Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus

Abstract

Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. One monoclonal antibody, E16, neutralized 10 different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity and neutralizing activity. In postexposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and may therefore be a viable treatment option against WNV infection in humans.

Figure 1. Mapping of monoclonal antibodies to DIII with yeast.

(a) Surface display of WNV E protein on yeast. A fusion protein is composed of the ectodomain or DIII of WNV E protein and the yeast Aga2 protein, which becomes attached to the Aga1 protein on the yeast cell wall. Yeast were transformed with the vector alone (pYD1; top row), the entire WNV E ectodomain (amino acids 1–415; middle row), or DIII alone (amino acids 296–415; bottom row). 24 h after induction, yeast were stained with the indicated monoclonal antibodies (negative control, anti-SARS ORF7a) and processed by flow cytometry. Data for a representative neutralizing (E16) and non-neutralizing (E18) antibody are shown. (b) Flow cytometric enrichment for DIII-expressing yeast variants that lose binding of E24. After each round, an increased percentage of DIII expressing yeast are recognized by the polyclonal WNV E–specific antibody but not by E24. After the final round, DIII-expressing variants (boxed region) were harvested.

Figure 2. Fine epitope mapping of DIII neutralizing and non-neutralizing monoclonal antibodies.

(a) Flow cytometry profiles for immunoreactivity by non-neutralizing (E2), weakly neutralizing (E1) and strongly neutralizing (E34) monoclonal antibodies with yeast expressing wild-type and variant DIII. The red, yellow and green arrows point to mutations that abolish yeast surface binding of individual monoclonal antibodies and correspond to distinct regions of DIII (see b). (b) Mapping of neutralizing and non-neutralizing monoclonal antibodies on the surface of WNV DIII. A molecular surface representation is depicted based on the nuclear magnetic resonance structure of WNV DIII. The indicated amino acid residues associated with binding of neutralizing, weakly neutralizing and non-neutralizing monoclonal antibodies are shown in red, yellow and green, respectively.

Figure 3. Therapeutic effect of DIII-neutralizing monoclonal antibodies.

(a–c) Dose-response analysis at day 2 after WNV infection. At 2 d after WNV infection, mice were passively transferred a single dose (0.8, 4, 20, 100 or 500 μg) of (a) E16, (b) E24, or (c) E34 monoclonal antibodies. As controls, mice were independently administered saline (PBS) or a negative control monoclonal antibody (anti-SARS ORF7a, 500 μg). The survival curves were constructed using data from two independent experiments. The number of animals for each antibody dose ranged from 20 to 30. The difference in survival curves was statistically significant for all WNV-specific monoclonal antibody doses shown (P < 0.0001). (d) WNV burden in the brain of 5-week-old wild-type mice. At days 4, 5 and 6 after WNV infection, brains were harvested and viral burdens were determined by plaque assay. The following percentage of mice had viral burdens below detection (<20 PFU/g): day 4, 33%; day 5, 22%; day 6, 17%. (e,f) Efficacy of WNV-specific monoclonal antibody therapy at days 4 (e) and 5 (f) after infection. A single dose (0.5 mg at day 4 or 2 mg at day 5) of monoclonal antibody (E16, E24, E34 or anti-SARS ORF7a) was administered either 4 or 5 d after WNV infection. Data reflect approximately 20 mice per condition. The difference in survival curves was statistically significant for all WNV-specific monoclonal antibody doses shown at day 4 (P < 0.0001) and day 5 (E16, P = 0.0009; E24, P = 0.027). (g) Effect of E16 therapy on viral burden. Mice were treated with 2 mg of E16 or PBS on day 5 after WNV infection. On day 9, brains were recovered, homogenized and subjected to plaque assay. For a subset (6) that received PBS treatment, brains were harvested at days 7 and 8 from moribund mice. The data is expressed as PFU/g. Of 16 mice treated with E16 68% (11) had undetectable viral loads in the brain at day 9 whereas all mice (14 of 14) treated with PBS had detectable virus at the time of harvest. The dotted line represents the limit of sensitivity of the assay and the dark bars represent the mean of the log values. The differences were statistically different (P < 0.0001).

Figure 4. Construction and efficacy of humanized E16.

(a,b) Sequence and alignment of VH and VL of mouse and humanized E16 (Hm-E16.1). Residues VL-49 and VH-71 were selected for back-mutation in version 16.2 (VL-Y49L) and 16.3 (VL-Y49L, VH-T71A) from the human to the mouse sequence to improve avidity and potency. (c) Binding curves and kinetic parameters of DIII binding to chimeric and humanized E16. One representative surface plasmon resonance experiment for Hm-E16.3 is shown. (d) Neutralization studies with different E16 antibodies. Neutralizing activity was determined by a PRNT assay. The indicated amount of mouse, humanized, chimerized or aglycosyl chimerized E16 were mixed with WNV before addition to BHK-21 cells. Samples were performed in duplicate and the experiment is one representative of three. (e) Therapeutic activity of Ch-E16 and aglycosyl Ch-E16. At 2 d after WNV infection, mice were passively transferred saline or a single intraperitoneal inoculation of increasing doses (4, 20 or 100 μg) of Ch-E16 or aglycosyl Ch-E16 N297Q that differs by a single amino acid. The survival curves were constructed using data from two independent experiments with 13 mice for each arm. At the 4 μg dose, the survival difference between Ch-E16 and aglycosyl Ch-E16 was statistically different (P = 0.008). (f) Therapeutic activity of Hm-E16 in wild-type mice. At 2 d after WNV infection, mice were passively transferred saline, or a single dose (4, 20 or 100 μg) of three versions of humanized E16 (16.1, 16.2 or 16.3). The survival curves were constructed using data from two or three independent experiments with at least 15 animals per treatment arm. All doses of Hm-E16 provided statistically significant protection compared to treatment with saline alone (P < 0.007).

Figure 5. Efficacy of mouse E16 in complement and Fcgr1- and Fcgr3-deficient mice.

Dose-response analysis at day 2 after WNV infection. (a) Fcgr1- and Fcgr3-deficient, (b) C1qa-deficient, or (c) C4-deficient 8-week-old C57BL/6J mice were inoculated with 10² PFU of WNV. At 2 d after infection, mice were passively transferred a single dose (4, 20, 100 or 500 μg) of mouse E16. As controls, mice were independently administered saline (PBS). The number of animals for each antibody dose ranged from 10 to 15. The difference in survival curves was statistically significant for all WNV-specific monoclonal antibody doses shown in the C1qa- and C4-deficient mice (P ≤ 0.001). The difference in survival curves for the Fcgr1- and Fcgr3-deficient mice was significant for 500 μg (P = 0.01), but not for 100 μg (P = 0.14) or 20 μg (P = 0.4) of mouse E16.