Rationale divinorum officiorum

[Editores] en a\b1\s Gabriel Brunnus (O.F.M.), en G\b5\s Joannes Aloisius TuscanusSign.: a-e\p8\s,f\p6\s, g-h\p8\s, i-z\p6\s, A-E\p6\s, F-G\p8\sLetra góticaTexto a dúas columnas e iniciais xilográfica

Magnitude and quality of vaccine-specific CD4⁺ T-cell responses induced by 4C-Staph/T7-alum and 4C-Staph/alum.

<p>Splenocytes from single mice (n = 16) vaccinated with 4C-Staph/T7-alum, 4C-Staph-alum, T7-alum or alum by 12 days were stimulated or not with vaccine antigens <i>in vitro</i>, stained and analyzed by intracellular cytokine staining. CD4⁺CD44^high T cells producing IL-2, TNF, IL-4/IL-13, IFN-γ or IL-17A were identified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147767#pone.0147767.s001" target="_blank">S1 Fig</a> for gating strategy). The response of unstimulated cells was subtracted from that of stimulated cells. Data are the merge of four independent experiments. (<b>A</b>) Percentages of CD4⁺CD44^high T cells producing any combination of IL-2, TNF, IL-4/IL-13, IFN-γ or IL-17A in response to vaccine protein stimulation (CD4⁺CD44^high total CYT⁺ cells) were calculated applying Boolean gates. Bars represent mean ± SEM. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 by one-way ANOVA and Sidak post-test. (<b>B</b>) Percentages of CD4⁺CD44^high T cells producing IL-2, TNF, IL-4/IL-13, IFN-γ and/or IL-17A in each of the possible combinations (CD4⁺CD44^high CYT⁺) in response to vaccine proteins stimulation calculated applying Boolean gates. No cells expressing more than 3 cytokines at once were detected. Bars represent mean ± SEM. *<i>p</i> < 0.05 by unpaired Student <i>t</i> test, two-tailed, and a partial permutation test.</p

One Dose of <i>Staphylococcus aureus</i> 4C-Staph Vaccine Formulated with a Novel TLR7-Dependent Adjuvant Rapidly Protects Mice through Antibodies, Effector CD4⁺ T Cells, and IL-17A

<div><p>A rapidly acting, single dose vaccine against <i>Staphylococcus aureus</i> would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent <i>S</i>. <i>aureus</i> candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4⁺ T cells not only reduced survival but also increased <i>S</i>. <i>aureus</i> load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by <i>in vivo</i> neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against <i>S</i>. <i>aureus</i> through the combined actions of antibodies, CD4⁺ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.</p></div

One dose of 4C-Staph/T7-alum induces functional antibodies.

<p>BALB/c mice (n = 16) were immunized once with 4C-Staph/T7-alum or 4C-Staph/alum. Control mice were injected with T7-alum or alum alone. (<b>A</b>) Vaccine-specific serum IgG titers measured 12 (d12) and 32 (d32) days after vaccination. IgG concentrations in control sera (open symbols) are reported only for d32. Each symbol represents one mouse, and data are the merge of two independent experiments. Median with interquartile range of each group is also shown. *<i>p</i> < 0.05, ****<i>p</i> < 0.0001 by Kruskal-Wallis test and Dunn's multiple comparisons test. (<b>B</b>) Hla neutralizing activity of pooled sera from vaccinated mice (n = 16, same animals as in A) was assessed on rabbit RBCs and expressed as effective dilution that neutralized 50% of Hla lytic activity (ED₅₀). No hemolysis inhibition was detected (ED₅₀ < 6) in pre-immune sera or in sera from adjuvant-treated mice. Lack of overlap in the 95% confidence intervals between the ED₅₀ of sera from mice vaccinated with 4C-Staph/alum (30.3 to 51.4) vs. 4C-Staph/T7-alum (114.6 to 318.7) by 32 days indicates a difference significant with <i>p</i> < 0.05. Bars represent SEM. (<b>C</b>) Vaccine-specific IgG1 and IgG2a. Columns represent median MFI with interquartile range of pooled sera from vaccinated mice (n = 16, same pools as in B) bled at d32. **<i>p</i> < 0.01, ***<i>p</i> < 0.001 by unpaired Student <i>t</i> test, two-tailed. (<b>D</b>) Hla_H35L-specific IgM. Columns represent median MFI with interquartile range of sera from vaccinated mice (n = 12) bled at d12. IgM specific for EsxAB, FhuD2 and Csa1A were at the limit of detection (data not shown). Data shown are the merge of two independent experiments.</p

One dose of 4C-Staph/T7-alum induces protective antibodies.

