Evaluation of in vitro antiviral activity of SARS-CoV-2 Mpro inhibitor pomotrelvir and cross-resistance to nirmatrelvir resistance substitutions.

The unprecedented scale of the COVID-19 pandemic and the rapid evolution of SARS-CoV-2 variants underscore the need for broadly active inhibitors with a high barrier to resistance. The coronavirus main protease (Mpro) is an essential cysteine protease required for viral polyprotein processing and is highly conserved across human coronaviruses. Pomotrelvir is a novel Mpro inhibitor that has recently completed a phase 2 clinical trial. In this report, we demonstrated that pomotrelvir is a potent competitive inhibitor of SARS-CoV-2 Mpro with high selectivity against human proteases. In the enzyme assay, pomotrelvir is also active against Mpro proteins derived from human coronaviruses CoV-229E, CoV-OC43, CoV-HKU1, CoV-NL63, MERS, and SARS-CoV. In cell-based SARS-CoV-2 replicon and SARS-CoV-2 infection assays, pomotrelvir has shown potent inhibitory activity and is broadly active against SARS-CoV-2 clinical isolates including Omicron variants. Many resistance substitutions of the Mpro inhibitor nirmatrelvir confer cross-resistance to pomotrelvir, consistent with the finding from our enzymatic analysis that pomotrelvir and nirmatrelvir compete for the same binding site. In a SARS-CoV-2 infection assay, pomotrelvir is additive when combined with remdesivir or molnupiravir, two nucleoside analogs targeting viral RNA synthesis. In conclusion, our results from the in vitro characterization of pomotrelvir antiviral activity support its further clinical development as an alternative COVID-19 therapeutic option.

Safety and efficacy of vebicorvir administered with entecavir in treatment-naïve patients with chronic hepatitis B virus infection.

BACKGROUND & AIMS: Nucleos(t)ide reverse transcriptase inhibitors do not completely suppress HBV DNA in chronic HBV infection (cHBV). Vebicorvir (VBR) is an investigational core inhibitor that interferes with multiple aspects of HBV replication. This phase II trial evaluated the safety and efficacy of VBR in combination with entecavir (ETV) in treatment-naïve patients with cHBV. METHODS: HBeAg-positive, treatment-naïve patients without cirrhosis were randomised 1:1 in a double-blind manner to once-daily VBR 300 mg+ETV 0.5 mg or placebo (PBO)+ETV 0.5 mg for 24 weeks. The primary endpoint was change in mean log10 HBV DNA from Baseline to Week 12 and 24. RESULTS: All patients in both treatment groups (PBO+ETV: 12/12; VBR+ETV: 13/13) completed the study. At Week 12, VBR+ETV led to a greater mean (SD) reduction from Baseline in log10 IU/ml HBV DNA (-4.45 [1.03]) vs. PBO+ETV (-3.30 [1.18]; p = 0.0077). At Week 24, VBR+ETV led to a greater reduction from Baseline in log10 IU/ml HBV DNA (-5.33 [1.59]) vs. PBO+ETV (-4.20 [0.98]; p = 0.0084). Greater mean reductions in pregenomic RNA were observed at Week 12 and 24 in patients receiving VBR+ETV vs. PBO+ETV (p <0.0001 and p <0.0001). Changes in viral antigens were similar in both groups. No drug interaction between VBR and ETV was observed. Two patients experienced HBV DNA rebound during treatment, with no resistance breakthrough detected. The safety of VBR+ETV was similar to PBO+ETV. All treatment-emergent adverse events and laboratory abnormalities were Grade 1/2. There were no deaths, serious adverse events, or evidence of drug-induced liver injury. CONCLUSIONS: In this 24-week study, VBR+ETV provided additive antiviral activity over PBO+ETV in treatment-naïve patients with cHBV, with a favourable safety and tolerability profile. CLINICAL TRIAL NUMBER: NCT03577171 LAY SUMMARY: Hepatitis B is a long-lasting viral infection of the liver. Current treatments can suppress hepatitis B virus but do not offer the opportunity of cure, hence, new treatment approaches are required. Herein, we show that the combination of the novel core inhibitor vebicorvir with an existing antiviral (entecavir) in treatment-naïve patients chronically infected with hepatitis B virus demonstrated greater antiviral activity than entecavir alone. Additionally, vebicorvir was safe and well tolerated. Thus, further studies evaluating its potential role in the treatment of chronic hepatitis B are warranted.

