Bulevirtide Combined with Pegylated Interferon for Chronic Hepatitis D.

BACKGROUND: In a phase 3 trial, bulevirtide monotherapy led to a virologic response in patients with chronic hepatitis D. Pegylated interferon (peginterferon) alfa-2a is recommended by guidelines as an off-label treatment for this disease. The role of combination therapy with bulevirtide and peginterferon alfa-2a, particularly with regard to finite treatment, is unclear. METHODS: In this phase 2b, open-label trial, we randomly assigned patients to receive peginterferon alfa-2a alone (180 µg per week) for 48 weeks; bulevirtide at a daily dose of 2 mg or 10 mg plus peginterferon alfa-2a (180 µg per week) for 48 weeks, followed by the same daily dose of bulevirtide for 48 weeks; or bulevirtide at a daily dose of 10 mg alone for 96 weeks. All the patients were followed for 48 weeks after the end of treatment. The primary end point was an undetectable level of hepatitis D virus (HDV) RNA at 24 weeks after the end of treatment. The primary comparison was between the 10-mg bulevirtide plus peginterferon alfa-2a group and the 10-mg bulevirtide monotherapy group. RESULTS: A total of 24 patients received peginterferon alfa-2a alone, 50 received 2 mg and 50 received 10 mg of bulevirtide plus peginterferon alfa-2a, and 50 received 10 mg of bulevirtide monotherapy. At 24 weeks after the end of treatment, HDV RNA was undetectable in 17% of the patients in the peginterferon alfa-2a group, in 32% of those in the 2-mg bulevirtide plus peginterferon alfa-2a group, in 46% of those in the 10-mg bulevirtide plus peginterferon alfa-2a group, and in 12% of those in the 10-mg bulevirtide group. For the primary comparison, the between-group difference was 34 percentage points (95% confidence interval, 15 to 50; P<0.001). At 48 weeks after the end of treatment, HDV RNA was undetectable in 25% of the patients in the peginterferon alfa-2a group, in 26% of those in the 2-mg bulevirtide plus peginterferon alfa-2a group, in 46% of those in the 10-mg bulevirtide plus peginterferon alfa-2a group, and in 12% of those in the 10-mg bulevirtide group. The most frequent adverse events were leukopenia, neutropenia, and thrombocytopenia. The majority of adverse events were of grade 1 or 2 in severity. CONCLUSIONS: The combination of 10-mg bulevirtide plus peginterferon alfa-2a was superior to bulevirtide monotherapy with regard to an undetectable HDV RNA level at 24 weeks after the end of treatment. (Funded by Gilead Sciences; MYR 204 ClinicalTrials.gov number, NCT03852433.).

Switching to tenofovir alafenamide in patients with virologically suppressed chronic hepatitis B and renal or hepatic impairment: final week 96 results from an open-label, multicentre, phase 2 study.

BACKGROUND: Phase 3 studies in patients with chronic hepatitis B have shown tenofovir alafenamide to have non-inferior efficacy to tenofovir disoproxil fumarate, with improved renal and bone safety. We conducted this study to evaluate the safety and efficacy of switching to tenofovir alafenamide in participants with chronic hepatitis B and renal or hepatic impairment. METHODS: This open-label, multicentre, phase 2 study was done in eight countries or territories at 30 sites. We recruited adults (≥18 years) with chronic hepatitis B who were virally suppressed on nucleoside or nucleotide analogues and had renal impairment (part A: moderate or severe in cohort 1 [estimated glomerular filtration rate by the Cockcroft-Gault formula (eGFRCG) 15-59 mL/min] or end-stage renal disease [eGFRCG <15 mL/min] on haemodialysis in cohort 2) or hepatic impairment including decompensation (part B: Child-Turcotte-Pugh score 7-12). Participants switched to 25 mg of tenofovir alafenamide given orally once daily for 96 weeks. The primary endpoint was the proportion of participants with viral suppression (HBV DNA <20 IU/mL) at week 24 by missing-equals-failure analysis. Efficacy (full analysis set) and safety (safety analysis set) analyses included all enrolled participants who received at least one dose of the study drug. Week 96 safety was assessed, including renal and bone parameters. This trial is registered at ClinicalTrials.gov, NCT03180619, and is completed. FINDINGS: 124 participants (93 in part A [78 in cohort 1 and 15 in cohort 2] and 31 in part B) were enrolled between Aug 11, 2017, and Oct 17, 2018, and included in the full and safety analysis sets. 106 (85%) participants completed the study. There were 69 (74%) men and 24 (26%) women in part A and 21 (68%) men and ten (32%) women in part B. At week 24, 91 (97·8%, 95% CI 92·4 to 99·7) of 93 individuals in part A (76 [97·4%, 91·0 to 99·7] of 78 in cohort 1 and 15 [100·0%, 78·2 to 100·0] of 15 in cohort 2) and 31 (100·0%, 88·8 to 100·0) in part B had HBV DNA of less than 20 IU/mL. By week 96, the most common adverse event was upper respiratory tract infection, which occurred in 14 (15%) participants in part A and in six (19%) participants in part B. Serious adverse events occurred in 20 (22%) part A participants and in ten (32%) part B participants; none were related to treatment. No treatment-related deaths occurred. At week 96, median change in estimated glomerular filtration rate (Cockcroft-Gault method) was 1·0 mL/min (IQR -2·8 to 4·5) in cohort 1 and -2·4 mL/min (-11·4 to 10·7) in part B. Mean changes in spine and hip bone mineral density were 1·02% (SD 4·44) and 0·20% (3·25) in part A and -0·25% (3·91) and 0·28% (3·25) in part B. INTERPRETATION: Tenofovir alafenamide might offer continued antiviral efficacy and a favourable safety profile for patients with renal or hepatic impairment and chronic hepatitis B switching from tenofovir disoproxil fumarate or other antivirals. FUNDING: Gilead Sciences.

