Production and characterization of hybrid mAbs.

<p>(A) Schematic representation of the full length ASP-2 protein and of the portion fused to the αDEC205 and Iso mAbs. (B) Approximately 1 μg of each mAb or recombinant protein were separated under reduced conditions. Gel was stained with Coomassie Blue dye. The heavy (HC) and light (LC) chains of αDEC-ASP2, Iso-ASP2 or αDEC-CS (used as control) and rec. ASP2 protein are shown. (C) Western blotting using the same concentration as in (B) and the anti-ASP-2 mAb K₂2. MW, molecular weight marker in kDa.</p

Immunization with the hybrid DEC-ASP2 mAb induces IFN-γ production and CD4⁺ T cell proliferation.

<p>BALB/c mice were immunized with two doses of the hybrid mAbs or the rec. ASP2 protein as described in materials and methods. Fifteen days after the administration of the second dose, mice were euthanized and their splenocytes were plated in the presence or absence of rec. ASP2 or ovalbumin, as a control. (A) The number of IFN-γ producing cells was detected by ELISPOT and each bar represents the mean ± SD of triplicates of pooled splenocytes. Numbers of IFN-γ producing cells in the absence of any stimulus were subtracted from the stimulated samples. (B) The percentage of CD3⁺CD4⁺ T cells that proliferated after 5 days of re-stimulation with the rec. ASP2 or ovalbumin is shown. Bars represent the mean ± SD of pooled animals performed in triplicates. Percentages of proliferating cells in the absence of any stimulus were subtracted from the stimulated samples. Experiment was analyzed by one-way ANOVA followed by the post-test HSD Tukey. * refers to p<0.05. The graph is representative of 2 (A) or 3 (B) independent experiments.</p

Aminoacid sequences and localization within the ASP-2 protein of the peptide pools used in this study.

<p>Aminoacid sequences and localization within the ASP-2 protein of the peptide pools used in this study.</p

Screening of the peptide pools comprising ASP-2 amino acids 261–380.

<p>BALB/c mice were immunized with two doses of the αDEC-ASP2 or Iso-ASP2 mAbs as described in material and methods. Fifteen days after the administration of the second dose, mice were euthanized and their splenocytes were plated in the presence or absence of the three peptide pools (5 μg/mL) shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117778#pone.0117778.t001" target="_blank">Table 1</a>. The number of IFN-γ producing cells was detected by ELISPOT and the bars represent the mean ± SD of pooled animals performed in triplicates. Numbers of IFN-γ producing cells in the absence of any stimulus were subtracted from the stimulated samples. Experiment was analyzed by one-way ANOVA followed by the post-test HSD Tukey. * refers to p<0.05. The graph is representative of 2 experiments.</p

Mapping the specific T-cell epitope responsible for the response to the ASP-2 protein.

<p>BALB/c mice were immunized with two doses of the hybrid mAbs or the rec. ASP2 protein as described in materials and methods. Fifteen days after the administration of the second dose, mice were euthanized and their splenocytes were plated in the presence or absence of the peptides FESRDMGKTWTEAIGTLSGV or WVMSQPGVRLYKIFRVGALIT. (A) The number of IFN-γ producing cells was detected by ELISPOT and the bars represent the mean ± SD of pooled animals performed in triplicates. Numbers of IFN-γ producing cells in the absence of any stimulus were subtracted from the stimulated samples. (B) The percentage of CD3⁺CD4⁺ T cells that proliferated after 5 days of re-stimulation with each peptide is shown. Bars represent the mean ± SD of pooled animals performed in duplicates. Percentages of proliferating cells in the absence of any stimulus were subtracted from the stimulated samples. Experiments were analyzed by one-way ANOVA followed by the post-test HSD Tukey. * refers to p<0.05. The graphs are representative of 2 experiments.</p

CD4⁺ and CD8⁺ T cells play important roles on the protective responses induced in mice immunized with the αDEC-NS1 mAb.

<p>Mice were immunized as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002330#pntd-0002330-g003" target="_blank">figure 3</a>. One day before the challenge, mice were intravenously injected with 100 µg of αCD4 or αCD8 purified antibodies to deplete the respective T cell populations. Survival (A) and morbidity (B) were monitored daily for 21 days after the challenge. n = 8 mice/group. * Refers to p<0.05 when compared to the αDEC-NS1+αCD4 mAb, adjuvant and saline groups.</p

The fusion αDEC-NS1 and αDCIR2-NS1 mAbs bind to the DC DEC205 and DCIR2 receptors, respectively.

<p>(A) A density of 10⁵ CHO cells expressing either DEC205 or DCIR2 receptors were incubated on ice with 10, 1 or 0.1 µg/mL of αDEC-NS1 or αDCIR2-NS1 fusion antibodies. Binding was detected on 30.000 cells using an anti-mouse IgG1 antibody. (B) A density of 5×10⁶ splenocytes were incubated on ice with the same concentrations of the fusion antibodies described in (A). Gates on CD11c⁺CD8α⁺ (DEC205 rich population) or CD11c⁺CD8α⁻ (DCIR2 rich population) are shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002330#pntd.0002330.s001" target="_blank">figure S1</a>. Binding was detected on 1×10⁶ cells using an anti-mouse IgG1 antibody. One experiment representative of three is shown.</p

Induction of anti-NS1 antibody responses in mice immunized with the fusion αDEC-NS1 and αDCIR2-NS1 mAbs.

<p>(A) Immunization protocol describing the order of immunizations, bleedings and challenge. Mice were immunized i.p. with 2 doses of 5 µg of each fusion antibody in the presence of 50 µg of poly (I:C) or 10 µg of recombinant NS1 co-administered with 50 µg of poly (I:C). (B) Anti-NS1 antibody titers 4 days before (white symbols) or 14 days after (black symbols) the administration of the second dose. (C) Anti-NS1 antibody titers obtained 14 days after the second dose were detected using intact (white symbols) or boiled (black symbols) NS1 protein. Symbols represent individual mice and horizontal lines represent the mean value for each group. Fusion antibodies used for immunizations are depicted below. Data were analyzed by a one-way ANOVA followed by the post-test HSD Tukey. P-value indicators * and *** refer to p<0.05 and p<0.01, respectively, while ns = not significant.</p