HBV-STOP study: Large hepatitis B virus flares off nucleot(s)ide analog therapy cause large innate immune response.

Abstract

Background and Aim: Current guidelines recommend indefinite nucleot( s)ide analog (NA) therapy for patients with HBeAg-negative chronic hepatitis B virus (HBV). However, sustained virological response (SVR) has been described in patients after discontinuation of long-term NA therapy. The HBV-STOP study is a prospective multicenter study of NA discontinuation in patients who have achieved long-term virological suppression on treatment. In addition to previously describing clinical outcomes after treatment discontinuation, we also aim to detail the immunological response in this scenario, especially that of the innate immune system. Method(s): Stimulation study: To gauge the potential innate activation of the peripheral blood cells by the flare, we stimulated peripheral blood mononuclear cell (PBMC) samples of HBV-STOP study patients by toll-like receptor (TLR)-specific ligands (TLR-2, TLR-3, TLR-4, TLR-7/8, and TLR-9) ex vivo. These patients either had a large biochemical flare (alanine aminotransferase [ALT] level > 10 x ULN) or did not have a biochemical flare (ALT < ULN). PBMCs were tested at baseline and peak ALT level time points, and matched non-flare samples were used as controls. Cytokine levels (IL-6, IL-8, IL-10, TNF, CCL-2, and CXCL-10) were measured after PBMC stimulation. Flow cytometry study: Additionally, we performed flow cytometry on PBMC samples of HBV-STOP study patients who either had a large biochemical flare (ALT > 10 x ULN) or did not have a biochemical flare. We did these studies at baseline and peak ALT level time points. Non-flare samples were matched to the closest respective week. Flow cytometry was used to isolate specific natural killer (NK) cell and monocyte populations using CD 56, CD 3, CD 16, and CD 14 cell surface markers. Expression of various immune markers was assessed in each cell population, including NKP46, NKG2D, TLR2, TLR4, and TREM1. Result(s): The stimulation study cohort consisted of 13 patients with flares and 12 patients without flares. Eight of the flare patients and six of the non-flare patients were also used in the flow cytometry study. Stimulation study: Comparisons between ratios of change from baseline to peak ALT samples with baseline to baseline samples were made for large flare and non-flare patients. Large flare values were superior to all non-flare values (Table 1), except for CXCL-10/TLR9 (0.79 vs 2.58), IL-6/ unstimulated (0.58 vs 1.13), and TNF/unstimulated (0.79 vs 1.08). Large flare ratios were all > 1, except for CXCL-10/TLR9, CXCL-10/TLR3, and unstimulated samples. Comparisons between large flare ratios had P values < 0.05, except for IL-8/TLR4, IL-10/TLR4, TNF/TLR3, CXCL-10 (TLR3, TLR 7/8, and TLR9), and unstimulated samples. A P value < 0.05 was seen in comparison between non-flare ratios for CXCL-10/TLR9, for which the baseline-flare ratio was superior to that of the large flare ratio. Flow cytometry study: Comparisons between ratios of change from baseline to peak ALT samples with baseline to baseline samples were made for large flare and non-flare patients. Large flare values were superior to all non-flare values (Table 2), except for classical monocytes/TLR2 (0.941 vs 0.942) and non-classical monocytes/TLR2 (1.07 vs 1.25). However, large flare ratios were all < 1 for classical and intermediate monocytes (TLR2, TLR4, and TREM1), non-classical monocytes (NKG2D, NKP46, andTLR4), NK Bright/NKG2D, and NK Dim/NKG2D. Comparisons between large flare ratios had significant P values < 0.05 for all NK and monocyte populations. Conclusion(s): In this analysis, we have shown that large HBV flares off NA therapy are associated with a significant inflammatory response from the innate immune system, including multiple proinflammatory cytokines. This is also associated with activation of the TLR receptors on NK cells and monocytes. Our study shows that TLRs, especially TLR2, appear to play a key role in this innate immune response to HBV flares, which highlights their ongoing importance as potential immunotherapeutic options for chronic hepatitis B management. Interestingly, TLR2 activation has been previously shown to increase production of proinflammatory cytokines IL-6 and TNF in hepatocytes, which are able to inhibit HBV replication in primary hepatocytes. We also showed that proinflammatory receptors and cytokines are indeed activated during the large inflammatory response that is associated with a large HBV flare, which has not been shown previously in a cohort of patients experiencing HBV flares after NA cessation.