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. 1999 Mar 2;96(5):2367-72.
doi: 10.1073/pnas.96.5.2367.

Interferon gamma expressed by a recombinant respiratory syncytial virus attenuates virus replication in mice without compromising immunogenicity

A Bukreyev  1 , S S Whitehead, N Bukreyeva, B R Murphy, P L Collins
Affiliations

Interferon gamma expressed by a recombinant respiratory syncytial virus attenuates virus replication in mice without compromising immunogenicity

A Bukreyev et al. Proc Natl Acad Sci U S A. .

Abstract

Interferon gamma (IFN-gamma) has pleiotropic biological effects, including intrinsic antiviral activity as well as stimulation and regulation of immune responses. An infectious recombinant human respiratory syncytial virus (rRSV/mIFN-gamma) was constructed that encodes murine (m) IFN-gamma as a separate gene inserted into the G-F intergenic region. Cultured cells infected with rRSV/mIFN-gamma secreted 22 microg mIFN-gamma per 10(6) cells. The replication of rRSV/mIFN-gamma, but not that of a control chimeric rRSV containing the chloramphenicol acetyl transferase (CAT) gene as an additional gene, was 63- and 20-fold lower than that of wild-type (wt) RSV in the upper and lower respiratory tract, respectively, of mice. Thus, the attenuation of rRSV/mIFN-gamma in vivo could be attributed to the activity of mIFN-gamma and not to the presence of the additional gene per se. The mice were completely resistant to subsequent challenge with wt RSV. Despite its growth restriction, infection of mice with rRSV/mIFN-gamma induced a level of RSV-specific antibodies that, on day 56, was comparable to or greater than that induced by infection with wt RSV. Mice infected with rRSV/mIFN-gamma developed a high level of IFN-gamma mRNA and an increased amount of interleukin 12 p40 mRNA in their lungs, whereas other cytokine mRNAs tested were unchanged compared with those induced by wt RSV. Because attenuation of RSV typically is accompanied by a reduction in immunogenicity, expression of IFN-gamma by an rRSV represents a method of attenuation in which immunogenicity can be maintained rather than be reduced.

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Figures

Figure 1
Figure 1
Construction of the rRSV/mIFN-γ chimeric genome. The mIFN-γ cDNA (shaded rectangle, with initiation and termination codons shown on either side in lowercase) was modified to be flanked by RSV gene-start and gene-end signals as an XmaI fragment. This transcription cassette was inserted into an antigenome cDNA that previously had been modified by the insertion of an 8-nt XmaI linker (bold, italicized) into the unique StuI site (the two halves of the StuI site, AGG and CCT, are underlined) present in the G-F intergenic region. XmaI sites are underlined.
Figure 2
Figure 2
Growth kinetics for rRSV/mIFN-γ, rRSV/CAT, and wt RSV in HEp-2 cells. Cell monolayers were infected with 2 pfu per cell (two replicate wells per virus), and 200 μl aliquots of supernatant were taken at the indicated time, adjusted to contain 100 mM magnesium sulfate and 50 mM Hepes buffer (pH 7.5), flash-frozen, and stored at −70°C until titration. Each aliquot taken was replaced with an equal amount of fresh medium. Each single-step growth curve represents the average of the virus titers from the two infected cell monolayers. The cell monolayer infected with wt RSV was more than 90% destroyed at 72 h postinfection (∗), whereas that infected with rRSV/mIFN-γ or rRSV/CAT was almost intact, reflecting attenuation of the chimeric virus in vitro.
Figure 3
Figure 3
Kinetics of accumulation of mIFN-γ in culture fluids of HEp-2 cells infected with rRSV/mIFN-γ or rRSV/CAT. Cell monolayers were infected with 2 pfu per cell (two replicate wells per virus), samples were taken at the indicated time points, and the mIFN-γ content was determined in each sample by ELISA using the Quantikine M Mouse IFN-γ Immunoassay (R & D Systems).
Figure 4
Figure 4
Kinetics of virus replication in the upper and lower respiratory tract of BALB/c mice inoculated intranasally with 106 pfu of rRSV/mIFN-γ, rRSV/CAT, or wt RSV. Five mice from each group were sacrificed on the indicated day, the nasal turbinates and lung tissues were removed and homogenized, and the concentration of infectious virus was determined by plaque assay of individual tissue specimens. Mean log10 titer per gram tissue with SD is shown. The limit of virus detection in the upper and lower respiratory tract is indicated.
Figure 5
Figure 5
Detection of pulmonary mIFN-γ, IL-12 p40, and L-32 (housekeeping gene) mRNAs by ribonuclease protection assay. Mice (five animals per group) were inoculated intranasally with medium alone (mock) or 106 pfu per animal of rRSV/mIFN-γ or wt RSV, and on day 4 after immunization the lungs were harvested and total RNA was purified. RNA was hybridized with radioactive RNA probes synthesized using mCK-2B template set (PharMingen RiboQuant Multi-Probe RNase Protection Assay System), treated with ribonuclease A, purified, and electrophoresed in a 5% denaturing acrylamide gel. Autoradiographs are shown for the region of the gel containing protected species corresponding to the indicated mRNAs. Different exposure times were used for each of the three mRNAs. Normal mouse RNA and yeast were used as additional negative controls (not shown).
Figure 6
Figure 6
Levels of cytokine mRNAs in the lungs of mice after primary infection with rRSV/mIFN-γ or wt RSV and after challenge with wt RSV. Mice were immunized intranasally with 106 pfu per animal of rRSV/mIFN-γ, wt RSV, or medium alone. On day 1 or 4 after immunization five animals from each group were sacrificed and pulmonary RNA was isolated. Additional immunized animals were challenged on day 28 with 106 pfu of wt RSV, and total lung RNA was isolated individually from five animals from each group on day 29 or 32. The accumulation of mRNA for selected cytokines was measured by the ribonuclease protection assay described in the legend to Fig. 5 (PharMingen; template sets mCK-1 and mCK-2B). Radioactivity for each mRNA was quantified by PhosphorImager analysis, backgrounds were subtracted, and each value was calculated as a percentage of radioactivity relative to the L-32 housekeeping gene mRNA and displayed as an average of five animals with the SD. Samples that lacked detectable mRNA for the indicated cytokine are marked with asterisks.
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