A Novel Bifunctional Fusion Protein, Vunakizumab-IL22, for Protection Against Pulmonary Immune Injury Caused by Influenza Virus

Abstract

Influenza A virus infection is usually associated with acute lung injury, which is typically characterized by tracheal mucosal barrier damage and an interleukin 17A (IL-17A)-mediated inflammatory response in lung tissues. Although targeting IL-17A has been proven to be beneficial for attenuating inflammation around lung cells, it still has a limited effect on pulmonary tissue recovery after influenza A virus infection. In this research, interleukin 22 (IL-22), a cytokine involved in the repair of the pulmonary mucosal barrier, was fused to the C-terminus of the anti-IL-17A antibody vunakizumab to endow the antibody with a tissue recovery function. The vunakizumab-IL22 (vmab-IL-22) fusion protein exhibits favorable stability and retains the biological activities of both the anti-IL-17A antibody and IL-22 in vitro. Mice infected with lethal H1N1 influenza A virus and treated with vmab-mIL22 showed attenuation of lung index scores and edema when compared to those of mice treated with saline or vmab or mIL22 alone. Our results also illustrate that vmab-mIL22 triggers the upregulation of MUC2 and ZO1, as well as the modulation of cytokines such as IL-1β, HMGB1 and IL-10, indicating the recovery of pulmonary goblet cells and the suppression of excessive inflammation in mice after influenza A virus infection. Moreover, transcriptome profiling analysis suggest the downregulation of fibrosis-related genes and signaling pathways, including genes related to focal adhesion, the inflammatory response pathway, the TGF-β signaling pathway and lung fibrosis upon vmab-mIL22 treatment, which indicates that the probable mechanism of vmab-mIL22 in ameliorating H1N1 influenza A-induced lung injury. Our results reveal that the bifunctional fusion protein vmab-mIL22 can trigger potent therapeutic effects in H1N1-infected mice by enhancing lung tissue recovery and inhibiting pulmonary inflammation, which highlights a potential approach for treating influenza A virus infection by targeting IL-17A and IL-22 simultaneously.

Keywords: H1N1 influenza A virus; IL-17A; IL-22; anti-inflammatory effects; bifunctional fusion protein; lung injury; tissue repair.

Figure 1

Cloning, expression, and characterization of vmab-mIL22, vmab and mIL22Fc. (A) Schematic representation of the cloning strategy and expression data for vmab-mIL22. Mouse IL22 fused to the C-terminus of the heavy chain of vunakizumab (vmab) via a 15-amino acid (aa) linker. (B) Schematic representation of the cloning strategy and expression data for vmab. (C) Schematic representation of the cloning strategy and expression data for mIL22Fc (mouse IL22 and IgG4 Fc fusion protein). (D) SDS-PAGE analysis of mIL22Fc (lane 1), vmab (lane 2) and vmab-mIL22 (lane 3) under either reducing or nonreducing conditions. (E) SEC-HPLC analysis of mIL22Fc, vmab and vmab-mIL22. VH: antibody variable heavy chain. VL: antibody variable light chain. CH: antibody heavy chain constant region. CL: antibody light chain constant region. mIL22: mouse IL-22. RT: retention time.

Figure 2

Stability analysis of mIL22-Fc, vmab and vmab-mIL22 at 37°C for 10 days. HPLC-SEC profiles of (A) vmab-mIL22, (B) vmab and (C) mIL22Fc at 0°C or 37°C for 10 days. (D) Compared with that observed at the 0 day timepoint, the measurable aggregation (agg) of mIL22-Fc, vmab and vmab-mIL22 did not increase evidently or show measurable degradation after 10 days at 37°C (clip). (E) Thermal stability analysis of vmab-mIL22, vmab and mIL22-Fc using intrinsic protein fluorescence measurements. The final values were averages of three independent measurements. Increasing Tm and Tagg values reflect an increase in macroscopic conformational stability. Tonset (°C), protein unfolding temperature; Tm, melting temperature; Tagg, aggregation temperature.