Safety and efficacy of vebicorvir in virologically suppressed patients with chronic hepatitis B virus infection.

BACKGROUND & AIMS: HBV nucleos(t)ide reverse transcriptase inhibitors (NrtIs) do not completely suppress HBV replication. Previous reports indicate persistent viremia during NrtI treatment despite HBV DNA being undetectable. HBV core inhibitors may enhance viral suppression when combined with NrtIs. This phase II trial (NCT03576066) evaluated the efficacy and safety of the investigational core inhibitor, vebicorvir (VBR), in virologically- suppressed patients on NrtIs. METHODS: Non-cirrhotic, NrtI-suppressed patients with chronic HBV were randomised to VBR 300 mg once daily or matching placebo (PBO) for 24 weeks. Treatment was stratified by hepatitis B e antigen (HBeAg) status. The primary endpoint was change from Baseline in serum HBeAg or hepatitis B surface antigen (HBsAg) after 24 weeks. RESULTS: Of 73 patients enrolled, 47 were HBeAg positive and 26 were HBeAg negative. In HBeAg-positive and -negative patients, there were no differences in the change from Baseline at Week 24 for HBsAg or HBeAg. Using a novel, high-sensitivity assay to detect HBV DNA, a greater proportion of patients with detectable HBV DNA at Baseline achieved undetectable HBV DNA at Week 24 in the VBR+NrtI vs. PBO+NrtI group. In HBeAg-positive patients, a greater change from Baseline in HBV pregenomic (pg)RNA was observed at Week 24 with VBR+NrtI vs. PBO+NrtI. Treatment-emergent adverse events (TEAEs) in VBR+NrtI patients included upper respiratory tract infection, nausea, and pruritus. No serious adverse events, Grade 4 TEAEs, or deaths were reported. CONCLUSIONS: In this 24-week study, VBR+NrtI demonstrated a favourable safety and tolerability profile. While there were no significant changes in viral antigen levels, enhanced viral suppression was demonstrated by greater changes in DNA and pgRNA with the addition of VBR compared to NrtI alone. CLINICAL TRIALS NUMBER: NCT03576066. LAY SUMMARY: Core inhibitors represent a novel approach for the treatment of chronic hepatitis B virus (HBV) infection, with mechanisms of action distinct from existing treatments. In this study, vebicorvir added to existing therapy reduced HBV replication to a greater extent than existing treatment and was generally safe and well tolerated.

Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production.

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.

Safety, pharmacokinetics, and antiviral effects of ABI-H0731, a hepatitis B virus core inhibitor: a randomised, placebo-controlled phase 1 trial.

