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

Materials

2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MuNANA), fetuin, carbonate/bicarbonate buffer, Tween 20, Bovine Serum Albumin (BSA), Clostridium perfrigens NA, horse radish peroxidase-labelled peanut agglutinin (HRP-PNA), 3,3′,5,5′-Tetramethylbenzidine (TMB), HCl, CelLytic M Reagent and Skim Milk were purchased from Sigma-Aldrich (St. Louis, MO). Polyclonal sheep sera specific for anti-N1 A/California/04/2009 (cat. N° 10/218), anti-N1 A/turkey/Turkey/1/2005 (cat. N° 8/126), anti-N1 A/NewCaledonia/20/99 (cat. N° 4/230), anti-N2 A/Wyoming/3/2003 (cat. N° 4/258), anti-NA B/Malaysia/2506/2004 (cat. N° 5/252), anti-NA B/Florida/4/2006 (cat. N° 9/316) were purchased from National Institute for Biological Standards and Control (NIBSC) (London, England). Influenza virus H1N1 A/California/04/2009 NA X-181 master seed was kindly provided by Novartis Flu Seed Facility (Basel, Switzerland).

Construction of NA-expressing vectors

Swine A/California/07/2009 (H1N1) (GenBank accession N° {"type":"entrez-nucleotide","attrs":{"text":"GQ377078.1","term_id":"253828632","term_text":"GQ377078.1"}}GQ377078.1) and avian A/turkey/Turkey/1/2005 (H5N1) (GenBank accession N° {"type":"entrez-nucleotide","attrs":{"text":"EF619973.1","term_id":"148340538","term_text":"EF619973.1"}}EF619973.1) NA are composed, from the N-terminal to the C-terminal, of a cytoplasmic domain (CD), a transmembrane and tetramerization domain (TM), a stalk region and a globular head domain that is responsible for their sialidase activity (Fig 1A, top). The nucleotide sequences of the globular heads plus the artificial N-terminal domain of 98 residues were human codon optimized for expression in mammalian cells and sub-cloned into prs5a vector (Novartis) using HindIII and NotI restriction enzymes. The artificial N-terminal domain contains a murine Ig κ-light chain secretion sequence (Igκ) followed by a 6 histidine purification tag (6xHis), a Tetrabranchion tetramerization domain (tetrabrachion) from the bacterium Staphylothermus marinus and two glycine residues as linker with the globular head sequence (Fig 1A, bottom). The identity of the resulting vectors was checked by sequencing.

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Fig 1

Design and expression of soluble, enzymatically active NA of H1N1 and H5N1 influenza viruses.

(A) Schematic representation of wild type (WT) and recombinant NA (rNA). The cytoplasmic (CD), tetramerization (TM) and stalk domains of the WT NA were replaced by an Ig-k secretion sequence, a 6xHis-tag, and a tetrabrachion domain for protein tetramerization. (B) Reducing SDS-PAGE followed by WB of Expi293 supernatants and lysates collected every 24 h post-transfection. Anti-His tag (left) and anti-NA (right) stainings were used to specifically detect avian H5N1 rNA. (C, D) Titration of specific sialidase activity in culture supernatants harvested every 24 h post-transfection by ELLA and expressed as the supernatant dilution corresponding to an OD_450nm = 2. Data show mean±SD and are representative of at least three independent experiments.

Expression and purification of rNAs

To produce rNAs, the expression vectors were transfected into Expi293F cells according to the manufacturer’s instructions (Life Technologies). Briefly, 30 μg of prs5a NA-expressing vectors were transfected into 30 ml culture containing 75 x 10⁶ Expi293F cells using ExpiFectamine 293 Reagent. Cells were incubated at 37°C, 120 rpm, 8% CO₂ and after 24 h, ExpiFectamine 293 Transfection enhancer 1 and 2 were added. Cells were further incubated at 37°C for 144 h. Aliquots of cultures were harvested every 24 h and analyzed for NA expression and activity by Western Blot (WB) and ELLA, respectively. Seventy-two and 144 h after transfection, cell cultures were centrifuged at 1000 rpm for 7 min and the NA-containing supernatants were harvested, pooled, clarified by centrifugation, filtered through a 0.22 μm filter, and stored a 4°C until purification. Affinity chromatography with Ni²⁺ was used to purify rNAs from culture supernatants using HisTrap FF crude column (GE Healthcare). Fractions of interest were pooled and were concentrated by using 10 kDa cutoff spin concentrator (Millipore Amicon Ultra); sodium dodecyl sulphate-poly-acrylamide gel electrophoresis (SDS-PAGE) was performed to check purity. Tetrameric rNAs were purified by size exclusion chromatography (SEC) with a HiLoad Superdex columns (GE Healthcare) in a mobile phase containing Tris 50 mM, 150 mM NaCl pH8, CaCl₂ 10 mM using Äkta Purifier (GE Healthcare). A high molecular weight standard kit (Biorad, 151–1901) containing Thyroglobulin (bovine) 670 kDa, γ-globulin (bovine) 158 kDa, Ovalbumin (chicken) 44 kDa, Myoglobin (horse) 17 kDa, and Vitamin B12 1.350 kDa was run to calibrate the columns and to estimate rNAs sizes. All the collected fractions were checked for NA content by SDS-PAGE and NA-containing fractions were pooled, aliquotated and stored at -20°C for further analysis.

