Role of alpha/beta interferons in the attenuation and immunogenicity of recombinant bovine respiratory syncytial viruses lacking NS proteins

Abstract

Alpha/beta interferons (IFN-alpha/beta) are not only a powerful first line of defense against pathogens but also have potent immunomodulatory activities. Many viruses have developed mechanisms of subverting the IFN system to enhance their virulence. Previous studies have demonstrated that the nonstructural (NS) genes of bovine respiratory syncytial virus (BRSV) counteract the antiviral effects of IFN-alpha/beta. Here we demonstrate that, in contrast to wild-type BRSVs, recombinant BRSVs (rBRSVs) lacking the NS proteins, and those lacking NS2 in particular, are strong inducers of IFN-alpha/beta in bovine nasal fibroblasts and bronchoalveolar macrophages. Furthermore, whereas the NS deletion mutants replicated to wild-type rBRSV levels in cells lacking a functional IFN-alpha/beta system, their replication was severely attenuated in IFN-competent cells and in young calves. These results suggest that the NS proteins block the induction of IFN-alpha/beta gene expression and thereby increase the virulence of BRSV. Despite their poor replication in the respiratory tract of young calves, prior infection with virus lacking either the NS1 or the NS2 protein induced serum antibodies and protection against challenge with virulent BRSV. The greater level of protection induced by the NS2, than by the NS1, deletion mutant, was associated with higher BRSV-specific antibody titers and greater priming of BRSV-specific, IFN-gamma-producing CD4(+) T cells. Since there were no detectable differences in the ability of these mutants to replicate in the bovine respiratory tract, the greater immunogenicity of the NS2 deletion mutant may be associated with the greater ability of this virus to induce IFN-alpha/beta.

FIG. 1.

Replication of NS deletion mutants of BRSV in simian Vero cells and in low-passage-number (5P) bovine NT cells. Vero or NT cells were infected at an MOI of 0.01 with WT rBRSV, BRSV Snook (Snk), rBRSVΔNS1, rBRSVΔNS2, or rBRSVΔNS1/2, and levels of infectious virus produced each day were determined as described in Materials and Methods. Values for Vero cells were obtained from two independent experiments, each performed in duplicate, and for NT cells from one experiment in duplicate.

FIG. 2.

NS deletion mutants of BRSV induce more IFN-α/β (Type I IFN) than do WT viruses in bovine NT cells. NT cells at passes 3 to 14 were either mock infected or infected at an MOI of 0.1 with rBRSV (wt); the Snook (Snk), 391-2, or 127 strain of BRSV; or rBRSVΔNS1 (ΔNS1), rBRSVΔNS2 (ΔNS2), or rBRSVΔNS1/2 (ΔNS1/2) in triplicate. At various times after infection, levels of IFN-α/β, analyzed by using the MxA-CAT bioassay (A, C, and E), and infectious virus titers (B, D, and F) were determined as described in Materials and Methods. As controls, viruses were either UV inactivated (not shown) or were neutralized with MAb B4. Induction of IFN-α/β (C) and virus titers (D) at 24 h after infection and induction of IFN-α/β (E) and virus titers (F) at 48 h after infection are shown. Figure shows representative data from six (A and B), three (C and D), or two (E and F) independent experiments that gave similar results.

FIG. 3.

NS deletion mutants of BRSV induce more IFN-α/β (Type I IFN) than do WT viruses in BAM. Freshly isolated BAM from a 6-week-old calf were either mock infected or infected at an MOI of 0.1 with rBRSV (WT); the Snook (Snk), 391-2, or 127 strain of BRSV; or rBRSVΔNS1 (ΔNS1), rBRSVΔNS2 (ΔNS2), or rBRSVΔNS1/2 (ΔNS1/2) in triplicate. As controls, viruses were also UV inactivated (not shown). Levels of IFN-α/β (A), analyzed in the MxA-CAT bioassay, and infectious virus titers (B) were determined as described in Materials and Methods at 24 h (open bars) and 48 h (solid bars) after infection. (C) Growth of different strains of WT BRSV in BAM. The figure shows representative data from one of three independent experiments that gave similar results.

FIG. 4.

Induction of IFN-α/β by WT rBRSV and rBRSVΔNS2 in BAM from animals of different ages. Freshly isolated BAM were obtained from cattle, aged from 6 weeks to 7 years, were infected at an MOI of 0.1 with WT rBRSV or rBRSVΔNS2 and were cultured in Teflon pots in triplicate. Levels of IFN-α/β were analyzed by using the MxA-CAT bioassay, as described in Materials and Methods, 48 h after infection.

