Detection of citrullinated histone 3 and myeloperoxidase in serially diluted BAL.

<p>Detection of citrullinated histone 3 (a) and myeloperoxidase (b) by dotblot in bronchoalveolar lavage (BAL) from BRSV-infected calves with clinical signs of disease and high levels of virus shedding (k-o and D1-D5) compared to that in BRSV-ISCOM-vaccinated, BRSV-infected calves with no or little clinical signs of disease and no or low levels of virus shedding (a-e, Experiment I), or non-vaccinated, non-infected calves (E1-E5, Experiment II). The proteins were selected based on being present in neutrophil extracellular traps, incriminated to be important in the pathogenesis of RSV [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186594#pone.0186594.ref030" target="_blank">30</a>].</p

Animals and experimental design.

<p>Animals and experimental design.</p

Scores of neutrophilic infiltration of alveolar septa in BRSV-infected calves.

<p>HE stained sections. a) score 0 (vaccinated calf a, experiment I), b) score 1 (vaccinated calf b, experiment I), c) score 2 (non-vaccinated, BRSV-infected calf l, experiment I) and d) score 3 (non-vaccinated, BRSV infected calf D5, experiment II). Lesions are examples of neutrophil scores and not representative for the overall inflammation regarding exudate and consolidation.</p

Correlations (Spearman’s rho) between relative expression level of selected proteins in bronchoalveolar lavage from calves six days post BRSV infection, BRSV-induced disease and presence of neutrophils in alveolar septa.

<p>Correlations (Spearman’s rho) between relative expression level of selected proteins in bronchoalveolar lavage from calves six days post BRSV infection, BRSV-induced disease and presence of neutrophils in alveolar septa.</p

Bronchoalveolar proteomes of calves.

<p>Proteins were identified by LC-MS/MS in bronchoalveolar lavage of calves and were classified as detected at group level if identified in at least 4 out of 5 calves per group. The groups consisted of non-vaccinated, BRSV-infected calves with clinical signs of disease and high levels of virus shedding (a and b, right circles), or ISCOM-vaccinated, BRSV-infected calves with no or little clinical signs of disease and no or low levels of virus shedding (a, experiment I, left circle), or non-vaccinated, non-infected calves (b, experiment II, left circle). Protein names are provided as supplemental data (*) or in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186594#pone.0186594.t001" target="_blank">Table 1</a> (**).</p

Semi-quantification of selected proteins involved in processes that were affected by BRSV-infection.

<p>Proteins were detected in bronchoalveolar lavage from non-vaccinated, BRSV-infected calves with clinical signs of disease and high levels of virus shedding (black bars) and/or in BRSV-ISCOM-vaccinated, BRSV-infected calves with no or little clinical signs of disease and no or low levels of virus shedding (a, experiment I, grey bars), and/or non-vaccinated, non-infected calves (b, experiment II, grey bars). Proteins were identified by LC-MS/MS and semi-quantified by label-free analysis. Statistically significant differences are indicated by asterisks; p≤0.05(*); p≤0.01 (**); p≤0.001 (***). Proteins were selected based on being related to neutrophil activation and chemotaxis or detoxification of reactive oxygen species: biological processes identified by protein pathway analysis.</p

Proteins associated with BRSV-induced disease.

<p>Proteins associated with BRSV-induced disease.</p

Mucosal IgA antibodies in the upper and lower airways, before and after BRSV challenge.

<p>Four groups of 5 calves were vaccinated as described in Fig. 1 and challenged with BRSV, 5 weeks after vaccination. Two weeks before challenge, one calf (c5) was euthanized due to traumatic injury. BRSV-specific IgA antibodies were analyzed by ELISA. (A) shows group mean levels of BRSV-specific IgA in nasal secretions on post-infection day (PID) 0 and 7, whereas (B) and (C) show group mean titers of total BRSV- and HRSV-N-specific IgA in bronchoalveolar lavage (BAL) on PID 7, respectively. BAL samples were titrated, whereas antibody levels in nasal secretions were semi-quantitatively determined and expressed as a percentage of a positive control sample, due to lack of sample material. Standard deviations are indicated by upward deflecting lines. Statistically significant differences between PID 0 and PID 7 in panel A are indicated by a horizontal line, whereas in all panels significant differences between groups for the same time-point are indicated by asterisks and the corresponding group letter; p≤0.05 (*); p≤0.01 (**); p≤0.005 (***); p≤0.001 (****).</p

BRSV-specific lymphocyte proliferative response in vaccinated calves.

<p>Four groups of 5 calves were vaccinated as described in Fig. 1 and challenged with BRSV, 5 weeks after vaccination, on post-infection day (PID) 0. Two weeks before challenge, one calf (c5) was euthanized due to traumatic injury. Peripheral blood mononuclear cells (PBMC) were purified from blood two weeks after first and second vaccination, as indicated in Fig. 1, and stimulated <i>ex-vivo</i> with either BRSV-infected or uninfected cell lysate. (A) Corrected optical density (COD) of Alamar Blue (Invitrogen, Sweden), indicating proliferative response after seven days of incubation. (B) IFNγ and IL-4 in supernatant from PBMC restimulated with BRSV-infected cell lysate, expressed as group means (ng/ml). Standard deviations are indicated by upward deflecting lines. Statistically significant differences are indicated by asterisks and the corresponding group; p≤0.05 (*); p≤0.01 (**); p≤0.001 (****).</p

Vaccination reduces virus load in upper and lower airways following virulent BRSV challenge.

<p>Four groups of 5 calves were vaccinated as described in Fig. 1 and challenged with BRSV, 5 weeks after vaccination, on post-infection day (PID) 0. Two weeks before challenge, one calf (c5) was euthanized due to traumatic injury. The figure presents mean viral load in nasal swabs collected from PID 0 to PID 7 in panel A and post-mortem bronchoalveolar lavage (BAL) in panel B, as determined by BRSV F-gene RT-PCR after total RNA extraction, and is expressed as TCID₅₀ equivalent, calculated from standard dilution series of virus with a known TCID₅₀. The area under mean curves in panel A represents the accumulated detected virus shed (AVS): calves immunized with either ΔSHrBRSV or SUMont had significantly lower AVS (1.4±2.2 eqTCID₅₀, p≤0.005 and 3.6±2.6 eqTCID₅₀, p≤0.05 respectively), compared to calves immunized with either SUAbis (7.9±3.4 eqTCID₅₀) or adjuvant alone (11.0±2.2 eqTCID₅₀). Statistically significant difference with Student’s <i>t</i>-test are indicated by asterisks and the corresponding groups; p≤0.05 (*); p≤0.01 (**); p≤0.005 (***); p≤0.001 (****).</p