Effects of alveolar macrophage depletion on liposomal vaccine protection against respiratory syncytial virus (RSV)

Abstract

Little is known about the identities and roles of antigen-presenting cells upon exposure to antigens of respiratory syncytial virus (RSV). Here, we focused on elucidating the importance of alveolar macrophages in conferring protective immunity in mice administered a liposome-encapsulated recombinant fragment of the RSV G protein. Mice were depleted of alveolar macrophages by intranasal inoculation of liposome-encapsulated dichloromethylenediphosphonic acid (DMDP). Mice depleted of alveolar macrophages prior to immunization developed reduced levels of serum RSV-neutralizing antibody and showed dramatically impaired protection against RSV challenge. The severity of interstitial inflammation was also markedly reduced in macrophage-depleted mice. In conclusion, this study demonstrates a pivotal role for alveolar macrophages during exposure to liposome-encapsulated RSV antigen in initiating both protective and histopathological responses against RSV.

Fig. 1

Leucocyte differential in bronchoalveolar lavage fluid of mice administered liposomal dichloromethylenediphosphonic acid (DMDP). Mice were killed 2 days after intranasal administration of varying doses of liposomal DMDP. From left to right, cells are macrophages, lymphocytes, neutrophils, eosinophils and unidentified. Data are represented as mean ± standard deviation and statistical analysis was performed using the Bonferroni multiple comparison test (***P < 0·001) for comparison of selected columns where n = 5 for each group.

Fig. 2

Effects of liposomal dichloromethylenediphosphonic acid (DMDP) treatment on interstitial macrophages (a) or dendritic cells (b). Mice were killed at the times indicated following intranasal administration of 24 µg/mouse of liposome-encapsulated DMDP (closed squares) or liposomes alone (closed circles). Lung tissue sections were stained with cell-specific antibodies and examined by light microscopy.

Fig. 3

Protection against respiratory syncytial virus (RSV) challenge in alveolar macrophage-depleted and non-depleted mice immunized with RSV G liposomal vaccine. (a) Lung RSV titres 4 days after RSV challenge in alveolar macrophage-depleted and non-depleted BALB/c mice using dichloromethylenediphosphonic acid (DMDP) (12 µg/mouse) followed by two intranasal immunizations at 14-day intervals with phosphate-buffered saline (PBS) or thioredoxin (Trx)-G-encapsulated liposomes. (b) RSV neutralizing antibody titres in the sera of alveolar macrophage-depleted and normal lung population in mice immunized intranasally with liposomes encapsulating Trx-G or inoculated with PBS followed by RSV challenge. The plaque reduction neutralization titre50 (PRNT₅₀) is the reciprocal dilution of sera required to neutralize 50% of RSV plaques on HEp-2 cells. Data are represented as mean ± standard deviation and the Mann–Whitney test (**P = 0·0079; *P = 0·0159) was performed for statistical analysis where n = 5 for each group.

Fig. 4

Leucocyte differential (a) and total numbers of recovered cells (B) in the bronchoalveolar lavage fluid of respiratory syncytial virus (RSV) challenged alveolar macrophage-depleted and non-depleted mice administered RSV G liposomal vaccine. Mice were administered dichloromethylenediphosphonic acid (DMDP) intranasally 2 days before two sessions of intranasal immunization occurring at 14-day intervals, followed by RSV challenge. Both alveolar macrophage-depleted and non-depleted mice were administered PBS or thioredoxin (Trx)-G-encapsulated liposomes intranasally. Mac, macrophages; Lym, lymphocytes; Neut, neutrophils; Eos, eosinophils; Unid, unidentified. Data are represented as mean ± standard deviation and selected columns were statistically analysed using the Bonferroni multiple comparisons test (***P < 0·001; **P < 0·01; *P < 0·05) where n = 5 for each group.

Fig. 5

Cellular infiltration surrounding bronchioles and vessels of alveolar macrophage-depleted and non-depleted mice immunized with respiratory syncytial virus (RSV) G liposomal vaccine, followed by RSV challenge. Alveolar macrophage-depleted and non-depleted mice were intranasally administered phosphate-buffered saline (PBS) and thioredoxin (Trx)-G-encapsulated liposomes twice at 14-day intervals and challenged with RSV. Lung sections were prepared as previously described and cellular infiltration surrounding bronchioles (a) and vessels (b) were determined using the semiquantitative scoring systems defined by Murphy et al. [26] and Connors et al. [27]. Data are represented as mean ± standard deviation and selected columns were analysed statistically using the Bonferroni multiple comparisons test (***P < 0·001; **P < 0·01; *P < 0·05).