DCs loaded with CD11c-scFv-IDRgag induced proliferation of FV-specific CD8⁺ T cells.

<p>(<b>A</b>) To study proliferation of FV-specific CD8⁺ T cells induced by CD11c-scFv-IDRgag treated bmDCs, isolated FV-specific TCRtg CD8⁺ T cells were stained with CFSE prior to co-culture with bmDCs. After 4 days of co-culture both control-scFv-IDRgag and CD11c-scFv-gagID-loaded bmDCs induced proliferation of FV-specific CD8⁺ T cells. (<b>B</b>) The proliferation of CD8⁺ T cells induced by CD11c-scFv-IDRgag was significantly higher compared to DCs with control-scFv-IDRgag. Data represent mean±SD of 5 independent experiments. Data were analyzed with unpaired <i>t</i>-test.</p

Binding of CD11c-scFv-IDRgag to bmDCs and spleen DCs.

<p>(<b>A</b>) Binding of CD11c-scFv-IDRgag to bmDCs at different concentrations. Data represent mean±SD values of 4 independent experiments. (<b>B</b>) At 10 µg/ml concentration, CD11c-scFv-IDRgag bound to bmDCs with significantly higher capacity as compared to the control-scFv-IDRgag. Data represent mean±SD values of 4 independent experiments. Data were analyzed with unpaired <i>t</i>-test. (<b>C</b>) Using spleen cell suspensions, we investigated binding-specificity of CD11c-scFv-IDRgag on DCs. By gating on CD11b and MHC class II we defined a population of CD11c expressing DC without staining for CD11c itself. Whereas CD11b⁺/MHC class II^- cells did not express CD11c, the CD11b/MHC class II double-positive population represented CD11c expressing DCs. We found that CD11c-scFv-IDRgag selectively targets CD11c positive cells <i>in vitro</i>. Results of one representative experiment are shown.</p

Analysis of FV-specific CD8⁺ T cell activation induced by targeting viral Ag to CD11c on DC.

<p>(<b>A</b>) Activation induced by DCs loaded with 10 µg/ml of CD11c-scFv-IDRgag was equivalent with DCs loaded with 20 times molar excess of non-targeted FV matrix protein p15 (MA) extended with the same immunodominant CCLCLTVFL FVgag epitope (IDRgag-Map15). Data represent mean±SD of 2 independent experiments. (<b>B</b>) We tested the receptor-specificity of IDRgag targeting by incubating DCs with CD11c-scFv-IDRgag either in the presence of parental anti-CD11c mAb clone N418 or control mAb (anti-human β-gal mAb) as well as isotype control Ab (Armenian hamster IgG). Whereas anti-CD11c parental mAb was able to significantly reduce CD11c-scFv-IDRgag induced CD8⁺ activation in co-culture experiments, no decrease of CD8⁺ activation was observed using the parental control mAb or isotype control Ab. Data represent mean±SD of 5 independent experiments. Data were analyzed with unpaired <i>t</i>-test. (<b>C</b>) Maturation of DCs pulsed with either 10 µg/ml control- or CD11c-scFv-IDRgag as well as IDRgag-MAp15 was measured after 24 hours by determining the expression of co-stimulatory molecule CD86 on the cell-surface. Data represent mean±SD of 3 independent experiments. (<b>D</b>) Effect of the addition of 10 ng/ml LPS to DCs pulsed with either control- or CD11c-scFv-IDRgag on their capacity to activate FV-specific CD8⁺ T cells. Data represent mean±SD of 3 independent experiments.</p

Immunization of mice with DCs loaded with CD11c-scFv-IDRgag provides a better control of acute viremia.

<p>(<b>A</b>) When animals were immunized twice with bmDCs loaded with CD11c-scFv-IDRgag or control-scFv-IDRgag in one week interval and challenged one week after the second immunization, animals immunized with bmDCs loaded with CD11c-scFv-IDRgag were protected significantly better against FV induced splenomegaly, which correlated with a significantly lower frequency of infected cells in the spleen (<b>B</b>). Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. Significance was labeled with * if p<0.05, ** if p<0.01, *** if p<0.001. Data represents results from one experiment.</p

Improved induction of FV-specific CD8⁺ T cells by vaccination with CD11c-scFv-IDRgag treated DCs followed by FV challenge.

<p>(<b>A and B</b>) By gating on CD43/CD44 double positive effector CD8⁺ T cell population the frequency of FVgag tetramer⁺ cells was measured at 4 dpi. We detected significant induction of FV-specific CTL-response in mice injected with DCs loaded with CD11c-scFvIDRgag (5.08±0.57 percent specific FVgag tetramer⁺ CD8⁺ T cells), when compared to mice inoculated with control-scFv-IDRgag loaded DCs (2.08±0.43 percent specific FVgag tetramer⁺ CD8⁺ T cells). At 4 days post FV challenge mice injected with non-loaded DC showed nearly no detectable FV-specific CTL responses. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. Significance was labeled with * if p<0.05, ** if p<0.01, *** if p<0.001. Data represents results from one experiment. (<b>C</b>) Investigating the total number of FV-specific CD8⁺ T cells per 10⁷ CD8⁺ T cells, we found significant higher number of FV-specific CTLs in mice vaccinated with bmDC/CD11c-scFv-IDRgag (11803±1855) compared to both mice inoculated non-loaded or control-scFv-IDRgag loaded DCs (4020±599 and 5782±916, respectively). Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. Significance was labeled with * if p<0.05, ** if p<0.01, *** if p<0.001. Data represents results from one experiment.</p

Improved rejection of FBL-3 tumor in B6 mice by CD11c-scFv-IDRgag loaded DCs.