TLR8 agonist selgantolimod regulates Kupffer cell differentiation status and impairs HBV entry into hepatocytes via an IL-6-dependent mechanism.

OBJECTIVE: Achieving HBV cure will require novel combination therapies of direct-acting antivirals and immunomodulatory agents. In this context, the toll-like receptor 8 (TLR8) agonist selgantolimod (SLGN) has been investigated in preclinical models and clinical trials for chronic hepatitis B (CHB). However, little is known regarding its action on immune effectors within the liver. Our aim was to characterise the transcriptomic changes and intercellular communication events induced by SLGN in the hepatic microenvironment. DESIGN: We identified TLR8-expressing cell types in the human liver using publicly available single-cell RNA-seq data and established a method to isolate Kupffer cells (KCs). We characterised transcriptomic and cytokine KC profiles in response to SLGN. SLGN's indirect effect was evaluated by RNA-seq in hepatocytes treated with SLGN-conditioned media (CM) and quantification of HBV parameters following infection. Pathways mediating SLGN's effect were validated using transcriptomic data from HBV-infected patients. RESULTS: Hepatic TLR8 expression takes place in the myeloid compartment. SLGN treatment of KCs upregulated monocyte markers (eg, S100A12) and downregulated genes associated with the KC identity (eg, SPIC). Treatment of hepatocytes with SLGN-CM downregulated NTCP and impaired HBV entry. Cotreatment with an interleukin 6-neutralising antibody reverted the HBV entry inhibition. CONCLUSION: Our transcriptomic characterisation of SLGN sheds light into the programmes regulating KC activation. Furthermore, in addition to its previously described effect on established HBV infection and adaptive immunity, we show that SLGN impairs HBV entry. Altogether, SLGN may contribute through KCs to remodelling the intrahepatic immune microenvironment and may thus represent an important component of future combinations to cure HBV infection.

Bulevirtide monotherapy in patients with chronic HDV: Efficacy and safety results through week 96 from a phase III randomized trial.

BACKGROUND & AIMS: Bulevirtide (BLV), a first-in-class entry inhibitor, is approved in Europe for the treatment of chronic hepatitis delta (CHD). BLV monotherapy was superior to delayed treatment at week (W) 48, the primary efficacy endpoint, in the MYR301 study (NCT03852719). Here, we assessed if continued BLV therapy until W96 would improve virologic and biochemical response rates, particularly among patients who did not achieve virologic response at W24. METHODS: In this ongoing, open-label, randomized phase III study, patients with CHD (N = 150) were randomized (1:1:1) to treatment with BLV 2 mg/day (n = 49) or 10 mg/day (n = 50), each for 144 weeks, or to delayed treatment for 48 weeks followed by BLV 10 mg/day for 96 weeks (n = 51). Combined response was defined as undetectable hepatitis delta virus (HDV) RNA or a decrease in HDV RNA by ≥2 log10 IU/ml from baseline and alanine aminotransferase (ALT) normalization. Other endpoints included virologic response, ALT normalization, and change in HDV RNA. RESULTS: Of 150 patients, 143 (95%) completed 96 weeks of the study. Efficacy responses were maintained and/or improved between W48 and W96, with similar combined, virologic, and biochemical response rates between BLV 2 and 10 mg. Of the patients with a suboptimal early virologic response at W24, 43% of non-responders and 82% of partial responders achieved virologic response at W96. Biochemical improvement often occurred independently of virologic response. Adverse events were mostly mild, with no serious adverse events related to BLV. CONCLUSIONS: Virologic and biochemical responses were maintained and/or increased with longer term BLV therapy, including in those with suboptimal early virologic response. BLV monotherapy for CHD was safe and well tolerated through W96. IMPACT AND IMPLICATIONS: In July 2023, bulevirtide was fully approved for the treatment of chronic hepatitis delta (CHD) in Europe based on clinical study results from up to 48 weeks of treatment. Understanding the efficacy and safety of bulevirtide over the longer term is important for healthcare providers. In this analysis, we demonstrate that bulevirtide monotherapy for 96 weeks in patients with CHD was associated with continued improvements in combined, virologic, and biochemical responses as well as liver stiffness from week 48 at both the 2 mg and 10 mg doses. Patients with suboptimal virologic responses to bulevirtide at week 24 also benefited from continued therapy, with the majority achieving virologic response or biochemical improvement by week 96. GOV IDENTIFIER: NCT03852719.

