Priming with a secreted form of the fusion protein of respiratory syncytial virus (RSV) promotes interleukin-4 (IL-4) and IL-5 production but not pulmonary eosinophilia following RSV challenge

Abstract

The attachment (G) protein of respiratory syncytial virus (RSV) is synthesized as two mature forms: a membrane-anchored form and a smaller secreted form. BALB/c mice scarified with vaccinia virus (VV) expressing the secreted form develop a greater pulmonary eosinophilic influx following RSV challenge than do mice scarified with VV expressing the membrane-anchored form. To determine if a soluble form of an RSV protein was sufficient to induce eosinophilia following RSV challenge, a cDNA that encoded a secreted form of the fusion (F) protein of RSV was constructed and expressed in VV (VV-Ftm(-)). Splenocytes and lung lymphocytes from mice primed with VV-Ftm(-) produced significantly more of the Th2 cytokines interleukin-4 (IL-4) and IL-5 than did mice vaccinated with VV expressing either the native (membrane-anchored) form of the F protein or the G protein. Although mice scarified with VV-Ftm(-) developed a slight increase in the number of pulmonary eosinophils following RSV infection, the increase was not as great as that seen in VV-G-primed mice. Despite the increased IL-4 and IL-5 production and in contrast to mice primed with VV-G, mice primed with VV-Ftm(-) developed RSV-specific cytotoxic T lymphocytes (CTL) and maintained high levels of gamma interferon production. These data demonstrate that recombinant VV strains expressing soluble forms of RSV proteins induce immune responses that are more Th2-like. However, this change alone does not appear sufficient to induce vaccine-augmented disease in the face of active CD8(+) CTL populations.

FIG. 1

Construction of rVV strains and expression of F proteins. (A) Scheme of F protein insertion. The locations of hydrophobic regions (black rectangles), cleavage site for the generation of F2 and F1 subunits (↓), potential sites for N glycosylations (▴), and cysteine residues (●) are all denoted. (B) Expression of F proteins encoded by rVV strains. F proteins expressed by rVV were immunoprecipitated and analyzed by SDS-PAGE as described in Materials and Methods. The position of the F1 subunit is indicated on the left. Numbers in the middle refer to molecular weight markers (in thousands). The F2 subunit is not detected under these conditions. Note that the F1 subunit of the Ftm⁻ mutant has a higher mobility than wild-type F, in agreement with its smaller size. Lanes: 1, cells infected with VV-F; 2, cells infected with control vRB12; 3, cells infected with VV-Ftm⁻.

FIG. 2

Immunofluorescence of rVV-infected cells. HEp-2 cells were infected with rVV expressing either wild-type F (a and c) or soluble F (b and d) proteins. At 24 h later, the cells were fixed with methanol-acetone (a and b) or formaldehyde (c and d) and stained by indirect immunofluorescence with a pool of anti-F MAbs.

FIG. 3

Kinetics of RSV clearance from the lungs of mice vaccinated with VV-F, VV-Fsig⁻, or VV-βgal. To determine if i.n. vaccination with VV-Fsig⁻ induced better immunity than that induced by i.p. vaccination, mice were vaccinated with 2 × 10⁵ PFU of rVV i.n. (a) or 2 × 10⁶ PFU of rVV i.p. (b) with VV-F (⧫), VV-Fsig⁻ (■), or VV-βgal (▴). The mice were challenged i.n. with RSV 3 weeks later. RSV titers in the lungs of the mice were assayed on days 4 to 8 after challenge.

FIG. 4

Effect of the site of F protein expression on neutrophil and eosinophil recruitment into the lungs of mice 5 days after RSV challenge. Cytospin preparations and differential cell counts of the cell population in BAL fluid were made. The percentage of each cell type was converted into cells per milliliter based on the total cell count in each BAL sample. Significant differences in the numbers of eosinophils between the different groups were observed (see the text).

FIG. 5

Effect of the subcellular site of expression of the F protein on priming for RSV-specific CTL. Splenocytes from mice immunized by dermal scarification with VV-F, VV-Ftm⁻, VV-Fsig⁻, VV-G, or VV-βgal were stimulated in vitro with RSV-infected naive splenocytes for 5 days. The RSV-specific cytolytic activity of these cultures was assessed by a standard Cr⁵¹ release assay with labelled BCH4 cells or uninfected BALB/c fibroblasts at different effector-cell-to-target-cell (E:T) ratios.

FIG. 6

Cytokine production by spleen cells. Mice were vaccinated by scarification and challenged i.n. with RSV 3 to 4 weeks later. At 5 days after challenge, lymphocytes were isolated from the spleen and 1.5 × 10⁷ cells were stimulated in vitro with RSV. Supernatants were harvested daily (days 1 to 4) and assayed for cytokines by capture ELISA. Data from one representative experiment of two are shown as the mean and standard deviation of triplicate samples from cultures of RSV-restimulated splenocytes.