Human Monoclonal Fab Antibodies Against West Nile Virus and its Neutralizing Activity Analyzed in Vitro and in Vivo

Abstract

The disease progression with West Nile virus (WNV) infection in humans leads to meningitis or encephalitis and may cause death, particularly among elderly and immunocompromised individuals. Passive immunity using immunoglobulins has shown efficacy in treating some patients with WNV infection, which makes the development of human anti-WNV antibodies significant. The goal of this study was to construct a Fab-specific phage display library against WNV, and to identify and select clones with neutralizing activities. Total RNA was extracted from peripheral blood lymphocytes (PBLs) of two immunized individuals, and RT-PCR was used to amplify the Fab fragments containing the heavy (V(H)) and light (V(L)) chains. The amplified genes were sequentially cloned into the recombinant antibody expression vector pComb3-H, and the Fab-specific phage display library was packaged with helper phage VCS-M13. Five rounds of panning were carried out with WNV E protein domain III, and then binding antibodies were selected by ELISA. Antigen binding specificity, complementarity determining region (CDR) sequence of V(H) and V(L), and neutralizing activity against WNV were analyzed in vitro and in vivo. Eight Fab monoclonal antibodies recognized E protein domain III from a library of 7×10(7) clones/ml. Of the eight, one (Fab 1), exhibited significant neutralizing activity, and completely blocked 100 pfu WNV infection in Vero cells at a concentration 160 μg/ml. In contrast, Fab 13 and Fab 25, showed weaker neutralizing activities, and modestly blocked 100 pfu WNV infections at concentrations of 320 μg/ml and 160 μg/ml, respectively. However, animal studies showed that Fab 1 failed to protect mice from death at the concentration of 160μg/ml indicating that the neutralizing potential of an antibody in vivo is determined by the strength of binding and the abundance of its epitope for the virion.

Figure 1

ELISA analysis of Fab antibodies against WNV E protein domain III.

Figure 2

Amino acid sequences of V_L and V_H of anti-WNV-E specific human Fabs Amino acid sequences were derived from the DNA sequences of the Fabs. Shown are the framework regions 1 to 4 (FWR1 to FWR4) and complementarity-determining regions 1 to 3 (CDR1 to CDR3) for V_H and V_L.

Figure 3

A. Western blotting analysis of Fab antibodies against WNV domain III, B. IFA assay to detect WNV in Vero cells. M: protein standards; P: positive control, human convalescent WNV serum (T-35582); N: human pooled immune globulin (without WNV antibody).

Figure 4

PRNT for virus neutralization by anti-WNV Fab antibodies. The “virus control” represents the result obtained by infection of virus only without addition of antibody. The “Negative Serum Control”, “Mock Control”, and “Positive Serum Control” represent the results obtained by the mixtures of virus with no anti-WNV antibody serum, supernatant of lytic XL1-blue, and anti-WNV serum, respectively. The final concentration of antibody in the virus-antibody mixture is indicated.