Last data update: Jun 03, 2024. (Total: 46935 publications since 2009)
Records 1-4 (of 4 Records) |
Query Trace: Shore DA [original query] |
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Structural characterization of a protective epitope spanning A(H1N1)pdm09 influenza virus neuraminidase monomers
Wan H , Yang H , Shore DA , Garten RJ , Couzens L , Gao J , Jiang L , Carney PJ , Villanueva J , Stevens J , Eichelberger MC . Nat Commun 2015 6 6114 A(H1N1)pdm09 influenza A viruses predominated in the 2013-2014 USA influenza season, and although most of these viruses remain sensitive to Food and Drug Administration-approved neuraminidase (NA) inhibitors, alternative therapies are needed. Here we show that monoclonal antibody CD6, selected for binding to the NA of the prototypic A(H1N1)pdm09 virus, A/California/07/2009, protects mice against lethal virus challenge. The crystal structure of NA in complex with CD6 Fab reveals a unique epitope, where the heavy-chain complementarity determining regions (HCDRs) 1 and 2 bind one NA monomer, the light-chain CDR2 binds the neighbouring monomer, whereas HCDR3 interacts with both monomers. This 30-amino-acid epitope spans the lateral face of an NA dimer and is conserved among circulating A(H1N1)pdm09 viruses. These results suggest that the large, lateral CD6 epitope may be an effective target of antibodies selected for development as therapeutic agents against circulating H1N1 influenza viruses. |
Structural stability of influenza A(H1N1)pdm09 virus hemagglutinins
Yang H , Chang JC , Guo Z , Carney PJ , Shore DA , Donis RO , Cox NJ , Villanueva JM , Klimov AI , Stevens J . J Virol 2014 88 (9) 4828-38 The non-covalent interactions that mediate trimerization of the influenza hemagglutinin (HA) are important determinants of its biological activities. Recent studies have demonstrated that mutations in the HA trimer interface affect the thermal and pH sensitivities of HA, suggesting a possible impact on vaccine stability (Farnsworth et al. 2011. Vaccine 29:: 1529-1533). We used size exclusion chromatography analysis of recombinant HA ectodomain to compare the differences among recombinant trimeric HA proteins from early 2009 pandemic H1N1 viruses, which dissociate to monomers, with those of more recent virus HAs that can be expressed as trimers. We analyzed differences amongst the HA sequences and identified inter-molecular interactions mediated by the residue at position 374 (HA0 numbering) of the HA2 sub-domain as critical for HA trimer stability. Crystallographic analyses of HA from the recent H1N1 virus A/Washington/5/2011 highlight the structural basis for this observed phenotype. It remains to be seen whether more recent viruses with this mutation will yield more stable vaccines in the future. IMPORTANCE: Hemagglutinins from the early 2009 H1N1 pandemic viruses are unable to maintain a trimeric complex when expressed in a recombinant system. However HAs from 2010 and 2011 strains are more stable and our work highlights the improvement in stability can be attributed to an E47K substitution in the HA2 subunit of the stalk that emerged naturally in the circulating viruses. |
Quantitation of influenza virus using field flow fractionation and multi-angle light scattering for quantifying influenza A particles
Bousse T , Shore DA , Goldsmith CS , Hossain MJ , Jang Y , Davis CT , Donis RO , Stevens J . J Virol Methods 2013 193 (2) 589-96 Recent advances in instrumentation and data analysis in field flow fractionation and multi-angle light scattering (FFF-MALS) have enabled greater use of this technique to characterize and quantitate viruses. In this study, the FFF-MALS technique was applied to the characterization and quantitation of type A influenza virus particles to assess its usefulness for vaccine preparation. The use of FFF-MALS for quantitation and measurement of control particles provided data accurate to within 5% of known values, reproducible with a coefficient of variation of 1.9%. The methods, sensitivity and limit of detection were established by analyzing different volumes of purified virus, which produced a linear regression with fitting value R2 of 0.99. FFF-MALS was further applied to detect and quantitate influenza virus in the supernatant of infected MDCK cells and allantoic fluids of infected eggs. FFF fractograms of the virus present in these different fluids revealed similar distribution of monomeric and oligomeric virions. However, the monomer fraction of cell grown virus has greater size variety. Notably, beta-propialactone (BPL) inactivation of influenza viruses did not influence any of the FFF-MALS measurements. Quantitation analysis by FFF-MALS was compared to infectivity assays and real-time RT-PCR (qRT-PCR) and the limitations of each assay were discussed. |
T cell receptors are structures capable of initiating signaling in the absence of large conformational rearrangements
Fernandes RA , Shore DA , Vuong MT , Yu C , Zhu X , Pereira-Lopes S , Brouwer H , Fennelly JA , Jessup CM , Evans EJ , Wilson IA , Davis SJ . J Biol Chem 2012 287 (16) 13324-35 ![]() Native and non-native ligands of the T cell receptor (TCR), including antibodies, have been proposed to induce signaling in T cells via intra- or intersubunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found for such postulated structural changes during TCR triggering induced by antibodies, using crystallographic and mutagenesis-based approaches. The crystal structure of murine CD3epsilon complexed with the mitogenic anti-CD3epsilon antibody 2C11 enabled the first direct structural comparisons of antibody-liganded and unliganded forms of CD3epsilon from a single species, which revealed that antibody binding does not induce any substantial rearrangements within CD3epsilon. Saturation mutagenesis of surface-exposed CD3epsilon residues, coupled with assays of antibody-induced signaling by the mutated complexes, suggests a new configuration for the complex within which CD3epsilon is highly exposed and reveals that no large new CD3epsilon interfaces are required to form during antibody-induced signaling. The TCR complex therefore appears to be a structure that is capable of initiating intracellular signaling in T cells without substantial structural rearrangements within or between the component subunits. Our findings raise the possibility that signaling by native ligands might also be initiated in the absence of large structural rearrangements in the receptor. |
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