Utility of a Telephone Triage Hotline in Response to the COVID-19 Pandemic: Longitudinal Observational Study.

BACKGROUND: During the initial months of the COVID-19 pandemic, rapidly rising disease prevalence in the United States created a demand for patient-facing information exchanges that addressed questions and concerns about the disease. One approach to managing increased patient volumes during a pandemic involves the implementation of telephone-based triage systems. During a pandemic, telephone triage hotlines can be employed in innovative ways to conserve medical resources and offer useful population-level data about disease symptomatology and risk factor profiles. OBJECTIVE: The aim of this study is to describe and evaluate the COVID-19 telephone triage hotline used by a large academic medical center in the midwestern United States. METHODS: Michigan Medicine established a telephone hotline to triage inbound patient calls related to COVID-19. For calls received between March 24, 2020, and May 5, 2020, we described total call volume, data reported by callers including COVID-19 risk factors and symptomatology, and distribution of callers to triage algorithm endpoints. We also described symptomatology reported by callers who were directed to the institutional patient portal (online medical visit questionnaire). RESULTS: A total of 3929 calls (average 91 calls per day) were received by the call center during the study period. The maximum total number of daily calls peaked at 211 on March 24, 2020. Call volumes were the highest from 6 AM to 11 AM and during evening hours. Callers were most often directed to the online patient portal (1654/3929, 42%), nursing hotlines (1338/3929, 34%), or employee health services (709/3929, 18%). Cough (126/370 of callers, 34%), shortness of breath (101/370, 27%), upper respiratory infection (28/111, 25%), and fever (89/370, 24%) were the most commonly reported symptoms. Immunocompromised state (23/370, 6%) and age >65 years (18/370, 5%) were the most commonly reported risk factors. CONCLUSIONS: The triage algorithm successfully diverted low-risk patients to suitable algorithm endpoints, while directing high-risk patients onward for immediate assessment. Data collected from hotline calls also enhanced knowledge of symptoms and risk factors that typified community members, demonstrating that pandemic hotlines can aid in the clinical characterization of novel diseases.

Safety, pharmacokinetics and pharmacodynamics of selgantolimod, an oral Toll-like receptor 8 agonist: a Phase Ia study in healthy subjects.

BACKGROUND: Selgantolimod is a novel oral, selective Toll-like receptor 8 (TLR8) agonist in development for the treatment of chronic hepatitis B (CHB). TLR8 is an endosomal innate immune receptor and a target for treatment of viral infections. This first-in-human study investigated the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of selgantolimod in healthy volunteers. METHODS: Of 71 subjects enrolled, 59 received a single dose of selgantolimod (0.5, 1.5, 3 or 5 mg) or placebo, and 12 were evaluated for food effect. Safety, PK and PD activity by induction of cytokines, chemokines and acute phase proteins were assessed. PK/PD analyses were conducted. RESULTS: Single doses of 0.5-5 mg were generally safe. No serious adverse events (AEs) or AEs leading to discontinuation were reported, and most were Grade 1 in severity. Selgantolimod displayed rapid absorption and dose-proportional PK and PD activity. Food had minimal effect on PK but resulted in diminished PD activity. In PK/PD analyses, near-saturation of induction for most evaluated biomarkers occurred at the 5-mg dose. CONCLUSIONS: Single doses of up to 5 mg selgantolimod were safe and induced dose-dependent PD responses. These data support evaluation of selgantolimod in combination with other agents in future clinical studies of CHB. Australian New Zealand Clinical Trials Registration: ACTRN12616001646437.

Switching from tenofovir disoproxil fumarate to tenofovir alafenamide in virologically suppressed patients with chronic hepatitis B: a randomised, double-blind, phase 3, multicentre non-inferiority study.