<p>Sera from mice immunized with 4C-Staph/T7-alum (immune serum), or T7-alum as negative control (control serum), by 32 days were pooled and injected i.v. (150 μl/mouse) in naïve BALB/c mice (n = 16) 24 h before i.p. challenge with <i>S</i>. <i>aureus</i>. <b>(A</b>) Survival was monitored for 15 days post challenge. Data are the merge of two independent experiments. ***<i>p</i> < 0.001 by Log-rank test. <b>(B</b>) Fifteen days after <i>S</i>. <i>aureus</i> infection, survivors were euthanized, both kidneys were homogenized and CFU enumerated. Each symbol represents one mouse. <b>(C</b>) B cell/antibody-deficient J_H mice, or BALB/c (wt) as control, were immunized with 4C-Staph/T7-alum or T7-alum alone. Twelve days after vaccination, mice (n = 25 for 4C-Staph/T7-alum; n = 15 for T7-alum) were challenged i.p. with <i>S</i>. <i>aureus</i> and their survival was monitored for 15 days. Data are the merge of four independent experiments. ***<i>p</i> < 0.001 by Log-rank test.</p

One dose of 4C-Staph/T7-alum vaccine protects better than 4C-Staph/alum in kidney abscess and peritonitis models of <i>S</i>. <i>aureus</i> infection.

<p>BALB/c mice were immunized once i.m. with 4C-Staph/T7-alum or 4C-Staph/alum. Control mice were injected with T7-alum or alum alone. After 12 days, mice were challenged with <i>S</i>. <i>aureus</i> Newman strain. (<b>A</b>) Kidney abscess model. Mice (n = 28–32) were injected i.v. with 2 x 10⁷ CFU. Four days later, both kidneys of each mouse were homogenized in pool and CFU enumerated. Each symbol represents one mouse, and data are the merge of three independent experiments. Mean ± SEM of each group are shown. The dotted line indicates the lower limit of detection (LLD). *<i>p</i> < 0.05, **<i>p</i> < 0.01 by one-way ANOVA and Sidak's multiple comparisons test. Number of survivors with non-detectable CFU (CFU ND) in kidneys/total number of survivors and corresponding percentages are reported above the graph. (<b>B-C</b>) Peritonitis model. Mice (n = 32) were injected i.p. with 5 x 10⁸ CFU. Survival was monitored for 30 days after challenge. Data are the merge of three independent experiments. ***<i>p</i> < 0.001 by Log-rank test. (<b>C</b>) Thirty days after <i>S</i>. <i>aureus</i> infection, survivors were euthanized, both kidneys were homogenized and CFU enumerated. Each symbol represents one mouse. Mean ± SEM of each group is shown. **<i>p</i> < 0.01 by unpaired Student <i>t</i> test, one-tailed. Number of survivors with CFU ND in kidneys/total number of survivors and corresponding percentages are reported above the graph.</p

IL-17A neutralization in mice vaccinated with 4C-Staph/T7-alum has no effect on survival but increases the bacterial load in kidneys upon i.p. <i>S</i>. <i>aureus</i> challenge.

<p>Twelve days after vaccination with 4C-Staph/T7-alum or T7-alum, BALB/c mice (n = 15–16) were challenged i.p. with <i>S</i>. <i>aureus</i>. Mice were injected i.p. with neutralizing mAb (neutr. Ab): (<b>A</b>) anti-IL-17A; (<b>B</b>) anti-IFN-γ; or (<b>C</b>) anti-IL-17A and anti-IFN-γ 3 h before challenge and every other day for 15 days after challenge. Control mice were injected with isot. ctr. (<b>A-C</b>, left panels) Survival was monitored for 15 days after challenge. Data are the merge of two independent experiments. No statistically significant differences between mice vaccinated with 4C-Staph/T7-alum treated with neutr. Ab or isot. ctr. (Log-rank test). (<b>A-C</b>, right panels) Fifteen days after <i>S</i>. <i>aureus</i> inoculation, survivors (n = 7–8) were euthanized, both kidneys homogenized and CFU enumerated. Each symbol represents a mouse. Mean ± SEM. One representative experiment out of two is shown. The dotted line indicates the lower limit of CFU detection. *<i>p</i> < 0.01; ***<i>p</i> < 0.001 by unpaired Student <i>t</i> test, one-tailed.</p