Differences in Western Blotting and human miRNA analyses between immunoprecipitated HBsAg particles and control immunoprecipitations.

<p>Left-most: Western blotting analysis of protein lysates from HBsAg positive immunoprecipitates (HBs-IP+) and control HBsAg negative immunoprecipitates (ctrl-IPs) for detection of Ago2 protein. IP+ lanes contain pooled protein lysates from samples # 1–13 and single lysates from samples # 1 and 9 respectively. Ctrl-IPs lanes contain protein lysates from immunoprecipitates obtained from HBsAg positive sera with mouse monoclonal anti-human c-myc antibody (unrelated-IP+) and HBsAg negative sera using anti-HBs monoclonal antibody (HBs-IP−). HEK is the protein lysate from HEK cells used as positive control for Ago2 protein. The figure is representative of 3 independent experiments. Right-most: HCL is unsupervised hierarchical cluster analysis of detected miRNAs (DC_T values) in both HBs-immunoprecipitated fractions from HBsAg positive immunoprecipitates (HBs-IP+) and control HBsAg negative immunoprecipitates (ctrl-IPs). GDM is supervised gene distance matrix correlating DCt values of HBs-IP+ vs ctrl-IP samples.</p

Identification of human miRNAs associated with serum HBsAg.

<p>(a) The circulating HBsAg-miRNA signature: average RQs were obtained from the comparative DDC_T analysis. Values are reported in a bar plot as a logarithmic scale base 10 along with SD. (b) Differentially detected miRNAs between HBsAg positive immunoprecipitates (HBs-IP+) samples (left-most, n = 11; right-most, n = 4) and the group of control HBsAg negative immunoprecipiates (ctrl-IPs) (n = 4) were selected by Mann-Whitney test on –DCt values (left-most, p<0.1; right-most, p<0.05), and an unsupervised hierarchical cluster analysis was finally performed. Venn diagrams indicate the comparison among the pool of HBsAg-associated miRNAs obtained from the comparative DDC_T analysis of panel a (blu circle) and the HBsAg-associated miRNAs obtained from the Mann-Whitney tests (red circle).</p

HBs-immunoprecipitation led to a significant change of detection of some human miRNAs in examined sera.

<p>Heatmap of differentially detected miRNAs in whole HBsAg sera (S+) and HBsAg positive flowthroughs after immunoprecipitation (HBs-F+) was obtained by Mann-Whitney test (p<0.05) followed by hierarchical clustering (-DC_T are represented). Venn diagram: the 157 differentially abundant serum miRNAs between whole HBsAg positive sera (S+) and HBsAg positive flowthroughs (HBs-F+) were compared to miRNAs of clusters 1-to-5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone-0031952-g003" target="_blank">Figure 3b</a> (right panel).</p

Demographic and Virologic Characteristics of Individuals and Sera.

<p>Abbreviations: VP = viral particles, virion; SVP = subviral HBsAg particles; IU = International Units; ng = nanograms; ALT = serum alanine amino trasferase.</p><p>Quantitative values for VP and SVP were obtained as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone.0031952-Gerlich1" target="_blank">[14]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone.0031952-Desire1" target="_blank">[15]</a>: 1 IU of HBsAg corresponds to 1,1E+06 IU HBV DNA and1 ng of HBsAg corresponds to 2,08E+08 SVP or 5,0E+07 VP.</p><p>HBV infection and disease phases were characterized as previuosly reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone.0031952-Brunetto1" target="_blank">[8]</a>: IC = Inactive HBsAg Carriers with serum HBV-DNA persistently below 2000 IU and without liver disease; AC1 = Active HBsAg Carriers with serum HBV-DNA fluctuating below 20.000 IU with normal liver histology; AC2 = Active HBsAg carriers with serum HBV-DNA fluctuating above 20.000 IU with chronic active hepatitis at histology, patients with chronic hepatitis B (CHB).</p

Canonical pathways enriched by predicted targets of HBsAg-associated miRNAs.