Figure 3

vmab-mIL22 exhibits dual functionality, and the functional integrity of the cytokine payloads is preserved. (A, B) Biacore sensorgrams of the interaction of vmab with mIL17A and BIAcore sensorgrams of the interaction of vmab-mIL22 with mIL17A. vmab-mIL22 retained a high affinity for the cognate antigen, similar to that of the parental antibody Vmab, which was confirmed by surface plasmon resonance analysis. Sensorgrams display the response values at IL17A concentrations of 0.01 to 0.2 μg/mL, with a CM5 chip and capture with 100 RU vmab or vmab-mIL22. The equilibrium dissociation constant (K_D) was calculated using k_d/k_a for each measurement. k_d, dissociation rate constant or off-rate constant; k_a, association rate constant or on-rate constant. Binding was biphasic, and the data were fit with heterogeneous ligand models. (C) In vitro inhibition of functional activity by vmab-mIL22. Inhibition of Groα release from HT-29 cells as a measure of IL-17A inhibition. HT29 cells were stimulated with IL-17A (5 nM) in the presence of various concentrations of vmab-mIL22. vmab was used as a positive control. Groα levels in the supernatants were measured by ELISA from R&D systems. (D) Western blot analysis of STAT3 phosphorylation (p-STAT3) induced by 10 nM vmab-hIL22 in HepG2 cells at different times. (E) Western blot analysis of STAT3 phosphorylation (p-STAT3) induced by 10 nM mIL-22Fc or vmab-mIL22 in HepG2 cells at 1.5 h Serum-starved cells (3×10⁶/mL) were stimulated with PBS, 10 nM mIL22-Fc or Vmab-mIL22 for 1.5 h at 37°C. Whole-cell lysates were resolved on denaturing gels, transferred, and immunoblotted with a polyclonal anti-phospho-STAT3 antibody.

Figure 4

The therapeutic efficacy of vmab-mIL22 against H1N1-induced lung immune injury. (A) Representative pulmonary edema showing the pathology of the lung. (B, C) Assessment of the therapeutic effect of vmab-mIL22 on pulmonary edema and lung index scores H1N1 infection, compared with saline, vmab, mIL22Fc, the combination of vmab and mIL22Fc and healthy mice. Mice were randomly divided into six groups. There are seven mice in each group (n = 7). Values were expressed as mean ± SEM. Multiple group comparisons were performed using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test using the software of SPSS. p-value of less than 0.05 was considered to be significant. (D) Representative histological sections of the lung tissues from H1N1 virus-infected mice receiving each therapy or saline treatment were stained with hematoxylin and eosin. Bars, 50 μm. Wedge indicates accumulation of mononuclear cells, and black arrow indicates the alveolar wall. *P < 0.05 or ***P < 0.001. NS, not significant.

Figure 5

Vmab-mIL22 treatment increased epithelial repair and modulated inflammatory cytokine levels. (A) Recovery of pulmonary goblet cells and the mucus layer in the vmab-mIL22 treatment group. Pulmonary goblet cells are visualized by PAS staining. Black arrows indicate the goblet cells. MUC2 and ZO1 protein expression was assayed via immunohistochemistry. (B) Cytokines in the serum or lung homogenate were assayed using ELISA.There are seven mice in each group (n = 7). Values were expressed as mean ± SEM. Multiple group comparisons were performed using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test using the software of SPSS. p-value of less than 0.05 was considered to be significant. *P < 0.05, **P < 0.01 or ***P < 0.001. NS, not significant.

Figure 6

Transcriptomics changes of H1N1 infection and vmab-mIL22 treatment. (A, B) The differential expressed gene (DEG) analysis results were shown in volcano plots and differential expressed (DE) genes (|log2-fold change| >=1.0 and BH-adjusted P <= 0.05) between H1N1-infected and normal mice were selected for cluster analysis in heatmap. Among the DE genes, a set of 1823 genes showed upregulated expression, and the remaining 1340 genes showed downregulated expression. Each row represents an individual gene, and each column represents an individual sample. (C) Pathway analysis of genes found to be significantly upregulated in H1N1-infected mice. The top 29 significantly affected signaling pathways are shown. (D, E) The DE genes between vmab-mIL22-treated and H1N1-infected mice were selected for cluster analysis in heatmap. Among the DE genes, a set of 349 genes were upregulated, and the remaining 253 genes were downregulated. (F) Pathway analysis of genes found to be significantly downregulated in vmab-mIL22-treated mice. Eight significantly affected signaling pathway is shown.