BACKGROUND: Therapies with novel mechanisms of action against hepatitis B virus (HBV) infection are being explored with the goal of achieving a functional cure (sustained off-treatment response) without requiring lifelong therapy. We aimed to evaluate the pharmacokinetics, safety, and antiviral activity of ABI-H0731, an investigational inhibitor of the HBV core protein. METHODS: This phase 1, randomised, placebo-controlled study was done in two parts. In part 1, healthy adults without hepatitis B aged 18-65 years at one clinical research centre in New Zealand (eight participants per dose cohort) were randomly assigned (3:1) to receive single oral doses of ABI-H0731 (100, 300, 600, or 1000 mg) or matching placebo, or once-daily or twice-daily doses of ABI-H0731 800 mg or matching placebo for 7 days. In part 2, adults aged 18-65 years at clinical research centres in New Zealand, Australia, the UK, Hong Kong, Taiwan, and South Korea with chronic HBV (12 participants per dose cohort) were randomly assigned (5:1) to receive ABI-H0731 (100, 200, 300, or 400 mg) or matching placebo once daily for 28 days. In part 2, participants were required to have HBeAg-positive or HBeAg-negative chronic HBV infection, with serum HBV DNA concentrations of at least 2 × 104 IU/mL (HBeAg-positive) or 2 × 103 IU/mL (HBeAg-negative) and serum alanine aminotransferase concentrations less than seven times the upper limit of normal. Both parts used simple randomisation, with study participants, site personnel, and study monitors masked to treatment assignments. The primary study objective was dose-related safety and tolerability of ABI-H0731 in healthy volunteers and in participants with chronic HBV infection, assessed in all treated participants. Key secondary assessments included pharmacokinetic analyses and virological responses. This study is registered with ClinicalTrials.gov, identifier NCT02908191 and is completed. FINDINGS: 48 [61%] of 79 healthy volunteers were enrolled in the single-ascending or multiple-ascending dose phase of part 1 between Nov 16, 2016, and Jan, 27, 2017. 38 [55%] of 69 HBV-infected participants were enrolled in part 2 between June 15, 2017, and March 15, 2018. All adverse events were non-specific and of mild or moderate intensity apart from a single HBV-infected participant given the 400 mg dose who developed a severe (grade 3) maculopapular rash and terminated treatment. Overall, the most frequent adverse events of any grade among the 74 participants who received ABI-H0731 were headache (11 [15%]), influenza-like illness (seven [9%]), and dizziness (six [8%]); the most frequent adverse events considered treatment-related were rash (four [5%]) and dizziness (three [4%]). In part 1, ABI-H0731 reached maximum plasma concentrations (Tmax) in 2·50-4·17 h; the mean plasma half-life (t1/2) was 23·5-28·4 h. In part 2, mean maximum HBV DNA declines from baseline were 1·7 log10 IU/mL in the 100 mg dose cohort, 2·1 log10 IU/mL in the 200 mg dose cohort, and 2·8 log10 IU/mL in the 300 mg dose cohort. Across dose cohorts, serum HBV RNA declines correlated with HBV DNA declines. INTERPRETATION: No pattern of treatment-emergent adverse events was observed at ABI-H0731 doses up to 300 mg in individuals with chronic hepatitis B. ABI-H0731 was rapidly absorbed and exhibited a plasma half-life supportive of once-daily dosing. Dose-dependent decreases in serum HBV DNA and RNA concentrations are consistent with the proposed mechanism of action. FUNDING: Assembly Biosciences.

Drugs in the Pipeline for HBV.

Chronic hepatitis B remains a significant cause of morbidity and mortality worldwide. Most hepatitis B virus (HBV)-infected individuals are neither diagnosed nor treated. In those treated, nucleos(t)ide polymerase inhibitors persistently suppress viremia to the limits of quantitation; however, few achieve a "functional cure," defined as sustained off-treatment loss of detectable serum HBV DNA with or without loss of hepatitis B surface antigen. The low cure rate has been attributed to an inability to eliminate the viral reservoir of covalently closed circular DNA from hepatocytes. This review focuses on the diverse therapeutic approaches currently under development that may contribute to the goal of HBV cure.

Viral minority variants in the core promoter and precore region identified by deep sequencing are associated with response to peginterferon and adefovir in HBeAg negative chronic hepatitis B patients.

BACKGROUND AND AIM: Precore (PC) and basal core promoter (BCP) mutations are associated with responses to interferon-based treatment in HBeAg-positive chronic hepatitis B (CHB) patients. Here, we identify viral minority variants in these regions and assess association with response to peginterferon-alfa (Peg-IFN) and adefovir combination therapy. PATIENTS AND METHODS: Ultra-deep pyrosequencing analysis of the BCP and PC region was performed for 89 CHB patients (42 HBeAg-positive; 47 HBeAg-negative), at baseline and during treatment. Specifically, associations of individual positions with the HBeAg-negative phenotype were studied, as well as the association of the most prevalent mutations with combined response in HBeAg-positive and -negative patients at week 72 (HBeAg negativity, HBV-DNA <2000 IU/mL and ALT normalization at 24 weeks of treatment-free follow-up). RESULTS: The mutations most strongly correlated with the HBeAg-negative phenotype were at positions 1762/1764 and 1896/1899 in the BCP and PC region, respectively. No major changes in nucleotide composition of these positions were observed during treatment. In HBeAg-negative patients, a combined presence of 1764A and 1896A was correlated with lower ALT levels (p = 0.004), whereas the presence of 1899A was correlated with higher age (p = 0.030), lower HBV-DNA level (p = 0.036), and previous IFN therapy (p = 0.032). The presence of 1764A/1896A or the absence of 1899A at baseline, was associated with lower response rates, after adjustment for HBV genotype (p = 0.031 and p = 0.017) and HBsAg level (p = 0.035 and p = 0.022). CONCLUSION: We identified novel correlations between common BCP and PC variants with response to Peg-IFN and adefovir in HBeAg-negative patients. Ultimately, this may guide the selection of those patients most likely to benefit from Peg-IFN-based treatment.