SDS-PAGE and western blotting

SDS-PAGE and WB analyses were performed to monitor NA expression in Expi293F cells transfected with p5RSa vectors, and to evaluate NA purity and identity during purification. Supernatants and pellets were obtained from centrifugation at 1200 rpm for 7 min of collected culture aliquots. Cells were lysed with CelLytic M Reagent at room temperature for 15 min and lysates were collected after centrifugation for 10 min at 8000 rpm, 4°C. Samples were mixed with 1X NuPage LDS loading buffer and 1X NuPage Sample reducing agent (Life Technologies), were heated at 100°C for 5 min and 20 μl were loaded onto a 4–12% NuPAGE Bis-Tris gel (Life Technologies). Novex Sharp Pre-stained Protein Standard or SeeBlue Prestained Standard markers were run onto each gel. In order to check the purity level of rNAs gels were stained with Comassie Blue while for rNA identity proteins were electro-transferred onto nitrocellulose membranes with iBlot 2 Dry Blotting System (Life Technologies). The membranes were blocked for 90 min with PBS + 0.1% Tween 20 + 5% milk and then incubated for 1 h with specific sheep polyclonal anti-N1 antibodies diluted 1:3000 or mouse anti-His-tag antibody diluted 1:1000 in blocking buffer.

After three washes in PBS + 0.1% Tween 20 (T-PBS) the membrane were incubated with HRP-labelled donkey anti-sheep or anti-mouse IgG at 1:5000 dilution, for 1 h at room temperature. Membranes were washed thrice with T-PBS, twice with PBS and then incubated with SuperSignal West Pico Chemiluminescent Substrate (ECL; Thermo Fisher Scientific, Rockford, IL) for 2 min. In the dark, photographic hyperfilms (GE Healthcare, Rydalmere, NSW, Australia) were placed against the membrane for increasing times and then developed using Curix60 (AGFA HealthCare, Greenville, USA) films processor.

Peptide-N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) treatment

For glycosylation analysis, N-linked oligosaccharides removal was carried out using recombinant PNGase F and Endo H (New England BioLabs, Beverly, Massachusetts) according to the manufacturer’s instruction. Briefly, 2.25 μg of purified swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/1/2005 (H5N1) rNAs were combined with 1 μl of 10X Glycoprotein Denaturing Buffer and then denaturated by heating reaction at 95°C for 5 min. Proteins were chilled on ice and centrifuged for 10 sec before adding 2 μl of 10X G7 Reaction Buffer, 2 μl 10% NP40, 6 μl H₂O and 1 μl PNGase F to achieve a final volume of reaction of 20 μl. Samples were gently mixed and incubated at 37°C for 1 h. To assess the extent of deglycosylation, the proteins were mixed with 1X NuPage LDS loading buffer and 1X NuPage Sample reducing agent (Life Technologies), heated at 100°C for 5 min and finally loaded onto a 4–12% NuPAGE Bis-Tris gel (Life Technologies) subjected to SDS-PAGE. The mobility shifts were checked by Comassie staining.

Transmission Electron Microscopy (TEM) on ghost membranes

5 μl aliquot of the purified rNA (0.6 mg/ml) sample was loaded onto glow-discharged (15 mA, 286 V) 300-square mesh copper grids coated with a thin carbon film (TED PELLA, Redding, CA) and let stand for 30 sec. Excess of solution was blotted by Whatman filter paper (Cat. N° 1001150). The grids were negatively stained with a solution of 1% buffered phosphotungsten acid (PTA) pH 7.2 for 30 sec. The excess of liquid was soaked off by Whatman filter paper. The grids were observed using a TEM Tecnai G2 spirit operating at voltage of 100 kV and a magnification of 87000X. Images were collected with a CCD camera Olympus SIS Morada 2K*4K.

