Single-dose administration of nanovaccines induced long-term antibody titers with high avidity.

<p>(<b>A</b>) Kinetics of IgG antibody titer throughout 23 weeks post-vaccination. (<b>B</b>) Antibody isotype induced by various immunization regimens. Optical density was determined by ELISA at a 1∶1000 dilution. (<b>C</b>) IgG antibody avidity throughout 23 weeks post-vaccination. Avidity was determined via ELISA at a 1∶200 dilution. Data is presented as the mean ± SEM (n = 7 per group) and is representative of two independent experiments. <b>*</b>  =  p<0.02, <b>#</b>  =  p<0.005 and <b>+</b>  =  p<0.0001 (compared to S₅₀ + MPLA).</p

Material properties of 50∶50 CPTEG:CPH nanoparticles.

<p>Representative scanning electron photomicrographs of (<b>A</b>) blank and (<b>B</b>) 2% F1-V loaded 50∶50 CPTEG:CPH nanoparticles (scale bar  =  1 µm). (<b>C</b>) Particle size distribution as determined by QELS for blank (204±62) and 2% F1-V loaded 50∶50 CPTEG:CPH nanoparticles (196±77) with n  =  3. (<b>D</b>) <i>In vitro</i> cumulative release of F1-V from 50∶50 CPTEG:CPH nanoparticles in pH 7.4 PBS analyzed by micro bicinchoninic acid assay (n  =  2, representative of two separate nanoparticle batches).</p

Vaccination regimens.

<p>*Quantities indicate the amounts of immunogen or adjuvant delivered to each mouse in the indicated group. S  =  soluble protein; E  =  encapsulated protein. Subscripts indicate amount of soluble or encapsulated protein (in µg) administered per dose.</p

Histopathological analysis of lungs, spleens, and livers 6 weeks post-vaccination at 72 h post-challenge from mice vaccinated six weeks earlier.

<p>Lungs of S₄₀ + E₁₀ vaccinated mice (v) were free of any histopathological lesions and bacteria and were similar to lung tissue from unimmunized and uninfected control mice (i). No bacteria, necrosis or edema were present in spleens of the S₄₀ + E₁₀ vaccinated mice (x) and histology was similar to healthy spleen tissue (vi). The livers of the S₄₀ + E₁₀ vaccinated mice (xv) did not show any lesions similar to control mice (xi). Av - Alveoli, F - Follicle, Mz - Marginal zone, and RP - Red pulp. Arrow - bacteria, black arrowhead - neutrophilic infiltration, yellow arrowhead - lymphocytic infiltrations, * – edema, and + - necrotic cells. Objective lens magnification is 100X. Scale bar  = 20 µm.</p

Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the <i>B</i>. <i>burgdorferi</i> P66 Protein

<div><p>Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living <i>Cd1d</i>^<i>-/-</i>mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of <i>B</i>. <i>burgdorferi</i> involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of <i>B</i>. <i>burgdorferi</i> mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration.</p></div

The effect of <i>bbK32</i> deletion in an infectious strain background on vascular transmigration and clearance in <i>Cd1d</i>^<i>-/-</i> mice.

<p><b>A)</b> GFP-expressing <i>B</i>. <i>burgdorferi</i> strains, infectious (GCB966), a <i>bbk32</i> deletion strain (GCB971) and a high-passage non-infectious strain (GCB706) were injected into the tail vein of <i>Cd1d</i>^<i>-/-</i> mice (n = 6/group, 4x10⁸ spirochetes were injected per mouse). After 24 hours, vascular transmigration was scored in the knee joint-proximal tissue in the living mice by intravital microscopy using a spinning disk laser confocal microscope. Blood vessels were stained with PE-conjugated or Alexa Fluor 555-conjugated PECAM-1 antibody and green fluorescent spirochetes outside of the vasculature were counted in at least five fields of view (FOV) per mouse. Statistical significance was analyzed using the non-parametric Mann-Whitney test; ns denotes not significant (P-values >0.05). <b>B)</b> Concentrations of <i>B</i>. <i>burgdorferi</i> in mouse plasma after iv inoculation. <i>Cd1d</i>^<i>-/-</i> mice were injected with <i>B</i>. <i>burgdorferi</i> through the tail vein and blood was withdrawn at 5 and 60 minutes post-inoculation (n = 3/group). Blood cells were allowed to settle overnight as described in Materials and Methods and spirochetes in the plasma were directly counted by dark-field microscopy. The change in spirochete concentration between 5 and 60 minutes was determined for each mouse as the percentage of spirochetes present at 60 minutes relative to the initial 5 minute time point. Statistical significance was analyzed using the non-parametric Mann-Whitney test; ns denotes not significant (P-values >0.05).</p

The effect of <i>p66</i> site-directed integrin binding mutants on vascular transmigration and clearance in <i>Cd1d</i>^<i>-/-</i> mice.