<p>(<b>A</b>) CD8 TCRtg B6 mice injected with CD11c-scFv-IDRgag-treated bmDCs showed a significant higher level of CD8⁺ T cell activation, compared to mice receiving bmDCs loaded with control-scFv-IDRgag as measured by the percentage of CD69 expressing CD8⁺ T cells in the spleen. Data represent mean±SD values from 6 mice each group collected from 2 independent experiments. Data were analyzed with unpaired <i>t</i>-test. (<b>B</b>) B6 mice received bmDCs loaded either with CD11c-scFv-IDRgag or control-scFv-IDRgag. Control mice were injected with non-loaded bmDCs. 24 hours after receiving DCs mice were injected i.v. with FV-specific TCRtg CD8⁺ T cells. FBL-3 cells were inoculated i.d. 48 h after bmDCs injection. Tumor development was monitored daily for the next 9 days by measuring tumor size. Animals injected with DCs alone rejected the tumor within 11 days (data not shown) with a maximum tumor size of 3.81±0.61 mm at day 6. A significant difference in size of the tumors was observed at day 2, 4, 5, 6 and 7 (unpaired <i>t</i>-test) between the groups of animals receiving DCs loaded either with control-scFv-IDRgag or CD11c-scFV-IDRgag with a maximum tumor size of 2.85±0.54 mm or 1.00±0.41 mm at day 5, respectively. Data represent mean±SD values from 12 mice from each group collected from 3 independent experiments.</p

Epidemiology and clinical profile of pathogens responsible for the hospitalization of children in Sousse area, Tunisia

<div><p>This study aimed to identify a broad spectrum of respiratory pathogens from hospitalized and not-preselected children with acute respiratory tract infections in the Farhat Hached University-hospital of Sousse, Tunisia. Between September 2013 and December 2014, samples from 372 children aged between 1 month and 5 years were collected, and tested using multiplex real-time RT-PCR by a commercial assay for 21 respiratory pathogens. In addition, samples were screened for the presence of <i>Streptococcus pneumoniae</i> 16S rDNA using real-time PCR. The viral distribution and its association with clinical symptoms were statistically analyzed. Viral pathogens were detected in 342 (91.93%) of the samples of which 28.76% were single positive and 63.17% had multiple infections. The most frequent detected viruses were rhinovirus (55.64%), respiratory syncytial virus A/B (33.06%), adenovirus (25.00%), coronavirus NL63, HKU1, OC43, and 229E (21.50%), and metapneumovirus A/B (16.12%). Children in the youngest age group (1–3 months) exhibited the highest frequencies of infection. Related to their frequency of detection, RSV A/B was the most associated pathogen with patient’s demographic situation and clinical manifestations (p<0.05). Parainfluenza virus 1–4 and parechovirus were found to increase the risk of death (p<0.05). Adenovirus was statistically associated to the manifestation of gastroenteritis (p = 0.004). Rhinovirus infection increases the duration of oxygen support (p = 0.042). Coronavirus group was statistically associated with the manifestation of bronchiolitis (p = 0.009) and laryngitis (p = 0.017). <i>Streptococcus pneumoniae</i> DNA was detected in 143 (38.44%) of tested samples. However, only 53 samples had a concentration of C-reactive protein from equal to higher than 20 milligrams per liter, and 6 of them were single positive for <i>Streptocuccus pneumoniae</i>. This study confirms the high incidence of respiratory viruses in children hospitalized for acute respiratory tract infections in the Sousse area, Tunisia.</p></div

Detection of membrane bound complement regulators CD59, CD55 and CD46 associated on patient-derived HCV isolates.

<p>Purified HCV particles isolated from HCV infected patients were subjected to virus capture assay using 96 well plate coated with either 5 µg anti-human CD59, anti-human CD55 or anti-human CD46<b>.</b> RNA isolated from the captured wells was analyzed using Roche T<i>aq</i>Man assay. Figures represent the individual results from 9 different patient serum samples. Significance was calculated using unpaired <i>t</i>-test.</p

Characteristics of <i>S</i>. <i>pneumoniae</i> infected subjects in relation to viral infection (negative, single and multiple) and CRP concentrations^a.

<p>Characteristics of <i>S</i>. <i>pneumoniae</i> infected subjects in relation to viral infection (negative, single and multiple) and CRP concentrations<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188325#t003fn001" target="_blank">^a</a>.</p

Impact of individual viral infection on patient’s clinical data and demographic information^a.

<p>Impact of individual viral infection on patient’s clinical data and demographic information<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188325#t006fn001" target="_blank">^a</a>.</p