BACKGROUND: Treatment with tenofovir disoproxil fumarate has been associated with renal toxicity or reductions in bone mineral density, or both, in some patients with chronic hepatitis B virus (HBV) infection. Tenofovir alafenamide is a tenofovir prodrug with high intrahepatic concentrations of active drug and reduced systemic tenofovir exposures compared with tenofovir disoproxil fumarate. In patients with chronic HBV, tenofovir alafenamide has shown efficacy non-inferior to that of tenofovir disoproxil fumarate with improved renal and bone safety. With this non-inferiority study, we aimed to evaluate the efficacy and safety of tenofovir alafenamide in patients with HBV infection switching from tenofovir disoproxil fumarate who are virally suppressed. METHODS: Patients with chronic HBV infection who had been receiving tenofovir disoproxil fumarate for 48 weeks or more and who had HBV DNA less than the lower limit of quantification (LLOQ) for at least 12 weeks were recruited to this randomised, multicentre, double-blind, phase 3 non-inferiority study. Patients were randomly assigned in a 1:1 ratio to receive tenofovir alafenamide 25 mg once a day or to continue tenofovir disoproxil fumarate 300 mg once a day. The primary efficacy endpoint was loss of virological control, defined as the proportion of patients who received at least one dose of study drug who had HBV DNA of at least 20 IU/mL at week 48 by the modified US Food and Drug Administration (FDA) snapshot algorithm. Key safety endpoints were changes in hip and spine bone mineral density, estimated creatinine clearance by Cockcroft-Gault, and markers of bone turnover and renal tubular function. The study was powered for non-inferiority in efficacy of tenofovir alafenamide versus tenofovir disoproxil fumarate with a 4% margin. Investigators and patients were unaware of treatment allocation and on-treatment results. This trial is ongoing and is registered with ClinicalTrials.gov, number NCT02979613. FINDINGS: Participants in this study were enrolled between Dec 29, 2016, and Oct 20, 2017. 541 patients were screened and 490 patients were randomly assigned to switch to tenofovir alafenamide or to stay on tenofovir disoproxil fumarate. Two patients assigned to receive tenofovir alafenamide did not receive treatment; thus the full analysis set for efficacy and safety analyses consisted of 243 patients in the tenofovir alafenamide group and 245 in the tenofovir disoproxil fumarate group. At week 48, one patient from each treatment group (both <1%) had HBV DNA of at least 20 IU/mL (difference in proportion 0·0%, 95% CI -1·9 to 2·0), thereby showing non-inferior efficacy of tenofovir alafenamide to tenofovir disoproxil fumarate. Patients who received tenofovir alafenamide had significantly increased bone mineral density at hip (mean change 0·66% [SD 2·08] vs -0·51% [SD 1·91]; difference in least square means 1·17% [95% CI 0·80 to 1·54; p<0·0001]) and at spine (mean change 1·74% [3·46] vs -0·11% [3·13]; difference in least square means 1·85% [1·24 to 2·46; p<0·0001]), creatinine clearance by Cockcroft-Gault relative to tenofovir disoproxil fumarate (median change 0·94 mL/min [IQR -4·47 to 6·24] vs -2·74 mL/min [-7·89 to 1·88]; p <0·0001), and improved markers of bone turnover and tubular function at week 48. The most common treatment-emergent adverse events were upper respiratory tract infection (18 [7%] of 243 patients in the tenofovir alafenamide group and 16 [7%] of 245 patients in the tenofovir disoproxil fumarate group) and nasopharyngitis (13 [5%] of 243 patients in the tenofovir alafenamide group and 12 [5%] of 245 patients in the tenofovir disoproxil fumarate group). The incidence of grade 3 and above adverse events and serious adverse events was low and similar between groups. No viral resistance was observed in patients who qualified for viral sequencing. INTERPRETATION: These findings suggest that tenofovir alafenamide can be substituted for tenofovir disoproxil fumarate in patients with HBV infection for improved safety without a loss of efficacy. FUNDING: Gilead Sciences.

Combined GS-4774 and Tenofovir Therapy Can Improve HBV-Specific T-Cell Responses in Patients With Chronic Hepatitis.

