Neil E. Motter

Increased Blood-Brain Barrier Permeability Is Not a Primary Determinant for Lethality of West Nile Virus Infection in Rodents

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.

Design, Synthesis, and Biological Evaluation of Triazolo-pyrimidine Derivatives as Novel Inhibitors of Hepatitis B Virus Surface Antigen (HBsAg) Secretion

The high levels of hepatitis B virus (HBV) surface antigen (HBsAg)-bearing subviral particles in the serum of chronically infected individuals play an important role in suppressing HBV-specific immune response and are only mildly affected by the current small molecule therapies. Thus, a therapy that specifically reduces HBsAg serum levels could be used in combination therapy with nucleos(t)ide drugs or permit therapeutic vaccination for the treatment of HBV infection. Herein, we report the design, synthesis, and evaluation of novel triazolo-pyrimidine inhibitors (1, 3, and 4) of HBsAg cellular secretion, with activity against drug-resistant HBV variants. Extensive SAR led to substantial improvements in the EC(50) of the parent compound, 5 (HBF-0259), with the best being 3c, with EC(50) = 1.4 ± 0.4 μM, SI ≥ 36. The lead candidates, both 1a (PBHBV-001) and 3c (PBHBV-2-15), were well-tolerated in both normal and HBV-transgenic mice and exhibited acceptable pharmacokinetics and bioavailability in Sprague-Dawley rats.

Persistent West Nile Virus Associated with a Neurological Sequela in Hamsters Identified by Motor Unit Number Estimation

ABSTRACT To investigate the hypothesis that neurological sequelae are associated with persistent West Nile virus (WNV) and neuropathology, we developed an electrophysiological motor unit number estimation (MUNE) assay to measure the health of motor neurons temporally in hamsters. The MUNE assay was successful in identifying chronic neuropathology in the spinal cords of infected hamsters. MUNE was suppressed at days 9 to 92 in hamsters injected subcutaneously with WNV, thereby establishing that a long-term neurological sequela does occur in the hamster model. MUNE suppression at day 10 correlated with the loss of neuronal function as indicated by reduced choline acetyltransferase staining (R2 = 0.91). Between days 10 and 26, some α-motor neurons had died, but further neuronal death was not detected beyond day 26. MUNE correlated with disease phenotype, because the lowest MUNE values were detected in paralyzed limbs. Persistent WNV RNA and foci of WNV envelope-positive cells were identified in the central nervous systems of all hamsters tested from 28 to 86 days. WNV-positive staining colocalized with the neuropathology, which suggested that persistent WNV or its products contributed to neuropathogenesis. These results established that persistent WNV product or its proteins cause dysfunction, that WNV is associated with chronic neuropathological lesions, and that this neurological sequela is effectively detected by MUNE. Inasmuch as WNV-infected humans can also experience a poliomyelitis-like disease where motor neurons are damaged, MUNE may also be a sensitive clinical or therapeutic marker for those patients.

Efficacy of cationic lipid–DNA complexes (CLDC) on hepatitis B virus in transgenic mice

Cationic lipid-DNA (non-coding) complexes (CLDC) are activators of the innate immune response that increase survival of rodents with some acute viral infections and cancers. CLDC were evaluated for their ability to impact viral DNA levels in transgenic mice carrying an infectious clone of hepatitis B virus (HBV). Mice used in the studies were diet-restricted as nursing pups from solid food, because the expression of HBV DNA in the liver was increased above background levels in some mice with this restriction. Survival surgery was performed on these mice to obtain liver biopsies from which to select animals with suitable levels of liver HBV DNA for entry into the experimental protocols. Intravenous administration of 5 microg/mouse of CLDC on days 1, 7 and 13 reduced liver HBV DNA to similar low levels achieved with the positive control, adefovir dipivoxil. In a subsequent experiment, the same treatment schedule was used to determine that the minimal effective CLDC dose was between 0.5 and 0.05 microg/mouse. Selective cytokines were increased in the livers of CLDC-treated compared to placebo-treated mice in a dose-responsive manner. CLDC were effective in reducing liver HBV DNA and could be considered for further evaluation in other hepatitis models.

