BTLA contributes to acute-on-chronic liver failure infection and mortality through CD4+ T-cell exhaustion.

B- and T-lymphocyte attenuator (BTLA) levels are increased in patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF). This condition is characterized by susceptibility to infection and T-cell immune exhaustion. However, whether BTLA can induce T-cell immune exhaustion and increase the risk of infection remains unclear. Here, we report that BTLA levels are significantly increased in the circulating and intrahepatic CD4+ T cells from patients with HBV-ACLF, and are positively correlated with disease severity, prognosis, and infection complications. BTLA levels were upregulated by the IL-6 and TNF signaling pathways. Antibody crosslinking of BTLA activated the PI3K-Akt pathway to inhibit the activation, proliferation, and cytokine production of CD4+ T cells while promoting their apoptosis. In contrast, BTLA knockdown promoted their activation and proliferation. BTLA-/- ACLF mice exhibited increased cytokine secretion, and reduced mortality and bacterial burden. The administration of a neutralizing anti-BTLA antibody reduced Klebsiella pneumoniae load and mortality in mice with ACLF. These data may help elucidate HBV-ACLF pathogenesis and aid in identifying novel drug targets.

Comparison of Pegylated Interferon Alfa Therapy in Combination with Tenofovir Alafenamide Fumarate or Tenofovir Disoproxil Fumarate for Treatment of Chronic Hepatitis B Patients.

Purpose: The study aims to evaluate the effectiveness of a tenofovir alafenamide fumarate (TAF) and pegylated interferon alfa (PegIFN-α) regimen compared to a tenofovir disoproxil fumarate (TDF) and PegIFN-α therapy in patients with chronic hepatitis B (CHB). Patients and Methods: Patients who were treated with PegIFN-α in combination with TAF or TDF were retrospectively enrolled. The primary outcome measured was the HBsAg loss rate. The rates of virological response, serological response for HBeAg, and normalization of alanine aminotransferase (ALT) were also calculated. The cumulative incidences of response rates were compared between the two groups using Kaplan-Meier analysis. Results: A total of 114 patients were retrospectively enrolled in the study, with 33 receiving TAF plus PegIFN-α treatment and 81 receiving TDF plus PegIFN-α treatment. The HBsAg loss rate for the TAF plus PegIFN-α group was 15.2% at 24 weeks and 21.2% at 48 weeks, while the TDF plus PegIFN-α group had rates of 7.4% at 24 weeks and 12.3% at 48 weeks (P=0.204 at 24 weeks, P=0.228 at 48 weeks). In subgroup analysis of HBeAg positive patients, the TAF group had a higher HBsAg loss rate of 25% at week 48, compared to 3.8% in the TDF group (P=0.033). According to Kaplan-Meier analysis, the TAF plus PegIFN-α group achieved virological response more quickly than the TDF plus PegIFN-α group (p=0.013). There was no statistical difference in HBeAg serological rate or ALT normalization rate. Conclusion: There was no significant difference in the HBsAg loss between the two groups. However, subgroup analysis revealed that TAF plus PegIFN-α treatment had a higher HBsAg loss rate than TDF plus PegIFN-α treatment in HBeAg-positive patients. Additionally, TAF plus PegIFN-α treatment demonstrated better virological suppression for CHB patients. Therefore, TAF plus PegIFN-α treatment regimen is recommended for CHB patients who aim to achieve functional cure.

Reduction of Hepatitis B Surface Antigen May Be More Significant in PEGylated Interferon-Alpha Therapy Combined with Nucleotide Analogues than Combined with Nucleoside Analogues in Chronic Hepatitis B Patients: A Propensity Score Matching Study.

Background: Nucleotide analogues (NTs) monotherapy may have a more significant effect on reducing hepatitis B surface antigen (HBsAg) than nucleoside analogues (NSs) due to their immunomodulatory function. However, this superiority remains unknown when combined with PEGylated interferon α (PegIFNα). Therefore, this study aimed to explore whether NTs have more significant antiviral effects than NSs in combination therapy with PegIFNα. Methods: Chronic hepatitis B (CHB) patients treated with PegIFNα plus nucleos(t)ide analogues (NAs) were retrospectively recruited. Efficacy and the predictors of hepatitis B surface antigen (HBsAg) reduction >1 log10 IU/mL after 48 weeks were analyzed. Results: A total of 95 patients were included and divided into the PegIFNα + NTs group and the PegIFNα + NSs group. Propensity score matching (PSM) was performed. The PegIFNα + NTs group had a greater reduction of HBsAg (-3.52 vs. -2.33 log10 IU/mL, P=0.032) and a higher proportion of patients with HBsAg reduction >1 log10 IU/mL (100.0% vs. 72.2%, P=0.003) even after PSM. However, HBsAg and hepatitis B e-antigen (HBeAg) loss rates, HBeAg seroconversion rates, degree of HBeAg and hepatitis B virus (HBV) DNA decline, HBV DNA undetectable rates, and alanine aminotransferase (ALT) normalization rates showed no significant differences. Subgroup analyses showed the difference in the reduction of HBsAg was particularly evident in HBeAg-positive and the "add-on" subgroups. PegIFNα plus NTs (OR = 36.667, 95% CI = 3.837-350.384) was an independent predictor for HBsAg reduction >1 log10 IU/mL after 48 weeks. Conclusion: This study suggests that PegIFNα plus NTs may lead to more HBsAg reduction, especially in HBeAg-positive and "add-on" patients.

