Seroprevalence of HBsAg in all blood donors according to their socio-demographic characteristics from 2009 to 2016.

<p>Seroprevalence of HBsAg in all blood donors according to their socio-demographic characteristics from 2009 to 2016.</p

Immunoassays used in the screening of HBsAg in blood donors from 2009 to 2016.

<p>Immunoassays used in the screening of HBsAg in blood donors from 2009 to 2016.</p

HBsAg seroprevalence according to age, sex and donor type.

<p>HBsAg seroprevalence according to age, sex and donor type.</p

Cases and HBsAg frequency per year.

<p>Cases and HBsAg frequency per year.</p

Sociodemographic characteristics of blood donors from 2009 to 2016.

<p>Sociodemographic characteristics of blood donors from 2009 to 2016.</p

Diverse MUC1 Antigen Preparations Generate Specific Immune Responses in Tolerant MUC1.Tg and Non-Tolerant WT Mice.

<p>MUC1.Tg mice were given three immunizations with vaccines containing 9mers, 22mers or rotating tumor lysates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145920#pone.0145920.g001" target="_blank">Fig 1</a>). Lymph node-derived T-cells were culture expanded for 7–14 days with DCs pulsed with the immunizing antigens. Antigen-specific T-cells were enumerated for intracellular IFN-γ production when re-stimulated with DCs pulsed with short peptides <b>(A)</b> and long peptides <b>(B)</b>. Data are shown after subtracting background from unpulsed DCs to facilitate visual comparisons. See Fig 2D for examples of representative unsubtracted backgrounds. A representative of 2 experiments is shown; pools of 7 mice were used. <b>(C) Wide specificity of the lysate-sensitized T-cells</b>: Lysate sensitized T-cells from MUC1.Tg mice or WT mice showed specificity against 19 out of 19 MUC1 peptides from both TR and non-TR regions. <b>(D)</b> MUC1.Tg mice were immunized twice with vaccines containing either long peptides from TR, APG 22mer (APGSTAPPAHGVTSAPDTRPAP) or the CT peptide (SLSYTNPAVAATSANL). After <i>in vitro</i> stimulation with DCs pulsed with immunizing peptides, antigen specific T-cells were analyzed for intracellular IFN-γ against dendritic cells pulsed with peptides (APG 22mer or CT) or no peptide (UP). One representative of three experiments is shown; pools of 7 mice were used in each experiment.</p

Aspects of Antigen Presentation Relevant to Tumor Recognition are Shown.

<p><b>(A)</b> Tumor cell expression of MUC1 was stable <i>in vitro</i> (C57mg.MUC1: 86%; KCM: 73%; EL4.MUC1: 79%; B16.MUC1 and Panc02.MUC1: 95%; MC38.MUC1: 99%) <b>(B)</b> Irradiated tumor digests passed in syngeneic mice prior to processing were used to assess T-cell recognition of tumor-associated MUC1. Representative flow data for Panc02.MUC1 is shown. MUC1 was predominantly expressed on the tumor cell population, while both MHC class I and II molecules were expressed by CD45⁺ host cells as well as tumor cells. <b>(C) Glycosylation alters DC processing of MUC1</b>. DCs were pulsed overnight with non-glycosylated APG 22mer or one of three glycoforms of APG 22mer (5-Tn; 4, 5-Tn or 18-Tn). The DCs were then analyzed for presentation of the C-terminal peptide SAPDTRPAP (PDTR) by flow cytometry with anti-MUC1 (BC2-Alexa488) specific for PDTR. Cells staining positively for PDTR also co-stained for CD11c⁺, K^b (Fig 3C) and I-A^b (not shown). Detection of PDTR on DCs was only observed if APG 22mer was glycosylated at 4-Tn or 4, 5-Tn (Fig 3C). <b>(D) Glycosylation promotes co-localization of SAPDTRPAP with class I molecules</b>. DCs in chamber slides were stimulated overnight with either non-glycosylated (APG 22mer) or glycosylated (APG 22mer 5-Tn) peptides and stained with anti-MUC1 (BC2-Alexa488; green) and anti-H-2K^b followed by secondary goat anti-mouse IgG-labeled-Alexa633 (red). Representative confocal images showed stronger co-localization (yellow) of epitope SAPDTRPAP with H-2K^b on the DCs pulsed with the glycopeptide. The experiment was repeated two times. <b>(E) Individual forms of antigen during vaccination elicited differential recognition of MUC1-expressing tumors</b>. Effector T-cells from MUC1.Tg mice immunized with vaccines containing either 9mers, 22mers or rotating tumor lysates were stimulated <i>in vitro</i> for 7–14 days with DCs pulsed with immunizing peptides or B16.MUC1-expressing tumor cell lysate. The stimulated T-cells were co-cultured with various MUC1-expressing or MUC1 non-expressing irradiated tumor digests (C57mg.MUC1, C57 WT; KCM, KCKO; EL4.MUC1, EL4.WT; B16.MUC1, B16.neo; Panc02.MUC1, Panc02.neo and MC38.MUC1, MC38.neo) and stained for intracellular IFN-γ. Data showed MUC1-specific responses that were determined by subtraction of background reactivity of the corresponding MUC1 non-expressing tumor digests. Representative data of two independent experiments are shown; pools of 7 mice were used.</p

Diagram of MUC1 Domains and Peptides Chosen for Study.

<p>The domains of MUC1 are shown on left side of the diagram with the sequences studied listed below the domains and the peptide name on the right side. Immunizing peptides and serines (S) and threonines (T) that are <i>O</i>-glycosylated with N-Acetylgalactosamine (Tn) are shaded.</p

Tumor Progression Following Immunization with Either Peptides or Rotating Lysates Prior to Tumor Challenge Consistently Resulted in Decreased Expression of Tumor MUC1 and MHC Proteins.

<p>The resistant B16.MUC1 tumors that grew after peptide immunizations were excised, digested and analyzed by flow cytometry with anti-CD45 APC-Cy7, anti-MUC1 FITC (CD227), anti-H-2K^b PE or anti-I-A^b PE. Cell surface MUC1 and MHC expression were significantly decreased following peptide vaccination compared to non-immunized mice. <b>(A)</b> Each bar represents the average fluorescent index (FI) of surface MUC1 expression for each treatment group. A typical experiment with n = 3 mice per group is shown; experiments were repeated at least 3 times. <b>(B)</b> Representative histograms are shown. The peptide shown is SAP 22mer, which is representative of the results for 9mer peptides. <b>(C)</b> Similar results were seen for Panc02.MUC1 in those groups where prior lysate immunization failed to prevent tumor outgrowth. <b>(D)</b> The cell line generated from Panc02.MUC1 tumor <i>in vitro</i> in G418 maintained low MUC1 surface expression when expanded <b>(right panel)</b> as compared to the parent cell line (<b>D, left panel</b>).</p

Peptide Vaccine is Rendered Therapeutically Effective if Repetitive Delivery is Continued Post Tumor Challenge.

<p>MUC1.Tg mice were given two peptide immunizations before and two after tumor challenges. Three out of four peptide vaccinations significantly delayed the growth of B16.MUC1 tumors (APG+SAP 9mer, p = 0.001; APG+SAP Tn-9mer, p = 0.001; SAP 22mer, p = 0.001) and <b>(B)</b> showed complete protection in 4 out of 10 mice (SAP 22mer) and 3 out of 10 mice (9mers and Tn-9mers), if immunizations were continued post tumor challenges. Experiment was repeated 2 times, n = 5 mice/group.</p