Targeted Nasal Vaccination Provides Antibody-Independent Protection Against Staphylococcus aureus

Despite showing promise in preclinical models, anti-Staphylococcus aureus vaccines have failed in clinical trials. To date, approaches have focused on neutralizing/opsonizing antibodies; however, vaccines exclusively inducing cellular immunity have not been studied to formally test whether a cellular-only response can protect against infection. We demonstrate that nasal vaccination with targeted nanoparticles loaded with Staphylococcus aureus antigen protects against acute systemic S. aureus infection in the absence of any antigen-specific antibodies. These findings can help inform future developments in staphylococcal vaccine development and studies into the requirements for protective immunity against S. aureu

The Long-Baseline Neutrino Experiment: Exploring Fundamental Symmetries of the Universe

The preponderance of matter over antimatter in the early Universe, the dynamics of the supernova bursts that produced the heavy elements necessary for life and whether protons eventually decay --- these mysteries at the forefront of particle physics and astrophysics are key to understanding the early evolution of our Universe, its current state and its eventual fate. The Long-Baseline Neutrino Experiment (LBNE) represents an extensively developed plan for a world-class experiment dedicated to addressing these questions. LBNE is conceived around three central components: (1) a new, high-intensity neutrino source generated from a megawatt-class proton accelerator at Fermi National Accelerator Laboratory, (2) a near neutrino detector just downstream of the source, and (3) a massive liquid argon time-projection chamber deployed as a far detector deep underground at the Sanford Underground Research Facility. This facility, located at the site of the former Homestake Mine in Lead, South Dakota, is approximately 1,300 km from the neutrino source at Fermilab -- a distance (baseline) that delivers optimal sensitivity to neutrino charge-parity symmetry violation and mass ordering effects. This ambitious yet cost-effective design incorporates scalability and flexibility and can accommodate a variety of upgrades and contributions. With its exceptional combination of experimental configuration, technical capabilities, and potential for transformative discoveries, LBNE promises to be a vital facility for the field of particle physics worldwide, providing physicists from around the globe with opportunities to collaborate in a twenty to thirty year program of exciting science. In this document we provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess.Comment: Major update of previous version. This is the reference document for LBNE science program and current status. Chapters 1, 3, and 9 provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess. 288 pages, 116 figure

Nasal Colonisation by <em>Staphylococcus aureus</em> Depends upon Clumping Factor B Binding to the Squamous Epithelial Cell Envelope Protein Loricrin

<div><p><em>Staphylococcus aureus</em> asymptomatically colonises the anterior nares, but the host and bacterial factors that facilitate colonisation remain incompletely understood. The <em>S. aureus</em> surface protein ClfB has been shown to mediate adherence to squamous epithelial cells <em>in vitro</em> and to promote nasal colonisation in both mice and humans. Here, we demonstrate that the squamous epithelial cell envelope protein loricrin represents the major target ligand for ClfB during <em>S. aureus</em> nasal colonisation. <em>In vitro</em> adherence assays indicated that bacteria expressing ClfB bound loricrin most likely by the “dock, lock and latch” mechanism. Using surface plasmon resonance we showed that ClfB bound cytokeratin 10 (K10), a structural protein of squamous epithelial cells, and loricrin with similar affinities that were in the low µM range. Loricrin is composed of three separate regions comprising GS-rich omega loops. Each loop was expressed separately and found to bind ClfB, However region 2 bound with highest affinity. To investigate if the specific interaction between ClfB and loricrin was sufficient to facilitate <em>S. aureus</em> nasal colonisation, we compared the ability of ClfB⁺<em>S. aureus</em> to colonise the nares of wild-type and loricrin-deficient (Lor^−/−) mice. In the absence of loricrin, <em>S. aureus</em> nasal colonisation was significantly impaired. Furthermore a ClfB⁻ mutant colonised wild-type mice less efficiently than the parental ClfB⁺ strain whereas a similar lower level of colonisation was observed with both the parental strain and the ClfB⁻ mutant in the Lor^−/− mice. The ability of ClfB to support nasal colonisation by binding loricrin <em>in vivo</em> was confirmed by the ability of <em>Lactococcus lactis</em> expressing ClfB to be retained in the nares of WT mice but not in the Lor^−/− mice. By combining <em>in vitro</em> biochemical analysis with animal model studies we have identified the squamous epithelial cell envelope protein loricrin as the target ligand for ClfB during nasal colonisation by <em>S. aureus</em>.</p> </div

Inhibition of bacterial adherence and rClfB binding to immobilized ligands.

