Virally delivered cytokines alter the immune response to future lung infections

Abstract

Respiratory syncytial virus (RSV) is an important cause of infant morbidity and mortality worldwide and is increasingly recognized to have a role in the development and exacerbation of chronic lung diseases. There is no effective vaccine, and we reasoned that it might be possible to skew the immune system towards beneficial nonpathogenic responses by selectively priming protective T-cell subsets. We therefore tested recombinant RSV (rRSV) candidates expressing prototypic murine Th1 (gamma interferon [IFN-gamma]) or Th2 (interleukin-4 [IL-4]) cytokines, with detailed monitoring of responses to subsequent infections with RSV or (as a control) influenza A virus. Although priming with either recombinant vector reduced viral load during RSV challenge, enhanced weight loss and enhanced pulmonary influx of RSV-specific CD8+ T cells were observed after challenge in mice primed with rRSV/IFN-gamma. By contrast, rRSV/IL-4-primed mice were protected against weight loss during secondary challenge but showed airway eosinophilia. When rRSV/IL-4-primed mice were challenged with influenza virus, weight loss was attenuated but was again accompanied by marked airway eosinophilia. Thus, immunization directed toward enhancement of Th1 responses reduces viral load but is not necessarily protective against disease. Counter to expectation, Th2-biased responses were more beneficial but also influenced the pathological effects of heterologous viral challenge.

FIG. 1.

Primary infection of mice with recombinant RSV expressing murine cytokines. BALB/c mice (4 weeks old) were infected i.n. with wt RSV, rRSV/IFN-γ, or rRSV/IL-4. (A) Viral titer during primary infection, measured by TaqMan quantification of the RSV L gene. (B and C) Bronchoalveolar (airway) lavage cytokine levels after infection measured by ELISA for IFN-γ (B) and IL-4 (C). Symbols represent means ± SEM of values obtained with mice (n ≥ 4). *, P < 0.05 between outlier group and other group values.

FIG. 2.

Effects of primary infection with cytokine-expressing virus on secondary RSV challenge. Mice infected i.n. with wt RSV, rRSV/IFN-γ, or rRSV/IL-4 were challenged i.n. with wt RSV 4 weeks later. (A) Weight loss following second challenge (2°). (B and C) Airway cell numbers 4 days (B) and 7 days (C) after challenge. (D and E) Percent airway lymphocyte (D) and eosinophil (E) cell types 7 days after challenge. Symbols and bars represent means ± SEM of values obtained with mice (n ≥ 4). *, P < 0.05 between outlier group and other group values.

FIG. 3.

Lymphocyte subsets in lungs after RSV rechallenge. Mice infected i.n. with wt RSV, rRSV/IFN-γ, or rRSV/IL-4 were challenged i.n. with wt RSV 4 weeks later (2°). Lung cells were analyzed by flow cytometry using CD8⁺ T cells (A), CD4⁺ T cells (B), and NK cells (C). Symbols represent means ± SEM of values obtained with mice (n ≥ 4). *, P < 0.05.

FIG. 4.

rRSV/IFN-γ-primed mice show increased levels of RSV-specific IFN-γ-secreting CD8⁺ cells in lungs. Mice infected i.n. with RSV, rRSV/IFN-γ, or rRSV/IL-4 were challenged i.n. with wt RSV 4 weeks later. Lung cells from mice obtained at day 7 after challenge were stimulated ex vivo with RSV M2 peptide. (A) Sample fluorescence-activated cell sorter plots for individual mice. (B) Pooled data. Symbols represent individual mice; lines represent means ± SEM of values obtained with mice (n ≥ 4). *, P < 0.05.

FIG. 5.

Priming with rRSV/IL-4 alters the subtype of RSV-specific serum antibody. RSV-specific antibody levels were measured by ELISA in sera of mice infected i.n. with wt RSV, rRSV/IFN-γ, or rRSV/IL-4. (A and B) Primary infection. Total values for anti-RSV Ig (A) and IgG subtypes IgG2a and IgG1 (B) measured at 15 days postinfection are shown. (C and D) RSV rechallenge. Total values for anti-RSV Ig (C) and IgG subtypes IgG2a and IgG1 (D) 15 days after RSV rechallenge are shown. Symbols and bars represent means ± SEM of values obtained with mice (n ≥ 4). *, P < 0.05. A₄₉₀ values were obtained at 1:400 dilution.

FIG. 6.

RSV is partially protective against challenge with influenza A virus. BALB/c mice (4 weeks old) were infected i.n. with wt RSV (gray squares), rRSV/IFN-γ (white circles, broken line), or rRSV/IL-4 (black diamonds) or left as naïve mice (white squares). At 4 weeks later the mice were infected with X31 influenza virus i.n. (A) Weight loss following rechallenge. (B to F) Airway cell numbers (B), anti-influenza A virus and anti-RSV total immunoglobulin levels (C), CD4⁺ T-cell numbers (D), CD8⁺ T-cell numbers (E), and airway eosinophilia levels (F) on day 8 after challenge are shown. (G to I) BAL cytokine levels (in picograms per milliliter) for IL-4 (G), IL-5 (H), and IFN-γ (I) on day 8 after challenge are shown. Symbols and bars represent means ± SEM of values obtained with mice (n ≥ 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001. 1°, initial challenge.