BACKGROUND & AIMS: One strategy to treat chronic hepatitis B virus (HBV) infection could be to increase the functions of virus-specific T cells. We performed a multicenter phase 2 study to evaluate the safety and efficacy of GS-4774, a yeast-based therapeutic vaccine engineered to express HBV antigens, given with tenofovir disoproxil fumarate (TDF) to untreated patients with chronic HBV infection. METHODS: We performed an open-label study at 34 sites in Canada, Italy, New Zealand, Romania, South Korea, and United States from July 2014 to August 2016. Adults who were positive for HB surface antigen (HBsAg) > 6 months and levels of HBV DNA ≥2000 IU/mL who had not received antiviral treatment for HBV within 3 months of screening were randomly assigned (1:2:2:2) to groups given oral TDF 300 mg daily alone (n = 27; controls) or with 2, 10, or 40 yeast units GS-4774 (n = 168), administered subcutaneously every 4 weeks until week 20 for a total of 6 doses. Blood samples were collected and analyzed and patients received regular physical examinations. Efficacy was measured by decrease in HBsAg from baseline to week 24. Specific responses to HBV (production of interferon gamma [IFNG], tumor necrosis factor [TNF], interleukin 2 [IL2], and degranulation) were measured in T cells derived from 12 HBeAg-negative patients with genotype D infections, after overnight or 10 days of stimulation of peripheral blood mononuclear cells with peptides from the entire HBV proteome. T-regulatory cells were analyzed for frequency and phenotype. Data from studies of immune cells were compared with data on reductions in HBsAg, HBV DNA, and alanine aminotransferase in blood samples from patients. RESULTS: GS-4774 was safe and well tolerated but did not produce significant decreases in levels of HBsAg. Production of IFNG, TNF, and IL2 increased significantly at weeks 24 and 48, compared with baseline, in HBV-specific CD8+ T cells from patients given GS-4774 but not from controls. GS-4774 had greater effects on CD8+ than CD4+ T cells, which were not affected at all or very weakly by TDF with or without GS-4774. GS-4774 did not affect responses of T cells to other viruses tested. HBV core peptides induced the greatest production of IFNG by T cells following overnight stimulation, whereas HBV envelope antigens did not induce a response. Following 10 days of stimulation, production of IFNG and TNF increased with time of exposure to GS-4774; the greatest levels of responses were to HBV envelope antigens followed by core and polymerase peptides. We observed a correlation in patients given GS-4774 between increased T-cell functions and reductions in numbers of T-regulatory cells. CONCLUSIONS: In a phase 2 study of patients with chronic HBV infection given TDF with or without GS-4774, we found that vaccination can increase production of IFNG, TNF, and IL2 by CD8+ T cells exposed to antigenic peptides, with little effect on CD4+ T cells. Although GS-4774 did not reduce levels of HBsAg in patients, its strong immune stimulatory effect on CD8+ T cells might be used in combination with other antiviral agents to boost the antivirus immune response. Clinicaltrials.gov no: NCT02174276.

Evaluating for Human Herpesvirus 6 in the Liver Explants of Children With Liver Failure of Unknown Etiology.

BACKGROUND: Liver failure of unknown etiology (LFUE) has a transplant-free survival rate <25%. Human herpesvirus 6 (HHV-6) may be associated with LFUE, but studies are limited by small sample size. METHODS: We identified all children who underwent liver transplant for LFUE at a single quaternary children's hospital; 51/65 cases could be age matched with controls (children who underwent liver transplant for metabolic liver disease). Quantitative polymerase chain reaction for HHV-6 was performed on DNA from formalin-fixed paraffin-embedded liver explant tissue. RESULTS: HHV-6 was detected in 34/51 cases (66.7%) and 19/51 controls (37.3%) (P = .005). Average HHV-6 viral load was 213207 copies/106 cells in positive cases (range: 7293-1102030) and 38115 copies/106 cells in positive controls (range: 1382-122375) (P = .0008). HHV-6 was present significantly more often in cases compared to controls in patients younger than 6 years. In particular, in patients younger than 3 years, HHV-6 was present in 13/27 cases (48.1%) and 2/27 controls (7.4%) (P = .0009). CONCLUSIONS: HHV-6 was detected in liver explants significantly more often and in higher quantities in children transplanted for LFUE compared to controls, suggesting HHV-6 should be evaluated in young children who present with LFUE.

Up-regulated expression of spherical body protein 2 truncated copy 11 in Babesia bovis is associated with reduced cytoadhesion to vascular endothelial cells.

The factors involved in gain or loss of virulence in Babesia bovis are unknown. Spherical body protein 2 truncated copy 11 (sbp2t11) transcripts in B. bovis were recently reported to be a marker of attenuation for B. bovis strains. Increased cytoadhesion of B. bovis-infected red blood cells (iRBC) to vascular endothelial cells is associated with severe disease outcomes and an indicator of parasite virulence. Here, we created a stable B. bovis transfected line over-expressing sbp2t11 to determine whether up-regulation of sbp2t11 is associated with changes in cytoadhesion. This line was designated sbp2t11up and five B. bovis clonal lines were derived from the sbp2t11up line by limiting dilution for characterisation. We compared the ability of iRBCs from the sbp2t11up line and its five derivative clonal lines to adhere to bovine brain endothelial cells, using an in vitro cytoadhesion assay. The same lines were selected for in vitro cytoadhesion and the levels of sbp2t11 transcripts in each selected line were quantified. Our results demonstrate that up-regulation of sbp2t11 is accompanied by a statistically significant reduction in cytoadhesion. Confirmed up-regulation of sbp2t11 in B. bovis concomitant with the reduction of iRBC in vitro cytoadhesion to bovine brain endothelial cell is consistent with our previous finding that up-regulation of sbp2t11 is an attenuation marker in B. bovis and suggests the involvement of sbp2t11 transcription in B. bovis virulence.

