 1,000 nM) when JEV strain Nakayama was used. The FabTJE12B02 recognized E protein of JEV strain Nakayama, and the dissociation equilibrium constant (Kd) of the FabTJE12B02 against purified JEV antigen was calculated as 1.21 × 10–8 M. Sequence analysis demonstrated that TJE12B02 used a VH sequence homologous to the VH3 family showing 88.8% homology to germline VH3–23, and used a VK sequence homologous to the VκII subgroup showing 92.8% homology to germline A17.

Humanized Monoclonal Antibodies Derived from Chimpanzee Fabs Protect against Japanese Encephalitis Virus In Vitro and In Vivo

“…There have been growing efforts to recover antibodies against DENV, WNV, and other flaviviruses from chimpanzees and humans (3,10,30,60). These studies have identified a number of MAbs that are useful for investigating viral antigenic structures and mechanisms of virus neutralization and for possible protection against flavivirus infections.…”

Isolation and characterization of novel human monoclonal antibodies possessing neutralizing ability against rabies virus

“…The Kd of the established huMAbs to rabies antigen was measured using VeroRab vaccine, which has a known potency and protein concentration, and calculated as the antibody concentration that gave 50% maximum binding by ELISA (41). In brief, 100 μg/ml VeroRab vaccine diluted with PBS was coated to a 96‐well plate.…”

Identification of a human monoclonal Fab with neutralizing activity against H3N2 influenza A strain from a newly constructed human Fab library

“…Transformed E. coli cultures (150 μl) were prepared as described elsewhere (19, 21), and analyzed for expression of human Fab molecules and their binding activities to influenza virus vaccine antigens by ELISA. Fifty μl of PBS‐diluted (1:1000) anti‐human F(ab′) 2 (Goat, Pierce, Rockford, IL, USA) or bicarbonate buffer (pH 8.8) diluted‐influenza vaccine (1.25 μg per well) was coated on ELISA plates and the centrifuged E. coli cultures were added and incubated.…”