Inhibition of Neuraminidase Inhibitor-Resistant Influenza Virus by DAS181, a Novel Sialidase Fusion Protein

Antiviral drug resistance for influenza therapies remains a concern due to the high prevalence of H1N1 2009 seasonal influenza isolates which display H274Y associated oseltamivir-resistance. Furthermore, the emergence of novel H1N1 raises the potential that additional reassortments can occur, resulting in drug resistant virus. Thus, additional antiviral approaches are urgently needed. DAS181 (Fludase®), a sialidase fusion protein, has been shown to have inhibitory activity against a large number of seasonal influenza strains and a highly pathogenic avian influenza (HPAI) strain (H5N1). Here, we examine the in vitro activity of DAS181 against a panel of 2009 oseltamivir-resistant seasonal H1N1 clinical isolates. The activity of DAS181 against nine 2009, two 2007, and two 2004 clinical isolates of seasonal IFV H1N1 was examined using plaque number reduction assay on MDCK cells. DAS181 strongly inhibited all tested isolates. EC50 values remained constant against isolates from 2004, 2007, and 2009, suggesting that there was no change in DAS181 sensitivity over time. As expected, all 2007 and 2009 isolates were resistant to oseltamivir, consistent with the identification of the H274Y mutation in the NA gene of all these isolates. Interestingly, several of the 2007 and 2009 isolates also exhibited reduced sensitivity to zanamivir, and accompanying HA mutations near the sialic acid binding site were observed. DAS181 inhibits IFV that is resistant to NAIs. Thus, DAS181 may offer an alternative therapeutic option for seasonal or pandemic IFVs that become resistant to currently available antiviral drugs

A two-step workflow based on plasma p-tau217 to screen for amyloid β positivity with further confirmatory testing only in uncertain cases

Cost-effective strategies for identifying amyloid-β (Aβ) positivity in patients with cognitive impairment are urgently needed with recent approvals of anti-Aβ immunotherapies for Alzheimer’s disease (AD). Blood biomarkers can accurately detect AD pathology, but it is unclear whether their incorporation into a full diagnostic workflow can reduce the number of confirmatory cerebrospinal fluid (CSF) or positron emission tomography (PET) tests needed while accurately classifying patients. We evaluated a two-step workflow for determining Aβ-PET status in patients with mild cognitive impairment (MCI) from two independent memory clinic-based cohorts (n = 348). A blood-based model including plasma tau protein 217 (p-tau217), age and APOE ε4 status was developed in BioFINDER-1 (area under the curve (AUC) = 89.3%) and validated in BioFINDER-2 (AUC = 94.3%). In step 1, the blood-based model was used to stratify the patients into low, intermediate or high risk of Aβ-PET positivity. In step 2, we assumed referral only of intermediate-risk patients to CSF Aβ42/Aβ40 testing, whereas step 1 alone determined Aβ-status for low- and high-risk groups. Depending on whether lenient, moderate or stringent thresholds were used in step 1, the two-step workflow overall accuracy for detecting Aβ-PET status was 88.2%, 90.5% and 92.0%, respectively, while reducing the number of necessary CSF tests by 85.9%, 72.7% and 61.2%, respectively. In secondary analyses, an adapted version of the BioFINDER-1 model led to successful validation of the two-step workflow with a different plasma p-tau217 immunoassay in patients with cognitive impairment from the TRIAD cohort (n = 84). In conclusion, using a plasma p-tau217-based model for risk stratification of patients with MCI can substantially reduce the need for confirmatory testing while accurately classifying patients, offering a cost-effective strategy to detect AD in memory clinic settings

Novel Pandemic Influenza A(H1N1) Viruses Are Potently Inhibited by DAS181, a Sialidase Fusion Protein