<p>Target genes of HBsAg-associated miRNAs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone-0031952-g003" target="_blank">Figure 3a</a>) were predicted (TargetScan) and ranked. Functional analysis (IPA) was then applied on predicted target genes, and canonical pathways were finally ranked according to their significance in a Fisher exact test (p-value≤0.05, horizontal threshold line in the plot). Left axis: –log(p-values) as histogram boxes; the asterisk (*) marks the three pathways that resulted as significantly enriched by the analyzed gene-list; right axis: for each pathway, black dots indicate the ratio of pathway-defining genes, which is calculated as the number of involved target genes over the total number of genes in that pathway reference dataset.</p

No association between <i>IFNL3 (IL28B)</i> genotype and response to peginterferon alfa-2a in HBeAg-positive or -negative chronic hepatitis B

<div><p>Background & aims</p><p>It has yet to be firmly established whether host <i>IFNL3</i> (<i>IL28B</i>) genotype influences interferon responsiveness in patients with chronic hepatitis B. We investigated associations between single-nucleotide polymorphisms (SNPs) in the <i>IFNL3</i> region and response to peginterferon alfa-2a in 701 patients enrolled in three large, randomized, international studies.</p><p>Methods</p><p>Responses were defined as hepatitis B surface antigen (HBsAg) loss and/or hepatitis B e antigen (HBeAg) seroconversion plus hepatitis B virus (HBV) DNA <2000 IU/ml in HBeAg-positive patients, and HBsAg loss and/or HBV DNA <2000 IU/ml in HBeAg-negative patients (24 weeks after end of treatment). Associations between treatment response and the number of copies of the poor-response allele at three SNPs (rs8099917, rs12980275, rs12979860) were explored with logistic regression models in Asian and white patients.</p><p>Results</p><p>The HBeAg-positive and -negative populations comprised 465 (92% Asian, 50% HBV genotype C) and 236 (79% Asian, 41% HBV genotype C) patients, respectively, and had respective response rates of 26% and 47%. The <i>IFNL3</i> genotype was strongly associated with ethnicity. There was no association between <i>IFNL3</i> genotype and treatment response in HBeAg-positive or -negative patients. Independent predictors of treatment response were: sex, HBV DNA level and alanine aminotransferase level in HBeAg-positive Asian patients; age in HBeAg-negative Asian patients; and HBV DNA in HBeAg-negative white patients.</p><p>Conclusions</p><p>This is the largest analysis to date of associations between <i>IFNL3</i> genotype and peginterferon response in patients with chronic hepatitis B. The data suggest that <i>IFNL3</i> polymorphism is not a major determinant of the response to peginterferon alfa-2a in either HBeAg-positive or HBeAg-negative patients.</p></div

Rates of HBeAg seroconversion in HBeAg-positive patients according to genotype at rs12980275, rs12979860, and rs8099917.

<p>Some patients did not have data available for all genotypes, and percentages are calculated on the basis of the number of patients with data.</p

Associations between baseline covariates and treatment outcome in univariate logistic regression analyses.

<p>Associations are shown for HBeAg-positive Asian (A) and white (B) patients, and in HBeAg-negative Asian (C) and white (D) patients. ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus. After adjusting for significant baseline covariates, there were no statistically significant associations between <i>IFNL3</i> genotypes and response in the final logistic regression models (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199198#pone.0199198.g003" target="_blank">Fig 3</a>).</p

Demographic and clinical characteristics of patients Included in the analyses.

<p>Demographic and clinical characteristics of patients Included in the analyses.</p

Response rates according to host <i>IFNL3</i> genotype.

<p>Response rates according to genotype at rs12980275, rs12979860, and rs8099917 in HBeAg-positive Asian (A) and white (B) patients, and in HBeAg-negative Asian (C) and white (D) patients. Some patients did not have data available for all genotypes, and response rates are calculated on the basis of the number of patients with data.</p