Improved Bone Safety of Tenofovir Alafenamide Compared to Tenofovir Disoproxil Fumarate Over 2 Years in Patients With Chronic HBV Infection.

BACKGROUND & AIMS: Long-term use of tenofovir disoproxil fumarate (TDF) reduces bone mineral density (BMD). Tenofovir alafenamide (TAF), a new prodrug of tenofovir, has shown non-inferior efficacy to TDF in patients with chronic hepatitis B virus (HBV) infection, with improved bone effects at 48 weeks. We performed a randomized trial to evaluate the bone safety of TAF compared with TDF over 2 years, assessing baseline risk factors for bone loss, were evaluated after 2 years of treatment. METHODS: In a double-blind study, hepatitis B e antigen (HBeAg)-positive patients (n = 873) and HBeAg-negative patients (n = 425) were randomly assigned (2:1) to groups given TAF (25 mg; n = 866) or TDF (300 mg; n = 432) once daily. We assessed bone safety, including hip and spine BMD, using dual-energy X-ray absorptiometry and measured changes in serum markers of bone turnover over 96 weeks. RESULTS: At baseline, treatment groups were well matched. At week 96, patients receiving TAF had significantly smaller decreases in hip BMD (mean reduction of 0.33%) than patients receiving TDF (mean reduction of 2.51%) (P < .001) and spine BMD (reduction of 0.75% in patients receiving patients receiving TAF vs reduction of 2.57% in patients receiving TDF) (P < .001). For hip BMD, the magnitude of difference in bone loss between the TAF and TDF groups increased at week 96 compared to week 48 (P < .001). The TAF group had minimal changes in markers of bone turnover by 12 weeks of treatment, but the TDF group had significant changes, compared to baseline. Risk factors for bone loss had fewer effects in patients receiving TAF than TDF at week 96. CONCLUSIONS: In double-blind randomized trials, we found that after 2 years of treatment, patients receiving TAF had continued improvements in bone safety compared with patients receiving TDF. Clinicaltrial.gov ID NCT01940471 and NCT01940341.

96 weeks treatment of tenofovir alafenamide vs. tenofovir disoproxil fumarate for hepatitis B virus infection.

BACKGROUND & AIMS: Tenofovir alafenamide (TAF) is a new prodrug of tenofovir developed to treat patients with chronic hepatitis B virus (HBV) infection at a lower dose than tenofovir disoproxil fumarate (TDF) through more efficient delivery of tenofovir to hepatocytes. In 48-week results from two ongoing, double-blind, randomized phase III trials, TAF was non-inferior to TDF in efficacy with improved renal and bone safety. We report 96-week outcomes for both trials. METHODS: In two international trials, patients with chronic HBV infection were randomized 2:1 to receive 25 mg TAF or 300 mg TDF in a double-blinded fashion. One study enrolled HBeAg-positive patients and the other HBeAg-negative patients. We assessed efficacy in each study, and safety in the pooled population. RESULTS: At week 96, the differences in the rates of viral suppression were similar in HBeAg-positive patients receiving TAF and TDF (73% vs. 75%, respectively, adjusted difference -2.2% (95% CI -8.3 to 3.9%; p = 0.47), and in HBeAg-negative patients receiving TAF and TDF (90% vs. 91%, respectively, adjusted difference -0.6% (95% CI -7.0 to 5.8%; p = 0.84). In both studies the proportions of patients with alanine aminotransferase above the upper limit of normal at baseline, who had normal alanine aminotransferase at week 96 of treatment, were significantly higher in patients receiving TAF than in those receiving TDF. In the pooled safety population, patients receiving TAF had significantly smaller decreases in bone mineral density than those receiving TDF in the hip (mean % change -0.33% vs. -2.51%; p <0.001) and lumbar spine (mean % change -0.75% vs. -2.57%; p <0.001), as well as a significantly smaller median change in estimated glomerular filtration rate by Cockcroft-Gault method (-1.2 vs. -4.8 mg/dl; p <0.001). CONCLUSION: In patients with HBV infection, TAF remained as effective as TDF, with continued improved renal and bone safety, two years after the initiation of treatment. Clinicaltrials.gov number: NCT01940471 and NCT01940341. LAY SUMMARY: At week 96 of two ongoing studies comparing the efficacy and safety of tenofovir alafenamide (TAF) to tenofovir disoproxil fumarate (TDF) for the treatment of chronic hepatitis B patients, TAF continues to be as effective as TDF with continued improved renal and bone safety. Registration: Clinicaltrials.gov number: NCT01940471 and NCT01940341.

TLR7 Agonist Increases Responses of Hepatitis B Virus-Specific T Cells and Natural Killer Cells in Patients With Chronic Hepatitis B Treated With Nucleos(T)Ide Analogues.

