Induction of IFN-γ and IL-2 in splenocytes from RSV F peptide immunized HLA-A*0201 transgenic mice upon secondary recall.

<p>Splenocytes harvested on day 30 from HLA-A-Tg B6 mice primed and boosted at 20 days interval intranasally with 10⁷ pfu of rAd-F0 (□) or 10⁴ pfu of RSV-B1 (▪)were restimulated with 2 µg of the individual RSV F peptide or 10 µg/ml Con A for 5 days in the presence of murine IL-2 . After stimulation, 5×10⁵ splenocytes were seeded to anti-IFN-γ (A) or anti-IL-2 (B) capture antibody coated ELISPOT plates for 2 days for ELISPOT assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025500#s2" target="_blank">Materials and Methods</a>. Cytokine-positive immunospots were developed and the results are expressed as the number of immunospots +/−2 standard deviations for each experimental group. Data is representative of results derived from two independent experiments, each with five mice per group.</p

Expression of proinflammatory chemokines in the lungs of HLA-A*0201 transgenic mice immunized with CD8 epitopes and challenged with RSV.

<p>At day 4 post RSV challenge, lung RNA was extracted from the individual mice subcutaneously immunized twice with IFA-emulsified peptides or vehicle (IFA only). RNA was subjected to quantitative expression analysis of CCL11 (A), CCL17 (B), CCL22 (C), IL-13 (D), IL-17 (E), and IL-18 (F) by real-time RT-PCR using specific primers. GAPDH was used as internal control. The results are representative of the relative expression of the target gene normalized to GAPDH expression for the individual mouse. *(p<0.05) indicates the treatment is significantly different from the vehicle-immunized control. Similar results were obtained from two independent experiments, each with six mice per group, and one of them is shown.</p

Determination of lung viral load and gain of lost body weight.

<p>Mice were immunized twice intranasally with vehicle (▪), peptide 3 (◊), peptide 13 (▴),peptide 14 (▽), or peptide 23 (□) before being intranasally challenged with 10⁷ pfu of live RSV B1. (A) The viral load in the lungs of individual mice was determined 4 days after challenge by real-time RT-PCR to quantitate RSV N gene expression as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025500#s2" target="_blank">Materials and Methods</a>. 10 mice per group were used and the results are expressed as the relative expression of N gene normalized to GAPDH gene expression for each mouse. *(p<0.05) indicates they are significantly different from the vehicle-immunized group. (B) The body weight of each mouse was recorded daily for 9 days after virus challenge. Results are expressed as % (mean) for 5 mice in each experimental group. Two independent experiments were performed and data from one is shown. P value <0.05 calculated for peptide 3, peptide 13, peptide 14, and peptide 23 indicates they are significantly different compared to vehicle-immunized control.</p

Characteristics of HLA-A*0201-restricted epitopes of peptide spanning RSV F protein^*.

<p>*Twenty-five HLA-A*0201-restricted 9-mer synthetic peptides from F protein of RSV-B1 strain were tested their binding ability with HLA-A*0201 by T2-stabilization assay, which has been described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025500#s2" target="_blank">Materials and Methods</a>. The study is a representative of results derived from two independent experiments, each with five mice per group. Results of IFN-γ and IL-2 ELISPOT assays have been described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025500#pone-0025500-g001" target="_blank">figure 1</a>.</p><p>**N/a, not assayed.</p

Induction of pulmonary IFN-γ and CTL activity in peptide immunized transgenic mice challenged with RSV.

<p>At day 4 post RSV infection, lung homogenates were prepared and the following were measured. (A) IFN-γ expression by ELISA, and (B) the number of CD8⁺ T cells in the lungs by flow cytometry using PE-cy5-labeled anti-CD8 antibody. (C). Enumeration of CTL activity in the lungs of mice immunized with CD8 peptide epitopes and challenged with RSV. Effector lymphocytes isolated from the lungs of mice immunized with peptide 3 (▪), peptide 13 (▴), peptide 14 (▾), or peptide 23 (•) were cultured and supplemented with murine IL-2 in the presence of 2 µg of the same peptide for 5 days. In parallel, cultured pulmonary lymphocytes from vehicle-immunized mice were stimulated with 2 µg of peptide 3 (□), peptide 13 (Δ), peptide 14 (▽), or peptide 23 (ο), respectively, for 4 days. DCs isolated from the tibia of HLA-B6 mice were pulsed with 20 µg per mL of the individual peptides for 2 hours at 37°C and labeled with CFSE and used as targets in the <i>in vitro</i> CTL assay. Un-pulsed DCs served as negative control. The viable effector cells were co-cultured with 10⁴ peptide-loaded target DCs cells at effector∶target ratios of 50∶1, 10∶1, and 0∶1 for five hours. The cell mixtures were labeled with 7-AAD and analyzed by flow cytometry. Results are expressed as mean percentage of 7-AAD/CFSE positive cells normalized with un-pulsed DCs. Six mice were taken in each group. The result is a representative of two independent experiments.</p

Epitope-specific CD4⁺ and CD8⁺ T-cell activation in peptide-immunized HLA-A*0201 transgenic mice.

<p>Splenocytes were isolated on day 17 from mice immunized twice subcutaneously with the peptides 3, 13, 14, 17, 23, or vehicle at day 0 and day 10. The splenocytes were labeled with CFSE and cultured in the presence or absence of 10 µg/mL of the respective peptides for 8 days. Proliferation of CD4⁺ (A) or CD8⁺ (B) lymphocytes in response to the different CD8 epitopes was analyzed by flow cytometry using anti-CD4 or CD8 antibodies conjugated with PE-Cy5. Results are presented as cell division index (CDI) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025500#s2" target="_blank">Materials and Methods</a>. (C) The splenocytes were stimulated <i>in vitro</i> with or without the peptides and were stained with anti-CD8 antibody conjugated with FITC, and then fixed and stained for intracellular IFN-γ using PE-conjugated anti-IFN-γ antibody. The percentage of CD8⁺ IFN-γ⁺ T cells was calculated. *(<i>p</i><0.05) and **(<i>p</i><0.01) indicate they are significantly different from the unstimulated splenocytes. Data is representative of results derived from three independent experiments.</p

Eosinophil infiltration into the lungs of peptide-immunized transgenic mice upon RSV challenge.

<p>Immunohistochemical analysis of lung sections with anti-major basic protein antibody specific for eosinophils followed by the HRP-conjugated anti-rat antibody was performed. (A) Pictures from the section of normal mouse lung (a), or vehicle- (b), peptide 3- (c), 13- (d), 14- (e) and 23- (f) immunized mouse lung section. (B) Quantitative representation of eosinophil count per section of peptide- or vehicle-immunized HLA-transgenic mice at day 4 post RSV challenge. Twenty bright field pictures from each lung mesenchymal region were examined and the number of eosinophils was counted under 200× magnification. The mean number of eosinophils in each group, six mice per group, is represented. Similar results from two independent experiments were obtained and one of the results is shown.</p