Vaccination of calves using the BRSV nucleocapsid protein in a DNA prime-protein boost strategy stimulates cell-mediated immunity and protects the lungs against BRSV replication and pathology

Abstract

Respiratory syncytial virus (RSV) is a major cause of respiratory disease in both cattle and young children. Despite the development of vaccines against bovine (B)RSV, incomplete protection and exacerbation of subsequent RSV disease have occurred. In order to circumvent these problems, calves were vaccinated with the nucleocapsid protein, known to be a major target of CD8(+) T cells in cattle. This was performed according to a DNA prime-protein boost strategy. The results showed that DNA vaccination primed a specific T-cell-mediated response, as indicated by both a lymphoproliferative response and IFN-gamma production. These responses were enhanced after protein boost. After challenge, mock-vaccinated calves displayed gross pneumonic lesions and viral replication in the lungs. In contrast, calves vaccinated by successive administrations of plasmid DNA and protein exhibited protection against the development of pneumonic lesions and the viral replication in the BAL fluids and the lungs. The protection correlated to the cell-mediated immunity and not to the antibody response.

Fig. 1

Polyacrylamide gel electrophoresis of the N protein, purified from recombinant baculovirus infected Sf9 insect cells (lane 2) and a molecular weight marker (Sigma, lane 1). The proteins were stained with Coomassie blue.

Fig. 2

In vitro BRSV-specific lymphoproliferation following vaccination and challenge. Blood samples were tested at the time of each vaccination (day 0, 29 and 60), on the day of challenge (week 80) and on days 6 and 12 after challenge (days 86 and 92). The results are expressed as stimulation index. The bars represent the mean value for each group and the vertical bars represent the standard deviation of the means. The horizontal dashed bar represents the cut-off value. The stars indicate the means that differ significantly from the mean of the mock-vaccinated group (p < 0.05).

Fig. 3

In vitro IFN-γ production after BRSV antigenic stimulation. Blood samples were tested before at the time of each vaccination (days 0, 29 and 60), at challenge (day 80) and at 6 and 12 days after challenge (days 86 and 92). The results are expressed as stimulation index. The bars represent the mean value for each group and the vertical bars represent the standard deviation of the means. The horizontal dashed bar represents the cut-off value. The stars indicate the means that differ significantly from the mean of the mock-vaccinated group.

Fig. 4

BRSV-specific antibody response. Sera taken at the time of each vaccination (days 0, 29 and 60), on the day of challenge (day 80) and on days 6 and 12 after challenge (days 86 and 92) were analyzed by indirect immunofluorescence. The results are expressed as the log 3 of the last positive dilution and they are presented as the mean value for each group. The vertical bars represent the standard deviation of the means.

Fig. 5

Total RNA was extracted from BAL fluids collected 3 days before and 3, 5, 7 and 11 days after challenge. The viral load was examined by quantitative real-time RT-PCR and expressed as the number of BRSV RNA copies per 10³ ACTB RNA copies. The bars represent the mean value for each group and the vertical bars represent the standard deviation of the means. The stars indicate the means that differ significantly from the mean of the control group.

Fig. 6

Pulmonary histopathology after BRSV challenge in the pNsyn–Npur vaccinated group (a, c and e) and in the other groups: mock-vaccinated group (b and d), pNsyn-vaccinated group (f). (b) Lymphoid peribronchial cuffing (arrows). (d) Epithelial hyperplasia and metaplasia (arrows) with desquamation of the cells in the lumen (arrowhead). (f) Consolidation of the alveoli with infiltration of mononuclear inflammatory cells. (a) Absence of lymphoid peribronchial cuffing. (c) Absence of epithelial proliferation. (e) Absence of infiltration of mononuclear inflammatory cells.