BACKGROUND & AIMS: The oral Toll-like receptor (TLR) 7 agonist GS-9620 has antiviral effects in woodchuck and chimpanzee models of chronic hepatitis B virus (HBV) infection. We investigated, in a clinical trial, the capacity of this agent to reconstitute protective immunity in patients with chronic HBV infection. METHODS: We performed a prospective study of 28 patients with suppression of HBV infection by nucleos(t)ide analogue therapy and who tested negative for hepatitis B e antigen at 4 medical centers in Italy. Patients were randomly assigned (1:3:3:3) to groups given placebo or different doses of GS-9620 (1, 2, and 4 mg, weekly for 12 weeks). We added data from 8 patients receiving nucleos(t)ide analogue therapy to the placebo group (controls); 13 treatment-naïve patients with chronic HBV infection and 15 subjects who spontaneously recovered from an acute HBV infection served as additional controls. Peripheral blood mononuclear cells were collected at baseline, during administration of GS-9620 or placebo, and 12 weeks afterward. Phenotype and function of natural killer (NK) and HBV-specific T cells were analyzed by flow cytometry. T cells were expanded by incubation with peptides from the entire HBV proteome and studied after overnight or 10 days culture. NK-cell inhibition of T-cell responses was measured by assessing cytokine production by T cells stimulated with peptides in the presence or absence of NK cells. RESULTS: T cells collected at baseline before addition of GS-9620, when patients were receiving only nucleos(t)ide therapy, had greater responses to HBV than T cells from treatment-naïve patients, based on cytokine production in response to HBV peptides. However, during or after administration of GS-9620, T cells produced higher levels of cytokines compared to baseline. NK-cell activation and function increased after patients were given GS-9620, but the ability of NK cells to suppress T-cell responses was lower during GS-9620 therapy than before. Changes in T-cell or NK-cell function did not correlate with levels of hepatitis B surface antigen. Serum levels of hepatitis B surface antigen did not decrease significantly compared to baseline in patients given any dose of GS-9620. CONCLUSIONS: Twelve weeks administration of GS-9620 had no significant effect on serum hepatitis B surface antigen levels, but did appear to increase T-cell and NK-cell responses and reduce the ability of NK to suppress T cells. GS-9620 might therefore be included in therapies to increase the immune response to HBV.

Safety, efficacy and pharmacodynamics of vesatolimod (GS-9620) in virally suppressed patients with chronic hepatitis B.

BACKGROUND & AIMS: Vesatolimod (GS-9620) is an oral agonist of toll-like receptor 7, an activator of innate and adaptive immune responses. Herein the safety and efficacy of vesatolimod is assessed after once-weekly treatment in patients with chronic hepatitis B (CHB) infection suppressed on oral antiviral treatment. METHODS: In a phase II, double-blind, randomized, placebo (PBO)-controlled study, 162 patients stratified by hepatitis B surface antigen (HBsAg) levels and serum hepatitis B e antigen (HBeAg) status were randomized 1:3:3:3 to once-weekly oral PBO or vesatolimod (1-, 2-, or 4-mg doses) for 4, 8 or 12 weeks per cohort. Efficacy was assessed by change in baseline HBsAg (log10 IU/ml) at the primary endpoint (Week 24). Safety assessments included adverse events (AE) and laboratory abnormality monitoring. Pharmacodynamic assessments included peripheral cytokine level quantification and interferon-stimulated gene (ISG) mRNA expression evaluation. RESULTS: The majority of patients were male (76%) and HBeAg-negative (79%) at baseline. Most (41-80%) experienced ≥1 AE during the study with the majority of AEs mild or moderate in severity. No significant declines in HBsAg were observed at the primary (Week 24) or secondary endpoints (Weeks 4, 8, 12, and 48). ISG15 induction was dose-dependent and consistent after repeat dosing, returning closer to baseline by one week after treatment at all dose levels; no patient demonstrated significant serum interferon alpha (IFNα) expression at any timepoint evaluated. Multivariate analyses showed that ≥2-fold ISG15 induction is associated with 2- or 4-mg vesatolimod dose and female sex. CONCLUSIONS: Vesatolimod was safe and well-tolerated in patients with CHB, demonstrating consistent dose-dependent pharmacodynamic induction of ISG15 without significant systemic induction of IFNα expression or related symptoms. However, no significant HBsAg declines were observed. LAY SUMMARY: In a phase II study, vesatolimod, an oral, once-weekly, experimental immune-activating drug for the treatment of hepatitis B virus (HBV), is safe and well-tolerated in chronic HBV patients who are virally suppressed on oral antiviral treatment. Despite demonstrating on-target biomarker responses in patients, no significant declines in hepatitis B surface antigen were observed. Clinical Trial Number: GS-US-283-1059; NCT 02166047.

Extended-spectrum antiprotozoal bumped kinase inhibitors: A review.

