SAP enhances the neutralizing activity of SP-D and MBL against WT but not SAP^R Mem/71-Bel.

<p>Dilutions of (i) SAP, SP-D and MBL were prepared in TBS supplemented with 10 mM Ca²⁺ alone, or (ii) SP-D and MBL were prepared in TBS supplemented with 10 mM Ca²⁺ and 0.60 µg/ml SAP, and incubated at 37°C for 15 mins to allow for interactions between different proteins. After this time, an equivalent volume of WT or SAP^R Mem71-Bel was added and incubated at 37°C for 30 mins and the amount of infectious virus remaining was determined by fluorescent-focus reduction assay. Final concentrations after incubation with virus were SAP alone (0.6 µg/ml), SP-D alone (0.3 µg/ml), MBL alone (2.5 µg/ml), SAP+SP-D (0.6 µg/ml +0.3 µg/ml) and SAP+MBL (0.6 µg/ml +2.5 µg/ml final). Neutralization data for WT (left panel) or SAP^R Mem/71-Bel (right panel) are expressed as the mean (±1 SD) percent of the number of fluorescent foci in each virus only control and are pooled data from two independent experiments, each performed in triplicate. **p<0.01, ***p<0.001, One-way ANOVA, Tukey’s post hoc.</p

Serum Amyloid P Is a Sialylated Glycoprotein Inhibitor of Influenza A Viruses

<div><p>Members of the pentraxin family, including PTX3 and serum amyloid P component (SAP), have been reported to play a role in innate host defence against a range of microbial pathogens, yet little is known regarding their antiviral activities. In this study, we demonstrate that human SAP binds to human influenza A virus (IAV) strains and mediates a range of antiviral activities, including inhibition of IAV-induced hemagglutination (HA), neutralization of virus infectivity and inhibition of the enzymatic activity of the viral neuraminidase (NA). Characterization of the anti-IAV activity of SAP after periodate or bacterial sialidase treatment demonstrated that α(2,6)-linked sialic acid residues on the glycosidic moiety of SAP are critical for recognition by the HA of susceptible IAV strains. Other proteins of the innate immune system, namely human surfactant protein A and porcine surfactant protein D, have been reported to express sialylated glycans which facilitate inhibition of particular IAV strains, yet the specific viral determinants for recognition of these inhibitors have not been defined. Herein, we have selected virus mutants in the presence of human SAP and identified specific residues in the receptor-binding pocket of the viral HA which are critical for recognition and therefore susceptibility to the antiviral activities of SAP. Given the widespread expression of α(2,6)-linked sialic acid in the human respiratory tract, we propose that SAP may act as an effective receptor mimic to limit IAV infection of airway epithelial cells.</p> </div

SAP^R mutants of H3 subtype IAV remain sensitive to inhibition by PTX3.

<p>(<b>A</b>) Binding of SAP or PTX3 to purified Mem/71-Bel WT or Mem/71-Bel SAP^R. Biotin-labelled (i) SAP (2 µg/ml) and (ii) PTX3 (2 µg/ml) diluted in BSA₅-TBST-Ca²⁺ were applied to wells coated with 1 µg/ml or 0.1 µg/ml (as indicated) of purified virus and binding of SAP or PTX3 was determined by ELISA. Equivalent coating levels of purified viruses was confirmed using mAb 165 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059623#s2" target="_blank">Materials and Methods</a> (data not shown). Data represent the mean of three independent experiments (±1 SD). **p<0.01, ***p<0.001, Student’s t-test, comparing SAP^R virus to WT virus for each pentraxin. (<b>B</b>) Neutralization of Mem/71-Bel WT and Mem/71-Bel SAP^R by SAP or PTX3. Viruses were mixed with an equal volume of PTX3 or SAP prepared in TBS/Ca (to a final concentration of 10 or 1 µg/ml of each pentraxin, as indicated), incubated for 30 min at 37°C and the amount of infectious virus remaining was determined by fluorescent-focus reduction assay. Results are expressed as a percent of the number of fluorescent foci in the virus only control and represent the mean (±1 SD) from three independent experiments. ***p<0.001, Student’s t-test, comparing SAP^R to WT virus for each pentraxin. (<b>C</b>) Inhibition of virus-induced hemagglutination by SAP and PTX3. Dilutions of PTX3 or SAP in TBS +10 mM Ca²⁺ were tested for their ability to inhibit hemagglutination by Mem/71-Bel WT (black bars) or Mem/71-Bel SAP^R (white bars). Results show the minimum inhibitory concentration (MIC) of SAP or PTX3 required to inhibit 4 HAU of virus. Dashed line represents the highest concentration of inhibitor tested (10 µg/ml); values above this line designated >10 µg/ml are indicated by a ‘+’. Data represent mean (±1 SD) from three independent experiments. (<b>D</b>) Mem/71-Bel SAP^R virus does not agglutinate erythrocytes treated with <i>S. pneumoniae</i> sialidase. Chicken erythrocytes were treated with 160 mU of <i>S. pneumoniae</i> sialidase for 1 hour at 37°C. Mem/71-Bel (WT, black bars) or Mem/71-Bel SAP^R (SAP^R, white bars) were adjusted to 64 HAU on untreated chicken erythrocytes and assayed for their ability to agglutinate sialidase-treated erythrocytes. Data represent mean (±1 SD) from three independent experiments.</p

