Clinical manifestations in the ILI and PH1N1 women.

<p>ILI: Pregnant women with influenza-like illness.</p><p>PH1N1: Confirmed H1N1pdm2009 virus-infected pregnant women.</p><p>*Fisher’s exact test.</p><p>Clinical manifestations in the ILI and PH1N1 women.</p

Demographic and obstetric characteristics of the study population.

<p>HW. Healthy women.</p><p>HP. Healthy pregnant women.</p><p>ILI. Pregnant women with influenza-like illness.</p><p>PH1N1. H1N1pdm2009 virus-infected pregnant women.</p><p>Gw. Gestational weeks.</p><p>n/a not applicable.</p><p>*Fisher’s exact test.</p><p>Demographic and obstetric characteristics of the study population.</p

Serum cytokine concentrations in HW, HP, ILI and PH1N1 women.

<p>Pro-inflammatory TNF-α (a), IL-1β (b) and IL-6 (c) and anti-inflammatory IL-10 (d) cytokines were quantified using a CBA system with flow cytometry. The Kruskal-Wallis test with Dunn’s multiple comparison post-test was performed using the GraphPad Software. The significance values were *p<0.05, **p<0.01, ***p<0.001.</p

Higher percentage of CD69+ lymphocytes in H1N1pdm2009 virus-infected pregnant women.

<p>The peripheral blood leukocytes from the HW, HP, ILI and PH1N1 women were immunostained with CD3-, CD19-, CD14- and CD69-specific antibodies and analyzed by flow cytometry. Representative data of each group are shown in the dot plots for CD69+CD3+ cells (a). The distribution of CD69 expression on CD3− (b), CD19− (c) or CD14− (d) gated cells. The Kruskal-Wallis test with Dunn’s multiple comparison post-test was performed using the GraphPad Software. The significance values were *p<0.05, **p<0.01, ***p<0.001.</p

Distribution of leukocytes in the blood.

<p>HW. Healthy women.</p><p>HP. Healthy pregnant women.</p><p>ILI. Pregnant women with influenza-like illness.</p><p>PH1N1. H1N1pdm2009 virus-infected pregnant women.</p><p>a. HW vs ILI.</p><p>b. ILI vs PH1N1.</p><p>c. HW vs PH1N1.</p><p>d. HP vs PH1N1.</p><p>e. HW vs HP.</p><p>f. HP vs ILI.</p>±<p>Kruskal-Wallis test with Dunn’s multiple comparison post-test.</p><p>P values: * = p<0.05; ** = p<0.01; *** = p<0.001.</p><p>Distribution of leukocytes in the blood.</p

Expression and purification of recombinant <i>Bacillus stearothermophilus</i> DNA polymerase (Bst) and reverse transcriptase (RT) from Moloney Murine Leukemia Virus.

Coomassie blue-stained 8% Tricine-SDS-PAGE electrophoresis gel analysis. (A) Expression of recombinant Bst enzyme and Ni2+-IMAC purification. Lane 1: clarified supernatant loaded; Lane 2: flow-through fraction; Lane 3–6: washing step fractions; Lane 7–9: eluted fractions containing Bst. (B) Heparin column purification of recombinant Bst. Lane 1: desalted sample loaded; Lane 2: flow-through fraction; Lane 3: washing step fraction; Lane 4–6: elution fractions; Lane 7–8: concentrated Bst-containing fractions. (C) Final Bst formulations from three different purification batches (B1, B2, B3). (D) Expression of recombinant RT enzyme and Ni2+-IMAC purification. Lane 1–3: flow-through fractions; Lane 4–5: washing step fractions; Lane 6–10: elution fractions containing RT. (E) Cation exchange column purification of recombinant RT. Lane 1: IMAC elution fraction; Lane 2: desalted sample loaded; Lane 3–5: flow-through fractions; Lane 6–7: washing step fractions; Lane 8–13: elution fractions. (F) Final RT formulations from three different purification batches (B1, B2, B3). M: molecular weight marker; C+: previously purified Bst or RT enzyme, employed as control positive; I: insoluble fraction; S: soluble fraction; C: clarified supernatant. Violet or green arrows indicate the expected size for Bst and RT enzymes, respectively.</p

Effect of additives in colorimetric end-point RT-LAMP assay performance.

(A) Colorimetric RT-LAMP reactions under optimized conditions using N1 primer set and 40 mM of guanidine hydrochloride (GuHCl) in the reaction buffer. The figure shows the colorimetric determination of each reaction (upper panel) and the electrophoretic profile of the amplification reaction products (lower panel). (B) Colorimetric RT-LAMP reactions under optimized conditions using N1 primer set in absence of GuHCl in the reaction buffer. The figure shows the colorimetric determination of each reaction (upper panel) and the electrophoretic profile of the amplification reaction products (lower panel). (C) Colorimetric RT-LAMP reactions under optimized conditions using N1 primer set and 0.8M of betaine in the reaction buffer. The figure shows the electrophoretic profile of the amplification reaction products. (D) Colorimetric RT-LAMP reactions under optimized conditions using N1 primer set in absence of betaine in the reaction buffer. The figure shows the electrophoretic profile of the amplification reaction products. C+: 1x104 copies of N1 in vitro transcript used as positive control; NTC: non-template control; M: DNA molecular weight marker 1 Kb Plus DNA Ladder (Invitrogen). (TIF)</p

Chromatographic profiles of the purification steps of Bst and RT by fast protein liquid chromatography (FPLC).

(A) Chromatogram of Bst purification by Ni+2-IMAC. (B) Chromatogram of RT purification by Ni+2-IMAC. (C) Chromatogram of the desalting step of the Bst-containing fractions. (D) Chromatogram of the desalting step of the RT-containing fractions. (E) Chromatogram of the second purification step by heparin affinity chromatography for RT. (F) Chromatogram of the second purification step by cation exchange chromatography for RT. Values expressed in mAU are shown in purple (Bst) or green (RT). The dotted lines correspond to the concentration of the elution buffer used in each case: EB-AI (A), EB-BI (B), DB-A (C), DB-B (D), EB-AII (E), EB-B-II (F). Black arrows indicate the peaks of the chromatograms selected for the following purification steps. (TIF)</p

Seasonal trivalent inactivated vaccination boosted VLP-induced antibody titres to A(H1N1)pdm09 virus.

<p>Haemagglutination inhibition (HI) titres from the VLP or placebo recipients (P) who did or did not receive seasonal inactivated vaccine (IIV) after VLP were evaluated in the Part A (a) and Part B subjects (b). Scatter dot plots with GMT with 95% CI for the HI titres are shown for each group. The antibody titres were analysed with the nonparametric 1-way ANOVA (Kruskal-Wallis test) and with the U-Mann-Whitney post-hoc multiple comparisons test (*<i>P</i><0.05, ****<i>P</i><0.0001).</p

Study population demographics and characteristics.

<p>Study population demographics and characteristics.</p