HBsAg loss in patients treated with peginterferon alfa-2a and adefovir is associated with SLC16A9 gene variation and lower plasma carnitine levels.

BACKGROUND & AIMS: Achievement of HBsAg loss remains the hallmark of chronic hepatitis B treatment. In order to identify host factors contributing to treatment-induced HBsAg loss, we performed a genome-wide screen of single nucleotide polymorphisms (SNPs) and studied its immunological consequence. METHODS: Chronic hepatitis B patients (40 HBeAg-positive and 44 HBeAg-negative) treated with peginterferon alfa-2a and adefovir were genotyped for 999,091 SNPs, which were associated with HBsAg loss at week 96 (n = 9). Plasma carnitine levels were measured by tandem-mass spectrometry, and the effect of carnitine on the proliferative capacity of hepatitis B virus (HBV)-specific and non-specific CD8 T cells was studied in vitro. RESULTS: One polymorphism, rs12356193 located in the SLC16A9 gene, was genome-wide significantly associated with HBsAg loss at week 96 (p = 1.84 × 10(-8)). The previously reported association of rs12356193 with lower carnitine levels was confirmed in our cohort, and baseline carnitine levels were lower in patients with HBsAg loss compared to patients with HBsAg persistence (p = 0.02). Furthermore, we demonstrated that carnitine suppressed HBV-specific CD8 T cell proliferation. CONCLUSIONS: In chronic hepatitis B patients treated with peginterferon and adefovir, we identified strong associations of SLC16A9 gene variation and carnitine levels with HBsAg loss. Our results further suggest that a lower baseline plasma carnitine level increases the proliferative capacity of CD8 T cells, making patients more susceptible to the immunological effect of this treatment. These novel findings may provide new insight into factors involved in treatment-induced HBsAg loss, and play a role in the prediction of treatment outcome.

Reduction of HBV replication prolongs the early immunological response to IFNα therapy.

BACKGROUND & AIMS: The interaction between HBV replication and immune modulatory effects mediated by IFNα therapy is not well understood. We characterized the impact of HBV DNA replication on the early IFNα-induced immunomodulatory mechanisms. METHODS: We interrogated the transcriptional, serum cytokine/chemokine and cellular immune profiles of 28 patients with HBeAg+ chronic HBV infection (CHB) randomly assigned to one of 4 treatment cohorts (untreated n=5, weekly dosing of 360 µg Pegasys [PegIFNα] n=11, daily dose of 300 mg Viread [tenofovir disoproxil fumarate, TDF] n=6, or a combination of both n=6). Samples were characterized at multiple early time points through day 14 of therapy, after which all patients were given standard of care (180 µg Pegasys injected subcutaneously, weekly). RESULTS: PegIFNα induced a distinct and rapid up-regulation of IFN signaling pathway that coincided with increase detection of distinct serum cytokines/chemokines (IL-15, IL-6, and CXCL-10) and the up-regulation of the frequency of proliferating NK and activated total CD8+ T cells. IFNα treatment alone did not result in rapid decay of HBV replication and was not able to restore the defective HBV-specific T cell response present in CHB patients. In addition, the IFNα immune-stimulatory effects diminished after the first dose, but this refractory effect was reduced in patients where HBV replication was simultaneously inhibited with TDF. CONCLUSIONS: We present here the first comprehensive description of the early effects of IFNα treatment on immune and viral biomarkers in HBeAg+ CHB patients. Our results show that PegIFNα-induced innate immune activation directly benefits from the suppression of HBV replication.

Safety, pharmacokinetics and pharmacodynamics of GS-9620, an oral Toll-like receptor 7 agonist.

