Phenotypic and genotypic characterization of influenza virus mutants selected with the sialidase fusion protein DAS181.

BACKGROUND: influenza viruses (IFVs) frequently achieve resistance to antiviral drugs, necessitating the development of compounds with novel mechanisms of action. DAS181 (Fludase), a sialidase fusion protein, may have a reduced potential for generating drug resistance due to its novel host-targeting mechanism of action. METHODS: IFV strains B/Maryland/1/59 and A/Victoria/3/75 (H3N2) were subjected to >30 passages under increasing selective pressure with DAS181. The DAS181-selected IFV isolates were characterized in vitro and in mice. RESULTS: despite extensive passaging, DAS181-selected viruses exhibited a very low level of resistance to DAS181, which ranged between 3- and 18-fold increase in EC(50). DAS181-selected viruses displayed an attenuated phenotype in vitro, as exhibited by slower growth, smaller plaque size and increased particle to pfu ratios relative to wild-type virus. Further, the DAS181 resistance phenotype was unstable and was substantially reversed over time upon DAS181 withdrawal. In mice, the DAS181-selected viruses exhibited no greater virulence than their wild-type counterparts. Genotypic and phenotypic analysis of DAS181-selected viruses revealed mutations in the haemagglutinin (HA) and neuraminidase (NA) molecules and also changes in HA and NA function. CONCLUSIONS: results indicate that resistance to DAS181 is minimal and unstable. The DAS181-selected IFV isolates exhibit reduced fitness in vitro, likely due to altered HA and NA functions.

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.

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.

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.

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.

Pre- and postsynaptic actions of L1-CAM in nicotinic pathways.

Cell adhesion molecules (CAMs) have long been known to guide axon outgrowth and pathfinding. More recent evidence indicates they contribute to synapse formation as well. The L1 family of IgCAMs has been implicated in long-term potentiation, learning, and some features of synaptic structure. We show here that L1 is localized in nicotinic pathways at both pre- and postsynaptic sites. In the chick ciliary ganglion, postsynaptic L1 is associated with nicotinic receptors and potentiates their downstream signaling. Postsynaptic L1 is also important for aligning presynaptic structures over the postsynaptic cell. Dominant negative experiments suggest this latter effect depends on homophilic interactions with presynaptic L1. At the neuromuscular junction L1 is also found presynaptically where dominant negative experiments again indicate a role in aligning presynaptic structures over postsynaptic receptors, both in culture and in vivo. These findings identify new roles for L1 at nicotinic synapses and underscore the multipotency of L1-CAMs.

Cytoplasmic domain of protocadherin-alpha enhances homophilic interactions and recognizes cytoskeletal elements.

Cell adhesion molecules of the protocadherin-alpha (pcdh-alpha), -beta, and -gamma families have been proposed to be synaptic specifiers. Pcdh-alpha and -gamma family members localize in part to synapses, and deletion of all pcdh-gammas in mouse affects synaptogenesis. Little is known, however, about the binding specificities and intracellular signaling of protocadherins. Using heterologous expression of tagged constructs, immunostaining, and biotinylation of surface components followed by Western blots we demonstrate that pcdh-alphas undergo homophilic interactions that are significantly enhanced by the cytoplasmic domain. Pcdh-alphas cloned from chick ciliary ganglion have one of two cytoplasmic constant regions (A- and B-types). Screening a yeast two-hybrid library of ciliary ganglion cDNA with the A-type domain yielded a fragment of neurofilament M (NFM); screening with B-type domain yielded a fragment of the actin-bundling protein fascin. Cotransfection of HEK cells with the constructs indicated that the NFM and A-type fragments codistributed as did the fascin and B-type fragments, and the latter could be coimmunoprecipitated. Antibody-induced clustering of full-length pcdh-alphas on the surface of transfected HEK cells induced coclustering of the interacting NFM fragment. Native full-length NFM in tissue extracts bound specifically to the A-type domain on beads, while native full-length fascin in tissue extracts specifically coimmunoprecipitated with pcdh-alpha. Immunostaining neurons demonstrated codistribution of full-length pcdh-alpha with both NFM and actin filaments. These findings suggest cytoskeletal links for pcdh-alphas and identify candidate targets. They also demonstrate homophilic interactions for pcdh-alphas as described for classical cadherins.

Alpha-protocadherins are presynaptic and axonal in nicotinic pathways.

The protocadherin families pcdh-alpha, beta, and gamma have been proposed to mediate synaptic specificity via homophilic interactions. Here we report isolation of two pcdh-alpha family members from chick. We find pcdh-alpha mRNA in multiple regions of chick CNS including cerebellum, tectum, olfactory bulb, and forebrain, and in the autonomic nervous system. Immunoblots identify major components of 120 and 140 kDa both in brain and ciliary ganglion extracts. Immunohistochemistry reveals pcdh-alphas in axons and perisynaptically in preganglionic terminals, adjacent to transmitter release sites. Pcdh-alphas appear to be absent from postsynaptic sites: They are nonoverlapping with postsynaptic receptor clusters in the ganglion and are rapidly lost after ganglionic denervation. Similar pcdh-alpha patterns are found in motor axons and at neuromuscular junctions of birds and mammals, and persist into adulthood. The results indicate that pcdh-alphas are widely expressed in nicotinic cholinergic pathways and may engage in heterophilic interactions at synapses and on axons.