Additional file 1: of Knowledge sharing in infection prevention in routine and outbreak situations: a survey of the Society for Healthcare Epidemiology of America Research Network

Knowledge Sharing in Infection Prevention in Routine and Outbreak Situations: A Survey of the Society for Healthcare Epidemiology of America Research Network . (DOCX 81 kb

Additional file 1 of Association between the introduction of a national targeted intervention program and the incidence of surgical site infections in Swiss acute care hospitals

Additional file 1. Supplementary Information

WT virus-induced antibody responses neutralize preferentially GP-1 variants that lack specific glycans.

<p>(A-C) We infected C57BL/6 mice i.v. with rLCMV (wt GP, 2x10⁵-4x10⁶ PFU, A), rLCMV/LAS (5x10⁵ PFU, B) or rLCMV/JUN (2x10⁵ PFU, C). Serum samples were collected during the indicated time windows after infection and were tested for their neutralizing capacity against the respective immunizing viruses or their partially deglycosylated variants. (A) Bars represent the mean ± SEM of 23–39 mice per group up to day 50 and of 8 mice per group between days 60–85. A two-way ANOVA followed by Bonferroni’s post-test for multiple comparisons was performed. * <i>p</i><0.05, ** <i>p</i><0.01. Combined data from six independent experiments are shown. (B-C) Symbols represent the mean ± SEM of four to five mice per group. One out of two similar experiments is shown. (A-C) Neutralizing titers were determined in 10-fold (A), 5-fold (B) or 20-fold (C) pre-diluted serum. (D) Convalescent sera of nine individual Lassa patients were tested for neutralizing activity against rLCMV/LAS and its partially deglycosylated variants rLCMV/LASΔGlc5 or rLCMV/LASΔGlc5,9. Neutralization of rLCMV was included as a specificity control. Each graph represents one LASV-convalescent subject. “1-2-19”, “8-2-24”, “10-2-22”, “8-1-10”, “2-4-31”, “13-5-27”, “9-2-15”, “10-3-35” and “TCC donor” represent patient identification codes as previously published [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005276#ppat.1005276.ref042" target="_blank">42</a>]. For each virus, the number of N-linked glycosylation motifs in GP-1 is indicated in brackets.</p

Monoclonal Abs neutralize preferentially GP-1 variants that lack specific glycans.

<p>(A) We infected C57BL/6 mice with rLCMV (10⁵−10⁶ PFU) and collected serum after virus clearance (day 40–80). We assessed neutralizing activity against the immunizing rLCMV and against the partially deglycosylated variant rLCMVΔGlc9. Each point represents an individual mouse serum sample. Data from 53 mice in nine independent experiments are summarized. The Pearson’s correlation coefficient and two-tailed <i>p</i>-value are indicated. ** <i>p</i><0.01. (B-D) We quantified the neutralization potency of KL25 and WEN3 mAbs against rLCMV (wt GP) and rLCMVΔGlc9, respectively (B-C), and determined half-maximal inhibitory concentrations (IC₅₀, D). The mean ±SEM of 7 data points recorded in 5 independent experiments is represented. Unpaired student’s t-tests were used for statistics. ** <i>p</i><0.01. (E) The neutralization potency of 5 mAbs elicited by WT JUNV [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005276#ppat.1005276.ref045" target="_blank">45</a>] was individually assessed against rLCMV/JUN (wt GP; four GP-1 glycosylation motifs) or rLCMV/JUN-vacc (three GP-1 glycosylation motifs). Symbols represent the mean ±SEM of 3 replicates per group. One representative out of three experiments is shown. (F) We used KL25 and WEN3 mAbs in neutralization assays against rLCMV (wt GP) and an rLCMV variant in which Glc5 was artificially introduced (rLCMV+Glc5). Symbols represent the mean ±SEM of 3 replicates.</p

Viral variants lacking select GP-1 glycans elicit a potent but largely variant-specific nAb response.

<p>We infected C57BL/6 mice with the indicated viruses and variants expressing partially glycan-deficient GP versions and determined neutralizing serum activity against the immunizing virus (A-C) or against heterologous virus in comparison to the immunizing virus (D-F), as indicated. Doses of 4x10⁶ PFU (A), 5x10⁵ PFU (B, E) or 2x10⁵ PFU (C, D, F) were given as a single i.v. injection on day 0. For each virus, the number of GP-1 N-linked glycosylation motifs on GP-1 of each virus’ GP is indicated in brackets. Symbols represent the mean ± SEM of four to five mice per group. One of two similar experiments is shown. Neutralizing titers were determined in 12.5-fold (A), 10-fold (B-C, E-F) or 8-fold (D) pre-diluted serum.</p

Mapping LASV GP-1 N-linked glycan sequons onto the structure of MACV GP-1.

