Transmission electron microscopy analysis of N1-PPs.

<p>Culture supernatants of 293T cells transfected with no DNA (Mock) and with both pNL4-3luc and pI.18_ N1 from the A/turkey/Turkey/01/2005 influenza strain (A/tk/TK N1-PPs) plasmids, 48 h post-transfection. Negative staining of A/tk/TK N1-PPs (A) and Mock (B), and immunogold labeling of NA in A/tk/TK N1-PPs (C) and Mock (D) samples with mouse polyclonal anti-N1 A/turkey/Turkey/01/2005 serum. Arrows represent gold-labeled Ab bound to NA. Scale bar is 200 nm.</p

Titration of NA activity and its specific inhibition by ELLA.

<p>(A) NA activity in A/CA and A/tk/TK N1-PPs, and Mock samples was titrated incubating fetuin-coated plates with serial dilutions of the culture supernatants. A NA working dilution corresponding to the beginning of the linear part of the titration curve (OD_450nm = 2) was selected to standardize NA activity in the inhibition test. Each data point represents the mean and the standard deviation from at least two independent experiments. Non-parametric Mann-Whitney test was performed to compare ODs values at each PPs dilution; *** p < 0.001, ** p < 0.005, and * p < 0.05. (B) Inhibition of A/CA and A/tk/TK N1-PPs enzymatic activity by sera specific for N1 (A/California/07/2009, A/turkey/Turkey/01/2005, A/NewCaledonia/20/99), N2 (A/Wyoming/3/2003) and B (B/Malaysia/2506/2004 and B/Florida/4/2006) NAs. Each bar represents the mean and the standard deviation from at least two independent experiments.</p

Detection of NA, p24 and p17 proteins in A/tk/TK N1-PPs.

<p>Culture supernatants of 293T cells transfected without plasmids (Mock) and with different combinations of the two plasmids (ΔN1-PPs or pI.18_N1) were resolved by 4–12% SDS-PAGE in reducing conditions, transferred to nitrocellulose membrane and immunoblotted with (A) sheep polyclonal anti-N1 A/Turkey, (B) mouse monoclonal anti-p24 and, (C) rabbit polyclonal anti-p55+p17 Ab. Arrows identify the corresponding protein. Data shown are representative of two independent experiments.</p

NI titers measured in a panel of sera using different sources N1 from A/California/07/2009 (H1N1) virus.

<p>(A) Sheep polyclonal anti-N1 A/California/07/2009 and anti-NA B/Florida/04/2006 sera, (B) mouse polyclonal anti-HA sera with low and high HI titers, and (C) mouse polyclonal preimmune and anti-A/California/07/2009 subunit vaccine sera and normal human serum were tested <i>vs</i> the whole live and Triton X-100 treated A/California/07/2009 (H1N1) viruses and <i>vs</i> N1-PPs, with or without the mismatched HA from A/Vietnam/1194/04 (H5N1) influenza virus. Each bar represents the mean and the standard deviation from at least two independent experiments. Parametric One-Way ANOVA test was performed to compare NI titers; *** p < 0.001, ** p < 0.005, and * p < 0.05.</p

Expression and Characterization of Recombinant, Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay

<div><p>Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. Hemagglutinin (HA) and neuraminidase (NA) are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments.</p><p>The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA) was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA).</p></div

rNAs as sources of sialidase in ELLA.

<p>(A) NI titers determined in a panel of NIBSC sheep polyclonal sera specific for A/turkey/Turkey/01/2005, A/California/07/2009 and A/Caledonia/22/99 N1, A/Wyoming/3/2003 N2, and B/Malaysia/2506/2004 and B/Florida/4/2006 B NAs. (B) NI titers in sera of mice immunized with swine H1N1 and avian H5N1 rNAs adjuvanted with MF59. Data show mean±SD from three independent experiments performed in duplicate. NA = not assayed.</p

Purification of tetrameric swine H1N1 and avian H5N1 rNAs.

<p>(A) Avian H5N1 rNA purification by ion metal affinity chromatography; SDS-PAGE followed by Coomassie staining. MW: molecular weight marker (kDa); lane 1: pooled crude supernatants; lane 2: flow-through; lane 3: fraction eluted after washing with 10 mM imidazole; lane 4: fraction eluted after wash with 20 mM imidazole; lane 5: fraction eluted with 300 mM imidazole. (B) Gel filtration chromatogram of His-purified avian H5N1 rNA recorded at 280 nm wavelength. (C) SDS-PAGE followed by Coomassie staining of final purified, soluble, tetrameric swine H1N1 (lane 1) and avian H5N1 (lane 2) rNAs.Data shown are representative of at least three independent experiments.</p

DSF analysis of avian H5N1 rNA.

<p>The thermostabilizing effect of Ca²⁺ ions binding to rNA was detected by DSF in the presence of Sypro orange. The graph shows fluorescence intensity <i>vs</i> temperature for increasing amount of Ca²⁺, 0.079–20 mM, in 25 mM Tris pH 8, 150 mM NaCl buffer.</p

Enzymatic properties of swine H1N1 and avian H5N1 rNAs.

<p>Enzymatic properties of swine H1N1 and avian H5N1 rNAs.</p

Sialidase activity of swine H1N1 and avian H5N1 rNAs.

<p>(A) Kinetic analyses of rNAs. Triplicate data sets for each experiment were used to calculate the steady-state velocity at decreasing concentrations of MuNANA substrate for each enzyme, and were expressed as initial rates (μM/s) <i>vs</i> concentration of substrate. The reactions containing 0.2 nM of enzyme and 0.59–600 μM of MuNANA were performed at 37°C in 200 mM NaOAc, 20 mM CaCl₂, 0,01 mg/ml BSA, pH5.5. (B) Titration of rNAs activity by ELLA. Decreasing amount of rNAs were incubated with a fixed amount of fetuin overnight at 37°C and OD detected were graphed <i>vs</i> the protein concentration. Data represent mean±SD of 3 independent experiments performed in duplicate.</p