RSV specific IgG in mice after vaccination with RSV virosomes and RSV-MPLA virosomes.

<p>Mice were vaccinated twice with RSV virosomes, RSV-MPLA virosomes or controls (HNE, live virus and FI-RSV). Each injection contained 5 µg of protein. (A) RSV-specific IgG titers in serum 14 days after prime and 14 days after booster vaccination. (B) RSV-specific IgG1 and IgG2a subtype levels in serum 14 days after booster vaccination. (C) IgE levels were determined at 14 days after booster vaccination. (D) RSV neutralizing antibody titers in serum obtained 5 days after challenge. Bars represent the GMT (panels A and C), mean concentration of RSV-specific IgG1/2a (panel B) or mean neutralization titer (panel D) of 6 mice per group. Error bars represent the SEM. Statistical differences were calculated using the Mann-Whitney-U test. *p<0.05, **p<0.01, ***p<0.001. Statistical differences in IgE levels were calculated with an ANOVA with Bonferroni correction for multiple testing ***p<0.001. The data shown are a representative of two individual experiments.</p

<i>In vitro</i> analysis of RSV and RSV-MPLA virosomes.

<p>(A,B) RSV virosomes and RSV-MPLA virosomes were spun on an equilibrium density sucrose gradient. Subsequently, density, protein concentration, and phosphate concentrations of each fraction was determined. (C,D) Fractions from A and B were analyzed for their TLR4-signaling ability using Hek-Blue TLR4 cells. To assess non-TLR specific activation of cells, control cells (Null2 cells) were incubated with the same virosome fractions. As a control for activation both Hek blue TLR4 and Hek blue null2 cells were stimulated with 100 ng/ml TNF-α. Bars represent TLR activation relative to that of the TNF-α control (E) Upregulation of DCs costimulatory molecules CD40, CD86, CD80. Unfractionated virosome preparations were used to stimulate <i>ex vivo</i> cultured mouse DCs overnight. Cells were stained for expression of costimulatory molecules using specific monoclonal antibodies and analyzed by FACS. Bars represent the percentage of positive cells. The data shown are a representative of three individual experiments.</p

Lung pathology in mice after immunization and RSV infection.

<p>Mice were immunized and challenged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036812#pone-0036812-g002" target="_blank">Figure 2</a> and the lungs were harvested, sliced and stained with H&E and assessed for pathology using light microscopy. Panels represent the lungs of (A) FI-RSV, (B) live virus, (C) RSV virosomes, (D) RSV MPLA virosomes (E) buffer immunized mice. Black arrows point to alveolar infiltrates, grey arrows to peribronchial infiltrates and white arrows to perivascular infiltrates. (F) Eosinophils in BAL expressed as percentage of total BAL cells. Data points represent values from individual mice. Statistical differences were calculated using the ANOVA test with Bonferroni correction for multiple testing. ***p<0.001. The data shown are a representative of two individual experiments.</p

IFNγ and IL5 concentrations in RSV-stimulated splenocyte cultures and lung tissue homogenates.

<p>Mice were vaccinated twice with RSV virosomes, RSV-MPLA virosomes and control vaccines as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036812#pone-0036812-g002" target="_blank">Figure 2</a>, and subsequently challenged with live RSV. Four days after challenge, IFNγ and IL5 responses were determined. (A) IFNγ concentrations in splenocyte cultures restimulated with BPL-inactivated RSV for three days. (B) IFNγ concentrations in homogenated lung tissue, four days after challenge. (C) IL5 concentrations in splenocyte cultures, restimulated with BPL-inactivated RSV for three days. (D) IL5 concentrations in homogenated lung tissue, four days after challenge. Bars represent the mean cytokine concentration of 6 mice per group and error bars represent the SEM. Statistical differences were calculated using a Mann-Whitney-U test. *p<0.05, **p<0.01, ***p<0.001. The data shown are a representative of two individual experiments.</p

Protection against live virus challenge and infiltration of eosinophils.

