Patient demographics.

<p>^a Healthcare-associated infections were defined as (i) index positive blood culture collected ≥48hrs after hospital admission, and no signs or symptoms of the infection noted at time of admission; OR (ii) index positive blood culture collected <48hrs after hospital admission if any of the following criteria are met: received intravenous therapy in an ambulatory setting in the 30 days before onset of BSI, attended a hospital clinic or haemodialysis in the 30 days before onset of BSI, hospitalised in an acute care hospital for ≥ 2 days in the 90 days prior to onset of BSI, resident of nursing home or long-term care facility.</p><p>^b<i>Staphylococcus aureus</i> bacteraemia was defined as uncomplicated if all of the following criteria were met: exclusion of endocarditis; no evidence of metastatic infection; absence of implanted prostheses; follow-up blood cultures at 2–4 days culture-negative for <i>S</i>. <i>aureus</i>; defervescence within 72 h of initiating effective therapy. Percentages shown are of entire <i>S</i>. <i>aureus</i> BSI population.</p><p>^† Three patients had chronic diabetic foot ulcers as a source of their <i>S</i>. <i>aureus</i> BSI, and in all cases the contiguous underlying bone was also found to be infected.</p><p>MRSA = methicillin-resistant <i>Staphylococcus aureus</i>. NA = not applicable. BSI = bloodstream infection.</p><p>Data are displayed as median (interquartile range) and number (percentage). <i>P</i> values are calculated by Mann-Whitney and Fisher’s exact test respectively.</p

Human <i>S</i>. <i>aureus</i> bloodstream infection induces <i>S</i>. <i>aureus</i> antigen-specific effector memory Th1 cells and anti-ClfA antibodies.

<p>PBMCs were isolated from patients, CFSE-labelled and incubated with heat-killed <i>S</i>. <i>aureus</i> PS80 strain (1μg/ml ≈ 1x10⁷ CFU/ml) or media alone for 10 d. Proportions of <i>S</i>. <i>aureus</i> antigen-specific effector memory cells were assessed by gating on IFNγ⁺CD45RO⁺ cells in the CFSE_lo CD4⁺ population (A). For each patient, media only responses were subtracted from responses to heat-killed <i>S</i>. <i>aureus</i> to determine the antigen-specific response. n = 5–12 per group. IgG antibody binding to ClfA was measured in patient sera using a bead-based flow cytometry technique (B). n = 11–24 per group. Results shown as box-and-whiskers plots where the horizontal line indicates the median, boundaries of the box the IQR, and whiskers indicate the highest and lowest values of the results, and representative FACS plots of CD4⁺CFSE_lo cells (A). SA = <i>S</i>. <i>aureus</i>, EC = <i>E</i>. <i>coli</i>, BSI = bloodstream infection. * p<0.05.</p

Vaccine-induced type 1 immunity protects against <i>S</i>. <i>aureus</i> infection.

<p>Mice were vaccinated with CpG (50μg/mouse), ClfA (1μg/mouse), or CpG+ClfA via s.c. injection on d 0, 14 and 28. On d 63 mice were challenged with <i>S</i>. <i>aureus</i> (5x10⁸ CFU) via i.p. injection alongside a control group of sham-immunised (with PBS) mice. At 72 h post-infection, bacterial burden was assessed in the peritoneal cavity, kidneys and spleen. Results expressed as log₁₀ CFU/ml with means indicated by bars. n = 5–10 per group. **p<0.005, ***p<0.001.</p

Human <i>S</i>. <i>aureus</i> bloodstream infection is associated with increased IFNγ production.

<p>Serum from <i>S</i>. <i>aureus</i> and <i>E</i>.<i>coli</i> bloodstream infection patients was collected on day 7 ± 2 post-initial bacteraemia and assessed for IFNγ (A) and IL-17A (B) by ELISA. Results expressed as individual patient values with median indicated by bar, n = 11–24 per group. *p<0.05.</p

Prior exposure to <i>S</i>. <i>aureus</i> increases IFNγ secretion by CD4⁺ and CD8⁺ T cells during subsequent infection.

<p>Groups of mice were exposed to <i>S</i>. <i>aureus</i> (5x10⁸ CFU) via an i.p. injection on d 0, 7 and 14. Prior exposed mice were then re-challenged with an i.p. injection of <i>S</i>. <i>aureus</i> (5x10⁸ CFU) on d 35 alongside a control group of naïve mice. At indicated time points post-challenge the peritoneal cavity was lavaged with PBS to assess IFNγ secretion by ELISA (A). n = 15 per group. At 3 h post challenge peritoneal cells were isolated to assess the proportions of IFNγ-producing CD4⁺ and CD8⁺ T cells using flow cytometry (B). Results expressed as mean ± SEM and representative FACS plots. n = 5 per group. *p<0.05, **p<0.005, ***p<0.001.</p

Transfer of <i>S</i>. <i>aureus</i> antigen-specific peritoneal Th1 cells protects against subsequent <i>S</i>. <i>aureus</i> infection via enhanced macrophage responses.

