Design and evaluation of novel interferon lambda analogs with enhanced antiviral activity and improved drug attributes

Abstract

Type III interferons (IFNs) (also called IFN-λ: IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4) are critical players in the defense against viral infection of mucosal epithelial cells, where the activity of type I IFNs is weak, and unlike type I IFNs that are associated with severe and diverse side effects, type III IFNs cause minimal side effects due to the highly restricted expression of their receptors, and thus appear to be promising agents for the treatment and prevention of respiratory and gastrointestinal viral infection. However, the antiviral potency of natural type III IFNs is weak compared to type I and, although IFN-λ3 possesses the highest bioactivity among the type III IFNs, IFN-λ1, instead of IFN-λ3, is being developed as a therapeutic drug due to the difficulty to express IFN-λ3 in the prokaryotic expression system. Here, to develop optimal IFN-λ molecules with improved drug attributes, we designed a series of IFN-λ analogs by replacing critical amino acids of IFN-λ1 with the IFN-λ3 counterparts, and vice versa. Four of the designed analogs were successfully expressed in Escherichia coli with high yield and were easily purified from inclusion bodies. Interestingly, all four analogs showed potent activity in inducing the expression of the antiviral genes MxA and OAS and two of them, analog-6 and -7, displayed an unexpected high potency that is higher than that of type I IFN (IFN-α2a) in activating the IFN-stimulated response element (ISRE)-luciferase reporter. Importantly, both analog-6 and -7 effectively inhibited replication of hepatitis C virus in Huh-7.5.1 cells, with an IC₅₀ that is comparable to that of IFN-α2a; and consistent with the roles of IFN-λ in mucosal epithelia, both analogs potently inhibited replication of H3N2 influenza A virus in A549 cells. Together, these studies identified two IFN-λ analogs as candidates to be developed as novel antiviral biologics.

Keywords:

• type III interferon
• IFN- λ
• IL-29
• IL-28B
• analog
• antiviral

Supplementary materials

Figure S1 Amino acid sequences of the nine designed analogs.

Note: Interferon (IFN)-λ1 sequence is shown in red and IFN-λ3 sequence in blue.

Figure S2 Stability of analog protein preparations.

Notes: (A) A representative graph of HPLC analysis: an aliquot of PEG-analog-6 (0221) was kept at 40°C for 39 days. (B) Purity of PEG-analog-6 (0221, 0225, 0302) kept at 40°C for up to 39 days. (C) Concentrations of PEG-analog-6 (0221, 0225, 0302) kept at 40°C for up to 39 days.
Abbreviation: HPLC, high-performance liquid chromatography.

Figure S3 qRT-PCR analysis of MxA and OAS gene expression.

Notes: Cells were treated by 10 ng/mL proteins for 12 hours; MxA and OAS induction activity was significantly reduced after the proteins were treated at 95°C for 5 minutes, and a similarly prepared unrelated protein, human growth hormone, showed no activity.
Abbreviations: IL, interleukin; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure S4 MTT assay.

Notes: (A and B) HEK293-ISRE-luciferase cells, (C) Huh-7.5.1 cells: cells were treated by serial dilutions of analog-6, analog-7, or IFN-λ1 (A), or PEG-analog-6, PEG-analog-7, PEG-IFN-λ1, or PEG-IFN-α2a (B and C).
Abbreviations: IFN, interferon; ISRE, IFN-stimulated response element; PEG-IFN, pegylated-interferon.

Figure S5 qRT-PCR measurement of changes in HCV RNA levels.

Notes: HCVcc-infected Huh-7.5.1 cells were treated by different concentrations of PEG-analog-6, PEG-analog-7, PEG-IFN-λ1, or PEG-IFN-α2a for 48 hours and HCV RNA levels in Huh-7.5.1 cells were measured by qRT-PCR using relative quantification and the 2^−ΔΔC(t) method. The results were expressed as fold of changes relative to untreated controls.
Abbreviations: HCV, hepatitis C virus; IFN, interferon; qRT-PCR, quantitative real-time polymerase chain reaction; PEG-IFN, pegylated-interferon.

Acknowledgments

We would like to thank Francis V Chisari of Scripts Research Institute for the Huh-7.5.1 cell line; Jin Zhong of the Institut Pasteur of Shanghai – Chinese Academy of Sciences, Shanghai, People’s Republic of China for assisting us to get the Huh-7.5.1 cell line; Ruifeng Xu of the National Institute of Metrology, Beijing, People’s Republic of China, for help with LC/Q-TOF/MS; and Xiaoguang Zhang of the National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, for assistance with anti-H3N2 assay. This study was wholly funded by Prosit Sole Biotechnology Co., Ltd., Beijing, People’s Republic of China.

Disclosure

H Liu, H Sun and M Zhang are employees of and M Zhao and Z Zou are shareholders in Prosit Sole Biotechnology Co., Ltd. The other authors report no conflicts of interest in this work.