A dual drug regimen synergistically blocks human parainfluenza virus infection.

International audienceHuman parainfluenza type-3 virus (hPIV-3) is one of the principal aetiological agents of acute respiratory illness in infants worldwide and also shows high disease severity in the elderly and immunocompromised, but neither therapies nor vaccines are available to treat or prevent infection, respectively. Using a multidisciplinary approach we report herein that the approved drug suramin acts as a non-competitive in vitro inhibitor of the hPIV-3 haemagglutinin-neuraminidase (HN). Furthermore, the drug inhibits viral replication in mammalian epithelial cells with an IC50 of 30 μM, when applied post-adsorption. Significantly, we show in cell-based drug-combination studies using virus infection blockade assays, that suramin acts synergistically with the anti-influenza virus drug zanamivir. Our data suggests that lower concentrations of both drugs can be used to yield high levels of inhibition. Finally, using NMR spectroscopy and in silico docking simulations we confirmed that suramin binds HN simultaneously with zanamivir. This binding event occurs most likely in the vicinity of the protein primary binding site, resulting in an enhancement of the inhibitory potential of the N-acetylneuraminic acid-based inhibitor. This study offers a potentially exciting avenue for the treatment of parainfluenza infection by a combinatorial repurposing approach of well-established approved drugs

Novel sialic acid derivatives lock open the 150-loop of an influenza A virus group-1 sialidase

This work was supported by the Medical Research Council and the Scottish Funding Council.Influenza virus sialidase has an essential role in the virus’ life cycle. Two distinct groups of influenza A virus sialidases have been established, that differ in the flexibility of the ‘150-loop’, providing a more open active site in the apo form of the group-1 compared to group-2 enzymes. In this study we show, through a multidisciplinary approach, that novel sialic acid-based derivatives can exploit this structural difference and selectively inhibit the activity of group-1 sialidases. We also demonstrate that group-1 sialidases from drug-resistant mutant influenza viruses are sensitive to these designed compounds. Moreover, we have determined, by protein X-ray crystallography, that these inhibitors lock open the group-1 sialidase flexible 150-loop, in agreement with our molecular modelling prediction. This is the first direct proof that compounds may be developed to selectively target the pandemic A/H1N1, avian A/H5N1 and other group-1 sialidase-containing viruses, based on an open 150-loop conformation of the enzyme.Publisher PDFPeer reviewe

Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors

Mammals express the sialic acids ​N-acetylneuraminic acid (​Neu5Ac) and ​N-glycolylneuraminic acid (​Neu5Gc) on cell surfaces, where they act as receptors for pathogens, including influenza A virus (IAV). ​Neu5Gc is synthesized from ​Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). In humans, this enzyme is inactive and only ​Neu5Ac is produced. Ferrets are susceptible to human-adapted IAV strains and have been the dominant animal model for IAV studies. Here we show that ferrets, like humans, do not synthesize ​Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret CMAH gene that is shared by the Pinnipedia and Musteloidia members of the Carnivora. Interactions between two human strains of IAV with the sialyllactose receptor (sialic acid—α2,6Gal) confirm that the type of terminal sialic acid contributes significantly to IAV receptor specificity. Our results indicate that exclusive expression of ​Neu5Ac contributes to the susceptibility of ferrets to human-adapted IAV strains

Novel sialic acid derivatives lock open the 150-loop of an influenza A virus group-1 sialidase

Influenza virus sialidase has an essential role in the virus' life cycle. Two distinct groups of influenza A virus sialidases have been established, that differ in the flexibility of the '150-loop', providing a more open active site in the apo form of the group-1 compared to group-2 enzymes. In this study we show, through a multidisciplinary approach, that novel sialic acid-based derivatives can exploit this structural difference and selectively inhibit the activity of group-1 sialidases. We also demonstrate that group-1 sialidases from drug-resistant mutant influenza viruses are sensitive to these designed compounds. Moreover, we have determined, by protein X-ray crystallography, that these inhibitors lock open the group-1 sialidase flexible 150-loop, in agreement with our molecular modelling prediction. This is the first direct proof that compounds may be developed to selectively target the pandemic A/H1N1, avian A/H5N1 and other group-1 sialidase-containing viruses, based on an open 150-loop conformation of the enzyme