Breaking B and T cell tolerance using cationic lipid--DNA complexes (CLDC) as a vaccine adjuvant with hepatitis B virus (HBV) surface antigen in transgenic mice expressing HBV.

Cationic lipid DNA complexes (CLDC), referred to here as JVRS-100, were evaluated as an adjuvant for hepatitis B surface antigen (HBsAg) for eliciting B and T cell responses in transgenic mice expressing hepatitis B virus (HBV). To confirm the immunogenicity of HBsAg+JVRS-1000, a study was conducted in C57BL/6 mice, the genetic background of the HBV transgenic mice used in the study. HBsAg+JVRS-100 elicited a T cell response and B cell response as evidenced by interferon-gamma (IFN-γ) secretion by re-stimulated splenocytes and anti-HBsAg IgG induction, respectively, whereas, HBsAg only elicited a B cell response. In HBV transgenic mice, HBsAg did not elicit either T or B cell responses, unlike the HBsAg+JVRS-100 that elicited both. Energix-B vaccine did perform better than the HBsAg by eliciting a B cell response in the transgenic mice, but it did not perform as HBsAg+JVRS-100 since it did not elicit a T cell response. The response by HBsAg+JVRS-100 was not sufficient to cause destruction of infected liver cells, but it did suppress HBV DNA non-cytolytically. From these results, JVRS-100 might be considered for further development as an adjuvant for HBV therapeutic vaccines.

Neurological suppression of diaphragm electromyographs in hamsters infected with West Nile virus

To address the hypothesis that respiratory distress associated with West Nile virus (WNV) is neurologically caused, electromyographs (EMGs) were measured longitudinally from the diaphragms of alert hamsters infected subcutaneously (s.c.) with WNV. The EMG activity in WNV-infected hamsters was consistently and significantly (P ≤ .001) less than that of sham-infected animals, beginning with suppression at day 3 and continuing to beyond day 17 after viral challenge. Of the tissues known to affect respiration, i.e., lung, diaphragm, cervical spinal cord, brain stem, and the carotid or aortic bodies, foci of WNV-immunoreactive neurons were only observed in the brain stems and some cervical spinal cords from EMG-suppressed animals. To confirm the involvement of the brain stem and spinal cord, WNV was injected directly in the ventrolateral medulla containing respiratory functions using stereotaxic surgery and into the cervical cord at the C4 vertebral level. As with subcutaneous WNV challenge, hamsters developed EMG suppression of the diaphragm within 4 days. Because WNV-positive neurons were only sporadically identified in EMG-suppressed animals as early as day 3, a plausible mechanism of EMG suppression may involve regulation of diaphragm activity via vagal afferents acting on respiratory control system neurons in the brain stem. Brain auditory evoked response (BAER) was performed to determine if generalized brain stem neuropathy was the cause of diaphragmatic EMG suppression. Because deficiencies of BAER were only observed after day 11, which is long after diaphragm EMGs became suppressed, multiple phases of WNV-induced neurological disease are likely. These data establish that WNV infection of hamsters causes electrophysiological suppression of the diaphragm either directly by lesions in the brain stem and cervical spinal cord, or indirectly by altered vagal afferent function. This WNV-induced EMG suppression may be analogous to conditions leading to respiratory distress of WNV-infected human patients.

Fatal Neurological Respiratory Insufficiency Is Common Among Viral Encephalitides

Background. Neurological respiratory insufficiency strongly correlates with mortality among rodents infected with West Nile virus (WNV), which suggests that this is a primary mechanism of death in rodents and possibly fatal West Nile neurological disease in human patients. Methods. To explore the possibility that neurological respiratory insufficiency is a broad mechanism of death in cases of viral encephalitis, plethysmography was evaluated in mice infected with 3 flaviviruses and 2 alphaviruses. Pathology was investigated by challenging the diaphragm, using electromyography with hypercapnia and optogenetic photoactivation. Results. Among infections due to all but 1 alphavirus, death was strongly associated with a suppressed minute volume. Virally infected mice with a very low minute volume did not neurologically respond to hypercapnia or optogenetic photoactivation of the C4 cervical cord. Neurons with the orexin 1 receptor protein in the ventral C3–5 cervical cord were statistically diminished in WNV-infected mice with a low minute volume as compared to WNV-infected or sham-infected mice without respiratory insufficiency. Also, WNV-infected cells were adjacent to neurons with respiratory functions in the medulla. Conclusions. Detection of a common neurological mechanism of death among viral encephalitides creates opportunities to create broad-spectrum therapies that target relevant neurological cells in patients with types of viral encephalitis that have not been treatable in the past.

