Enhanced disease and pulmonary eosinophilia associated with formalin-inactivated respiratory syncytial virus vaccination are linked to G glycoprotein CX3C-CX3CR1 interaction and expression of substance P

Abstract

Vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine or RSV G glycoprotein results in enhanced pulmonary disease after live RSV infection. Enhanced pulmonary disease is characterized by pulmonary eosinophilia and is associated with a substantial inflammatory response. We show that the absence of the G glycoprotein or G glycoprotein CX3C motif during FI-RSV vaccination or RSV challenge of FI-RSV-vaccinated mice, or treatment with anti-substance P or anti-CX3CR1 antibodies, reduces or eliminates enhanced pulmonary disease, modifies T-cell receptor Vbeta usage, and alters CC and CXC chemokine expression. These data suggest that the G glycoprotein, and in particular the G glycoprotein CX3C motif, is key in the enhanced inflammatory response to FI-RSV vaccination, possibly through the induction of substance P.

FIG. 1.

The BAL cell numbers in the lungs of untreated FI-A2-vaccinated mice (A), anti-SP antibody (aSP)-treated or NRS-treated FI-A2-vaccinated mice (B), anti-CX3CR1 antibody (aCX3CR1)-treated or NRS-treated FI-A2-vaccinated mice (C), and anti-CX3CR1 antibody-treated or NRS-treated FI-R10C7G-vaccinated mice (D) were determined at 0, 8, 18, 24, and 40 h after A2, B1, CP52, PIV-3, or R10C7G challenge or following treatment with uninfected Vero cell lysate (VCL). The mean number of BAL cells (± standard error) from three independent experiments examining three mice per treatment is shown.

FIG. 2.

ELISA was used to determine the level of SP expression in cell-free BAL fluid from untreated FI-A2-vaccinated mice (A) or FI-A2-vaccinated mice treated with anti-SP antibody (aSP), anti-CX3CR1 antibody (aCX3CR1), or NRS (B) at 0, 8, 18, 24, and 40 h after A2, B1, CP52, PIV-3, or R10C7G challenge. The mean level of SP expression (± standard error) from three independent experiments examining three mice per treatment is shown.

FIG. 3.

The BAL cell types in the lungs of untreated FI-A2-vaccinated mice challenged with A2 (A) or B1 (B), challenged with A2 or B1 and treated with NRS (C and D, respectively), or challenged with A2 or B1 and treated with anti-SP antibody (E and F, respectively), were determined at 8, 18, 24, and 40 h after A2 or B1 challenge. The mean percentage of each BAL cell type (± standard error) was determined by H&E staining and microscopic visualization from three independent experiments examining three mice per treatment. LYM, lymphocytes; EOS, eosinophils; MAC, macrophages.

FIG. 4.

The BAL cell types in the lungs of untreated FI-A2-vaccinated mice challenged with CP52 (A), FI-A2-vaccinated mice challenged with CP52 and treated with NRS or anti-SP antibody (B and C, respectively), FI-A2-vaccinated mice challenged with A2 and treated with anti-CX3CR1 antibody (D), FI-A2-vaccinated mice challenged with R10C7G (E), or FI-A2-vaccinated mice challenged with PIV-3 (F) were determined at 8, 18, 24, and 40 h after CP52, R10C7G, or PIV-3 challenge. The mean percentage of each BAL cell type (± standard error) was determined by H&E staining and microscopic visualization from three independent experiments examining three mice per treatment. LYM, lymphocytes; EOS, eosinophils; MAC, macrophages.

FIG. 5.

The BAL cell types in the lungs of FI-R10C7G-vaccinated mice challenged with A2 (A), FI-R10C7G-vaccinated mice challenged with A2 and treated with NRS or anti-CX3CR1 antibody (B and C, respectively), FI-R10C7G-vaccinated mice challenged with R10C7G (D), FI-R10C7G-vaccinated mice challenged with R10C7G and treated with NRS (E) or FI-R10C7G-vaccinated mice challenged with R10C7G and treated with anti-CX3CR1 antibody (F) were determined at 8, 18, 24, and 40 h after A2 or R10C7G challenge. The mean percentage of each BAL cell type (± standard error) was determined by H&E staining and microscopic visualization from three independent experiments examining three mice per treatment. LYM, lymphocytes; EOS, eosinophils; MAC, macrophages.

FIG. 6.

CD4⁺ TCR Vβ (Vb) usage by BAL cells in the lung following 6340WT, 6340ΔG (6340DG), or R10C7G infection of naive mice (A) or FI-A2-vaccinated mice (B) was determined at day 7 p.i. by flow cytometry. The results represent the mean (± standard error) of CD4⁺ TCR Vβ expression in total BAL cells collected and pooled from three mice in three individual experiments.