BACKGROUND: GS-9620 is a novel oral agonist of Toll-like receptor 7 (TLR7) in development for the treatment of chronic viral hepatitis. TLR7 is a highly conserved innate immune receptor expressed primarily on plasmacytoid dendritic cells and B lymphocytes. The aim of this double-blind, placebo-controlled, single ascending-dose study was to investigate the safety, tolerability, pharmacokinetics and pharmacodynamics of GS-9620 in healthy volunteers. METHODS: In total, 75 healthy volunteers (8 subjects in each of the 10 cohorts; 5 subjects participated in two cohorts) were randomized (6:2) to receive a single dose of GS-9620 (0.3, 1, 2, 4, 6, 8 or 12 mg) or placebo. RESULTS: GS-9620 was well-absorbed and well-tolerated in oral doses up to 12 mg. Minimal treatment-related adverse events were seen at doses up to 8 mg. Serum interferon (IFN)-α was only detected in subjects who received 8 or 12 mg doses, and the adverse event profile at 8 and 12 mg doses was generally consistent with that associated with IFN-α exposure (flu-like symptoms), consistent with the mechanism of TLR7 agonism. All adverse events resolved within 72 h. Induction of chemokines/cytokines and IFN-stimulated genes were seen at GS-9620 doses ≥ 2 mg, well below doses that induced serum IFN-α or led to clinical adverse events. CONCLUSIONS: GS-9620 demonstrates safety and pharmacodynamic activity at doses up to 12 mg. Pharmacodynamic activity is seen before adverse events, suggesting the potential for induction of an antiviral response without systemic adverse events in subjects with chronic viral hepatitis.

Lymphotoxin alpha-deficient mice clear persistent rotavirus infection after local generation of mucosal IgA.

Rotavirus is a major cause of pediatric diarrheal illness worldwide. To explore the role of organized intestinal lymphoid tissues in infection by and immunity to rotavirus, lymphotoxin alpha-deficient (LTα(-/-)) mice that lack Peyer's patches and mesenteric lymph nodes were orally infected with murine rotavirus. Systemic rotavirus was cleared within 10 days in both LTα(-/-) and wild-type mice, and both strains developed early and sustained serum antirotavirus antibody responses. However, unlike wild-type mice, which resolved the intestinal infection within 10 days, LTα(-/-) mice shed fecal virus for approximately 50 days after inoculation. The resolution of fecal virus shedding occurred concurrently with induction of intestinal rotavirus-specific IgA in both mouse strains. Induction of intestinal rotavirus-specific IgA in LTα(-/-) mice correlated with the (late) appearance of IgA-producing plasma cells in the small intestine. This, together with the absence of rotavirus-specific serum IgA, implies that secretory rotavirus-specific IgA was produced locally. These findings indicate that serum IgG responses are insufficient and imply that local intestinal IgA responses are important for the clearance of rotavirus from intestinal tissues. Furthermore, they show that while LTα-dependent lymphoid tissues are important for the generation of IgA-producing B cells in the intestine, they are not absolutely required in the setting of rotavirus infection. Moreover, the induction of local IgA-producing B cell responses can occur late after infection and in an LTα-independent manner.

Transcriptomic analysis of the woodchuck model of chronic hepatitis B.

UNLABELLED: The Eastern woodchuck (Marmota monax) is naturally infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to the human hepatitis B virus (HBV). The woodchuck is used as an animal model for studying chronic hepatitis B (CHB) and HBV-associated hepatocellular carcinoma (HCC) in humans, but the lack of sequence information has hitherto precluded functional genomics analysis. To address this major limitation of the model, we report here the sequencing, assembly, and annotation of the woodchuck transcriptome, together with the generation of custom woodchuck microarrays. Using this new platform, we characterized the transcriptional response to persistent WHV infection and WHV-induced HCC. This revealed that chronic WHV infection, like HBV, is associated with (1) a limited intrahepatic type I interferon response; (2) intrahepatic induction of markers associated with T cell exhaustion; (3) elevated levels of suppressor of cytokine signaling 3 (SOCS3) in the liver; and (4) intrahepatic accumulation of neutrophils. Underscoring the translational value of the woodchuck model, this study also determined that WHV-induced HCC shares molecular characteristics with a subtype of human HCC with poor prognosis. CONCLUSION: Our data establish the translational value of the woodchuck model and provide new insight into immune pathways which may play a role either in the persistence of HBV infection or the sequelae of CHB.

Effect of IL28B genotype on early viral kinetics during interferon-free treatment of patients with chronic hepatitis C.