<p><b>A)</b> GFP-expressing <i>B</i>. <i>burgdorferi</i> strains, infectious wild type (GCB847), <i>p66</i>^{<i>D205A</i>,<i>D207A</i>} (GCB3003) and <i>p66</i>^{<i>Δ202–208</i>} (GCB3004) were injected into the tail vein of <i>Cd1d</i>^<i>-/-</i> mice (n = 3/group, 4x10⁸ spirochetes were injected per mouse). After 24 hours, vascular transmigration was scored in the knee joint-proximal tissue in the living mice by intravital microscopy using a spinning disk laser confocal microscope. Blood vessels were stained with PE-conjugated PECAM-1 antibody and green fluorescent spirochetes outside of the vasculature were counted in at least five fields of view (FOV) per mouse. Statistical significance was analyzed using non-parametric Kruskal-Wallis ANOVA followed by Dunn's multiple comparisons test. <i>P</i>-values for select pairwise comparisons are shown; ns denotes not significant (P-values >0.05). <b>B)</b>. Concentrations of <i>B</i>. <i>burgdorferi</i> in mouse plasma after iv inoculation. <i>Cd1d</i>^<i>-/-</i> mice were injected with <i>B</i>. <i>burgdorferi</i> through the tail vein and blood was withdrawn at 5 and 60 minutes post-inoculation (n = 3/group). Blood cells were allowed to settle overnight as described in Materials and Methods and spirochetes in the plasma were directly counted by dark-field microscopy. The change in spirochete concentration between 5 and 60 minutes was determined for each mouse as the percentage of spirochetes present at 60 minutes relative to the initial 5 minute time point. Statistical significance was analyzed using non-parametric Kruskal-Wallis ANOVA followed by Dunn's multiple comparisons test. <i>P</i>-values for select pairwise comparisons are shown; ns denotes not significant (P-values >0.05).</p

Progression of Lyme disease in humans following <i>B</i>. <i>burgdorferi</i> infection.

<p>Spirochetes are inoculated in the skin through the bite of an infected hard-shelled <i>Ixodes</i> tick. CNS, central nervous system; PNS, peripheral nervous system. Reprinted from Trends in Microbiology, Vol. 21, No. 8, Coburn, J., Leong, J. and Chaconas, G., Illuminating the roles of the <i>Borrelia burgdorferi</i> adhesins, Pages 372–379, Copyright 2013, with permission from Elsevier. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005333#ppat.1005333.ref007" target="_blank">7</a>]</p

Visualization of β₃ integrin in post-capillary venules in knee joint-proximal tissue by multi-channel spinning disk intravital microscopy.

<p>GFP-expressing infectious <i>B</i>. <i>burgdorferi</i> (GCB847) was injected into the jugular vein of BALB/c mice as noted for other intravital experiments and data were acquired between 5–60 minutes postinfection. Blood vessels were stained with PE-conjugated PECAM-1 antibody and Alexa Fluor 647-conjugated β₃ integrin antibody. The upper panel shows tethering and stationary interactions of <i>B</i>. <i>burgdorferi</i> (green), with PECAM-1 expressing endothelium visualized inside the blood vessels (red). The lower panel shows tethering and stationary interactions of the <i>B</i>. <i>burgdorferi</i> (green), with β₃ integrin visualized inside the blood vessels (blue). The β₃ integrin staining is also visible in smooth muscles (blue) surrounding the blood vessels.</p

The effect of <i>p66</i> deletion on vascular adhesion and clearance in a high- passage <i>B</i>. <i>burgdorferi</i> strain in BALB/c mice.

<p>Non-infectious GFP-expressing <i>B</i>. <i>burgdorferi</i> wild type (GCB3212), <i>Δp66</i> (GCB3214), and a strain where the wild-type <i>p66</i> gene was reintroduced into the <i>p66</i> deletion mutant <i>(Δp66/comp</i>, GCB3218) were injected into the jugular vein of BALB/c, 4x10⁸ spirochetes per mouse (n = 7/group). Over a period of up to 45 minutes, microvascular interaction rates <b>A)</b> (tethering + dragging), and stationary adhesions <b>B)</b> were enumerated in the knee joint-proximal tissue by intravital microscopy using spinning disk laser confocal microscopy as described in Materials and Methods. Blood vessels were stained with PE-conjugated PECAM-1 antibody. Statistical significance was analyzed using the non-parametric Kruskal-Wallis test; ns denotes not significant (P-values >0.05). <b>C)</b> Concentrations of <i>B</i>. <i>burgdorferi</i> in mouse plasma after iv inoculation. BALB/c mice were inoculated with <i>B</i>. <i>burgdorferi</i> through the tail vein as above and blood was withdrawn at 3 and 18 minutes post-inoculation (n = 7/group). Blood cells were allowed to settle overnight as described in Materials and Methods and spirochetes in the plasma were directly counted by dark-field microscopy. The change in spirochete concentration between 3 and 18 minutes was determined for each mouse as the percentage of spirochetes present at 18 minutes relative to the initial 3 minute time point. Statistical significance was analyzed using the non-parametric Kruskal-Wallis test; ns denotes not significant (P-values >0.05).</p