Background: The recent emergence of a novel pandemic influenza A(H1N1) strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. However, resistance to antivirals can be a formidable problem as evidenced by the currently widespread oseltamivir- and adamantane-resistant seasonal influenza A viruses (IFV). Additional antiviral approaches with novel mechanisms of action are needed to combat novel and resistant influenza strains. DAS181 (Fludase)™) is a sialidase fusion protein in early clinical development with in vitro and in vivo preclinical activity against a variety of seasonal influenza strains and highly pathogenic avian influenza strains (A/H5N1). Here, we use in vitro, ex vivo, and in vivo models to evaluate the activity of DAS181 against several pandemic influenza A(H1N1) viruses. Methods and Findings: The activity of DAS181 against several pandemic influenza A(H1N1) virus isolates was examined in MDCK cells, differentiated primary human respiratory tract culture, ex-vivo human bronchi tissue and mice. DAS181 efficiently inhibited viral replication in each of these models and against all tested pandemic influenza A(H1N1) strains. DAS181 treatment also protected mice from pandemic influenza A(H1N1)-induced pathogenesis. Furthermore, DAS181 antiviral activity against pandemic influenza A(H1N1) strains was comparable to that observed against seasonal influenza virus including the H274Y oseltamivir-resistant influenza virus. Conclusions: The sialidase fusion protein DAS181 exhibits potent inhibitory activity against pandemic influenza A(H1N1) viruses. As inhibition was also observed with oseltamivir-resistant IFV (H274Y), DAS181 may be active against the antigenically novel pandemic influenza A(H1N1) virus should it acquire the H274Y mutation. Based on these and previous results demonstrating DAS181 broad-spectrum anti-IFV activity, DAS181 represents a potential therapeutic agent for prevention and treatment of infections by both emerging and seasonal strains of IFV.published_or_final_versio

Nicotinic synapse formation between neurons : novel roles for cell adhesion molecules

Nicotinic cholinergic synapses between neurons are thought to be involved in numerous aspects of nervous system development, function, and degeneration. Despite their importance, almost nothing is known about how these neural connections are formed, modulated, and maintained. To address these issues the chick ciliary ganglion (CG), a central component of a classic nicotinic pathway, was used to identify and characterize some of the key transmembrane molecules responsible for formation of interneuronal nicotinic synapses. Immunostaining and biochemistry were used to characterize the temporal and spatial expression patterns of a recently discovered family of cell adhesion molecules (CAMs) in the CG, protocadherin-alphas (pcdh- [alpha]s). Furthermore, intracellular binding interactions for pcdh-[alpha]s in the CG were defined using a yeast-two -hybrid screen and immunoprecipitations. Previous reports have suggested a role for pcdh-[alpha]s in synaptic specificity during target cell selection, and the data presented here corroborates this hypothesis. Several other CAMs have been reported to play more central roles in directing synapse formation in glutamatergic pathways. Using complex cell culture assays to dissect aspects of synaptogenesis this work shows that the IgCAM L1 is necessary and sufficient to accumulate and align presynaptic specializations in cholinergic neurons. Further examination revealed that a transsynaptic homophilic interaction between distinct L1 populations likely mediates this affect, and also facilitates nAChR signaling to the nucleus. The presence of two other synaptogenic CAMs in the CG, neuroligin and SynCAM, is described here. To address the relative roles of L1, neuroligin, and SynCAM in CG neurons a novel electroporation protocol was generated to transfect CG neurons in situ. This technique revealed that L1 and NL are each required for normal presynaptic organization in the ganglion. Furthermore, these two CAMs are not redundant in this function, yet act at the same synapse population and in the same functional pathway. This work identifies novel functions for CAMs beyond simple adhesion. The results also suggest an emerging theme of multipotency of CAM function, as well as the existence of numerous synaptic CAMs on the same cholinergic neurons. These conclusions suggest that CAMs play crucial, inductive, and complex roles in the formation of cholinergic nicotinic synapses between neuron

