Modulation of protective immunity, eosinophilia, and cytokine responses by selective mutagenesis of a recombinant G protein vaccine against respiratory syncytial virus

Abstract

Using an Escherichia coli-grown plasmid vector encoding a fragment of thioredoxin (Trx) fused to a central region (amino acids 128 to 229) of the respiratory syncytial virus (RSV) (Long strain) G protein, we employed site-directed mutagenesis to investigate the importance of selected amino acids to vaccine efficacy. Mice were immunized with a total of 10 wild-type or mutant Trx-G proteins and challenged intranasally with RSV. Striking differences in the induction of RSV G-protein-specific antibodies, protection against RSV challenge, cytokine RNA responses, and induction of RSV-associated eosinophilic inflammation were observed among the mutant proteins examined. Taken together, the results identify a critical role for specific amino acids in the induction of protective immunity and priming for eosinophilia against RSV.

FIG. 1.

Effects of G-protein mutations on the induction of serum antibodies which recognize authentic RSV G protein. Sera were collected from groups of seven to nine mice 14 days after the second of two subcutaneous administrations of the indicated immunogen in alum. For immunoblotting, extracts from RSV-infected HEp-2 cells (A) or Trx-E fusion protein containing amino acids 304 to 404 of the dengue virus type 2 E protein (B) were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% acrylamide), and proteins were electroblotted to polyvinylidene difluoride membranes. After overnight blocking at room temperature with 4% skim milk powder and 0.5% casein (Hammerstein grade) in TBST (0.8% NaCl, 0.1% Tween-20, 20 mM Tris [pH 7.6]), membranes were incubated for 1 h at room temperature with pooled mouse sera from the experimental groups (diluted 1:100 in TBST) and washed with TBST, followed by 1 h of incubation at room temperature with horseradish peroxidase-conjugated goat antimouse antibody (1:5,000 dilution; Amersham, Oakville, Canada), with subsequent detection using a mixture of diaminobenzidine (1 mg/ml), 0.03% NiCl₂, and 0.1% H₂O₂. Data are from one of two experiments which showed close agreement with each other. wt, wild type.

FIG. 2.

Effects of G-protein mutations on protection against RSV challenge. Groups of seven to nine mice were immunized twice subcutaneously at 14-day intervals with PBS-alum or alum-adjuvanted wild-type (wt) or mutant Trx-G128-229 protein, followed by RSV challenge. Titers of RSV in lung homogenates were determined (by plaque assay on HEp-2 cells) 4 days after RSV challenge. Results are shown as means ± standard deviation. Significant differences (P < 0.05) from results with the wild-type protein are marked with asterisks, while significant differences from results with PBS are marked with daggers. Data are from one of two experiments which showed close agreement with each other.

FIG. 3.

Effects of G-protein mutations on lung eosinophilia. Groups of seven to nine mice were immunized twice subcutaneously at 14-day intervals with PBS-alum or alum-adjuvanted wild-type (wt) or mutant Trx-G128-229 protein, followed by RSV challenge. Bronchoalveolar lavage eosinophils (as a percentage of total cells) were measured 4 days after RSV challenge. Results are shown as means ± standard deviations. Significant differences (P < 0.05) from results with the wild-type protein are marked with asterisks, while significant differences from results with PBS are marked with daggers. For reference, absolute numbers of eosinophils recovered in the bronchoalveolar lavage per mouse were as follows: for PBS, 11 ± 6; for the I185A mutant, 1,092 ± 497; for the C186A mutant, 6,158 ± 3,377; for the K187A mutant, 4,072 ± 894; for the R188A mutant, 2,880 ± 1,191; for the I189A mutant, 20,361 ± 5,065; for the P190A mutant, 7,747 ± 5,165; for the N191A mutant, 5,959 ± 1,092; for the K192A mutant, 13,110 ± 3,277; for the K193A mutant, 14,600 ± 2,185; for the wild type, 10,031 ± 2,284. Data are from one of two experiments which showed close agreement with each other.

FIG. 4.

RPA of lung RNA, illustrating relative levels of cytokine mRNA in lungs of mice assayed 4 days after RSV challenge, having been previously immunized twice subcutaneously at 14-day intervals with PBS-alum or an alum-adjuvanted, wild-type (wt) or mutant Trx-G128-229 protein. RNA was isolated from individual mouse lungs by using the RNeasy minikit (QIAGEN, Mississauga, Canada) and then pooled per group of seven to nine mice. RNA was quantitated and subjected to RPA by using a transcription kit (BD-Pharmingen, Mississauga, Canada) to synthesize probe from a cytokine (MCK-1) template (BD-Pharmingen) and radiolabeled by using [α-³²P]UTP, followed by hybridization and RNase digestion, using an RPAIII kit (Ambion, Austin, Tex.). Reaction mixtures were resolved on a 5% polyacrylamide 8 M urea gel according to the manufacturer's instructions, followed by drying and autoradiography at −70°C, using an intensifying screen. Controls include yeast RNA, (similarly subjected to the entire RPA procedure), as well as ³²P-labeled cytokine probe (not subjected to RNase digestion). Panels A and B show different regions of the polyacrylamide gel, which was autoradiographed for 3 days (A) or 1 h (B). Panel C shows relative levels of selected cytokines, IL-4, IL-5. IL-10, IL-13 and IFN-γ (normalized with respect to L-32 and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) obtained by densitometric analysis of the autoradiograms. For each cytokine, relative levels are normalized to 100 for the wild type Trx-G128-229 protein.