Humans infected with West Nile virus (WNV) may clinically present with symptoms that are suggestive of neurological infection. Nearly all treatments of WNV disease have been effective in animal models only if administered before or soon after viral challenge. Here, we evaluated whether a potent neutralizing anti-WNV humanized monoclonal antibody (MAb), hE16, could improve the course of disease in a hamster model when administered after the virus had infected neurons in the brain. Five days after viral injection, WNV was detected in the brains of hamsters by cytopathic assay, quantitative reverse-transcription polymerase chain reaction, and immunohistochemical staining of WNV envelope in neurons. Notably, 80%-90% of the hamsters treated 5 days after viral injection by intraperitoneal injection with hE16 survived the disease, compared with 37% of the placebo-treated hamsters (P< or =.001). The hamsters that received hE16 directly in the brain also exhibited markedly improved survival rates, compared with those in the placebo-treated hamsters. In prospective experiments, hamsters with high levels of infectious WNV in their cerebrospinal fluid were also protected by hE16 when administered 5 days after viral injection. These experiments suggest that humanized MAbs with potent neutralizing activity are a possible treatment for human patients after WNV has infected neurons in the central nervous system.

Milk fat globule membrane is a protein-lipid complex that may strengthen the gut barrier. The main objective of this study was to assess the ability of a membrane-rich milk fat diet to promote the integrity of the gut barrier and to decrease systemic inflammation in lipopolysaccharide (LPS)-challenged mice. Animals were randomly assigned to one of 2 American Institute of Nutrition (AIN)-76A formulations differing only in fat source: control diet (corn oil) and milk fat diet (anhydrous milk fat with 10% milk fat globule membrane). Each diet contained 12% calories from fat. Mice were fed diets for 5 wk, then injected with vehicle or LPS (10mg/kg of BW) and gavaged with dextran-fluorescein to assess gut barrier integrity. Serum was assayed for fluorescence 24h after gavage, and 16 serum cytokines were measured to assess the inflammatory response. Gut permeability was 1.8-fold higher in LPS-challenged mice fed the control diet compared with the milk fat diet. Furthermore, mice fed the milk fat diet and injected with LPS had lower serum levels of IL-6, IL-10, IL-17, monocyte chemotactic protein (MCP)-1, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and IL-3 compared with LPS-injected mice fed the control diet. The results indicate that the membrane-rich milk fat diet decreases the inflammatory response to a systemic LPS challenge compared with corn oil, and the effect coincides with decreased gut permeability.

Blood-brain barrier (BBB) permeability was evaluated in mice and hamsters infected with West Nile virus (WNV, flavivirus) as compared to those infected with Semliki Forest (alphavirus) and Banzi (flavivirus) viruses. BBB permeability was determined by measurement of fluorescence in brain homogenates or cerebrospinal fluid (CSF) after intraperitoneal (i.p.) injection of sodium fluorescein, by macroscopic examination of brains after i.p. injection of Evans blue, or by measurement of total protein in CSF compared to serum. Lethal infection of BALB/c mice with Semliki Forest virus and Banzi virus caused the brain : serum fluorescence ratios to increase from a baseline of 2-4 % to as high as 11 and 15 %, respectively. Lethal infection of BALB/c mice with WNV did not increase BBB permeability. When C57BL/6 mice were used, BBB permeability was increased in some, but not all, of the WNV-infected animals. A procedure was developed to measure BBB permeability in live WNV-infected hamsters by comparing the fluorescence in the CSF, aspirated from the cisterna magnum, with the fluorescence in the serum. Despite a time-dependent tendency towards increased BBB permeability in some WNV-infected hamsters, the highest BBB permeability values did not correlate with mortality. These data indicated that a measurable increase in BBB permeability was not a primary determinant for lethality of WNV infection in rodents. The lack of a consistent increase in BBB permeability in WNV-infected rodents has implications for the understanding of viral entry, viral pathogenesis and accessibility of the CNS of rodents to drugs or effector molecules.

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 g/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.Since patients infected with West Nile virus (WNV) often present for medical attention with symptoms that suggest possible central nervous system (CNS) infection (9), therapies for WNV neurological disease should work even after the virus has infected the CNS. One possible therapy, immune immunoglobulin G (IgG), is being evaluated in a phase IIB clinical trial (NIH identifier NCT00068055) that assesses safety and efficacy in patients with known or suspected WNV infection. However, the product (Omr-IgGam) was generated from pools of nonimmune and immune serum from Israeli donors and has a relatively low neutralizing activity against the strains of WNV that currently circulate in North America (2, 6).A mouse monoclonal antibody (MAb), E16, specific for domain III (DIII) of the envelope protein, has been identified to have potent WNV-neutralizing activity (7,19,20). This MAb engaged 16 residues positioned on four loops of DIII and formed a consensus neutralizing epitope in virtually all WNV strains tested (18). Structural and virological studies suggest that E16 blocks infection at a postattachment state, possibly by inhibiting virus-endosome fusion and nucleocapsid release into the cytoplasm (18). A humanized version of E16 (hE16) that retained its antigen specificity, avidity, and neutralizing activity was generated. Studies with mice showed that treatment was effective even at 5 days after viral injection (16,19), a time at which infectious virus was identified in homogenized mouse brain.Studies with a hamster model of WNV infection subsequently confi...

Acute flaccid polio-like paralysis occurs during natural West Nile virus (WNV) infection in a subset of cases in animals and humans. To evaluate the pathology and the possibility for therapeutic intervention, the authors developed a model of acute flaccid paralysis by injecting WNV directly into the sciatic nerve or spinal cord of hamsters. By directly injecting selected sites of the nervous system with WNV, the authors mapped the lesions responsible for hind limb paralysis to the lumbar spinal cord. Immunohistochemical analysis of spinal cord sections from paralyzed hamsters revealed that WNV-infected neurons localized primarily to the ventral motor horn of the gray matter, consistent with the polio-like clinical presentation. Neuronal apoptosis and diminished cell function were identified by TUNEL (terminal deoxynucleotidyl transferase-mediated BrdUTP nick end labeling) and choline acetyltransferase staining, respectively. Administration of hE16, a potently neutralizing humanized anti-WNV monoclonal antibody, 2 to 3 days after direct WNV infection of the spinal cord, significantly reduced paralysis and mortality. Additionally, a single injection of hE16 as late as 5 days after WNV inoculation of the sciatic nerve also prevented paralysis. Overall, these experiments establish that WNV-induced acute flaccid paralysis in hamsters is due to neuronal infection and injury in the lumbar spinal cord and that treatment with a therapeutic antibody prevents paralysis when administered after WNV infection of spinal cord neurons.