<p>(A) The predicted secondary structure composition of LASV GP-1 (above the sequence) was compared to the secondary structure observed in the crystal structure of MACV GP-1 (below the sequence, [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005276#ppat.1005276.ref048" target="_blank">48</a>]). Arrows represent beta-sheets and spirals represent helices, the rainbow code corresponds to panel B. Stars indicate identical residues. The number and position of predicted N-linked glycans are indicated above black shaded sequons. The alignment and annotations were extracted from the arenavirus GP-1 sequences alignment presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005276#ppat.1005276.s001" target="_blank">S1 Fig</a>. (B) Cartoon diagram of MACV GP-1 colored as a rainbow ramped from blue (N-terminus) to red (C-terminus). Sites of N-linked glycosylation observed in the MACV GP-1 crystal structure are annotated and shown as pink sticks. The locations of putative N-linked glycosylation sequons from LASV GP-1 were mapped by sequence alignment (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005276#ppat.1005276.s001" target="_blank">S1 Fig</a>) and are shown as grey spheres. (C) Surface of MACV GP-1 shown in van der Waals surface representation. Residues on MACV GP-1 which form contacts with human TfR1 are colored green. N-linked glycan sites and sequons are colored as in panel B.</p

nAb response kinetics against Clade B arenavirus envelopes correlate with GP-1 glycan density.

<p>(A) Neutralization titer data published by Trapido <i>et al</i>. in 1971 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005276#ppat.1005276.ref037" target="_blank">37</a>] are plotted in relation to the number of N-linked glycosylation motifs in the respective virus’ GP-1 (according to Genbank accession numbers as listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005276#ppat.1005276.t001" target="_blank">Table 1</a>). Trapido <i>et al</i>. had tested the antiviral neutralizing potency of hamster serum obtained upon hyperimmunization with the respective viruses. (B-C) We infected C57BL/6 mice i.v. with 2x10⁵ PFU of the indicated recombinant LCM viruses carrying a range of clade B arenavirus GPs. rLCMV/LAS data originate from a separate experiment and are included for comparison. The number of GP-1 N-linked glycosylation motifs is indicated in brackets. (B) Serum samples were tested in neutralization assays against the respective virus used for infection. Symbols represent the mean ± SEM of five mice per group. One of two similar experiments is shown. (A-B) Neutralizing titers were determined in 8-fold (A) or 10-fold (B) pre-diluted serum. (C) LCMV-NP specific IgG titers were determined in 100-fold pre-diluted serum on d35. Individual data points and the mean ± SEM of five mice per group are shown.</p

Glc9-mediated nAb evasion promotes protracted LCMV infection in mice.

<p>(A-D) We infected T11μMT and C57BL/6 mice i.v. with 4x10⁶ PFU of rLCMV or rLCMVΔGlc9. Blood and serum samples were collected over time. (A-B) Serum samples were tested for their neutralizing capacity against the respective immunizing viruses. Symbols represent the mean ± SEM of four to five mice per group. Neutralizing titers were determined in 10-fold pre-diluted serum. (C-D) Viremia was measured in whole blood samples. Symbols represent the mean ± SEM of four to five mice per group. Differences between rLCMV and rLCMVΔGlc9 viral loads were assessed by two-way ANOVA, followed by a Bonferroni post-test for individual time points if the F-test of ANOVA indicated statistically significant differences. ** <i>p</i><0.01.</p

Facilitated binding of neutralizing mAb to Glc9-deficient LCMV-GP-1.

<p>(A) We transfected 293T cells with plasmids for expression of either LCMV-GPwt or LCMV-GPΔGlc9. We then incubated these cells with titrated concentrations of KL25 or WEN3 mAbs, and saturation of LCMV-GP binding was characterized by flow cytometry. The mean fluorescence intensity (MFI) is plotted as percentage of maximum staining intensity. Untransfected cells were used for reference. The 50% effective concentrations (EC₅₀) are displayed in the chart. One out of two representative experiments is shown. (B-C) Binding kinetics of KL25 and WEN3 Fab fragments to LCMV-GPwt or LCMV-GPΔGlc9 were characterized by surface plasmon resonance (SPR). The association constant (<i>k</i>a), dissociation constant (<i>k</i>d) and the resulting equilibrium dissociation constant (<i>K</i>_D = <i>k</i>d/<i>k</i>a) were determined. Each measurement was done in triplicates for WEN3 and quadruplicates for KL25, respectively. In panel (B) means ± SEM are indicated. * <i>p</i><0.05, ** <i>p</i><0.01 by unpaired student’s <i>t</i> tests. (C) <i>k</i>a and <i>k</i>d values obtained by SPR were plotted on a two-dimensional graph such that identical KD values are located along iso-affinity lines (diagonals).</p