Construction of human fab (γ1/κ) library and identification of human monoclonal fab possessing neutralizing potency against Japanese encephalitis virus

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 x 10(8) 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 TJE12BO2 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 mu g/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 FabTJE12BO2 against purified JEV antigen was calculated as 1.21 x 10(-8) m. Sequence analysis demonstrated that TJE121302 used a V. sequence homologous to the V(H)3 family showing 88.8% homology to germline VH3-23, and used a V kappa sequence homologous to the VicIl subgroup showing 92.8% homology to germline A17.