<p>(A) <i>L. lactis</i> NZ9800 (pNZ8037), <i>L. lactis</i> NZ9800 (pNZ8037:<i>clf</i>B) and <i>L. lactis</i> NZ9800 (pNZ8037:<i>clf</i>BQ235A) were added to wells containing immobilized GST-tagged Hlor, MLor, HK10, MK10 (0.5 µM). Bacterial adherence was measured by staining with crystal violet and measurement of the absorbance at 570 nm. The data shown is representative of two individual experiments (B). <i>S. aureus</i> Newman pre-incubated with GST or HK10 (2 µM) was added to loricrin-coated microtitre wells (0.5 µM). Bacterial adherence was measured by staining with crystal violet and measurement of the absorbance at 570 nm and was expressed as a percentage of total binding. Values represent the mean ± SD of triplicate wells. The data shown is representative of two individual experiments. (C) Recombinant ClfB N23_201–542 was pre-incubated with GST, HK10 or L2v (14 µM) before being added to loricrin-coated microtitre wells (0.5 µM). Bound protein was detected using HRP-conjugated anti-his antibodies and was expressed as a percentage of total bound protein. Values represent the mean ± SD of triplicate wells. The values shown are representative of 3 individual experiments. Statistical analysis was performed using an unpaired t-test. *** p<0.0005 versus binding of ClfB-expressing bacteria (A) or pre-incubation with GST (B, C).</p

Affinities of ClfB N2N3_201–542 for loricrin, keratin and fibrinogen using surface plasmon resonance.

*<p>Data representative of n = 3 individual experiments.</p

HLor region L2v blocks ClfB-mediated adherence of <i>S. aureus</i> to human desquamated epithelial cells.

<p><i>S. aureus</i> strains were grown to exponential phase in TSB (A) or in RPMI (B). Washed cells were incubated with recombinant GST or recombinant L2v-GST, or just resuspended in PBS, before being incubated with human nasal epithelial cells. Adherent bacteria were enumerated by microscopy and were expressed as a percentage of the positive control. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003092#s2" target="_blank">Results</a> are expressed as the mean ± SD of 3 independent experiments. Statistical analysis was performed using an unpaired t test.</p

Systemic infection in Lor^−/− mice.

<p><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003092#s2" target="_blank">Results</a> expressed as mean Log CFU/ml of fluid or homogenised tissue ± SEM, n = 4 per group.</p

Nasal colonisation of <i>L. lactis</i> expressing ClfB and Newman Δ<i>clfB</i>⁻ in the FVB wild-type and Lor^−/− mice.

<p>(A) Mice were inoculated intra-nasally with <i>L. lactis</i> MG1363 (pKS80) or <i>L. lactis</i> MG1363 (pKS80:<i>clf</i>B) (2×10¹¹ CFU). Mice were euthanized after 24 hours and the bacterial burden in noses was established. Inoculation with the empty vector (pKS80) did not result in significant colonisation (>5 CFU per nose) in either WT or Lor^−/− mice. Statistical analysis was performed using the Mann-Whitney test. (B) Mice were inoculated intra-nasally with Newman or Newman Δ<i>clfB</i> (2×10⁸ CFU). After 10 days, mice were euthanized and bacterial burden in the noses was established. Each dot indicates the number of CFU/nose for a single mouse. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003092#s2" target="_blank">Results</a> expressed as Log CFU per nose, median indicated by bar (n = 15–20 per group). Statistical analysis was performed using the Krustal-wallis test and Dunns Multiple Comparisons test. *p<0.05, **p<0.005.</p

Surface Plasmon Resonance analysis of the interaction of ClfB with loricrin and keratin.

<p>Representative sensorgrams display binding of rClfB_201–542 to and dissocation from (A) GST-HLor, (B) GST-HK10, (C) GST-MLor and (D) GST-MK10 in a single cycle kinetics assay. GST-tagged ligands were captured onto a CM5 chip coated with anti-GST IgG and were exposed to increasing concentrations of rClfB_201–542. Binding is measured as response units (RU) against time. The affinities were calculated from curve fitting to a plot of the RU values against concentrations of rClfB_201–542. Arrows indicate the time at which rClfB_201–542 is injected. The data shown is representative of 3 individual experiments.</p