Thyroid dysfunction incidence and risk factors in Chinese chronic hepatitis B patients treated with pegylated interferon alpha: A long-term follow-up study.

The long-term impact, incidence and risk factors of thyroid dysfunction in chronic hepatitis B (CHB) patients receiving pegylated interferon (IFN) alpha (PegIFN-alpha) therapy remain unclear. We aim to investigate the long-term safety of thyroid dysfunction in CHB patients receiving PegIFN-alpha. A retrospective observational study of 425 CHB patients with normal baseline thyroid function was carried out. Patients were followed up over 10 years to assess thyroid function after receiving IFN. At the end of the IFN therapy, 67 patients (15.8%) had developed thyroid dysfunction, 31 patients (46.3%) had hyperthyroidism and 64.4% presented with subclinical thyroid dysfunction. In follow-up of thyroid dysfunction patients, 37 patients (74.0%) spontaneously regained normal thyroid function. Pretreatment thyroid-stimulating hormone (TSH) level, thyroid peroxidase antibody (TPOAb) positivity and free thyroxine (FT4) were independent risk factors associated with thyroid dysfunction incidence. High TSH level (OR = 9.866, 95%CI, 3.245-29.998) was associated with a greater likelihood of hypothyroidism. High FT4 levels (OR = 0.464, 95%CI, 0.248-0.868) indicate a low likelihood of thyroid dysfunction. Thyroid dysfunction is a common but acceptable side effect of IFN therapy for CHB. Most thyroid dysfunction is reversible. Pretreatment TSH level and TPOAb positivity are risk factors for thyroid dysfunction development during IFN therapy. A high TSH level predicts an increased incidence of hypothyroidism. Moreover, FT4 may be a protective factor for thyroid dysfunction.

Hepatitis B e antigen induces the expansion of monocytic myeloid-derived suppressor cells to dampen T-cell function in chronic hepatitis B virus infection.

Chronic hepatitis B virus (HBV) infection is associated with functionally impaired virus-specific T cell responses. Although the myeloid-derived suppressor cells (MDSCs) are known to play a critical role in impairing antiviral T cell responses, viral factors responsible for the expansion of MDSCs in chronic hepatitis B (CHB) remain obscure. In order to elucidate the mechanism of monocytic MDSCs (mMDSCs) expansion and T cell function suppression during persistent HBV infection, we analyzed the circulation frequency of mMDSCs in 164 CHB patients and 70 healthy donors, and found that the proportion of mMDSCs in HBeAg (+) CHB patients was significantly increased compared to that in HBeAg (-) patients, which positively correlated with the level of HBeAg. Furthermore, exposure of peripheral blood mononuclear cells (PBMCs) isolated from healthy donors to HBeAg led to mMDSCs expansion and significant upregulation of IL-1ß, IL-6 and indoleamine-2, 3-dioxygenase (IDO), and depletion of the cytokines abrogated HBeAg-induced mMDSCs expansion. Moreover, HBeAg-induced mMDSCs suppressed the autologous T-cell proliferation in vitro, and the purified mMDSCs from HBeAg (+) subjects markedly reduced the proliferation of CD4+ and CD8+ T cells and IFN-γ production, which could be efficiently restored by inhibiting IDO. In summary, HBeAg-induced mMDSCs expansion impairs T cell function through IDO pathway and favors the establishment of a persistent HBV infection, suggesting a mechanism behind the development of HBeAg-induced immune tolerance.

Adding an Artificial Tail-Anchor to a Peptide-Based HIV-1 Fusion Inhibitor for Improvement of Its Potency and Resistance Profile.