Use of plethysmography in assessing the efficacy of antivirals in a mouse model of pandemic influenza A virus

The recently emerged swine-origin H1N1 influenza A virus (IAV) caused a pandemic outbreak in 2009 with higher risk of severe disease among children and pregnant women in their third trimester (Van Kerkhove et al., 2011), and is continuing to be important seasonal IAV strain. Mice are commonly used in antiviral studies as models of influenza disease, which utilize morbidity and mortality to assess the efficacy of a test compound. Here, we investigated the utility of unrestrained plethysomography to quantify the lung function of IAV-infected BALB/c mice. Administration of a lethal dose (∼30X LD(50)) of pandemic H1N1 IAV resulted in a rapid decline in breath volume, as determined by a significant (P<0.001) decrease in the pressure associated with inspiration and expiration detected as early as 2 days after virus challenge. Severe disease was also accompanied by a significant (P<0.05) increase in breath time on 8 dpi. Plethysmography parameters correlated with weight loss and other parameters of disease such as gross pathology and the weight of the lung. Breath time was reduced in surviving mice challenged with a sublethal dose of virus as compared with normal controls, and is a predictive indicator of outcome in these mice. In antiviral studies, the use of plethysmography resulted in the detection of a clear and rapid treatment response, which was similar to other non-invasive parameters, such as weight change. Oseltamivir and ribavirin significantly (P<0.001) improved parameters of lung function, particularly mean breath volume, as early as 2 dpi and in a dose-dependent manner. Moreover, a combination of these two drugs further improved these parameters. Plethysmography provides a sensitive evaluation of lung function in IAV-infected mice in response to antiviral therapy.

Activity of nucleic acid polymers in rodent models of HBV infection

ABSTRACT Nucleic acid polymers (NAPs) block the release of HBsAg from infected hepatocytes. These compounds have been previously shown to have the unique ability to eliminate serum surface antigen in DHBV‐infected Pekin ducks and achieve multilog reduction of HBsAg or HBsAg loss in patients with chronic HBV infection and HBV/HDV coinfection. In ducks and humans, the blockage of HBsAg release by NAPs occurs by the selective targeting of the assembly and/or secretion of subviral particles (SVPs). The clinically active NAP species REP 2055 and REP 2139 were investigated in other relevant animal models of HBV infection including woodchucks chronically infected with WHV, HBV transgenic mice and HBV infected SCID‐Hu mice. The liver accumulation of REP 2139 in woodchucks following subcutaneous administration was examined and was found to be similar to that observed in mice and ducks. However, in woodchucks, NAP treatment was associated with only mild (36–79% relative to baseline) reductions in WHsAg (4/10 animals) after 3–5 weeks of treatment without changes in serum WHV DNA. In HBV infected SCID‐Hu mice, REP 2055 treatment was not associated with any reduction of HBsAg, HBeAg or HBV DNA in the serum after 28 days of treatment. In HBV transgenic mice, no reductions in serum HBsAg were observed with REP 2139 with up to 12 weeks of treatment. In conclusion, the antiviral effects of NAPs in DHBV infected ducks and patients with chronic HBV infection were weak or absent in woodchuck and mouse models despite similar liver accumulation of NAPs in all these species, suggesting that the mechanisms of SVP assembly and or secretion present in rodent models differs from that in DHBV and chronic HBV infections. HighlightsNAPs display similar liver accumulation in ducks, mice, non‐human primates and woodchucks.In woodchuck and mouse models of HBV infection, WHsAg or HBsAg response to NAP treatment is weak or absent.SVP assembly/secretion occurring chronic HBV infection may differ from that in rodent models of HBV infection.