The search for novel markers predicting the functional cure of chronic hepatitis B : proteomic profiling and immune studies

Abstract

This thesis aimed to identify potential proteins or immune factors that may be associated with hepatitis B surface antigen (HBsAg) seroclearance. Plasma protein profiling of HBsAg seroclearance are described in the Chapters 4 to 6 studies, with immune factors being described in the Chapter 7. Chapter 4 investigated protein profiling of HBsAg seroclearance using isobaric tags for relative and absolute quantitation (iTRAQ) based quantitative proteomics. Twenty chronic hepatitis B virus (HBV) infection patients achieving spontaneous HBsAg seroclearance with undetectable anti-HBs titre (experimental group), as well as 20 age- and sex-matched, treatment-naïve, hepatitis B e antigen-negative patients (control group) were enrolled. A total of 487 proteins were identified, of which 97 proteins showed altered expression. Eighty-eight proteins were found to be upregulated (abundance ratio >1.2), and 9 proteins down-regulated (abundance ratio <0.8). Bioinformatics tools including gene ontology (GO) enrichment analysis and Ingenuity Pathway Analysis (IPA) were applied to annotate the biological function of these differentially expressed proteins. The tandem mass spectrometry (MS/MS) spectrum were retrieved to confirm the intensity ratios of candidate proteins. Four proteins including fibronectin, CD44, Aldolase A (ALODA), and S100 calcium-binding protein A9 (S100A9) were finally regarded as candidate proteins. The plasma expressions of these four candidate proteins were then validated in Chapter 5 using a separate cohort with 90 similarly matched patients from each of the experimental (n=45) and control (n=45) groups. Enzyme-linked immunosorbent assay results showed that plasma expression of fibronectin was higher in patients achieving HBsAg seroclearance than that in control group. Chapter 6 examined whether fibronectin could be act as a potential biomarker for predicting HBsAg seroclearance using a historical cohort. One hundred and sixty-four patients (82 patients achieving HBsAg seroclearance and 82 age and gender-matched HBsAg-positive controls) had available archived plasma at Year -3, Year -1 prior to HBsAg seroclearance and at the time point of HBsAg seroclearance. In the subgroup patients with annual HBsAg log reduction >0.5 (N=60), the addition of plasma fibronectin level at Year -1 achieved the highest predictive value, with the area under the receiver operating characteristics (AUROC) of 0.884. Finally, Chapter 7 delineated the changes of regulatory T cells (Tregs) in chronic HBV infection patients achieving spontaneous HBsAg seroclearance. Twenty-seven patients achieving spontaneous HBsAg seroclearance as well as 27 age and gender-matched controls were recruited. Patient with spontaneous HBsAg seroclearance had a significantly downregulated expression of programmed cell death protein 1(PD-1) in CD4+ Tregs and CD4+ T cells, compared with that in control subjects. The expressions of glucocorticoid-induced tumor necrosis factor-related receptor (GITR) in CD8+ Tregs and in CD4+ T cells in patient with spontaneous HBsAg seroclearance were significantly reduced compared with that in control subjects. In this present thesis, plasma fibronectin level was identified to be associated with HBsAg seroclearance in chronic HBV infection; the phenotyping of Tregs and effector T cells was also altered in peripheral blood in such patients. The increased expression of fibronectin, as well as reduced expression of PD-1 and/or GITR, may act as novel biomarkers of the “functional cure” of chronic HBV.