Neutralization of RG viruses by SAP or horse serum.

<p>Dilutions of WT or SAP^R PR8-PC/73 HA and PR8-Ud/72 HA viruses in TBS were incubated for 30 min at 37°C with an equivalent volume of (<b>A</b>) SAP, at a final concentration of 20 µg/ml or 2 µg/ml in TBS/Ca, or (<b>B</b>) horse serum (HS), at a final dilution factor (DF) of 100 or 1000 in TBS/Ca, and the amount of infectious virus remaining was determined by fluorescent-focus reduction assay. Data are expressed as a percent of the number of fluorescent foci in the virus only control. Data show the mean of three independent experiments (±1 SD). *p<0.05, **p<0.01, ***p<0.001, Student’s <i>t</i>-test, comparing each SAP^R virus to its appropriate WT control.</p

Antiviral activities of SAP and PTX3 against IAV and IAV-infected cells.

<p>(<b>A</b>) Inhibition of NA activity by SAP and PTX3. A dilution of HKx31 was mixed with a final concentration of 10 or 1 µg/ml of SAP (white bars) or PTX3 (black bars) in BSA₅-TBS-Ca²⁺ and assayed for NA activity as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059623#s2" target="_blank">Materials and Methods</a>. Results are expressed as the percent of NA activity compared to a virus only control. Data shown are the mean (±1 SD) from three independent experiments. *p<0.05, Students <i>t</i>-test, significant difference in levels of NA activity in the presence of SAP compared to an equivalent concentration of PTX3. (<b>B</b>) Neutralization of IAV by SAP and PTX3. Pentraxins were treated with sialidase from <i>Vibrio cholerae</i> (50 mU) in TBS containing 10 mM Ca²⁺ or mock-treated (M) in buffer alone for 30 min at 37°C, and then heated to 56°C for 30 min to inactivate sialidase. Sialidase- (S) or mock- (M) treated pentraxins were mixed with a dilution of HKx31 to give a final concentration of 10 or 1 µg/ml of PTX3 or SAP, and the amount of infectious virus remaining determined by fluorescent-focus reduction assay. Results are expressed as a percent of the number of fluorescent foci in the virus only control. Data show the mean (±1 SD) from three independent experiments. *p<0.05, ***p<0.001, Student’s <i>t</i>-test, significant difference between sialidase-treated sample compared to appropriate mock-treated control. (<b>C</b>) Binding of SAP and PTX3 to IAV-infected MDCK cells. Binding of SAP and PTX3 to uninfected (i, iii) or HKx31-infected MDCK cells (ii, iv) was determined at 6 hrs post-infection. Aliquots of 10⁶ cells were incubated with biotin-labelled SAP (10 µg/ml) or PTX3 (2 µg/ml) in BSA₅-TBS-Ca²⁺ (white histograms) or BSA₅-TBS-EDTA (grey histograms). Binding of biotin-labelled pentraxins was determined by flow-cytometry. Histograms are representative of two independent experiments.</p

(A/B) Binding of PTX3, SAP and CRP to influenza virus HKx31 and to C1q in presence or absence of calcium.

<p>Increasing concentrations of biotin-labelled (i) PTX3 (○,•), (ii) SAP (□,▪) or (iii) CRP (△,▴) in BSA₅-TBST supplemented with 10 mM Ca²⁺ (open symbols) or 5 mM EDTA (closed symbols) were applied to wells coated with (<b>A</b>) 1 µg/ml IAV strain HKx31, or (<b>B</b>) 5 µg/ml C1q and binding determined by ELISA. Data are representative of at least two independent experiments.</p

Sialic acids expressed by SAP are resistant to hydrolysis by the viral NA.