Many life-cycle processes in parasites are regulated by protein phosphorylation. Hence, disruption of essential protein kinase function has been explored for therapy of parasitic diseases. However, the difficulty of inhibiting parasite protein kinases to the exclusion of host orthologues poses a practical challenge. A possible path around this difficulty is the use of bumped kinase inhibitors for targeting calcium-dependent protein kinases that contain atypically small gatekeeper residues and are crucial for pathogenic apicomplexan parasites' survival and proliferation. In this article, we review efficacy against the kinase target, parasite growth in vitro, and in animal infection models, as well as the relevant pharmacokinetic and safety parameters of bumped kinase inhibitors.

Absence of miR-182 Augments Cardiac Allograft Survival.

BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate the posttranscriptional expression of target genes and are important regulators in immune responses. Previous studies demonstrated that the miRNA, miR-182 was significantly increased during allograft rejection. Further, the transcription factor Forkhead box (FOX) protein 1, (FOXO1) was shown to be a target of miR-182. The aim of this study is to further examine the role of miR-182 in alloimmune responses. METHODS: Transplantation of BALB/c cardiac allografts was performed in C57BL/6, miR-182, B6.129S-H2 (MHC II and CD4 T cell-deficient) and B6.129S2-Tap1 (MHC I and CD8 T cell-deficient) mice, with or without CTLA-4Ig administration. T cell phenotype, FOXO1 protein levels and graft infiltrating lymphocytes were determined in C57BL/6 or miR-182 mice by flow cytometric analysis, Western blot, and immunohistochemistry, respectively. RESULTS: We now show that T cells, mainly CD4 are the main cellular source of miR-182 during allograft rejection. In the absence of miR-182, CTLA-4Ig treatment significantly increased allograft survival (31.5 days C57BL/6 vs 60 days miR-182; P < 0.01). Further, CTLA4-Ig treatment inhibits miR-182 expression, increases FOXO1 levels, and reduces the percentage of CD4CD44 T cells after transplantation. Fewer T cells infiltrate the cardiac allografts, and memory T cells are significantly decreased in allograft recipients deficient in miR-182 with CTLA4-Ig treatment (P < 0.01). CONCLUSIONS: Our findings suggest that miR-182 contributes to the T-cell responses to alloantigen especially under costimulation blockade. Therapeutics that target specific miRNAs may prove beneficial in transplantation.

Mass cytometry reveals a distinct immunoprofile of operational tolerance in pediatric liver transplantation.

Long-term IS in transplant patients has significant morbidity, poorer quality of life, and substantial economic costs. TOL, defined as graft acceptance without functional impairment in the absence of IS, has been achieved in some pediatric LT recipients. Using mass cytometry, peripheral blood immunotyping was performed to characterize differences between tolerant patients and patients who are stable on single-agent IS. Single-cell mass cytometry was performed using blood samples from a single-center pediatric LT population of operationally tolerant patients to comprehensively characterize the immune cell populations in the tolerant state compared with patients on chronic low-dose IS. Specific T-cell populations of interest were confirmed by flow cytometry. This high-dimensional phenotypic analysis revealed distinct immunoprofiles between transplant populations as well as a CD4+ TOT (CD4+ CD5+ CD25+ CD38-/lo CD45RA) that correlates with tolerance in pediatric LT recipients. In TOL patients, the TOT was significantly increased as compared to patients stable on low levels of IS. This TOT cell was confirmed by flow cytometry and is distinct from classic Treg cells. These results demonstrate the power of mass cytometry to discover significant immune cell signatures that have diagnostic potential.

Expression of 6-Cys Gene Superfamily Defines Babesia bovis Sexual Stage Development within Rhipicephalus microplus.

Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the causative agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) where their orthologues in Plasmodium parasite have been shown to encode for proteins required for the development of sexual stages. The current study identified four additional 6-Cys genes (G, H, I, J) in the B. bovis genome. These four genes are described in the context of the complete ten 6-Cys gene superfamily. The proteins expressed by this gene family are predicted to be secreted or surface membrane directed. Genetic analysis comparing the 6-Cys superfamily among five distinct B. bovis strains shows limited sequence variation. Additionally, A, B, E, H, I and J genes were transcribed in B. bovis infected tick midgut while genes A, B and E were also transcribed in the subsequent B. bovis kinete stage. Transcription of gene C was found exclusively in the kinete. In contrast, transcription of genes D, F and G in either B. bovis infected midguts or kinetes was not detected. None of the 6-Cys transcripts were detected in B. bovis blood stages. Subsequent protein analysis of 6-Cys A and B is concordant with their transcript profile. The collective data indicate as in Plasmodium parasite, certain B. bovis 6-Cys family members are uniquely expressed during sexual stages and therefore, they are likely required for parasite reproduction. Within B. bovis specifically, proteins encoded by 6-Cys genes A and B are markers for sexual stages and candidate antigens for developing novel vaccines able to interfere with the development of B. bovis within the tick vector.