Peptides derived from the C-terminal heptad repeat (CHR) of human immunodeficiency virus type 1 (HIV-1) envelope protein transmembrane subunit gp41, such as T20 (enfuvirtide), can bind to the N-terminal heptad repeat (NHR) of gp41 and block six-helix bundle (6-HB) formation, thus inhibiting HIV-1 fusion with the target cell. However, clinical application of T20 is limited because of its low potency and genetic barrier to resistance. HP23, the shortest CHR peptide, exhibits better anti-HIV-1 activity than T20, but the HIV-1 strains with E49K mutations in gp41 will become resistant to it. Here, we modified HP23 by extending its C-terminal sequence using six amino acid residues (E6) and adding IDL (Ile-Asp-Leu) to the C-terminus of E6, which is expected to bind to the shallow pocket in the gp41 NHR N-terminal region. The newly designed peptide, designated HP23-E6-IDL, was about 2- to 16-fold more potent than HP23 against a broad spectrum of HIV-1 strains and more than 12-fold more effective against HIV-1 mutants resistant to HP23. These findings suggest that addition of an anchor-tail to the C-terminus of a CHR peptide will allow binding with the pocket in the gp41 NHR that may increase the peptide's antiviral efficacy and its genetic barrier to resistance.

Creating an Artificial Tail Anchor as a Novel Strategy To Enhance the Potency of Peptide-Based HIV Fusion Inhibitors.

20 (enfuvirtide) and other peptides derived from the human immunodeficiency virus type 1 (HIV-1) gp41 C-terminal heptad repeat (CHR) region inhibit HIV fusion by binding to the hydrophobic grooves on the N-terminal heptad repeat (NHR) trimer and blocking six-helix-bundle (6-HB) formation. Several strategies focusing on the binding grooves of the NHR trimer have been adopted to increase the antiviral activity of the CHR peptides. Here, we developed a novel and simple strategy to greatly enhance the potency of the existing peptide-based HIV fusion inhibitors. First, we identified a shallow pocket adjacent to the groove in the N-terminal region of NHR trimer as a new drug target, and then we designed several short artificial peptides to fit this target. After the addition of IDL (Ile-Asp-Leu) to the C terminus of CHR peptide WQ or MT-WQ, the conjugated peptides, WQ-IDL and MT-WQ-IDL, showed much more potent activities than WQ and T20, respectively, in inhibiting HIV-1 IIIB infection. WQ-IDL and MT-WQ-IDL were also more effective than WQ in blocking HIV-1 Env-mediated membrane fusion and had higher levels of binding affinity with NHR peptide N46. We solved the crystal structure of the 6-HB formed by MT-WQ-IDL and N46 and found that, besides the N-terminal MT hook tail, the IDL tail anchor of MT-WQ-IDL also binds with the shallow hydrophobic pocket outside the groove of the NHR trimer, resulting in enhanced inhibition of HIV-1 fusion with the target cell. It is expected that this novel approach can be widely used to improve the potency of peptidic fusion inhibitors against other enveloped viruses with class I fusion proteins. IMPORTANCE: The hydrophobic groove of the human immunodeficiency virus type 1 (HIV-1) gp41 NHR trimer has been known as the classic drug target to develop fusion inhibitors derived from the gp41 CHR. Here, we developed a novel and simple strategy to improve the existing peptide-based HIV fusion inhibitors. We identified a shallow pocket adjacent to the groove in the NHR trimer and added a short artificial peptide consisting of three amino acids (IDL) to the C terminus of a fusion inhibitor to fit this new target. The inhibition activity of this new conjugated peptide was significantly enhanced, by 77-fold, making it much more potent than T20 (enfuvirtide) and suggesting that the IDL tail can be adopted for optimizing existing HIV-1 CHR peptide fusion inhibitors. This new approach of identifying a potential binding pocket outside the traditional target and creating an artificial tail anchor can be widely applied to design novel fusion inhibitors against other class I enveloped viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV).

Rational improvement of gp41-targeting HIV-1 fusion inhibitors: an innovatively designed Ile-Asp-Leu tail with alternative conformations.

Peptides derived from the C-terminal heptad repeat (CHR) of HIV gp41 have been developed as effective fusion inhibitors against HIV-1, but facing the challenges of enhancing potency and stability. Here, we report a rationally designed novel HIV-1 fusion inhibitor derived from CHR-derived peptide (Trp628~Gln653, named CP), but with an innovative Ile-Asp-Leu tail (IDL) that dramatically increased the inhibitory activity by up to 100 folds. We also determined the crystal structures of artificial fusion peptides N36- and N43-L6-CP-IDL. Although the overall structures of both fusion peptides share the canonical six-helix bundle (6-HB) configuration, their IDL tails adopt two different conformations: a one-turn helix with the N36, and a hook-like structure with the longer N43. Structural comparison showed that the hook-like IDL tail possesses a larger interaction interface with NHR than the helical one. Further molecular dynamics simulations of the two 6-HBs and isolated CP-IDL peptides suggested that hook-like form of IDL tail can be stabilized by its binding to NHR trimer. Therefore, CP-IDL has potential for further development as a new HIV fusion inhibitor, and this strategy could be widely used in developing artificial fusion inhibitors against HIV and other enveloped viruses.