Image Processing and 3D Reconstruction

Images were first analyzed for defocus (1.1–3.2 μm) and Contrast Transfer Function (CTF) using the Medical Research Council (MRC) program CTFFIND3 [25]. Direct measurement of rNA tetramer diameters using command measure from the software EMAN [26, 27] clearly showed that rNA tetramers displayed similar external diameter corresponding to 100 Ǻ. Image phases were corrected for the effect of the contrast transfer function in IMAGIC5 [28] at a box of 64x64 pixels (corresponding to a 246x246 Ǻ box, 3.84 Ǻ/pixel step size in IMAGIC5). The different views of the tetramer were collected using the command “boxer” from the software EMAN. Images were then band pass filtered with a Gaussian edge at 17–200 Å to remove background noise and normalized. Filtered images were first pre-aligned interactively and subsequently using alignments with only limited angular ranges (-5°, +5°), finally vertical and translational alignments were performed. Centered rNA tetramers were than classified by Multivariate Statistical Analysis (MSA) to sort images into class average with similar features. The class averages obtained confirmed that rNA tetramers presented a small size variation and a typical C4 symmetry. The images were sorted into size groups by a combination of Multi Reference Alignment (MRA), MSA and classification applying the strategy described by White and coworkers [29]. After several iterations of MRA and MSA, the best class averages that contained the most of the raw data were used to generate 3D reconstruction. A total of 974 images where used to generate the 3D structure of the rNA tetramer. All the top-view class averages clearly showed rNA tetramers as structures made up by 4 subunits arranged around the same z-axis forming a ring like structure. The final 3-D was refined at 24 Å resolution (FSC = 0.5) [30] by iterating procedures of alignment and classification. 3D rendered surface was visualized in UCSF CHIMERA [31].

Differential scanning fluorimetry (DSF)

To investigate the thermal stability of rNAs in presence of calcium ions, the NA protein solution was first mixed with 150 mM EDTA and then incubated at 25°C for 1 h. Then EDTA was removed from protein sample by buffer exchange (PD10 column, GE Healthcare), and different CaCl₂ concentrations (from 0 to 20 mM) were added to the protein solution, followed by DSF analysis. Purified Avian H5N1 rNA was diluted to a final concentration of 20 μM in DSF buffer (25 mM Tris pH 8, 150 mM NaCl), containing different calcium concentrations. 4 μl of freshly prepared 100X solution of Sypro Orange from a 5000X stock (Invitrogen) were added to all protein solutions. The final volume of the reaction mixture was 40 μl in a 96-well plate (Thermo Fast 96-ABgene). The plate also included a baseline control containing Sypro Orange with DSF buffer only. The melting point (T_m) of each protein-CaCl₂ solutions was measured by ramping from 25°C to 95°C with 1°C per min increase. A quantitative PCR thermo cycler (Stratagene) has been applied to monitor and record the unfolding profile and the melting temperature (Ericsson, et al., 2006). The experiment has been performed as triplicated. Fluorescence intensities were plotted as a function of temperature and the reported T_m is the inflection point of the sigmoid curve.

Kinetic parameters

The kinetic parameters of the purified rNAs were measured using a fluorescence-based assay that rely on the chemical conversion of the non-fluorescent MuNANA substrate into the fluorescent product 4-methylumbelliferone (4-MU) after NA action. Briefly, in a flat-bottom 96-well opaque black plate (Corning, Tewksbury, MA) serial dilutions, ranging from 0.59 μM to 600 μM, of MuNANA substrate were performed in 100 μl/well of reaction buffer (200 mM NaOAc, 20 mM CaCl₂, pH 5.5, 0.01 mg/ml BSA) in duplicate. 100 μl/well of rNAs at 0.01 μg/ml (0.2 nM) concentration were added to all wells and plates were immediately incubated at 37°C. Fluorescence was measured every 10 min for 60 min with Infinite M200 Spectrophotometer (Tecan, Mannedorf, Switzerland) microplate reader using excitation and emission wavelengths of 355 nm and 450 nm, respectively. As a control, the highest MuNANA concentration was incubated without the enzyme. The background was calculated as the mean fluorescence of well incubated with reaction buffer only and was subtracted to all the other fluorescence values. Initial velocities were calculated as the difference of fluorescence values at T_3600” and T_0’ versus time (3600”) and plotted against MuNANA concentration. The data were fitted to the Michaelis-Menten equation using non-linear regression analysis of data with GraphPad Prism 6.04. The best fits of the data produced the V_max, K_m, K_cat, K_cat/K_m values that are reported in Table 1.