<p>Mice were vaccinated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036812#pone-0036812-g002" target="_blank">figure 2</a> and challenged with live virus 14 days after the booster vaccination. Four days after challenge, lungs were removed and the viral titer was determined and expressed as TCID₅₀. RSV TCID₅₀ titers from the lungs of challenged animals. Statistical differences were calculated using the Mann-Whitney-U test. *p<0.05. The data shown are a representative of two individual experiments.</p

Nanoparticles target lymph node dendritic cells better after i.d. vs. i.m. delivery.

<p>(<b>a</b>) Blood concentrations of Dy649-labeled NPs after i.v., i.m. and i.d. administration. (<b>b</b>) Heat maps representing the median percentage of NP⁺ cells for indicated cell populations. Note that maxima vary from 10% in total leukocytes to 100% in monocytes. <i>P</i> values were computed by comparing the adjusted means of each organ between i.d. and i.m. for each cell type with a two-tailed Student's t-test. (<b>c</b>) Importance of route of administration for each cellular subtype. The log-likelihood ratio represents the likelihood of the alternate model, i.e. the model without taking account the route of administration, over the likelihood of the full factorial model. <i>P</i> values were computed using the <i>Chi</i> Square test between the alternate model and the full model for each population. For 144 h, <i>n</i> = 2, for all else, <i>n</i>≥4. Leukocytes: CD45⁺, mature myeloid DCs: CD11c⁺CD11b⁺I/A^b+, cross-presenting DCs: CD11c⁺CD8α⁺ I/A^b+, immature myeloid DCs: CD11c⁺CD11b⁺I/A^b−, immature lymphoid DCs: CD11c⁺CD11b⁻I/A^b−, medullary/red pulp (RP) macrophages (MØ): CD11b⁺F4/80⁺, monocytes: CD11b⁺GR1^midSSC^lowF4/80⁺, granulocytes: CD11b⁺GR1^highSSC^high, T cells: CD3ε⁺, B cells: B220⁺. Draining lymph nodes are indicated by Ax: axillary, Br: brachial, In: inguinal, Po: popliteal; Sp: spleen. *<i>p</i>≤0.05, **<i>p</i><0.01, ***<i>p</i><0.005.</p

Monocytes internalize nanoparticles via macropinocytosis while B and T cell associate externally.

<p>(<b>a</b>) Representative flow cytometry plots of <i>in vivo</i> NP-Dy649⁺ uptake kinetics after intradermal administration: monocytes (CD11b⁺GR1^midSSC^lowF4/80⁺) and B cells (B220⁺) in the spleen. (<b>b</b>) Characteristic flow cytometry plots of biotinylated nanoparticle (NP-biotin) association with splenic (B, CD4, and CD8 cells) and bone marrow (CD11b⁺Ly6c⁺ and CD11b⁺Ly6g⁺) cells after 12 h incubation <i>in vitro</i>. To distinguish surface-associated- from internalized-NPs, cells were incubated before permeabilization with streptavidin-A488 (for extracellular association) and after permeabilization with streptavidin-A647 (for intracellular uptake). (<b>c</b>) Percentage of fluorescently labeled NPs (NP-Dy649) taken up by bone marrow cells as a function of the PI3K inhibitor (Ly294002) concentration. Bone marrow cells were incubated with increasing concentrations of Ly294002 (maximum 50 µM) for 45′ prior to the addition of NP-Dy649 for 12 h. Cells were subsequently stained and analyzed by flow cytometry. Open circles: CD11b⁺Ly6c⁺, filled squares: CD11b⁺Ly6g⁺, continuous line: vehicle control (VH, DMSO) for CD11b⁺Ly6c⁺, dashed line: VH for CD11b⁺Ly6g⁺.</p

Nanoparticle biodistribution in tissues and cells show secondary lymphoid organ accumulation.

