Characterization of an experimental vaccine for bovine respiratory syncytial virus

Abstract

Bovine respiratory syncytial virus (BRSV) and human respiratory syncytial virus (HRSV) are major causes of respiratory disease in calves and children, respectively, and are priorities for vaccine development. We previously demonstrated that an experimental vaccine, BRSV-immunostimulating complex (ISCOM), is effective in calves with maternal antibodies. The present study focuses on the antigenic characterization of this vaccine for the design of new-generation subunit vaccines. The results of our study confirmed the presence of membrane glycoprotein (G), fusion glycoprotein (F), and nucleoprotein (N) proteins in the ISCOMs, and this knowledge was extended by the identification of matrix (M), M2-1, phosphoprotein (P), small hydrophobic protein (SH) and of cellular membrane proteins, such as the integrins αVβ1, αVβ3, and α3β1. The quantity of the major protein F was 4- to 5-fold greater than that of N (∼77 μg versus ∼17 μg/calf dose), whereas G, M, M2-1, P, and SH were likely present in smaller amounts. The polymerase (L), M2-2, nonstructural 1 (NS1), and NS2 proteins were not detected, suggesting that they are not essential for protection. Sera from the BRSV-ISCOM-immunized calves contained high titers of IgG antibody specific for F, G, N, and SH. Antibody responses against M and P were not detected; however, this does not exclude their role in protective T-cell responses. The absence of immunopathological effects of the cellular proteins, such as integrins, needs to be further confirmed, and their possible contribution to adjuvant functions requires elucidation. This work suggests that a combination of several surface and internal proteins should be included in subunit RSV vaccines and identifies absent proteins as potential candidates for differentiating infected from vaccinated animals.

FIG 1

Negative-stain transmission electron microscopy image of BRSV-ISCOMs.

FIG 2

BRSV protein content in BRSV-ISCOMs, BRSV proteins, and Vero-ISCOMs. (a) Coomassie blue-stained SDS-PAGE gel showing the bands analyzed by mass spectrometry and tandem mass spectrometry; (b) dot blots using monoclonal antibodies against BRSV proteins; (c) Western blots using monoclonal antibodies against HRSV F and BRSV G and M, rabbit polyclonal antiserum against recombinant HRSV M2-1, N, and P, and serum from an unvaccinated experimentally BRSV-infected calf. Arrows indicate BRSV proteins.

FIG 3

Reactivity of calf serum to BRSV proteins. Calves with various levels of BRSV-specific maternal antibodies were immunized twice subcutaneously with BRSV-ISCOMs containing 188 μg proteins (A), 188 μg BRSV proteins (B), 390 μg AbISCO-300 (C), or PBS (D), 33 and 12 days before blood sampling. The results are presented as a group mean of IgG antibody titers (log₁₀) obtained by ELISAs using lysates of cells infected with BRSV Snook (BRSV SNK), wild-type fowlpox vector (FPV-WT), fowlpox vectors expressing BRSV G, F, M, P, or SH protein, or purified BRSV N protein expressed by E. coli (N SRS) as antigens. The titers presented against N are of the IgG1 isotype, whereas titers against G, F, M, P, or SH represent IgG. Standard deviations are shown by upward deflection lines. The asterisks indicate statistically significant differences between groups: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.