Enzyme-Linked Lectin Assay

Recombinant NA ability to cleave N-acetylneuraminic acids from larger substrate was assayed according to ELLA protocol, previously described by Lambré et al. 1990. Briefly, Maxisorp Nunc 96well plates (Thermo Fisher Scientific) were coated with 7.5 μg/well of fetuin and incubated overnight at 4°C. Plates were washed 4X with 350 μl/well of T-PBS, covered and stored at 4°C at least for 1 month. Duplicates of rNA were serially diluted in incubation buffer (DPBS containing Ca²⁺ and Mg²⁺ and 1% BSA) and incubated on fetuin-coated plates overnight at 37°C. Serial dilutions of NA from Clostridium perfrigens were loaded on the same plate and used as internal positive control. Plates were washed 4X with 350 μl/well T-PBS and incubated with 100 μl of HRP-PNA. After 1 h incubation at room temperature, the plates were washed again and incubated with 100 μl/well of TMB for 30 min at room temperature. The reaction was stopped with 100 μl/well of 0.5 M HCl and absorbance was read at 450 nm using EnVision Multilabel Plate Reader (Perkin Elmer, Waltham, MA, USA). The standard dose of rNA that yields an OD_450nm = 2 was calculated and used for NI antibodies detection. Fifty-five μl/well of recombinant NA was added to 55 μl/well of heat-inactivated sera, serially diluted in incubation buffer in U-bottom 96-well plates. As virus positive control, 8 wells were incubated with rNA only. After 2 h incubation at 37°C, 100 μl/well were transferred into corresponding wells of a fetuin-coated plate and incubated overnight at 37°C. The plates were then washed and developed using the same procedure followed for NA activity titration. NI titers were defined as the reciprocal of serum dilution at which the mean absorbance was ≤ 50% of the mean signal of virus control (ID50); samples with a titer < 50 were assigned a value of 25. Data represents mean +/- SD of 3 independent experiments performed in duplicate.

Production of rNA-specific antisera

All animal experiments were performed in accordance with Institutional Animal Care and Use Committee protocols and mice were housed in the Novartis Vaccines Animal Facility. 10 μg of either swine H1N1 or avian H5N1 rNA adjuvanted with MF59 in 100 μl of total volume were injected in female CD1 mice (5 weeks aged) three times intramuscularly. The second and the third shots were performed 30 and 45 days after the first dose, respectively. Pre-immune, post 2 (15 days after second dose) and post 3 (15 days after third dose) bleedings were collected for each mouse. To detect functional anti-NA antibodies the sera were pooled and inactivated for 30 min at 56°C.

rNAs form stable tetramers and are glycosylated

To confirm rNA assembly in a tetrameric structure, the purified rNAs were examined by gel filtration analysis and were shown to have a single sharp peak (Fig 2B). Based on the comparisons of the elution volumes (Ev) of both swine and avian rNAs with the Ev of molecular weight (MW) standards run in the same conditions, the calculated MW of these species consistent with a NA tetramer (≈240 kDa). No peaks indicative of rNA monomers, dimers, or trimers were detected during the SEC purification step. A minor amount of NA aggregates was represented by a small peak that eluted prior to the high molecular weight standard. The rNAs were obtained with a purity>90% by SDS-PAGE. (Fig 2C).

Mammalian cells are a suitable viral NA expression system, compared to baculovirus or yeast expression systems, because of the protein glycosylation patterns that are physiologically relevant in the context of human infections. Purified rNAs were deglycosylated with PNGase F or Endo H and their glycosylation patterns were analyzed by SDS-PAGE (Fig 3). Untreated NAs migrated at a MW of ≈70 kDa, higher than their theoretical MW of ≈52 kDa, suggesting post-translational modifications including glycosylation. PNGase F deglycosylated NAs ran at ≈50 kDa while Endo H treated NAs ran at ≈65 kDa. These data indicated that rNAs produced in mammalian cells contain N-linked glycosylations.

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Fig 3

Glycosylation pattern of swine H1N1 and avian H5N1 rNAs.

rNAs were deglycosylated with PNGase F or Endo H and molecular weights of treated and untreated samples were detected by SDS-PAGE followed by Coomassie staining. Data shown are representative of two independent experiments.

Calcium binding induces a large increase in the stability of rNA

The several crystal structures of influenza neuraminidase show the presence of five calcium binding sites, one with high affinity close to the active site of each monomer and one with low affinity on the molecular 4-fold symmetry axis [32]. Calcium binding has been reported to be important for NA enzymatic activity and thermostability [33].

The thermostability of A/turkey/Turkey/1/2005 (H5N1) rNA was measured by DSF using Sypro Orange as the external fluorescent probe. The thermostabilizing effect of Ca²⁺ binding to avian rNA was investigated by incubating the purified protein with increasing concentrations of Ca²⁺. A T_m shift from 44°C to 59°C was observed as the Ca²⁺ concentration in the solution was increased (Fig 4). This result indicated that higher concentrations of Ca²⁺ contribute to the NA thermal stability.