BACKGROUND & AIMS: Although interleukin 28B (interferon, lambda 3) (IL28B) genotype affects the response of patients with chronic hepatitis C to peginterferon and ribavirin, little is known regarding its effect on response to direct-acting antivirals in interferon-free combinations. We analyzed the effects of IL28B genotype on the viral kinetic (VK) response to an interferon-free combination of the nucleoside polymerase inhibitor mericitabine (RG7128) and the hepatitis C virus (HCV) protease inhibitor danoprevir. METHODS: We performed a double-blind, dose-escalation study of patients with chronic HCV genotype 1 infection who were interferon treatment naive or had not responded to previous therapy with peginterferon and ribavirin. Patients were sequentially assigned to 1 of 7 cohorts then randomly assigned to groups that received up to 13 days of treatment with mericitabine (500 or 1000 mg, twice daily) plus danoprevir (100 or 200 mg, every 8 hours, or 600 or 900 mg, twice daily) or placebo. Eighty-three of 87 patients were genotyped for the IL28B single-nucleotide polymorphism rs12979860. VKs were analyzed only in patients who received 13 days of treatment, at optimal doses, using a biphasic model to describe first- and second-phase slopes of viral decay during therapy. RESULTS: At day 14 (the end of interferon-free treatment), the mean reduction in the serum level of HCV RNA was slightly greater in patients with the CC polymorphism (5.01 log(10) IU/mL) than those without (4.59 log(10) IU/mL). Modeling revealed that patients with the CC polymorphism had slightly better early VKs, most apparent in the ß-phase of viral decay. A mixed effect on the α-phase was observed, which was reduced in magnitude but prolonged in patients with CC, who also had better on-treatment response to peginterferon and ribavirin during follow up. CONCLUSIONS: IL28B genotype appears to affect early VKs in patients with chronic hepatitis C receiving interferon-free treatment.

Targeting hepatitis B virus-infected cells with a T-cell receptor-like antibody.

Virus-specific CD8 T cells are activated when their T-cell receptors (TCRs) recognize the specific viral peptide/major histocompatibility complex (MHC) class I (pMHC) complexes present on the surface of infected cells. Antibodies able to recognize the specific pMHC can mimic TCR specificity and both represent a valuable biological tool to visualize pMHC complexes on infected cells and serve as a delivery system for highly targeted therapies. To evaluate these possibilities, we created a monoclonal antibody able to specifically recognize a hepatitis B virus (HBV) envelope epitope (Env at positions 183 to 91 [Env183-91]) presented by the HLA-A201 molecule, and we tested its ability to recognize HBV-infected hepatocytes and to deliver a cargo to a specific target. We demonstrate that this antibody detects and visualizes the processed product of HBV proteins produced in naturally HBV-infected cells, is not inhibited by soluble HBV proteins present in patient sera, and mediates the intracellular delivery of a fluorescent molecule to target cells. Additionally, compared to CD8 T cells specific for the same HBV epitope, the TCR-like antibody has both a superior sensitivity and a specificity focused on distinct amino acids within the epitope. These data demonstrate that a T-cell receptor-like antibody can be used to determine the quantitative relationship between HBV replication and specific antigen presentation to CD8 T cells and serves as a novel therapeutic delivery platform for personalized health care for HBV-infected patients.

Oral combination therapy with a nucleoside polymerase inhibitor (RG7128) and danoprevir for chronic hepatitis C genotype 1 infection (INFORM-1): a randomised, double-blind, placebo-controlled, dose-escalation trial.

