Respiratory syncytial virus (RSV) G glycoprotein is not necessary for vaccine-enhanced disease induced by immunization with formalin-inactivated RSV

Abstract

Following respiratory syncytial virus (RSV) challenge, mice immunized with RSV G or with formalin-inactivated RSV (FI-RSV) exhibit severe disease associated with type 2 cytokine production and pulmonary eosinophilia. This has led to the proposal that the presence of RSV G is the factor in FI-RSV that induces disease-enhancing T-cell responses. Therefore, we evaluated the role of RSV G and its immunodominant region in the induction of aberrant immune responses during FI-RSV immunization. BALB/c mice were immunized with FI preparations of wild-type (wt) RSV or recombinant RSV (rRSV) containing deletions of (i) the entire G gene, (ii) the region of the G gene encoding amino acids 187 to 197 of the immunodominant region, or (iii) the entire SH gene. After challenge, illness, RSV titers, cytokine levels, and pulmonary eosinophilia were measured. Peak RSV titers postchallenge were significantly greater in mice immunized with FI preparations of the deletion viruses than in those immunized with FI-rRSV wt, suggesting that the absence of G or SH in FI-RSV reduced its protective efficacy. Deletion of G or its epitope did not reduce illness, cytokine production, or eosinophilia relative to that in mice immunized with FI-rRSV wt. While cytokine levels and eosinophilia were similar, illness was reduced in mice immunized with SH-deleted FI-RSV. These data suggest that G-specific immune responses may be important for vaccine-induced protection and are not solely the basis for FI-RSV vaccine-enhanced illness. These data suggest that the method of RSV antigen delivery, rather than the protein composition, influences the phenotype of the induced immune responses and that RSV G should not necessarily be excluded from potential vaccine strategies.

FIG. 1.

Weight loss in FI-rRSV-immunized mice following challenge with live RSV. Mice were immunized with FI preparations of rRSV and challenged with live RSV 6 weeks later. FI-Vero, consisting of formalin-treated supernatant from mock-infected Vero cells, was used as a negative control. Following RSV challenge mice were weighed daily, and weights were normalized to the base weight at day 0. n = 10 at days 0 to 7 and 5 at days 8 to 10. *, value for FI-rRSV ΔSH-immunized mice statistically less than that for FI-rRSV wt-immunized mice, P < 0.05; **, values for all FI-rRSV-immunized groups statistically greater than that for FI-Vero-immunized mice, P < 0.05; ***, value for FI-rRSV ΔG-immunized mice statistically less than that for FI-rRSV wt-immunized mice, P < 0.05.

FIG. 2.

RSV titers in FI-rRSV-immunized mice following challenge with live RSV. Five mice from each group of FI-rRSV-immunized mice were euthanized on day 4 and on day 7 postchallenge. Viral titers in the lung tissue were measured by plaque assay on subconfluent HEp-2 monolayers. Data represent the means ± standard deviations of the log₁₀ PFU per gram of lung tissue. *, value for FI-Vero-immunized mice statistically greater than those for all FI-rRSV-immunized groups at day 4, P < 0.05; **, value statistically greater than that for FI-rRSV wt-immunized mice at day 4, P < 0.05.

FIG. 3.

BAL eosinophilia in FI-rRSV-immunized mice following challenge with live RSV. Seven days after RSV challenge FI-rRSV-immunized mice were euthanized, and BAL was performed. Cytospins were prepared with the BAL cells and differentially stained with HemaStain. Data are represented as the means ± standard deviations of the percentages of eosinophils present in at least 300 cells counted. n = 5 mice per group. All FI-rRSV-immunized mice had statistically greater percentages of eosinophils than did FI-Vero-immunized controls (P < 0.05).

FIG. 4.

Histopathology in FI-rRSV-immunized mice following challenge with live RSV. Seven days after RSV challenge mice were euthanized, and the left lungs were removed and fixed in formalin. Thin sections of paraffin-embedded tissue were cut and stained with hematoxylin and eosin or with Giemsa stain. The degree of inflammation was evaluated in hematoxylin-and-eosin-stained tissue at a ×16 magnification (left panels). The extent of eosinophilia was evaluated in Giemsa-stained tissue at a ×100 magnification (right panels). A representative section (of five per group) is shown at each magnification. Full-size photos of the Giemsa-stained sections with eosinophils indicated can be viewed at http://www.vrc.nih.gov/vrc/johnson.htm.