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

Figures & data

Table 1 Primers used in the construction of IFN-λ analogs and qPCR assays

λ1-U-F	5′-TATACATATGAAGCCGACTACTACTG-3′
λ1-D-R	5′-CCGGATCCTCAGGTGCTCTCCGGGTG-3′
λ3-U-F	5′-TATACATATGCGTCTGCGTGGTGCAC-3′
λ3-D-R	5′-CCGGATCCTCAAACAGACAGGTCGCCAGAAGC-3′
Analog-1-M-F	5′-CGTACTTGGGACCTGCGTCTGCTGCAGGTGCGTGAG-3′
Analog-1-M-R	5′-CTCACGCACCTGCAGCAGACGCAGGTCCCAAGTACG-3′
Analog-2-M-F	5′-CTGAAAGTGCTGGAAGCAGCAGCTGGTCCGGCTCTG-3′
Analog-2-M-R	5′-CAGAGCCGGACCAGCTGCTGCTTCCAGCACTTTCAG-3′
Analog-3-M-F	5′-GCAACTGCAGATACCGACGGTCCGGCTCTGGAAGAC-3′
Analog-3-M-R	5′-GTCTTCCAGAGCCGGACCGTCGGTATCTGCAGTTGC-3′
Analog-4-M-F	5′-GCTCCGAAAAAGGAATCCGCGGGTTGCCTGGAAGC-3′
Analog-4-M-R	5′-GCTTCCAGGCAACCCGCGGATTCCTTTTTCGGAGC-3′
Analog-5-M-F	5′-GCACCGAAGAAAGAATCTCCGGGTTGCCTGGAAGC-3′
Analog-5-M-R	5′-GCTTCCAGGCAACCCGGAGATTCTTTCTTCGGTGC-3′
Analog-6-M-F	5′-CCGGTTTTCCCGGGTAACTGGGACCTGCGTCAGCTG-3′
Analog-6-M-R	5′-CAGCTGACGCAGGTCCCAGTTACCCGGGAAAACCGG-3′
Analog-7-M-F	5′-GGTAACTGGGATCTGCGTCAGCTGCAGGTTCGTGAAC-3′
Analog-7-M-R	5′-TTCACGAACCTGCAGCTGACGCAGATCCCAGTTACC-3′
Analog-8-M-F	5′-CTGGAAGCCGCAGCTGGTGATACCGACCCGGCACTG-3′
Analog-8-M-R	5′-CAGTGCCGGGTCGGTATCACCAGCTGCGGCTTCCAG-3′
Analog-9-M-F	5′-GCACCGAAGAAAGAATCTCCGGGTTGCCTGGAAGC-3′
Analog-9-M-R	5′-GCTTCCAGGCAACCCGGAGATTCTTTCTTCGGTGC-3′
MxA-F	5′-ATCCTGGGATTTTGGGGCTT-3′
MxA-R	5′-CCGCTTGTCGCTGGTGTCG-3′
OAS-F	5′-CATCCGCCTAGTCAAGCACTG-3′
OAS-R	5′-CCACCACCCAAGTTTCCTGTAG-3′
HCV-F	5′-TCTGCGGAACCGGTGAGTA-3′
HCV-R	5′-TCAGGCAGTACCACAAGGC-3′
GAPDH-F	5′-GAAGGCTGGGGCTCATTT-3′
GAPDH-R	5′-CAGGAGGCATTGCTGATGAT-3′

Note: The underlined nucleotides are NdeI or BamHI restriction enzyme sites.

Abbreviations: HCV, hepatitis C virus; IFN, interferon; qPCR, quantitative polymerase chain reaction.

Figure 1 Design of IFN-λ analogs.

Notes: (A) Alignment of amino acid sequences of IFN-λ1 (IL-29S) and IFN-λ3: conserved amino acids were labeled in red, and the helices A–F were marked underneath the sequence, while the arbitrarily divided fragments a, b, c, and d were labeled above the sequence. (B) Sequence structure of the nine analogs: IFN-λ1 fragments were labeled in red and IFN-λ3 fragments in blue.
Abbreviation: IFN, interferon.

Figure 2 Analysis and characterization of expressed proteins.

