Interleukin-27 inhibits vaccine-enhanced pulmonary disease following respiratory syncytial virus infection by regulating cellular memory responses

Abstract

Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract disease in young children. In the 1960s, infants vaccinated with formalin-inactivated RSV developed a more severe disease characterized by excessive inflammatory immunopathology in lungs upon natural RSV infection. The fear of causing the vaccine-enhanced disease (VED) is an important obstacle for development of safe and effective RSV vaccines. The recombinant vaccine candidate G1F/M2 immunization also led to VED. It has been proved that cellular memory induced by RSV vaccines contributed to VED. Interleukin-27 (IL-27) and IL-23 regulate Th1, Th17, and/or Th2 cellular immune responses. In this study, mice coimmunized with pcDNA3-IL-27 and G1F/M2 were fully protected and, importantly, did not develop vaccine-enhanced inflammatory responses and immunopathology in lungs after RSV challenge, which was correlated with moderate Th1-, suppressed Th2-, and Th17-like memory responses activated by RSV. In contrast, G1F/M2- or pcDNA3-IL-23+G1F/M2-immunized mice, in which robust Th2- and Th17-like memory responses were induced, developed enhanced pulmonary inflammation and severe immunopathology. Mice coimmunized with G1F/M2 and the two cytokine plasmids exhibited mild inflammatory responses as well as remarkable Th1-, suppressed Th2-, and Th17-like memory responses. These results suggested that Th1-, Th2-, and Th17-like memory responses and, in particular, excessive Th2- and Th17-like memory responses were closely associated with VED; IL-27 may inhibit VED following respiratory syncytial virus infection by regulating cellular memory responses.

Fig 1

Percentage of CD44⁺ CD62L⁺ cells of CD4⁺ or CD8⁺ spleen cells in immunized mice. Mice were immunized i.p. three times as described in Materials and Methods. Spleens from immunized mice were removed 3 weeks after the last immunization. Splenocytes were incubated with anti-CD4-FITC/anti-CD8-FITC, anti-CD44-PE, and anti-CD62L-Pecy5 (PC5) and then detected by flow cytometry. Percentages of CD4⁺ CD44⁺ CD62L⁺ T cells (A) and CD8⁺ CD44⁺ CD62L⁺ T cells (B) in the splenocytes of mice immunized with pIL-23+G1F/M2, pIL-27+G1F/M2, pIL-23+pIL-27+G1F/M2, pcDNA3+G1F/M2, G1F/M2, or PBS are shown.

Fig 2

Specific cellular memory induced by the vaccine candidate in BALB/c mice. Mice were immunized i.p. three times as described in Materials and Methods. Spleens from immunized mice were removed 3 weeks after the last immunization. (a) Frequency of G1F/M2-specific IFN-γ- or IL-4-secreting T cells in 10⁶ splenocytes of immunized mice. Splenocytes were restimulated for 48 h with 20 μg G1F/M2. The numbers of specific IFN-γ- and IL-4-secreting T cells were evaluated using an ELISPOT assay. Results are shown as the mean value ± SD of the number of spots observed for 10⁶ spleen cells of 6 mice per group, obtained from triplicate wells. *, P < 0.05. (b) Ratio of IFN-γ- and IL-4-secreting T cells. (c) Induction of G1F/M2-specific CTL responses in immunized mice. Splenocytes were restimulated in vitro for 5 days with 0.5 μM G1F/M2 to produce effectors. Target cells were SP2/0 cells pulsed with UV-unactivated RSV. Data represent the percentage of specific lysis at an effector-to-target cell ratio of 50:1. Results are the means ± SDs of 5 or 6 mice per group. *, P < 0.05.

Fig 3

Humoral immunity induced by the vaccine candidate in BALB/c mice. Mice were immunized i.p. three times with pIL-23+G1F/M2, pIL-27+G1F/M2, pIL-23+pIL-27+G1F/M2, pcDNA3+G1F/M2, G1F/M2, or PBS. Serum samples were collected at 14 days after each immunization. G1F/M2-specific IgG (A) and IgG1 and IgG2a (B) antibodies 14 days after the third immunization were determined by ELISA. Results are expressed as geometric means of serum antibody titers and the means ± SDs from triplicate wells. *, P < 0.05. (C) Serum neutralizing antibody titers against RSV obtained in a neutralization assay. Results are expressed as the geometric mean titer of sera that neutralized 60% of plaques on RSV-infected HEp-2 cells.

Fig 4

Immunization with the vaccine candidate protects BALB/c mice against RSV challenge. Mice were immunized i.p. three times as described in Materials and Methods. Three weeks after the final immunization, mice were challenged i.n. with 10⁵ TCID₅₀s/100 μl RSV A. At 5 days postchallenge, lungs were removed. RSV replication in lungs was measured using RT-PCR. (A) Semiquantitative RT-PCR products. Lane 1, DL2000 DNA maker; lane 2, PBS group; lane 3, pIL-23+G1F/M2 group; lane 4, pIL-27+G1F/M2 group; lane 5, pIL-23+ pIL-27+G1F/M2 group; lane 6, G1F/M2 group. (B) Relative expression of RSV N mRNA by qRT-PCR. The fold reduction of RSV N-gene expression in each experimental group was calculated by comparison to that in PBS-treated mice (100%). Results are means ± SDs of 5 or 6 mice per group and are representative of two experiments.

Fig 5

RSV challenge of pIL-27+G1F/M2-immunized mice activated moderate Th1, few Th2, and no Th17 memory responses in lungs. Mice were immunized i.p. three times as described in Materials and Methods. Three weeks after the final immunization, mice were challenged i.n. with 10⁵ TCID₅₀s/100 μl RSV A. At 5 days postchallenge, lungs were removed. The relative expression of the transcription factors and cytokines in lungs was measured using qRT-PCR. Naïve mice neither immunized nor challenged were used as controls (Ctr). The results are represented as the fold expression of each gene in experimental mice relative to control mice. (A and B) Th1-type transcription factors and cytokines; (C to E) Th2-type transcription factors and cytokines; (F) Th17 typical cytokine. Results are means ± SDs of 5 or 6 mice per group and are representative of two experiments.

Fig 6

pIL-27+G1F/M2-immunized mice showed infiltration of few inflammatory cytokines in lungs following RSV challenge. Mice were immunized i.p. three times as described in Materials and Methods and then challenged i.n. with 10⁵ TCID₅₀s/100 μl RSV A 3 weeks after the final immunization. Lungs were removed at 5 days postchallenge. The relative expression of the inflammatory cytokines GRO-α and eotaxin (A), MCP-1 (B), IL-6 (C), and TNF-α (D) in lungs was measured using qRT-PCR. Naïve mice neither immunized nor challenged were used as controls (Ctr). Results are means ± SDs of 5 or 6 mice per group and are representative of two different experiments.

Fig 7

pIL-27+G1F/M2 immunization prevented pulmonary pathology following RSV infection. (A) Lung histology was determined by H&E staining at 5 days postchallenge. An image for a representative animal is shown for each experimental group or for naïve mice (control). (B) Scores for pulmonary inflammation. Tissue sections obtained from each mouse were scored for inflammation on a scale of from 0 to 3 or 4 as described in Materials and Methods. (C) Relative expression of gob5 gene in lungs at 5 days postchallenge. Preimmunized mice were challenged with RSV A 3 weeks after the final immunization. Total RNAs were extracted from the lungs. The relative expression of gob5 mRNA was measured by qRT-PCR. Naïve mice neither immunized nor challenged were used as controls. Results are means ± SDs of 5 to 6 mice/group and are representative of two experiments.