Glycan based detection and drug susceptibility of influenza virus

ABSTRACT: We have developed a panel of synthetic glycans as receptor mimics for the specific capture of influenza viruses. The glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. The microarray was evaluated for its ability to capture three different strains of influenza A virus, two H1N1, A/Brisbane/59/2007 and A/Solomon Islands/3/2006 and one H3N2, A/Aichi/2/1968. We observed an excellent detection ability with some compounds exhibiting clinically relevant (101 plaque forming units) limit of detection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using this microarray and could determine antiviral resistance for these strains

Characterization of the N-glycans of female Angiostrongylus cantonensis worms

Glycoconjugates play a crucial role in the host-parasite relationships of helminthic infections, including angiostrongyliasis. It has previously been shown that the antigenicity of proteins from female Angiostrongylus cantonensis worms may depend on their associated glycan moieties. Here, an N-glycan profile of A. cantonensis is reported. A total soluble extract (TE) was prepared from female A. cantonensis worms and was tested by western blot before and after glycan oxidation or N- and O-glycosidase treatment. The importance of N-glycans for the immunogenicity of A. cantonensis was demonstrated when deglycosylation of the TE with PNGase F completely abrogated IgG recognition. The TE was also fractionated using various lectin columns [Ulex europaeus (UEA), concanavalin A (Con A), Arachis hypogaea (PNA), Triticum vulgaris (WGA) and Lycopersicon esculentum (LEA)], and then each fraction was digested with PNGase F. Released N-glycans were analyzed with matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) and MALDI-TOF/TOF-MS/MS. Complex-type, high mannose, and truncated glycan structures were identified in all five fractions. Sequential MALDI-TOF-TOF analysis of the major MS peaks identified complex-type structures, with a α1-6 fucosylated core and truncated antennas. Glycoproteins in the TE were labeled with BodipyAF558-SE dye for a lectin microarray analysis. Fluorescent images were analyzed with ProScanArray imaging software followed by statistical analysis. A total of 29 lectins showed positive binding to the TE. Of these, Bandeiraea simplicifolia (BS-I), PNA, and Wisteria floribunda (WFA), which recognize galactose (Gal) and N-acetylgalactosamine (GalNAc), exhibited high affinity binding. Taken together, our findings demonstrate that female A. cantonensis worms have characteristic helminth N-glycans.Office of the Snr Dep Vice Chancellor, Institute for GlycomicsNo Full Tex

Prediction of Directional Changes of Influenza A Virus Genome Sequences with Emphasis on Pandemic H1N1/09 as a Model Case

Influenza virus poses a significant threat to public health, as exemplified by the recent introduction of the new pandemic strain H1N1/09 into human populations. Pandemics have been initiated by the occurrence of novel changes in animal sources that eventually adapt to human. One important issue in studies of viral genomes, particularly those of influenza virus, is to predict possible changes in genomic sequence that will become hazardous. We previously established a clustering method termed ‘BLSOM’ (batch-learning self-organizing map) that does not depend on sequence alignment and can characterize and compare even 1 million genomic sequences in one run. Strategies for comparing a vast number of genomic sequences simultaneously become increasingly important in genome studies because of remarkable progresses in nucleotide sequencing. In this study, we have constructed BLSOMs based on the oligonucleotide and codon composition of all influenza A viral strains available. Without prior information with regard to their hosts, sequences derived from strains isolated from avian or human sources were successfully clustered according to the hosts. Notably, the pandemic H1N1/09 strains have oligonucleotide and codon compositions that are clearly different from those of human seasonal influenza A strains. This enables us to infer future directional changes in the influenza A viral genome

Structural and Functional Analysis of Laninamivir and its Octanoate Prodrug Reveals Group Specific Mechanisms for Influenza NA Inhibition

