DataYETI10

The data are percentages of Tetramer+ YFV-specific CD8+ T cells prospectively in primary vaccinated individuals as well as in primo vs. boosted individuals, and the antibodies for all volunteers in IU/mL

Percentages of YF-tetramer positive CD8⁺ T-cells over time in 13 healthy individuals that received a single vaccination of whom 11 were HLA A02, 2 HLA B27, 1 HLA B35 and 1 HLA B07 (2 donors had 2 HLA types compatible with tetramer reactivity).

<p>Seven donors, 3 HLA A02, 2 HLA B07 and 2 HLA B35 had received a booster vaccination. On the x-axis the number of years since last vaccination until PBMC collection is shown. On the y-axis the percentage of YF-tetramer⁺ cells gated on total CD8⁺ T cells is shown. Black, closed symbols depict single vaccinated individuals; red, open symbols depict boosted individuals. In 4 donors tetramer⁺ CD8⁺ T cells could not be detected directly ex-vivo but only after in vitro expansion by culturing for 9 days in the presence of IL-2 and a YF-peptide pool. Analysis of the correlation between YF-tetramer⁺ CD8+ T cells of singly vaccinated HLA-A2⁺ donors and time since vaccination showed a significant negative correlation (r = -0.76, p = 0.0086, Spearman’s Rank Correlation Coefficient).</p

Frequency of YF-tetramer positive CD8⁺ T-cells in 6 singly vaccinated individuals (one individual (# 10) had 2 tetramer compatible HLA types, therefore seven lines are depicted).

<p>A. Dot plots of a representative donor B. Frequency of YF-tetramer⁺CD8⁺ T-cells expressed as percentage of YF-tetramer positive CD8⁺ T-cells directed against the NS2b, NS4b and NS5 epitopes in HLA-B35, HLA-A02 and HLA-B07 positive individuals at days 0, 3, 5, 12, 28 and 180 after vaccination.</p

Correlation between YF-Antibody titers in 99 individuals that received a single vaccination and time since vaccination.

<p>The y-axis shows the time since vaccination and the x-axis shows the YF-serum antibody titer. The correlation between serum titer and time since vaccination was calculated with Spearman’s Rank Correlation coefficient. As a reference, the red line depicts the YF antibody serum level threshold of protection (0.5 IU/mL).</p

Longitudinal analysis of T-bet eomes expression in singly vaccinated individuals.

<p>A Dot plots of a representative donor. Total CD8⁺ T-cells are depicted in grey and YF-tetramer positive CD8⁺ T-cells in black. B-C T-bet:Eomes ratios on days 12, 28 and 180 in CD8⁺ tetramer⁺ cells. D Correlation between Granzyme K and Eomes expression and Granzyme B and Tbet expression on days 12, 28 and 180 after vaccination. E. T-bet:Eomes ratios of YF-tetramer positive at T = 180 in different subsets. Comparisons were performed with a paired Wilcoxon Rank sum test. ns = not significant.</p

Longitudinal analysis of the phenotype of YF-tetramer positive CD8⁺ T-cells on days 12, 28 and 180 in singly vaccinated individuals.

<p>A Dot plots of a representative donor. Cells are gated on total CD8⁺ T-cells (in grey) and YF-tetramer positive cells (in black). B-D Summary of percentages of tetramer positive cells expressing CD45RA, CD27, granzyme K, granzyme B and Ki67 in 6 donors (1 donor had 2 matching HLA types). Comparisons were performed with a paired Wilcoxon Rank sum test. ns = not significant.</p

Immunostaining for S100A9 in the liver and spleen of mice during <i>S</i>. Typhimurium infection.

<p>Positive immunostaining for S100A9 in liver parenchyma (A) and the red pulp of the spleen (C) tissue was observed in uninfected control animals. Five days after infection with <i>S</i>. Typhimurium (10⁶) there was a marked increase of S100A9 in both liver (B) and spleen (D) corresponding with inflammatory cell influx. Magnification 10 × and 40 ×.</p

S100A8/A9 inhibits bacterial growth of <i>S</i>. Typhimurium and <i>S</i>. Typhi in vitro.

<p>Growth of <i>S</i>. Typhimurium (14028; A) and <i>S</i>. Typhi (Vivotif, Crucell; B) was assessed for a maximum of 24 hours in the presence of recombinant S100A8/A9 (50 μg/ml; grey bars) or control (white bars). Bacterial growth was dose dependently inhibited by S100A8/A9. Growth arrested, CFSE-labelled heat-inactivated <i>S</i>. Typhimurium bacteria were incubated with peripheral blood neutrophils (C) from wildtype (WT; white) and <i>S100A9</i>^<i>-/-</i> mice (grey) (n = 4 per mouse strain) for 0, 15 and 60 minutes respectively after which phagocytosis was quantified (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003663#sec005" target="_blank">Methods</a>). Data are expressed, as mean ± SD. **** P<0.0001, determined via non-parametric <i>t</i> tests comparing different dosages per time point.</p

Patient characteristics.

<p>Baseline characteristics of patients with acute typhoid fever (admitted to the hospital, Bangladesh study) or during convalescence (treated in an outpatient setting, Indonesian study). Days of fever prior to admission or when sample was taken, median. Specific complications were: pneumonia (n = 3); encephalopathy (n = 2); gastrointestinal bleeding (n = 1); severe anemia requiring blood transfusion (n = 1); acute hepatitis (n = 1); hepatic abscess (n = 1); septic arthritis (n = 1); and renal failure, cardiopulmonary arrest and death (n = 1). N/A: not applicable.</p><p>Patient characteristics.</p

S100A8/A9 complexes are abundantly present in plasma and feces from patients with typhoid fever and correlate with duration of fever.

<p>Increased levels of S100A8/A9 (ng/ml) complexes are measured in plasma of patients with confirmed typhoid fever caused by <i>S</i>. Typhi admitted to the hospital (A; n = 28 for admission) and compared to healthy controls (n = 38). However S100A8/A9 levels were not yet normalized at time of discharge (n = 15). In a community based control study during convalescence (3 weeks post infection) S100A8/A9 remains elevated (B; n = 60) compared to healthy controls (n = 60). Increased levels of S100A8/A9 (μg/g) measured in feces from hospitalized patients (C; n = 14) compared to healthy controls (n = 36). No differences were seen in levels of S100A8/A9 between hospitalized patients with complicated or uncomplicated typhoid fever as detailed in method section (D). Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation. Correlation curve log alanine aminotransferase (ALT) versus log plasma S100A8/A9 (E). Correlation curve days of fever prior to admission versus log plasma S100A8/A9 (F). For linear regression analysis, P value and Spearman rho are reported.</p