<p>Heat maps show nanoparticle (NP)-positive percentages of each indicated cell type in lymph nodes (LN) or blood-filtering organs 12 h after i.d. injection as analyzed by flow cytometry. (<b>a</b>) Overall leukocyte (CD45+) in different tissues. leukocyte subpopulations with (<b>b</b>) low to medium levels (0–15%) or (<b>c</b>) high levels (up to 98%) of NP accumulation. B cells: B220⁺, T cells: (CD3ε⁺ then CD4⁺CD25⁺, CD4⁺CD25⁻, CD8⁺), TCRγδ: CD3ε⁺CD4⁻CD8⁻ TCRγδ⁺, immature myeloid dendritic cells (DCs): CD11c⁺CD11b⁺I/A^b−, immature lymphoid DCs: CD11c⁺CD11b⁻I/A^b−. (<b>c</b>) Granulocytes: CD11b⁺GR1^highSSC^high, monocytes: CD11b⁺GR1^lowSSC^lowF4/80⁺, mature myeloid DCs: CD11c⁺CD11b⁺I/A^b+, CD11c⁺CD8α⁺I/A^b+, CD11c⁺CD11b⁻I/A^b+, medullary macrophages (MØ): CD11b⁺F4/80⁺. Draining LNs are indicated by Ax: axillary, Br: brachial, In: inguinal, Po: popliteal; Sp: spleen, Bl: blood, Kd: kidneys, Li: liver, Lu: lungs. Heatmap color scales indicated on the right. Refer to gating strategies in Figures S2 and S3.</p

Nanoparticles are taken up by MDSCs in draining nodes, spleen and tumor.

<p>Mice were inoculated subcutaneously with 10⁶ E.G7-OVA thymoma cells underneath the left shoulder blade (dorsoanterior left lateral side). After tumors reached 100 mm³, mice were injected with Dy649-labeled nanoparticles (NPs). Flow cytometry plots illustrating targeting of (<b>a</b>) monocytic (MO) MDSCs and (<b>b</b>) polymorphonuclear (PMN) MDSCs in the tumor draining lymph node (TDLN), the spleen and the tumors. (<b>c</b>) Three-dimensional flow-cytometry representation of the MDSC compartment (MO and PMN) of the tumor. Comparison between different organs of interest of the (<b>d</b>) MO-MDSCs and (<b>e</b>) PMN-MDSCs subpopulation accumulating NPs. One-way ANOVA followed by Bonferroni post test. n = 3 *<i>p</i>≤0.05, **<i>p</i>≤0.01. Tu: Tumor; Sp: Spleen.</p

Lymphatic drainage is required for nanoparticle targeting of the lymph node and spleen after i.d. administration.

<p>(<b>a</b>) Bioavailability of Dy649-nanoparticles (NPs) in the blood compartment after i.d. administration in mice that lack peripheral lymphatics (K14-VEGR-3-Ig) and their wild type littermates. VH: vehicle control (non-fluorescently labeled NPs). Two-way repeated measures ANOVA followed by Bonferroni post test. (<b>b</b>) Comparison of NP⁺ association as assessed by flow cytometry in the brachial lymph node (LN) and the spleen 24 h post-i.d. administration. n = 4 *<i>p</i>≤0.05, ***<i>p</i>≤0.005. (<b>c</b>) 9 µm thick section of a Dy649-NP (red) draining wild type popliteal LN stained with nuclei (DAPI, blue). Scale bar, 200 µm. (<b>d</b>) 9 µm thick section of the NP-Dy649 (red) draining wild type brachial LN 12 h after i.d. administration, stained for lymphatic endothelium (LYVE-1, green), the T cell zone stroma (ERTR7, white). Scale bar, 40 µm. (<b>e</b>) 40 µm section of the wild type anterior spleen stained with DAPI (blue) and NP-Dy649 (red) shows NP accumulation in the red pulp (RP) and the marginal zone (MZ), as well as surrounding the B cell follicles (FO). Scale bar, 300 µm. (<b>f</b>) Enlarged region of the central arteriole of the spleen (white filled arrow) (<b><i>i</i></b>) immunofluorescence image (NP-Dy649, red and DAPI, blue). (<b><i>ii</i></b>) Hematoxylin & eosin staining of the same section of the spleen; dark blue FO, purple red pulp and pink blood vessels. Scale bar, 100 µm.</p