<p>Groups of mice received transfers of 5x10⁶<i>S</i>. <i>aureus</i> specific Th1 cells originating from the peritoneal cavity of previously exposed mice via i.p. injection. Another group of mice received a transfer of 5x10⁶ naive splenic CD3⁺ cells as a control. At 3 h post transfer both groups of mice were challenged with <i>S</i>. <i>aureus</i> (5x10⁸ CFU) via i.p. injection. At 72 h post-bacterial challenge the bacterial burden was assessed in the peritoneal cavity, kidneys and spleen (A). Results expressed as log₁₀ CFU/ml with mean indicated by bars. At indicated time points post-bacterial challenge, the peritoneal cavity was lavaged with PBS to assess CXCL1 and CCL5 secretion by ELISA (B,C). Results expressed as mean ± SEM. At indicated time points post-challenge, the absolute numbers of macrophages (F4/80⁺Ly6G^-) were assessed in the peritoneal cavity by flow cytometry (D). MHCII expression by infiltrating macrophages was determined 24 h post infection (E). Absolute numbers of neutrophils (Ly6G⁺CD11b⁺) in the peritoneal cavity were assessed at the indicated time points post-challenge (F). Results expressed as mean ± SEM. n = 5–8 mice per group. Data pooled from 3 independent experiments. *p<0.05, **p<0.005.</p

Human CD4⁺ T cells show ClfA-specific type 1 immune responses.

<p>Purified CD4⁺ T cells and irradiated APCs from healthy blood donor buffy coats were CFSE-labelled and co-cultured with ClfA (5μg/ml), heat-killed <i>S</i>. <i>aureus</i> PS80 strain (1μg/ml ≈ 1x10⁷ CFU/ml), or media alone. After 10 d, proliferation was assessed by gating on CFSE_lo cells in the CD4⁺ population (A). At 96 h cell supernatants were collected to assess IFNγ secretion by ELISA (B). ClfA-specific Th1 cell division was assessed at d 10 by gating on CFSE_lo IFNγ⁺ cells in the CD4⁺ population (C). Results expressed as mean ± SEM. n = 9–14 per group.</p

Vaccination with CpG and ClfA induces ClfA-specific cellular and humoral responses.

<p>Mice were vaccinated with CpG (50μg/mouse), CpG+ClfA (50μg+1μg/mouse) or PBS via s.c. injection on d 0, 14 and 28. On d 63 sera was collected from vaccinated mice. ClfA-specific IgG titres were determined using ELISA (A). Neutralising antibodies were determined by measuring the ability of serum to inhibit staphylococcal adherence to fibrinogen via ClfA (B). Results expressed as mean ± SEM. n = 5 per group. *p<0.05, **p<0.005, ***p<0.001.</p

Human <i>S</i>. <i>aureus</i> bloodstream infection induces <i>S</i>. <i>aureus</i> antigen-specific Th1 cells.

<p>PBMCs were isolated from patients, CFSE-labelled and incubated with heat-killed <i>S</i>. <i>aureus</i> PS80 strain (1μg/ml ≈ 1x10⁷ CFU/ml) or media alone for 10 d before assessing <i>S</i>. <i>aureus</i> antigen-specific proliferation by gating on CFSE_lo cells of the CD4⁺ population using flow cytometry (A). <i>S</i>. <i>aureus</i> antigen-specific Th1 and cytotoxic T cell division was assessed by gating on CFSE_lo IFNγ⁺ cells of the CD4⁺ and CD8⁺ populations respectively (B). <i>S</i>. <i>aureus</i> antigen-specific Th1 and Th17 proportions were compared by gating on CFSE_lo IFNγ⁺ or IL-17A⁺ cells in the CD4⁺ population (C). For each patient, media only responses were subtracted from responses to heat-killed <i>S</i>. <i>aureus</i> to determine the antigen-specific response. Results shown as box-and-whiskers plots where the horizontal line indicates the median, boundaries of the box indicate the IQR, and whiskers indicate the highest and lowest values of the results, and representative FACS plots of CD4⁺ or CD8⁺ lymphocytes (A, B). n = 5–17 per group. SA = <i>S</i>. <i>aureus</i>, EC = <i>E</i>. <i>coli</i>, BSI = bloodstream infection. *p<0.05, **p<0.005, ***p<0.001.</p