Absence of vaccine-enhanced RSV disease and changes in pulmonary dendritic cells with adenovirus-based RSV vaccine

Abstract

The development of a vaccine against respiratory syncytial virus (RSV) has been hampered by the risk for vaccine-enhanced RSV pulmonary disease induced by immunization with formalin-inactivated RSV (FIRSV). This study focuses on the evaluation of vaccine-enhanced pulmonary disease following immunization with AdF.RGD, an integrin-targeted adenovirus vector that expresses the RSV F protein and includes an RGD (Arg-Gly-Asp) motif. Immunization of BALB/c mice with AdF.RGD, resulted in anti-RSV protective immunity and induced increased RSV-specific IFN-γ T cell responses compared to FIRSV. RSV infection 5 wk after immunization with FIRSV induced pulmonary inflammatory responses in the lung, that was not observed with AdF.RGD. Additionally, In the FIRSV-immunized mice following infection with RSV, pulmonary DC increased and Tregs decreased. This suggests that distinct responses of pulmonary DC and Tregs are a features of vaccine-enhanced RSV disease and that immunization with an RGD-modified Ad vaccine does not trigger vaccine-enhanced disease.

Figure 1

Frequency of RSV F protein-specific Th2-type CD4 and CD8 T cells and immunity against RSV in BALB/c mice following intramuscular immunization with AdF or AdF.RGD (10¹⁰ pu/mouse). A, B. Ten days following immunization, spleen CD4 and CD8 T cells were co-cultured with splenic DC alone or with DC pulsed with recombinant smt3-RSV F protein or F peptide (DC-smt3-RSV F; DC-F85-93, respectively) and RSV F-specific cytokine responses were measured after 48 h by ELISPOT assay. A. RSV-specific IL-4 production in CD4 T cells; B. F epitope-specific production in CD8 T cells. C. RSV titer in the lungs of mice challenged by intranasal administration of RSV (10⁶ cfu) 4 wk after immunization. Data represent mean ± SD of 5 animals/group from one of two independent experiments. ***, ** and * denote significance of p < 0.001, p < 0.01 and p < 0.05, respectively.

Figure 2

Protective immunity induced by AdF.RGD. BALB/c mice were immunized intramuscularly with AdNull, AdF.RGD (both at 10¹⁰ pu), formalin-inactivated RSV (FIRSV, 10⁵ pfu) or intranasally with RSV (10⁶ pfu) and neutralizing serum anti-RSV Titer was measured 4 wk following immunization. Data represent mean ± SD of 5 animals/group from one of two independent experiments.* denotes significance p < 0.05.

Figure 3

Absence of RSV-induced inflammatory responses in lung and BAL following immunization with AdF.RGD. BALB/c mice were immunized subcutaneously with AdNull, AdF.RGD (both at 10¹⁰ pu), formalin-inactivated RSV (FIRSV, 10⁵ pfu) or intranasally with RSV (10⁶ pfu). Five wk later the mice were challenged intranasally with RSV (10⁶ pfu) and lungs were harvested after 6 days. A-C. Lung histology (H+E stain): A. RSV, B. FIRSV, C. AdF.RGD. D. Quantification of cells in BAL. Data for D are shown as mean ± SEM of duplicate measurements of n = 4 mice/group and represent one of two independent experiments.

Figure 4

Absence of RSV-induced inflammatory responses in lung and BAL following immunization with AdF.RGD. BALB/c mice were immunized subcutaneously with AdNull, AdF.RGD (both at 10¹⁰ pu), formalin-inactivated RSV (FIRSV, 10⁵ pfu) or intranasally with RSV (10⁶ pfu). Five wk later the mice were challenged intranasally with RSV (10⁶ pfu) and lungs were harvested after 6 days. A-D. Cytokine levels in homogenates: A. Eotaxine levels in lung homogenate. B. IL-4 levels in lung homogenate. C. IL-13 levels in lung homogenate. D. IL-10 levels in lung homogenate Data are shown as mean ± SEM of duplicate measurements of n = 4 mice/group and represent one of two independent experiments.*** and * denote significance of p < 0.001 and p < 0.05, respectively.

Figure 5

Frequency of RSV-specific Th2-type CD4 and CD8 T cells and protection. BALB/c mice were immunized subcutaneously with AdNull (10¹⁰ pu/mouse), AdF.RGD (10¹⁰ pu/mouse) or FIRSV (10⁵ pfu) A, B. Ten days following immunization spleen CD4 and CD8 T cells were cultured with splenic DC pulsed with UV-inactivated RSV (10⁵ pu/ml) for 48 h. RSV-specific cytokine response were determined by ELISPOT assay. A. RSV-specific IL-4 production in CD4 T cells. B. RSV-specific IFN-γ production in CD8 T cells. C. RSV titer in the lungs of mice challenged by intranasal administration of RSV (10⁶ cfu) 4 wk after immunization. Data represent mean ± SD of 5 animals/group from one of two independent experiments. *** and ** denote significance of p < 0.001 and p < 0.01, respectively.

Figure 6

Lung DC and IFN-α secretion following infection with RSV in immunized mice. BALB/c mice were immunized subcutaneously with AdF.RGD (10¹⁰ pu/mouse) or FIRSV (10⁵ pfu). A-C. Five wk later the mice were challenged intranasally with RSV (10⁶ pfu) for 6 days and the lung suspensions were analyzed for cDC (CD11b⁺,CD11c^high), pDC (CD11b^low, CD11c^high, B220⁺, PDCA-1⁺) and IFN-α secretion by flow cytometry and intracellular cytokine staining. A. cDC. B. pDC. C. IFN-α secretion by pDC. Data are presented as mean ± SEM of 4 mice/group from one of two independent experiments.** and * denote significance of p < 0.01 and p < 0.05, respectively.

Figure 7

Treg cells (CD4⁺, CD25^bright, FoxP3⁺) and IL-17 level following infection with RSV in immunized mice. BALB/c mice were immunized subcutaneously with AdF.RGD (10¹⁰ pu/mouse) or FIRSV (10⁵ pfu). A, B. Five wk later the mice were challenged intranasally with RSV (10⁶ pfu) for 6 days and lung suspensions were analyzed for Treg cells by flow cytometry and IL-17 cytokine level. A. Treg cells. B. IL-17. Data are presented as mean ± SEM of 4 mice/group from one of two independent experiments.* and ** denote significance of p < 0.05 and p < 0.01, respectively.