Multiple cell adhesion molecules shaping a complex nicotinic synapse on neurons

Neuroligin, SynCAM, and L1-CAM are cell adhesion molecules with synaptogenic roles in glutamatergic pathways. We show here that SynCAM is expressed in the chick ciliary ganglion, embedded in a nicotinic pathway, and, as shown previously for neuroligin and L1-CAM, acts transcellularly to promote synaptic maturation on the neurons in culture. Moreover, we show that electroporation of chick embryos with dominant negative constructs disrupting any of the three molecules in vivo reduces the total amount of presynaptic SV2 overlaying the neurons expressing the constructs. Only disruption of L1-CAM and neuroligin, however, reduces the number of SV2 puncta specifically overlaying nicotinic receptor clusters. Disrupting L1-CAM and neuroligin together produces no additional decrement, indicating that they act on the same subset of synapses. SynCAM may affect synaptic maturation rather than synapse formation. The results indicate that individual neurons can express multiple synaptogenic molecules with different effects on the same class of nicotinic synapses.status: publishe

DAS181, a sialidase fusion protein, protects human airway epithelium against influenza virus infection: an in vitro pharmacodynamic analysis

OBJECTIVES: The influenza virus (IFV) infection models commonly used to evaluate antiviral agents (e.g. MDCK cell line and mice) are limited by physiological differences from the human respiratory tract in vivo. Here we report the pharmacodynamics of DAS181, a sialidase fusion protein that inhibits influenza infection, in the model systems of well-defined human airway epithelium (HAE) culture and ex vivo culture of fresh human bronchial tissue, both of which are close mimics of the human respiratory tract in vivo. METHODS: HAE culture and ex vivo human bronchi were used to evaluate the sialic acid removal and regeneration efficiency and IFV inhibition after various DAS181 treatment levels and regimens. RESULTS: DAS181 effectively desialylates HAE cultures and ex vivo bronchi tissues and therefore potently inhibits replication of different IFV strains. The treatment effect of DAS181 occurs immediately upon application to the epithelial surface and is unaffected by the respiratory mucus. In both HAE and human bronchial tissue, the inhibitory effect of DAS181 treatment lasts for at least 2 days. Approximately 80% epithelial surface desialylation and significant anti-IFV efficacy can be achieved at topical concentrations of DAS181 in the range of 5-10 microg/cm(2) when applied once daily. An additional treatment or a higher loading dose of DAS181 on the first day provides significant additional treatment benefit. Comparing the effect of DAS181 versus its two analogues, DAS180 and DAS185, has confirmed that sialidase function is critical for DAS181, and the cell-binding domain (amphiregulin tag) prolongs DAS181 retention and potentiates its function. CONCLUSIONS: These results provide valuable insights into DAS181 treatment dose and potential regimens in the clinical setting.link_to_subscribed_fulltex

DAS181 inhibits pandemic IFV A(H1N1): viral yield in 2009 A(H1N1) infected HAE culture.

<p>Well differentiated primary human airway epithelium cultures (HAE) were pretreated with DAS181 for 2 hours (at 10 µM) before infection with either A/Mexico/4604/2009 or A/California/04/2009 at indicated infection levels. After 24 hrs the apical wash was collected and viral yield was determined on MDCK cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007788#s4" target="_blank">Materials and Methods</a>. Values represent mean±SD of quadruplicate analysis of each sample.</p

DAS181 inhibits pandemic IFV A(H1N1) viral replication in mice.

<p>BALB/c mice were inoculated with the pandemic influenza A(H1N1) virus A/Mexico/4108/2009 (1000 MID₅₀/mouse, <i>i.n.</i>). The mice were treated with PBS or DAS181 (0.3, 0.6, or 1 mg/kg) <i>q.d.x5</i>, beginning 6 hours post-infection. Lungs were harvested on day 3 and day 6 p.i.. Viral titer in lung homogenate was determined by standard plaque assay on MDCK cells. Values represent mean±SEM viral titer amongst 3 mice per group/day. PFU = plaque forming units. No virus was detectable on day 6 in the DAS181 1.0 mg/kg treatment group. Statistical significance from PBS treatment determined by ANOVA with Bonferroni post-test, * = p<0.05, ** = p<0.01, *** = p<0.001. The limit of detection is 100 PFU/ml.</p