Vaccination protects against clinical signs of BRSV disease.

<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. Following challenge, calves were examined daily until euthanization on PID 7, and the severity of clinical signs of diseases were scored as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100392#pone.0100392-Hgglund1" target="_blank">[12]</a>. (A) presents the mean square root of clinical scores per day (to approximate normal distribution for statistical analysis), and (B) the accumulated clinical score from PID 0 to PID 7, with standard deviations indicated by upward deflecting lines. Statistically significant differences are indicated by asterisks p≤0.05 (*); p≤0.01 (**); p≤0.005 (***); p≤0.001 (****); p≤0.0001 (*****).</p

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

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

Animals and experimental design.

<p>Animals and experimental design.</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

RSV proteins and epitopes used in subunit vaccine formulation.

a<p>Protein product included in subunit vaccine formulations, as abbreviated in the current paper.</p>b<p>HRSV Long strain (GenBank accession no. AY911262) was used to construct recombinant protein.</p>c<p>Full length HRSV M2-1 protein (Long strain).</p>d<p>Full length HRSV P protein (Long strain).</p>e<p>BRSV strain 9402022 (Larsen et al. 1998) was used to construct recombinant protein.</p>f<p>Selected residues were recombinantly attached to the N or C terminus of HRSV N protein (Long strain) and co-expresses with a fragment of HRSV P protein (Long strain, AA residues 161–241) to form N-nanorings with attached epitopes (eN) on each of 10 or 11 protomers.</p>g<p>Antigenic site II (AA residues 422–438) on F, as described by Chargelegue et al. (1998).</p

Vaccination reduces the extent of lung lesions following 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. Lungs were removed after exsanguination, lesions were recorded on a lung chart after visual examination and palpation, and the proportion of lung showing pneumonic consolidation was calculated. Formalin-fixed tissue samples from each lobe in the right lung were analyzed for the severity of histopathological changes and scored as either normal (0), mild (1), moderate (2) or severe (3). (A) shows the extent of macroscopic lesions on the y-axes, and the microscopic severity of inflammation (mean score of four sections per calf) on the x-axes. Statistically significant difference is indicated by asterisks (p≤0.05). (B) shows the percent of pneumonic consolidation in each animal (also depicted as filled areas in lung-charts), and emphysema (outlined areas in calves d3 and d4). Panels C (I–IV) show representative histological images from each of the four groups of calves. Bar indicate 100 µm. Panels C (I) (ΔSHrBRSV), C (II) (SUMont), C (III) (SUAbis) and C (IV) (Control) show lung parenchyma with minimal, mild, moderate and severe pathological changes, respectively.</p

Experiment timeline, vaccination and sampling.

<p>Twenty calves with moderate titers of BRSV-specific serum antibodies (MDA) were allocated into 4 groups and vaccinated as indicated in the figure; all were vaccinated on post-vaccination day (PVD) 0 (Vacc. I, white arrow) with either (a) 5×10⁶ pfu of ΔSHrBRSV intranasally (i.n.); (b) BRSV and HRSV recombinant protein subunits (SU) adjuvanted by Montanide (SUMont) intramuscularly (i.m.), (c) SU adjuvanted by AbISCO-300 (SUAbis) subcutaneously (s.c.), or (d) adjuvant alone s.c. (Controls). On PVD 20, all animals except those immunized with ΔSHrBRSV, were boosted with the same formulation and route as for Vacc. I (Vacc. II, gray arrow). Three BRSV-seronegative calves were housed in contact with ΔSHrBRSV-infected animals to determine transmission of the vaccine virus (Sentinel calves), and monitored until euthanized (†) on PVD 22. On PVD 20, one calf in group c was euthanized due to traumatic injury. On post-infection day (PID) 0, all calves were challenged i.n. with 10⁴ pfu virulent BRSV (black arrow), and clinically scored daily until PID 7. Throughout the experiment, samples were collected, as indicated in the figure, to analyze antibodies in serum and nasal secretions, <i>ex-vivo</i> response of peripheral blood mononuclear cells (PBMC) to restimulation with BRSV, and virus shedding in nasal secretions (Nasal swab). At post-mortem examination (PM), lung lesions were recorded and tissue samples collected, as well as bronchoalveolar lavage (BAL) samples for antibody, BRSV RT-PCR and virus isolation.</p

RSV-specific serum antibodies in calves before and after immunization and subsequent challenge with virulent BRSV.

<p>Four groups of 5 calves were vaccinated as described in Fig. 1 (white and grey arrows) and challenged with BRSV, 5 weeks after vaccination (black arrow) on post-infection day (PID) 0. Two weeks before challenge, one calf (C5) was euthanized due to traumatic injury. Panels show group mean log₁₀ serum titers of: (A) BRSV-specific IgG₁ (by ELISA); (B) IgG directed against BRSV G on PID 0 and PID 7 (by ELISA); (C) IgG directed against HRSV F on PID 0 and PID 7 (by ELISA); (D) IgG directed against HRSV N (by ELISA); (E) IgG directed against HRSV P (by ELISA); and (F) IgG directed against HRSV M2-1 (by ELISA) (G) BRSV-neutralizing antibodies (by plaque reduction assay). Note that the scale of the y-axis is not uniform between panels. Statistically significant difference on PID 7 is indicated by asterisks and the corresponding group; p≤0.05 (*); p≤0.01 (**); p≤0.005 (***); p≤0.001 (****).</p