Defining limits of treatment with humanized neutralizing monoclonal antibody for West Nile virus neurological infection in a hamster model

Abstract

A potent anti-West Nile virus (anti-WNV)-neutralizing humanized monoclonal antibody, hE16, was previously shown to improve the survival of WNV-infected hamsters when it was administered intraperitoneally (i.p.), even after the virus had infected neurons in the brain. In this study, we evaluated the therapeutic limit of hE16 for the treatment of WNV infection in hamsters by comparing single-dose peripheral (i.p.) therapy with direct administration into the pons through a convection-enhanced delivery (CED) system. At day 5 after infection, treatments with hE16 by the peripheral and the CED routes were equally effective at reducing morbidity and mortality. In contrast, at day 6 only the treatment by the CED route protected the hamsters from lethal infection. These experiments suggest that hE16 can directly control WNV infection in the central nervous system. In support of this, hE16 administered i.p. was detected in a time-dependent manner in the serum, cerebrospinal fluid (CSF), cerebral cortex, brain stem, and spinal cord in CSF. A linear relationship between the hE16 dose and the concentration in serum was observed, and maximal therapeutic activity occurred at doses of 0.32 mg/kg of body weight or higher, which produced serum hE16 concentrations of 1.3 microg/ml or higher. Overall, these data suggest that in hamsters hE16 can ameliorate neurological disease after significant viral replication has occurred, although there is a time window that limits therapeutic efficacy.

FIG. 1.

Efficacy of humanized MAb hE16 when it is delivered i.p. or by the CED route to hamsters infected with WNV by the s.c. route. Artif. CSF, artificial CSF (solution of sodium, potassium calcium, and magnesium in CSF) (16). (A) Eight and nine animals were used in the hE16- and placebo-treated groups, respectively. The animals were infected with 10^5.3 50% cell culture infectious doses of WNV (17). hE16 (5 mg/hamster/day, 43 mg/kg/day, 116 g/hamster) was administered by the CED route. (B) Twelve animals were used in each group, and the animals were infected with 10^7.1 50% cell culture infectious doses of WNV. hE16 (5 mg/hamster/day, 31 mg/kg/day, 160 g/hamster) was administered by the CED route. (C) Ten and nine animals were used in the hE16- and placebo-treated groups, respectively. The animals were infected with 10^7.1 50% cell culture infectious doses of WNV. hE16 (5 mg/hamster, 48 mg/kg/day, 103 g/hamster) was administered by the CED route. (D) Ten animals were used in each group, and the animals were infected with 10^5.7 50% cell culture infectious doses of WNV. hE16 (32 mg/kg) was administered i.p. (E) Thirty animals were used in each group, and the animals were infected with 10^7.1 50% cell culture infectious doses of WNV. hE16 (32 mg/kg) was administered i.p. **, P ≤ 0.01 by log rank survival analysis.

FIG. 2.

Temporal expression of WNV and neutralizing antibody in serum and CSF of hamsters infected s.c. with 10^5.3 50% cell culture infectious doses of WNV. PRNT, plaque reduction neutralization test for measurement of neutralizing antibody; dashed lines, limit of detection.

FIG. 3.

Dose-response of MAb hE16 for survival of WNV-infected hamsters and concentrations in serum. Five days after s.c. injection of WNV (10^5.1 50% cell culture infectious doses), one-half log doses (100, 1.0, 0.32, 0.10, 0.032, 0.01, 0 mg/kg) of hE16 were administered i.p. Fifteen animals were included in each group except the 1.0-mg/kg group, which had 20 animals. On the next day, sera were obtained by bleeding of the orbital sinuses of anesthetized hamsters and were assayed for human IgG. (A) Percent survival plotted against hE16 dose. (B) Serum hE16 concentration plotted against hE16 dose. The dosage of hE16 that yielded half-maximal survival (MED₅₀), depicted in panel A, was calculated by using a linear equation. (C) The dosage of 0.13 mg/kg was extrapolated to derive the serum hE16 concentration reflecting half-maximal therapeutic activity (MEC₅₀) of 0.44 μg/ml.

FIG. 4.

Concentrations of MAb hE16 in serum and CSF at 7, 12, and 30 dpi for WNV- or sham-infected hamsters treated i.p. at 5 dpi with 32 mg/kg hE16. The animals were injected s.c. with 10^7.1 50% cell culture infectious doses of WNV or were sham treated. Six animals each were used in the WNV-infected and sham-infected groups. Dashed line, limit of detection (0.6 μg/ml).

FIG. 5.

Correlation of hE16 concentration in serum with hE16 concentrations in neurological tissues and CSF at 7 dpi for WNV- and sham-infected hamsters treated i.p. at 5 dpi with 32 mg/kg hE16. The animals were injected with 10^7.1 50% cell culture infectious doses WNV or were sham treated. The results for the WNV-infected and sham-infected hamsters were combined because there was no difference in the hE16 concentration between these groups. Dashed lines, limit of detection (0.06 μg/ml).