BACKGROUND: Present interferon-based standard of care treatment for chronic hepatitis C virus (HCV) infection is limited by both efficacy and tolerability. We assessed the safety, tolerability, and antiviral activity of an all-oral combination treatment with two experimental anti-HCV drugs-RG7128, a nucleoside polymerase inhibitor; and danoprevir, an NS3/4A protease inhibitor-in patients with chronic HCV infection. METHODS: Patients from six centres in New Zealand and Australia who were chronically infected with HCV genotype 1 received up to 13 days oral combination treatment with RG7128 (500 mg or 1000 mg twice daily) and danoprevir (100 mg or 200 mg every 8 h or 600 mg or 900 mg twice daily) or placebo. Eligible patients were sequentially enrolled into one of seven treatment cohorts and were randomly assigned by interactive voice or web response system to either active treatment or placebo. Patients were separately randomly assigned within each cohort with a block size that reflected the number of patients in the cohort and the ratio of treatment to placebo. The random allocation schedule was computer generated. Dose escalation was started in HCV treatment-naive patients; standard of care treatment-experienced patients, including previous null responders, were enrolled in higher-dose danoprevir cohorts. Investigators, personnel at the study centre, and patients were masked to treatment allocation. However, the pharmacist who prepared the doses, personnel involved in pharmacokinetic sample analyses, statisticians who prepared data summaries, and the clinical pharmacologists who reviewed the data before deciding to initiate dosing in the next cohort were not masked to treatment allocation. The primary outcome was change in HCV RNA concentration from baseline to day 14 in patients who received 13 days of combination treatment. All patients who completed treatment with the study drugs were included in the analyses. This study is registered with ClinicalTrials.gov, NCT00801255. FINDINGS: 88 patients were randomly assigned to a study drug treatment regimen (n=74 over seven treatment groups; 73 received at least one dose of study drug) or to placebo (n=14, all of whom received at least one dose). The median change in HCV RNA concentration from baseline to day 14 ranged from -3·7 to -5·2 log(10) IU/mL in the cohorts that received 13 days of combination treatment. At the highest combination doses tested (1000 mg RG7128 and 900 mg danoprevir twice daily), the median change in HCV RNA concentration from baseline to day 14 was -5·1 log(10) IU/mL (IQR -5·6 to -4·7) in treatment-naive patients and -4·9 log(10) IU/mL in previous standard of care null responders (-5·2 to -4·5) compared with an increase of 0·1 log(10) IU/mL in the placebo group. The combination of RG7128 and danoprevir was well tolerated with no treatment-related serious or severe adverse events, no grade 3 or 4 changes in laboratory parameters, and no safety-related treatment discontinuations. INTERPRETATION: This oral combination of a nucleoside analogue polymerase inhibitor and protease inhibitor holds promise as an interferon-free treatment for chronic HCV. FUNDING: Roche Palo Alto.

Actinomyces in chronic granulomatous disease: an emerging and unanticipated pathogen.

BACKGROUND: Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system that causes defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections, mostly by catalase-producing organisms. We report for the first time, to our knowledge, chronic infections with Actinomyces species in 10 patients with CGD. Actinomycosis is a chronic granulomatous condition that commonly manifests as cervicofacial, pulmonary, or abdominal disease, caused by slowly progressive infection with oral and gastrointestinal commensal Actinomyces species. Treatment of actinomycosis is usually simple in immunocompetent individuals, requiring long-term, high-dose intravenous penicillin, but is more complicated in those with CGD because of delayed diagnosis and an increased risk of chronic invasive or debilitating disease. METHODS: Actinomyces was identified by culture, staining, 16S ribosomal DNA polymerase chain reaction, and/or a complement fixation test in 10 patients with CGD. RESULTS: All 10 patients presented with a history of fever and elevated inflammatory signs without evident focus. Diagnosis was delayed and clinical course severe and protracted despite high-dose intravenous antibiotic therapy and/or surgery. These results suggest an unrecognized and unanticipated susceptibility to weakly pathogenic Actinomyces species in patients with CGD because these are catalase-negative organisms previously thought to be nonpathogenic in CGD. CONCLUSIONS: Actinomycosis should be vigorously sought and promptly treated in patients with CGD presenting with uncommon and prolonged clinical signs of infection. Actinomycosis is a catalase-negative infection important to consider in CGD.

Reactivation of hepatitis B with reappearance of hepatitis B surface antigen after chemotherapy and immunosuppression.