<p>(<b>A</b>) Wells coated with 1 µg/ml of HKx31 were incubated with increasing concentrations of fetuin or asialofetuin (ASF) for 1 hr before addition of 5 µg/ml biotin-labelled SAP. Binding of biotin-labelled SAP to HKx31 was then determined by ELISA. All incubations and washes were performed at 4°C using chilled buffers to inhibit the enzymatic activity of the viral NA. Data show a single titration (left panel) as well as triplicate wells at a specific concentration of fetuin/ASF (right panel). The absorbance of biotin-labelled SAP binding in the absence of fetuin/ASF was 1.558 (±0.105). ***p<0.001, Student’s t-test for equivalent concentrations of fetuin compared to ASF. (<b>B</b>) Purified PTX3 and SAP were subjected to 12% SDS-PAGE under reducing conditions followed by lectin blot using DIG-conjugated MAA (panel i) or SNA (panel ii) for detection of α(2,3)- or α(2,6)-linked SA, respectively. Fetuin was included as a positive control for each SA linkage. (<b>C</b>) SAP (2.5 µg) was incubated for 1 hr at 37°C with sialidase from <i>V. cholerae</i> (25 mU), HKx31 (1/20 dilution of allantoic fluid) or with buffer alone and then analysed by SDS-PAGE and DIG-lectin blot using DIG-SNA (panel i). Ponceau S stain confirmed similar amounts of SAP were transferred to the PVDF membrane prior to the lectin blot (panel ii). (<b>D</b>) ELISA plates coated with (i) fetuin (20 µg/ml), or (ii) SAP (40 µg/ml) were incubated with HKx31 (1/10 dilution of allanotic fluid) or sialidase from <i>V. cholerae</i> (0.5 mU) for 30 min at 37°C and removal of SA was determined using NA assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059623#s2" target="_blank">Material and Methods</a>. Uninfected allantoic fluid (AF) was included as a negative control. Data shows the mean absorbance (±1 SD) of triplicate wells and is representative of at least two independent experiments. ***p<0.001, One-way ANOVA compared to uninfected allantoic fluid control or buffer alone control.</p

SAP binds to the HA glycoprotein of IAV to mediate its antiviral activities.

<p>SAP was tested for its ability to mediate a range of anti-IAV activities against Ud/72, PR8, PR8-Ud/72 HA or PR8-Ud/72 NA. (<b>A</b>) Inhibition of virus-induced hemagglutination. Dilutions of SAP in TBS/Ca were tested for anti-IAV activity in standard HI assays. MIC values are the mean (±1 SD) concentration of SAP required to inhibit 4 HAU of virus. Dotted line indicates the maximum concentrated tested (5 µg/ml) and ‘+’ indicates a MIC value >5 µg/ml. (<b>B</b>) Inhibition of viral NA activity. Dilutions of virus were mixed with SAP (at a final concentration of 20 µg/ml or 2 µg/ml and assayed for NA activity. Results are expressed as the percent of NA activity compared to a virus only control. (<b>C</b>) Virus neutralisation. A dilution of each virus was mixed with SAP diluted in TBS/Ca to a final concentration of 20 µg/ml or 2 µg/ml and then incubated for 30 min at 37°C. The amount of infectious virus remaining was then determined by fluorescent-focus reduction assay. Results are expressed as a percent of the number of fluorescent foci in the virus only control. Data shown in A, B and C are the mean of three independent experiments (±1 SD). *p<0.05, ***p<0.001, One-way ANOVA, significantly different to viruses expressing Ud/72 HA (i.e. Ud/72 and PR8-Ud/72 HA).</p

HI activity of PTX3, SAP and MBL against IAV.

a<p>MIC are representative of 2 or more independent experiments using HKx31.</p>*<p>≥4-fold difference in MIC relative to results in +10 mM CaCl₂.</p

HA receptor specificity of H3 subtype IAV strains correlates with sensitivity to SAP and PTX3.

<p>(<b>A</b>) Human erythrocytes were desialylated and subsequently resialylated with α(2,3)- or α(2,6)-sialyltransferases and used in hemagglutination assays. All viruses were adjusted to 64 HAU on native erythrocytes and showed no hemagglutinating activity (<1 HAU) against desialylated erythrocytes (data not shown). (<b>B</b>) Inhibition of HA activity of H3 subtype IAV by pentraxins. SAP or PTX3 were diluted in TBS containing 10 mM Ca²⁺ and used in a standard HI assay. Bars indicate the minimum concentration of SAP or PTX3 required to inhibit 4 HAU of each virus tested. Dashed line represents the highest concentration of inhibitor tested (10 µg/ml). ‘+’ indicates a value of >10 µg/ml. Data are representative of 2 or more independent experiments.</p