Impact of Interferon Lambda 4 Genotype on Interferon‐Stimulated Gene Expression During Direct‐Acting Antiviral Therapy for Hepatitis C

New directly acting antivirals (DAAs) provide very high cure rates in most patients infected by hepatitis C virus (HCV). However, some patient groups have been relatively harder to treat, including those with cirrhosis or infected with HCV genotype 3. In the recent BOSON trial, genotype 3, patients with cirrhosis receiving a 16‐week course of sofosbuvir and ribavirin had a sustained virological response (SVR) rate of around 50%. In patients with cirrhosis, interferon lambda 4 (IFNL4) CC genotype was significantly associated with SVR. This genotype was also associated with a lower interferon‐stimulated gene (ISG) signature in peripheral blood and in liver at baseline. Unexpectedly, patients with the CC genotype showed a dynamic increase in ISG expression between weeks 4 and 16 of DAA therapy, whereas the reverse was true for non‐CC patients. Conclusion: These data provide an important dynamic link between host genotype and phenotype in HCV therapy also potentially relevant to naturally acquired infection. (Hepatology 2018; 00:000‐000).

HCV genotyping was based on whole genome sequences compared to a set of reference sequences annotated by ICTV(2)

IFNL4 genotyping
To perform the genotyping analysis, genomic DNA was extracted from PBMC collected in 0.5M EDTA. Genotyping was performed using Affymetrix UK Biobank array. IFNL4 SNP rs12979860 is directly typed in this array.

RNA extraction:
Total RNA from blood collected in Paxgene tubes were extracted using the Paxgene Blood RNA kit (Qiagen 762174)following manufacturers instruction.
Briefly, the frozen blood collection tubes were thawed at RT and let to further incubate for 2 hrs, and centrifuged for 10 min 4000 xg.
The supernatant was discarded and the washed with water, centrifuged and the pellet was treated with proteinase K incubated at 55 o C for 10 mins on a shaker heat block. The resulting lysate was then passed through the Shredder spin column, topped up with 0.5 vol of 100% ethanol. This mix was then passed through the RNA spin column washed once and the column was incubated with DNase, washed further and finally the RNA was isolated using 100 ul of elution buffer. The eluate RNA was then incubated at 65 o C incubator for 5 mins and immediately chilled on ice. The resulting RNA was then assessed for yield and quality using a Nanodrop and frozen at -20 o C or -70 o C.

Globin clear:
As globins are predominant in the RNA sample, we used the Invitrogen globin clear kit (Ambion AM1980) to improve the global expression screening of samples prior to analysis by microarray analysis following manufacturers instruction.
Briefly, 1-10ug total RNA was hybridised with biotinylated Globin capture oligo beads for 15 mins . This step tags globin mRNA in the sample with biotinylated beads. The tagged RNA is then washed and further incubated with Streptavidin magnetic beads at 50 o C for 15 mins. The streptavidin-biotin tagged RNA was then washed with buffer and placed on a magnetic stand that separates the labelled Globin RNA from the rest of the RNA which is collected by aspiration, quantified and stored at -80 o C until ready for microarray analysis.

Real time PCR validation:
Reverse Transcription: Approximately 500ng of total RNA from blood was reverse transcribed using the Superscript reverse transcriptase III using OligodT and random hexamer primers. (Invitrogen). The resulting cDNA was used for qPCR as described below.
qPCR: 5ul of a 1/20th dilution of the cDNA was used as a template for qPCR on the Roche light cycler 480 to detect expression of selected ISGs: IFIT1, MX1, RSAD2, ISG15, IFIT3, IFI44L and HERC5. Primers were synthesized as suggested by the universal probe library assay design centre. Primer and probe sequences are provided in Table below. Relative quantification of genes were performed (48) using the ΔΔCT method normalizing to GAPDH or beta-actin as reference genes and further normalized to control RNA from healthy individuals. Values were analysed using GraphPad Prism (version 7) and statistical analysis on data were generated by t-test analysis within Prism software. For

Supplimentary table 1
Suppl Our data could be explained by a model whereby the CC genotype undergoes "damping" at higher viral loads and the non-CC genotype does not. As infection progresses, if it is not cleared, a steady state would be reached where, as seen, ISG levels are lower in the CC genotype (figure is traversed left to right). As virus is cleared by DAA therapy, the ISG response is "undamped" and a peak is seen as the figure is traversed right to left. The shape of the curves could be determined by the differential cross-regulation of Type III and Type I interferons driven by the underlying genotype.    Table 1b: List of genes differentially regulated at baseline between non-CC vs Control sorted by ladj p value in ascending order. Adjusted P value was generated using moderated t-test in limma package