BACKGROUND & AIMS: HBV infection may reactivate in the setting of immunosuppression, although the frequency and consequences of HBV reactivation are not well known. We report 6 patients who experienced loss of serologic markers of hepatitis B immunity and reappearance of HBsAg in the serum as a result of a variety of acquired immune deficiencies. METHODS: Between 2000 and 2005, six patients with reactivation of hepatitis B were seen in consultation by the Liver Diseases Branch at the Clinical Center, National Institutes of Health. The course and outcome of these 6 patients were reviewed. RESULTS: All 6 patients developed reappearance of HBsAg and evidence of active liver disease after stem cell transplantation (n = 4), immunosuppressive therapy (n = 1), or change in human immunodeficiency virus antiretroviral regimen (n = 1), despite having antibody to HBsAg (anti-HBs) or antibody to hepatitis B core antigen (anti-HBc) without HBsAg before. All 6 patients developed chronic hepatitis B, 2 patients transmitted hepatitis B to their spouses, and 1 patient developed cirrhosis. The diagnosis of hepatitis B reactivation was frequently missed or delayed and often required interruption of the therapy for the underlying condition. None of the patients received antiviral prophylaxis against HBV reactivation. CONCLUSIONS: Serologic evidence of recovery from hepatitis B infection does not preclude its reactivation after immunosuppression. Screening for serologic evidence of hepatitis B and prophylaxis of those with positive results by using nucleoside analogue antiviral therapy should be provided to individuals in whom immunosuppressive therapy is planned.

Interactions among dendritic cells, macrophages, and epithelial cells in the gut: implications for immune tolerance.

The intestine is described as an immune privileged site where immunoregulatory mechanisms simultaneously defend against pathogens, yet preserve tissue homeostasis to avoid immune-mediated pathology in response to environmental challenges. Additionally, tolerance to ingested antigens promotes the development of systemic unresponsiveness towards the same antigens. It is increasingly clear that this tolerance is a complex process that derives from the coordinated action of both canonical immune and non-immune cells at mucosal sites, including dendritic cells, macrophages and epithelial cells. Recent evidence suggests that dysregulation in gut-induced tolerance and commensal bacterial handling affects both local and systemic compartments and contributes to autoimmune disease. Understanding how tolerance is achieved at mucosal sites may thus be exploited to re-establish tissue homeostasis.

Severe community-acquired pneumonia due to Staphylococcus aureus, 2003-04 influenza season.

During the 2003-04 influenza season, 17 cases of Staphylococcus aureus community-acquired pneumonia (CAP) were reported from 9 states; 15 (88%) were associated with methicillin-resistant S. aureus (MRSA). The median age of patients was 21 years; 5 (29%) had underlying diseases, and 4 (24%) had risk factors for MRSA. Twelve (71%) had laboratory evidence of influenza virus infection. All but 1 patient, who died on arrival, were hospitalized. Death occurred in 5 (4 with MRSA). S. aureus isolates were available from 13 (76%) patients (11 MRSA). Toxin genes were detected in all isolates; 11 (85%) had only genes for Panton-Valentine leukocidin. All isolates had community-associated pulsed-field gel electrophoresis patterns; all MRSA isolates had the staphylococcal cassette chromosome mec type IVa. In communities with a high prevalence of MRSA, empiric therapy of severe CAP during periods of high influenza activity should include consideration for MRSA.

Severe community-onset pneumonia in healthy adults caused by methicillin-resistant Staphylococcus aureus carrying the Panton-Valentine leukocidin genes.

BACKGROUND: Recent worldwide reports of community-onset skin abscesses, outbreaks of furunculosis, and severe pneumonia associated with methicillin-resistant Staphylococcus aureus (MRSA) carrying Panton-Valentine leukocidin (PVL) genes and the staphylococcal cassette chromosome mec (SCCmec) type IV indicate that MRSA infections are evolving into a community-related problem. The majority of cases reported to date involve skin and soft-tissue infections, with severe pneumonia representing a relatively rare phenomenon. During a 2-month period in the winter of 2003-2004, four healthy adults presented to 1 of 2 Baltimore hospitals with severe necrotizing MRSA pneumonia in the absence of typical risk factors for MRSA infection. METHODS: Patients' MRSA isolates were characterized by strain typing with use of pulsed-field gel electrophoresis and SCCmec typing with use of a multiplex polymerase chain reaction (PCR) assay and detection of PVL genes by PCR. RESULTS: All 4 patients' MRSA isolates carried the PVL genes and the SCCmec type IV element and belonged to the USA300 pulsed-field type. These 3 findings are among the typical characteristics of community-onset MRSA strains. In addition, 2 of our patients had concomitant influenza A diagnosed, which likely contributed to the severity of their presentation. CONCLUSIONS: To our knowledge, these patients represent the first reported North American adults with severe community-onset MRSA pneumonia caused by strains carrying the PVL genes.