The 2009 H1N1 influenza pandemic (pH1N1) led to record sales of neuraminidase (NA) inhibitors, which has contributed significantly to the recent increase in oseltamivir-resistant viruses. Therefore, development and careful evaluation of novel NA inhibitors is of great interest. Recently, a highly potent NA inhibitor, laninamivir, has been approved for use in Japan. Laninamivir is effective using a single inhaled dose via its octanoate prodrug (CS-8958) and has been demonstrated to be effective against oseltamivir-resistant NA in vitro. However, effectiveness of laninamivir octanoate prodrug against oseltamivir-resistant influenza infection in adults has not been demonstrated. NA is classified into 2 groups based upon phylogenetic analysis and it is becoming clear that each group has some distinct structural features. Recently, we found that pH1N1 N1 NA (p09N1) is an atypical group 1 NA with some group 2-like features in its active site (lack of a 150-cavity). Furthermore, it has been reported that certain oseltamivir-resistant substitutions in the NA active site are group 1 specific. In order to comprehensively evaluate the effectiveness of laninamivir, we utilized recombinant N5 (typical group 1), p09N1 (atypical group 1) and N2 from the 1957 pandemic H2N2 (p57N2) (typical group 2) to carry out in vitro inhibition assays. We found that laninamivir and its octanoate prodrug display group specific preferences to different influenza NAs and provide the structural basis of their specific action based upon their novel complex crystal structures. Our results indicate that laninamivir and zanamivir are more effective against group 1 NA with a 150-cavity than group 2 NA with no 150-cavity. Furthermore, we have found that the laninamivir octanoate prodrug has a unique binding mode in p09N1 that is different from that of group 2 p57N2, but with some similarities to NA-oseltamivir binding, which provides additional insight into group specific differences of oseltamivir binding and resistance

Pneumococcal Phasevarions Control Multiple Virulence Traits, Including Vaccine Candidate Expression

Published online 10 May 2022Streptococcus pneumoniae is the most common cause of bacterial illness worldwide. Current vaccines based on the polysaccharide capsule are only effective against a limited number of the .100 capsular serotypes. A universal vaccine based on conserved protein antigens requires a thorough understanding of gene expression in S. pneumoniae. All S. pneumoniae strains encode the SpnIII Restriction-Modification system. This system contains a phase-variable methyltransferase that switches specificity, and controls expression of multiple genes—a phasevarion. We examined the role of this phasevarion during pneumococcal pathobiology, and determined if phase variation resulted in differences in expression of currently investigated conserved protein antigens. Using locked strains that express a single methyltransferase specificity, we found differences in clinically relevant traits, including survival in blood, and adherence to and invasion of human cells. We also observed differences in expression of numerous proteinaceous vaccine candidates, which complicates selection of antigens for inclusion in a universal protein-based pneumococcal vaccine. This study will inform vaccine design against S. pneumoniae by ensuring only stably expressed candidates are included in a rationally designed vaccine.Zachary N. Phillips, Claudia Trappetti, Annelies Van Den Bergh, Gael Martin, Ainslie Calcutt, Victoria Ozberk, Patrice Guillon, Manisha Pandey, Mark von Itzstein, W. Edward Swords, James C. Paton, Michael P. Jennings, John M. Atac

Production of α-L-iduronidase in maize for the potential treatment of a human lysosomal storage disease

Lysosomal storage diseases are a class of over 70 rare genetic diseases that are amenable to enzyme replacement therapy. Towards developing a plant-based enzyme replacement therapeutic for the lysosomal storage disease mucopolysaccharidosis I, here we expressed α-L-iduronidase in the endosperm of maize seeds by a previously uncharacterized mRNA-targeting-based mechanism. Immunolocalization, cellular fractionation and in situ RT-PCR demonstrate that the α-L-iduronidase protein and mRNA are targeted to endoplasmic reticulum (ER)-derived protein bodies and to protein body-ER regions, respectively, using regulatory (5′-and 3′-UTR) and signal-peptide coding sequences from the γ-zein gene. The maize α-L-iduronidase exhibits high activity, contains high-mannose N-glycans and is amenable to in vitro phosphorylation. This mRNA-based strategy is of widespread importance as plant N-glycan maturation is controlled and the therapeutic protein is generated in a native form. For our target enzyme, the N-glycan structures are appropriate for downstream processing, a prerequisite for its potential as a therapeutic protein.9 page(s