Notes: (A) SDS-PAGE gel analysis of small-scale expression of the nine designed analogs: the arrow on the right marks the position of the expected protein band; M: protein marker; A: IPTG-induced; B: uninduced. (B) Representative chromatographs of analog-6 (left) and PEG-analog-6 (right) eluted from the SP HP column. (C) SDS-PAGE gel analysis of purified proteins (left) and purified PEGylated proteins (right): the samples were undenatured and run in nonreducing conditions. (D) HPLC analysis of purified IFN-λ1 (IL-29S) (left) and analog-6 (right): the peak on the left represents the PEGylated protein, and the peak on the right represents the unmodified protein.
Abbreviations: IFN, interferon; IPTG, isopropyl-β-D-thiogalactopyranoside; PEG-IFN, pegylated-interferon; SDS-PAGE, polyacrylamide gel electrophoresis.

Table 2 Characterization of purified proteins by LC/Q-TOF/MS

	Number of amino acids	Number of cysteines	Theoretical MW	Detected MW	MW difference
IFN-λ1	176	4	19,590.58	19,590.20	Normal
Analog-6	178	4	19,869.79	19,869.40	Normal
Analog-7	169	5	18,782.63	18,783.00/18,902.10	Normal/+119
Analog-8	170	5	18,794.69	18,781.10/18,900.20	−14/+105.5
Analog-9	167	5	18,503.42	18,503.90/18,623.00	Normal/+119

Abbreviations: IFN, interferon; LC/ESI-QTOF-MS, liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry; MW, molecular weight.

Figure 3 Stimulation of antiviral gene expression.

Notes: (A–C) qRT-PCR analysis of MxA and OAS gene expression: cells were treated by different concentrations of proteins for 12 hours, and induction of MxA and OAS expression by analog-6, -7, -8, and -9, and IFN-λ1 showed good dose-dependency (A); cells were treated by 10 ng/mL proteins for 3, 12, and 24 hours, and induction of MxA and OAS expression showed good time-dependency (B); cells were treated by 10 ng/mL PEGylated proteins for 12 hours, and PEG-analog-6 and PEG-analog-7 showed similar inducing activity as PEG-IFN-λ1 and PEG-IFN-α2a (C). (D) Representative image of Western blot analysis of STAT1 phosphorylation: cells were treated by 10 ng/mL PEGylated protein for 12 hours.
Abbreviations: IFN, interferon; PEG-IFN, pegylated-interferon; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 4 Activation of the ISRE-luciferase reporter.

Notes: (A) Representative response curves of anolog-6, analog-7, and IFN-λ1. (B) Representative response curves of PEG-analog-6, PEG-analog-7, PEG-IFN-λ1, and PEG-IFN-α2a. Data were analyzed using the GraphPad Prism software.
Abbreviations: IFN, interferon; ISRE, IFN-stimulated response element; PEG-IFN, pegylated-interferon.

Figure 5 Anti-HCV assay.

Notes: (A) HCVcc-infected Huh-7.5.1 cells were treated by 0, 0.5, 5, and 50 ng/mL of PEG-analog-6, PEG-analog-7, PEG-IFN-λ1, or PEG-IFN-α2a for 48 hours and stained by an anti-HCV core antibody (green, upper panels) and DAPI (blue, lower panels); scale bar: 50 μm. (B) Quantification of HCV positive cells: number of HCV foci in three wells was counted manually, and the average number of HCV foci per well was shown.
Abbreviations: HCV, hepatitis C virus; IFN, interferon; PEG-IFN, pegylated-interferon.

Figure 6 Quantification of anti-HCV and anti-H3N2 potency.

Notes: (A) Representative curves of HCV inhibition: HCVcc-infected Huh-7.5.1 cells were treated by PEG-analog-6, PEG-analog-7, PEG-IFN-λ1, or PEG-IFN-α2a for 48 hours and values representing relative HCV RNA levels were fitted into the GraphPad Prism software to generate the inhibition curves and IC₅₀. (B) Representative curves of H3N2 inhibition: A549 cells were treated by analog-6, analog-7, IFN-λ1, or IFN-α2a for 24 hours and then infected by H3N2 influenza A virus for 90 minutes, and the amount of H3N2 virus was quantified by ELISA 72 hours later.
Abbreviations: HCV, hepatitis C virus; IFN, interferon; ELISA, enzyme-linked immunosorbent assay; IC₅₀, half maximal inhibitory concentration; PEG-IFN, pegylated-interferon.

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.