FIG. 5.

Induction of IFN-α and -β mRNA by NS deletion mutants of BRSV in bovine NT cells and BAM. Total RNA was extracted from NT cells or BAM that were either mock infected or infected with rBRSV (WT), rBRSVΔNS1 (ΔNS1), rBRSVΔNS2 (ΔNS2), rBRSVΔNS1/2 (ΔNS1/2), or the Snook (Snk) strain of BRSV, treated with DNase I, reverse transcribed, and amplified as described in Materials and Methods. Quantification of IFN-α/β mRNA and 28S rRNA was based on changes in fluorescence detected by the ABI PRISM 7700 Sequence Detection system, as described in Materials and Methods. Results are expressed as the number of copies of IFN mRNA/10⁶ copies of 28S rRNA. (A) Effect of virus neutralization with MAb B4 on levels of IFN-α and -β mRNA in NT cells, used for Fig. 2C and D, 24 h after infection. (B) Levels of IFN-α and -β mRNA in BAM, used for Fig. 3A and B, 24 and 48 h after infection. The mean number of copies for IFN-α or -β mRNA for BAM exposed to UV-inactivated virus for 24 or 48 h was 0.04569 ± 0.1003. The figure shows representative data from one of two independent experiments that gave similar results.

FIG. 6.

Inhibition of rBRSVΔNS1/2-mediated IFN-α/β production by WT rBRSV or rBRSVΔNS1 in bovine NT cells. Triplicate cultures of NT cells were infected with rBRSVΔNS1/2 at an MOI of 0.05 and 4 h later with rBRSV (WT), rBRSΔNS1 (ΔNS1), or rBRVΔNS2 (ΔNS2) at an MOI of 0.5. Levels of IFN-α/β were analyzed by using the MxA-CAT bioassay, as described in Materials and Methods. Results are expressed as the percent inhibition of IFN-α/β in rBRSVΔNS1/2-infected cells superinfected with the WT or ΔNS1 or ΔNS2 mutant 48 h after infection. The figure shows representative data from one of two independent experiments that gave similar results.

FIG. 7.

BRSV-specific antibody and CD4⁺-T-cell responses induced by mucosal immunization with rBRSV in calves. Gnotobiotic calves were inoculated i.n. and i.t. with approximately 4 × 10⁶ PFU of rBRSV (WT) (n = 2), rBRSVΔNS1 (ΔNS1) (n = 3), rBRSVΔNS2 (ΔNS2) (n = 3), or noninfected Vero cell lysate (mock) (n = 3). Calves were challenged 6 weeks later with 5 × 10³ PFU of the Snook strain of BRSV in BAL. BRSV-specific IgG1 (A) and IgG2 (B) serum antibody responses were determined by ELISA. (C) BRSV-specific IgG1 and IgA antibody titers were determined by ELISA in BAL from immunized calves 6 days after challenge with BRSV Snook. (D) Number of BRSV-specific, IFN-γ⁺-secreting CD4⁺ T cells, determined by ELISPOT assay, in peripheral blood 6 weeks after immunization. sfc, spot-forming cells.

FIG. 8.

Immunization with rBRSV protects against the development of gross pneumonic lesions following challenge with the Snook strain of BRSV. Given is the percentage of lung showing macroscopic lung lesions 6 days after challenge with the Snook strain of BRSV in BAL in calves immunized by i.n. and i.t. inoculation with approximately 4 × 10⁶ PFU of rBRSV (wt), rBRSVΔNS1 (ΔNS1), rBRSVΔNS2 (ΔNS2), or noninfected Vero cell lysate (Mock).

FIG. 9.

Effect of immunization with NS deletion mutants on induction of IFNs-α/β in the respiratory tract following challenge with BRSV Snook. Gnotobiotic calves were inoculated i.n. and i.t. with approximately 4 × 10⁶ PFU of rBRSVΔNS1 (ΔNS1) (n = 3), rBRSVΔNS2 (ΔNS2) (n = 3), or noninfected Vero cell lysate (Mock) (n = 3). Calves were challenged 6 weeks later with 5 × 10³ PFU of the Snook strain of BRSV in BAL. Levels of IFN-α/β analyzed by using the MxA-CAT bioassay as described in Materials and Methods were determined in supernatants from nasopharyngeal swabs obtained after challenge with BRSV Snook (A) and in BAL (B) obtained 1 day prior to (D-1) or 6 days (D6) after challenge.