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Fig 4

DSF analysis of avian H5N1 rNA.

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

Soluble, tetrameric rNAs are enzymatically active

To determine and compare the specific activity of both swine H1N1 and avian H5N1 rNAs, a MuNANA activity assay was performed calculating the Michaelis-Menten steady state kinetic constants (K_m, K_cat, K_cat/K_m) (Fig 5A and Table 1). As previously reported [34], the kinetic parameters for the two rNAs were substantially different. The rNA derived from the avian H5N1 at 0.2 nM, corresponding to 0.01 μg/ml, catalyzed more efficiently the MuNANA substrate than swine H1N1 rNA at the same concentration, as indicated by the K_cat/K_m ratios of 1.679 μM s^-1 and 1.025 μM s^-1, respectively. In addition, avian H5N1 rNA V_max was 15.09 μM s^-1, higher than the swine H1N1 rNA that had a V_max of 6.116 μM s^-1. Interestingly, the affinity of the avian rNA for the MuNANA substrate was lower than the swine rNA, as demonstrated by the K_m constants of 44.93 μM and 29.82 μM, respectively.

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Fig 5

Sialidase activity of swine H1N1 and avian H5N1 rNAs.

(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) vs 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 vs the protein concentration. Data represent mean±SD of 3 independent experiments performed in duplicate.

Next, the activity of both purified rNAs was compared using fetuin, a larger substrate containing N-acetylneuraminic acid, used in the ELLA assay. Avian H5N1 rNA was more active than swine H1N1 rNA (Fig 5B), judging from the amounts of rNAs that yielded an OD_{450 nm} = 2, in agreement with the data obtained by MuNANA assay.

Visualization and structural features in 3D reconstructions of recombinant NAs

An additional confirmation that recombinant NA forms stable tetramers in solution was obtained by visualizing the purified protein using negative stain TEM. As shown in Fig 6A, avian H5N1 rNA sample appeared as differentially oriented homogeneous population of ring-like structures, with a uniform external diameter of 90 Å, an internal diameter of 20 Å and a height of 50 Å. Single particle reconstruction method was applied to TEM images in order to generate the three-dimensional structure of the tetrameric head. Single boxed rNAs tetramers (box size 64x64 pixel) [26, 27] (Fig 6B, top) were firstly band pass filtered in order to increase the signal-to noise ratio, than rotationally and translationally aligned, and finally centered before undergoing MSA for classification [28, 29]. Fig 6B shows a selection of rNA tetramers class averages, representative of the different orientations of the oligomer particle on the carbon film support. The 3D-EM structure (Fig 6C) [31] of the soluble tetrameric head generated confirm to be a ring-like structure composed by four lobes and presenting an internal hole of 30 Å in diameter. The reference-free reconstruction obtained without imposing tetrameric symmetry, shows a clear C4 symmetry, already visible in the class-averages.

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Fig 6

Structural characterization of avian H5N1 rNA using TEM with single particles reconstruction.

(A) Typical negative staining TEM image of avian H5N1 rNA. Scale bar corresponds to 200 nm. (B) Representative class averages of avian H5N1 rNa tetramers. Each class average (~50 images per class) contains particles selected from several micrographs and represents the different orientations of the enzyme. Scale bar corresponds to 10 nm. (C) Top (upper panel) and side (lower panel) surface views of the reconstructed avian H5N1 rNA globular head obtained at a resolution of 24 Å (FSC = 0.5).

Both swine and avian rNAs are suitable sources of NA in ELLA

To investigate the suitability of soluble swine H1N1 and avian H5N1 rNAs as sources of enzyme in ELLA inhibition test, a standard dose of NA corresponding to an OD_450nm = 2 was calculated from the titration curve (Fig 5B) and incubated with serial dilutions of a panel of sheep polyclonal sera specific for different NAs. Swine H1N1 and avian H5N1 rNA’s activity were specifically inhibited by sera raised against the homologous N1 (Fig 7A). Furthermore, functional cross-reactive antibodies were detected in sera raised against heterologous A/N1, but not against A/N2 or B/NA. The strength of antibody cross-reactivity correlated with the degree of identity between the different A/N1 strains (S1 Fig). NI titers measured using the whole live A/California/07/2009 (H1N1) virus as source of NA followed the same trend.

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Fig 7

rNAs as sources of sialidase in ELLA.

(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.

The authors would like to thanks Novartis Animal Care facility and Miguela Vieru for formulating the recombinant proteins.